SYMPOSIA - ComBio · repressed and male differentiation was not promoted similar to the case of NANOS2-null. On the other hand, some NANOS2-null properties, meiosis initiation and

ComBio2018 s Darling Harbour, Sydney s 23 - 26 September, 2018 Page 21

symposia

Monday - Wednesday

Page 22 ComBio2018 s Darling Harbour, Sydney s 23 - 26 September, 2018

SymPoSia moNDay

SYM-01-01 SYM-O2-01

SYM-02-02 SYM-02-03

ASSESSMENT, RESEARCH, AND PUBLICATION: NAVIGATING THE STAGES OF EDUCATION SCHOLARSHIP

Dolan E.L. University of Georgia, Athens, GA, USA.

Are you just learning what assessment means and wondering how to do it? Have you collected data in your class and are thinking about how to share it with colleagues who may be interested? Have you conducted an education study and are trying to figure out how to publish the results? This session will touch on key issues related to all three of these education scholarship topics: classroom assessment, education study design, and publication, ending with discussion about challenges conferees have faced in designing, conducting, and reporting on their educational work. Bring your questions!

COTTON SEED FIBRE MUTANTS PROVIDE INSIGHTS INTO CRITICAL DETERMINANTS OF CELL WALL DEVELOPMENTAL PROCESSES

Pettolino F., Liu S., Moncuquet P., Wilson I., White R., Macmillan C., Yulia D. and Llewellyn D. CSIRO Agriculture & Food, Canberra, ACT 2601, Australia.

Plant cell wall composition and structure are important for fundamental processes and agronomic traits. The composition and structure of the developing cotton seed fibre cell wall, and that of mature fibre, are critical determinants of fibre properties including length, fineness, strength and maturity that define commercial fibre quality. For example, fibre cells can reach great lengths due to the controlled flexibility of the walls which allows them to elongate but not widen, and the deposition and organisation of the cellulosic secondary cell wall is essential for strength. We are using multiple approaches to understand and control cell wall composition and its impact on cotton fibre properties. We have undertaken a comprehensive analysis of cell wall composition and gene expression throughout seed fibre development (MacMillan & Birke et al., 2017. BMC Genomics, 18:539); manipulated the expression of cell wall genes involved in fibre composition and quality; and generated fibre quality mutants to identify genes and pathways involved in determining fibre properties. I will present our current findings from the study of a short seed fibre mutant including phenotyping, from the whole plant to the cell wall; transcript profiling; genome sequencing; and gene mapping to identify the mutated gene and understand pathways associated with fibre and cell wall development.

MARCHANTIA: A SIMPLE MODEL SYSTEM TO STUDY CELL WALL BIOSYNTHESIS

Lampugnani E.R.1, Golz J.F.1, Flores-Sandoval E.2, Roberts E.3, Ullah M.O.4, Khan G.A.1, Bacic A.5, Bulone V.4, Bowman J.L.2and Persson S.1 1School of BioSciences, University of Melbourne, Australia. 2School of Biological Sciences, Monash University, Australia. 3Department of Biology, Rhode Island College, USA. 4ARC Centre of Excellence in Plant Cell Walls, and School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, Australia. 5La Trobe Institute for Agriculture and Food, School of Life Sciences, La Trobe University, Australia.

The liverwort Marchantia polymorpha is a basal land plant with many of the key innovations seen in angiosperms. With little genetic redundancy in its genome, Marchantia is an ideal system for studying cell wall biosynthesis in plants. Its walls are mostly composed of cellulose with the main non-cellulosic polysaccharides being xyloglucan and heteromannan. To facilitate dissection of the genes involved in cell wall biosynthesis, we have constructed a library of Marchantia glycosyltransferases (GTs) and used it to unravel processes involved in cellulose biosynthesis. In angiosperms, such as Arabidopsis, cellulose is produced by large CELLULOSE SYNTHASE A (CESA) complexes (CSCs) that are embedded in the plasma membrane. Each CSC is heteromeric, being composed of the products of three or more CESA genes. By contrast, Marchantia has only two CESA genes with non-overlapping expression profiles. Freeze-fracture electron microscopy experiments show rosette-like structures in the plasma membrane, suggesting that Marchantia CSCs are homomeric. Loss of Marchantia CESA function leads to plantlets with severe growth defects linked to the loss of cellulose from their cell walls. Together, these data indicate that cellulose synthesis in Marchantia requires only a single protein, indicating that the heterotrimeric CESA dogma may need revision.

IDENTIFICATION OF A NOVEL POLYSACCHARIDE IN LAND PLANTS AND SYNTHESIS BY MEMBERS OF THE CELLULOSE SYNTHASE-LIKE F GENE FAMILY

Little A.1, Lahnstein J.1, Jeffery D.W.2, Khor S.F.1, Schwerdt J.G.1, Shirley N.J.1, Hooi M.3, Xing X.1, 3, Burton R.A.1 and Bulone V.1, 3 1ARC Centre of Excellence in Plant Cell Walls, School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia. 2School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia. 3Adelaide Glycomics, School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia.

As a significant component of monocot cell walls, (1,3;1,4)-β-glucan has conclusively been shown to be synthesised by the cellulose synthase-like F6 protein. In this study, we investigated the synthetic activity of other members of the barley CslF gene family using heterologous expression. As expected, the majority of the genes encode proteins that are capable of synthesising detectable levels of (1,3;1,4)-β-glucan, however, overexpression of HvCslF3 and HvCslF10 genes resulted in the synthesis of a novel polysaccharide. A simple diagnostic assay has been developed and the polysaccharide has been biochemically characterised. To demonstrate that this product was not an aberration of the heterologous system, the characteristic polysaccharide linkages were confirmed to be present in wild type barley tissues known to contain HvCslF3 and HvCslF10 transcripts. The finding of this linkage in land plants has significant implications for defining the cell wall content of many crop species and challenges the concept that members of a single Csl family possess the same carbohydrate synthetic activity.


SymPoSia moNDay

SYM-02-04 SYM-O2-05

SYM-03-01 SYM-03-02

PHI THICKENINGS IN BRASSICA ROOTS - AN ADAPTION TO WATER STRESS?

Collings D.A., Aleamotu’a M. and McCurdy D.W. The University of Newcastle, Callaghan NSW 2308 Australia.

With the Earth’s population to pass 9 billion by 2050, and with climate change increasing rainfall variability, breeding crop varieties more efficient in water usage and resistant to water stress is essential. We have investigated phi thickenings, unusual secondary cell wall thickenings found in the root cortex where only thin, primary cell walls normally occur. These bands extend around radial cell walls, with synthesis coordinated between adjacent cells so that in transverse view they look similar to the Greek letter phi. Surprisingly little research into phi thickenings has been conducted, despite the observations that they can be induced by abiotic stresses in a wide variety of evolutionarily-diverse species. Currently, their function(s) remain unclear. We investigated phi thickenings in Brassica roots grown on agar plates. Confocal microscopy demonstrated the lignified cell walls of phi thickenings forming a complex network of thick reinforcements surrounding the inner cortical cells of the root, and a more delicate, reticulate network on the inner face of these cells adjoining the endodermis. Quantification of phi thickenings showed induction in response to water stress caused by salt (40 mM or higher) or sucrose (1% or higher). However, induction strongly depended on cultivar: of more than 20 commercial Brassica oleracea cultivars, some such as “golden acre” lacked inducible phi thickenings whereas others such as “marathon F1” induced strongly. Similar strong cultivar differences occur in B. napus roots where the winter varieties (for example “edimax”) lacked phi thickening induction whereas spring varieties such as “hyola474CL” induced strongly. These observations provide a platform to test phi thickening functions, and discover the genetic pathways leading to their formation. Such information might be applicable to crop breeding strategies in Brassica, and other crops where phi thickenings occur, to develop varieties with improved resistance to water stress.

COLD, ANTIOXIDANT AND OSMOTIC PRE-TREATMENTS MAINTAIN THE STRUCTURAL INTEGRITY OF MERISTEMATIC CELLS AND IMPROVE PLANT REGENERATION IN CRYOPRESERVED KIWIFRUIT SHOOT TIPS

Mathew L.1, 2, McLachlan A.1, Jibran R.1, Burritt D.J.2 and Pathirana R.1 1The New Zealand Institute for Plant and Food Research Limited, Private Bag 11600, Palmerston North 4442, New Zealand. 2Department of Botany, University of Otago, PO Box 56, Dunedin 9054, New Zealand.

Cryopreservation is a reliable and cost-effective method for the long-term preservation of clonally propagated plants. The number of clonally propagated species conserved by cryopreservation is increasing as vitrification-based methods are developed; droplet vitrification is becoming the preferred method for many species, as it ensures fast freezing and thawing rates. We investigated whether cold, antioxidant and osmotic pre-treatments could maintain the structural integrity of cells, thus aiding in developing a droplet vitrification protocol for kiwifruit, using Actinidia chinensis var. chinensis ‘Hort16A’ as a model. Cold acclimation of donor plantlets at 4°C for 2 weeks followed by sucrose pre-culture of shoot tips and supplementing all media used throughout the procedure with ascorbic acid (0.4 mM) resulted in 40% regeneration after cryopreservation. Transmission electron microscopy was used to examine meristematic cell structure at every critical step of droplet vitrification. After treatment in vitrification solution, meristematic cells from cold-acclimated plantlets pre-treated with sucrose and ascorbic acid exhibited severe plasmolysis and some disruption of membrane and vacuoles. In contrast, cells without pre-treatments exhibited minimal changes even after exposure to vitrification solution. However, after cryopreservation and recovery, all shoot-tip cells not pre-treated showed rupturing of the plasma membrane, loss of cytoplasmic contents and organelle distortion. By comparison, most pre-treated shoot-tip cells from cold-acclimated plantlets retained their structural integrity after cryopreservation, suggesting that only dehydrated and plasmolysed cells can withstand cryopreservation by vitrification.

REQUIREMENT OF DDX6 MEDIATED P-BODY FORMATION FOR THE FUNCTION OF NANOS2 IN MALE GERM CELL DIFFERENTIATION

Saga Y. National Institute of Genetics, Mishima, Japan.

In embryonic male gonads, germ cells produce abundant mRNA-protein complex called P-body. The formation of P-bodies is one of sex specific characteristics and is associated with male germ cell differentiation in the embryonic stage. We reported previously that an RNA binding protein NANOS2 is recruited to P-body and plays an essential role for germ cell differentiation. However, it is not clear whether P-body formation is necessary for NANOS2 to control the target RNAs and whether all NANOS2 functions are dependent on P-bodies. To understand relationship between P-body formation and NANOS2 function, we deleted P-bodies by knocking out DDX6 gene, essential for P-body formation in a germ cell-specific manner. For this purpose, we produced an XY-ES cell line which contains a germ-cell specific inducible Cre recombinase together with Cre reporter and introduced floxed DDX6 alleles via Cas9-mediated homologous recombination. The results show that NANOS2-target Dazl was not repressed and male differentiation was not promoted similar to the case of NANOS2-null. On the other hand, some NANOS2-null properties, meiosis initiation and germ cell escape from seminiferous tubules are not observed in DDX6-KO germ cells, indicating that NANOS2 also works in a P-body-independent manner. This study also demonstrates that ES-mediated chimera method offers a powerful method as a standard genetic tool.

YAP AND TAz IN TISSUE REGENERATION AND CANCER

Thompson, B.

ABSTRACT NOT AVAILABLE AT TIME OF PUBLICATION


SymPoSia moNDay

SYM-03-03 SYM-O3-04

SYM-03-05 SYM-04-01

UNRAVELING COMMON PATHOGENIC PATHWAY UNDERLYING THE FORMATION OF HYPOPLASTIC LEFT HEART SYNDROME

Fonoudi H.1, 2, Bosman A.1, Humphreys D.1, Patrick R.1, Blue G.3, Hill A.1, Ho J.1, Winlaw D.3 and Harvey R.1, 2 1Victor Chang Cardiac Research Institute, Sydney. 2St. Vincent׳s Clinical School, University of New South Wales, Sydney. 33. The Heart Centre for Children, The Children’s Hospital at Westmead, Sydney.

Hypoplastic left heart syndrome (HLHS) is a genetically complex disease, characterized by hypoplasia of the left side of the heart. Although being one of the most severe forms of congenital heart defects, our knowledge of the molecular underpinnings is very limited. Here, we have generated an in vitro model of HLHS using human induced pluripotent stem cells (hiPSCs) to uncover disease-causing factors. hiPSCs were generated from 10 HLHS patients and their parents (3 clones per individual; 87 hiPSC lines in total). To investigate differences during early cardiovascular development, hiPSCs were differentiated using both embryoid body and small moleculte cardiac-directed differentiation methods, and their cellular populations and gene expression were studied. Gene expression analysis of spontaneously differentiated cells showed lower expression of both cardiac and vascular smooth muscle markers in patients compared to controls. Flow cytometry analysis performed on hiPSC cultures after directed cardiac differentiation at 5-day intervals (day 0-30) showed cardiomyocyte differentiation in HLHS-hiPCSs was perturbed. Time-course RNA-seq of 5 HLHS families revealed down-regulation of cell cycle. This was confirmed using another 5 indipendent HLHS families. Cell phenotyping also indicated that beating cardiomyocytes derived from patients were immature and their calcium flux properties were significantly different. In summary, our findings suggest that the progression of cardiogenesis and vasculogenesis in HLHS-hiPSCs is perturbed, which may include problems in the cell cycle. Furthermore, the functionality of cardiomyocytes derived from HLHS-hiPSCs with respect to calcium flux properties was altered, suggestive of cardiomyocyte immaturity.

A HIGHLY SELECTIVE MECHANISM TO SUPPRESS RETROTRANSPOSON EXPRESSION IN DROSOPHILA

Hayashi R.1, Handler D.2, Brandstaetter S.2, Helmrath S.2, Felder A.K.2 and Brennecke J.2 1The John Curtin School of Medical Research (JCSMR), The Australian National University, 131 Garran Road, Acton ACT 2601, Australia. 2Institute of Molecular Biotechnology of Austrian Academy of Sciences (IMBA), Dr Bohr-Gasse 3, 1030 Vienna, Austria.

A selective and plastic mechanism to silence parasitic genetic elements is pivotal to maintain the integrity of eukaryotic genomes. In the somatic compartment of fruit fly ovaries, gypsy class LTR-retrotransposons are suppressed by a class of small RNA called Piwi-interacting RNAs (piRNAs). piRNAs in this tissue are predominantly derived from a single precursor RNA called flamenco (flam), which is transcribed from the ~300 kb genomic locus that is enriched of gypsy-family transposon sequences. Although piRNAs are also derived from other cytoplasmic transcripts, flam RNA produces piRNAs far more efficiently (10 ~ 10^3 fold). The mechanism that underlies the selectivity is not known. In the present work, we show that the piRNA biogenesis factor Fs(1)Yb selects flam RNA against other RNAs. Upon depletion of Fs(1)Yb, flam-derived piRNAs are largely lost while other cellular RNAs including mRNAs become preferentially processed into piRNAs. Based on a computational analysis, we were able to uncover the underlying features in piRNA-precursor transcripts to recruit Fs(1)Yb. Our work therefore sheds light on the fundamental question of how the cell distinguishes self from non-self transcripts.

ELEVATED WNT SIGNALLING DISRUPTS HEART DEVELOPMENT AND MAY UNDERLIE SOME CASES OF HUMAN HETEROTAXY

Diamand K.E.M., Barratt K.S., Alzahrani A.S., Walsh K., Ahmed J.N. and Arkell R.M. John Curtin School of Medical Research, The Australian National Univeristy, Canberra, ACT, 2601, Australia.

Congenital heart disease (CHD) is the most common type of birth defect and can occur in isolation or as part of a syndrome such as Heterotaxy, in which the laterality of internal organs is disrupted. Many cardiovascular abnormalities are associated with low heritability, hindering investigations into the genetic causes of CHD. Heterotaxy is the most highly heritable cardiovascular abnormality and is frequently shown to arise from mutation of the ciliome. Mutation of the X-linked transcription factor ZIC3 is associated with both isolated CHD and Heterotaxy but the cellular and molecular cause of ZIC3-associated Heterotaxy remains unknown. A genetic screen for mutations that affect murine embryogenesis identified a novel null allele of Zic3, called katun (Ka). The mutant embryos exhibit Heterotaxy and also incompletely penetrant, partial (posterior) axis duplications and anterior truncation. These latter two phenotypes are redolent of elevated canonical Wnt signalling. ZIC3 is a member of the Zic family of transcriptional regulators and previous work has shown that the ZICs can interact with TCF proteins to inhibit Wnt/β-catenin mediated transcription when overexpressed in cell lines. This raises the possibility that dysregulated WNT signalling may underlie some cases of Heterotaxy and CHD. We have investigated this notion using mouse genetics and find that ZIC3 loss-of-function leads to elevated WNT signalling, that elevated WNT signalling is consistently associated with heart defects and Heterotaxy during embryogenesis in the absence of pronounced cilia defects. Precisely how elevated WNT signalling interferes with the laterality of internal organs is being investigated.

STRUCTURE OF A CHOLINERGIC POSTSYNAPTIC MEMBRANE

Unwin P.N.T. MRC Laboratory of Molecular Biology, Cambridge UK.

Cholinergic postsynaptic membranes are specialized membranes of the nerve-muscle synapse, designed to depolarise rapidly when activated by the transmitter acetylcholine (ACh) released from a nerve terminal into the synaptic cleft. The resident postsynaptic ion channels, nicotinic ACh receptors, mediate the electrical response by opening cation-selective pathways across the muscle cell membrane, signaling the muscle to contract. Membrane lipids are known to play a vital role in this process, which we are currently investigating by cryo-EM, using postsynaptic membranes from the Torpedo electric ray. We find that cholesterol segregates away from the phospholipids in the vicinity of the ACh receptors, associating robustly with specific transmembrane sites and forming microdomain bridges between neighbouring protein molecules. The cholesterol-interacting parts of the receptor are those most directly implicated in controlling cation conductance and gating of the channel. Since cholesterol harbors a sterol group it would confer local rigidity to the membrane, particularly when packed side-by-side as in a microdomain. Our results suggest that such structural support is needed to stabilize the transmembrane architecture and to restrict mobility to relevant regions of the protein so that a productive conformational change occurs upon acetylcholine release, ensuring a maximal depolarizing response.


SymPoSia moNDay

SYM-04-02 SYM-O4-03

SYM-04-04 SYM-04-05

STRUCTURE OF HUMAN TELOMERASE

Rhodes D. NTU Institute of Structural Biology, Nanyang Technological University, Experimental Medicine Building, 6-03, 59 Nanyang Drive, Singapore 636921.

Telomeres, the protein-DNA complexes that cap the ends of eukaryotic chromosomes, are essential for genome stability and cell viability. In eukaryotes, telomere length is maintained by telomerase, a specialized reverse transcriptase capable of de novo DNA synthesis. Telomere length maintenance in stem cells and the majority of cancers is carried out by the specialized reverse transcriptase, telomerase. I will describe our recent 3D-structural analysis of human telomerase using single-particle cryo-EM as well as the biochemical and structural characterization of the telomerase recruitment complex.

STRUCTURAL BASIS FOR IMPORTIN ALPHA 3 BINDING SPECIFICITY OF W PROTEINS IN HENDRA AND NIPAH VIRUSES

Forwood J.K.1, Smith K.1, Tsimbulyak S.1, Cross E.1, Soares Dacosta T.2, Aragao D.3, Edwards M.4 and Basler C.4 1Charles Sturt University. 2La Trobe University. 3Australian Synchrotron. 4Georgia State University.

Nucleocytoplasmic transport of proteins is an essential cellular process that mediates gene expression, cell differentiation, and a wide range of disease pathways, including cancer and virus infection. Nuclear translocation of proteins via the classical import pathway is initiated upon recognition of a nuclear localization signal (NLS) by a member of the importin α subfamily. There are seven human isoforms of importin α that mediate nuclear import of cargo in a tissue- and isoform-specific manner. Our understanding as to how nuclear import adaptors differentially interact with cargo harbouring the same NLS remains poorly understood as all importin α isoforms are conserved in the domain responsible for NLS recognition. Here, we provide a structural basis for the nuclear import specificity of the W proteins of Hendra virus (HeV) and Nipah virus (NiV). Using structural approaches, we identify the interaction interfaces between importin α1 and α3 for the W proteins of both HeV and NiV, highlighting marked differences including a 2.4-fold more extensive interface and >50-fold binding affinity for importin α3. Using structure-guided design of importin α1 and α3 chimeric and mutant proteins, together with structures of importin α1 and α3 isoforms without cargo in the binding site, we establish that the molecular basis of specificity resides in the differential positioning of the armadillo (ARM)-repeats 7 and 8. Overall, our study provides new mechanistic insights into a range of important cellular and disease processes that are reliant on isoform specificity of nuclear import adaptors.

REGULATION OF FAT MASS AND OBESITY-ASSOCIATED (FTO) FUNCTION BY PROTEIN UBIQUITINATION

Widagdo J., Zhu T. and Anggono V. Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, Brisbane, Queensland, 4072, Australia.

The fat mass and obesity-associated (FTO) protein is an RNA demethylase that dynamically reverses the methylation of adenosine base at the N6 position (m6A). Genetic polymorphisms in FTO gene have been strongly associated with obesity in humans. The cellular level of FTO is tightly regulated, with alterations in its expression influencing energy metabolism, food intake and body weight. More recently, I have discovered a role for FTO in regulating m6A dynamics and memory consolidation in the mouse prefrontal cortex (Widagdo et al., J. Neurosci., 2016). Accumulating evidence has demonstrated the physiological importance of FTO proteostasis, however, the cellular mechanism underlying FTO protein turnover remains unknown. Here, I will present evidence that FTO undergoes post-translational ubiquitination on the evolutionary conserved Lys-216. CRISPR/Cas-9-mediated knock-in cells harboring the ubiquitin-deficient K216R mutation displayed a slower rate of FTO turnover, resulting in an increase in the level of FTO as well as enhanced phosphorylation of the ribosomal S6 kinase. Surprisingly, K216R mutation reduced the level of nuclear FTO and completely abolished the nuclear translocation of FTO in response to amino acid starvation. Collectively, my results reveal the functional importance of ubiquitination in controlling FTO expression and localization, which may be crucial for determining body mass and composition, as well as synaptic plasticity, learning and memory.

STRUCTURAL BASIS OF NAD+ CLEAVAGE ACTIVITY BY MAMMALIAN AND PLANT TIR DOMAINS

Horsefield S.1, Burdett H.1, Zhang X.2, 1, Shi Y.3, Manik M.K.1, Gu W.1, Chen J.1, Ve T.3, Dodds P.N.2 and Kobe B.1 1University of Queensland, Brisbane, Qld 4072, Australia. 2Agriculture and Food, Commonwealth Scientific and Industrial Research Organisation, Canberra, ACT 2601, Australia. 3Institute for Glycomics, Griffith University, Southport, QLD 4222, Australia.

TIR (Toll/interleukin-1 receptor, resistance protein) domains are key components of innate immunity and cell-death signaling pathways in animals and plants. Signaling depends on self-association and homotypic association of TIR domains. We have recently reconstituted large assemblies of the TLR (Toll-like receptor) adaptor TIR domains and determined the structure of the filamentous assembly of TLR adaptor MAL by cryo-electron microscopy. As an unexpected twist, we found that the TIR domains involved in cell-death pathways, including those from the mammalian TLR adaptor SARM, involved in axon degeneration, and those from plant immune receptors (NLRs), possess self-association-dependent NAD+-cleavage activity. Crystal structures of human SARM TIR domain and grapevine NLR Run1 TIR domain in complex with small-molecule ligands shed light on the structural basis of this enzymatic activity. Our studies unify the mechanism of function of TIR domains as “signaling by cooperative assembly formation (SCAF)” with prion-like features that leads to the activation of effector enzymes, and show that some TIR domains can themselves function as effector enzymes.


SymPoSia moNDay

SYM-05-01 SYM-O5-02

SYM-05-03 SYM-05-04

CHARACTERISATION OF IMPAIRMENTS IN GLUCOSE METABOLISM IN MODELS OF NEUROLOGICAL DISEASE AND TREATMENTS THEREOF

Borges K. University of QLD.

Energy is needed to prevent brain damage and seizures. In the CNS most ATP is generated by oxidative metabolism of glucose. In this talk I will highlight biochemical techniques to study CNS glucose metabolism in rodent models of neurological disease. Data using 13C-glucose injection followed by subsequent quantification of 13C-glucose derived metabolites in CNS tissue will be shown. This can be coupled with studying maximal activities of enzymes involved in glycolysis and the Krebs cycle. Applying these techniques in a chronic epilepsy model, my laboratory found that entry of glucose-derived carbons into the Krebs cycle is impaired along with reductions in pyruvate dehydrogenase (PDH) activity in the epileptogenic hippocampus. This indicates that ATP generation in the epileptogenic hippocampus will be reduced, which likely contributes to seizure generation, as ATP is vital to keep neuronal membrane potentials stable and to regulate neuronal signalling. To improve ATP generation, treatments should be aimed at either increasing PDH activity or circumventing the need of PDH for ATP generation. Thus, fuels that can enter the CNS, such as ketones or medium chain fats are likely to decrease seizure generation. Promising data from a clinical trial with medium chain fats in people with epilepsy will be shown.

MATHEMATICAL MODELLING OF AMINO ACID TRANSPORTER FLUXES IN ASTROCYTES

Todd A.C., Hulme S.R., Broer S. and Billups B. The Australian National University, Canberra, ACT 2600.

Astrocytes in the brain are closely associated with synapses. They support neurotransmission by sequestering the released glutamate and converting it to glutamine for recycling back to presynaptic terminals. We have previously demonstrated that astrocytic glutamate sequestration via excitatory amino acid transporters (EAATs) stimulates the release of glutamine via the System N neutral amino acid transporter SNAT3. Since the released glutamine plays a vital role in resupplying the presynaptic neurotransmitter pool, it is important to know how this process is regulated to ensure a continuous supply of neurotransmitters. Therefore, the aim of our current work is to understand how the different transporters function cooperatively to control amino acid fluxes. To investigate this, we have established a mathematical model of EAAT and SNAT3 fluxes based on kinetic rate equations. Parameters for the model were determined using electrophysioloical recording and fluorescence imaging of astrocytes in rat brain slices. Using this model, we show that EAAT-mediated fluxes of amino acids and sodium stimulate SNAT3-mediated glutamine release and that modulation of the intracellular sodium concentration is the main regulator of astrocytic glutamine efflux. Our results provide a clear demonstration of how these two transporters interact to regulate the release of amino acids from astrocytes. The model we have employed can be extended to include multiple simultaneous transport processes, and we will develop it further to understand how different plasma membrane transporters coordinate the homeostasis of amino acids in brain cells. This will provide significant benefit in enabling the prediction of the role of individual transporters in essential functions such as neurotransmission, cell growth and survival.

METABOLIC DYSFUNCTION IN MOTOR NEURONE DISEASE (MND): INSIGHTS GAINED FROM STUDIES IN THE LAB AND THE CLINIC

Ngo S.T.1, 2, 3 1Australian Institute for Bioengineering and Nanotechnology, University of Queensland. 2Queensland Brain Institute, University of Queensland. 3UQ Centre for Clinical Research, University of Queensland.

Characterised by the irreversible death of upper and lower motor neurones, MND is a disease for which there is no effective treatment, and no cure. In recent years, there have been an increasing number of reports that describe altered metabolic regulation in MND patients. While it is proposed that changes in energy homeostasis is a modifier of disease outcome, the clinical impact of metabolic changes on disease progression and survival has remained mostly undefined. In this talk, I will present data from mouse studies where we seek to characterise the impact of metabolic changes on disease course, while also interrogating the origin of metabolic dysregulation. I will then present data from our prospective, multi-centre study that highlights, for the first time, the negative prognostic impact of altered energy expenditure in people living with MND. Finally, I will touch on some of our most recent work, in which we are investigating the relationship between whole body energy expenditure and cellular bioenergetics using MND patient derived cells.

SLEEP AND CANCER: A CELL CULTURE MODEL OF SLEEP APNOEA ALTERS GENE EXPRESSION IN THE HYPOXIC, INFLAMMATORY AND CIRCADIAN RHYTHM PATHWAYS

Martinez C., Kerr B., Kataria N., Cistulli P. and Cook K.M. University of Sydney, Charles Perkins Centre, Sydney, NSW.

Obstructive sleep apnoea (OSA) affects a significant proportion of the population and is characterised by episodic upper airway obstruction resulting in systemic intermittent hypoxia. Recent epidemiological studies have shown OSA is associated with higher rates of cancer development and cancer mortality. Animal models of OSA have found increased metastasis and tumour growth. However, the molecular mechanisms associated with the altered tumour behaviour seen in OSA is poorly understood. Hypoxia inducible factor (HIF) is a transcription factor that can influence cancer growth and metabolism. In OSA, increased activity of HIF and NF-κB transcriptional activity is proposed to occur systemically and hence may affect tumours. However, we have yet to understand how HIF or NF-κB may be activated by rapid oxygen fluctuations, as compared to chronic tumour hypoxia, and how this may lead to altered cancer outcomes. We have established a cell-based model of OSA tissue oxygenation in order to study the effects of rapid, intermittent cycles of hypoxia in HCT116 colorectal cancer cells. Using quantitative rtPCR and western blotting, we found that HIF-1α increases during cycles of intermittent hypoxia and that expression of HIF target genes increase in response. We have also seen changes in inflammatory gene expression and changes in the pathways that regulate circadian rhythm. Inflammation and disruption of circadian rhythm have both been linked to cancer. Finally, we identified that the HIF-mediated response appears to be different in rapid intermittent hypoxia when compared to chronic tumour hypoxia. These differences may be the key to understanding how rapid intermittent hypoxia, as occurs in OSA, may influence tumour growth and metastasis.


SymPoSia moNDay

SYM-05-05 SYM-O6-01

SYM-06-02 SYM-06-03

COPPER(II) PREVENTS NEUROKININ B FUNCTIONAL AMYLOID FORMATION AND DISASSEMBLES PREFORMED FIBRILS

Jayawardena B.M. and Jones C.E. School of Science and Health, Western Sydney University, Locked bag 1797, Penrith, NSW, 2751.

Neurokinin B (NKB) belongs to a family of neuropeptides that have diverse roles in cognition, neuroprotection and neuroinflammation. NKB has a key role in reproduction and consequently is found in high concentrations in hypothalamic neurons that descend into the pituitary gland. Some evidence suggests NKB is packed into secretory granules as an amyloid, termed ‘functional amyloid’, which is thought to allow high peptide concentrations to be achieved in the granule. Several neuropeptides and hormones are known to form functional amyloids but interact with receptors in the monomeric form. The mechanisms underlying the disassembly of the amyloid are not clear, although dilution, pH and ionic strength are all thought to contribute. NKB is known to bind copper with reasonably high affinity. Metal binding does not affect receptor activation and it has remained unclear as to why NKB binds copper. In this work we use a variety of biophysical techniques to show that copper(II) has an important role in NKB fibrillogenesis. At stoichiometric concentrations, copper will completely inhibit formation of NKB fibrils and is the only biological metal that can achieve this. Importantly, the metal can also promote disassembly of preformed fibrils. These results will be discussed in the context of NKB’s function and compared to the role of metals in amyloidogenic neurodegenerative diseases.

TOOLS FOR THE DETECTION AND NETWORK CONTEXTUALISATION OF PROTEIN METHYLATION

Tay A., Pang C.N.I., Winter D.L., Hart-Smith G., Hamey J. and Wilkins M.R. School of Biotechnology and Biomolecular Sciences, University of New South Wales.

Protein methylation has recently emerged as one of the most widespread post-translational modifications of proteins in the eukaryotic cell. Its detection by mass spectrometry, however, is affected by high false discovery rates. Functionally, it is poorly understood yet it is known to modulate many protein-protein interactions and, in some cases, can interplay with other modifications. Here we describe two new tools that assist with the identification of methylated peptides from tandem mass spectrometry analyses (MS2 Deisotoper, MethylQuant). The first increases peptide identification rates through the deisotoping of fragmentation spectra, whereas the second takes advantages of heavy isotopic labelling (methyl-SILAC) to unequivocally identify sites of protein methylation. We also describe a new Cytoscape app (PTMoracle). This facilitates the contextualisation of methylation in protein interaction networks, with structural data associated with protein-protein interactions and with all other known post-translational modifications. Case studies will be given to illustrate the utility of these tools.

DIFFERENTIAL CORRELATION ACROSS RANKED SAMPLES FOR SINGLE CELL RNA-SEQUENCING DATA

Ghazanfar S., Strbenac D., Ormerod J.T., Yang J.Y.H. and Patrick E. The University of Sydney.

Genes act as a system and not in isolation. Thus, it is important to consider coordinated changes of gene expression rather than single genes when investigating biological phenomena. We have developed an approach for quantifying how changes in the association between pairs of genes may inform the outcome of interest called Differential Correlation across Ranked Samples (DCARS). Modelling gene correlation across a continuous sample ranking does not require the dichotomisation of samples into two distinct classes and can identify differences in gene correlation across early, mid or late stages of the outcome of interest. We have recently demonstrated the utility of DCARS in the context of assessing differential correlation across survival ranking in TCGA, and further explore the use of DCARS in the context of single cell RNA-Sequencing data. Furthermore, we demonstrate that DCARS can be used in conjunction with network analysis techniques to extract biological meaning from multi-layered and complex data.

MODELLING BREAST CANCER PROGRESSION USING SINGLE-CELL RNA-SEQ

Gloss B., Valdes-Mora F., Salomon R., Colino-Sanguino Y., Roden D., Ormandy C. and Gallego-Ortega D. Garvan Institute, 384 Victoria St Darlinghurst, NSW Australia.

Cancer cell diversity constitutes a challenge for cancer treatment and deeply impact the outcome of cancer patients. A simultaneous overview of cancer cells and associated stromal cells is critical for the design of improved therapeutic regimes. Single-cell RNA-seq has emerged as a powerful method to unravel heterogeneity of complex biological systems; this has enabled in vivo characterization of cell type compositions through unsupervised sampling and modelling of transcriptional states in single cells. Here we use the cell type agnostic, high-throughput microfluidic-based, single-cell RNA-seq method Drop-seq to elucidate the function and cellular composition of breast tumours. We use the MMTV-PyMT ± Elf5 mouse mammary tumour model to provide high-resolution landscapes of the disease and highlight cellular events that result in the acquisition of the metastatic phenotype. We show breast cancer cell composition and tumour heterogeneity with unprecedented definition, elucidating the cellular and molecular complexity of tumour progression within the context of a complex multicellular environment.


SymPoSia moNDay

SYM-06-04 SYM-O6-05

SYM-07-01 SYM-07-02

REMAPPING OUR RECENT EVOLUTIONARY HISTORY

Upton K.R. School of Chemistry and Molecular Biosciences, University of Queensland.

Transposons are mobile DNA sequences that replicate within a host genome and compose around half of the human genome. Although they were originally described as ‘controlling elements’ that are able to regulate gene expression, they are often derided as ‘Junk DNA’ with minimal benefit to the host. This belief has been reinforced by the technical limitation of uniquely identifying individual transposons in short-read sequencing data. Most reads that identify these elements align to multiple locations throughout the genome (multimapping reads). Traditional bioinformatic approaches have taken a conservative approach, only including reads that map to a unique location, resulting in an underrepresentation of transposons in functional models of genome regulation and reinforcing the Junk DNA hypothesis. Mappability analysis indicates ~40% of the human genome is adversely affected by the removal of multimapping reads, effectively masking the last 10 million years of our evolution from functional analysis. To address this gap in knowledge, my lab has developed and validated ReMapQ, a GPU-based machine learning algorithm incorporating a deep neural network to resolve the placement of multimapping reads. ReMapQ is able to incorporate all mappable reads, even those with 500 possible alignment locations. In-Silico validation with known-truth data sets has shown excellent performance, with ~90% precision in read placement. In a pilot analysis of Differentially Methylated Regions (DMRs) in triple negative breast cancer we have shown that biological signals identified by traditional analyses (including only high confidence single mapping reads) are highly conserved within ReMapQ processed data and have excellent correlation in log-fold-change values. Further, ReMapQ is able to identify around 50% more DMRs, not identified in single-mapping read analysis. We are working to refine this algorithm and apply it to integrated functional data sets to elucidate the functional impact of transposons and their role in disease and development in the human genome.

GENOMICS FOR COMMUNICABLE DISEASES SURVEILLANCE AND CONTROL IN AUSTRALIA

Howden B. The Microbiological Diagnostic Unit Public Health Laboratory.The University of Melbourne at the Doherty Institute for Infection and Immunity.

Microbial genomics is revolutionising the surveillance and response to communicable diseases in many countries around the world, however there are many challenges to ensure the full potential of genomics in this area is realised. Key areas of need include platforms for rapid, reproducible and accredited bioinformatics analyses; processes and regulatory changes to ensure effective data sharing at jurisdictional, national and international levels; and effective engagement and training of relevant public health professionals and epidemiologists. Through a coordinated approach, the implementation of genomics into routine communicable diseases surveillance and control in Australia will significantly enhance the quality of this system.

USING MACHINE LEARNING TO PERSONALIzE CRISPR-CAS9 APPLICATIONS

Wilson L.1, O’Brien A.1, Reti D.1, Horlbacher M.1, Dunne R.2 and Bauer D.1 1CSIRO, Sydney, NSW, Australia. 2CSIRO, Data61, NSW, Australia.

Numerous studies have sought to build machine learning models that predict general CRISPR-Cas9 activity and while great progress has been made, these approaches are still limited. Small sequence variations can have a dramatic effect on the CRISPR-Cas9 system, leading to changes in on-target activity or increases the number of off-targets. Despite this risk, current tools are not accounting for genetic variation among a population. To address this, we developed VARiant-aware detection and SCoring of Off-Targets (VARSCOT), which allows researchers to design personalized CRISPR-Cas9 applications for specific individuals or populations. VARSCOT is able to use variant information to identify CRISPR-Cas9 target sites unique to a specific individual or population. We find our tool to be the most sensitive detection method for off-targets, finding 40% to 70% more experimentally verified off-targets compared to other popular software tools. VARSCOT uses a machine learning model to score off-target activity, leading to a 98% reduction in false positives when predicting which off-targets are active. As off-target activity varies with CRISPR-Cas9 concentration, VARSCOT’s model provides a probabilistic scores that accounts for different conditions.

DEVELOPMENT OF AN OPTIMIzED NICOTIANA BENTHAMIANA HOST LINE FOR TRANSIENT EXPRESSION OF HUMANIzED IGG

Leblanc z., Mortimer C., Naim F. and Waterhouse P. Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Australia.

Production of biologics in plants is forecast to be a disruptive technology in the pharmaceutical industry. An ideal plant system for this application is N. benthamiana, which is a relative of the tobacco plant and native to Australia. A diminished viral defence response in N. benthamiana facilitates its transient transformation by the plant pathogen Agrobacterium tumefaciens. In commercial biologics production systems this pathogen/host interaction is exploited to produce large amounts of a desired recombinant protein. Genome editing technologies such as CRISPR/Cas9 provide a means to further improve N. benthamiana as a pharmaceutical and general biologics production host by increasing yield and human compatibility of recombinant proteins synthesized in this system. Leveraging the increased editing efficiencies seen in plant protoplast transformation as well as the granularity provided by this transformation medium, this study aims to generate ideal plant host lines for the production of biologics through multiplex editing of genes involved in glycosylation and pathogen defence.


SymPoSia moNDay

SYM-07-03 SYM-O7-04

SYM-07-05 SYM-O8-01

MODELLING CANCER MUTATIONS USING CRIPSR/CAS9 GENOME EDITINGLannagan T.R.M.1, Lee Y.K.1, Wang T.1, Roper J.2, Bettington M.3, 4, Fennell L.4, Vrbanac L.1, Jonavicius L.5, Somashekar R.1 Gieniec K.1, Yang M.1, Ng J.Q.1, Suzuki N.1, Ichinose M.1, Wright J.A.1, Kobayashi H.1, Putoczki T.L.6, Abud H.E.7, Marker J.8, Kleve S.5, Wirapati A.9, Tejpar S.10, Leggett B.A.4, Whitehall V.L.4, Worthley, D.L.1* and Woods S.L.1* 1School of Medicine, University of Adelaide and South Australian Health and Medical Research Institute, Adelaide, SA Australia. 2Tufts Medical Center & Tufts University, School of Medicine, Boston, MA USA. 3Envoi Specialist Pathologists, Brisbane, QLD Australia. 4QIMR Berghofer Medical Research Institute, Brisbane, QLD Australia. 5Department of Anatomical Pathology, Flinders Medical Centre, Bedford Park, SA Australia. 6Department of Medical Biology, University of Melbourne and the Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC Australia 7Cancer Program, Monash Biomedicine Discovery Institute and the Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia8Cancer Voices SA, Adelaide, South Australia, Australia 9Swiss Institute of Bioinformatics, Bioinformatics Core Facility, Lausanne, Switzerland. 10Digestive Oncology Unit, Department of Oncology, University Hospitals Leuven, Leuven, Belgium. *=contributed equally In this age of next generation sequencing we are fast accruing more information on cancer associated genetic alterations than ever before. How do we translate this new knowledge into better outcomes for cancer patients? Clearly we must prioritise genetic alterations for study from this wealth of data. Here we utilise the organoid culture technique, combined with CRISPR/Cas9 genome engineering, to sequentially introduce genetic alterations associated with the serrated pathway to colorectal cancer (CRC). Our novel preclinical models enable therapeutic evaluation in known, complex genetic landscapes. These models can also be readily personalised to investigate the many leads generated by next generation sequencing of our patients.

MOUSE KNOCKOUT OF NUCLEAR FACTOR I GENES CAUSE CORTICAL MALFORMATIONS THAT PERSIST INTO ADULTHOOD

Bunt J.1, Boogert S.1, Lim J.W.C.1, Huth S.F.1, Dean R.J.1, Bridges C.1, Gronostajski R.M.2 and Richards L.J.1 1The University of Queensland, Queensland Brain Institute, Brisbane, Australia. 2State University of New York at Buffalo, Department of Biochemistry- Program in Genetics- Genomics and Bioinformatics- Center of Excellence in Bioinformatics and Life Sciences, Buffalo, USA.

The Nuclear factor I (NFI) family of transcription factors are required for the development of multiple organ systems. Using mouse knockout models, we previously demonstrated that family members NFIA, NFIB and NFIX are important for normal brain development. These proteins have overlapping biological functions in regulating the transition of progenitor cells from proliferation to differentiation and therefore result in overlapping neurodevelopmental defects in embryos. Nfia and Nfib knockout mice die at birth due to kidney and lung defects, respectively. To investigate whether the phenotypes observed in embryonic development persist into adulthood, we generated cortex-specific conditional knockout mouse models of Nfia and Nfib. These Nfiaflox or Nfibflox; Emx1-Cre mice are viable and fertile. Their postnatal cortical development is delayed, but no major defects are observed. In adulthood, these mutants have enlarged brains due to increased volume of the cerebral cortex and, in particular, the cingulate cortex. Preliminary assessment revealed only a minor behavioural phenotype. These observed phenotypes are comparable to those in humans with a deletion or mutation of NFI. Hence, we now have a model to further study the aetiology and the functional defects in the human disorders.

KEEP CALM AND CRISPR: JOINING THE GENOME EDITING REVOLUTION

Adikusuma A.1, Piltz S.1, 2, White M.1, 2, Robertson L.1, Dawson R.1, Hughes J.1 and Thomas P.Q.1, 2 1University of Adelaide, SA, Australia 5005. 2South Australian Health & Medical Research Institute, Adelaide, SA, Australia 5000.

CRISPR genome editing technology enables targeted genetic modification of virtually any species with unprecedented efficiency. For biomedical research, CRISPR technology offers unparalleled opportunities to develop accurate and sophisticated cell and animal disease models using virtually any species or cell type. Importantly, CRISPR can also be used to modify the human genome in vivo, enabling functional correction of disease-causing mutations for precision medicine applications. Prof Paul Thomas is Director of the SA Genome Editing (SAGE) facility and the Genome Editing Laboratory (GEL) at SAHMRI. He was an early adopter of CRISPR technology and his lab has generated over 60 mutant mouse lines using CRISPR editing. Prof Thomas will provide an overview CRISPR editing and describe novel applications and unexpected outcomes of this relatively new technology.

APPLYING FOR A TEACHING AWARD - INTERACTIVE WORKSHOP

Costabile M.1, 2 1School of Pharmacy and Medical Sciences, University of South Australia. 2Molecular Signalling Laboratory, University of South Australia Cancer Research Institute.

This session is aimed at academic staff interested in applying for a Teaching Award in the near future. The session will be run by Dr. Maurizio Costabile, who has been awarded teaching awards at University (UniSA Citation, 2013 and UniSA Digital Citation, 2015), National (Office of Learning and Teaching Citation, 2014) and International (Online Learning Consortium, Effective practice award, 2016, New Orleans) as well as being a finalist in the SA Science Excellence Awards for STEM Educator of the Year: Tertiary Teaching in 2017. The session will cover how to identify “excellence” in teaching, the forms of evidence required in an application and how it can be presented. How to craft your application, including creating and developing your narrative will be covered. The session will be run in an interactive workshop style with ample opportunity for questions and feedback during the session. Attendees that are at the start of their journey in applying for an award through to staff that have won local awards and are now seeking broader recognition are encouraged to attend.


SymPoSia moNDay

SYM-09-01 SYM-09-02

SYM-09-03 SYM-09-04

DYNAMIC CELLULAR INTERPLAY IN TISSUE REGENERATION

Rosenthal N.1, 2 1The Jackson Laboratory, Bar Harbor Maine USA. 2Imperial College London UK.

Regeneration, the restoration of original tissue structure and function in response to damage or disease, differs from tissue repair, in which collagen deposition and scar formation often lead to functional impairment. In both scenarios, an early inflammatory response is essential to clear damaged cells and initiate organ repair, but the quality and extent of immune engagement affects the outcome, with distinct responses underlying the repair of heart, skeletal muscle and skin. The diverse regenerative capacity of adult mammalian organs is also reflected in the tissue-specific composition and transcriptomic profiles of resident immune cell subsets and the stromal cells with which they interact. In the damaged heart, immune cells sense and modulate inflammation through a dynamic interplay with stromal cells in the cardiac interstitium, which either leads to recapitulation of cardiac morphology by rebuilding functional scaffolds to support muscle regrowth, or fails to resolve the inflammatory response and forms fibrotic scar tissue. Investigation into the mechanistic basis of homeostasis and restoration of cardiac function in an unbiased screen of recombinant inbred mouse panels has yielded dramatic strain-specific variation, offering unexpected insights into the functions of cardiac interstitial components and highlighting the potential for more genetically precise, organ-specific treatments of degenerative diseases.

ANALYSIS OF CARDIAC DIFFERENTIATION AT SINGLE CELL RESOLUTION REVEALS A REQUIREMENT OF HYPERTROPHIC SIGNALING FOR HOPX TRANSCRIPTIONFriedman C.1, Nguyen Q.1, Lukowski S.1, Chiu H.1, Bar-Joseph Z.5, Tam P.4, Murry C.E.3, Ruohola Baker H.3, Powell J.2 and Palpant N.J.1 1University of Queensland. 2Garvan Institute. 3University of Washington. 4University of Sydney. 5Carnegie Mellon University.

Differentiation into diverse cell lineages requires the orchestration of gene regulatory networks guiding diverse cell fate choices. Utilizing human pluripotent stem cells, we measured expression dynamics of 17,718 genes from 43,168 cells across five time points over a thirty day time-course of in vitro cardiac-directed differentiation. We used unsupervised clustering to identify transcriptional networks underlying lineage derivation of 15 subpopulations including mesoderm, definitive endoderm, vascular endothelium, cardiac precursors, and definitive cardiac fates including contractile cardiomyocytes and non-contractile derivatives. Utilizing customized machine learning algorithms, we analyzed scRNA-seq data to identify transcription factor regulatory networks linking the trajectory of subpopulations in vitro with cell types derived during cardiac development in vivo. We leveraged this data to study gene networks governing cardiomyocyte differentiation in vivo to advance translational applications of stem cells in disease modelling and therapies. Among a network of known genetic drivers of differentiation, we identified dysregulation of the non-DNA binding homeodomain protein, HOPX as a candidate cause for the immature state of in vitro derived cardiomyocytes. While HOPX is expressed in cardiac progenitor cells (CPC) in vivo, we show during in vitro differentiation that HOPX is expressed in only 16% of hPSC-derived cardiomyocytes. Using genetic models we determined the mechanisms underlying transcriptional regulation of HOPX. We show that HOPX is situated downstream from hypertrophic signaling, HOPX directly drives hypertrophic growth, and is required for expression of myofibrillar genes involved in cardiomyocyte maturation. Through genetic dissection underlying the HOPX transcriptional landscape, we show that the distal HOPX transcriptional start site is the primary regulatory driver of HOPX expression underlying hypertrophic stimulation. Taken together, we utilized single cell analysis of cardiac in vitro differentiation to identify mechanisms for activating gene networks in cardiac differentiation as they occur during in vivo heart development that enhance the utility of hPSCs for cardiac translational applications.

THE MECHANOSENSOR YAP DRIVES CUTANEOUS TYPE 2 INFLAMMATION AND ECzEMA DEVELOPMENT

Mendoza-Reinoso V.1, Corley S.2, Lim J.Y.1, Goh L.F.3, Tong P.4, Wilkins M.2, Common J.E.3, Roediger B.4 and Beverdam A.1, 5 1School of Medical Sciences, UNSW Sydney, NSW 2052, Australia. 2School of Biotechnology and Biomolecular Sciences, UNSW Sydney, NSW 2052, Australia. 3Institute of Medical Biology, A(⁎)STAR, 8A Biomedical Grove, Immunos 06-08, Singapore 138648, Singapore. 4Skin Inflammation Group, Centenary Institute, The University of Sydney, NSW 2050, Australia. 5School of Biomedical Sciences, The University of Queensland, QLD 4072, Australia.

One in five people in the Western world is affected by atopic dermatitis (AD)/eczema. This allergic skin disease develops as result of intrinsic epidermal barrier defects. These drive a local and systemic type 2 immune response, resulting in a pathological cycle of itching, scratching and inflammation, and eventually in AD pathogenesis. The exact mechanism of how epidermal barrier dysfunction, itch and type 2 inflammation connect at the molecular level remains poorly understood. Yes-associated protein (YAP) is a mechanosensor that responds to mechanical stimuli to control tissue homeostasis. Our transcriptomics analyses demonstrated a strong activation of the type 2 immune response in skin of transgenic mice expressing the dominant active YAP2-5SA-ΔC protein the basal epidermis. Re-assessment of the phenotype of YAP2-5SA-ΔC mice indeed demonstrated behavioral, histological, immunological and genetic features of atopic dermatitis. Interestingly, we also found that YAP was activated in an independent AD mouse model and in skin biopsies of human AD patients. Furthermore, we identified that YAP activity in keratinocytes drives IL-33 and CTSS expression in vitro and in the murine skin in vivo. We propose the YAP2-5SA-ΔC transgenic mouse line as a new mouse model for AD development. Furthermore, we revealed that YAP drives type 2 inflammation and itch, eventually leading to AD pathogenesis, through activation of IL33 and CTSS production in the epidermis. Our studies identify YAP as a hitherto unrecognized upstream inducer of cutaneous atopic inflammation. This provides fundamental insights into the mechanisms of allergic sensitization in vivo, thereby defining novel treatment strategies for individuals with established atopic dermatitis.

REPROGRAMMING HUMAN IPSC INTO SENSORY NEURONS TO STUDY RETT SYNDROME

Sharmin S.1, 2, Lesperance L.S.1, Wei W.1, Piekna A.1, Prescott S.A.1 and Ellis J.1 1The Hospital for Sick Children, Toronto, ON, Canada. 2The University of Queensland, Brisbane, QLD, Australia.

There is a large and growing population of individuals diagnosed with neurodevelopmental disorders. Rett syndrome is one of the major neurodevelopmental disorders. Symptoms include difficulties in social interaction, communication and repetitive behaviours as well as altered touch and pain sensitivity. Sensory neurons in the peripheral nervous system respond to somatosensory input including touch and pain. Our studies are focused on evaluating sensory neurons from control subjects for comparison neurons from subjects with Rett syndrome. To this end we adapted a previously published protocol (Chambers et al., Nature Biotechnology, 2012) to produce populations of sensory neurons from induced Pluripotent Stem Cell (iPSC) lines derived from healthy control and Rett syndrome individuals (Cheung et al., Human Molecular Genetics, 2011). Our modification of the growth factor and media composition in the Chambers et al. protocol resulted in 85% of the cells exhibiting a repetitive spiking pattern at 5 weeks consistent with nociceptor-like neurons. In contrast, preliminary lentivirus-mediated NGN2 infections and addition of neurotrophin-3 (NT-3) may accelerate the formation of single spiking mechanoreceptor / proprioceptor cell populations with repetitive spiking nociceptor population in sensory neuron culture. Western blots for TRK family proteins and immunostaining for sensory neuron markers support these conclusions and FACS analysis is underway. Preliminary screening of Rett syndrome patient lines using the adapted protocol suggests TRKA and glutamate receptor 1(GLUR1) are down regulated whereas gamma amino-butyric acid (GABA) receptor seems unchanged in western blot which indicates potential synaptic / morphological abnormalities. In future, electrophysiology and morphological analysis will be performed for more depth analysis.


SymPoSia moNDay

SYM-09-05 SYM-10-01

SYM-10-02 SYM-10-03

THE INTEGRATED STRESS RESPONSE IN SKELETAL DEVELOPMENT AND DISEASE

Cheah K.S.E. School of Biomedical Sciences, University of Hong Kong, 5 Sassoon Rd, Hong Kong.

The integrated stress response (ISR) has a central role in maintaining homeostasis in cells experiencing many forms of cellular stress such as biomechanical loading, oxidative stress, hypoxia, endoplasmic reticulum (ER) stress. Induction of the ISR is important for maintaining cells in normal development and is associated with diverse congenital and common diseases. Human congenital skeletal dysplasia caused by disrupted development of the growth plate is often associated with mutations that trigger ER stress that induces the Unfolded Protein Response (UPR). Here, we exploited a mouse model of Metaphyseal Chondrodysplasia type Schmid (MCDS) to provide mechanistic insight into the impact of the ISR on cell fate. We show that the ISR core protein kinase RNA-like ER kinase (PERK) signaling pathway that modulates translation, dominates in causing dysplasia by reverting chondrocyte differentiation via ATF4 directed transactivation of Sox9. Treatment of mutant mice with a chemical inhibitor of PERK signaling prevents the differentiation defects and ameliorates chondrodysplasia. By preventing aberrant differentiation, titrated inhibition of the ISR may be a therapeutic strategy for stress-induced skeletal disorders.

ABERRANT PROTEIN FOLDING AND AGGREGATION AND INCREASED VULNERABILITY IN MOTOR NEURONS IN ALS

Ooi L.1 1University of Wollongong, Wollongong, NSW 2522, Australia. 2Australia and Illawarra Health and Medical Research Institute, Wollongong, NSW 2522, Australia.

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease that causes the degeneration of motor neurons in the brain and spinal cord. The proteostasis network comprises pathways that regulate the biogenesis, folding, trafficking and degradation of proteins. The role of protein aggregates in the pathogenesis of ALS remains unclear. However misfolded and aggregated proteins contribute to a failure in proteostasis, which is thought to underlie motor neuron vulnerability in ALS. The aim of this research was to identify mechanisms underlying vulnerability of motor neurons in ALS. To address this we have used biochemical and electrophysiological tools to analyse motor neurons from induced pluripotent stem cells (iPSCs) from ALS patients, a SOD1G93A mouse model and post mortem tissue from ALS cases. We have identified alterations in membrane proteins, alterations in protein solubility and an increased vulnerability to cell stress in motor neurons in ALS. Further we have identified mechanisms that contribute to increased vulnerability and aberrant folding and aggregation of proteins in motor neurons in ALS.

DYNAMIC STRUCTURAL PROPERTIES OF 14-3-3 zETA PROTEIN UNDERPIN ITS MOLECULAR CHAPERONE ACTION AGAINST AMORPHOUS PROTEIN AGGREGATION

Woodcock J.M.1, Goodwin K.L.2, Sandow J.3, Coolen C.1, Rekas A.4 and Carver J.A.2, 5 1Centre for Cancer Biology, SA Pathology and University of South Australia, SA. 2School of Physical Sciences, University of Adelaide, SA. 3Walter and Eliza Hall Institute of Medical Research, Parkville, VIC. 4Australian Nuclear and Science Technology Organisation, NSW. 5Research School of Chemistry, Australian National University, Canberra, ACT.

The family of 14-3-3 proteins are dimeric phospho-serine binding proteins that function as adaptors with important roles in the regulation of many signaling responses in eukaryotic cells. Less well described, 14-3-3 proteins also exhibit molecular chaperone activity that attenuates the amorphous aggregation of proteins. This property may explain the occurrence of the 14-3-3 zeta isoform in the pathological protein aggregation associated with neurodegenerative conditions including Alzheimer’s and Parkinson’s diseases. To better understand this aspect of 14-3-3 proteins’ function, we have examined the regions of 14-3-3 zeta that play a role in its molecular chaperone action. We determined that neither the flexible C-terminus region nor the amphipathic phospho-serine binding groove contribute to molecular chaperone action. Published studies using mutant forms of 14-3-3 zeta that are engineered to disrupt the dimeric state of the protein suggest that monomeric 14-3-3 zeta represents the chaperone-competent form of the protein. However, our recent results suggest that this is a simplistic view and that the dimer interface of 14-3-3 zeta represents a structurally dynamic region that is involved simultaneously in both 14-3-3 protein dimer formation and molecular chaperone function.

STRUCTURAL CAPACITANCE IN PROTEIN EVOLUTION AND HUMAN DISEASES

Buckle A.M. Department of Biochemistry and Molecular Biology, Monash University.

Canonical mechanisms of protein evolution include the duplication and diversification of pre-existing folds through genetic alterations that include point mutations, insertions, deletions, and copy number amplifications, as well as post-translational modifications that modify processes such as folding efficiency and cellular localization. Following a survey of the human mutation database, we have identified an additional mechanism, that we term ‘structural capacitance’, which results in the de novo generation of microstructure in previously disordered regions. We suggest that the potential for structural capacitance confers select proteins with the capacity to evolve over rapid timescales, facilitating saltatory evolution as opposed to exclusively canonical Darwinian mechanisms. Our results implicate the elements of protein microstructure generated by this distinct mechanism in the pathogenesis of a wide variety of human diseases. The benefits of rapidly furnishing the potential for evolutionary change conferred by structural capacitance are consequently counterbalanced by this accompanying risk. The phenomenon of structural capacitance has implications ranging from the ancestral diversification of protein folds to the engineering of synthetic proteins with enhanced evolvability.


SymPoSia moNDay

SYM-10-04 SYM-10-05

SYM-11-01 SYM-11-02

A BIOSENSOR BASED FLIM-FRET PHASOR APPROACH TO MEASURE PROTEOSTASIS CAPACITY IN CELLS

Moily N.S., Hinde E. and Hatters D. Department of Biochemistry and Molecular Biology, University of Melbourne, Australia.

The pool of quality control proteins that maintain protein-folding (proteostasis) is dynamic but can become quickly depleted in cellular stress and disease. The ability of these quality control chaperones to maintain the proteome in a folded state in health and response to stressors is not yet defined quantitatively. We have developed a family of barnase FRET-based biosensors with differing folding stabilities that engage primarily with HSP70 and HSP90 family proteins and modify its foldedness and aggregation[1]. Here we quantify the ability of these critical cellular chaperones to bind to the barnase biosensor using the phasor approach to FLIM analysis of FRET in a living cell. The phasor method is a fit free approach to fluorescence lifetime analysis that has the capacity to quantify the FRET efficiency of the barnase sensor in each pixel of a FLIM image and therefore spatially map protein foldedness in a living cell[2]. Using phasor FLIM-FRET analysis we are able to calculate the chaperone occupancy rates in folded and unfolded barnase biosensors fractions, thereby quantifying their holdase abilities in baseline conditions and when challenged through stress. By multiplexing this technology with image correlation spectroscopy, we also hope to understand the stickiness of unfolded proteins in cells by measuring barnase diffusion rates and predict how cells react to excess unfolded protein load and aggregate formation. References: 1. Wood, R.J., et al., A biosensor-based framework to measure latent proteostasis capacity. Nature Communications, 2018. 9(1): p. 287. 2. Hinde, E., et al., Biosensor FRET detection by the phasor approach to fluorescence lifetime imaging microscopy (FLIM). Microsc Res Tech, 2012. 75(3): p. 271-81.

CONTRIBUTION OF THE RESIDUE AT POSITION 4 WITHIN CLASSICAL NUCLEAR LOCALIzATION SIGNALS TO MODULATING INTERACTION WITH IMPORTINS AND NUCLEAR TARGETINGSmith K.M.1, Di Antonio V.2, Bellucci L.3, Thomas D.R.4, Caporuscio F.5, Wagstaff K.M.4, Forwood J.K.1, Jans D.A.4, Palu G.2and Alvisi G.2 1School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, New South Wales 2650, Australia. 2Department of Molecular Medicine, University of Padua, Via Gabelli 63, 35121 Padua,Italy. 3NEST, Istituto Nanoscienze del CNR and Scuola Normale Superiore, P.zza S. Silvestro 12, 56127 Pisa, Italy. 4Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, Victoria 3800, Australia. 5Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 103, 41125 Modena, Italy.Nuclear import involves the recognition by importin (IMP) superfamily members of nuclear localization signals (NLSs) within protein cargoes destined for the nucleus, the best understood being recognition of classical NLSs (cNLSs) by the IMPα/β1 heterodimer. Although the cNLS consensus [K-(K/R)-X-(K/R) for positions P2–P5] is generally accepted, recent studies indicated that the contribution made by different residues at the P4 position can vary. Here, we apply a combination of microscopy, molecular dynamics, crystallography, in vitro binding, and bioinformatics approaches to show that the nature of residues at P4 indeed modulates cNLS function in the context of a prototypical Simian Virus 40 large tumor antigen-derived cNLS (KKRK, P2–5). Indeed, all hydrophobic substitutions in place of R impaired binding to IMPα and nuclear targeting, with the largest effect exerted by a G residue at P4. Substitution of R with neutral hydrophobic residues caused the loss of electrostatic and van der Waals interactions between the P4 residue side chains and IMPα. Detailed bioinformatics analysis confirmed the importance of the P4 residue for cNLS function across the human proteome, with specific residues such as G being associated with low activity. Furthermore, we validate our findings for two additional cNLSs from human cytomegalovirus (HCMV) DNA polymerase catalytic subunit UL54 and processivity factor UL44, where a G residue at P4 results in a 2–3-fold decrease in NLS activity. Our results thus showed that the P4 residue makes a hitherto poorly appreciated contribution to nuclear import efficiency, which is essential to determining the precise nuclear levels of cargoes.

METABOLOMIC ANALYSIS OF INSULIN RESISTANCE ACROSS DIFFERENT MOUSE STRAINS AND DIETS

Stoeckli J.1, Fisher-Wellman K.H.2, Muoio D.M.2 and James D.E.1 1Charles Perkins Centre, School of Life and Environmental Sciences, The University of Sydney, Australia. 2Duke Molecular Physiology Institute, Duke University, Durham, NC, USA.

Insulin resistance is a major risk factor for many diseases. However, its underlying mechanism remains unclear in part because it is triggered by a complex relationship between multiple factors including genes and the environment. Here we used metabolomics combined with computational methods to identify factors that classified insulin resistance across individual mice derived from three different mouse strains fed two different diets. Three inbred ILSXISS strains were fed high fat or chow diets and subjected to metabolic phenotyping and metabolomics analysis of skeletal muscle. There was significant metabolic heterogeneity between strains, diet and individual animals. Distinct metabolites were changed with insulin resistance, diet and between strains. Computational analysis revealed 113 metabolites that were correlated with metabolic phenotypes. Using these 113 metabolites, combined with machine learning to segregate mice based on insulin sensitivity we identified C22:1-CoA, C2-carnitine and C16-ceramide as the best classifiers. Strikingly, when these three metabolites were combined into one signature, they classified mice based on insulin sensitivity more accurately than each metabolite on its own or other published metabolic signatures. Furthermore, C22:1-CoA, was 2.3-fold higher in insulin resistant mice and correlated significantly with insulin resistance. We have identified a metabolomic signature comprised of three functionally unrelated metabolites that accurately predicts whole body insulin sensitivity across three mouse strains. These data indicate the power of simultaneous analysis of individual, genetic and environmental variance in mice for identifying novel factors that accurately predict metabolic phenotypes like whole body insulin sensitivity.

INCREASED NUCLEAR NAD+ BIOSYNTHESIS ALTERS SKELETAL MUSCLE PHYSIOLOGY

Samsudeen A.F.1, Fiveash C.E.1, Brandon A.E.2, Das A.3, Kiriaev L.4, Araki T.5, Head S.I.4, Cooney G.J.2, Osborne B.1 and Turner N.1 1Mitochondrial Bioenergetics Laboratory, UNSW Sydney. 2The Charles Perkins Centre, University of Sydney. 3Molecular Biology of Ageing Laboratory, UNSW Sydney. 4Department of Physiology, UNSW Sydney. 5National Center of Neurology and Psychiatry, Tokyo, Japan.

Nicotinamide adenine dinucleotide (NAD+) is a ubiquitous co-substrate used in a multitude of cellular reactions. Recent recognition of the role of NAD+ in obesity and ageing has sparked a surge in interest in NAD+ biology. Nicotinamide mononucleotide adenylyltransferase (NMNAT) is a key enzyme regulating NAD+ levels, however the metabolic consequences of NMNAT manipulation has not been explored. Our studies investigated transgenic mice overexpressing NMNAT1 (the nuclear NMNAT isoform) where NMNAT1Tg mice had a reduced lean mass compared to wild-type (WT) littermates, primarily driven by a marked reduction (~30-40%) in skeletal muscle mass. Functionally, NMNAT1Tg mice showed reduced forelimb grip strength in comparison to WT littermates, but surprisingly no difference in exercise endurance. Immunohistochemical analysis showed a decrease in the average cross-sectional area of muscle fibres underpinned the reduced muscle mass. NMNAT1Tg muscle was characterised by an increase in more oxidative myosin heavy chain (MHC) isoforms (MHC1, MHC2a) and decreased fast-twitch MHC2b expression. A potential shift to a more oxidative phenotype in NMNAT1Tg vs. WT mice was confirmed by twitch characteristics and force/fatigue experiments in isolated extensor digitorum longus and soleus muscles. At a whole-body level, NMNAT1Tg mice showed higher energy expenditure, improved glucose tolerance and greater clearance of glucose into skeletal muscle in hyperinsulinaemic-euglycaemic clamp experiments. Overall, our findings indicate that enhancing nuclear NAD+ biosynthesis invokes widespread changes in skeletal muscle physiology.


SymPoSia moNDay

SYM-11-03 SYM-11-04

SYM-11-05 SYM-12-01

METABOLISM REGULATES MUSCLE STEM CELL SELF RENEWAL BY CONNECTING THE MICROENVIRONMENT AND HISTONE ACETYLATION

Ly C.H.1, Su S.2, Tian L.2, Zalcenstein D.2, Naik S.2, Ritchie M.2, Lynch G.S.1 and Ryall J.G.1 1Centre for Muscle Research, Department of Physiology, The University of Melbourne. 2The Walter and Eliza Hall Institute of Medical Research.

Skeletal muscle contains a resident population of somatic stem cells which are capable of both self-renewal and differentiation. The signals that regulate this important decision have yet to be fully elucidated. Here we use scRNAseq to identify the innate metabolic signature of muscle stem cells. We show that committed muscle progenitor cells exhibit an enrichment of glycolytic and TCA cycle genes and that extracellular carbohydrate availability regulates intracellular citrate levels and global histone acetylation. Muscle stem cells exposed to a reduced (or altered) carbohydrate environment demonstrate reduced global histone acetylation and transcription of myogenic determination factors (including myod1). Importantly, reduced carbohydrate availability was directly linked to increased rates of asymmetric division and muscle stem cell self-renewal. Our results reveal an important role for the extracellular metabolic environment in the decision to undergo self-renewal or myogenic commitment, suggesting local metabolite production may be a therapeutic target to improve muscle regeneration.

MITOCHONDRIAL COQ DEFICIENCY DRIVES INSULIN RESISTANCE BY INCREASING MITOCHONDRIAL OXIDANTSFazakerley D.J.1, Chaudhuri R.1, Yang P.2, Maghzal G.J.3, Krycer J.R.1, Minard A.Y.1, Samocha-Bonet D.4, Murphy M.P.5, Stocker R.3, 6 and James D.E.1, 7 1Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Camperdown, NSW, Australia. 2School of Mathematics and Statistics, University of Sydney, Camperdown, NSW, Australia. 3Vascular Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia. 4Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW, Australia. 5MRC Mitochondrial Biology Unit, Hills Road, University of Cambridge, Cambridge, UK. 6St Vincent’s Clinical School, University of New South Wales, Sydney, Australia. 7Charles Perkins Centre, Sydney Medical School, University of Sydney, Camperdown, NSW, Australia.Insulin resistance in muscle, adipose and liver tissue is a gateway to a number of metabolic diseases including type 2 diabetes. Alterations in several facets of mitochondrial biology are implicated in insulin resistance including impaired oxidative phosphorylation and increased mitochondrial reactive oxygen species (ROS). However, disentangling the respective roles of these processes in insulin resistance has been difficult since they often occur in tandem. We have used a new small molecule (mitochondria-targeted paraquat) to acutely generate superoxide within mitochondria, without disrupting the respiratory chain, to show that mitochondrial oxidants alone are sufficient to induce insulin resistance. Increased ROS, specifically in mitochondria, are a common feature of an array of in vitro and in vivo models of insulin resistance, yet the drivers of mitochondrial ROS under these conditions are not completely understood. To address this, we assessed the proteome of insulin resistant 3T3-L1 adipocytes and adipose tissue from mice and humans. We found lower expression of mevalonate/Coenzyme Q (CoQ) biosynthesis pathway proteins in insulin resistant samples. Analysis of subcellular CoQ content revealed selective depletion of CoQ from mitochondria in both insulin resistant adipose and muscle tissue. Given its role in electron transport, we investigated whether loss of CoQ caused insulin resistance via mitochondrial ROS. Pharmacologic or genetic manipulations that decreased mitochondrial CoQ triggered insulin resistance through increased mitochondrial ROS, while CoQ supplementation in either insulin resistant cell models or mice lowered ROS and restored insulin sensitivity. Our data place loss of mitochondrial CoQ upstream of mitochondrial ROS in the pathway to insulin resistance and suggest that interventions that restore mitochondrial CoQ may be effective therapeutic targets for treating insulin resistance.

MITOCHONDRIAL ENERGY GENERATION DISORDERS: GENES AND MECHANISMSThorburn D.R.1, 2, 3 1Murdoch Childrens Research Institute, Parkville, VIC 3052. 2Victorian Clinical Genetics Services, Parkville, VIC 3052. 3Department of Paediatrics, University of Melbourne, Melbourne, VIC 3052.

Inherited disorders of oxidative phosphorylation (OXPHOS) cause a clinically and genetically heterogeneous range of mitochondrial diseases. Mutations causing these disorders have been identified in 35 of the 37 genes encoded by mitochondrial DNA (mtDNA) and over 250 nuclear genes, with many still to be discovered. Apart from primary defects affecting OXPHOS subunits, assembly factors, electron carriers, and mtDNA maintenance or expression, many patients with mitochondrial disease have secondary defects related to enzyme cofactors, metabolite transport or other aspects of mitochondrial biogenesis, homeostasis and quality control. Several examples of recent collaborative studies illustrate the challenges of elucidating disease mechanisms. Firstly, we identified four families with Leigh syndrome with bi-allelic mutations that destabilized the MRPS34 protein, located in the foot of the small 28S mitoribosome subunit. Ribosome profiling and quantitative proteomic analyses of patient fibroblasts showed this led to loss of most of the 30 proteins of the 28S subunit, relative sparing of the large subunit, failure to assemble actively translating mitoribosomes and loss of many subunits of OXPHOS complexes I and IV. Secondly, we identified large deletions and gene conversions in a highly repetitive chromosomal region refractory to conventional analyses. The resulting ATAD3B/ATAD3A gene fusions cause lethal pontocerebellar hypoplasia and lead to mtDNA aggregation in patient fibroblasts, with multiple indicators of altered cholesterol metabolism. ATAD3 appears to be a key protein linking formation of mitochondrial nucleoids in cholesterol-rich mitochondrial inner membrane domains to cellular cholesterol metabolism. A final example is the SURF1 gene, in which patients typically have bi-allelic knockout mutations and suffer severe neurological and other symptoms but mouse knockouts show no clear phenotype. We used gene editing to generate human embryonic stem cell SURF1 knockouts. These differentiate well to cardiomyocytes and show marked abnormalities of OXPHOS function and calcium signaling as well as decreased contractility in 3D organoids.

THE USE OF RECOMBINANT HONEYBEE SILK FOR RATIONAL DESIGN OF ADVANCED MATERIALS

Sutherland T.D. and Rapson T.D. CSIRO, Health and Biosecurity.

Precise control of a polymers composition is required for the rational design of advanced materials such as stimuli responsive or multifunctional materials. Recombinant protein systems provide this control, and in contrast to synthetic equivalents are compatible with large scale production. However, despite the promise of proteins for advanced material design, protein-based materials are underrepresented in materials science. Here we describe our efforts to develop recombinant silk proteins that are tolerant of amino acid modifications without compromising the ability to produce the proteins at large scale in recombinant systems or fabricate them into material forms. We describe design of the silk proteins into materials with diverse functional properties ranging from recoverable heme-silk sponges with peroxidase activity, stable nitric oxide-sensing protein films and in electrodes capable of fully reducing oxygen to water.


SymPoSia moNDay

SYM-12-02 SYM-12-03

SYM-12-04 SYM-13-01

TISSUE ENGINEERED MULTICELLULAR STRUCTURES FOR AQUATIC DETOXIFICATIONPollak N.M.1, 2, 3, Glass N.R.2, Suzuki K.G.H.2, Cooper-White J.J.2, 4 and Macdonald J.1, 5, 6 1Genecology Research Centre, School of Science and Engineering, University of the Sunshine Coast, Sippy Downs, Queensland, Australia. 2Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia. 3CSIRO Synthetic Biology Future Science Platform. 4UQ Centre for Stem Cell Ageing and Regenerative Engineering, The University of Queensland, Brisbane, Queensland, Australia. 5Inflammation and Healing Research Cluster, School of Science and Engineering, University of the Sunshine Coast, Sippy Downs, Queensland, Australia. 6Division of Experimental Therapeutics, Department of Medicine, Columbia University, New York, USA.

The development of new systems to target environmental pollution is critically important for improved bioremediation, and synthetic biology holds potential to revolutionize such approaches. Our group is exploring the expansion of synthetic biology into the field of tissue engineering, to produce novel multicellular structures, which can move and sense their environment in an organism-like fashion. These multicellular structures, also called “pseudo-organisms”, are constructed from biological and synthetic hybrid components. We are engineering them to detoxify toxins in water by ligand-induced activation of an enzyme scavenger, and to trigger warnings. Our approach may revolutionize aquatic detoxification systems in industry by offering a mobile scavenging system that is highly-specific, yet without harmful side products. Importantly, these pseudo-organisms cannot reproduce, representing a unique solution to ethical and social impact deliberations compared to use of GMOs, and creating new possibilities in water management. Benefits ultimately include novel technologies for water purification for industrial applications, such as reducing biochemically active pharmaceuticals in waste water and harmful pesticide-run off from agricultural activities. In addition, our research is seeking to study cell-to-cell communication for signal amplification between sender and receiver cells and aims to engineer quorum sensing to initiate directional movement of multicellular structures.

MEASURING AND MODIFYING TRANSLATION IN ESCHERICHIA COLI THROUGH START CODON AND ORTHOGONAL TRNA ENGINEERING

Vincent R.M.1, Wright B.M.1, Hecht A.2, 3, 4, Glasgow J.2, 3, 4, Bawazer L.2, 3, 4, Munson M.S.2, 3, 4, Cochran J.R.2, 4, Endy D.2, 4, Salit M.2, 3, 4 and Jaschke P.R.1 1Department of Molecular Sciences, Macquarie University, Sydney, NSW. 2Joint Initiative for Metrology in Biology, Stanford, USA. 3Genome-scale Measurements Group, National Institute of Standards and Technology, Stanford, USA. 4Department of Bioengineering, Stanford, USA.

Efficient translation initiation in bacteria requires the specific interaction between the start codon on the mRNA and the anticodon on the initiator tRNA. Recently, we measured the interaction strength of E. coli initiator tRNA with all 64 possible start codons in vivo. We found a surprising number of non-canonical start codons leading to translation initiation. Based on these findings, we have been working towards creating an orthogonal system that specifically initiates translation at UAG codons. An interesting feature of the standard genetic code is the dual function of AUG as both the dominant initiation codon as well as a methionine codon internal to genes. The AUG codon duality can create issues in gene prediction and genetic design. For example, in recombinant protein design, in-frame AUG codons can behave as start codons if upstream sequence resembles a Shine-Dalgarno site. Therefore, an orthogonal translation initiation system using a codon uniquely reserved for translation initiation could overcome this problem as well as provide other engineering benefits. In the genetic code, UAG normally signals “stop translation”. This UAG can be freed up in engineered strains where all native UAG codons are recoded to the UAA stop codon. Here, we will describe deployment of an orthogonal initiator tRNA within a UAG-less E. coli strain called C321 and effects on fitness and proteome.

BIOMACROMOLECULES WITHIN MOLLUSK SHELLS: GATEWAY TO BIOMIMETIC COMPLEX SUPERSTRUCTURES

Agbaje O.B.A. and Jacob D.E. Department of Earth and Planetary Sciences, Macquarie University, NSW 2109 Australia.

Organisms precipitate a large variety of biominerals which are biogenic hierarchical nanocomposite materials consisting of inorganic-organic hybrid components, each with its structural motifs, inorganic crystal formation - shape, and micro- or macroscopic properties. Shells of mollusk, for instance, are formed with different ratios of inorganic-organic materials and different structural motifs resulting in a large variety of calcareous biocomposites with material properties outperforming those of their synthetic counterparts. Amongst all different shell microstructures, nacre is the most studied to date, but comparable knowledge is lacking for non-nacre shell structures such as homogeneous and crossed-lamellar structures. While the organic matrix occluded in shells is a minor component, ca 5 wt% of the total, significant fractions of the shell macromolecules have been presumed to be chitin and/or proteins. Identifying the biopolymer phase is, therefore, a crucial step in improving our understanding of design principles relevant to biominerals. We have used different bioanalytical and biochemical techniques including solid state-NMR, FTIR, Raman spectroscopy, scanning electron microscopy, ACQUITY ultraperformance liquid chromatography and SDS-electrophoresis to explore the composition of the organic part of mollusk shells and examine its interface with the inorganic part. The biopolymer content consists primarily of proteinaceous matter with structural motifs as silk-like β-sheets in nacre and collagenous gel-like matrix in non-nacre shells. Though other motifs persist in non-nacre, the composition in nacre is strikingly similar to the constituents of spider dragline silk but does not conform to the prevalent model that the biopolymer component in shells consists of well-arranged polysaccharide-chitin1. 1 Y. Levi-Kalisman, G. Falini, L. Addadi, S. Weiner, J. Struct. Biol., 2001, 135, 8-17.

USING CALCIUM PHOSPHATE LIPID-COATED NANOPARTICLES TO DELIVER ANTISENSE OLIGONUCLEOTIDES TO MOTOR NEURONS IN MOTOR NEURONE DISEASEVine K.L.1, 2, Watson C.1, 2, Chen L.1, 2, Saunders D.3, Morsch M.4, Chung R.4, Cole N.4 and Yerbury J.J.1, 2 1School of Biological Sciences, Faculty Science, Medicine and Health, University of Wollongong, NSW, 2522, Australia. 2Illawarra Health and Medical Research Institute, Wollongong, NSW, 2522, Australia. 3School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, 2052, Australia. 4Department of Biomedical Sciences, Macquarie University, Sydney, NSW, 2113, Australia.

Introduction: Abnormal accumulation of mutant superoxide dismutase I (SOD1) in motor neurons is a pathological hallmark of some forms of ALS. Considering that SOD1 can propagate from cell-to-cell in a prion-like fashion, potentially contributing to the orderly progression of the disease [1], reducing levels of SOD1 is a promising therapeutic approach. Antisense oligonucleotides (ASOs) can efficiently silence proteins with gain-of-function mutations. However, naked ASOs have a short circulation half-life and are unable to cross the blood brain barrier (BBB) warranting the use of a drug carrier for effective delivery. We therefore aimed to improve the delivery of gene therapies to motor neurons in the context of ALS [2], using solid core calcium phosphate lipid-coated nanoparticles (CaP-lipid NPs), encapsulating an ASO directed to SOD1. Here we report the manufacture and biophysical characterization of CaP-lipid NPs that encapsulate a SOD1 ASO and describe their in vitro uptake and in vivo distribution in a zebrafish model. Methods: CaP-lipid NPs were prepared as described in our paper [3] and characterized for and size (nm), zeta potential (mV), polydispersity index (PDI), encapsulation effiencicy (%) and particle concentration. The in vitro cellular uptake and gene silencing were assessed by confocal microscopy and Western blotting, respectively. The in vivo distribution was assessed using a well-established ALS zebrafish model [4]. Results: Specifically, we demonstrated that the delivery of CaP-lipid NPs is efficacious in a motor-neuron-like established cell line (NSC-34) and in primary motor neuron cultures isolated from murine spinal cords [5] by confocal microscopy. Significant down-regulation of SOD1 protein expression was confirmed by immunoblotting following the delivery of SOD1 ASO-loaded CaP-lipid NPs. We also describe for the first time nanoparticle distribution in the brain, spinal cord and blood circulation of zebrafish, a powerful experimental vertebrate model for studying ALS. Conclusions: Our results suggest that CaP-lipid NPs could be an effective and safe system for the improved delivery of SOD1 ASOs to affected motor neurons in ALS. Acknowledgements This work has been supported by the Motor Neuron Disease Research Institute of Australia (2016-2017) and the US Department of Defence (AL150057; 2016-2018). References [1] Grad et al. 2014 PNAS, 111 (9), 3620-3625 [2] Foust et al. 2013 Molecular Therapy, 21(12): 2148–2159 [3] Chen et al. 2017 Frontiers in Neuroscience, 11:476 [4] Morsch et al. 2017 Journal of Visualized Experiments, (120): 54983 [5] Kieran & Greensmith 2004 Neuroscience (125): 427-439.


SymPoSia moNDay

SYM-13-02 SYM-13-03

SYM-13-04 SYM-13-05

A STRATEGY TO PROTECT THE HEART AGAINST DOXORUBICIN INDUCED CARDIOTOXICITY

Kok C.Y.1, Rao R.1, Ghossein G.1, Igoor S.1, Skelton R.1, Chong J.1, 2 and Kizana E.1, 2 1Center for Heart Research, The Westmead Institute for Medical Research, The University of Sydney. 2Department of Cardiology, Westmead Hospital.

Doxorubicin is an anti-cancer drug used in treating a variety of malignancies. However, its major adverse effect is cardiotoxicity, which is dose dependent and can be either acute or chronic. Doxorubicin causes injury by DNA damage, formation of free reactive oxygen radicals and induction of apoptosis. Our aim is to induce expression of the multiple drug transporter gene (MRP1) in cardiomyocytes derived from human iPS cells (iPSC-CM), to determine whether this will allow cells to effectively remove doxorubicin. To determine the dose of lentiviral vector required for efficient gene delivery to iPSC-CM, a dose titration of GFP vector (LV.GFP) was performed. We found that an MOI of 20 was sufficient for transduction of >90% of cells. Having determined the optimal dose, we then generated a lentivirus vector for inducing expression of MRP1 (LV.MRP1) and validated its function in iPSC-CM by qPCR and western blot. We successfully showed increase of MRP1 mRNA and protein in transduced cells. The activity of the overexpressed MRP1 was also tested, by quantifying the amount of dye exported from the cell by the transporter. We demonstrated reduced dye sequestration in cells overexpressing MRP1. In conclusion, we have optimised the conditions for gene delivery to human iPSC-CM in vitro. We have also shown that we can successfully over-express MRP1 protein in iPSC-CM, with functional transporter activity. The next step is to determine the dose of doxorubicin which induces cell toxicity, and then to assess the protective effect of MRP1 in those cells.

LIPOSOME-BASED NANOSENSORS FOR CHEMICAL AND BIOLOGICAL SENSING

Chandrawati R. School of Chemical Engineering and Australian Centre for NanoMedicine (ACN), The University of New South Wales, Sydney, NSW 2052, Australia.

The detection of target chemical and biological molecules in a specific and sensitive manner is critical for the development of disease diagnostic devices. Membrane fusion is a key biological event that involves a highly selective recognition mechanism for molecular trafficking, facilitating communication between and within cells. The highly evolved fusion process can occur on a sub-millisecond timescale. The rapid response, specificity, and sensitivity make membrane fusion an attractive mechanism for sensing. This talk will describe our recent developments in mimicking lipid membrane fusion mechanism using liposomes for the detection of disease biomarkers. This system does not require washing, amplification or separation steps, and present a unique sensing mechanism inspired by nature.

CELLULAR NANOVESICLES: EXOSOMES REDUCE PRIMARY TUMOR BURDEN BUT ACCELERATE METASTASIS

Samuel M.1, Timpson P.2, Parker B.1 and Mathivanan S.1 1La Trobe University, Melbourne. 2Garvan Institute for Medical Research, Sydney.

It has been proposed that exosomes from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and exert phenotypic changes. Here, we orally administered bovine milk-derived exosomes to mice and demonstrate that milk-derived exosomes can survive the harsh degrading conditions of the gut and subsequently be detected in multiple organs. Interestingly, oral administration of milk-derived exosomes reduced the primary tumor burden in various cancer models and attenuated cancer cachexia. Intriguingly, in spite of the reduction in primary tumor growth, milk-derived exosomes accelerated metastasis in breast and pancreatic cancer mice models. Timing of exosome administration was critical as oral administration after resection of the primary tumor reversed the pro-metastatic effects of milk-derived exosomes in breast cancer. Taken together, our study provides novel context-based and opposing role of milk-derived exosomes as metastasis inducers and as metastasis blocker.

EMERGING TECHNOLOGIES FOR POINT-OF-CARE DIAGNOSIS OF SOIL-TRANSMITTED HELMINTH INFECTIONS

Soni S.K.1, Ravindran V.B.1, Traub R.2 and Ball A.S.1 1School of Science, RMIT University, Melbourne. 2Faculty of Veterinary and Agricultural Sciences, Melbourne University, Melbourne.

Parasitic worms (helminths) cause some of the most neglected tropical diseases. They infect ~ 1 billion people globally and contribute substantially to poor physical and cognitive development in children, and poor maternal birth outcomes. No commercial vaccines are available and treatment relies heavily on only a small number of drugs. Because drug resistance is a major threat, there is an urgent need to develop new and innovative methods of diagnosis to contribute toward achieving the Millennium Goals of the London Declaration. Currently, coproscopic diagnostic methods such as Kato Katz thick smear is performed, however this technique is antiquated and its sensitivity is very poor. Although PCR-based methods are an alternative to coproscopy, constraints relate to the transport of samples to a central laboratory, high cost and inability to estimate infection intensity. Therefore, a need for radically new and innovative tools is essential for the specific diagnosis of helminths. Here, we coupled propidium monoazide with isothermal amplification to effectively quantify the viability of Ascaris and Trichuris eggs. Also, we distinguished 3 different helminth ova based on surface acoustic wave drive microfluidics. Furthermore a highly selective and sensitive lateral flow based strip assay (Microbial Detection Assay Strips-MIDAS) also has been developed, that can detect 2 genera of STHs on 1 strip in 30 min. These studies will be validated further on a point-of-care basis to support mass treatment programs in the field.


SymPoSia moNDay

SYM-14-01 SYM-14-02

SYM-14-03 SYM-14-04

HOW AUTOTRANSPORTER PROTEINS MODULATE BACTERIA-HOST INTERACTIONS

Paxman J.J.1, Lo A.2, Vo J.1, Martinez Ortiz C.1, Scembri M.A.2 and Heras B.1 1Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC. 2Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane QLD 4072, Australia.

Bacterial pathogens deploy an arsenal of virulence factors to establish infection and cause disease. At the front line of the infection process are bacterial surface components, which are responsible for host colonisation and pathogen adhesion. Autotransporter (AT) proteins are the largest group of surface adhesins in Gram-negative bacteria. These proteins play a central role in controlling bacterial interactions with their environment; they allow bacteria to aggregate with other bacteria, adhere to human cells, and form biofilms all key facilitators of bacterial persistence and pathogenesis. We previously elucidated the mechanism by which the AT adhesin Antigen43 (Ag43) from uropathogenic E. coli (UPEC) promotes bacterial aggregation/biofilm formation, by means of self-association between neighbouring cells. We have produced the structures of a further AT proteins and we are now starting to elucidate the mechanisms of action for diverse ATs. So far, we have found that different AT adhesins promote bacterial aggregation using subtle variations in this self-association mechanism compared to Ag43. We are also beginning to uncover in atomic detail how AT adhesins like UpaB and TibA bind epithelial surfaces. TibA is a multifunctional AT from enterotoxigenic E. coli(ETEC), the leading bacterial cause of diarrhea. This surface protein was known to be glycosylated by the cognate glycosyltransferase TibC. Our work is beginning to uncover how glycosylation regulates the function of multifunctional AT adhesins. This may represent a general mechanism for bacteria to regulate the virulence functions of the vast number of ATs expressed on their cell surface. Finally, we are using this new knowledge to successfully develop methods for disrupting AT function.

INFLAMMASOME ASSEMBLY AND ACTIVATION MECHANISMSVajjhala P.R.1, Lu A.2, 3, Sagulenko V.1, Wong P.Y.1, Brown D.L.4, Fu T.M.2, 3, Sester D.P.1, Stow J.L.4, Wu H.2, 3 and Stacey K.J.1 1School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD 4072, Australia. 2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. 3Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA. 4Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia.Pattern recognition receptors (PRR) of the innate immune system mediate the first line of defence against infections and cellular stress signals. The inflammasome complex initiated by a subset of PRRs forms a platform for activation of procaspases-1 and -8 to mediate inflammation and cell death responses. Inflammasomes initiated by the AIM2 PRR in response to cytosolic DNA are relevant to viral and bacterial infections and autoimmune diseases caused by recognition of self DNA. Inflammasomes initiated by the NLRP3 PRR, activated by many diverse stimuli, contribute to the pathology of many common diseases including diabetes and atherosclerosis. Inflammasome assembly is mediated by death-fold domains, which include pyrin domains (PYDs), caspase recruitment domains (CARDs) and death effector domains (DEDs). Activation of the PRR induces oligomerisation and PYD clustering, which recruits the adaptor protein ASC and nucleates polymerization of ASC PYD into a helical filament. ASC CARDs condense the complex to form a speck that is synonymous with inflammasome activation and also recruit procaspase-1. We showed that ASC recruits procaspase-8 to the inflammasome to mediate apoptosis. The interaction is mediated by interaction between ASC PYD and procaspase-8 DEDs and nucleates formation of procaspase-8 DED filaments. Studies with reconstituted inflammasomes in HEK cells show that procaspase-8 DED filaments extend from the ASC speck while full-length procaspase-8 is condensed within the ASC speck suggesting inter-filament catalytic domain interactions. Procaspase-1 CARD did not form extensive filaments from the ASC speck in HEK cells thus a similar mechanism cannot be presumed. Time course analysis of endogenous inflammasomes to characterise the mechanism of inflammasome activation indicates rapid activation upon stimulation while live cell imaging indicates rapid ASC filament elongation. Our current models of inflammasome assembly and activation based on our data and insights from structural studies of death-fold domain filaments will be presented.

IMMUNOSUPPRESSIVE ACTIVITY OF HUMAN CD52 VIA SPECIFIC SIALOFORMS

Shathili A.M.1, Bandala-Sanchez E.2, Goddard-Borger E.D.2, John A.2, Thaysen-Andersen M.1, Everest-Dass A.3, Harrison L.C.2 and Packer N.H.1, 3 1Dept. of Molecular Sciences and ARC Centre of Excellence in Nanoscale BioPhotonics, Macquarie University, Sydney, New South Wales, Australia. 2Walter & Eliza Hall institute of Medical Research and University of Melbourne, Parkville. Victoria, Australia. 3Institute for Glycomics, Griffith University, Gold Coast, Queensland, Australia.

Homeostatic mechanisms are required to limit immune T-cell proliferation and prevent autoimmune diseases. Human CD52 is a small glycoprotein (12 amino acid residues), with an N-linked glycan at Asn3 and possible O-glycosylation. Glycosylated and soluble CD52 contributes to immune homeostasis by ligating the inhibitory sialic acid-binding immunoglobulin-like lectin-10 (Siglec-10) receptor on the surface of activated T-cells. We aimed to define the bioactive glycoforms of CD52. Our initial analysis of purified human spleen CD52 confirmed the presence of multi-antennary sialylated N-glycans with abundant polyLacNAc extensions, together with some O-glycan structures. To gain insight into the molecular basis of CD52 binding mechanism, recombinant soluble CD52-Fc expressed from HEK293 or Expi293 cells, but not the Fc alone, suppressed T-cell function and was used to relate bioactivity with the CD52 glycoform structure. Glycomics (porous graphitised carbon-liquid chromatography-ESI-MS) and intact glycopeptide (high resolution C8-ESI-MS) analyses of recombinant CD52 revealed that only specific type of N-glycans contributed to the bioactivity of CD52. Interestingly, the relative abundance of a specific sialic acid linkage correlated with higher bioactivity, which was verified by de- then re-sialylation experiments. Anion exchange chromatography on a MonoQ-GL column fractionated CD52-Fc into glycoforms with increased capacity to suppress T-cell function. Fractions with suppressive activity confirmed previous results. O-glycans were assigned their site localisation in the active fractions. These findings define glycans involved in the immune suppressive bioactivity of CD52 and resulted in CD52 fractions with higher suppressive activity than the biologically isolated CD52.

TACKLING ANTIMICROBIAL RESISTANCE (AMR) USING SINGLE PARTICLE CRYOEM

Belousoff M.J.1, Yonath A.3, Lupton D.2 and Lithgow T.1 1School of Microbiology, Monash University. 2School of Chemistry, Monash University. 3Weizmann Institute of Science, Israel.

The World Health Organization states that by 2050, deaths from antibiotic or drug-resistant microbes could exceed all other current global health risks. These predictions rest on the assumption that there will be no new treatments come to the clinic. Our lab is determined to tackle a facet of the AMR problem by exploring an important target of antimicrobials; the bacterial ribosome. Ribosomal interfering antibiotics are used to commonly treat serious infections by methicillin resistant S. aureus (MRSA) and vancomycin resistant Enterococcus (VRE). One of the more effective ribosomal interfering antibiotics is the first fully synthetically produced antimicrobial; linezolid. Our work attempts to address two main questions: is it possible to understand how bacteria modify their ribosomal structure as they evolve linezolid resistance and is it possible to use this structural information to redesign the linezolid chemical structure to keep it active against resistant strains of bacteria? I will present the structure of the MRSA ribosome and the structure from a clinical isolate from the Alfred hospital which became resistant to linezolid during the course of treatment [1]. Our cryoEM studies clearly show how the binding site of the antibiotic is drastically remodeled due to a single point mutation in the resistant Staph. strain. Using the structural information as to how MRSA evades linezolid treatment we rationally redesigned linezolid and chemically synthesised new antibiotic derivatives which remained active against the linezolid resistant strains of Staph. Moreover, I will present high resolution (2.8 Å resolution) structures of these new drugs in complex with the MRSA ribosome to confirm our rational drug designs. Excitingly, we have shown that utilizing a pragmatic approach to antibiotic design that it is possible to make minor chemical changes to an existing antibiotic platform to keep it active in the face of antibiotic resistance. [1] Matthew J. Belousoff, et al. Structural basis for linezolid binding site rearrangement in the Staphylococcus aureus ribosome. MBio, 2017, 8:e00395-17.


SymPoSia moNDay

SYM-14-05 SYM-15-01

SYM-15-02 SYM-15-03

HAEM AT THE INTERFACE BETWEEN PATHOGENIC AND COMMENSAL BACTERIAL SPECIES IN THE HUMAN RESPIRATORY TRACT

Latham R.D.1, Del Rey M.T.2, Walshe J.2, Brianna A.1, Guss J.M.2, Mackay J.P.2, Tristram S.G.1 and Gell D.A.1 1University of Tasmania, TAS, Australia. 2University of Sydney, NSW, Australia.

Non-typeable Haemophilus influenzae (NTHi) is an important opportunistic pathogen of the human respiratory tract that has proved recalcitrant to vaccine development and shows increasing prevalence of antibiotic resistance, prompting us to search for alternative anti-microbial strategies. Haemophilus haemolyticus is closely related to NTHi, and also colonises the respiratory tract, but is recognised as a non-pathogenic commensal species. We identified two H. haemolyticus isolates secreting a 27-kDa protein that inhibited the growth of NTHi in vitro, but did not inhibit a range of other respiratory flora that were tested, suggesting a level of species-specificity against NTHi. A gene knockout established that the gene product of interest was responsible for inhibitory activity. Spectroscopic and x-ray crystallographic analysis of the recombinant protein identified a haem binding site. The protein shares structural features with some non-haem iron scavenging proteins, but the haem-binding site is unique. Insights into the biological function, including NTHi inhibitory actions, of this protein, based on structure, biochemistry and bioactivity assays are presented. The work is ongoing in the context that strains of H. haemolyticus might be developed as respiratory probiotics to combat colonisation and infection with NTHi.

CREATING OPPORTUNITIES FOR STUDENT SELF-REFLECTION ON THE DEVELOPMENT OF GRADUATE CAPABILITIES IN A LARGE FIRST YEAR MOLECULAR BIOLOGY SUBJECT

Kuit T. and Skropeta D. University of Wollongong.

Every university aims to develop graduates that are highly sought after by industry. Programs of study aim for graduates to achieve a set of core learning outcomes that develop key skills such as communication and teamwork. These are often referred to as graduate capabilities. These skills are then typically demonstrated to employees through selection criteria. Currently, at UOW science undergraduate students have little opportunity to engage in self-reflection, which is key to effectively meeting these criteria. One way to effect this change is to encourage student e-Portfolio development and maintenance. E-Portfolios are a collection of digital evidence that validates learning and development through a process of self-reflection. This enhances the meaning of students’ work and encourages insight and higher learning. Thus, a peer-assessed e-Portfolio component was designed and successfully implemented in a molecular biology subject to a large cohort of first-year science students (700+) with diverse educational and cultural backgrounds. We successfully introduced the necessary skill foundations to students who inturn gained a greater understanding into their own learning, particularly through the guided peer review process. In this presentation I will share the outcomes of this project and key lessons learnt along the way with a chance for discussion on how others are supporting students to evidence the development of graduate capabilities.

FLIPPING THE LABORATORY IN PLANT ANATOMY, BIOCHEMISTRY AND GLOBAL CHANGE BIOLOGY

Loveys B.R. School of Agriculture, Food and Wine, University of Adelaide, Adelaide, South Australia.

Practical classes are generally compulsory and all teachers hope their students will arrive in the laboratory prepared for the class; the reality, of course, is that students are often not prepared and are therefore disengaged and confused in class. Many students do not read the relevant material in their laboratory manual to ensure they understood of the principles they are about to learn. This makes it difficult for students to make the link between theory and application. To address this problem interactive, online pre-class activities were developed and deployed in plant anatomy, biochemistry and global climate change practical classes, thus Flipping the Laboratory to encourage students to prepare for practical classes. Using this approach I have developed multiple pre-practical online activities for my students on topics such as plant anatomy, enzyme kinetics, photosynthetic reactions and carbohydrate metabolism. Central to the flipped classroom learning model is that pre-class learning should be used to introduce foundational concepts and focus on lower level Blooms Taxonomy such as remember and understand. The pre-practical activities provide students with examples and activities to encourage them to prepare for practical sessions before class. Information is presented in a multi-media format with videos of demonstrations of commonly used lab techniques. Check-point questions are presented by way of multiple choice answers with unlimited attempts so student can gain confidence. Understanding foundational key concepts is critical for deeper learning. This presentation will show how sustained improvement in average and distribution of practical grades has been achieved. Failure rates have also declined despite increasing student enrolments. Students indicated the pre-practical activities encouraged independent learning, and, 70% of students enjoyed the pre-practical activities. Improved engagement in practical classes extends beyond the laboratory. Once students are engaged in a course it is easier maintain their interest in difficult and challenging content.

EFFECT OF CONTEXT-BASED UNDERGRADUATE BIOCHEMISTRY FOR HEALTH SCIENCES (CUBHS) INSTRUCTION ON STUDENT PERCEPTION OF RELEVANCE, ACHIEVEMENT AND ATTITUDEFernandez K.1, Overton T.2, Thompson C.3 and Samarawickrema N.4 1School of Chemistry/Department of Biochemistry & Molecular Biology, Monash University. 2School of Chemistry, Monash University. 3School of Chemistry, Monash University. 4Department of Biochemistry & Molecular Biology, Monash University.

There is an ongoing debate on the relevance of foundational biochemistry in the health sciences. It stems from the fact that much of biochemistry instruction has focused on the didactic delivery of concepts and theories with little emphasis on clinical applications. This has resulted in a foundational-clinical gap and to the negative perception of biochemistry among health science students. Notwithstanding, the inclusion of biochemistry in the health science curricula remains valid. As Gwee, Samarasekera and Chay-Hoon (2010) assert, since clinical practice is based on scientific knowledge, biochemistry remains indispensable. The recommendation is for biochemistry to be taught in the context of clinical practice (1, 2). Considering this, the study aims to determine the effects of a researcher developed Context-based Undergraduate Biochemistry for Health Sciences (CUBHS) instruction on student perception of relevance, achievement and attitude. It is a two-group posttest design wherein cohorts will be health science students (nursing, pharmacy, psychology and medical laboratory science). The control group will be 2018 students who are currently studying biochemistry in its traditional didactic form while, the experimental group will be 2019 students who will be studying biochemistry in CUBHS instruction. Scores on relevance, attitude and achievement in biochemistry will be measured through adapted tools: Message Content Relevance Scale (MCRS), Attitude towards Biochemistry Inventory (ABI) measure and Biochemistry Achievement Tool (BAT). Finally, the significant difference of scores between groups will be tested. References: (1) Gwee, M., Samarasekera, D and Chay-Hoon, T.(2010). Role of Basic Sciences in 21st Century Medical Education: An Asian Perspective. Medical Science Educator, 20(3). (2) Bandierra, G.; Boucher, A.; Neville, A.; Kuper, A.; Hodges, B. (2013). Integration and timing of basic and clinical sciences education. Journal of Medical Teacher, 35(5).


SymPoSia moNDay

SYM-15-04 SYM-16-01

SYM-16-02 SYM-16-03

EMBEDDING SCIENCE COMMUNICATION SKILLS IN FIRST YEAR BIOLOGY STUDENTS USING A MULTI-STAGE TEAM PROJECT

Galea A.M., LeBard, R. and Wilson J.E. School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia.

Communication skills, information literacy and the capacity for self-reflection are all highly desirable graduate attributes that can be developed and honed from the very beginning of a student’s university program. But building the optimal learning tasks for achieving these goals in both general and discipline-specific contexts can be very challenging, especially in large first year courses which are typically populated by students with very diverse interests and educational backgrounds. Here we describe the design and implementation of a new team-based learning activity that aims to embed a range of general and discipline-specific skills and capabilities in the students of a large first year biology course at the University of New South Wales, Sydney. The BABS1201 Science Communication Project is a semester-long group task with multiple components. Working in teams of 3 to 4 people, students select a modern biology topic or discovery from an extensive list of subjects that align with the core concepts and themes of the course. For each team, the primary objective is to design and develop a presentation (in any format) that will effectively communicate their subject and its core biological concepts to their peers. The project is scaffolded by several other components, including a scientific literature essay, a project pitch with peer feedback, a project diary, a reflective essay, and peer evaluation. Creativity and innovation are encouraged, acknowledged and rewarded throughout the project. Initial outcomes and feedback from students and staff have indicated that the project effectively engages students with modern biology and permits them to develop, exercise and enhance a range of communication skills.

SHOOT BRANCHING - ROLE OF STRIGOLACTONES AND INTERACTIONS WITH OTHER SIGNALS

Beveridge C.1, Chabikwa T.1, Kerr S.1, Han F.1, Fichtner F.2, Lunn J.2 and Barbier F.1 1The University of Queensland, School of Biological Sciences, Brisbane, Australia. 2Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1.

Shoot branching occurs due to the regulation of the outgrowth of axillary buds which are embryonic shoots in the axil of leaves. Long-distance signaling is central to this regulation and mainly involves strigolactones, cytokinins, auxin and sugars. The sugar role may be at least partly due to sugar signalling and to involve trehalose 6-phosphate. It also appears that the growth of axillary buds from a state of very slow growth or dormancy, to sustained growth involves a number of stages during which the emerging shoots show differential sensitivity to growth stimulus and inhibition. For example, there are substantial differences in responses to different hormones at different periods after shoot tip removal. This could be due to differences in hormone signaling and downstream responses as well as due to changes in the vasculature of the growing buds. We will present our latest unpublished findings on the interaction of signals during bud outgrowth. In addition to providing a new mechanism for how plants respond to shoot tip removal, this work is provides a better understanding of how plants achieve diverse architecture in response to the environment.

MIX AND MATCH: EPF/EPFL PEPTIDE SIGNALING IN PLANT DEVELOMENT

Torii K.U.1, 2 1Investigator, Howard Hughes Medical Institute, Department of Biology, University of Washington. 2Principal Investigator, Institute of Transformative Biomolecules, Nagoya University.

Plant peptide signals mediate both local cell-cell communications and long-distance root-to-shoot signaling. Secreted cysteine-rich peptides in the EPIDERMAL PATTERNING FACTOR (EPF)- EPF LIKE (EPFL) family regulate diverse aspects of plant development, including shoot meristem size, inflorescence architecture, vascular differentiation, and stomatal development. The EPFL peptides are versatile: The same peptide could function in an autocrine or paracrine manner. Moreover, related EPF/EPFL family members could work in a unique, redundant, or even in an antagonistic manner, depending on the specific developmental programs or tissue-specific contexts. Thus far, ERECTA-family receptor kinases are the known receptors for EPF/EPFL peptides. As such, this peptide hormone-receptor system poses an important question of how the same receptor could perceive multiple peptide ligands to elicit such diverse developmental outcomes. Our recent study on the developmental functions of EPFL2 peptide revealed a feedback circuit between the peptide-receptor system and auxin response as a mechanism for maintaining proper auxin maxima during leaf margin morphogenesis. Our study highlights the intersection of localized peptide signaling and universal small chemical hormone auxin in shaping plant development and morphogenesis.

IDENTIFICATION OF REGULATORY PATHWAYS CONTROLLING CELL DIFFERENTIATION DURING BARLEY GRAIN DEVELOPMENT

Aubert M.K.1, Shirley N.J.1, Houston K.2, Burton R.A.1 and Tucker M.R.1 1School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Urrbrae, South Australia, Australia. 2Cell and Molecular Sciences, The James Hutton Institute, Dundee, UK.

Barley is a diploid cereal crop used in the feed and brewing industries. The benefits of the barley grain are derived mainly from the endosperm, which is produced after fertilisation. During early stages of seed development, the endosperm differentiates along a radial axis to form two prominent cell types; the peripheral aleurone and the inner starchy endosperm. We have been studying early grain development in barley with a view to understanding how aleurone differentiation is regulated. Microscopic assays were used to measure sub-epidermal details of grain development in a panel of ~200 barley cultivars. Association mapping identified multiple genomic regions that contribute to variation. Candidate genes underlying variation in aleurone development were identified using a combination of RNAseq profiling, laser capture microdissection and plant transformation. The results of these assays will be discussed. The fundamental knowledge generated in this project is providing insight into how different tissues and cells contribute to grain development. This knowledge may be applied in future to tailor specific improvements in grain composition.


SymPoSia moNDay

SYM-16-04 SYM-16-05

SYM-17-01 SYM-17-02

AN APOCAROTENOID RETROGRADE SIGNAL POST-TRANSCRIPTIONALLY CONTROLS PROTEIN LEVELS, PLASTID BIOGENESIS AND PLANT DEVELOPMENT DURING EXTENDED PERIODS OF DARKNESSCazzonelli C.I.1, Hou X.2, Alagoz Y.1, Rivers J.2, Dhami N.1, Lee J.3, Shashikanth M.2 and Pogson B.2 1Hawkesbury Institute for the Environment, University of Western Sydney, Hawkesbury Campus, Bourke Street, Richmond, NSW AUSTRALIA 2753. 2Australian Research Council Centre of Excellence in Plant Energy Biology, College of Medicine, Biology and Environment, Research School of Biology, The Australian National University, Canberra, ACT 26. 3Centre for Advanced Microscopy, The Australian National University, Canberra, ACT 2601, Australia.

Cleavage products of cis-carotenes are reported to regulate nuclear gene expression, carotenoid homeostasis and leaf development in plants. Environmental conditions that affect their biosynthesis, their physiological functions and regulatory targets remain unknown. Carotenoid isomerase (crtiso) mutants display various virescent phenotypes depending upon the environmental conditions. Here we demonstrate a physiological link between day length (extended darkness), the accumulation of a cis-carotenes and control over plastid biogenesis. Chemical inhibition of carotenoid cleavage dioxygenase activity restored prolamellar body (PLB) formation crtiso/ccr2 etioplasts during skotomorphogenesis providing evidence for a novel cis-carotene derived apocarotenoid signal (ACS) that controls plastid biogenesis A forward genetic screen identified an epistatic interaction between the ζ-carotene isomerase (ziso-155) and Arabidopsis ccr2 mutants that blocked the biosynthesis of specific cis-carotenes, restored PLB formation in ccr2 etioplasts during skotomorphogenesis, and prevented leaf virescence in plants grown under shorter photoperiod. Transcriptomic analysis of ccr2 ziso-155 mutant tissues revealed down-regulation of repressors of photomorphogenesis and up-regulation of photosynthesis associated nuclear gene (PhANG) expression that correlated with normal plastid development. We next identified a mutation in DEETIOLATED 1 (det1-154) that restored PLB formation in ccr2 and reduced specific cis-carotenes to levels below a threshold that promoted the efficient biogenesis of plastids in plants grown under extended periods of darkness. det1-154 reduced transcription of protochlorophyllide oxidoreductase (POR) and ACS post-transcriptionally maintained POR protein levels. We describe a model whereby a retrograde ACS acts via an alternative signaling pathway downstream of DET1 to post-transcriptionally regulate POR, ELONGATED HYPOCOTYL5 (HY5) and PHYTOCHROME-INTERACTING FACTOR3 (PIF3) thereby coordinating PhANG expression and plastid development during extended periods of darkness.

INVESTIGATING THE FUNCTIONS AND INTERACTIONS OF FLAS IN SECONDARY CELL WALLS

Ma Y.1, 2, Zeng W.3, Bacic A.1, 2 and Johnson K.L.1, 2 1University of Melbourne. 2La Trobe University. 3Zhejiang Agriculture and Forestry University.

Our natural and plantation forests represents 90% of the captured carbon and is our most renewable bio-resource. The bulk of the biomass consists of secondary cell walls, which are thick, rigid and deposited on the inner side of the primary walls in specialized tissues. Secondary cell wall development is an area of intense interest given its importance for plant growth, water conductance and human applications such as biofuels, bio-inspired materials/bio-composites, construction and paper. A group of plant cell wall glycoproteins, the Fasciclin-Like Arabinogalactan proteins (FLAs) have been implicated in regulating secondary cell wall development and influencing their biomechanical properties. FLAs are a sub-class of the arabinogalactan-proteins (AGPs), glycoproteins implicated in cell wall sensing and signalling and proposed to cross-link to pectins in the wall. The fasciclin (FAS) domain has been shown to be involved in protein-protein interactions and development in mammals, insects and algae. Glycoproteins such as FLAs are therefore fascinating ‘chimeric’ molecules with a number of interesting properties; being able to potentially form protein-protein, protein-carbohydrate and carbohydrate-carbohydrate interactions in the wall. Using Arabidopsis as a model system, we have focused on a subset of FLAs (FLA11, FLA12 and FLA16) that are expressed in cells undergoing secondary cell wall development. Preliminary evidence suggests these FLAs influence the amount and angle of cellulose deposition, the most abundant cell wall polymer that forms the structural basis for all cell walls. Through mutant studies, biomechanical analysis, examination of FLA location and bioinformatics we are gaining insight into the function(s) of these complex glycoproteins.

SELF-ASSEMBLING HYDROGEL SUBSTRATES FOR NEURAL NETWORKS

Martin A.D. University of New South Wales.

Primary neuronal cultures are a powerful tool to understand neuronal maturation, aging and neurodegeneration. They have been used to screen the effects of drugs and misfolded proteins on neural networks in vitro. However, culturing primary neurons in vitro is notoriously difficult, owing to their high sensitivity to their environment. Currently, primary neurons are cultured on glass coverslips coated with poly-D-lysine (PDL). However, it is well known that significant differences exist in cell behaviour in a 2D versus 3D environment, which more accurately mimics in vivo conditions. Hydrogels have significant potential biomedical applications, including in cell culture, owing to their similarity to the extracellular matrix. We have previously used short peptides capped at their N-terminus with an aromatic group to form biocompatible hydrogels with tuneable stiffnesses, pore sizes and chemical functionalities. Here, we present a collaborative, multidisciplinary effort where short peptide hydrogels which support the growth of primary neurons in a 2D and 3D environment have been developed. Neurons cultured atop these hydrogels display initial development and maturation comparable to that on PDL, complete with synapse formation and electrical activity. Neurons can also be cultured within the hydrogels, with these 3D neuronal cultures having potential in identifying neurodegenerative disease biomarkers, better screening of drug molecules, modelling CNS damage and insights into aging.

UNDERSTANDING FORCE SENSING MECHANISMS IN MECHANOSENSITIVE PIEzO CHANNELS

Cox C.D., Bavi N. and Martinac B. Victor Chang Cardiac Research Institute.

Mechanosensitive channels are essential molecular components of mechanosensory systems in all organisms. Bacterial mechanosensitive channels are gated directly by bilayer tension. Recent work has demonstrated that bilayer tension may also activate eukaryotic mechanosensitive channels such as TREK-1/2. Using bleb-based electrophysiology we have shown that Piezo1 can also be gated by membrane tension, supported by the fact that purified mouse Piezo1 can be gated in pure lipid systems. More recently we have explored the influence of cytoskeletal proteins on Piezo1 and how these components modulate the channels inherent tension sensitivity using patch fluorometry. Moreover, we measure the mechanical properties of the cells expressing these channels concomitantly while we estimate the membrane tension necessary for channel activation. Changes in Piezo1 sensitivity are not exclusively correlated to changes in bulk mechanical properties of the cell. In the presence of STOML3 and tropomyosin4.2 the membrane tension required to gate Piezo1 is ≤ 0.5 mN/m, which is well within a physiological range. In contrast, the activation threshold of Piezo1 in the presence of Filamin A increases. Our results shed light on the ability of different structural scaffold proteins to sensitize or desensitize Piezo1 channels to mechanical stimuli by modulating their tension sensitivity. This model holds general applicability for sensitization/desensitization of all inherently mechanosensitive ion channels by cytoskeletal elements.


SymPoSia moNDay

SYM-17-03 SYM-17-04

SYM-17-05 SYM-18-01

SPECIALISATION OF THE GLIOMA CYTOSKELETON FOR NAVIGATING THE SOFT TISSUE ENVIRONMENT OF THE BRAIN

O’Neill G.M.1, 2 1Childrens Cancer Research Unit, Kids Research Institute, The Childrens Hospital at Westmead, Westmead, New South Wales, Australia. 2Discipline of Childhood and Adolescent Health, The University of Sydney, Sydney, New South Wales, Australia.

Glioma brain tumours are characteristically highly infiltrative into the surrounding healthy brain tissue, which is a mechanically soft tissue environment. Thus mechanisms of cancer cell invasion that have been defined using hard plastic dishes may not apply for the dissemination of glioma brain cancer cells through brain tissue. Indeed, high grade gliomas characteristically lose expression of the high molecular weight tropomyosins, a class of actin-associating proteins that are essential regulators of the actin stress fibres and focal adhesions that underpin cell migration on hard 2-dimensional surfaces. Thus in the present study we have investigated how loss of the high molecular weight tropomyosins affects glioma cell morphology using soft matrices that better recapitulate the biomechanical attributes of the brain. We show that Tpm 2.1 is down-regulated in glioma cells grown on soft brain-like environments. Next we demonstrate that Tpm 2.1 depletion by siRNA induces cell spreading and elongation in soft 3D hydrogels, irrespective of matrix composition, and further that this mimics the effects of Rho kinase inhibition. Tpm 1.7, a second high molecular weight tropomyosin is also down-regulated when cultured on soft brain-like surfaces and we show that effects of this isoform are matrix dependent, with Tpm 1.7 inducing cell rounding in 3D collagen gels. Finally, we show that the absence of Tpm 2.1 from primary patient derived high grade glioma cells correlates with elongated, mesenchymal invasion in 3D. We propose that Tpm 2.1 down-regulation overcomes the rigidity-sensing mechanism that would otherwise prevent the cells from spreading throughout the soft brain environment. The unique organisation of the glioma actin cytoskeleton organisation that is highly suited to the soft brain like environment may provide novel therapeutic targets for arresting invasion of high grade glioma cells.

NEWLY IDENTIFIED MOLECULAR MECHANISM OF GLUCOCORTICOID ACTION IN ARTHRITIS

Achuthan A., Lupancu T., Lee M.C., Fleetwood A., Cook A. and Hamilton J. Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Australia.

Rheumatoid arthritis is a chronic inflammatory autoimmune disease, which leads to poor quality of life due to the debilitating effect of inflammation. Clinical trials in rheumatoid arthritis targetting the cytokine, granulocyte macrophage-colony stimulating factor (GM-CSF) are showing promise although its mode of action remains largely unknown. We have recently shown that that GM-CSF drives CCL17 production via a new interferon regulatory factor 4 (IRF4)-dependent pathway in human monocytes and murine macrophages, as well as in vivo. Importantly, in arthritis and pain models IRF4-regulated CCL17 formation mediates the proinflammatory and algesic actions of GM-CSF. Glucocorticoids (GCs) are potent anti-inflammatory and immunosuppressive agents broadly used in anti-inflammatory therapy, albeit with adverse side effects associated with long-term usage. The negative consequences of GC therapy provide an impetus for research into gaining insights into the molecular mechanisms of GC action on immune cells. We report here that GM-CSF-induced CCL17 expression is inhibited by GCs in human monocytes and mouse macrophages. Moreover, we provide evidence for the first time that GCs suppress GM-CSF-induced IRF4 expression via regulating the expression and activity of JMJD3, which demethylases trimethylated-H3K27. Significantly, a recent study reported that synovial fluid from patients with rheumatoid arthritis had elevated levels of CCL17 as compared to healthy controls. We will provide molecular evidence for the anti-inflammatory actions of GCs in rheumatoid arthritis patient samples. The delineated pathway potentially provides new therapeutic options for the treatment of inflammatory diseases and their associated pain.

ROLE OF ARP2/3 IN BLEBBING MIGRATION OF T LYMPHOCYTES IN VIVO

Obeidy P.1, Ju L.2, Oehlers S.1, Zulkhernain N.S.1, Galeano Nino L.G.3, Tikoo S.1, Jackson S.P.2, Biro M.3, Roediger B.1 and Weninger W.1 1The Centenary Institute of Cancer Medicine and Cell Biology, Newtown, NSW 2042, Australia. 2Heart Research Institute, Charles Perkins Centre, University of Sydney, Camperdown, NSW 2006, Australia. 3Cell Motility and Mechanobiology, School of Medical Sciences, University of New South Wales, Sydney, 2050 NSW, Australia.

Cytotoxic T lymphocytes (CTL) rely on the precise rearrangement of the actin cytoskeleton to provide immunosurveillance against invading pathogens and malignant cells. Constant remodelling of the cytoskeleton, particularly at the leading edge, is associated with efficient T cell migration and function. The consequences of modulating Arp2/3, a macromolecular machine that nucleates branched actin filaments, at the leading edge of migrating T cells are incompletely understood. We report that modulation of Arp3 (one of the main subunits of Arp2/3) profoundly affects CTL actin cortex integrity, surveillance in vivo and effector function in vitro. We also demonstrate a significant reduction in the total F-actin resulting in decreased cortical tension and disruption of lamellipodia formation. As a result, Arp3 knockdown CTL switched from lamellipodia-based migration to the blebbing mode characterised by transient, membrane balloon-like protrusions at the leading edge both in vitro and in a zebrafish model. Our study established that optimal mechano-physical and biochemical properties of the actomyosin cortex, as maintained by the Arp2/3 complex, are essential for the proper functioning and effective migration of CTL. These unforeseen findings pave the way for a deeper understanding of actin nucleators in T cell cytoskeleton and are crucial for the development of improved therapies for cancer and inflammatory diseases.

CRYO-EM STUDIES OF E. COLI ATP SYNTHASE

Sobti M.1, Smits C.1, Wong A.S.W.2, Ishmukhametov R.3, Stock D.1, 4, Sandin S.5 and Stewart A.G.1, 4 1Molecular, Structural and Computational Biology Division, The Victor Chang Cardiac Research Institute, Darlinghurst 2010, Australia. 2NTU Institute of Structural Biology, Nanyang Technological University, Singapore, Singapore. 3Department of Physics, Clarendon Laboratory, University of Oxford, Oxford OX1 3PU, UK. 4Faculty of Medicine, The University of New South Wales, Sydney 2052, Australia. 5School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

Here we present our cryo-EM maps of the intact ATP synthase complex from Escherichia coli. This essential enzyme synthesises the bulk of cellular ATP, the energy currency of the cell. The structures highlight unique features of this ATP synthase complex, such as the bifurcation of the peripheral stalk homodimer and the position of the inhibitory subunit ε. Further studies on this complex reveal a possible partially active conformation, which points to the molecular events that may inhibit this marvellous motor.


SymPoSia moNDay

SYM-18-02 SYM-18-03

SYM-18-04 SYM-18-05

THE TUMBLEWEED: CONSTRUCTION OF A SYNTHETIC PROTEIN MOTOR

Davies R.1, Bromley E.2, Niman C.3, Blab G.4, Woolfson D.5, Zuckermann M.6, Forde N.6, Linke H.3 and Curmi P.1 1School of Physics, UNSW Sydney, Australia. 2Department of Physics, University of Durham, UK. 3Solid State Physics and Nanometer Structure Consortium, Lund University, Sweden. 4Molecular Biophysics, Universiteit Utrecht, The Netherlands. 5School of Biochemistry, Bristol University, UK. 6Department of Physics, Simon Fraser University, Canada.

Molecular motors and machines are highly complex, multi-subunit proteins that use chemical energy to perform a multitude of critical, mechanical tasks in cells. The physical mechanisms by which motor proteins (such as myosin and kinesin) transduce chemical energy into mechanical work are still poorly understood. Traditionally, scientists have taken a “top down” approach to addressing this question by determining crystal structures of motor proteins, characterizing mutants and making single molecule measurements of performance. The goal of our work is to take a “bottom up” approach and design an artificial motor based on non-motor protein components. In this way, we can test our understanding of motor protein operation by including components that have well characterized functional properties. Our current design, the Tumbleweed, consists of a three-legged clocked-walker protein that operates on a repetitive DNA track. Tumbleweed uses three discrete ligand-dependent DNA-binding domains (repressor proteins) to perform cyclical ligand-gated rectified diffusion along a synthetic DNA molecule. We have used a modular combination of molecular biology and synthetic biology to express and assemble the Tumbleweed motor where three different DNA-binding proteins are linked to an assembly hub via coiled-coil arms. The SpyCatcher-SpyTag system was used to create the covalently linked Tumbleweed motor. We are currently assaying Tumbleweed for motion on a DNA track.

NANOSCALE DNA ORIGAMI TOOLS TO STUDY MOLECULAR MACHINES

Wickham S. School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia.

DNA has huge potential as a programmable building material for biocompatible nanostructures, with many applications as tools for single molecule biophysics, platforms for diagnostics and therapeutics, and templates for nanofabrication. DNA origami is a method for making a diverse range of shapes by folding up a long single-stranded DNA scaffold. Custom DNA origami nanostructures can be designed as tools for specific biophysical measurements. For example, a DNA origami nanospring, to simultaneously measure force and position of the protein motor Myosin VI during stepping. DNA nanotechnology can also be used to build and test synthetic molecular machines, inspired by their biological counterparts. For example a molecular motor that can transport cargo through a maze of tracks. DNA origami structures can also serve as the substrate for future hybrid machines, which integrate functional protein components with synthetic DNA scaffolds.

NANOSCALE IMAGING OF PROTEIN SECRETION SYSTEMS USED BY BACTERIA AND THEIR VIRUSES

Hay I.D. and Lithgow T. Monash University, Infection & Immunity Program, Biomedicine Discovery Institute & Dept of Microbiology, Clayton, VIC, Australia.

Secretins form large (~150 Å), homo-oligomeric, gated pores in the outer membrane (OM) of bacteria. Each secretin complex comprises 12-16 copies of the secretin protein. Indicative of the quintessential nature of this family of proteins, secretins are ubiquitous among all didermic bacterial phyla and also found in some phage genomes. They form the OM component of the Type Four Pili (T4P), the Type Two Secretion System (T2SS), the Type Three Secretion System (T3SS/Injectisome) and are also required for the assembly and export of filamentous phage (Inoviridae). The T2SS is unique in that it exports a range of soluble folded exoproteins directly from the periplasm, commonly hydrolytic enzymes used to degrade biopolymers (proteins, carbohydrates, lipids) for nutrient acquisition, but also far more nefarious virulence factors such as the Cholera toxin, ETEC Heat-labile enterotoxin, or Pseudomonas Exotoxin A. Previously referred to as the terminal component of the general secretion system, it is now apparent that the T2SS is a more specialised secretion machine which must selectively recruit pre-exoproteins from the densely packed periplasm. Here we present our recent advances in characterising bacterial T2SS secretins. The distribution and classification of these proteins will be discussed and our recent structural insights into the function and assembly of these complex molecules will be provided.

THE NUCLEOSOME REMODELLING AND DEACETYLASE (NURD) COMPLEX HAS AN ASYMMETRIC, DYNAMIC AND MODULAR ARCHITECTURE

Silva A.P.G.1, Low J.K.K.1, Tabar M.S.1, Torrado M.1, Webb S.R.1, Parker B.L.1, Schmidberger J.W.1, Brillault L.2, Landsberg M.J.2 and Mackay J.P.1 1School of Life and Environmental Sciences, The University of Sydney, NSW, Australia. 2School of Chemistry and Molecular Biosciences, The University of Queensland, QLD, Australia.

The NuRD complex is essential for normal development and regulates both gene transcription and DNA damage repair. We have used structural, biophysical and biochemical data to define the architecture of the native mammalian complex. We showed that the complex displays considerable dynamics and we identified stable subcomplexes within NuRD, showing that the full complex is composed of two parts with separable enzymatic activities. A pseudo-symmetric deacetylase module comprising MTA, HDAC and RBBP subunits; whereas MBD, GATAD2 and CHD subunits form an asymmetric 1:1:1 arrangement with remodelling activity. The previously enigmatic GATAD2 both controls the asymmetry in the complex and recruits the ATP-dependent CHD remodeller. Taken together, our data define the architecture of the intact NuRD complex, revealing its structural dynamics and functional plasticity.


SymPoSia moNDay

SYM-19-01 SYM-19-02

SYM-19-03 SYM-19-04

DETECTION OF FLUORESCENTLY LABELED PROTEINS BY ELECTRON MICROSCOPY

Ariotti N.1, Rae J.2, Ferguson C.2, Hall T.E.2 and Parton R.G.2 1Electron Microscope Unit, The University of New South Wales. 2Institute for Molecular Bioscience, The University of Queensland.

Localising the position of proteins in the cell at high-resolution is critical for determining cellular function. As GFP revolutionized the detection of proteins by light microscopy, similarly new genetic tags for electron microscopy (EM) have great potential for EM visualisation of proteins in cells. We have developed a modular system for enzyme-based protein tagging that facilitates the detection of fluorescently-tagged proteins in the electron microscope by employing a modified soybean ascorbate peroxidase system termed APEX. This system allows for efficient analysis of subcellular protein distributions using existing GFP- and mCherry-tagged protein libraries. We demonstrate we can target APEX to any GFP- or mCherry-tagged protein of interest by engineering and genetically encoding a specific nanobody/binding peptide (BP) fused directly to the APEX-tag. We show that this system is robust, rapid and can be used in animal models. Moreover, this method compatible with correlative light and electron microscopy and can be coupled with sensitive methods for detecting protein-protein interactions through the use of split-GFP. The application of this method to a number of cell biological questions will be addressed in the talk.

CHLOROPLAST VOLUME IS UNDERESTIMATED FROM TWO-DIMENSIONAL CROSS SECTIONS

Harwood R.H. and Barbour M.B. University of Sydney, The Center For Carbon Water and Food, 380 Werombi Rd, Brownlow Hill NSW 2570.

Cell ultrastructure is predominantly studied by transmission electron microscopy (TEM), providing the user detailed two-dimensional (2D) information. Recent advances in microscopy have streamlined the acquisition of three-dimensional (3D) images. Scanning electron microscopy with an automated microtome (SBFSEM) produces serial micrographs that can be stacked and segmented to produce a three-dimensional volume data set. We are using SBFSEM on leaf cells to explore organelle size, shape and position, along with cell density and packing. The 3D anatomical data produced is being used to explore the relationship between leaf form and function. Models of key leaf processes, such as photosynthesis, sit at the heart of crop productivity and climate change models but include significant assumptions regarding the structure of leaves that ignore 3D complexity. SBFSEM allows us to challenge the idea of “textbook” leaf cell. To date we have focused on wheat and found that chloroplast volume estimates from 2D cross sections underestimated volume by 54% in mesophyll cells and 44% in bundle sheath cells. Chloroplast in meosphyll cells were 35% bigger then those in bundle sheath cells when measured in 3D, when estimated from 2D cross sections the difference was only 20%. Size, shape and relative distance of cells and organelles are critical in addressing leaf form and function, our research shows over simplification from 2D quantification limits our understanding of leaf function. Using volume microscopy, we can demonstrate uniform geometry assumptions can no longer be made in plant physiology.

MODULATION OF ROK MEMBRANE DISSOCIATION RATE TRIGGERS ROK PLANAR POLARISATION DURING MORPHOGENESIS

Sidor C.M.1, Stevens T.J.1, Boulanger J.1, Bailey M.J.2, Prehoda K.E.2, Harris T.J.3 and Roeper K.1 1MRC laboratory of Molecular Biology, Cambridge, UK. 2Department of Chemistry and Biochemistry at the University of Oregon, USA. 3Department of Cell & Systems Biology, University of Toronto, Canada.

The MyosinII activator Rok is involved in a variety of morphogenetic processes during embryonic development. We have shown previously in the Drosophila embryonic salivary gland placode that Rok is planar polarised at the tissue boundary through a negative regulation by the apical polarity proteins Crumbs (Crb) and aPKC. Intriguingly, despite Crb, aPKC and Rok being expressed in the whole tissue, this effect is specific to the boundary of the tissue. Using FRAP in embryos expressing endogenously tagged Rok, we find that Rok membrane dissociation rate is lower at the boundary of the tissue, where Crb and aPKC membrane levels are lower. Moreover, aPKC can phosphorylate the Rok membrane association region in vitro, suggesting Rok phosphorylation by aPKC might be responsible for the difference in Rok membrane dissociation rate. Finally, computer simulations show that such differences in Rok membrane dissociation rate are sufficient to explain Rok planar polarisation at the tissue boundary.

ADAPTIVE OPTICS AND ACTIVE PSF SHAPING ENABLE SUPER RESOLUTION FLUORESCENCE MICROSCOPY IN TISSUESMlodzianoski M.J.1, 9, Cheng-Hathaway P.J.2, 3, Bemiller S.M.4, McCray T.J.4, Liu S.1, Miller D.A.1, Lamb B.T.4, 5, 6, Landreth G.E.2, 3, 4 and Huang F.1, 7, 8 1Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana, USA. 2Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA. 3Department of Neurosciences, Case Western Reserve University, School of Medicine, Cleveland, Ohio, USA. 4Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, USA. 5Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana, USA. 6Department of Neurosciences, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, USA. 7Purdue Institute of Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, Indiana, USA. 8Purdue Institute for Integrative Neuroscience, Purdue University, West Lafayette, Indiana, USA. 9current address: The Walter and Eliza Hall Institute, Melbourne, VIC, Australia.Single molecule localization requires accurate and precise localization of the three-dimensional positions of single molecule point spread functions (PSFs) to reconstruct the 3D volume of a structure with high fidelity. Depth and sample induced optical aberrations make this task challenging when imaging more than just a few microns beyond the coverslip surface. These aberrations distort the PSFs of single molecules resulting in significant worsening of the localization precision, and therefore the resolution, while also introducing spatial localization biases. Optical aberrations can be compensated for using adaptive optics approaches, often with a deformable mirror, to restore high quality PSFs. We present here an efficient sensor-less adaptive optics approach using a deformable mirror for removal of aberrations for robust, 3D single molecule localization imaging. This method utilizes single molecule data as the base for the Nelder-Mead simplex algorithm to optimize the shape of the deformable mirror for removal of optical aberrations. We control the deformable mirror to include astigmatism for 3D localization information and adaptively control the magnitude of astigmatism to enforce a consistent, astigmatic PSF shape for a nearly uniform localization precision throughout the sample depth. We demonstrate this development by imaging through 30-μm thick brain tissue sections in order to visualize and reconstruct the 3D morphology and the nanoscale details of amyloid-β filaments in a mouse model of Alzheimer’s disease.


SymPoSia moNDay

SYM-19-05 SYM-20-01

SYM-20-02 SYM-20-03

MICROSTRUCTURE IMAGING FROM MACRO RESOLUTION MRI

Bourne R.1 and Panagiotaki E.2 1The University of Sydney. 2University College London.

Magnetic resonance imaging is increasingly being used to non-invasively detect diseases that have previously been characterised by biopsy and histopathology. However, the way underlying tissue microstructure affects the relatively low spatial resolution MRI signal is generally poorly understood. Further, many so-called ‘advanced’ imaging techniques are in fact extremely primitive and fail to exploit the potential of modern MRI technology. This presentation looks at two new approaches that enable the estimation of specific tissue microstructure changes from sophisticated image acquisition protocols and advanced data modeling.

GROUPER IRIDOVIRUS MEDIATED INHIBITION OF APOPTOSIS

Banjara S.1, Mao J.1, Ryan T.M.2, Caria S.1 and Kvansakul M.1 1Department of Biochemistry & Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia. 2SAXS/WAXS, Australian Synchrotron, 800 Blackburn Road, Clayton, VIC 3168, Australia.

Programmed cell death or apoptosis is a critical mechanism for the controlled removal of damaged or infected cells, and proteins of the Bcl-2 family are important arbiters of this process. Viruses have been shown to encode for functional and structural homologs of Bcl-2 to counter premature host cell apoptosis to ensure viral proliferation and/or survival. Grouper iridovirus (GIV) is a large DNA virus belonging to the iridoviridae family that harbors GIV66, a putative Bcl-2 like protein. GIV66 is a mitochondrially localized inhibitor of apoptosis, however the molecular and structural basis of apoptosis inhibition is currently not understood. To gain insight into the mechanism of action we systematically evaluated the ability of GIV66 to bind peptides spanning the BH3 domain of pro-apoptotic Bcl-2 family members. Our data reveal that GIV66 harbors an unusually high level of specificity for pro-apoptotic Bcl-2, and only displays affinity for Bim. We then determined crystal structures of both GIV66 on its own as well as bound to Bim BH3. Unexpectedly, GIV66 forms dimers via an interface that results in occluded access to the canonical Bcl-2 ligand binding groove, which breaks apart upon Bim binding. These data suggest that GIV66 dimerization may impacts on the ability of GIV66 to bind host pro-death Bcl-2 protein. Our findings provide a mechanistic understanding for the potent anti-apoptotic activity of GIV66 by identifying it as the first single specificity pro-survival Bcl-2 protein, and identifying a pivotal role of Bim for GIV mediated inhibition of apoptosis.

ROLE OF DYNAMIC COOPERATIVITY IN THE MECHANISM OF THE PLASMODIUM FALCIPARUM M17 AMINOPEPTIDASE

Drinkwater N.1, Yang W.1, Riley B.T.2, Malcolm T.R.1, Buckle A.M.2 and McGowan S.1 1Biomedicine Discovery Institute, Department of Microbiology, Monash University, Clayton Melbourne, VIC 3800, Australia. 2Biomedicine Discovery Institute, Department of Biochemistry, Monash University, Clayton Melbourne, VIC 3800, Australia.

The family of hexameric M17 aminopeptidases are conserved throughout all kingdoms of life, and are exciting drug targets for novel antimalarial and antibacterial agents. The M17 from P. falciparum (Pf-M17) is crucial to parasite survival, and a validated antimalarial drug target. We were interested to probe the role the conserved hexameric assembly plays in the function of Pf-M17, and to investigate the inherent dynamics of Pf-M17 and how they contribute to function. Towards this end, we undertook a comprehensive strategy composed of molecular dynamics simulations, X-ray crystallography, and mutational analyses to characterise the range of protein motions that Pf-M17 undergoes, and to probe the specific contribution of these motions to enzyme function. Based on these results, we propose a novel model for how Pf-M17 functions on an atomic level, whereby the two trimers within the hexamer operate in a mutually exclusive manner, and rely on a dynamic re-arrangement of bound metal ions and flexibility of a key regulatory loop. The regulatory loop possesses different characteristics in M17 aminopeptidases from other organisms, suggesting that the loop has undergone divergent evolution, and further, might be crucial to the emergence of new functions within this large and important enzyme family.

SUPER RESOLUTION: A CLOSER LOOK AT THE PLASMODIUM FALCIPARUM VIRULENCE COMPLEX

Looker O., Blanch A., McMillan P., Liu B., Dixon M. and Tilley L. Department of Biochemistry & Molecular Biology, The University of Melbourne, Bio21 Molecular Science and Biotechnology Institute, Victoria 3010.

After invading the human red blood cell (RBC), Plasmodium falciparum modifies the host cell surface by exporting proteins to the RBC periphery. The physical properties of the RBC are altered and parasite derived structures called knobs arrive at the cell periphery where they are anchored to the RBC membrane skeleton. These knobs are comprised mainly of the Knob-Associated Histidine Rich Protein (KAHRP), which acts as a scaffold for the presentation of the major virulence protein, P. falciparumErythrocyte Membrane Protein 1 (PfEMP1), through the membrane. A method has been developed for exposing the inner-surface of the infected RBC membrane allowing for the organisation of the RBC membrane skeleton to be visualised by scanning electron microscopy and for protein location to be examined using super resolution localisation microscopy. This has allowed us to visualise knob assembly at the RBC membrane skeleton. In a major advance, we have combined these two imaging modalities in a CLEM (correlative light and electron microscopy) based approach to investigate membrane remodelling and virulence complex assembly. The development of CLEM techniques is allowing further investigation into how RBC membrane skeleton remodelling facilitates knob formation and how PfEMP1 arrives at the knobs to drive parasite virulence. Through combining multiple imaging modalities with cellular biology and biophysical measurements we aim to understand the remodelling events that underpin parasite virulence.


SymPoSia moNDay

SYM-20-04 SYM-20-05

SYM-21-01 SYM-22-01

FINE MAPPING OF ADULT PLANT LEAF RUST RESISTANCE GENE LR49 IN WHEAT

Baranwal D., Bariana H. and Bansal U. The University of Sydney Plant Breeding Institute, School of Life and Environmental Sciences, Faculty of Science 107 Cobbitty Road Cobbitty NSW 2570.*Corresponding author: [email protected]

Sustainable wheat production is continuously affected by several biotic and abiotic stresses. Among biotic stresses, leaf rust caused by Puccinia triticina is considered as a major threat to sustain wheat production across the globe. Adult plant hypersensitive leaf rust resistance (R) genes such as Lr12, Lr22a, Lr22b, Lr35 and Lr49 have been characterised by necrotic flecks or small pustules which check the further proliferation of causing pathogen. Later, Lr49 was characterised as slow rusting resistance gene and is effective against all pre-dominating pathotypes of Australia and India. SNP were identified by aligning the sequences of flow sorted chromosome 4B of parental lines (VL404-Lr49 and WL711-susceptible parent) and converted into KASP markers. KASP markers sunKASP_21 and sunKASP_24, flanked Lr49 proximally and distally, respectively. Current study was planned to develop high-resolution map of Lr49 using 5120 F2 gametes from a cross of VL404/Avocet S to narrow down the physical interval between gene of interest and flanking markers to facilitate map-based cloning. DNA was extracted from leaf tissues from 2560 F2 plants using SDS-extraction method. KASP markers were tested of F2 DNA using real time PCR and LGC genomics protocol. Seventy-two recombinants were identified between flanking markers. These recombinants were screened against PBI leaf rust culture 539 (76-1, 3, (5), 10, 12) to confirm their disease response in F3 generation. Ref seq v 1.0 of Chinese Spring was used to map the flanking markers and these markers mapped in scaffold3450 representing 4Mb region. Markers will be developed to saturate the region.

MECHANISMS OF LIGAND SENSING IN THE NEWLY CHARACTERIzED, DOMINANT FAMILY OF PROKARYOTIC RECEPTORS

Roujeinikova A. Monash University, Melbourne, VIC 3800, Australia.

Chemotaxis, mediated by membrane-embedded chemotaxis receptors, plays an important role in bacterial ecology and pathogenesis. We investigate the structural basis of how the dominant family of bacterial chemoreceptors with an extracytoplasmic double Cache sensing domain (dCache SD) recognize chemical cues, and how they discriminate between attractants and repellents. We have determined the first representative crystal structures of the characterised dCache SDs of chemoreceptors from a broad range of bacteria of medical and biotechnological importance, including carcinogenic bacterium Helicobacter pyloriand important human and animal pathogen Campylobacter jejuni. Analysis of these structures, in conjunction with mutagenesis, biophysical and molecular simulation studies, provided an insight into diverse mechanisms of ligand recognition by this protein fold. In all previously characterised dCache SDs, direct sensing involved binding of the signal molecule to the membrane-distal, rather than membrane-proximal, subdomain. I will present data that changes this paradigm and reveal the first example of a chemoreceptor that directly recognises its ligand via the membrane-proximal subdomain, helping H. pylori to seek out lactate. In addition, I will present the results of our systematic study of the attractant-bound and repellent bound structures and discuss implications for the mechanism of discrimination between atrractants and repellents. I will then describe examples of very specific (Pseudomonas fluorescens CtaB) versus rather promiscuous (P. fluorescens CtaA, C. jejuni Tlp3) chemoreceptors with the same overall fold and discuss the structural basis behind this phenomenon. Finally, I will present an example of a dCache chemoreceptor (C. jejuni Tlp1) that recognises its signal molecule indirectly.

WRITING A TEACHING GRANT

Rowland S.1 and Kuit T.2 1The University of Queensland. 2The University of Wollongong.

In this interactive session Tracey Kuit and Susan Rowland will work with you to develop ideas for a teaching grant, pointing out the tips, tricks, and simple fixes you can use to increase your chance of success. The presenters are experienced writers and assessors of teaching grants. Bring your ideas!.

IMPROVING CRISPR-CAS9 MEDIATED GENE EDITING IN PLANTS

Naim F.1, Shand K.1, Roden S.1, Hayashi S.1, O’Brien M.2, Johnson A.2, Dugdale B.1 and Waterhouse P.1 1Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Australia. 2School of BioSciences, The University of Melbourne, Melbourne, Australia.

CRISPR-Cas9 driven gene editing of crops is rapidly advancing agricultural biotechnology. The process relies on DNA double stranded breaks at user defined genomic loci and repair by non-homologous end joining and/or homologous recombination. However, there are limitations in achieving precise and efficient editing of genes. These limitations include optimum design rules for gRNAs and delivery of Cas9 editing cassette. There are many gRNA design rules and online algorithms available to design gRNAs targeting genes in humans. We used a number of these tools to design and test the efficiency of gRNAs in Nicotiana benthamiana, banana, rice and tomato. Our results showed that there is no consensus between predictions by these algorithms and efficiency of obtaining mutations in plants. We discovered that targeting a single gene with two gRNAs increased the frequency of gene edits. To further improve editing, we also explored delivery of Cas9 plasmid using various DNA viruses. Here we report our exploration of the techniques and progress in efficiently editing plant genes.


SymPoSia moNDay

SYM-22-02 SYM-22-03

SYM-22-04 SYM-22-05

REVEALING THE ROLE OF SEPALLATA-LIKE GENES IN DETERMINING CEREAL INFLORESCENCE ARCHITECTURE USING GENOME EDITING APPROACH

Li G.1, Kuijer H.1, Wu D.2, Zhu W.2, Liang W.2, Burton R.1, Dreni L.2 and zhang D.1 1School of Agriculture, Food and Wine, University of Adelaide. 2School of Life Sciences and Biotechnology, Shanghai Jiao Tong University.

Over 50% of the global populations calories are obtained from cereal grains produced from spikelets on a flowering structure called an inflorescence. Improvements in crop yield could be made by increasing spikelet number. To achieve this goal, we need to gain a deeper understanding of the mechanisms underpinning inflorescence architecture. The shape of an inflorescence varies between cereals, ranging from highly branched in rice (Oryza sativa) termed a panicle, to a much more compact spike in barley (Hordeum vulgare) and wheat (Triticum spp.). One SEPALLATA (SEP) MADS domain transcription factor gene OsMADS34 has recently been identified that control inflorescence architecture and seed number in rice. To further understand the role of SEP genes, i.e. OsMADS1, OsMADS5 and OsMADS34 in the LOFSEP clade from rice and their counterparts in barley in regulating the inflorescence development, we are using gene-editing approaches in creating single mutants and high-order mutants of these genes in rice and barley respectively. Our preliminary data revealed that rice LOFSEPs play collaborative role in determining rice panicle branching, while barley homologs of rice OsMADS1, OsMADS5 and OsMADS34 showed conserved and divergent function in specifying barley inflorescence development.

CRISPR/CAS9-MEDIATED DISRUPTION OF THE RICE OSVIT1 AND OSVIT2 GENES TO IMPROVE GRAIN IRON DENSITY

O’Brien M.1, Kielnhofer E.O.F.1, Eftekhari F.N.2 and Johnson A.A.T.1 1School of Biosciences, The University of Melbourne, VIC 3010, Australia. 2Centre for Tropical Crops and Biocommodities, Queensland University of Technology, QLD 4001, Australia.

Rice provides 3.5 billion humans with more than 20% of their daily caloric intake. The commonly consumed white rice of modern rice cultivars is inherently poor in iron (Fe). Human Fe deficiency can cause mild to severe anaemia and is the most common nutritional disorder worldwide, estimated to affect up to two billion people. It is most common in developing countries where people depend upon micronutrient-poor staple crops such as rice. This has prompted the development of biofortified rice varieties with enhanced grain Fe density as a low-cost and sustainable strategy to tackle global human Fe deficiency. The rice VACUOLAR IRON TRANSPORTER 1 and 2 genes (OsVIT1/OsVIT2) are ubiquitously expressed at low levels in all tissues except the flag leaf where they are predominantly expressed. The OsVIT1 and 2 proteins are Fe transporters that are localised to the vacuole where they function to sequester Fe in the flag leaf. Disruption of OsVIT1 or OsVIT2 gene through T-DNA insertion lead to an increased in Fe concentrations in all tissues, including the grain1. In this study, we have used Agrobacterium-mediated transformation of rice with CRISPR/Cas9 plasmids targeting the OsVIT1 and OsVIT2 genes to generate a collection of osvit1 and osvit2 mutants. Eleven and fifteen independent mutant alleles were generated for OsVIT1 and OsVIT2, respectively. We present molecular and phenotypic analyses of homozygous osvit1 and osvit2 mutants, including an analysis of grain Fe concentration. 1 Zhang et al, 2012. Plant Journal 72, 400-410.

STREAMLINING CRISPR DELIVERY TO PLANTS

Jones B., Shueh S.Y., Soni K. and Lyu W. University of Sydney, School of Life and Environmental Sciences.

Developments in gene editing technologies have the potential to transform the way we research and improve our production plant species. The recalcitrance of crop species and elite cultivars to transformation and regeneration from explants and tissue cultured cells, however, continues to pose a significant bottleneck in the implementation of gene editing technologies in agriculturally important species. We are exploring ways of delivering CRISPR/Cas technologies to crop and model plant species in order to overcome these persistent difficulties.

CREATING SYNTHETIC GENE REGULATORY CIRCUITS IN PLANTS

Khan A., Kidd B. and Lister R. ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia.

Plants have an innate ability to respond to the environment to efficiently allocate resources and regulate their development in order to survive. To produce economically valuable chemicals and metabolites in plants and gain control of plant yield and development, new genetic tools and regulatory switches are required that allow sophisticated and precise control of plant gene expression, without impacting the endogenous genetic regulatory system and its ability to respond to stress. Existing genetic switches typically produce a control function in one direction (either on or off, but not both), and therefore there is a need for more sophisticated switches that can integrate signals from multiple inputs and enact a logic function to produce a desired outcome. Here we report our progress in constructing modular synthetic biology components for targeted control of plant gene expression with the aim of constructing orthogonal regulatory circuits that enact Boolean based logic in plants.


SymPoSia moNDay

SYM-23-01 SYM-23-02

SYM-23-03 SYM-23-04

A MOLECULAR DISSECTION OF NEURAL INDUCTION

Trevers K., Lu H.C., Anderson C., Strobl A.C., Palinkasova B., Filipkova L., Perez-Campos L., Moncaut N., de Almeida I.M. and Stern C.D. Dept Cell & Developmental Biology, University College London, United Kingdom.

Neural induction is the process, during normal development, when the future neural plate becomes specified and set aside from the rest of the ectoderm, under the influence of signals emanating from a special region of the embryo, the “organizer”. Neural induction is generally viewed as a switch, assuming a single event. To explore the nature of such a developmental switch, we have undertaken a detailed spatio-temporal analysis of the process at the molecular level. We establish that neural induction occurs over many hours and involves a hierarchical cascade of more than 200 transcription factors. Their expression is regulated by cooperation between several signalling pathways acting both sequentially and in parallel. We also uncover the epigenetic regulation of this cascade by mapping chromatin marks and revealing many regulatory elements associated with these transcription factors. We generate a dynamic Gene Regulatory Network, representing the interactions underlying the neural induction process. Therefore the switch between neural and non-neural ectoderm involves a highly complex network of events, regulated at many levels. This provides support for Waddington’s view of an “epigenetic landscape”: successive decisions made by cells over time, which gradually commit cells to their ultimate fate.

14-3-3zETA MODULATES NON CANONICAL SHH SIGNALLING TO CONTROL CORTICAL INTERNEURON DEVELOPMENT

Greenberg Z., Ramshaw H., Xu X. and Schwarz Q. Centre for Cancer Biology, University of South Australia.

Dysfunction in the formation and function of GABAergic cortical interneurons has been implicated as a central pathogenic mechanism in schizophrenia. 14-3-3ζ is part of a family of highly conserved intracellular proteins, that bind to the phosphoserine/theronine sites on target proteins and is highly expressed in the brain. Several findings in recent years implicate 14-3-3ζ as a candidate risk factor for schizophrenia including: 1) 14-3-3ζ is downregulated in post-mortem schizophrenic brain samples at the mRNA level; 2) 14-3-3ζ is downregulated across multiple neuroproteomic studies on schizophrenia patient samples; 3) linkage studies have implicated 14-3-3 family proteins in numerous neurodevelopmental disorders, and 4) genetic mutations in the gene encoding 14-3-3ζ have been found in schizophrenia patients. Previous studies have shown that 14-3-3ζ KO mice exhibit anatomical and behavioural traits akin to those seen in schizophrenia. Here we identify a novel role for 14-3-3ζ in interneuron development. We found a specific reduction in parvalbumin expressing interneurons throughout the cortex of 14-3-3ζ KO mice. Through a series of molecular, biochemical and morphological studies we further show that parvalbumin interneuron deficiency arises from defects in the specification and formation of interneurons during early brain development. Mechanistically, we found that 14-3-3ζ regulates non-canonical Shh signalling via controlling the activity of Rac1. Taken together, this work provides novel insight into the role of 14-3-3ζ in controlling interneuron development and identifies a novel role of 14-3-3ζ in the pathogenesis of schizophrenia.

GETTING CONNECTED: THE ROLE OF SEz6 FAMILY PROTEINS IN EXCITATORY SYNAPSE DEVELOPMENT AND MAINTENANCE

Munro K.M.1, Nash A.N.1, Teng K.S.-L.1, Carrodus N.L.1, 2, Barwood J.M.1, 2, Fuller S.J.1, 2, Eroglu C.3, Takeshima H.4, Power J.5

and Gunnersen J.M.1, 2 1Department of Anatomy and Neuroscience, The University of Melbourne, Parkville, 3010, Victoria, Australia. 2The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, 3010, Victoria, Australia. 3Duke University Medical Center, Durham, NC 27710, USA. 4Graduate School and Faculty of Pharmaceutical Sciences, Kyoto University, Japan. 5Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, UNSW Australia, Sydney, 2052, NSW, Australia.

The development of efficient synaptic connections and their refinement and maintenance is vital for cognition. Altered expression and/or function of proteins in the Seizure-related 6 (Sez6) family is associated with cognitive disorders and mice lacking Sez6 throughout development exhibit fewer neocortical excitatory synapses. Whether Sez6 proteins are required in the adult brain to maintain excitatory synaptic function, and the extent to which the three Sez6 family members functionally compensate for each other, is not known. These questions are highly relevant to the clinical trials of β-site amyloid precursor protein cleavage enzyme 1 (BACE1) inhibitors for Alzheimer’s disease because Sez6 proteins are major BACE substrates and, therefore, likely to contribute to mechanism-based side effects when BACE1 is chronically inhibited. The results of behavioural, electrophysiological, morphological and biochemical analyses of Sez6 family triple knockout and Sez6 conditional knockout mice reveal that Sez6 proteins are important, not only for synapse development but also for maintaining excitatory synapse structure and function in the adult brain.

AUTISM-LIKE SOCIAL INTERACTION DEFICITS CAN BE PREVENTED IN MICE HAPLOINSUFFICIENT FOR THE SULFATE TRANSPORTER SLC13A4 BY POSTNATAL ADMINISTRATION OF N-ACETYLCYSTEINE

Zhang Z.1, Dawson P.2, Simmons D.1 and Piper M.1 1The School of Biomedical Sciences, The University of Queensland. 2Mater Research Institute, The University of Queensland.

Mounting evidence suggests that dysregulated sulfate metabolism is associated with autism spectrum disorder (ASD). Here we reveal that the sulfate transporter SLC13A4 is a critical regulator of postnatal brain development, and that haploinsufficiency of Slc13a4 leads to deficits linked to ASD, including impaired social behaviours and altered neurogenesis. Importantly, conditional deletion of Slc13a4 demonstrates that the absence of this transporter during a critical postnatal period, but not afterwards, gives rise to the behavioural deficits. Excitingly, N-acetylcysteine, a safe, FDA-approved, amino acid derivative that can be metabolized to inorganic sulfate, rescues the ASD-like behavioural phenotypes when administered within this defined postnatal window. NAC may therefore have utility for the prevention of some forms of autism if administered in early life.


SymPoSia moNDay

SYM-24-01 SYM-24-02

SYM-24-03 SYM-24-04

VISUALIzING FUNCTIONAL ION CHANNELS AT THE CELL SURFACE

Irving H.R.1, 2, Abad I.2, Nguyen D.-T.2 and Manallack D.T.2 1La Trobe Institute for Molecular Science, La Trobe University Bendigo VIC 3550. 2Monash Institute of Pharmaceutical Sciences, Monash University Parkville VIC 3052.

Assembly of components of ion channels into functional heteromers at the cell surface is still mysterious. To begin to obtain clues about this process we use HTR3A and HTR3C receptor subunits that have each been tagged with separate fluorescent proteins in the second intracellular loop. The subunits are transiently expressed in HEK293T cells and visualised by high resolution microscopy. These subunits are processed normally through endoplasmic reticulum and Golgi pathways to reach the plasma membrane surface. Using TIRF microscopy we show that the subunit heteromers are formed intracellularly. Whole cell patch clamp reveals that the subunits are functional at the surface where they respond to 5-HT and this response is suppressed by ondansetron. Analysis of our TIRF images has allowed us to determine the proportion of different heteromers within the cell and at the surface. This finding is important as it will allow a more precise analysis of how different drugs contribute to subtle changes in heteromer versus homomer function that in turn may be related to individual receptor components.

HIGH THROUGHPUT PHENOTYPING OF HERG CHANNEL MUTATIONS

Ng C.A.1, 2, Perry M.D.1, 2 and Vandenberg J.I.1, 2 1Victor Chang Cardiac Research Institute, 405 Liverpool St, Darlinghurst, NSW 2010. 2St Vincent’s Clinical School, UNSW Sydney, Darlinghurst, NSW 2010.

The expression of hERG potassium channels at the plasma membrane of cardiac myocytes is critical for the coordinated propagation of the electrical signals that regulate the rhythm of the heartbeat. Reduced hERG function due to mutations increases the risk of sudden cardiac arrest and death. Mutations may affect synthesis, assembly, trafficking and/or function of hERG channels. The majority of mutants affect channel trafficking and traditionally this has been assessed using Western blot assays. Manual patch clamping is the gold standard for assessing the gating phenotype of hERG mutants and can also be used to assess current density. However, manual patch clamp assays are far too labour intensive for them to have clinical utility in assessing large numbers of mutants. In the era of precision medicine, large numbers of hERG mutations with unknown significance are going to be identified. Therefore, there is a need to develop higher throughput methods that are amenable to automation to substitute for the current Western blot and manual patch clamp assays. In this study, wild-type and 25 clinical hERG mutations were expressed in HEK293 cells to quantify their expression and gating phenotypes using ELISA assay and automated patch clamp assays (Syncropatch-384), respectively. The expression levels determined using the ELISA assay versus traditional Western blot analysis had a correlation coefficient of 0.86. The expression levels determined by current density measurements using the syncropatch (more than 1000 successful recordings) showed good correlation with the ELISA assays (correlation coefficient, 0.85). Our results indicate that both ELISA and the syncropatch methods can be used to assess channel expression. These methods not only have a higher throughput than traditional methods but they can also be applied to any ion channel including channels where the lack of glycosylation precludes the use of simple Western blot analysis.

CONTROL OF CELL MIGRATION AND SHAPE BY DUAL WATER AND ION CONDUCTING AQUAPORIN CHANNELS

Yool A.J., Pei J.V. and Kourghi M. University of Adelaide, SA 5005.

Structure-function analyses of human aquaporin-1 (AQP1) channels are defining the gating mechanisms and permeation pathways of these intriguing dual water- and ion-conducting channels, and identifying new pharmacological agents that differentially regulate the parallel ion and water pores. The AQP1 ion conductance is necessary for rapid migration in subtypes of aggressive cancer cells. A photoswitchable cation sensing probe developed by our team allows real-time imaging to localise AQP1-mediated cation entry in migrating cells. An arylsulfonamide agent AqB011 designed by our group selectively blocks the ion channel of AQP1 (IC50 14 μM) without altering water permeability (at doses up to 200 μM), providing a key tool for analysing the physiological roles of the AQP1 cation channel conductance in the ability of cells to move and maintain proper shape, in processes important for development, repair and survival in multicellular organisms.

NEW INSIGHTS INTO THE POST-TRANSLATIONAL REGULATION OF ABC LIPID TRANSPORTERS

Aleidi S.M.1, Yang A.1, Alrosan A.1, Sharpe L.2, Brown A.J.2 and Gelissen I.C.1 1School of Pharmacy, Faculty of Medicine and Health, University of Sydney. 2School of Biotechnology and Biomolecular Sciences, University of NSW, Sydney.

A number of ABC transporters, including ABCA1, ABCG1 and its close relative ABCG4, are essential regulators of cellular lipid homeostasis. ABCA1 and ABCG1 have been studied in the context of macrophage lipid homeostasis and atherosclerosis, insulin secretion in β-cells as well as brain lipid homeostasis, with substrates including cholesterol and phospholipids. ABCG4 on the other hand is thought to transport cholesterol, oxysterols and cholesterol synthesis intermediates exclusively in the brain, and has been linked to Alzheimer’s disease due to its potential to transport amyloid-β peptides from cells. These transporters are thought to be highly regulated at the post-translational level. Our group and collaborators have previously identified two separate avenues by which these transporters can be regulated i.e. via cellular cholesterol status as well as protein ubiquitination. The rate limiting step in protein ubiquitination is carried out by E3-ubiquitin ligases that have become of interest as potential therapeutic targets in a number of disease settings. We have identified a number of E3-ligases that are involved in the regulation of ABCA1, ABCG1 and ABCG4 (Aleidi et al 2015 and 2018). Here we describe under which circumstances these ligases regulate ABC lipid transporter protein levels and activity, and propose how these pathways may be exploited in the disease context in future.


SymPoSia moNDay

SYM-24-05 SYM-25-01

SYM-25-02 SYM-25-03

ENHANCING KCC2 AS A NOVEL STRATEGY FOR TREATING SEIzURES

Goulton C.S., Cheung D.L., Prikas E. and Moorhouse A.J. Department of Physiology, School of Medical Sciences, UNSW Sydney, Australia.

The K+Cl--cotransporter (KCC2) plays a key role in regulating intracellular Cl-, influencing the efficacy of GABAa-receptor mediated inhibition. To investigate how elevating KCC2 expression affects neuronal function, we used a transgenic mouse in which forebrain-restricted over-expression of KCC2 in pyramidal neurons could be regulated by doxycycline in the diet. In acute brain slices, field potentials were evoked from hippocampal CA1. It was found that KCC2 upregulation did not influence normal excitability, with similar input-output relationships, paired-pulse ratios, and concentration-response to muscimol observed. Interestingly, hyperexcitability was significantly reduced, as measured using the tetanus-induced afterdischarge and Zero-Mg2+ seizure models. This finding was subsequently supported in vivo, where seizures induced by kainic acid (up to 50mg/kg, i.p) were less likely to progress to status epilepticus in KCC2 upregulated mice (1/5 mice vs. 15/15 mice from control cohorts). A recent study found that the clinically available compound prochlorperazine (Stemetil) acutely increases KCC2 expression (Liabeauf et al, 2017). To explore the potential effects of Stemetil on hyperexcitability, we conducted preliminary experiments using the in vitroafterdischarge seizure model. It was found that a 60 min incubation with Stemetil (10μM) reduced afterdischarge bursts (p<0.01) with no effects on basal excitability. Overall, our data support the strategy of enhancing KCC2 to reduce neuronal hyperexcitability, without negatively affecting basal synaptic transmission. This means that increasing KCC2 may enable greater Cl- homeostasis and maintain more efficacious GABAergic inhibition, hence providing a novel strategy to enhance neuronal inhibition. Further, preliminary results support continued investigation into Stemetil as a therapeutically relevant KCC2 enhancer for the treatment of seizures.

UNDERSTANDING THE BIOSYNTHESIS OF THE GLYCOPEPTIDE ANTIBIOTICS

Cryle M.J. Department of Biochemistry & Molecular Biology, Monash University, Clayton, VIC 3800.

The glycopeptide antibiotics (GPAs) are a structurally complex and medically important class of peptide natural products that include the clinical antibiotics vancomycin and teicoplanin. They contain a large number of non-proteinogenic amino acids and are produced by a linear non-ribosomal peptide synthetase (NRPS) machinery comprising seven modules. Furthermore, GPAs are extensively crosslinked late in their biosynthesis on the NRPS assembly line by the actions of a cascade of Cytochrome P450 enzymes, a process which contributes to the rigidity and structural complexity of these compounds. Due to the challenge of synthesising GPAs, biosynthesis remains the only means of accessing GPAs for clinical use, which makes understanding the biosynthesis of GPAs of key importance. Here I will detail results from our studies into the NRPS machinery, the P450-cyclisation cascade and the interplay of these two important processes during GPA biosynthesis. These demonstrate how selectivity is mediated through the careful orchestration of critical modification steps and interactions between the peptide-producing NRPS machinery and trans-modifying enzymes.

PEPTIDE-INSPIRED INHIBITORS OF C-MANNOSYLTRANSFERASES

Goddard-Borger E.D.1, 2 1The Walter & Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia. 2Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia.

Tryptophan mannosylation is an unusual co-translational modification that promotes protein folding and enhances the stability of some type-I cytokine receptors. This protein modification is installed by two integral membrane enzymes that are localised to the ER and encoded by dpy19l1 and dpy19l3. Perturbing tryptophan mannosylation dramatically reduces the cell surface expression of TPOR and IL7R: cytokine receptors whose constitutive expression drives the proliferation of some blood cancers (e.g. MPNs and T-ALL). We have developed small molecule inhibitors of C-mannosyltransferases to determine if pharmacological inhibition of tryptophan mannosylation might be useful for treating some blood cancers. To accomplish this, we established in vitro C-mannosyltransferase activity assays and probed the enzyme’s peptide substrate preferences in detail. SAR studies then guided us in the conversion of a tetrapeptide substrate of the enzyme into a non-peptide inhibitor of tryptophan mannosylation with low micromolar activity in cells.

DNA-BASED INHIBITORS OF THE HUMAN APOBEC3B DNA CYTOSINE DEAMINASEBarzak F.1, Kvach M.V.1, Harjes S.1, Jameson G.B.1, 2, Aihara H.4, Harris R.S.4, 5, Filichev V.V.1, 2, Harki D.A.3 and Harjes E.1, 2 1Institute of Fundamental Sciences, Massey University, Private Bag 11 222, Palmerston North, New Zealand. 2Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, New Zealand. 3Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA. 4Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA. 5Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN 55455, USA.

The APOBEC3 (A3) protein subfamily of seven proteins (A3A-H) are cytidine deaminases that attack retroviruses and other pathogens by hypermutating cytidine residues of single-stranded DNA [1] while, at the same time some APOBEC3 family members are involved in carcinogenesis [2]. In particular, one member of the family, APOBEC3B promotes mutagenesis in several cancers and contributes to tumor evolution including the detrimental outcomes of metastasis and drug resistance [3-7]. Inhibition of APOBEC3B may therefore be used to augment existing anticancer therapies [8]. We designed and tested chemically modified APOBEC3 DNA substrates for their inhibitory potential and obtained the first DNA-based inhibitors of APOBEC3 enzymes with low micromolar inhibition constants. We found that neighbouring nucleotides in the DNA sequence influence the specificity of our inhibitor which, led to the development of the first inhibitor selectively targeting APOBEC3B. This provides a platform for further development of APOBEC3B inhibitors with cellular activity. REFERENCES 1. Harris, R.S. and J.P. Dudley, APOBECs and virus restriction. Virology, 2015. 479: p. 131-145. 2. Swanton, C., et al., APOBEC enzymes: mutagenic fuel for cancer evolution and heterogeneity. Cancer discovery, 2015. 5(7): p. 704-712. 3. Burns, M.B., et al., APOBEC3B is an enzymatic source of mutation in breast cancer. Nature, 2013. 494(7437): p. 366-370. 4. Burns, M.B., N.A. Temiz, and R.S. Harris, Evidence for APOBEC3B mutagenesis in multiple human cancers. Nature Genetics, 2013b. 45(9): p. 977-983. 5. Law, E.K., et al., The DNA cytosine deaminase APOBEC3B promotes tamoxifen resistance in ER-positive breast cancer. Science advances, 2016. 2(10): p. e1601737. 6. Sieuwerts, A.M., et al., Elevated APOBEC3B correlates with poor outcomes for estrogen-receptor-positive breast cancers. Hormones and Cancer, 2014. 5(6): p. 405-413. 7. Ding, Q., et al., APOBEC3G promotes liver metastasis in an orthotopic mouse model of colorectal cancer and predicts human hepatic metastasis. The Journal of clinical investigation, 2011. 121(11). 8. Olson, M.E., R.S. Harris, and D.A. Harki, APOBEC Enzymes as Targets for Virus and Cancer Therapy. Cell chemical biology, 2017.


SymPoSia moNDay

SYM-25-04 SYM-25-05

SYM-26-01 SYM-26-02

GENETIC INCORPORATION OF UNNATURAL AMINO ACIDS INTO SINGLE CHAIN VARIABLE FRAGMENTS FOR GENERATION OF STABLE ANTIBODY-IMAGING PROBE CONJUGATES FOR CANCER IMAGING

Subas Satish H.P.1, 2, 3, Howard C.B.3, Huda P.3, Fletcher N.3 and Thurecht K.3 1Walter and Eliza Hall Institute, 1G Royal Parade, Parkville, VIC 3052. 2University of Melbourne, Parkville, VIC 3010. 3University of Queensland, St Lucia, QLD 4072.

The linker chemistry utilised for the generation of antibody-imaging dye, nanomaterial or drug conjugates plays a crucial role in the development of stable and clinically successful antibody-targeted diagnostics or therapeutics. The incorporation of an unnatural amino acid having orthogonal chemical reactivity into single chain variable fragments (scFv) in vivo provides a chemical handle for the generation of such stable antibody conjugates. An unnatural amino acid having an azide functional group, 4-azido phenylalanine or p-azido phenylalanine (pAzF) was incorporated into anti-EGFR scFv expressed in Escherichia coli. A stable scFv-imaging dye conjugate was generated via a strong triazole linkage formed by strain-promoted copper-free click reaction between the azide functional group of the pAzF anti-EGFR scFv and DBCO functionalized fluorescent dyes, cyanine 5 or cyanine 7. The pAzF anti-EGFR scFv showed significant binding to recombinant EGFR in vitro and also, through flow cytometry, the target specificity of the pAzF anti-EGFR scFv-DBCO cy5 conjugates to native EGFR expressing MDA MB 468 breast cancer cells was validated in vitro. In addition, in vivo optical imaging studies performed with MDA MB 468 tumour xenograft models showed targeted accumulation of pAzF anti-EGFR scFv-DBCO cy7 in MDA MB 468 cancer, thereby establishing both the targeting and imaging potential of the conjugates. This study therefore demonstrates the incorporation of an unnatural amino acid with orthogonal chemical reactivity site-specificially into an scFv as well as the potential of unnatural amino acids to form stable antibody-imaging dye conjugates for use in tumour imaging.

STRUCTURAL VARIANTS OF A LIVER FLUKE DERIVED GRANULIN PEPTIDE POTENTLY STIMULATE WOUND HEALING

Dastpeyman M.1, Bansal P.S.1, Wilson D.1, Sotillo J.1, Brindley P.J.2, Loukas A.1, Smout M.J.1 and Daly N.L.1 1James Cook University, Cairns Australia. 2George Washington University, Washington USA.

Granulins are a family of growth factors involved in cell proliferation. The liver-fluke granulin, Ov-GRN-1, isolated from a carcinogenic liver fluke Opisthorchis viverrini, can significantly accelerate wound repair in vivo and in vitro. However, it is difficult to express Ov-GRN-1 in recombinant form at high yield, impeding its utility as a drug lead. A truncated analogue (Ov-GRN12-35_3s) promotes healing of cutaneous wounds in mice. NMR analysis of this analogue indicates the presence of multiple conformations, most likely as a result of proline cis/trans isomerisation. To further investigate whether the proline residues are involved in adopting the multiple confirmations we have synthesised analogues involving mutation of the proline residues. We have shown that the proline residues have a significant influence on the structure, activity and folding of Ov-GRN12-35_3s. These results provide insight into improving the oxidative folding yield and bioactivity of Ov-GRN12-35_3s, and might facilitate the development of a novel wound healing agent.

MECHANISMS CONTROLLING METABOLISM BY REGULATING PROTEIN TURNOVER

Christiano R., Farese R.J. and Walther T.C. Harvard University/Howard Hughes Medical Institute.

Many metabolic reactions are tightly controlled. One mechanism for such control occurs through regulating the stability of key enzymes of metabolism. Such regulation is mediated by ubiquitin ligases, sorting factors, and proteases. To elucidate regulatory pathways, we globally investigated protein turnover a large sets of mutants in the degradative machinery in S. cerevisiae. Analysis of the resulting turnover map (T-MAP) revealed targets for most ubiquitin ligases and identified the primary degradation routes for most short-lived proteins. Illustrating the power of this approach, we uncovered new insights into ERAD pathways governing sterol synthesis and numerous previously unknown nodes of regulation for sphingolipid synthesis. Expansion of the T-MAP strategy in yeast and mammalian cells provides a powerful tool to unravel the contributions of protein degradation to proteostasis.

USING AN OMICS APPROACH TO IDENTIFY NOVEL REGULATORS OF HEPATIC LIPID METABOLISM

Drew B.G.1, Parker B.L.2, Seldin M.3, Keating M.F.1, Meikle P.J.1, Tarling E.J.3, Lusis A.J.3, James D.J.2, De Aguiar Vallim T.Q.3

and Calkin A.C.1 1Baker Heart and Diabetes Institute. 2Charles Perkins Centre, University of Sydney. 3University of California, Los Angeles.

Background: The liver controls numerous pathways central to the maintenance of whole body lipid metabolism. Dysregulation of these pathways can result in increased levels of pathological lipids that can promote insulin resistance, fatty liver disease and cardiovascular disease. Thus, we need a greater understanding of the pathways regulating hepatic lipid metabolism. Methods: We utilised a trans-omics approach to analyse mouse livers across 100+ strains of genetically diverse mice, integrating genetics, phenomics, and proteomics (>8000 proteins; Orbitrap Fusion) as well as liver and plasma lipidomics (>300 lipids; API4000 Q/TRAP) to identify novel lipid signatures and pathways associated with metabolic diseases. Results: This approach has generated a powerful platform that we can interrogate to perform protein:protein correlations, which can give us insight into protein complexes and protein localisation within the cell; protein:lipid correlations, which can provide insight into novel regulators of known pathological lipids; lipid:lipid correlations, which can provide an avenue to identify plasma lipids that might predict hepatic accumulation of known pathological lipids linked to metabolic disease. Findings are validated in vitro, in preclinical models and in clinical cohorts such as the UK Biobank and the San Antonio Family Heart Study. Conclusions: We have established a high-resolution trans-omics platform for the identification of novel regulators of hepatic lipid metabolism, with implications for therapeutic intervention of metabolic diseases.


SymPoSia moNDay

SYM-26-03 SYM-26-04

SYM-26-05 SYM-27-01

THE ROLE OF MITOCHONDRIAL PHOSPHATIDYLETHANOLAMINE SYNTHESIS IN REGULATING SKELETAL MUSCLE LIPID HOMEOSTASIS

Bruce C.R. Institute for Physical Activity and Nutrition (IPAN), School of Exercise and Nutrition Sciences, Deakin University, Burwood, VIC 3125, Australia.

Phosphatidylethanolamine (PE) is the second most abundant phospholipid in mammals. PE is synthesized via two pathways, cytidine diphosphate (CDP)-ethanolamine pathway located in the endoplasmic reticulum and the phosphatidylserine decarboxylase (PSD) pathway in the mitochondria. While the CDP-ethanolamine pathway is considered the major route for PE production in most mammalian tissues, our recent observations suggest the mitochondrial PSD pathway could be an important site of PE synthesis in muscle (Selathurai et al., 2015). Here, the results from studies exploring the role of the mitochondrial PE synthetic pathway in regulating lipid homeostasis in skeletal muscle will be discussed and will reveal unique insight into the significance of mitochondrial phospholipid synthesis in skeletal muscle. Selathurai A et al. The CDP-Ethanolamine Pathway Regulates Skeletal Muscle Diacylglycerol Content and Mitochondrial Biogenesis without Altering Insulin Sensitivity. Cell Metab 21(5):718-30, 2015.

THE E3 UBIQUITIN LIGASE MARCH6 IS BOOSTED BY CHOLESTEROL

Sharpe L.J., Howe V. and Brown A.J. UNSW Sydney NSW 2052.

The E3 ligase membrane-associated RING finger (C3HC4) 6 (MARCH6) helps control protein levels of the two rate-limiting enzymes in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and squalene monooxygenase (SM). However, little is known about how MARCH6 itself is regulated. Considering MARCH6’s role in controlling cholesterol synthesis, we hypothesised that cholesterol may regulate MARCH6. Indeed, we found that cholesterol stabilises MARCH6 protein. Preliminary evidence suggests that Insigs also play a role in regulating MARCH6’s stability. Our ongoing investigations focus on the nature of the MARCH6-Insig interplay,and whether other intermediary proteins are involved. This work provides new insights into the complex feedback mechanisms underlying cholesterol homeostasis.

TACKLING LIPID DIVERSITY IN MEMBRANES: THE EFFECT ON MEMBRANE AND PROTEIN FUNCTIONS

Poger D., Corbett M.S.P. and Mark A.E. School of Chemistry & Molecular Biosciences, The University of Queensland, Brisbane QLD 4072, Australia.

Biological membranes regulate a myriad of cellular processes through the modulation of essential properties such as membrane fluidity and the formation of lipid microdomains. Such differences in turn affect the function of membranes and membrane proteins. The chemical and structural diversity of lipids is only being uncovered. For example, the repertoire of lipids in bacterial membranes is much broader than in eukaryotic membranes. In many if not most bacteria, membrane lipids include branched-chain fatty acids. Hopanoids have been identified in a range of bacteria. Branched-chain fatty acids have been proposed to protect membranes against hostile conditions and hopanoids have long been hypothesised to be surrogates of sterols, but, in fact, little is known about their actual effect on membranes. Using atomistic simulations, I showed that the different types of branching and hopanoids have specific effects on membrane fluidity and structure that allow bacteria to finely tune the sensitivity of their membranes to the environment. Furthermore, branched-chain lipids could affect the activity of commonly used disinfectants such as triclosan and para-chloroxylenol on membranes by modulating the interaction of the biocides with lipids and how deep they could insert into a membrane. The membrane composition also plays a critical role in the function of proteins. In simulations of the type-I cytokine receptors for growth hormone (GHR), prolactin (PRLR) and erythropoietin (EPOR) embedded in membranes, the presence of cholesterol altered the behaviour of the transmembrane domains, suggesting a key role of cholesterol in the mechanical coupling of the receptors through the plasma membrane upon receptor activation. The lipid composition is thus critical in the function of membrane and membrane proteins.

UNDERSTANDING SPATIAL AND TEMPORAL CONTROL OF GPCR SIGNALLING USING HIGH RESOLUTION IMAGING

Halls M.L. Monash Institute of Pharmaceutical Sciences, Monash University, 399 Royal Parade, Parkville, VIC 3052, Australia.

Cells endogenously express many different receptors that can activate the same second messenger, but with remarkably diverse physiological outcomes. This suggests a high degree of organisation and regulation of intracellular signalling, which is achieved by the spatiotemporal compartmentalisation of signals – the restriction of second messengers in space and time. The development of targeted Forster Resonance Energy Transfer (FRET)-based biosensors has increased the resolution at which we can measure the spatial and temporal signalling of GPCRs. Moreover, single molecule imaging techniques such as Total Internal Reflection Fluorescence (TIRF) and Fluorescence Correlation Spectroscopy (FCS) now allow us to correlate subtle changes in receptor organisation at the plasma membrane with large changes in spatiotemporal signalling. The complementary use of single cell and molecule imaging has revealed that GPCRs can activate very defined signals in limited sub-cellular compartments by assembling focused protein complexes. These complexes facilitate second messenger production, the organisation and scaffolding of effectors, and co-ordination of regulatory elements.


SymPoSia moNDay

SYM-27-02 SYM-27-03

SYM-27-04 SYM-27-05

PROTEIN DYNAMICS AND KINETICS OF GENOME-WIDE OCCUPANCY OF THE SOX18 TRANSCRIPTION FACTOR

McCann A.1, Lou J.2, Blum A.3, Moustaqil M.4, Fontaine F1, Sierecki E.4, Gambin Y.4, Meunier F.3, Liu J.Z.5, Hinde E.2 and Francois M.1 1Institute for Molecular Bioscience, The University of Queensland. 2Bio21 Institute, The University of Melbourne. 3Queensland Brain Institute, The University of Queensland. 4European Molecular Biology Laboratory, University of New South Wales. 5Janelia Farm, Howard Hughes Medical Institute.

Cell fate determination relies on the ability of transcription factors (TFs) to select protein partners and specific regulatory elements to instruct a particular transcriptional output. Central to endothelial cell fate acquisition during embryonic development, the SOX18 transcription factor is a key regulator of both blood vascular and lymphatic endothelial cell specification. This TF is transiently expressed in all vascular beds during embryogenesis, however, the mechanisms that drive its specific molecular mode of action to instruct the differentiation of distinct sub-population of endothelial cells are currently unknown. To identify a global mechanism that could drive a differential activity of this TF in discrete cell subtypes, we analysed chromatin occupancy dynamics of SOX18 by combining Halo-tag technology with single molecule tracking in vitro. This approach identified that SOX18 binds to the chromatin via a two-component model; which means that SOX18 binds transiently to several non-specific sites, before binding more stably to specific target sites. Lastly, taking advantage of a non-functional SOX18 dominant-negative protein that causes the human syndrome Hypotrichosis-Lymphedema-Telangiectasia (HLT), we show that this non-functional TF interferes with the search pattern of SOX18 to identify its target genes. Our study shed light onto molecular behaviours of SOX18 TF on a genome-wide scale, and reinforces the concept that protein-protein interactions are central to govern target gene selectivity. We also uncover a global mechanism that explains at a molecular level how a dominant-negative protein disrupts TF activity and drives the aetiology a rare human vascular disease.

PROBING MECHANISMS FOR FORCE-SENSING IN MECHANOSENSITIVE ION CHANNELS WITH ARTIFICIAL DROPLET BILAYER SYSTEMS

Jaggers O.1, Ridone P.2, Xiao B.3, Martinac B.2 and Baker M.A.B.1 1School of Biotechnology and Biomolecular Science, UNSW. 2Victor Chang Cardiac Research Institute. 3Tsinghua University.

Droplet Hydrogel Bilayers enable simultaneous single channel current and fluorescence measurements[1]. They have been used to characterise the functionality of alpha-haemolysin for use in nucleobase recognition in DNA sequencing and they have been arranged in multiple arrays to parallelise high throughput channel measurements. We recently used this platform to apply force and measure the response of bacterial mechanosensitive ion channel MscL[2]. We are now using this platform to investigate the force sensitive ion channel PIEZO1[3], in which single point mutations cause blood disorders such as xerocytosis and which is generally linked to cancer progression and post traumatic osteoarthritis. We observe that PIEZO1 in DHBs displays the same gating activity in DHBs as in patch-liposomes, and, similarly, is much more sensitive in the presence of cholesterol. We also investigate the use of DNA-origami to regulate lipid-lipid interactions. This serves to enable light-triggered insertion of membrane proteins into artificial bilayers and allows greater spatiotemporal control. [1.] A. J. Heron, et al., JACS. 131, 1652-1653 (2009). [2.] K. Rosholm et al., Scientific Reports. 7, 45180 (2017). [3.] Q Zhao et al., Nature, 554, 487-492 (2018).

METAL STARVATION TRIGGERS CETz1-DEPENDENT CELL SHAPE CHANGES IN HALOARCHAEA

De Silva R.T., Ithurbide S. and Duggin I.G. University of Technology Sydney.

Microbial cell shape is a significant attribute that affects survival, and many species can change morphology to adapt to environmental change and stress. Model haloarchaeon Haloferax volcanii cells transition from plates to rods to optimise swimming motility, which requires the tubulin-like cytoskeletal protein CetZ1. The signals that trigger cell shape change via CetZ1 are not yet clear. We analysed cell shape changes in various nutrient-depleted media, and found that cells developed highly irregular elongated forms in response to depletion of metal nutrients. These shapes were substantially more diverse compared to the regular rod-shaped cells seen in motile cells. Remarkably, the addition of a solution containing 8 metals to complex growth medium (HvYPC) significantly improved culture growth, and the cells showed a uniform plate-shaped morphology compared to non-supplemented HvYPC that produces mixed elongated- and plate-cell types. The formation of elongated cells during trace element limitation was dependent on CetZ1, and the cell elongation defect of an in-frame knock-out of cetZ1 was rescued by expression of CetZ1 from a plasmid. Towards the goal of understanding how CetZ1 functions to control cell shape, we have adapted a set of modern fluorescent proteins for use in H. volcanii. A CetZ1-mTurquoise2 fusion showed cell elongation capacity and revealed a highly dynamic localisation pattern associated with cell elongation during metal depletion, indicating that a dynamic remodelling of the cell envelope occurs during cell elongation. The improved tools and growth conditions can be utilized for working with H. volcanii in research and biotechnology.

SCF-BTRCP MEDIATES THE DEGRADATION OF CEP68 INTERCENTROSOMAL LINKER PROTEIN TO CONTROL THE DISASSEMBLY OF THE PERICENTRIOLAR MATERIAL IN MITOSIS

Pagan J.1, Jones M.2 and Pagano M.3 1University of Queensland, SBMS. 2Memorial Sloan Kettering Cancer Center. 3New York University Medical Center.

Skp1-Cul1-F-box protein (SCF) complexes are the best-characterised of the multisubunit E3 Ubiquitin ligases. Each SCF complex contains one of 69 exchangeable substrate-targeting subunits, called F-box proteins. Core SCF components, SKP1 and CUL1, localise to mitotic and interphase centrosome suggesting that SCF ligases mediate the turnover of centrosomal substrates. Indeed, several specific F-box proteins have been recently shown to regulate the stability of key centrosome proteins, including PLK4 kinase (by SCF-bTrCP), the master regulator of centriole duplication. We demonstrate that SCF-bTrCP also mediates the degradation of Cep68 intercentrosomal linker protein in mitosis. Cep68 degradation is initiated by PLK1 phosphorylation of Cep68 on Ser 332, allowing recognition by bTrCP. The removal of Cep68 mediated by PLK1 and bTrCP is the first in a series of relocalization and degradation mechanisms in mitosis required to reset the centriole to a duplication competent configuration.


SymPoSia moNDay

SYM-28-01 SYM-28-02

SYM-28-04SYM-28-03

NON-CANONICAL TGF-β/SMAD SIGNALLING ENHANCES CELL-TO-CELL SPREAD DURING VACCINIA VIRUS INFECTION

Gowripalan A., McKenzie C.D. and Newsome T.P. School of Life and Environmental Sciences, University of Sydney, NSW, Australia, 2006.

The ability of viruses to manipulate the hostile cell microenvironment is often crucial to ensuring their prolonged survival within the host. The pathways they influence range from those which mediate apoptosis and cell proliferation, to others that control migration and cytoskeletal structure. One commonly manipulated host system is the TGF-β signalling cascade. Observations in our lab suggest vaccinia virus (VACV) can exploit elements of this pathway during infection. Specifically, we found, via luciferase assay and western blots, that VACV can potently activate the TGF-β-associated, R-Smad transcription factors, Smad2 and Smad3, as well as the common Smad, Smad4. Using CRISPR-Cas9 and siRNA technologies, we have also demonstrated a role for Smad4 in a number of aspects of viral infection, including viral replication, cell-to-cell spread and cell migration. Global transcriptomic analysis uncovered a number of Smad4-dependent transcriptional targets which may play a role in enhancing VACV spread. Interestingly, it appears that activation of these Smad proteins occurs entirely independently of TGF-β receptor phosphorylation in the VACV context. To our knowledge, no other microbe is able to stimulate this pathway in the same manner. VACV seems to be unique in this ability, as closely related virus, Ectromelia virus is unable to activate this pathway. Understanding how VACV is able to induce this signalling cascade independently of the TGF-β receptor, could be critical in understanding regulation of non-canonical Smad signalling and also provide new insights into oncolytic virus research and tumour cell dynamics.

UNCOUPLING PKR ACTIVATION AND TRANSLATIONAL ARREST DURING NOROVIRUS INFECTION

Fritzlar S., Chao Y.W., Aktepe T.E. and Mackenzie J.M. Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, at the University of Melbourne.

Human norovirus (HuNoV) are positive sense RNA viruses belonging to the Caliciviridae family and are a major cause of acute gastroenteritis. However, the study of HuNoV is challenging due to the lack of effective tissue culture systems and small animal models. Despite its significant health burden, there are currently no effective treatments for HuNoV infections. Recently, the discovery of a closely related norovirus, Murine Norovirus (MNV) has advanced our understanding of norovirus biology and pathogenesis. Here we investigated the association between MNV infection, stress granules and protein translation. We observed that stress granules (SG) are not induced during MNV infection. Further, infected cells treated with sodium arsenite, a known oxidative stressor to induce SG formation, were restricted in their ability to form SGs, suggesting a potential viral control for delayed SG formation. We also demonstrated that during infection, there was a progressive increase in phosphorylated eukaryotic initiation factor 2 alpha (eIF2α), yet increased MNV translation still occurred under these conditions implying MNV may employ an alternative protein translation mechanism. To confirm the increasing host translation shutoff, we treated infected cells with puromycin and showed that there is increasing stalling of translation via western blotting with anti-puromycin antibodies, which correlated to the increasing phosphorylation of eIF2α. Our subsequent analyses suggested that the translational repression is mediated via Protein kinase-R (PKR), but further investigation revealed that PKR activation and translational arrest were uncoupled during infection. These results suggest MNV may regulate the PKR-mediated response by promoting eIF2α phosphorylation but inhibits cellular protein translation by a currently unknown mechanism. These observations may provide a link between MNV infection, stress granules, PKR and translational control.

SUBCELLULAR TRAFFICKING OF HOST RNA HELICASE DDX3X MODULATES INNATE ANTIVIRAL SIGNALLING AND PARAINFLUENzA VIRUS IMMUNITY

Heaton S.M., Atkinson S.A., Jans D.A., Sweeney M.N. and Borg N.A. Infection & Immunity Program, Biomedicine Discovery Institute, Department of Biochemistry & Molecular Biology, Monash University, Clayton, 3800, Australia.

DEAD-box RNA helicase 3, X-linked (DDX3X) multiply regulates the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) antiviral signalling cascade. Accordingly, manipulating DDX3X is crucial to the replication strategy of a growing list of evolutionarily diverse pathogens including influenza A virus, HBV, HCV, dengue virus and HIV-1. To fulfil these host- and pathogen-directed roles, DDX3X localises to various subcellular compartments. Interestingly, nuclear export of DDX3X was previously attributed to an exportin-1/CRM1-dependent mechanism that, uniquely, required neither a nuclear export signal (NES) nor the nucleocytosolic Ran-GTP/GDP gradient. We examined the DDX3X-exportin-1 interaction by analytical ultracentrifugation and confocal microscopy. We probed its role in immune signalling and human parainfluenza-3 (hPIV-3) replication in human lung A549 cells using plaque assays, luciferase gene reporter assays, NanoString transcriptome analysis, FACS and ELISA, then dissected its mechanism of immune regulation by constitutively activating RLR signalling proteins or treating with poly(I:C), LPS or IFN-β. Contrasting the current model, we find the region bound by exportin-1 bears no primary sequence homology to any recognised protein domain in evolution. We show exportin-1 is the bulk nuclear export receptor for DDX3X, which is specifically impaired by treatment with exportin-1 inhibitors, DDX3X point mutagenesis or removal of Ran-GTP. In response to viral RNA exposure during hPIV-3 infection, DDX3X accumulates inside the nucleus to support IFN-β expression. However, ectopic nuclear DDX3X impairs RLR signalling, deregulates dozens of genes relevant to immunity and fails to suppress hPIV-3 replication. Our results redefine the molecular mechanism of DDX3X nuclear export and reveal new modes of immune regulation.

BIOLOGICAL CONTROL OF RABBITS IN AUSTRALIA - AN ONGOING CO-EVOLUTIONARY ARMS RACE

Hall R.N. and Strive T. CSIRO Health & Biosecurity, GPO Box 1700, Canberra, Australian Capital Territory 2601, Australia.

Wild European rabbits have enormous impacts on Australia’s agricultural industries and environment, currently costing over $200 million in production losses annually, and threatening 304 native plants and animals. To manage the damaging rabbit plagues of the last century, Australia introduced two biological control agents, a poxvirus, Myxoma virus, in the 1950s and a calicivirus, rabbit haemorrhagic disease virus, in the 1990s. This has been tremendously successful, with the cumulative benefits of biocontrol agents to Australia’s agricultural industries estimated at $70 billion over the last 60 years. However, due to inevitable host-pathogen co-evolution, biocontrol agents must be used strategically to maximise and prolong their effectiveness. Australia has adopted a pipeline approach to rabbit biocontrol, which aims to successively introduce new viruses to boost control efforts, as part of a broader integrated rabbit management program. The introduction of biological control agents into naive populations from a single point source provides a unique opportunity to study the spread, establishment, and evolution of emerging pathogens. Of particular interest is how closely related viruses interact with each other - a viral competition experiment on a continental scale. For caliciviruses in particular, our data show that recombination appears to be an important mechanism for generating genetic diversity, with multiple recombinant viruses emerging in the last three years. Understanding the mechanisms behind the epidemiological fitness, or lack thereof, of different virus strains enables strategic, targeted use of biocontrol agents to maximise the impacts of control programs. This has the potential to prolong the effectiveness of existing virus strains in a manner analogous to strategic use of antibiotics to delay the development of antimicrobial resistance.


SymPoSia moNDay

SYM-28-05CAPSID DEPENDENT EVASION OF INNATE IMMUNE SENSING IN MACROPHAGES DISTINGUISHES PANDEMIC HIV-1(M) AND NON-PANDEMIC HIV-1(O)

Jacques D.A.1, 2, Hilditch L.3, Rasaiyaah J.3, James L.C.2 and Towers G.J.3 1UNSW Sydney, NSW, 2052 Australia. 2MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK. 3University College London, London, WC1E 6BT, UK.

Transmission of simian immunodeficiency virus from great apes into humans has occurred on at least four separate occasions giving rise to the four distinct HlV1 groups: M, N, O, and P. The most common group, HIV1(M), was transmitted by chimpanzees and is the only group that has resulted in pandemic levels of human-to-human spread despite a similar time since introduction into human populations. The next most common group is HIV1(O), which originated from gorillas, and has resulted in significant spread (~100,000 human infections) yet has not reached the levels seen for HIV1(M) (~60,000,000 human infections) suggesting a reduced capacity to replicate in or transmit between humans. We have previously shown that the HIV1(M) capsid confers the ability to evade innate immune sensing in primary human macrophages. This evasion is dependent on the capsid’s ability to recruit host cofactors. When comparing HIV1(M) with HIV1(O), we find the HIV1(O) capsid to be less effective at evading innate immune triggering, with viral DNA activating the expression of interferon-stimulated genes. While this is analogous to HIV1(M) mutants that fail to engage host cofactors, HIV1(O) retains the ability to bind to CPSF6 and CypA demonstrating that binding per se is not responsible for the different phenotypes. Rather, the consequence of binding is altered. Phylogenetic comparison of HIV1(M) sequences and HIV1(O) CA sequences identified two key residues that split the M and O groups. Comparison of HIV1(M) and HIV1(O) CA crystal structures reveals that these two residues result in a less dynamic CA structure in HIV1(O), suggesting that it may not have the same allosteric capability as HIV1(M). This reduction in dynamics can be recapitulated in HIV1(M) through point mutation. We hypothesise that HIV1(O) capsids are less responsive to host cofactor interaction leading to premature reverse transcription, release of DNA, and innate immune triggering in macrophages. We further hypothesise that a reduction in the ability to infect these cells reduces the sexual transmissibility of HIV1(O) relative to HIV1(M), resulting in diminished pandemic potential.


SymPoSia TUESDay

SYM-29-01 SYM-29-02

SYM-29-03 SYM-29-04

DYNAMICS OF TISSUE-SPECIFIC GENOME REGULATORY PROGRAMS IN THE GERMINATING BARLEY SEED

Liew L.C., Chen M., Wang Y., Whelan J. and Lewsey M.G. AgriBio, La Trobe University, Melbourne, VIC, Australia.

We are studying tissue-specific genome regulatory programs in germinating barley seeds so as to better understand how they are controlled by transcription factors. The roles of transcription factors in genome-wide regulation of gene expression are subject to much attention currently, but determing these roles is complex. Regulatory events occur dynamically over short time scales (minutes to hours), with many transcription factors interacting to regulate the output of any given gene and cascades of interacting factors operating across the wider program. Studies have historically depended on analysis of bulk tissue samples, due largely to the capabilites of available techniques. However, individual cell-types must be specified by distinct regulatory programs. To investigate cell-type specific genome regulation during germination we have applied laser-capture microdissection RNA sequencing to three tissues of barley seeds that have distinct functions (plumules, radicles and scutellum). We analysed complete transcriptomes from samples of 200 cells over a 36 h time series, enabling us to observe the dynamic changes in gene expression. The data allow us to identify modules of gene expression that are unique to and conserved between these three tissues and to identify associated groups of potential cis-regulatory motifs. By extrapolation from the extensive Arabidopsis transcription factor target datasets we are also able to infer likely families of regulatory factors. The outcome is an improved understanding of the direct regulation of gene expression and how this varies between tissues in a crop species with a relatively complex genome.

REGULATION SHOOT ANION LOADING VIA THE ATSLAH1-ATSLAH3 COMPLEX

Qiu J. and Gilliham M. ARC CoE PEB, University of Adelaide, South Australia, Australia.

AtSLAH1 and AtSLAH3, two Arabidopsis slow type anion channel-associated 1 (SLAC1) homologues form a protein complex to regulate root-to-shoot delivery of chloride (Cl-) and nitrate (NO3

-). Both proteins are expressed in Arabidopsis root pericycle and their expression is down-regulated by NaCl and ABA (Qiu et al., 2016). AtSLAH3 carries predominantly NO3

- when expressed by itself but has increased Cl- transport in a complex with SLAH1 (Cubero-Font et al., 2016). Here, we explore the regulation of this complex using heterologous expression in Xenopus laevis oocytes and two-electrode voltage clamp. We observed two ABA signaling associated protein kinases, SnRK2.2 and SnRK2.3, negatively regulate anion currents through AtSLAH1-AtSLAH3. Conversely, a positive guard cell anion channel regulator from the same kinase subgroup, SnRK2.6 (OST1), appears not to regulate the complex. To confirm whether AtSLAH1-AtSLAH3 can be targeted by SnRK2s, the only SnRK2s-specific substrate motif RxxT/S in AtSLAH1 was site-mutagenized (R176K) to disrupt the recognition by SnRk2s. When SLAH1R176 was co-injected with SLAH3 and SnRK2.2 the conductance inhibition was less pronounced compared to SLAH1-SLAH3-SnRK2.2. This result implies SnRk2.2 has an important role in mediating root-to-shoot Cl- transport by regulating AtSLAH1-AtSLAH3 activity. Previous studies have reported different protein kinases from the same subgroup might distinguish their roles with different tissue localization. As SnRK2.2 and SnRk2.3 are abundantly expressed within the plant including the root, while SnRK2.6 is highly expressed in the shoot but less detectable in the root; this is consistent with why SnRk2.6 does not affect the ionic conductance of AtSLAH1-AtSLAH3 complex. In addition, the AtSLAH1-AtSLAH3-SnRK2.2 was also found to be regulated by calcium-dependent protein kinase 21 (CPK21) by restoring the anion transport, suggesting a potential cross talk between kinases with/without Ca2+ dependency. As AtSLAH1-AtSLAH3 is involved in mediating root-to-shoot anion movement, multiple regulating mechanisms of the complex may help the plant to acclimate to stressful growth conditions by adjusted anion transfer to the shoot. Experiments to explore whether this regulation occurs in planta is underway.

A NOVEL MITOCHONDRIAL LYR PROTEIN IS REQUIRED FOR COMPLEX I ASSEMBLY IN ARABIDOPSIS

Ivanova A.1, Gille-Hill M.1, Branca R.2, Kmiec B.3, Teixeira P.3

and Murcha M.W.1 1School of Chemistry and Biochemistry & The ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, 35 Stirling Highway, Crawley, Perth 6009, Australia. 2Clinical Proteomics Mass Spectrometry, Department of Oncology-Pathology, Science for Life Laboratory and Karolinska Institutet, Stockholm, Sweden. 3Department of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories for Natural Sciences, Stockholm, Sweden.

Mitochondrial Complex I, a proton pumping NADH;ubiquinone oxidoreductase is the first and most complicated enzyme involved in the generation of ATP via oxidative phosphorylation. Located in the mitochondrial inner membrane it is composed of at least 44 subunits and requires the co-ordination of both nuclear and mitochondrial gene expression, protein import, co-factor biosynthesis and the assembly of each individual subunit into a 1000 kDa complexome. So far, only two bona fide Complex I assembly factors (GLDH and INDH) have been identified for plants. Here we functionally characterise a novel Complex I assembly factor that is located in the mitochondrial matrix and contains the conserved Complex I LYR Fe/S domain. Knock-out mutants lack the monomeric Complex I and the supercomplex I + III and exhibit the phenotypic characteristics typical of Complex I defective lines. BN-PAGE analysis shows the stalling and accumulation of the 650 kDa and 850 kDa Complex I assembly intermediates, with protein-protein interaction assays displaying specificity for subunits. Consequently, there is a general upregulation of mitochondrial biogenesis with regards to protein import ability and mitochondrial translation rates. Our data suggest that this novel LYR protein facilitates the biogenesis and assembly of particular subunits that are essential for the formation of a functional Complex I.

DIURNAL VARIATION IN THERMAL ACCLIMATION OF LEAF RESPIRATION IN RICE

Rashid F.A.A.1, Asao S.1, Taylor N.L.2, Fenske R.2 and Atkin O.K.1 1ARC Centre of Excellence in Plant Energy Biology, Research School of Biology, Building 134, The Australian National University, Canberra, ACT 2601, Australia. 2ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, Bayliss Building, The University of Western Australia, Crawley, WA 6009, Australia.

Past studies using plants sampled at a single time-point during the day have shown that cold-acclimated leaves exhibit higher rates of leaf dark respiration (R) at a common temperature than their warm/hot grown counterparts. What is unclear, however, is whether these differences are held throughout the day. To address this, we examined the effect of growth temperature on diurnal variations in leaf R at a set temperature (30°C) and associated metabolite pools of rice (Oryza sativa, IR64). Plants were grown in three temperature controlled glasshouses (25, 30 and 35°C), with leaf R measured at 8:30 am, 1 pm, 5:30 pm, 9:30 pm and 4:30 am; metabolites were also quantified at the same time points. As expected, rates of leaf R measured at 30°C were significantly higher in the 25°C grown plants than those grown at 30°C and 35°C. However, the relative difference in leaf R between the growth temperatures changed through the day, being greatest at the end of the day-light period and least during the night hours. Underpinning this pattern were diurnal variations in leaf R of the 25°C & 30°C grown plants (with rates highest at 5:30 pm, and lowest at 4:30 am), whereas leaf R did not vary diurnally in the 35°C grown plants. Starch concentrations were lowest at the end of the night and highest at the day period – by contrast, soluble sugar concentrations remained constant through the day-night cycle in all three growth treatments. Thus, availability of substrate pools to glycolysis did not account for the divergent effects of time observed among the growth treatments. The effect of growth temperature and time of day on glycolytic and TCA cycle metabolites will be reported. Collectively, the results highlight the importance of considering time of day when assessing thermal acclimation of respiratory energy metabolism in rice.


SymPoSia TUESDay

SYM-29-05 SYM-30-1

SYM-30-2 SYM-30-3

REACTIVE OXYGEN SPECIES CONTRIBUTE TO SUGAR SIGNALLING AND GROWTH IN ARABIDOPSIS

Roman A.1, 2, Deng D.1, Eastmond H.2, Arshad W.2, Davey J.2, James S.2, Ashton P.D.2, Graham I.A.2 and Haydon M.J.1, 2 1School of BioSciences, The University of Melbourne, Parkville VIC. 2Department of Biology, University of York, UK.

Plants produce sugars from photosynthesis to provide the stored energy and building blocks for all living cells. Sugars also act as dynamic signals to regulate growth and physiology. Thus, carbon status has wide-ranging effects on plant productivity but defining specific sugar signalling pathways can be challenging in the context of photoautrophic metabolism because it is difficult to separate responses to light and sugar. We have previously shown that sugar signals regulate circadian rhythms in plants: exogenous sugars can initiate robust circadian rhythms in dark-grown seedlings; and inhibition of photosynthesis in the light can adjust phase of the circadian oscillator. To dissect contributions of sugar and light signals on gene networks in Arabidopsis, we have performed an RNA-Seq time-series in dark-adapted seedlings treated with sucrose or mannitol in the dark or transferred to the light with or without an inhibitor of photosynthesis. We have used weighted gene co-expression network analysis (WCGNA) and GO-enrichment of this large dataset to identify functional classes of transcripts. As expected, inhibition of photosynthesis identified GO-enrichment for ‘circadian rhythm’ but also ‘response to absence of light’, including transcripts for major photoreceptors. The latter suggests a contribution of sugars to canonical light signalling and indicates our experimental design could deconvolute light and sugar signals in plants. The most significantly-enriched class of transcripts responding early to sugar was ‘response to oxygen-containing compound’, pointing to a potential role for reactive oxygen species (ROS) signalling. Using luciferase reporters and qPCR, we have shown that chemical inhibitors of ROS inhibit transcriptional responses to sugar. These inhibitors also inhibit promotive effects of sugars on root growth and shoot biomass, suggesting these contribute to meaningful sugar signalling pathways.

MODELING GENE EXPRESSION VARIABILITY TO UNDERSTAND STEM CELL REGULATION

Mar J.C.1, 2, 3 1Australian Institute for Bioengineering and Nanotechnology. 2University of Queensland. 3Albert Einstein College of Medicine.

When studying the transcriptome and its contribution to the regulation of stem cells, our inferences typically revolve around changes in average gene expression. For a wide range of stem cell populations, heterogeneity in gene expression is a recognized part of transcriptomic data, and recent studies have demonstrated how modeling variability has revealed more information than following average trends alone. This talk outlines some of the approaches my group has developed to investigate how variability of gene expression contributes to our understanding of transcriptional regulation using examples from human stem cell populations.

LIVER REGENERATIVE MEDICINE - TOWARDS CELL AND ORGANOID THERAPIES

Yap K.1, 2 1St Vincent’s Institute, Fitzroy, Victoria. 2University of Melbourne Department of Surgery at St Vincent’s Hospital Melbourne, Fitzroy, Victoria.

Liver disease is on the rise, particularly fatty liver disease which is commonly associated with obesity and diabetes. Chronic liver disease often leads to liver failure, which is fatal without a liver transplant. However, liver transplantation simply cannot meet the ongoing increase in demand. Strategies to better understand, diagnose, and treat liver disease are critically required. Our group has an interest in identifying the role and utility of cells within the liver that can contribute to liver regeneration, which can also be harnessed in cell and organoid therapies for liver disease. These include human liver progenitor cells and liver sinusoidal endothelial cells derived from adult human liver, as well as induced pluripotent stem cell (iPSC) produced counterparts. Organoids generated from these cell types have been established in culture and transplanted into a mouse model of liver disease, with demonstration of tissue assembly, functional maturation, and survival in vivo. Comparison between iPSC-derived cells/organoids and human adult liver derived cells/organoids are underway, and recent developments include upscaling of transplantation experiments from mouse to rat, representing a 10-fold increase in scale. It is hoped insights gained from these experiments will guide the development of cell and organoid-based therapies for liver disease.

HOW: THE LONG AND SHORT OF INTESTINAL STEM CELLS

Qi J., Dominado N., Savva E., Casagranda F., Siddall N.A. and Hime G.R. Department of Anatomy and Neuroscience, University of Melbourne, Parkville 3010, Australia.

Intestinal stem cells play a key role in maintaining the intestinal environment. Multiple signaling pathways regulate replication and differentiation of intestinal stem cells (ISCs). The midgut epithelium of Drosophila melanogaster is comprised of similar cell types to the vertebrate intestinal epithelium and has served as a model for identification of genes that regulate epithelial biology. We have recently used Drosophila to show that an RNA-binding protein, known as HOW (Held-out wings), is also involved in regulating ISCs. HOW can be found as 2 major isoforms; long and short proteins named HOW(L) and How(S) respectively. HOW(L) and HOW(S) have been shown to have opposing roles in regulation of tendon cell differentiation in the wings of the fly. Flies with this mutation have their wings perpendicular to their body which gave rise to the name, ‘held- out wings’. The vertebrate ortholog of HOW is known as QKI (Quaking) and has been associated with regulation of colorectal cancer cell differentiation. The QKI gene also produces multiple isoforms; 2 of which are very similar to HOW(S) and HOW(L). Despite research showing that HOW is present in intestinal stem cells, little research has been conducted investigating its role and its targets. Complete loss of HOW function in the midgut results in a decrease in ISC number. Ectopic expression of HOW(L) results in an ISC increase and a corresponding decrease in the proportion of differentiated enterocytes. I aim to determine whether HOW(L) and HOW(S) have their own individual roles in intestinal stem cell maintenance or have redundant functions.


SymPoSia TUESDay

SYM-30-04 SYM-30-05

SYM-31-01 SYM-31-02

THE ROLE OF THE PRORENIN RECEPTOR IN MALE FERTILITY

Ahmady S.1, Bernard P.2, Merriner D.J.3, Chan A.4, Bagheri-Fam S.1, Hobbs R.M.4, O’Bryan M.K.3, Pask A.J.2 and Wilhelm D.1 1Department of Anatomy & Neuroscience, University of Melbourne, Australia. 2School of BioSciences, University of Melbourne, Australia. 3School of Biological Sciences, Monash University, Australia. 4ARMI and the Department of Anatomy & Developmental Biology, Monash University, Australia.

The prorenin receptor (PRR) is best known for its tole in the renin-angiotensin-system (RAS) to regulate blood pressure and salt homeostasis. However, more recently, PRR has been shown to be a multi-functional protein, which is involved in a number of downstream pathways including MAPK signalling, protein sorting and folding and receptor-mediated endocytosis and recycling through its interaction with the vacuolar H+-ATPase (V-ATPase), as well as canonical and non-canonical WNT signalling. Given that MAPK and WNT signalling is important for ovary and testis differentiation, we hypothesised that PRR plays a role in gonadal development and function. To test this hypothesis, we deleted Prr specifically in gonadal somatic cells using the Nr5a1-Cre mouse. While these mice appear to develop normally and are born at the expected Mendelian ratio, both males and females are infertile. Here, we present our analysis to date of the testicular phenotype of the conditional Prr-null mice.

β-CATENIN DRIVES DISTINCT TRANSCRIPTIONAL NETWORKS IN REGENERATIVE AND NON-REGENERATIVE CARDIOMYOCYTESQuaife-Ryan G.A.1, 2, Lavers G.1, 2, Mills R.J.1, 2, Voges H.K.1, 2, Ramialison M.3, Hudson J.E.1, 2 and Porrello E.R.1, 4, 5 1School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, 4072, Australia. 2QIMR Berghofer Medical Research Institute, Brisbane, QLD, 4006, Australia. 3Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, 3800, Australia. 4Murdoch Children’s Research Institute, The Royal Children’s Hospital, Parkville, VIC, 3052, Australia. 5Department of Physiology, School of Biomedical Sciences, The University of Melbourne, Parkville, VIC, 3010, Australia.

The inability of the adult mammalian heart to regenerate following cardiac injury represents a major limitation in the management of heart failure. In comparison, the neonatal mouse heart regenerates following myocardial infarction (MI). We recently compared the neonatal and adult transcriptomes of multiple cardiac cell populations and uncovered a regenerative gene network associated with Wnt/β-catenin signalling. However, it is unclear what role Wnt/β-catenin signalling plays in driving the pro-regenerative network. Here, we study Wnt/β-catenin involvement in cardiomyocyte regeneration. We found that stimulation of β-catenin signalling by GSK3 inhibition (GSK3i) in immature human embryonic stem-cell-derived cardiomyocytes and 3D human cardiac organoids potently induced cardiomyocyte proliferation. Furthermore, β-catenin inhibition abrogated GSK3i-induced cardiomyocyte proliferation in vitro. To identify direct β-catenin transcriptional targets, we undertook RNA sequencing (RNA-seq) of GSK3i treated human cardiomyocytes combined with chromatin-immunoprecipitation sequencing (ChIP-seq) to reveal 22 direct β-catenin/TCF7L2 target genes that were shared in common between the human and mouse regenerative networks. Consistent with these results, delivery of constitutively active β-catenin (caBCAT) in vivo stimulated neonatal cardiomyocyte proliferation. Additionally, β-catenin inhibition limited neonatal cardiomyocyte cell cycle activity in vivo and downregulated β-catenin-target genes. However, in contrast to these effects in regenerative cardiomyocytes, caBCAT delivery to the adult mouse heart following MI did not induce cardiomyocyte proliferation, although cardiac function was improved. RNA-seq of purified adult cardiomyocytes treated with caBCAT uncovered a distinct transcriptional network associated with cardioprotection and modulation of the immune response that appears to be driven by FoxO. Therefore, β-catenin drives distinct transcriptional programs in regenerative and non-regenerative cardiomyocytes. Redirection of β-catenin to its pro-proliferative target genes could be exploited for regenerative applications in the future.

INFLAMMASOMES IN SEVERE ASTHMA

Kim R.Y.1, Pinkerton J.W.1, Essilfie A.T.1, Robertson A.A.B.2, Baines K.J.1, Brown A.C.1, Mayall J.R.1, Ali K.1, Starkey M.R.1, Hansbro N.G.1, Hirota J.A.3, Wood L.G.1, Simpson J.L.1, Knight D.A.1, Wark P.A.1, Gibson P.G.1, O’Neill L.A.J.4, Cooper M.A.2, Horvat J.C.1 and Hansbro P.M.1

1University of Newcastle, Newcastle, New South Wales, Australia. 2University of Queensland, Brisbane, Queensland, Australia. 3University of British Columbia, Vancouver, British Columbia, Canada. 4Trinity College Dublin, Dublin, Ireland.

Rationale: Severe, steroid-resistant (SSR) asthma is the major unmet need in asthma therapy. Disease heterogeneity and poor understanding of pathogenic mechanisms hampers the identification of therapeutic targets. Excessive NLRP3 inflammasome and concomitant IL-1β responses occur in COPD, respiratory infections and neutrophilic asthma. However, the direct contributions to pathogenesis, mechanisms involved and potential for therapeutic targeting are unknown in SSR asthma. Methods: We developed mouse models of Chlamydia, and Haemophilus, respiratory infection-mediated, ovalbumin-induced SSR allergic airways disease. These models share the hallmark features of human disease, including elevated airway neutrophils, and NLRP3 inflammasome and IL-1β responses. The roles and potential for targeting of NLRP3 inflammasome, caspase-1, and IL-1β responses in experimental SSR asthma were examined using a highly-selective NLRP3 inhibitor, MCC950, the specific caspase-1 inhibitor, Ac-YVAD-cho, and neutralizing anti-IL-1β antibody. Roles for IL-1β-induced neutrophilic inflammation were examined using IL-1β and anti-Ly6G. Results: Chlamydia and Haemophilus infections increase NLRP3, caspase-1, IL-1β responses that drive steroid-resistant neutrophilic inflammation and airways hyper-responsiveness (AHR). Neutrophilic inflammation, disease severity and steroid-resistance in human asthma correlates with NLRP3 and IL-1β expression. Treatment with anti-IL-1β, Ac-YVAD-cho, and MCC950 suppressed IL-1β responses and the important steroid-resistant features of disease in mice, whereas IL-1β administration recapitulated these features. Neutrophil depletion suppressed IL-1β-induced steroid-resistant AHR. Conclusions: NLRP3 inflammasome responses drive experimental SSR asthma and are potential therapeutic targets in this disease.

EPIGENETIC AND TRANSCRIPTIONAL REGULATION OF IL-4 INDUCED CCL17 PRODUCTION IN HUMAN MONOCYTES AND MURINE MACROPHAGES

Lupancu T.1, Hsu A.1, Lee M.1, Fleetwood A.1, Cook A.1, Hamilton J.1, 2 and Achuthan A.1 1University of Melbourne, Department of Medicine, Royal Melbourne Hospital. 2Australian Institute for Musculoskeletal Science (AIMSS), The University of Melbourne and Western Health.

Interleukin- 4 (IL4) is an anti- inflammatory cytokine and alternative macrophage activator, recognized as a hallmark cytokine of Th2- associated diseases. On the other hand, granulocyte- macrophage colony stimulating factor (GM-CSF) is a pro-inflammatory hematopoietic cytokine and involved in the development of autoimmune inflammatory diseases, such as rheumatoid arthritis. Despite the opposing inflammatory roles of these two cytokines, GM-CSF and IL4 are used together to enhance in vitro differentiation of monocytes into dendritic cells. We have previously reported that GM-CSF can induce the secretion of chemokine (C-C motif) ligand 17 (CCL17) from monocytes in an interferon regulatory factor 4 (IRF4)-dependent manner. Interestingly, CCL17 is also an upregulated marker of IL4 polarised M2 macrophages. We investigated whether the IL4 induced CCL17 pathway shared any common elements with the established GM-CSF pathway. We report here that, like GM-CSF, IL4 induces CCL17 expression in an IRF4 dependent manner. IL4 also enhances the expression and activity of Jumanji domain-containing protein 3 (JMJD3) demethylase which epigenetically upregulates IRF4 expression. Signal transducer and activator of transcription 6 (STAT6) is activated by IL4, not GM-CSF, and silencing the STAT6 gene led to a decrease in CCL17 formation, as well as that of its upstream regulators, JMJD3 and IRF4. Moreover, IL4 treatment of human monocytes resulted in an increased association of STAT6 to the promoter regions of the CCL17, IRF4 and JMJD3 genes. Thus, despite the different and almost opposing roles GM-CSF and IL4 play in inflammation, these 2 cytokines share a common signaling pathway in regulating CCL17 production in human monocytes and murine macrophages.


SymPoSia TUESDay

SYM-31-03 SYM-31-04

SYM-31-05 SYM-32-01

MITOCHONDRIAL FUNCTION DURING MAMALIAN OOCYTE DEVELOPMENT: GOING BEYOND BIOENERGETICS

Adhikari D.1, Liu J.1, Alzubaidi U.1, Zhang Q.H.1, Yuen W.S.1, Robker R.L.2 and Carroll J.1 1Department of Anatomy and Developmental Biology and Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia. 2The Robinson Research Institute, School of Medicine, The University of Adelaide, Australia 5005.

Due to limited glycolytic capabilities of mammalian oocytes, mitochondrial ATP production by OXPHOS is essential for oocyte and preimplantation embryo development. Mitochondrial biogenesis occurs throughout oocyte growth during which the number of mitochondria increases from about 1000 to up to 500,000. Mitochondrial DNA (mtDNA) amplifies and mitochondria undergo divisions during oocyte growth. mtDNA replication is driven by upregulation of Transcription Factor A, Mitochondrial (TFAM) and mitochondrial fission requires Dynamin-related Protein 1 (DRP1). Disruption of mitochondrial biogenesis by genetic ablation of TFAM or DRP1 during oocyte growth does not significantly reduce the levels of ATP in the fully grown oocyte. Furthermore, oocyte maturation, fertilization and early embryo development appear normal, although embryo development does not progress beyond day 15 of gestation. These findings suggest that the role of mitochondria in oocytes extends beyond that of a simple bioenergetics hub. Mitochondria are increasingly being recognized for coordinating multiple metabolic pathways and regulating nuclear modifications through metabolites. However, the roles of mitochondria in oocytes beyond ATP production are poorly understood. Primary or secondary mitochondrial defects in oocytes caused by maternal environment are known to be detrimental for embryo development and offspring health. Our analyses reveal altered metabolites, nuclear epigenetic modifications and nuclear gene expression profiles in TFAM and DRP1-deleted oocytes. Thus, the function of mitochondria in oocytes can be extended to include roles in regulating the levels of key metabolites that have potential to modify essential epigenetic modifications occurring during oocyte growth. In conclusion, our results show that oocyte mitochondria have roles that extend well beyond the traditional and widely held view that mitochondria act as a simple power supply for the purposes of maintaining oocyte function.

TLR4 AS AN INFLAMMATORY KEY TO SEVERE DENGUE DISEASE

Modhiran N., Vajjhala P.R., Watterson D., Young P.R. and Stacey K.J. Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Qld 4072.

Toll-like receptor 4 (TLR4) is the pattern recognition receptor responsible for mediating inflammatory effects of lipopolysaccharide (LPS), the major constituent of the Gram-negative bacterial cell wall. We have shown that the dengue virus secreted non-structural protein NS1 also activates TLR4, inducing the release of inflammatory cytokines from myeloid cells and loss of endothelial monolayer integrity. The serious complications of dengue virus infection include haemorrhage and shock, indicating a critical role for disruption of the endothelial barrier. We find that inhibition of TLR4 responses in dengue virus-infected mice substantially reduces vascular leak and is thus a potential therapeutic avenue. This suggests that at least in the later stages of infection, the host does not benefit from recognition of infection via TLR4. The striking similarities in cellular responses to LPS and NS1 via TLR4 suggest that NS1 is a viral counterpart of bacterial endotoxin. Similar to the role of LPS in septic shock, NS1 may contribute to vascular leak in dengue patients. A role for TLR4 in dengue infection opens up new therapeutic avenues for decreasing the severity of dengue pathology using established antagonists.

CELL DEATH AND AUTOINFLAMMATION

Lawlor K.E.1, Feltham R.1, Yabal M.2, Jost P.J.2 and Vince J.E.1 1The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia. 2III. Medical Department for Hematology and Oncology, Klinikum rechts der Isar,Technische Universität München, 81675 Munich, Germany.

Cytosolic inflammasome sensor proteins activate caspase-1, resulting in cleavage-induced maturation and secretion of the potent inflammatory cytokine Interleukin-1β (IL-1β). Consequently, activating mutations in inflammasome sensor proteins result in autoinflammatory disease that can be treated with anti-IL-1 therapeutics. Surprisingly, recent studies have highlighted that mutations in components of the Toll-like Receptor (TLR) and TNF Receptor (TNFR) signaling machinery can also result in pathological IL-1β production, and autoinflammatory disease with symptoms reminiscent of patients with activating inflammasome mutations. How this aberrant TLR and TNF Receptor signaling can activate cytosolic inflammasome complexes remains unclear. In our recent studies we have explored this conundrum using a murine model of X-linked Inhibitor of Apoptosis (XIAP) deficiency, which can trigger potentially fatal, pathogen-associated, hyperinflammation in humans. We define a novel TLR and TNF Receptor cooperative signaling mechanism that is essential for inflammasome and IL-1β activation in the absence of XIAP, and document how this pathway may also act to generate pathological IL-1β responses in models of endotoxic shock and inflammatory arthritis.

COMBATING ANTIBIOTIC RESISTANCE: STRUCTURAL AND BIOPHYSICAL STUDIES OF A COLISTIN RESISTANCE ENzYME INVOLVED IN ENDOTOXIN MODIFICATION

Vrielink A.1, Anandan A.1, Evans G.L.1, Condic-Jurkic K.2, O’Mara M.L.2 and Kahler C.M.1 1University of Western Australia, 35 Stirling Highway, Crawley, WA, 6009. 2The Australian National University, Canberra, ACT 2601.

Multiple drug resistance (MDR) in Gram-negative bacteria represents one of the most intractable problems facing modern medicine. Colistin and polymyxin are cationic antimicrobial peptide antibiotics which permeabilise the bacterial outer membrane and have been used to treat infections. Resistance to these antibiotics is conferred by the modification of the lipid A headgroups with phosphoethanolamine (PEA) moieties resulting in a reduced negative charge of the bacterial surface and exclusion of the drug. This modification is carried out by the enzyme, lipid A PEA transferase (EptA). Recently a mobile colistin resistance determinant, mcr-1, encoding an EptA homologue was identified in MDR Escherichia coli. The crystal structure of a full-length EptA from Neisseria sp. to 2.75Å resolution will be presented as well as molecular dynamics and biophysical studies. These studies help us to better understand the conformational flexibility of the protein and provide insights into the catalytic activity of the enzyme.


SymPoSia TUESDay

SYM-32-02 SYM-32-03

SYM-32-04 SYM-32-05

HOMODIMERIzATION REGULATES AN ENDOTHELIAL SPECIFIC SIGNATURE OF THE SOX18 TRANSCRIPTION FACTOR

Moustaqil M.1, Fontaine F.2, Overman J.2, MacCann A.2, Bailey T.L.3, Rudolffi Soto P.1, Gambin Y.1, Francois M.2 and Sierecki E.1 1EMBL Australia Node in Single Molecule Science School of Medical Sciences, The University of New South Wales, Sydney, Australia. 2Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia. 3Department of Pharmacology, School of Medicine, University of Nevada, Reno, USA.

During embryogenesis, vascular development relies on a handful of transcription factors that instruct cell fate in a distinct sub-population of the endothelium. The SOXF proteins that comprise SOX7, 17 and 18, are molecular switches modulating arterio-venous and lymphatic endothelial differentiation. We recently found that in the SOX-F family, SOX18 alone has the ability to switch between a monomeric and a dimeric form, using in vitro binding assays and a split-GFP reporter assay in a zebrafish model system in vivo. SOX18 dimerization is driven by a newly identified motif located in the vicinity of the C-terminus of the DNA binding region. Insertion of this motif in a SOX7 monomer forced its assembly into a dimer. Genome-wide analysis of SOX18 binding locations revealed enrichment for a SOX dimer binding motif on the chromatin, correlating with genes with a strong endothelial signature. Using a SOX18 small molecule inhibitor that disrupts dimerization, we revealed that dimerization is important for transcription. Overall, we show that dimerization is a specific feature of SOX18 that enables the recruitment of key endothelial transcription factors, and refines the selectivity of the binding to discrete genomic locations assigned to endothelial specific genes.

ORDERED LIM DOMAINS TUNE ASSOCIATION RATES OF DISORDERED PARTNERS TO REGULATE TRANSCRIPTION FACTOR COMPLEX FORMATION

Robertson N.O.1, Smith N.C.1, Manakas A.1, Mahjuob M.1, McDonald G.2, Kwan A.H.1 and Matthews J.M.1 1School of Life and Environmental Sciences, University of Sydney, Australia. 2Centre for Translational Data Science, University of Sydney, Australia.

Intrinsically disordered regions (IDRs) are overrepresented among transcription factors, where they often act as protein-protein interaction motifs. Despite the importance of IDR interactions there is little quantitative information about the role disorder plays in the thermodynamic and kinetic parameters of binding. We have used LIM domain transcription factors to understand the role disorder can play in complex formation. Transcription factors containing disordered LIM interacting domains (LIDs) interact with the ordered tandem LIM domains (LIM1+2) of LIM-only (LMO) and LIM-homeodomain (LIM-HD) proteins. The resulting complexes regulate gene expression and are important mediators of cell specification, proliferation and differentiation. We have used FRET-based assays to study the binding affinities and kinetics of different LID:LIM1+2 interactions. LDB1LID association rate constants can vary by up to 3-orders of magnitude depending on the interacting LIM1+2 domains. These disparate kinetics are not accounted for by differences in electrostatic attractions; rather they reflect differences in binding mechanisms of LDB1LID. LDB1LIDcan bind single LIM domains with high association rate constants, indicating that the differences in LIM1+2 association occur after the initial encounter. We have used our equilibrium and kinetic data to model LID:LIM1+2 transcription factor complex as it occurs in motor neuron development, showing that the disparate kinetics facilitate the exchange of high affinity binding partners over time. Together, our studies suggest that ordered domains can use the inherent flexibility of IDRs generate highly differential binding kinetics, which may provide a mechanism for temporally regulating transcriptional complex formation during development.

MOLECULAR ARCHITECTURE OF AN E. FAECALIS ANTITERMINATION PROTEIN BOUND TO RNA

Walshe J.L., Patel, K. and Ataide S.F. University of Sydney.

Regulated transcription termination provides an efficient and responsive means to control gene expression. Rho-independent termination occurs through the formation of a RNA stem-loop which disrupts the RNA polymerase elongation complex. To overcome this, bacteria may use anti-termination, whereby a mutually exclusive RNA structure is formed. In some cases this alternative structure is stabilised by ANTAR domains of proteins, preventing termination. We have determined the novel 3.8 Å crystal structure of the stabilising anti-terminator protein EutV, bound to RNA. Our studies highlight the key interactions between conserved EutV residues and the RNA, as well as protein conformational changes undergone upon RNA binding. This has allowed us to propose a broad model for ANTAR domain anti-termination.

CIRCULAR RNAS IN EPITHELIAL TO MESENCHYMAL TRANSITION (EMT)

Goodall, G.J.



SymPoSia TUESDay

SYM-33-01 SYM-33-02

SYM-33-03 SYM-33-04

BONE REGULATION OF ENERGY AND GLUCOSE METABOLISM

Brennan-Speranza T.C.1, Liu X.1, Brock K.1 and Levinger I.2 1School of Medical Sciences, University of Sydney. 2ISEAL, Victoria University.

The skeleton is an endocrine organ participating in energy metabolism and glucose homeostasis via the undercarboxylated form of the bone-derived protein, osteocalcin. Skeletal muscle is a major site of glucose uptake and disposal in response to both insulin and exercise. It was previously shown that osteocalcin may enhance whole body insulin sensitivity and glucose control via its action on the pancreatic beta cells by increasing beta cell proliferation and insulin secretion, as well as affecting adipocytes by enhancing adiponectin secretion. Both the increase in insulin and adiponectin levels can increase skeletal muscle glucose uptake. However, whether osteocalcin can enhance skeletal muscle insulin sensitivity and glucose uptake at rest and following muscle contraction/exercise via a direct pathway is not clear. Our recent evidence suggests that a direct pathway is plausible. In order to better understand the pathway by which osteocalcin may affect glucose uptake in skeletal muscle, a receptor for osteocalcin should be identified. The class C G protein-coupled receptor 6A (GPRC6A) is the postulated receptor for osteocalcin in several tissues including skeletal muscle. We now have evidence demonstrating a direct role of osteocalcin in enhancing skeletal muscle insulin sensitivity and that that GPRC6A may not be the only receptor for osteocalcin in this tissue. Furthermore, recent studies in humans have reported significant correlations between osteoclacin levels and insulin sensitivity.

THE MEVALONATE PATHWAY IN BONE BIOLOGY

Munoz M. and Rogers M.J. Bone Biology Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia.

The mevalonate pathway, necessary for the biosynthesis of cholesterol, is a conserved and ubiquitous process in eukaryotic organisms. It works via a step-wise condensation of isoprene units to form longer-chain isoprenoid lipids. It has two branches: one leads to the synthesis of sterols, and the other to the synthesis of isoprenoid lipids tags required for the post-translational modification of proteins known as prenylation. It is estimated that at least 300 proteins of the human proteome are prenylated, particularly signaling proteins such as Ras, Rho and Rab small GTPases. During prenylation, farnesyl or geranylgeranyl isoprenoid moieties are irreversibly added to a cysteine residue at the protein’s carboxy terminus, enabling subcellular membrane localisation and protein:protein interactions, and hence adequate signal transduction and regulation. The importance of the mevalonate pathway in bone biology emerged from the discovery that most bisphosphonate drugs, used worldwide to treat common diseases of excessive bone destruction such as osteoporosis, act by blocking protein prenylation in bone-degrading osteoclasts. Bisphosphonates (BPs) are non-hydrolysable analogues of naturally-occurring pyrophosphate. Having high avidity for calcium ions, BPs bind rapidly to bone mineral and are preferentially internalised by osteoclasts. Once in the cytosol, BPs lock farnesyl diphosphate synthase in an inactive state, thereby blocking the synthesis of farnesyl and geranylgeranyl isoprenoid tags and preventing protein prenylation. In this way, bisphosphonates interfere with cellular processes governed by prenylated small GTPases that are essential for bone resorption, such as cell polarisation, cytoskeletal organisation and vesicular trafficking. We are now using the tools developed to study the actions of bisphosphonates to provide new advances in the diagnosis and pathophysiology of a human autoinflammatory disease caused by defective protein prenylation.

EPIGENETIC REGULATION OF PROSTATE CANCER METASTASIS TO THE BONE

Wilkinson E.J.1, 2, Malley R.1, Dickinson J.L.2 and Holloway A.F.1 1School of Medicine, College of Health and Medicine, University of Tasmania, Hobart, Tasmania, Australia, 7000. 2Menzies Institute for Medical Research, College of Health and Medicine, University of Tasmania, Hobart, Tasmania, Australia, 7000.

Prostate cancer is the most commonly diagnosed cancer in Australian males with a reported five-year survival rate of 95%. Prostate cancers, however, primarily metastasise to the bone with a decreased five-year survival rate of 3%. Current diagnostic tools are unable to distinguish indolent disease from that with a propensity to become metastatic. We have taken complimentary single-gene and genome-wide approaches to identifying key genes that contribute to prostate cancer metastasis. Integrins, a group of adhesion receptors, are known to facilitate metastasis to the bone, however the pathway(s) through which this occurs are incompletely understood. Integrins, which act as receptors for the bone constituents laminin and collagen, are aberrantly expressed in prostate cancer. We have shown that methylation changes and associated expression changes are observed in several key integrins, including ITGB4, in cell lines representative of different stages of prostate tumour disease progression. Formalin-fixed paraffin tissue derived from matched localised and metastatic prostate tumours were examined to determine whether these changes are also observed in patient samples. We also performed genome-wide methylation analysis using the Infinium® MethylationEPIC array in matched normal and primary tumour samples, and matched tumour and metastasis samples to identify changes associated with disease progression. Preliminary analysis has revealed 10 significantly differentially methylated regions between matched normal and primary tumour samples, and over 2,300 significantly differentially methylated regions between matched tumour and metastasis samples. These data indicate that changes in methylation occur throughout disease progression, and suggest that wide-spread epigenetic changes accompany tumour metastasis.

REGULATION OF EXPRESSION OF VITAMIN D HYDROXYLASES

Morris H.A. School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia 5000.

Vitamin D is a secosteroid that is metabolically activated and degraded through the actions of three cytochrome P450 hydroxylase enzymes which are expressed in a wide range of tissues stimulating both endocrine and autocrine activities of vitamin D. Bioactivation occurs through the sequential actions of CYP2R1 and CYP27B1 enzymes, resulting in synthesis of the pleiotropic hormone 1,25-dihydroxyvitamin D (1,25D), which regulates over 100 genes whose actions include those associated with calcium homeostasis and immune responses as well as cellular growth, differentiation, and apoptosis. Inactivation of 1,25D occurs by C23/C24 oxidation pathways that are catalyzed by the multifunctional CYP24A1 enzyme. Both CYP27B1 and CYP24A1 are highly regulated genes whose differential expression is controlled in response to numerous cellular modulatory agents such as parathyroid hormone (PTH), calcitonin, interferon gamma, calcium, phosphorus, and pituitary hormones as well as the secosteroid hormone 1,25D. The upregulation of CYP27B1 by 1,25D has both a rapid nongenomic and a slower genomic component that are functionally linked. The rapid response involves protein kinase C and mitogen-activated protein kinase (MAPK) pathways that direct the phosphorylation of nuclear transcription factors. The slower genomic actions are linked to the binding of 1,25D to the vitamin D receptor (VDR) and the interaction of the VDR-1,25D complex with its heterodimer partner retinoid-X-receptor and associated coactivators. The regulatory complex is assembled on vitamin D response elements in the proximal promoter of the CYP24A1 gene and functions to increase the transcription rate.


SymPoSia TUESDay

SYM-34-01

SYM-34-02 SYM-34-03

SYM-33-05SPLICING FACTOR SRSF3 AS A NOVEL REGULATOR OF ONCOGENIC MIRNAS

Ratnadiwakara M.1, Jarde T.1, 2, Engel R.1, 3, Oliva J.3, Mcmurrick P.J.3, Abud H.E.1 and Anko M.L.1 1Monash University. 2Hudson Institute of Medical Research. 3Cabrini Health.

MicroRNAs (miRNAs) regulate gene expression post-transcriptionally by fine-tuning mRNA levels and translation. miRNA biogenesis is tightly regulated to maintain specific miRNA expression patterns in different tissues and developmental stages, misexpression leading to pathological conditions. miRNAs are transcribed as longer precursors that undergo multiple processing steps before the mature miRNAs reach their target mRNAs in the cytoplasm. Although the RNA binding proteins (RBPs) Drosha, DGCR8 and Dicer are the essential components of the miRNA processing pathway, multiple other RBPs, have recently been identified as critical regulators of miRNA biogenesis. The CNNC motif bound by the SR protein splicing factor SRSF3 specifies pri-miRNA hairpins and in vitro studies have demonstrated SRSF3 enhancing pri-miRNA processing in a CNNC dependant manner. However, the in vivo functional relevance of SRSF3 activity in miRNA processing has remained unexplored. Our analysis of SRSF3 binding sites in embryonic stem cells (ESCs) identified SRSF3 binding at the CNNC motif particularly in miRNAs located in polycistronic miRNA clusters. We demonstrate that SRSF3 depletion in ESCs leads to reduced levels of mature miRNAs without affecting the levels of the pri-miRNAs. Intriguingly, the processing of specific miRNAs within miRNA clusters is individually regulated, leading to differential expression of mature miRNAs derived from the same pri-miRNA. Furthermore, SRSF3 is frequently overexpressed in tumour cells and misexpression of miRNAs is a characteristic of many cancers. Our analysis of SRSF3, pri-miRNA, miRNA and miRNA-target expression in human tumour-normal pairs demonstrates that SRSF3 may confer some of its pathological properties through the control of miRNAs. Our work reveals a novel mechanism regulating the hallmark properties of cancer cells and highlights the roles of multifunctional RBPs in gene regulation in health and disease.

SYSTEMATIC FUNCTIONAL IDENTIFICATION OF CANCER DRUG RESISTANCE GENES

Lau M.T. and Neely G.G. The Dr. John and Anne Chong Lab for Functional Genomics, Charles Perkins Centre and School of Life & Environmental Sciences, The University of Sydney NSW 2006, Australia.

Cancer drug resistance is a major obstacle in cancer therapy. To elucidate the genetic factors that regulate sensitivity to anti-cancer drugs, we performed whole genome CRISPR/Cas9 knockout screens for resistance to a spectrum of anti-cancer drugs of varying compositions and general or targeted mechanisms of action. In addition to known targets and resistance mechanisms, this study revealed novel insights into drug mechanisms of action, including cellular transporters, drug target effectors, and genes involved in target-relevant pathways. Anti-cancer drugs could be classified based on resistance mechanisms, and we provide the first functional “phylogenecity” for these compounds. Importantly, we identified 49 multi-drug resistance genes, including a previously uncharacterised gene named here Required for Drug-induced Death 1; RDD1greg. Loss of RDD1 resulted in resistance to five anti-cancer drugs, primarily anti-tubulin agents, and RDD1 is required for an anti-tubulin drug to trigger MCL-1 degradation and cell death. Loss of RDD1 also conferred resistance to anti-tubulin therapy in a mouse xenograft model, and clinically, low RDD1 expression was associated with poor prognosis in multiple cancer cohorts, with the strongest association for ovarian cancer patient outcome. Together, we provide the first functional landscape of resistance mechanisms to a broad range of chemotherapeutic drugs and reveal new multi-drug resistance nodes. This information can guide personalised anti-cancer therapies or instruct rational drug combinations designed to minimise acquisition of resistance.

GENOMIC HETEROGENEITY IN COLORECTAL PRIMARY AND LIVER METASTASES SAMPLES

Patch A.-M.1, Kawamata F.2, 3, Behrenbruch C.4, 5, Nones K.6, Kazakoff S.6, Addala V.6, Pearson J.V.7, Hollande F.4, Waddell N.6 and Whitehall V.L.J.2 1Clinical Genomics, QIMR Berghofer Medical Research Institute, Queensland, Australia. 2Conjoint Gastroenterology Laboratory, QIMR Berghofer Medical Research Institute, Queensland, Australia. 3Hokkaido University Graduate School of Medicine, Sapporo, Japan. 4Department of Clinical Pathology, The University of Melbourne, Victoria, Australia. 5Department of Oncology, Sir Peter MacCallum Cancer Centre, Victoria, Australia. 6Medical Genomics, QIMR Berghofer Medical Research Institute, Queensland, Australia. 7Genome Informatics, QIMR Berghofer Medical Research Institute, Queensland, Australia.

Colorectal cancer (CRC) is the third most common malignancy diagnosed worldwide and in Australia, with an estimated >16,500 Australians affected in 2017. At diagnosis around 25% of CRC patients have metastatic (stage IV) disease and a further 25% of patients initially diagnosed at stage II/III will relapse with metastatic disease. Unfortunately, 75% of patients presenting with metastatic disease will have surgically unresectable disease and are incurable. The 5-year survival of this group of patients is approximately 5%, almost ten times lower than patients with resectable disease. Treatment is palliative and there are essentially only three available cytotoxic agents, 5-fluorouracil, oxaliplatin and irinotecan. Newer targeted agents and immunotherapy only show benefit in a subset of patients. Different sites of disease such as the primary tumour compared with the metastasis will have heterogeneity in treatment response making the choice of therapy more complex. A personalised medicine strategy that enables selection of the most effective therapy based on the tumour’s molecular characteristics could provide more effective treatment options for patient management especially for those with unresectable metastatic disease. Therefore, we are investigating the heterogeneity between matched primary CRC and liver metastasis samples in the context of targetable treatments.

MIR-496 EXPRESSION IS ALTERED IN OROPHARYNGEAL CANCERS BY HPV16 E6

Mason D.1, Zang X.2, 3, Monteiro Marques T.4, Gama-Carvalho M.4 and Tran N.1, 2 1School Biomedical Engineering. Centre for health Technologies. University of Technology Sydney, NSW, Australia. 2The Sydney Head and Neck Cancer Institute, Sydney Cancer Centre, Royal Prince Alfred Hospital, NSW, Australia. 3Department of Infectious Diseases and Immunology, University of Sydney, NSW, Australia. 4University of Lisboa, Faculty of Sciences, BiolSl- Biosystems and Integrative Sciences Institute, Campo Grande, Lisboa, Portugal.

HPV is a major risk factor for oropharyngeal cancers(OPC), with 75% being associated with HPV16. Infection by the virus is known to regulate small non-coding RNAs, known as miRNAs. MiRNAs have been shown to play a critical role in tumorigenesis. Using an LNA array we determined the expression levels of miRNAs in OPC positive for HPV. miR-33, miR-210, miR-142-3p and miR-496 were differentially expressed between HPV16(+) and HPV16(-) OPCs. Of these miR-496 and miR-33 were the most deregulated. Mechanistically we showed that E6, one of the oncoproteins of HPV16 modulates the expression of miR-496 and miR-33. We then revealed that miR-496 can regulate the transcription factor E2F2, which has been shown to be required for viral replication. A viral/miRNA interactome was constructed to map association between miRNAs and E6. From this analysis, we discovered a mechanistic link between miR-496, miR-33 and E2F2. We believe that E6 increases the expression of SREBF2 which harbours miR-33. The increase in miR-33 regulates transcription factors, (BACH1, STAT5A, and GATA2) which impacts the expression of miR-496. Our study has identified a specific regulatory pathway involving E6, miRNAs and E2F2. These events may be important for viral replication and transformation of the cell. These interactions uncovered between HPV and miRNAs may provide an insight into the cellular mechanisms in HPV positive oropharyngeal SCC, and the aetiology of the disease.


SymPoSia TUESDay

SYM-34-04 SYM-35-01

SYM-35-02 SYM-35-03

SYSTEMS BIOLOGY ANALYSIS OF EPITHELIAL-MESENCHYMAL TRANSITION IN PANCREATIC CANCER

Nagaraj Hiriyur, S.

CLINICAL BACTERIOPHAGE THERAPY IN THE 21ST CENTURY

Morales S. AmpliPhi Biosciences.

It is now unanimously accepted that the world has entered a dangerous phase in the treatment of multi-resistant bacterial infection. The problem seems to be that the speed and ability of the bacterial community to develop resistance far exceeds the capacity of the biomedical and scientific community to develop new antibacterials. Faced with this situation is it perhaps time to acknowledge that the current antibiotic paradigm, if not broken, is in need of review, and that new alternative treatment options are urgently needed. A new antibacterial treatment would ideally have the capability of matching, and if possible exceeding the mutation rate of the bacterial targets. In other words, a form of intelligent antibacterial that out-mutates the mutators. Bacteriophage therapy, the use of highly specific bacterial viruses to treat bacterial infections, is one alternative with the potential to achieve this. Historically, bacteriophages (phages) were discovered 100 years ago at the Pasteur Institute in Paris. The therapy was widely practised until the middle of last century, until the advent of small molecule antibiotics such as penicillin saw the practise fade into obscurity for the next 50 years, before a major renaissance in the late 1990s. This talk will concentrate on the extensive progress made since that time in understanding the biology of phages and their potential for clinical application. The results from both pre-clinical and human clinical trials will be discussed, along with the issues facing the fledgling phage therapy industry such as production, regulatory and financial constraints. Exciting new developments in the combined use of phages and antibiotics will be described which offer the tantalising possibility that phage therapy might not only be able to contain antibiotic resistant bacteria but even reverse it.

STRUCTURE, FUNCTION, AND BIOSYNTHETIC ORIGIN OF OCTAPEPTIN ANTIBIOTICS ACTIVE AGAINST EXTENSIVELY DRUG-RESISTANT GRAM-NEGATIVE BACTERIATony Velkov,1,10,11, Alejandra Gallardo-Godoy,2,10 James D. Swarbrick,3 Mark. A.T. Blaskovich,2 Alysha G. Elliott,2 Meiling Han,4 Philip E. Thompson,3 Kade D. Roberts,3 Johnny X. Huang,2 Bernd Becker,2 Mark S. Butler,2 Lawrence H. Lash,5 Sonia Troeira Henriques,2 Roger L. Nation,4 Sivashangarie Sivanesan,4 Marc-Antoine Sani,6 Frances Separovic,6 Haydyn Mertens,7 Dieter Bulach,8 Torsten Seemann,8 Jeremy Owen,9 Jian Li,4, and Matthew A. Cooper2

1Department of Pharmacology & Therapeutics, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville, VIC 3010, Australia.2Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia. 3Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, 3052 VIC, Australia. 4Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, 3052 VIC, Australia. 5Department of Pharmacology, Wayne State University, School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA 6School of Chemistry, Bio21 Institute, University of Melbourne, Parkville, VIC 3010, Australia. 7EMBL, Hamburg, Germany. 8Department of Immunology and Microbiology, University of Melbourne, Parkville, VIC 3010, Australia. 9School of Biological Sciences, Victoria University, Wellington 6012, New Zealand.

Resistance to the last-resort antibiotic colistin is now widespread and new therapeutics are urgently required. We report the first in toto chemical synthesis and pre-clinical evaluation of octapeptins, a class of lipopeptides structurally related to colistin. The octapeptin biosynthetic cluster consisted of threenon-ribosomal peptide synthetases (OctA, OctB, and OctC) that produced an amphiphilic antibiotic, octapeptin C4, which was shown to bind to and depolarize membranes. While active against multidrug resistant (MDR) strains in vitro, octapeptin C4 displayed poor in vivo efficacy, most likely due to high plasma protein binding. Nuclear magnetic resonance solution structures, empirical structure-activity and structure-toxicity models were used to design synthetic octapeptins active against MDR and extensively drug-resistant (XDR) bacteria. The scaffold was then subtly altered to reduce plasma protein binding, while maintaining activity against MDR and XDR bacteria. In vivo efficacy was demonstrated in a murine bacteremia model with a colistin-resistant Pseudomonas aeruginosa clinical isolate.

IN SEARCH OF NEW ANTIBIOTICS THAT EXPLOIT THE CELL WALL DEFICIENT L-FORM LIFESTYLE

Max D.O., Lazenby J.J. and Whitchurch C.B. The ithree institute, University of Technology Sydney, Ultimo, NSW, Australia.

Pseudomonas aeruginosa is a Gram negative bacterial pathogen that has been identified by the WHO as in critical need for the development of new antibiotics. P. aeruginosa has high levels of intrinsic resistance to a broad range of antibiotics due to possession of a highly impermeant outer-membrane (OM) and multiple, inducible multi-drug efflux pumps. We have previously shown that P. aeruginosa tolerates β-lactam antibiotics by undergoing a rapid and reversible en masse conversion from bacilli to cell wall deficient L-forms that lack the OM. We hypothesized that L-form P. aeruginosa may be susceptible to antibacterial compounds that are normally inhibited by the Gram negative cell wall. Indeed we found that L-form P. aeruginosa is rapidly killed by antimicrobial peptides (AMPs) that are normally ineffective against bacillary P. aeruginosa. These observations suggest that drug combinations that induce L-form transitions with compounds that exploit L-form susceptibility may be novel therapeutic options. We have developed high-throughput screens that enable the identification of compounds that induce or kill L-form bacteria. We have used our L-form kill assay to screen an FDA-approved drug library and have identified ~100 drugs with enhanced bactericidal activity against L-form P. aeruginosa. Interestingly, most of these drugs have not previously been identified as having any antibiotic activity indicating that there are a number of FDA-approved drugs that might be able to be repurposed for use as antibiotic therapeutics that target L-form bacteria. Our observations indicate that the ability of bacteria to transition into CWD deficient L-form lifestyle to avoid the effect of compounds that target the bacterial cell wall may be exploited to develop novel antibiotic therapeutic approaches.



SymPoSia TUESDay

SYM-35-04 SYM-35-05

SYM-36-01 SYM-36-02

STRUCTURAL AND KINETIC CHARACTERISATION OF CLASS III BIOTIN PROTEIN LIGASES; NOVEL ANTI FUNGAL DRUG TARGETSSternicki L.M.1, Nguyen S.1, Pukala T.L.2, Pendini N.R.1, 3, Beckham S.3, Wilce M.C.3, Booker G.W.1, Wegener K.L.1 and Polyak S.W.1 1School of Biological Sciences, The University of Adelaide, North Terrace, Adelaide, South Australia, Australia 5005. 2School of Physical Sciences, The University of Adelaide, North Terrace, Adelaide, South Australia, Australia 5005. 3School of Biological Sciences, Monash University, Clayton, Victoria, Australia 3800.The covalent post-translational attachment of biotin is necessary for the activity of certain metabolic enzymes. Biotin protein ligase (BPL) is responsible for this modification and has been proposed as a novel anti-infective target. Crystal structures of class I and II BPLs, present in archaea and bacteria, have been reported and have aided the design of inhibitors against bacterial BPLs. However, the class III BPLs, found in mammals, fungi and insects, have not been extensively characterised nor exploited for antifungal therapeutics. These BPLs differ as they contain a large N-terminal extension that is proposed to assist selection of appropriate biotinylation targets. Limited structural information, including the absence of a class III BPL crystal structure, has hindered the molecular understanding of substrate recognition by the N-terminal extension and the development of antifungal inhibitors. Initial kinetic characterisation of four class III BPLs, namely those from the fungi Saccharomyces cerevisiae, Candida albicans, Botrytis cinerea and Zymoseptoria tritici, revealed different KM values for the substrates. Likewise, inhibition constants for known substrate mimics varied between the enzymes. These data reveal subtle structural differences localised around the substrate binding sites between these class III enzymes, suggesting selective inhibition may be possible. The S. cerevisiae BPL was more thoroughly investigated for structural insights, as crystallography of these four BPLs has so far been unsuccessful. SAXS, ion mobility MS and hydrogen-deuterium exchange MS are being employed to investigate conformational changes and enzyme dynamics associated with ligand binding. Crosslinking MS is also being utilised to delineate how the N-terminal domain facilitates interactions with substrates targeted for biotinylation. This information will aid the refinement of homology models of S. cerevisiae BPL to provide a model of a class III BPL structure. This structural information is vital for the development of selective anti-fungal inhibitors that target pathogenic BPLs but not the human isoform.

NOVEL STRUCTURAL PROPERTIES OF A HAEMOPHORE-LIKE PROTEIN OFFER AN AVENUE FOR TARGETED ELIMINATION OF PORPHYROMONAS GINGIVALIS

Kwan A.1, Gao J.2, Yammine A.1, Zhou Z.2, Nguyen K.2, Hunter N.2 and Gell D.3 1School of Life and Environmental Sciences, University of Sydney, NSW 2006, Australia. 2Institute of Dental Research, Westmead Hospital, University of Sydney, NSW 2145, Australia. 3School of Medicine, University of Tasmania, TAS 7005, Australia.

Porphyromonas gingivalis is a keystone bacterium found in chronic periodontal disease, which is characterised by severe inflammation and destruction of supportive tooth structures. The disease has also been linked to systemic pathologies including cardiovascular diseases and autoimmune disorders1. P. gingivalis growth is dependent on the acquisition of environmental haem for its iron and porphyrin requirements. As such, the bacterium has an array of proteins for haem acquisition, including HusA (Heme uptake system protein A). In previous work2, we have shown that HusA directly binds haem and is essential for P. gingivalisto grow under haem-limiting conditions, such as those found inside plaques and in saliva. Therefore, HusA may represent a therapeutic target for treating chronic periodontitis and possibly other associated systemic diseases. To develop compounds that target HusA and P. gingivalis, we have determined the solution structure of HusA using Nuclear Magnetic Resonance (NMR) spectroscopy. Using NMR titration studies, mutagenesis and in silico docking, we have identified that haem binds in a hydrophobic groove on the α-helical structure that lacks the typical iron coordination seen in other haemophores. This mode of interaction allows HusA to bind a variety of abiotic and metal-free porphyrins with significantly higher affinity than to haem. We have exploited the unusual porphyrin binding activity of HusA to target a prototypical deuteroporphyrin-metronidazole conjugate with restricted antimicrobial specificity in a “Trojan horse” strategy that effectively kills intracellular P. gingivalis. Therefore, targeting and manipulating HusA:porphyrin interactions is a promising avenue to developing new therapeutics against P. gingivalis. 1. Seymour GJ, Ford PJ, Cullinan MP, Leishman S, Yamazaki K. (2007) Relationship between periodontal infections and systemic disease. Clin Microbiol Infec 13:3. 2. Gao JL, Nguyen KA, Hunter NJ. (2010) Characterization of a hemophore-like protein from Porphyromonas gingivalis. J Biol Chem 285:40028. .

SUPPORT NETWORKS FOR EMCRS

Bowden N.A.1, 2 1University of Newcastle, University Dr, Callaghan, NSW 2308. 2Hunter Medical Research Institute, Kookaburra Cct, New Lambton Heights, NSW 2305.

Building a network can seem difficult and many EMCRs don’t know where to start. Having a support network is crucial for researchers at all career stages, but is particularly important when starting a career and when transitioning into leading a team. Australia has several different types of networks open to EMCRs and I will discuss how to get involved and build your own network locally, nationally and internationally.

HOW TO MAKE VALUABLE CONNECTIONS BETWEEN ACADEMIA AND INDUSTRY

Oliver B. University of Technology Sydney and Woolcock Institute of Medical Research, Australia.

Industry is a broad term used for everything from spinout companies with 1 employee, to large multinational companies, and how you interact with each needs to be different. Smaller companies often paradoxically have more money to invest in partnerships and projects as they don’t have the capacity inhouse, but they often want their “pound of flesh”. Large companies are shrewder with their investments, but generally expect less in return (ie they are more realistic). In this short presentation, I will attempt to cover the question: So what should you do? For example, should you develop the relationship by doing Pro Bono research, quote $500,000 for 6 months work, or just not bother. Do you chase industry or do they come to you? What is that, that you have produced again - the worlds first recombinant monkey TNF? What exactly are you going to do together? I will also talk about barriers to effective interactions, e.g. oops I forgot about IP, I don’t have enough money (institutional overheads).


SymPoSia TUESDay

SYM-36-03 SYM-36-04

SYM-36-05 SYM-37-01

SHAMELESS SELF-PROMOTION: CAN YOU FAKE IT TILL YOU MAKE IT?

Bouveret R. University of New South Wales, Sydney, NSW 2052.

Self-promotion presents real benefits but also some risks. If well executed, it allows you to communicate your skills and interests and to influence how others, including employers, see you. If you can self-promote and influence how others perceive and remember you, you are more likely to get opportunities to do what you wish to do. However, self-promotion is also risky. When poorly done, it is often associated with bragging, obnoxiousness or narcissism. In this talk, I will share my own experience and views on how to build a personal brand with modesty and humility. Ultimately, what matters most is not getting better offers or being promoted, it is having talents and interests, and being remembered for doing well and doing good.

CREATING USEFUL MENTOR RELATIONSHIPS

Georgousakis M. Franklin Women, Sydney NSW 2065

Mentoring relationships are very valuable for research career progression however there is much confusion around what a mentor actually does and how you go about getting, and keeping, one. In this talk Melina will describe the different types of professional development relationships: Mentoring, Sponsorship and Coaching. She will also share her personal experiences with each of them in her own research career as well as lessons learnt after launching the first cross-organisational mentoring program in the health and medical research sector that specifically aims at support women aspiring to leadership roles in their careers.

HOW PLASTIC ARE THE C4 SUBTYPES? INVESTIGATING THE PLASTICITY OF C4 GRASSES THROUGH EXPOSURE TO LOW LIGHT AND LOW CARBON DIOXIDE

Watson-Lazowski A., Sagun J., Koller F., Papanicolaou A. and Ghannoum O. Hawkesbury Institute for the Environment, Western Sydney University, Locked Bag 1797, Penrith, NSW 2753, Australia.

C4 photosynthesis is an essential process which accounts for ~25% of total plant productivity, generated from a relatively small subset of plant species (~3%). This increased productivity is due to the evolution of a carbon concentrating mechanism between the mesophyll and bundle sheath cells of C4 plants, which allows for an increased concentration of carbon dioxide (CO2) around Rubisco. Three subtypes of C4 photosynthesis exist, each named after the main decarboxylase they utilise to release CO2: NADP-ME, NAD-ME and PEPCK. A number of grass species which utilise C4 photosynthesis are essential to maintaining increasing food and energy demands, highlighting them as an important subset of species for investigation. Firstly, the genetic make-up of the C4 cycle within each subtype requires some elucidation, especially in regards to the NAD-ME subtype. Therefore, we provide an updated model for NAD-ME photosynthesis within grasses via analysis of transcript expression and gene evolution. We can then begin to understand the plasticity of each subtype, both within the C4 cycle and on a whole plant basis. Understanding this plasticity is key to identifying both beneficial traits and targets for improvement. Utilising multiple C4 grass species, spanning two independent origins and all three subtypes, we subjected plants to ambient, low CO2 (180 ppm) and low light (200 PAR) conditions. By comparing phenotype, physiology, biochemistry and transcript expression we are able to dissect the plasticity of C4 grasses, as well as identify candidate genes which may be utilised to improve C4 crop productivity.

GOVERNANCE AND POLICY: WHY SUSTAINING LINKS WITH GOVERNMENT IS IMPORTANT TO YOUR FUTURE

Phillips, P.



SymPoSia TUESDay

SYM-37-02 SYM-37-03

SYM-37-04 SYM-37-05

PROFILING OF THYLAKOID COMPLEXES FROM THE MESOPHYLL AND BUNDLE SHEATH CELLS OF C4, C3 AND C2 PANICUM GRASSES BY BN/PAGE AND LC/MSHernandez-Prieto M.A.1, Foster C.2, Watson-Lazowski A.2, Ghannoum O.2 and Chen M.1 1ARC Centre of Excellence for Translational Photosynthesis, The University of Sydney, School of Life and Environmental Sciences, University of Sydney, NSW 2006. Australia. 2ARC Centre of Excellence for Translational Photosynthesis, Hawkesbury Institute for the Environment, Western Sydney University, Locked Bag 1797, Penrith NSW 2751, Australia.

Photosynthetic efficiency is largely limited because of the low specificity of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). C4 plants have higher efficiency compared to C3 plants because they use two carboxylation cycles, physically separated into two cell types: the mesophyll (MC) and the bundle sheath cells (BSC). This permits C4 plants to concentrate CO2 around RuBisCO, which favors the carboxylation pathway over oxygenation. The fact that C4 photosynthesis evolved independently more than 60 times (Osborne and Sack, 2012) is indicative of its success. Depending on the enzyme responsible for C4 decarboxylation, C4 plants can be divided into different biochemical subtypes: NADP-malic enzyme (ME), NAD-ME, and phosphoenolpyruvate carboxykinase (PEP-CK). The plant genus Panicum, similarly to Flaveria, contains species that perform typical C3 photosynthesis (P. bisulcatum), C2 intermediates (P. milioides), as well as representative of the main C4 biochemical subtypes: C4/NADP-ME (P. antidotale), C4/NAD-ME (P. miliaceum), and C4/PCK (P. maximumrenamed Megathyrsus maximus). Because of the interest in introducing some of the characteristic of C4 photosynthesis into C3 crops, the study of plant families representing the evolutionary and photosynthetic gradient from C3 to C4 is required. An important aspect of this transition that needs to be compared is the organization of the complexes involved in the light reactions to compensate for the higher demand of ATP in C4 plants. To compare the chloroplast organization of these species, we tested different methods to separate BSC chloroplasts from MC chloroplasts, and optimized the thylakoid preparation to obtain comparable band patterning in Native polyacrylamide gel electrophoresis (PAGE) under non-denaturing (native) conditions. Our results, comprising the analysis of 130 bands, showed that both C4 plants of the NADP-ME subtype analyzed have a higher proportion of PSI and NDH complexes with respect to PSII in BSC than in MC. No such imbalance was observed in C4 species of the other biochemical subtypes.

STRATEGIES FOR IMPROVING PHOTOSYNTHETIC ELECTRON TRANSPORT IN C4 PLANTS

Ermakova M.1, Furbank R.1, 2 and von Caemmerer S.1 1ARC Centre of Excellence for Translational Photosynthesis, Australian National University, Canberra, Australia. 2CSIRO Agriculture, Canberra, Australia.

Recent activities to improve photosynthetic performance in crop plants have focused primarily on C3 photosynthesis where there are clear identified targets such as improving Rubisco kinetics, installation of a CO2 concentrating mechanism and alleviating limitations in chloroplast electron transport. However, C4 plants that utilise the C4 photosynthetic pathway also play a key role in world agriculture and strategies to manipulate and enhance C4 photosynthesis thus have potential for major agricultural impacts. The C4 photosynthetic pathway is a biochemical CO2 concentrating mechanism that requires the coordinated functioning of mesophyll (M) and bundle sheath cells (BS) of leaves and species have evolved a complex blend of anatomy and biochemistry to achieve this. Chloroplast electron transport in C4 plants is shared between these two cell types and the diversity of thylakoid protein complexes of each cell type is defined by the requirements of the metabolic sub-type of C4 photosynthesis. Our recent work with the model monocot C4 species Setaria viridis (green foxtail millet) and transgenic S.viridis plants with altered amount of cytochrome (Cyt) b6f complex demonstrates the link between electron transport capacity of the leaves and CO2 assimilation. Overexpression of the Cyt b6f in both M and BS allows higher rates of assimilation in transgenic plants without affecting Rubisco content. However, increasing the amount of the Cyt b6f only in M, surprisingly, leads to a reduced rate of CO2 assimilation at low CO2. We link this observation to measurements of electron transport components and light harvesting capacity of BS.

UNDERSTANDING AQUAPORINS AS POTENTIAL ENHANCERS OF PHOTOSYNTHESIS AND PLANT PERFORMANCE

Groszmann M., De Rosa A., Mani-George A., Skinner S., von Caemmerer S. and Evans J.R. ARC Centre of Excellence for Translational Photosynthesis. Research School of Biology. The Australian National University. Canberra ACT, 2601.

Plants are in constant exchange with the surrounding environment, absorbing and transporting nutrients and gases essential for growth. Aquaporins represent a major class of channel proteins that aid in this process by increasing the permeability of biological membranes. Different aquaporin isoforms can selectively facilitate the transmembrane transport of not only water, but also a range of other small molecules and gases necessary for optimal plant performance. Amongst these is carbon dioxide (CO2), a key substrate for photosynthesis. For the majority of plants, photosynthetic efficiency is limited by the amount of CO2 diffusion from the atmosphere to the chloroplast where it is fixed into sugars. A strategy to increase photosynthetic efficiency could be to use aquaporins to enhance CO2 diffusion to the chloroplasts by increasing transport across the plasma membrane and chloroplast envelope (collectively termed mesophyll conductance). Of the few aquaporins so far shown to enhance membrane permeability to CO2, these all belong to the Plasma membrane Intrinsic Proteins (PIP) sub-family. PIP aquaporins also transport a range of other substrates necessary for optimal plant performance, making them exciting targets for engineering crop improvements. I will present our progress towards characterising PIP aquaporin biology and their potential translational use in crop species.

EXPLORATION OF SEASONAL CHANGE OF THERMAL TOLERANCE AND PROTEINS IN THREE AUSTRALIAN DESERT PLANTS

Milner K.V.1, Van Sluyter S.C.2, French K.3, Valenzuela S.M.1 and Leigh A.1 1School of Life Sciences, University of Technology Sydney, NSW. 2Department of Biological Sciences, Macquarie University, NSW. 3School of Biological Sciences, University of Wollongong, NSW.

Plants in Australia’s southern semi-arid zone experience a wide range of temperatures (4.6 °C mean minimum in winter and 34.2 °C mean maximum in summer) and show plasticity in their thermal tolerance thresholds with the ability to shift photosynthetic thresholds upwards 5 °C from winter to summer. An understanding of how they are able to make these threshold adjustments is required. With a new absolute protein quantification method, we have the opportunity to identify protein changes that may explain this ability. A selection of three species from different functional groups were used to explore species differences. Acacia ligulata a widespread nitrogen-fixing shrub, Myoporum montanum a widespread shrub and Solanum oligacanthum a perennial herb or subshrub were grown in an experimental garden in semi-arid South Australia. Sampling occurred in winter, spring and summer where the assessment of thermal tolerance included a series of temperature assays for photosynthetic thermal tolerance (T50using chlorophyll fluorescence) and membrane stability (Tcrit using electrical conductivity). Protein identification and absolute quantitation used a new extraction method and QconCAT-spiked samples coupled with MS/MS and SWATH acquisition. All species were able to adjust upwardly thermal tolerance thresholds from cooler to warmer months. However, the changes seen in proteins of interest differed depending on plant species. This early exploration of temporal protein changes provides insight into acclimatisation mechanisms Australian desert plants use to cope in a difficult climate.


SymPoSia TUESDay

SYM-38-01 SYM-38-02

SYM-38-03 SYM-38-04

PRECISION MEDICINE IN A DISH

Palmieri M., Hirokawa Y., Leong E., Burgess A. and Sieber O. The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia.

Despite the exponential increase in our knowledge of cancer genetics, our ability to effectively treat tumours based upon their genetics alone remains limited. While determining the sensitivity and resistance of an organism before treatment has been the standard of care in infectious diseases for many years, therapy selection in oncology continues to be initiated largely based on tumour histology. Patient-derived tumour organoid (PDTO) technology holds promise for changing the current treatment paradigm, providing a system amenable to culture and “real-time” drug sensitivity testing in clinical practice to inform decisions in a timeframe that is useful to an individual patient. This presentation will highlight the challenges and lessons learned from our efforts to establish high-throughput clinical and research workflows for PDTOs derived from human colorectal cancers.

‘NEXTGEN’ HUMAN BRAIN ORGANOIDS USING 3D PRINTED GELATIN METHACRYLATETomaskovic-Crook E.1, 2, Zhang B.1, Bourke J.L.3, 4, Gu Q.1, Kapsa R.M.3, Wallace G.G.1 and Crook J.M.1, 2, 5 1ARC Centre of Excellence for Electromaterials Science (ACES), AIIM, University of Wollongong, Innovation Campus, North Wollongong, NSW. 2IHMRI, University of Wollongong, Wollongong, NSW. 3ACES, St Vincent’s Hospital, University of Melbourne, Fitzroy, VIC. 4Dept Medicine, St Vincent’s Hospital, University of Melbourne, Fitzroy, VIC. 5Dept Surgery, St Vincent’s Hospital, University of Melbourne, Fitzroy, VIC.

The generation of brain organoids derived from human pluripotent stem cells (PSCs) is a significant step towards better in vitro modelling of neurodevelopment and disease. Brain organoids are discerned by their cellular and structural complexity, with characteristics of developing embryonic brains. Conventional methods of organoid formation are limited to small-scale operation and require multiple handling steps following stem cell aggregation, coating with expensive and undefined tumour-derived MatrigelTM basement membrane preparation, and arduous bioreactor based differentiation and expansion methods. We have initially demonstrated gelatin methacrylate (GelMA) to be a cell growth substrate for rapid and novel induction of brain organoids from human induced PSCs (iPSCs). GelMA is a versatile, 3D printable semisynthetic matrix that incorporates the intrinsic bioactivity of natural matrices with the fidelity of synthetic biomaterials for more defined and clinically-compliant cell support. Towards scaling up organoid production we have now 3D printed GelMA-based microwell arrays to generate large numbers of organoids for higher throughput R&D. Constructs consist of densely packed cell soma with regional divisions resembling cortical plate or rudimentary grey matter tissue with underlying white matter-like tissue, as well as hollow neural tube-like structures. With larger numbers of organoids we are progressing our understanding through immunofluorescent-based histochemistry of cortical lamination using layer-specific markers of cerebral neocortex and early progenitor regions; markers including RELN, CTIP2, TBR1, SATB2, PAX6, NES, and SOX2, as well as the forebrain specific marker, FOXG1. Moreover, we have demonstrated coordinated glutamate-responsive neural network activity of formed neurons by extracellular recordings using multi-electrode arrays (MEAs). The optimised ‘NextGen’ method provides a defined, simplified and higher throughput platform for ‘brain on a bench’ research and translation of iPSCs, neural derivatives and neural organoids, including in vitro modelling of brain development and disease, tissue engineering and regenerative medicine.

SCREENING IN HUMAN CARDIAC ORGANOIDS IDENTIFIES A REQUIREMENT FOR THE MEVALONATE PATHWAY IN CARDIOMYOCYTE PROLIFERATION

Hudson J.E. QIMR Berghofer Medical Research Institute.

Rationale: Induction of endogenous cardiomyocyte proliferation is a promising strategy for cardiac regeneration. Recent studies suggest that dual inhibition of glycogen synthase kinase 3 (GSK3) and serine/threonine protein kinase 4 (STK4 aka MST1) potently drives cardiomyocyte proliferation but the mechanisms of action are not defined. Objective: Decipher the effects of GSK3 and MST1 inhibition on cardiomyocyte proliferation and identify small molecules that can drive proliferation without detrimental effects on contractile force. Methods and Results: We coupled high throughput proteomics and functional screening of small molecules in single miniaturised human cardiac organoids (hCOs) to identify key pathways driving cardiomyocyte proliferation. We report that GSK3 inhibition activates a cell cycle network whereas MST1 inhibition drives the mevalonate pathway, with synergistic effects on proliferation. However, all GSK3 inhibitors tested also reduced contractile force in hCO. This was overcome by screening of 105 compounds, identifying a p38 inhibitor, which activated a cell cycle network without reducing force. The screen also identified a TGFBR inhibitor that induced the mevalonate pathway, increased protein prenylation and synergised with cell cycle activators to promote proliferation. Inhibition of the mevalonate pathway abolished the myocyte proliferative response and also reduced cell cycle activity in immature cardiomyocytes, consistent with downregulation of the mevalonate pathway during cardiac maturation in vivo. Conclusions: These findings implicate the mevalonate pathway in cardiomyocyte cell cycle control, which could have important ramifications for congenital heart disease and the development of regenerative therapeutic strategies for heart failure.

NEUREGULIN1 IS A KEY NICHE SIGNAL THAT SUPPORTS INTESTINAL STEM CELL PROLIFERATION

Jarde T.1, 2, 3, Rossello F.1, 2, 4, Kurian Arackal T.1, 2, Flores T.1, 2, Giraud M.1,

2, Prasko M.1, 2, Nefzger C.M.1, 2, 4, Abe S.5, Polo J.M.1, 2, 4 and Abud H.E.1, 2 1Department of Anatomy and Developmental Biology, Monash University, Clayton, Australia. 2Cancer Program, Monash Biomedicine Discovery Institute, Clayton, Australia. 3Centre for Cancer Research, Hudson Institute of Medical Research, Clayton, Australia. 4Australian Regenerative Medicine Institute, Monash University, Clayton, Australia. 5Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto, Japan.

Identifying strategies to enhance intestinal stem cell proliferation may enable regeneration of the epithelium to be manipulated in degenerative diseases and intestinal pathologies. Using intestinal organoids grown ex vivo, we have defined Neuregulin1/ErbB signalling as a strong driver of cell proliferation in the intestinal epithelium. We defined the localisation of Neuregulin1 and interacting receptors in the small intestine using immunofluorescence and qRT-PCR. We observed that supporting niche cells express Neuregulin1, while stem cells express ErbB receptors, supporting a model where Neuregulin1/ErbB signalling directly regulates stem cells. The role of Neuregulin1 was also investigated in vivo using both a gene knockout approach and a model where activation of Neuregulin1/ErbB signalling was achieved in mice injected with 15ug Nrg1 for 5 days. Elevation of signalling increased cell proliferation in crypts, altered cellular differentiation and promoted regeneration. Loss of Neuregulin1 resulted in a significant decrease in cell proliferation within crypts in both stem and progenitor cells. The molecular changes induced by Neuregulin1 were examined using RNA sequencing which defined a proliferative molecular signature in both stem and progenitor cells. This was reinforced by examining the ability of single intestinal stem cells to generate organoids. Neuregulin1 significantly promotes organoid growth and the formation of colonies from single stem cells.


SymPoSia TUESDay

SYM-38-05 SYM-39-01

SYM-39-02 SYM-39-03

NOTCH1/NRG1 CONTROL OF CARDIAC JELLY DYNAMICS DEFINES THE BUILDING PLAN FOR TRABECULATIONDel Monte-Nieto G.1, Ramialison M.2, Adam A.A.S.1, Wu B.3, Aharonov A.4, Bourke L.M.5, Harten S.K.5, Tzahor E.4, Zhou B.3and Harvey R.P.1 1Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales 2010, Australia. 2Australian Regenerative Medicine Institute. Monash University, Victoria 3800, Australia. 3Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, USA. 4Weizmann Institute of Science, Rehovot 7610001, Israel. 5QIMR Berghofer Medical Research Institute, Herston, Queensland 4006, Australia.

In vertebrate hearts, ventricular trabecular myocardium develops as a sponge-like network of cardiomyocytes critical for efficient cardiac contraction in the early embryo, and for ventricular conduction, septation, and wall thickening through the process of compaction in the foetal heart. Defective trabeculation at early developmental stages leads to embryonic lethality whereas defects at later stages can lead to Non-Compaction Cardiomyopathy, the third most commonly diagnosed cardiomyopathy in adults. There are divergent views on when and how trabeculation is initiated in different species. In mice, the onset of trabeculation has not been formally addressed, but is proposed to begin at embryonic day (E)9.0 as cardiomyocytes form radially-oriented ribs. Endocardium-myocardium communication is essential for trabeculation, with numerous signalling pathways identified, including Neuregulin1 (Nrg1) and Notch1. Late disruption of the Notch1 pathway causes Non-Compaction Cardiomyopathy. Although mutants in cardiac jelly extracellular matrix (ECM) genes Has2 and Vcan lack trabeculae, and matrix metalloprotease ADAMTS1 activity at E14.5 promotes total cardiac jelly degradation and trabecular growth arrest, the role of ECM dynamics and its molecular regulation during early trabeculation is poorly understood. Here we present a new model of cardiac trabeculation starting as early as the heart tube forms (E8.0), integrating for the first time dynamic endocardial and myocardial cell behaviours, and ECM remodelling, and revealing new epistatic relationships for known signalling pathways. Notch1 signalling promotes ECM degradation during formation of endocardial projections critical for individualization of trabecular units, while Nrg1 promotes myocardial ECM synthesis, necessary for trabecular rearrangement and growth. These systems interconnect through Nrg1 control of Vegfa, yet act antagonistically to establish trabecular architecture. Furthermore, these insights allowed the prediction of persistent cardiac jelly and trabecular growth as a potential cause of disease in a murine Non-Compaction Cardiomyopathy model, providing new understanding of congenital heart disease pathophysiology.

CONTROL OF RECEPTOR FUNCTION THROUGH MEMBRANE EMBEDDED SEQUENCES

Call M.J.1, 2 1Structural Biology Division, The Walter and Eliza Hall Institute. 2Department of Medical Biology, University of Melbourne.

To sense the external environment, cells rely on receptors that span the membrane and transmit signals to the cell interior. Our understanding of how single-pass receptors transmit signals is lacking in part because we do not have full-length structures for single-pass receptors. It is becoming more apparent that single-pass receptor systems act in homo- and hetero-oligomeric formats and that the transmembrane domains of receptors play important roles in mediating signal transduction across the membrane, providing contacts around which receptor chains are organised and are targets for regulation by other cellular components. We are interrogating the function of single-pass receptors by studying interactions among transmembrane domains to develop a more comprehensive view of receptor function and regulation.

TOWARDS CAPTURING AN ATOMIC VIEW OF THE ACTIVATION OF P-REX ONCOGENES AT THE MEMBRANE

Lucato C.M.1, Mitchell C.A.1, Whisstock J.C.1, Halls M.L.2 and Ellisdon A.M.1 1Biomedicine Discovery Institute, Monash University. 2Monash Institute of Pharmaceutical Sciences, Monash University.

P-Rex1 and 2 are guanine nucleotide exchange factors that activate a number of Rho family GTPases that are crucial in regulating cell growth and motility. Interestingly, P-Rex proteins regulate these cellular functions downstream of a number of GPCRs and RTKs and, in doing so, function as a membrane-localised signalling node in many cancer-associated pathways. Recently, P-Rex1 expression was shown to be necessary for melanoma dissemination and has been associated with increased metastatic phenotypes in both breast and prostate cancer. P-Rex1 is therefore a critical signal integrator in tumorigenesis and metastasis and subsequently a desirable therapeutic target, however currently there is little mechanistic understanding of its mode of activity. Crystallization and structural analysis of the catalytic DH-PH domains of P-Rex1 have allowed the elucidation of its mechanism of nucleotide exchange. This exchange activity is inhibited by interactions with P-Rex1 C-terminal domains which have been mapped with cross-linking and mass spectrometry. Interestingly, intra-domain cross-links move significantly upon effector binding and analysis of these movements has allowed for characterization of the multi-step P-Rex1 activation pathway. Further, molecular differences between autoinhibited P-Rex1 (185kDa) and effector-bound, partially activated P-Rex1 complexes (~230kDa) have been visualized with cryo-electron microscopy and resulting models have provided further molecular insight into the mode of P-Rex1 activation.

HOW ARE SIGNALS TO DEVELOPING T CELLS COORDINATED AT THE MEMBRANE?

Russell S.1, 2, Charnley M.1, 2 and Allam A.1, 2 1Peter MacCallum Cancer Centre. 2Swinburne University of Technology.

T cell development in the thymus is precisely orchestrated by a interactions with thymic stroma, but the mechanisms of these interactions are not yet clear. We find that developing T cells assemble a signaling platform at the interface with stromal cells that is analogous to the immunological synapse of mature T cells. This finding is intriguing because the developing T cells do not yet express the peptide-specific T Cell Receptors that are essential for immunological synapse formation. Here, we describe the mechanisms by which developing T cells assemble an antigen-independent immunological synapse, and the consequences of that synapse on subsequent T cell fate decisions.


SymPoSia TUESDay

SYM-39-04 SYM-39-05

SYM-40-01 SYM-40-02

ANNEXIN A6 DEPLETION RESCUES CHOLESTEROL EGRESS IN NPC1 MUTANT CELLS VIA FORMATION OF ENDOSOME-ER MEMBRANE CONTACT SITES

Grewal T.1, Meneses-Salas E.2, Garcia-Melero A.2, Bianco-MuñOz P.2, Egert A.1, Beevi S.S.1, Rentero C.2 and Enrich C.2 1School of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia. 2Departament de Biomedicina, Unitat de Biologia Cel·lular, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, 08036-Barcelona, Spain.

The accumulation of cholesterol in late endosomes (LE)/lysosomes of Niemann-Pick type C1 (NPC1) mutant cells critically disturbs intracellular cholesterol transport. Besides NPC1, oxysterol-binding proteins (ORP1L, ORP5), StAR related lipid transfer domain (STARD) proteins, and several Rab proteins also facilitate cholesterol export from LE. Some of these proteins are engaged in the formation of membrane contact sites (MCS), defined by the apposition of two cellular membranes, providing opportunity for non-vesicular transport of lipids, including cholesterol. Hence, yet unidentified players or gatekeepers in LE may control activation or fine-tune alternative LE-cholesterol transport routes other than NPC1. Annexin A6 (AnxA6), a member of the annexin family, has been implicated in endo- and exocytic pathways and cholesterol homeostasis. We previously demonstrated that AnxA6 is recruited to Low Density Lipoprotein (LDL) -cholesterol enriched LE. Moreover, similar to the loss of NPC1, AnxA6 overexpression led to LE-cholesterol accumulation. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 recruiting TBC1D15, a Rab7-GTPase activating protein (Rab7-GAP), to cholesterol-rich LE to inhibit Rab7 GTPase activity. In contrast, AnxA6 depletion elevated Rab7 activity, rescuing cholesterol export from LE in NPC1 mutant cells. This was associated with peripheral distribution and increased mobility of LE and enhanced lipid accumulation in lipid droplets (LDs) in an acetyl-coenzyme A acetyltransferase (ACAT)-dependent manner. Moreover, StARD3 depletion compromised LE-Chol export and lipid deposition in LD in AnxA6-deficient NPC1 mutant cells. Electron microscopy revealed a significant increase of MCS between LE and the endoplasmic reticulum (ER) in NPC1 mutant cells lacking AnxA6, suggesting LE-cholesterol transfer to the ER via StARD3-dependent MCS formation. Taken together, this study identifies an annexin, AnxA6, as a novel gatekeeper that controls cellular distribution of LE-Chol via two critical biological activities: regulation of a Rab7-GAP and MCS functioning.

RETROMER IS REQUIRED FOR THE RETROGRADE SORTING OF CATION-INDEPENDENT MANNOSE 6-PHOPHATE RECEPTOR INTO A SUBSET OF ENDOSOME TRANSPORT CARRIERS

Cui Y.1, Carosi J.2, 3, Yang Z.1, Kerr M.4, Sargeant T.2, 5 and Teasdale R.1 1School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, Brisbane, Queensland, Australia. 2Hopwood Centre for Neurobiology, Nutrition and Metabolism Theme, South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia. 3Centre for Cancer Biology, University of South Australia, Adelaide, South Australia, Australia. 4Institute for Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia. 5School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA, Australia.

Retromer is a peripheral protein complex that coordinates multiple vesicular trafficking events within the endo-lysosomal system. Here, we demonstrate that retromer is required for the maintenance of normal lysosomal function. At the whole cell level, the knockout of retromer Vps35 subunit reduces the lysosomal proteolytic capacity, as a consequence of the improper processing of lysosomal hydrolases, dependent on the cation-independent mannose 6-phophate receptor (CI-M6PR) trafficking. Moreover, we identify that CI-M6PR, sorted via the retromer-dependent process, is incorporated into a subset of endosome transport carriers (ETCs) tethered by a specific trans-Golgi protein. Finally, we show that this retromer-dependent retrograde cargo trafficking pathway also requires a specific retromer-associated endosomal protein.

UNANTICIPATED PROTEOLYSIS DIVERSIFIES TARGETS OF IMMUNITY

Purcell A.W.1, 2 1Department of Biochemistry, Monash University. 2Infection and Immunity Program, Biomedicine Discovery Institute, Monash University.

The cellular immune response relies upon T cell recognition of peptides presented on the cell surface in complex with HLA molecules. As such, it is the peptide cargo of these HLA molecules that dictates the quality of the immune response and ultimately the efficacy of protective immunity. Relatively simplistic models have been used to explain how these peptide antigens are generated and selected for presentation, however, such models fail to predict and explain the diversity and complexity of the immune response. Much of this unexplained complexity resides in degenerate and reconstructive proteolysis. We have recently highlighted the complex role of proteolysis in the generation and diversification of peptide antigens displayed for T cell recognition. For example, functional heterogeneity is observed during degenerate peptidase trimming of T cell epitopes such that peptides with ragged N- or C-termini (nested peptides) can be presented with different immune outcomes. Moreover, we have generated evidence that reinforces recent surprising studies that up to 30% of peptides presented by class I HLA molecules are generated by post-translational proteasomal splicing (i.e. the ligation of peptide fragments within the proteasome rather than peptide destruction). Using a novel bioinformatic workflow we show that these spliced peptides can be generated either from two regions of the same antigen (cis-splicing) or two distinct antigens (trans-splicing) by retrospectively interrogating datasets for several common HLA allotypes. This is the first demonstration that trans-spliced peptides are abundantly represented in the immunopeptidome. However, the role of spliced peptides in immune responses and the precise mechanism of their generation remains poorly understood and will be discuss.

DISSECTING THE SUBCELLULAR SECRETORY GLYCOPROTEOME WITH MASS SPECTROMETRY PROTEOMICS

zacchi L.F.1, Phung T.2 and Schulz B.L.1, 2 1Centre for Biopharmaceutical Innovation, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland. 2School of Chemistry and Molecular Biosciences, The University of Queensland.

N-glycosylation is a critical post-translational modification that influences the folding and function of 1/3rd of the cellular proteome. The biosynthesis of N-glycoproteins begins in the endoplasmic reticulum (ER), where an oligosaccharide is transferred to selected asparagine residues in nascent polypeptides by the enzyme oligosaccharyltransferase. The presence of N-glycans at specific sites is critical for productive protein folding in the ER, and defects in this process perturb glycoprotein folding, secretion, and function at a systems level. We have developed integrated subcellular fractionation and SWATH glycoproteomic workflows to understand the causes and consequences of changes in the N-glycosylation biosynthetic pathway. We combined biochemical subcellular fractionation methods with quantitative SWATH-MS glycoproteomic and proteomic workflows to measure the response to a range of genetic and chemical perturbations to N-glycoprotein biosynthesis. We optimized biochemical fractionation methods in yeast to enable precise analysis of the subcellular proteome and glycoproteome. This enabled quantitative measurement of subcellular proteomes and site-specific and global profiling of glycan occupancy and structure. We tested these methods in yeast with defined defects in N-glycosylation, and then expanded our analysis to profile the quantitative effects of combined defects in glycoprotein biosynthesis and protein quality control on glycoprotein maturation. Our results give key insights into the effect of site-specific glycosylation on glycoprotein quality control processes, and our methods will be useful in diverse applications in industrial and medical glycobiotechnology.


SymPoSia TUESDay

SYM-40-03 SYM-40-04

SYM-40-05 SYM-41-01

MULTI-OMIC PROFILING OF THE LIVER IN A RAT MODEL OF TYPE 2 DIABETES

Li D.K.1, Smith L.E.1, Koay Y.C.1, 2, McEwan H.1, 3, Don A.1, 3, O’Sullivan J.1, 2, Cordwell S.J.1 and White M.Y.1 1University of Sydney, NSW, Australia. 2Heart Research Institute, NSW, Australia. 3ACRF Centenary Cancer Research Centre, NSW, Australia.

Altered glucose metabolism via insulin resistance is a hallmark of type 2 diabetes (T2D), clinically observed as the inability to maintain postprandial blood glucose levels (BGL). Associated with energetic excess arising from caloric overload, T2D is linked to excess non-esterified fatty acid production and rising nutrient levels, which influence metabolic processes. The liver plays a pivotal role in the pathogenesis of T2D, via elevated gluconeogenesis, whereby glycogen stores are liberated, further elevating BGL. It is important to understand the molecular adaptations of the liver to the metabolic flux and insulin resistance arising from T2D. To achieve this we performed a multi-omic analysis including proteomics, lipidomics and metabolomics in a rat model of T2D combining the effects of high fat diet feeding (calorie overload) and streptozotocin (elevated BGL). To quantify alterations in protein abundance, samples were isobarically tagged prior to mass spectrometry (MS). Discovery lipidomics was achieved with relative quantitation by comparison with synthetic standards. Targeted metabolomics was performed using multiple reaction monitoring, in the presence of deuterated metabolite standards. We quantified close to 7,000 proteins, 300 lipid species and 100 metabolites in the course of this study. Proteomics revealed increased levels of proteins regulating phospholipid biosynthesis and fatty acid metabolism. A concurrent decrease in proteins involved in steroid biosynthesis was observed. Lipid analysis show increased sphingomyelin levels and decreased levels of phosphatidylcholines in T2D, both of which are components of cell membranes and can play a role in metabolic and apoptotic signalling. Elevated levels of branched chain amino acids as well as changes in metabolites indicative of altered energy and amino acid metabolism were detected by metabolomics. The current study has identified changes in protein, lipid composition and metabolite levels indicative of dysregulated energy utilisation and molecular adaptations that contribute to the pathogenesis of T2D.

GOOD AND BAD FAT: DISCOVERING KEY DISTINGUISHING FEATURES WITH MULTIFACTORIAL PROTEOMICS DATA

Deshpande V.1, Humphrey S.J.1, Yang P.2, Lo K.2, Healy M.E.1, Cooke K.C.1, Stoeckli J.1, Yang J.Y.H.2 and James D.E.1 1Charles Perkins Centre, School of Life and Environmental Sciences, The University of Sydney, NSW 2006, Australia. 2School of Mathematics and Statistics, The University of Sydney, NSW 2006, Australia.

Subcutaneous (SC) and visceral (VIS) adipocytes store energy as fat and regulate whole body metabolism. Excessive VIS fat is associated with insulin resistance, a precursor to Type 2 diabetes. In contrast, SC fat may be protective. Despite these important physiological functions, relatively little is known about the molecular features that distinguish these discrete adipose depots. We have used mass spectrometry to construct proteomes of mouse SC and VIS depots, which consist of >7,500 quantified proteins spanning six orders of magnitude. Our study consists of three experimental variables: depot (SC and VIS), diet (normal and high-fat) and sample type (adipocytes and whole tissue). Given this multifactorial experimental design, we devised a computational framework to comprehensively and systematically answer biological questions. This bioinformatic approach involved a series of ANOVA models to stratify the proteome into distinct classes defined by the variable(s) driving the changes in protein expression. We verified that positive control proteins were assigned to expected classes. Our results suggest that adipocytes, rather than the local microenvironment, drive major differences between SC and VIS depots. Of the total proteins, ~2% were upregulated in SC relative to VIS, and ~4% upregulated in VIS relative to SC. These included coenzyme Q and lipolysis proteins in SC, and collagens and cathepsins in VIS. Thus we demonstrate the utility of this proteomic resource and analytic approach in uncovering novel insights into adipocyte biology.

GLYCOCIN F: A BACTERIOSTATIC, GLYCOSYLATED BACTERIOCIN

Bisset S.W.1, 3, Amso Z.2, Yang S.H.2, Brimble M.A.2, 3, Patchett M.L.1 and Norris G.E.1, 3 1Institute of Fundamental Sciences, Massey University, Colombo Rd, Palmerston North 4442, New Zealand. 2School of Chemical Sciences, The University of Auckland, 23 Symonds St, Auckland 1142, New Zealand. 3Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, New Zealand.

Bacteriocins are a class of bacterial peptides that inhibit the growth of closely-related strains or species of bacteria. Glycocin F (GccF) is a glycosylated, 43 amino acid bacteriocin (glycocin) produced by a strain of the probiotic bacteria Lactobacillus plantarum. GccF contains two N-acetylglucosamine (GlcNAc) moieties required for full anti-bacterial activity, one O-linked through serine 18, and the other S-linked through the C-terminal cysteine 43 at the end of a flexible tail. Two nested disulfide bonds are also required for activity. Whereas most bacteriocins exhibit bactericidal effects on a narrow range of species, GccF displays a potent, immediate and reversible bacteriostatic activity towards a range of Gram positive bacteria, and at least one Gram negative strain, making it a potentially useful tool for combating antibiotic resistant bacteria. Although a GlcNAc-specific phosphoenolpyruvate:sugar phosphotransferase system (PTS) EIIC domain has been shown to be one target, there is evidence that another target may be involved. In order to understand how GccF works, we have chemically synthesised a number of GccF analogues designed to probe structure-activity relationships, have used genetic engineering techniques to probe potential receptor-GccF interactions, and are currently investigating the structure of one particularly interesting analogue using NMR. A transcriptomic study of susceptible cells has also been carried out in the absence and presence of GccF in an effort to narrow down the pathways exploited by GccF. These results will be presented, along with a model for the mechanism of action that best fits the data we have collected thus far.

UNDERSTANDING EARLY TOLL-LIKE RECEPTOR SIGNALLING THROUGH MYDDOSOME EXAMINATIONDe Nardo D.1, 2, Balka K.R.1, 2, Cardona Gloria Y.3, Rao V.R.4, Latz E.3, 5, 6 and Masters S.L.1, 2 1Inflammation Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria, 3052, Australia. 2Department of Medical Biology, The University of Melbourne, Parkville 3010, Australia. 3Institute of Innate Immunity, University Hospital, University of Bonn, Sigmund Freud Str. 25, 53127, Bonn, Germany. 4Inflammation and Immunology, Pfizer Inc., Cambridge, Massachusetts 02139, USA. 5Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. 6German Center for Neurodegenerative Diseases, Bonn 53175, Germany.Toll-like receptors (TLRs) form part of the host innate immune system, where they act as sensors of microbial and endogenous danger signals. Upon activation, the intracellular Toll/Interleukin-1 receptor (TIR) domains of TLR dimers form a platform for oligomerisation of a multiprotein signalling platform comprising MyD88 and members of the IRAK family, termed the Myddosome. Formation of the Myddosome complex initiates signal transduction pathways leading to the activation of transcription factors and ultimately, the production of inflammatory cytokines. Despite the critical role Myddosome formation plays in initiating TLR-induced signalling, the molecular mechanisms controlling Myddosome function remain poorly defined. Using immunoprecipitation approaches we successfully isolated Myddosome complexes from whole cell lysates of TLR activated primary mouse bone marrow-derived macrophages (BMDMs) and from IRAK-deficient immortalised BMDMs reconstituted with WT and mutant forms of IRAKs via retroviral transduction. Through the use of a selective IRAK4 inhibitor we were able to examine the role of IRAK4 kinase activity within the Myddosome. Immunoblot and immunofluorescence techniques were used to assess TLR signalling and NF-κB translocation respectively, while ELISAs were employed to examine the secretion of pro-inflammatory cytokines into the supernatant. Here we demonstrate the kinetics of the Myddosome upon TLR activation, revealing rapid assembly and slow disassembly. Furthermore, we show that inhibition of IRAK4 kinase activity leads to increased stability of the Myddosome complex as demonstrated by greater associations between MyD88 and IRAK4. Importantly, we found that the kinase activity of IRAK4 is dispensable for TLR-mediated NF-κB and MAPK signalling but essential for production of inflammatory cytokines. To our knowledge this is the first full examination of the kinetics of the Myddosome from macrophages. We further demonstrated that a loss of IRAK4 activity by either chemical inhibition or genetic manipulation resulted in a significantly more stable Myddosome structure. This increase in stability is suggestive of a prominent protein scaffold role of IRAK4, independent of its kinase activity, in which IRAK4 interacts with MyD88 and IRAK1, tethering them together into the Myddosome complex. Our findings may help explain why, to date, therapeutic targeting IRAK4 kinase activity has not been as successful as hoped, and highlights that targeting the scaffold function of IRAK4 may be an attractive alternative.


SymPoSia TUESDay

SYM-41-02 SYM-41-03

SYM-41-04 SYM-41-05

MACROPHAGE MIGRATION INHIBITORY FACTOR IS REQUIRED FOR NLRP3 INFLAMMASOME ACTIVATION

Lang T., Lee J.P.W., Deen N.S., Morand E.F. and Harris J. Centre for Inflammatory Diseases, Monash University.

Macrophage migration inhibitory factor (MIF) exerts multiple effects on immune cells, as well as having functions outside of the immune system. However, despite over 50 decades of research, the mechanisms by which it does so are not well understood. MIF can promote inflammation through the induction of other cytokines, including TNF, IL-6 and IL-1 family cytokines. We have found that inhibition of MIF regulates the release of IL-1α, IL-1β and IL-18 via activation of the NLRP3 inflammasome. MIF is required for the interaction between NLRP3 and the intermediate filament protein vimentin, which is critical for NLRP3 activation. Further, we demonstrate that MIF interacts with NLRP3, indicating a role for MIF in inflammasome activation independent of its role as a cytokine. These data advance our understanding of how MIF regulates inflammation and identify it as a factor critical for NLRP3 inflammasome activation.

WNT SIGNALLING AND HOST CONTROL OF BACTERIAL PATHOGENS

Thanh-Tran T.1, Gatica Andrades M.1, Nguyen T.T.K.1, Rollo R.F.1, Zamoshnikova A.1, Taveras C.1, Wyer O.K.1, Barnett T.2, Joseph S.1 , Simpson F.1, Brown D.3, Stow J.L.3, Kling J.C.1, Begun J.4 and Blumenthal A.1 1Diamantina Institute, The University of Queensland, Brisbane, Australia. 2School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia. 3Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia. 4Mater Research Institute, The University of Queensland, Brisbane, Australia.

Bacterial infections remain an important clinical challenge despite our extensive arsenal of antibiotics. This is exemplified by lengthy treatments of chronic infections, high mortality due to excessive inflammation, and an alarming increase in antibiotic resistance. One attractive strategy for improved treatments for challenging infections is to enhance the host anti-microbial defence. We and others have associated the WNT signalling pathway with bacterial infections in patients and model systems, implicating novel immune-related functions for this well-known developmental signalling pathway. However, the nature of its contribution to the host response to infection remains to be clearly defined. Focus of our work is defining infection-associated WNT responses and delineate functions of WNT signalling in tailoring host responses to acute bacterial infection.

PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE: AN EMERGING CELL DEATH MEDIATOR AND IMMUNE REGULATOR

Phan T.K., Lay F.T., Jarva M., Kvansakul M. and Hulett M.D. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University.

Phosphorylated phosphatidylinositol lipids, or phosphoinositides (PIPs), vitally regulate diverse cellular processes, including signalling transduction, cytoskeletal reorganisation, membrane dynamics and cellular trafficking. However, PIPs have been inadequately investigated in the context of cell death and inflammation, where they are mainly regarded as secondary messengers of PI3K-Akt signalling. Interestingly, recent studies implied their importance in mediating cell death by demonstrating that PIPs, particularly phosphatidylinositol 4,5-bisphosphate (PIP2), are essential effectors for different forms of programmed cell death. MLKL and gasdermin D require PIP binding to execute necroptosis and pyroptosis respectively. In our studies, we reported biphasic functions of PIP2 in mediating cytokine induction and necrotic cell death in response to low and high concentration of host defense peptides, particular human β-defensins (HBDs). Using multiple biochemical and cell biological approaches, we was able to show that the interaction of HBD-3 with PIP2 is important for receptor-independent PI3K-Akt-NF-κΒ-mediated cytokine induction at its sub-cytotoxic level. Higher concentration of HBD-3 also binds to PIP2, however leading to membrane blebbing and acute cell permeabilisation in mammalian cells. Furthermore, in our most recent study, it was found that PIP2, via a different mechanism, mediates pathogenic fungal cell killing by HBD-2, a closely-related defensin, by promoting a necrotic HBD-2:PIP2 oligomeric complex. Our findings highlight the critical roles of PIPs, particularly PIP2, in (i) orchestrating various cellular processes including a novel role in immune response and (ii) mediating cell death upon in different scenarios strategically targeting both host and pathogen cells.

INFLAMMASOME SIGNALLING IN THE HOST DEFENSE AGAINST INFECTIOUS DISEASE

Man S. Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia.

Recognition of pathogens by the host cell is of paramount importance for the initiation of an immune response and clearance of the pathogen. Intracellular and cytosolic bacteria must secure entry into the host cytoplasm in order to engage activation of cytosolic immune sensors and the inflammasome. However, microbial ligands from extracellular pathogens are also detected by cytosolic innate immune sensors. We discuss our latest findings on the role of inflammasomes and innate immune regulatory proteins in the recognition of bacteria. We also highlight novel anti-microbial host defense systems mediating cytosolic release of bacterial ligands for sensing by pattern-recognition receptors and inflammasomes.


SymPoSia TUESDay

SYM-42-01 SYM-42-02

SYM-42-03 SYM-42-04

MAGNIFYING THE CAREER PATH AFTER PHD

Le S. Olympus Australia and New Zealand.

Upon receiving my PhD, I dived into my postdoc life. Driven by my passion about microscopy and uncontrolled external factors, I joined Olympus to pursue a career outside academia. I am passionate about leveraging my expertise and company resources to provide the best solution of advanced microscopy to support research in life science. A career path of a PhD graduate is always deemed narrow, which is completely untrue. I would like to discuss the necessary preparation, practical steps and strategies that worked for me to transition from academic role into a position in life science related industry.

SPINNING OUT: TALES FROM A UNIVERSITY START UP

Burke C. School of Life Sciences, University of Technology Sydney, Broadway, NSW 2007.

I will talk about my experience as a founding member of UTS spin-out company Longas. From initial experiments as a post doctoral scientist to patents and board meetings, working with a spin-out has been an exciting, stressful, illuminating but overall positive experience. I have learned many lessons along the way about project management, strategy and the importance of maintaining a good team.

NAVIGATING THE IMPACT OF CAREER DISRUPTION

Caldon E. Garvan Institute of Medical Research.

Dr Liz Caldon is a group leader at the Garvan Institute, studying genomic instability and drug resistance in cancer. Over the last 6 years she has combined a 3 year career disruption with establishing her own research group. Our current system in academia provides support for career disruptions, but this does not always bridge important gaps which arise when combining career disruption with career progression. Liz will discuss strategies to minimise the long-term disruption to career, drawing on her personal experiences.

DOES AN EXPERIENCE IN THE PRIVATE SECTOR REALLY MAKE YOUR CAREER “NON-ACADEMIC”?

Delerue F. Macquarie University, Sydney, NSW.

In 2015 in the U.S., only 42% of people awarded a PhD in Science worked in Academia. Despite the competitiveness of the academic world and its constant decrease in grant funding, the so-called “non-traditional” career might end up being a good alternative to help researchers navigate their academic journey. Based on a personal experience, this presentation details how working in a private industry can help academics achieve their goals by managing the interface between business and science.

SYM-42-05SCIENCE TO COMMERCIALISATION — OH THE PLACES YOU’LL GO!

Chapman N. gemaker Pty Ltd, Sydney, NSW 2000.

Natalie is Co-Founder and Managing Director of gemaker, a science and technology commercialisation consultancy which provides expert advice, services and training to research organisations and innovative businesses. Natalie is also Corporate Communications Manager for ASX-listed Alkane Resources, a Director of the Commercialisation Studies Centre (CSC) and sits on the External Advisory boards for UNSW School of Chemistry and the UOW Faculty of Business. Her previous work, as General Manager of Commercialisation at the Smart Services CRC, resulted in two spin-off businesses. As Leader of Business Development and Marketing at ANSTO, she provided strategic advice and managed the commercialisation of the intellectual property portfolio, including incubating new businesses and growing technical consultancy services. In 2017, gemaker won three Asia-Pacific Stevie Awards for Innovation, and the NSW Telstra Business Award for Microbusiness and Natalie was a finalist in the Telstra Business Women’s Awards. In 2018 Natalie launched the AUSinnovates initiative to celebrate Australian technology commercialisation success and bring attention to Australian researchers and innovators who are changing our lives for the better, by creating new industries and jobs, enhancing our health and education and making the world safer.


SymPoSia TUESDay

SYM-43-01 SYM-43-02

SYM-43-03 SYM-43-04

DEFINING INITIAL MOLECULAR MECHANISMS OF HUMAN CATARACT FORMATION USING LIGHT-FOCUSING MICRO-LENSES

Umala Dewi C.1, Kabir M.D.H.1, Murphy P.1, Ho J.2, 3 and O’Connor M.D.1, 4 1Western Sydney University, Campbelltown, Australia. 2Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia. 3St. Vincent’s Clinical School, University of New South Wales, Sydney, NSW, Australia. 4Medical Sciences Research Group, Western Sydney University, Campbelltown, NSW, Australia.

Cataract, caused by opacification of the eye’s lens, affects over 65 million people worldwide. Surgery is the only effective treatment, however, there is great interest in anti-cataract drug development due to: i) the large number of cataract operations performed annually (millions); ii) the cost of these surgeries (billions of dollars); iii) the fact that, despite these surgeries, the number of people affected by cataract is increasing (50.5 million in 1990 to 65.2 million in 2015); and iv) the relatively high incidence of vision-impairing complications that arise from cataract surgery (such as posterior capsule opacification). Environmental risk factors have been associated with cataract formation. The molecular pathologies initiated by different risk factors are likely to be different, yet are poorly understood due to the inability to access human lenses during cataract initialization. To address this, we have established methods for large-scale production of human lens epithelial cells and light-focusing micro-lenses from pluripotent cells. Extensive characterization of these human lens cells and micro-lenses revealed significant functional, morphological and molecular similarities to primary human lenses. An initial study demonstrated the micro-lenses can be used to study clinically-relevant cataract, with assessment of transparency and focusing ability new and quantifiable readouts of cataract formation. Our recent studies suggest other environmental factors can be investigated using human micro-lenses. The micro-lens system also offers an opportunity to re-assess drugs that failed pre-clinical development due to an association with cataract formation in animals.

MOUSE AND HUMAN MICROGLIAL PHENOTYPES IN ALzHEIMER’S DISEASE ARE CONTROLLED BY PLAQUE PHAGOCYTOSIS THROUGH HIF1α

Grubman A.1,*, Choo X.Y.1,*, Chew G.2,*, Ouyang J.F.2, Sun, G.1, Croft N.P.1, Rossello F.1, Simmons R.3, Buckberry S.3, Vargas Landin D.3, Pflueger J.3, Vandekolk T.H.1, Abay Z.1, Chan J.1, Haynes J.1, Williams S.1, Chai S.1, Wilson T.4, Lister R.3, Pouton C.W.1, Purcell A.1, Rackham O.2, Petretto E.2 and Polo J.M.11Monash University, Clayton, Australia. 2Duke NUS, Singapore. 3University of Western Australia, Perth, Australia. 4MHTP Medical Genomics Facility, Clayton Australia. *Equal contribution.

Microglia are brain immune cells that remove cellular and extracellular debris and regulate synaptic plasticity, maturation and removal. Recently altered microglial genomics, epigenomics and functions emerged as key contributors to Alzheimer disease (AD). Nonetheless, whether toxic microglial inflammatory cytokine secretion and aberrant synapse overpruning outweigh the beneficial amyloid clearance functions of microglia in AD remains highly controversial. To address these questions, we explored whether functional differences in amyloid plaque phagocytosis in a plaque-depositing AD mouse result from or contribute to the underlying molecular and functional diversity of microglia in AD. Using a combination of bulk and single cell RNA-seq, and proteomics approaches, we showed that the amyloid plaque phagocytic subset of microglia are molecularly distinct from physiological microglia and from non-plaque containing microglia in AD brains. For instance, several later onset AD risk factors and their direct interacting partners are differentially expressed in plaque-containing microglia. We are now using stem cell derived microglia like cells to manipulate the signaling pathways involved in generating the plaque-associated microglial signature.

HUMAN PLURIPOTENT STEM CELL MODELS OF HEART DEVELOPMENT AND DISEASE

Elliott D.A. Murdoch Childrens Research Institute.

Congenital heart disease is the most common form of birth defect, with a prevalence approaching 1 in 100 children. Although the etiologies underlying congenital heart disease and cardiovascular disease differ, the development of new treatments for either condition will be critically dependent on a detailed understanding of how the human heart is formed and how it functions at the cellular and molecular level. Human pluripotent stem cell (hPSC) derived cardiomyocytes are the only tractable platform for illuminating the fine detail of the genetic networks that control human cardiomyocyte cell biology. We have developed a cellular framework to investigate the genetic regulation of human cardiac cell lineage specification. We are now utilizing these reagents and technologies to study congenital heart disease using differentiating hPSCs. In particular, we are examining the role of the important cardiac transcription factor NKX2-5 to determine the molecular mechanisms underlying congenital heart disease in individuals with NKX2-5 mutations. We have shown that NKX2-5 is essential for the activation of the ventricular cardiomyocyte commitment marker VCAM1 and for the coordinated contraction of hPSC derived cardiac monolayers. Through RNA-seq and ChIP-seq HEY2 was identified as a downstream mediator of NKX2-5. HEY2 was able to restore both VCAM1 expression and contractile synchronicity to NKX2-5 deficient cardiomyocytes. Thus, NKX2-5 and HEY2 are components of the genetic network controlling human ventricular cardiomyocyte differentiation.

REGULATION OF THE SKELETAL MUSCLE STEM CELL NICHE

Tajbakhsh S. Developmental & Stem Cell Biology, CNRS UMR 3738, Institut Pasteur, Paris.

The microenvironment is critical for the maintenance of stem cell populations, and it can be of cellular and non-cellular nature, including secreted growth factors and extracellular matrix (ECM) as well as intrinsic regulators. Skeletal muscle satellite (stem) cells are quiescent during homeostasis and they are mobilised to restore tissue function after muscle injury. Although certain signalling pathways that regulate quiescence have been identified, the mechanisms by which niche molecules regulate stem cell properties remain largely unknown. We have identified Notch signalling as a major regulator of the muscle stem cell niche. Specifically, Notch/RBPJ-bound regulatory elements are located adjacent to specific collagen genes in adult muscle satellite cells. These molecules are linked to the ECM and constitute putative niche components. Notably, satellite cell-produced collagen V (COLV) is a critical component of the quiescent niche, as conditional deletion of Col5a1 leads to anomalous cell cycle entry and differentiation of satellite cells. Strikingly, COLV, but not collagen I and VI, specifically regulated quiescence through Calcitonin receptor mediated activity, therefore, a Notch/COLV signalling cascade cell-autonomously maintains the stem cell quiescent state, and raises the possibility of a similar reciprocal mechanism acting in diverse stem cell populations. This novel mechanism of stem cell niche regulation implicates a reciprocal mechanism were a mechanotransduction ECM protein acts as a signalling molecule for cell autonomous regulation of stem cell quiescence. Notch signalling also acts in an intrinsic manner to regulate a microRNA pathway to modulate cell migration and stem cell quiescence. These findings point to two distinct modulatory mechanisms for maintaining stem cell and niche stability.


SymPoSia TUESDay

SYM-43-05 SYM-44-01

SYM-44-02 SYM-44-03

ADVANCES IN KIDNEY ORGANOID GENERATION; INSIGHTS GAINED THROUGH NOVEL HUMAN IPS CELL REPORTER LINE APPROACHES AND GENE EXPRESSION PROFILING

Vanslambrouck J.M.1, Howden S.E.1, 2, Wilson S.1, Tan K.S.1, Soo J.1 and Little M.H.1, 2 1Murdoch Children’s Research Institute, Parkville, Melbourne, 3052. 2Department of Pediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, 3052, Australia.

Development of directed differentiation techniques and stem cell-derived organoid culture methods are revolutionising the fields of regenerative medicine, disease modelling and drug screening. Based on our knowledge of human kidney organogenesis, we recently established a robust protocol to direct the differentiation of human pluripotent stem cells towards a kidney fate within complex, 3D kidney organoids (Takasato et al., 2014, 2016). These organoids display evidence of differentiation into many of the compartments present in early embryonic kidney, including nephrons, collecting duct, vasculature and interstitium. However, despite these impressive similarities, the structures within kidney organoids are still developmentally immature and they lack the continuous growth pattern seen in embryonic kidneys in vivo. Whilst we know that successful differentiation of pluripotent stem cells towards kidney progenitors and subsequent nephron patterning are critically reliant on the culture media and growth conditions, progress in this area is dependent on our ability to interrogate individual cell types and modify organoid conditions accordingly. Using CRISPR/Cas9-mediated gene-editing, we have generated a range of induced pluripotent stem cell lines carrying fluorescent reporters targeted to genes that mark specific kidney cell types and nephron segments, including nephron progenitors, proximal and distal nephron and collecting duct, enabling real-time monitoring of differentiation. Through the use of these reporter lines, in combination with detailed gene expression analyses, we have now been able to examine organoid development more deeply than ever before, enabling the perturbation of culture conditions to improve organoid growth and nephron patterning.

COMPUTATIONAL STUDIES OF IMMUNE STIMULATING COMPLEX (ISCOM) STRUCTURE

Chalmers D.K. Monash Institute of Pharmaceutical Sciences, Monash University.

Immune stimulating complexes (ISCOMs) are lipid nanoparticles mixtures of cholesterol, phospholipid, protein and saponin from the bark of Quillaja Saponiaria (the Chilean soap bark tree). ISCOMS, and the simpler ISCOM matrix (IMX) which lack protein, are complex systems with intriguing physical properties and biological properties, including applications as vaccine adjuvants. ISCOM and IMX particles consist of supramolecular structures, which appear from electron microscopy, to be ‘holey’ spheres with a diameter of approximately 30 nm, although the precise structure of the IMX particles and the processes driving their formation are currently not well understood. We have used large-scale molecular dynamics simulations to model the formation of IMX particles and have developed models that are consistent with the experimental information available. Our models raise the prospect for the design of new, simpler saponin analogues for the development of biologically active lipid particles.

THE EFFECT OF H3O+ ON MEMBRANE STRUCTURE AND HYDROGEN BONDING IN PHOSPHOLIPID BILAYERS

Deplazes E.1, Cranfield G.C.2, Sarami F.3, Poger D.4 and Cornell B.5, 2 1Curtin University, Perth, Australia. 2University of Technology Sydney, Sydney, Australia. 3University of Western Australia, Perth, Australia. 4University of Queensland, Brisbane, Australia. 5SDx Tethered Membranes Pty. Ltd., Sydney, Australia.

The cell membrane is critical for cells to adapt to changes in pH yet we know little about the molecular mechanisms of how hydronium ions (H3O

+) affect the structure of phospholipid membranes. A recent study showed changes membrane conduction and structure as a function of H3O

+ concentration. To gain molecular level insight into this effect we carried out μs-long unrestrained MD simulations of phospholipid bilayers in the presence of 10 mM and 100 mM H3O

+ and compared them to the same system in the absence of H3O

+. Results show that in the presence of H3O

+ the membrane undergoes a significant increase in bilayer thickness accompanied by a significant decrease in the area per lipid, in agreement with experimental data. Analysis of the density profiles shows that the H3O

+ ions accumulate close to the hydrophobic core of the membrane where they displace water and form hydrogen bonds with the carbonyl and phosphate oxygens of the lipids. These hydrogen bonds are, on average, shorter and longer-lived than hydrogen bonds with water molecules. Thus, our simulations confirm the hypothesis that H3O

+disrupts hydrogen bonding between phospholipids and that this is likely the cause of the reduced area per lipid. In addition, the layer of constrained water at the water-lipid interface known to influence membrane morphology and structure as well as proton transport appears to increase in the presence H3O

+.

UNDERSTANDING THE DYNAMIC SUBSTRATE BINDING MECHANISM OF HUMAN HEPARANASE

Ahmed F.H.1, 3, Marsavelski A.1, 2, Mohamed A.E.1, Jamieson E.1, Correy G.1 and Jackson C.J.1 1The Australian National University, Research School of Chemistry, Sullivan’s Creek Road, Acton, ACT 2601, Australia. 2Department of Chemistry, Faculty of Science, University of Zagreb, Zagreb, Croatia. 3Current address: CSIRO, Land and Water, Clunies Ross Street, Acton, ACT 2601, Australia.

Heparanase (HPSE) hydrolyzes heparan sulfate (HS) in the extracellular matrix. It mediates the release of regulatory molecules bound to HS, and hence is essential for processes that control cell growth, coagulation and inflammation. Inhibition of the increased HPSE activity observed in a variety of pathological conditions such as tumour metastasis, angiogenesis and auto-immune diseases attenuates disease progression, making HPSE a promising drug target. Although crystal structures of HSPE are now available, the dynamic nature of its interaction with its flexible polysaccharide substrate HS is not fully understood. We have explored the ligand binding mechanism of HPSE by analysing its conformational landscape with molecular dynamics (MD)-simulations and multi-conformer models generated by crystallographic ensemble refinement. We found that HS binding is a complex process, whereby the molecule is stabilized at the active site for catalysis, but the outer regions of the enzyme:substrate complex are dominated by ionic interactions and exhibit substantial conformational flexibility. This dynamic binding mechanism allows HSPE to retain high affinity while minimizing the entropic costs associated with binding highly flexible molecules. These findings have direct implications on efforts in identifying and optimising inhibitors for this promising drug target.


SymPoSia TUESDay

SYM-44-04 SYM-44-05

SYM-45-01 SYM-45-02

DYNAMIN STUCTURE AND FUNCTION HINGING ON RYNGOS

Cardoso D.1, Abdel-Hamid M.2, McCluskey A.2 and Robinson P.1 1Cell Signalling Unit, Children’s Medical Research Institute, University of Sydney, NSW, Australia. 2School of Environmental and Life Sciences, Faculty of Science, University of Newcastle, NSW, Australia.

Dynamins are GTPase enzymes responsible for performing the final scission of invaginated plasma membrane prior to completion of endocytosis. Pharmacological targeting in relevant mouse models has been shown to provide therapeutic relief for ailments as diverse as chronic kidney disease and epilepsy. We have generated a series of small molecule modulators (Ryngos) which ‘lock’ dynamin into a ‘ring’ oligomer that structurally differs from the ‘helical’ state required for endocytosis. Ryngos exhibit different actions on enzyme activity in vitro (Ryngo-1, mixed-mode; Ryngo-3, stimulation). Due to their chemical similarity, it can be surmised that these compounds share a common binding pocket. This study aims to establish the binding site of Ryngos to allow for targeted drug design. Advanced computer modelling predicted lead compounds; Ryngo-1-23 and Ryngo-3-32, independently localised to, and differentially interacted with Hinge 1, located between middle domain and bundle-signalling element of dynamin. Partial overlap of residues between Ryngo-1-23 and Ryngo-3-32 suggests drug binding to different sub-regions of Hinge 1 may be capable of imparting different actions (inhibition/stimulation) on dynamin activity. To validate this, mutagenesis of Hinge 1 residues was undertaken and mutants characterised. Functional assays largely support these predictions (i.e. single mutations selectively lost drug action) whilst highlighting a broader role for Hinge 1 in dynamin characteristics (activity, oligomerisation). To account for allosteric effects of mutation, a chemically dissimilar dynamin inhibitor (Dynole-34-2) revealed loss of Ryngo action to be specific to Hinge 1. The data supports the model of these compounds differentially interacting with a flexible hinge within dynamin, an exceptionally rare binding site in pharmacology.

SUPER-RESOLUTION IMAGING OF SUBCELLULAR REMODELLING BY VIRAL PROTEINS

Rozario A.M.1, Zwettler F.2, Rawlinson S.3, Brice A.3, Sauer M.2, Whelan D.1, Moseley G.3 and Bell T.D.M.1 1School of Chemistry, Monash University, Clayton, Victoria. 2Department of Biotechnology and Biophysics, University of Wuerzburg, Bavaria, Germany. 3School of Biomedical Sciences, Monash University, Clayton, Victoria.

Viruses are microscopic infectious agents capable of evading immune responses and causing fatal human disease. Investigating viral mechanisms and viral-host interactions using light microscopy is limited due to diffraction of light (~250 nm). Super-resolution fluorescence microscopy (SRFM) can achieve resolutions as good as 20 nm, making possible observation of nanoscale changes in virally altered cell structures. Lyssavirus phosphoproteins (P1-P5) interact with STAT1 to antagonize interferon-mediated antiviral responses. Previously, we observed rabies lyssavirus (RABV) P3 bind onto microtubules (MTs) and induce bundling. Mutations to P3 diminish MT bundling, correlating with improved interferon response and reduced lethality in mice models (Brice et al., Sci Rep. 2016, doi: 10.1038/srep33493). However, the precise role of MT bundling in lyssavirus disease progression is still unclear. Here we report on using SRFM to analyse bundling effects of P3 from other lyssaviruses, finding significant divergence in MT interactions between the related pathogens. To elucidate bundle structure, we have developed assays for expansion microscopy (ExM), a method that physically enlarges samples ~4-fold for improved imaging resolution, potentially down to <10 nm. Additionally, we have investigated pathogenic henipavirus matrix protein (HeV M) which localises in subnucleolar puncta and binds Treacle, a protein involved in DNA-damage response (DDR) machinery. We have imaged for the first time using SRFM subnucleolar Treacle puncta (100-200 nm) and effects thereon of HeV M, and show that M protein subverts Treacle and supresses rRNA synthesis to a similar extent as during a DDR (Rawlinson et al., doi: https://doi.org/10.1101/219071).

THE STRUCTURAL BASIS FOR SELECTIVE METAL ION IMPORT

McDevitt C.A. Research Centre for Infectious Diseases, The University of Adelaide.

Bacterial infection involves a constant tug-of-war between host and pathogen for the essential nutrients of life. Nevertheless, how bacteria selectively acquire the essential first-row transition metal ion manganese from the host environment remains poorly understood. Here, we investigated the manganese importing ATP-binding cassette (ABC) transporter PsaBCA of Streptococcus pneumoniae, the foremost human bacterial pathogen. By combining molecular microbiological, biochemical, biophysical and structural approaches we show that specificity is achieved not by selective binding of manganese, but by the inability of ions other than manganese to be released into the transporter. Our data show that the metal-recruiting component of the pathway, PsaA, is not restricted to binding manganese and is highly permissive for interaction with any divalent first-row transition metal ion. However, biochemical assays and single molecule FRET studies show that zinc and copper ions, which are highly abundant during infection, result in the formation of PsaA-metal complexes incapable of releasing these non-cognate metal ions. Further, these PsaA-metal complexes are incapable of stimulating ATP hydrolysis in proteoliposome-reconstituted PsaBC. In contrast, manganese can be readily released from PsaA-metal complexes, and manganese-bound PsaA stimulates ATP hydrolysis in proteoliposome-reconstituted PsaBC. Collectively, these findings provide a structural basis for how manganese ions are selectively imported by the PsaBCA importer.

EXPLORING THE PHYSIOLOGICAL SUBSTRATES OF THE PROTOTYPICAL PACE FAMILY EFFLUX PUMP ACEI

Hassan K.A.1, 2, 3, Naidu V.2, Liu Q.2, Edgerton J.3, Fahmy L.3, Li L.2, Mettrick K.A.1, Jackson S.M.3, Ahmad I.3, Sharples D.3, Henderson P.J.F.3 and Paulsen I.T.2 1School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW, Australia. 2Department of Molecular Sciences, Macquarie University, North Ryde, NSW, Australia. 3School of BioMedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.

Resistance to antimicrobials is one of the most pressing health issues of our time. Multidrug efflux pumps have gained notoriety as a major and highly promiscuous class of drug resistance determinants that contribute to the failure of antibiotic therapy and promote the persistence of pathogens in hospitals. Despite their widely-studied roles in drug resistance, for many multidrug efflux pumps drug transport is likely to be a fortuitous side reaction made possible by flexible substrate binding sites that have become beneficial to host organisms living under highly drug selective conditions in hospitals. The core functions of these pumps are likely to be linked to the physiology of the organism and the environments in which they evolved. This is almost certainly true for the AceI transport protein, the prototype for the novel PACE family of efflux pumps. The gene encoding AceI is conserved across all Acinetobacter baumannii strains to have had their genomes sequenced, indicating an ancient origin and long term pressure for gene maintenance. Paradoxically, its only characterised substrate is chlorhexidine, which, although widely used as an antiseptic today, is purely synthetic and has been produced only since last century. In this talk I will describe our progress in deciphering the core physiological functions of the AceI protein, as well as its mode of energisation.


SymPoSia TUESDay

SYM-45-03 SYM-45-04

SYM-45-05 SYM-46-01

PROGESTERONE RECEPTOR MEMBRANE COMPONENT 1: A CONSERVED EUKARYOTIC PROTEIN WITH MULTIPLE AND STRATIFIED DISEASE-RELEVANT FUNCTIONS IN CELL AND ORGANISMAL BIOLOGYThejer B.M.1, 2, Teakel S.L.1, Marama M.1, Fang J.1, Gurusinghe S.3, Quinn J.C.3, Weston P.A.3, Weston L.A.3, Forwood J.K.1and Cahill M.A.1 1School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW 2678, Australia. 2Department of Biology, College of Science, University of Wasit, Wasit, Iraq. 3Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga Wagga, NSW 2678, Australia.

PGRMC1 is the archetypical member of the Membrane Associated Progesterone Receptor (MAPR) family of cytochrome b5 (cytb5) domain proteins, of which animals have three gene subfamilies: pgrc, nenf (Neudesin) and neufc (Neuferricin). We show that all three MAPR genes were present in the ancestor of Opisthokonts: the eukaryotic clade including yeasts and animals. MAPR proteins are more closely related to archaebacterial than to alpha-proteobacterial cytb5 proteins. Eukaryotic cytb5 is related to alpha-proteobacterial genes. Lanosterol is the first sterol produced from squalene cyclisation. PGRMC1-like proteins regulate CyP51A1 lanosterol 14-alpha demethylase enzymes from yeast to humans. Both squalene cyclase and CyP51A1 are of bacterial origin, whereas PGRMC1 is of archaebacterial origin. Cholesterol dramatically affects the properties of mitochondrial membranes, and the first step of animal steroid hormone synthesis takes place there, where cholesterol is modified to the first progestogen, pregnenolone. PGRMC1 confers progestogen-responsiveness to cells, and is also a member of the Insig/SCAP complex which regulates SREBP1 to induce genes associated with fatty-acid catabolism, and SREBP2 to activate the mevalonate pathway leading to sterol production. This suggests a model where PGRMC1 function may reflect the regulatory interplay between the proto-eukaryotic archaebacterial host cell and its proto-mitochondrial bacterial endosymbiont at the root of eukaryotic evolution. PGRMC1 also acquired tyrosine phosphorylation sites during animal evolution concurrently with the appearance of striated muscle and nerve synapses, and before bilateral body plan. PGRMC1 phosphorylation differences exist between cancers, and it is tyrosine phosphorylated in post-synaptic densities. Migration of embryonic nerve cord axons requires PGRMC1 from nematodes to mammals, and PGRMC1 synaptic function is required for the synaptorestorative effects of ElaytaTM, a promising potential Alzheimers drug. Our data provide exciting new insights into PGRMC1 function in animal and disease biology.

MEMBERS OF THE CHLORIDE INTRACELLULAR ION CHANNEL PROTEIN FAMILY DEMONSTRATE CATALYSE PROTEIN DEGLUTATHIONYLATION

Ali H.A.1, Hossain K.R.1, D’Amario C.1, Jiang L.1 and Valenzuela S.M.1, 2 1School of Life Sciences, University of Technology Sydney, Sydney, NSW 2007, Australia,. 2Centre for Health Technologies, University of Technology Sydney, Sydney, NSW 2007Australia.

The chloride intracellular ion channel (CLICs) proteins are atypical anion selective channel proteins, with some members also known to have enzymatic activity. Human CLIC proteins found in most tissues and cells. (1).We have recently shown that members of CLIC proteins have intrinsic enzymatic activity (2, 3). Also, we have evidence that the expression of CLIC proteins by bacterial cells could provide increased tolerance to oxidative stress (unpublished study). Our current study demonstrates for the first time that CLICs possesses a significant deglutathionylation activity with a model peptide substrate. CLIC1 dependent glutathionylation of cellular proteins can also detect in cultured cell lines. In contrast, our study found that the expression of CLIC1 in cultured CHO-K1 cell lines promotes the rapid glutathionylation of cellular proteins. The deglutathionylation activity was determined via an in vitro assay using a glutathionylated model synthetic peptide as a substrate. Deglutathionylation of CLIC proteins was assessed using a real-time fluorescence-based method. CLIC proteins were found to have a significant deglutathionylation activity. Mutating the active site cysteine residue in CLICs largely eliminated their deglutathionylation activity. Furthermore, mutating one of the only two charged residues: arginine 29 (R29A) or lysine 37 (K37A) in the putative transmembrane region of CLIC1 resulted in a reduction of its deglutathionylation activity. The discovery that CLIC proteins participate in the glutathionylation cycle and targets specific proteins could explain the association of CLICs with a diverse range of clinical disorders and provide a novel therapeutic target. REFERENCES 1. Valenzuela, S. M. etal.,(1997). Molecular cloning and expression of a chloride ion channel of cell nuclei.J Biol Chem 272, 12575-12582. 2. Al Khamici, H. etal., (2015). Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity.PLoS One 10, e115699. 3. Juan R. Hernandez-Fernaud etal, (2017).Secreted CLIC3 drives cancer progression through its glutathione- dependent oxidoreductase activity. Received 11 Aug 2016. Accepted 6 Dec 2016. Published 15 Feb 2017. http://www.nature.com/naturecommunications.

HOW DOES P-GLYCOPROTEIN BIND SO MANY DRUGS?

Callaghan R. Division of Biomedical Science & Biochemistry, Research School of Biology & Medical School, Australian National University, Canberra 0200, ACT.

P-glycoprotein (P-gp) gained notoriety for its role in conferring drug resistance to an astonishing number of cytotoxic drugs used in oncology. The protein is able to confer resistance by preventing sufficient accumulation of chemotherapy drugs within cancer cells. P-gp is one of three multidrug efflux pumps in humans and their actions have been the focus of considerable research. Recently, an x-ray crystallography based structure has been presented for P-gp and has been touted as the “missing piece in the puzzle” to generate a mechanistic understanding of this protein. Has this been achieved yet and is there any benefit to further biochemical studies on P-gp? Our continuing research focus is to provide a dynamic understanding of the drug translocation process of P-gp. In particular, we aim to locate the drug binding site(s) on the protein and describe their communication with the energy providing domains. This information will be used to describe the precise steps involved in multidrug transport at a molecular level.

CHROMATIN INTERACTOME MAPPING IDENTIFIES TARGET GENES AT BREAST CANCER RISK SIGNALS

Sivakumaran H., Beesley J., Marjaneh M.M., Chenevix-Trench G., French J.D. and Edwards S.L. Cancer Division, QIMR Berghofer Medical Research Institute, Brisbane, Australia.

Genome-wide association studies (GWAS) for breast cancer have identified 196 independent signals associated with increased risk. The majority of risk-associated variants within these signals fall in regulatory sequences, such as enhancers, that control gene expression. We perform in situ Capture Hi-C using a high-resolution Variant Capture array (VCHi-C), which includes probes to cover all fine-mapped candidate causal variants. We apply VCHi-C and Promoter Capture Hi-C (PCHi-C) to link risk variants to their target genes in six human mammary epithelial and breast cancer cell lines. We use the CHiCAGO pipeline to assign confidence scores, apply a strict threshold, and identify between 10-27,000 interactions per cell type. Hierarchical clustering of interaction scores stratifies cell lines by estrogen receptor status. Global analysis of promoter-interacting regions (PIRs) shows strong enrichment for cell-type specific accessible chromatin, histone marks for active enhancers and transcription factor binding, supporting the regulatory potential of many PIRs. In total, reciprocally validated CHiCAGO-identified interactions results in 647 candidate target genes. To further prioritise the CHi-C-derived chromatin interactions, we use a recently developed Bayesian framework, to fine-map the direct contacts. Importantly, the combined PCHi-C and VCHi-C contact fine-mapping enables us to prioritize 1832 out of 7375 highly-correlated risk variants and lowers the total number of target genes to 393. One example which makes evident the utility of this dual approach is the 1p22 risk region, where contact fine-mapping decreases the number of risk variants from 34 to 8, and the candidate target genes from 14 to 2. Our results demonstrate the power of combining genetics, computational genomics and molecular studies to streamline the identification of key variants and target genes at GWAS-identified risk regions.


SymPoSia TUESDay

SYM-46-02 SYM-46-03

SYM-46-04 SYM-46-05

SUCCINATE DEHYDROGENASE AND HEREDITARY PARAGANGLIOMA SYNDROMES: LINKING KREBS CYCLE DYSFUNCTION TO CANCERClifton-Bligh R. Kolling Institute University of Sydney and Department of Endocrinology, Royal North Shore Hospital.

Succinate dehydrogenase (SDH) is located on the inner mitochondrial membrane and functions in the mitochondrial respiratory chain and the Krebs cycle. In the respiratory chain, SDH transports electrons to the ubiquinone pool, then to cytochrome c of complex III. In the Krebs cycle, SDH catalyses conversion of succinate to fumarate. Two predictable consequences therefore of SDH inactivation are succinate accumulation, and increased production of reactive oxygen species. Both outcomes have been suggested to contribute to cellular accumulation of hypoxia-inducible factors (HIFs) and tumours associated with SDH deficiency display notable upregulation of hypoxia-responsive genes. Mitochondrial dysfunction due to mutations in genes encoding the subunits of SDH (SDHA-D) leads to adrenal phaeochromocytomas (PCs), sympathetic and parasympathetic paragangliomas (PGLs), renal cell carcinomas (RCCs), gastrointestinal stromal tumours (GISTs), and pituitary tumours. SDHB mutations in particular are associated with metastatic PC/PGLs. We have developed several orthogonal models to test genotype-phenotype correlations of SDHB variants, including immunohistochemistry and metabolomic assays of tumour samples, structural modeling, and in vitro localization and enzymatic assays. Loss of SDHB staining in tumours is a reliable marker for mutations in any of the SDH subunit genes. This is corroborated by measurement of succinate in tumour samples by LC/MS-MS: high succinate measurement relative to fumarate in the tumors represents a direct link to functional aspects associated with SDH-deficiency. Elevated succinate:fumarate ratios are a consistent biomolecular phenotype of SDH-deficient tumors including PC/PGLs, GISTs and RCCs. A homology model for human SDH was developed from a crystallographic structure. Structural modelling showed that many mutations within SDHB are predicted to disrupt the electron path. In vitro assessment by immunoprecipation from transfected cells demonstrated that most SDHB mutations result in impaired mitochondrial localisation and/or SDH enzymatic activity. In conclusion, studying SDH mutations represent fertile ground for understanding the association between Krebs cycle dysfunction and cancer.

REGULATORY SMALL RNA NETWORKS AND BACTERIAL PATHOGENESIS

Tree J.J. School of Biotechnology and Biomolecular Sciences, UNSW, Sydney.

The transcriptomes of bacteria contain hundreds of short regulatory RNAs termed, small RNA (sRNA). These gene regulators control protein expression by modulating protein interactions with messanger RNAs (ribosomes, RNases, and termination factors), and by modulating mRNA structure. Small RNAs often base-pair with target mRNAs through regions of limited complementary making computational prediction of sRNA targets difficult. Recently, we have developed a proximity-dependant ligation technique termed RNase E-CLASH that allows recovery of bacterial sRNA-mRNA interactions occurring in vivo, profiling the sRNA interactome. We have used RNase E-CLASH to capture the sRNA interactome of the bacterial pathogen Enterohaemorrhagic E. coli (EHEC). We find that the major EHEC virulence factor, the Shiga toxin, unexpectedly encodes a regulatory sRNA within the toxin mRNA, that controls the general stress response pathway. RNase-CLASH is applicable to a broad range of bacteria and we demonstrate that the sRNA interactome can be recovered from multi-drug resistant Staphylococcus aureus, identifying novel regulators of antibiotic tolerance.

TINC: A METHOD TO DISSECT TRANSCRIPTIONAL COMPLEXES AT SINGLE LOCUS RESOLUTIONKnaupp A.S.1,2,3, Larcombe M.R.1,2,3, Ford E.4,5, Nguyen T.4,5., Mohenska M.1,2,3, Williams S.M.1,2,3, Firas J.1,2,3, Chen J.1,2,3, Pflueger J.4,5, Liu X.1,2,3, Lim S.M.1,2,3, Wong K.1,2,3, Sun Y.B.Y.1,2,3, Hodgson-Garms M.1,2,3, Holmes M.L.1,2,3, Nefzger C.M.1,2,3, Rossello F.J.1,2,3, Kleifeld O.6, Haigh J.J.3,7, Schittenhelm R.B.6, Lister R.4,5 and Polo J.M.1,2,3

1Department of Anatomy and Developmental Biology, Monash University, VIC, Australia. 2Development and Stem Cells Program, Monash Biomedicine Discovery Institute, VIC, Australia. 3Australian Regenerative Medicine Institute, Monash University, VIC, Australia. 4Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, WA, Australia. 5Harry Perkins Institute of Medical Research, WA, Australia. 6 Monash Biomedical Proteomics Facility, Monash University, VIC, Australia. 7Australian Centre for Blood Diseases, Monash University, VIC, Australia.Being able to determine the molecular composition of protein complexes that assemble at specific regulatory elements to activate or repress gene transcription is essential for a better understanding of how gene expression is controlled in normal and diseased states. We have developed a transcription activator-like effectors (TALE) based method termed TINC (TALE-mediated Isolation of Native Chromatin), which enables the isolation of a specific chromatin region from mammalian cells and consequent identification of associated proteins by mass spectrometry. For proof of concept, we targeted the Nanog proximal promoter in mouse embryonic stem cells and were able to identify transcription factors known to bind to this locus and most importantly novel proteins that play an essential role in pluripotency and reprogramming. As TINC does not require any genetic modification of the target sequence, a target-specific antibody nor high copy numbers of the target sequence, we strongly believe that this method is applicable to any scientific field and has immense potential to change the concept of how we study gene regulation.

DEFINING TISSUE SPECIFIC PROTEIN-PROTEIN INTERACTOMES IN VIVO AND IN DISEASE

Werner H., Chojnowski A., Sobota R., Burke B. and Stewart C.L. Institute of Medical Biology, 8A Biomedical Grove, Immunos, Singapore 138648.

The genomic era identified and defined the composition of the genome. The post-genomic era seeks to understand how the organism’s genome functions in both building and maintaining the organism through the actions of the proteins encoded by the genome. To accomplish these ends, proteins interact extensively with each other resulting in the establishment of complex protein-protein interaction (PPI) networks or interactomes. We are using the BioID technique to describe and decipher PPI to define the LaminA interactome and to understand how different mutations in the LMNA gene result in a range of tissue specific diseases called the laminopathies. BioID involves the fusion of a promiscuous variant of the biotinylation enzyme Bira Ligase (BirA) to the protein of interest. The resulting fusion protein is expressed in vivo in any cell. Proteins within the 10-20nm of the fusion protein are potentially biotinylated, isolated by streptavidin pull down, and identified by MassSpec, establishing them as potential interactors with the protein of interest. We inserted the BirA gene in frame into the N-terminus of the murine Lmna gene in ES cells. From these we derived a mouse line expressing a hybrid LaminA/C-BirA fusion protein that correctly localizes to the nuclear lamina in different tissues. Endogenous levels of biotin within the various murine tissues were sufficient to result in the biotinylation and identification of proteins that are known interactors of the A-type lamins. With these mice, we are defining the tissue specific interactome of the A-type lamins and find that the interactome varies between different tissues. We are also determining how the interactome is altered by lamins carrying specific mutations that result in disease.


SymPoSia TUESDay

SYM-47-01 SYM-47-02

SYM-47-03 SYM-47-04

HARNESSING THE SELF-ASSEMBLY OF PROTEINS FROM DIVERSE ORGANISMS TO BUILD FUNCTIONAL MATERIALS

Glover D. UNSW Sydney, School of Biotechnology and Biomolecular Sciences.

The intricate and ordered complexes that proteins adopt in nature is central to many biological processes, ranging from cellular scaffolding provided by cytoskeletal proteins to the encapsulation of nucleic acids in viral capsids. Exploiting this remarkable fidelity and precision in self-assembly is highly attractive for the fabrication of functional materials with nanometer dimensions. This talk will highlight recent engineering and standardisation of modular protein subunits for self-assembly into geometrically defined templates. The central protein building block in the creation of these templates is the gamma-prefoldin (gPFD), a chaperone filament isolated from a hyperthermophilic archaeon. Redesign of the gPFD subunit interface enabled the creation of two and three-way connectors that can link multiple gPFD filaments into macromolecular structures. These protein templates are now being applied to achieve more complex patterning, while expanding the applicability of modular protein templates to diverse enzymatic systems. Fusing different enzymes to each subunit enables periodic positioning of multiple enzymes along the filament to catalyse sequential reactions and metabolic pathways. In addition, metalloproteins can be aligned at high density along filaments to create conductive nanowires. Ultimately, these strategies will enable the design of smart biomaterials for complex applications that require multifunctionalities, such as drug delivery systems, biosensors, and bioelectronic devices.

CYBERNOSE AND CYBERTONGUE TECHNOLOGIES: A MICROFLUIDIC SENSING PLATFORM

Dacres H.1, Gel M.2, Wang J.1, Caron K.1, Anderson A.1 and Trowell S.1 1CSIRO Health & Biosecurity. 2CSIRO Manufacturing.

Objective quantitative measures of chemical composition are invaluable in many spheres of human activity, from healthcare to industrial processing. A number of very sophisticated analytical methods have been developed, for separating and identifying chemicals, including chromatography and mass spectroscopy. The sensitivity, resolving power and user-friendliness of these technologies are spectacular. However, these existing methods are, in general, expensive, slow and not very portable. They may generate large amounts of data, requiring time and expertise to interpret. Therefore they have not been widely adopted outside research or analytical laboratory environments. To allow chemical analysis to be easily carried out in “real world” situations, there is a need for faster and more portable methods that can be used by non-specialists. Highly evolved chemical senses are critical for the survival of most modern day organisms. We have drawn inspiration from biological systems to develop a sensitive, fast and easy-to-use chemical analyser. The CYBERTONGUE/NOSE biosensing platform can measure a wide range of analytes, continuously and rapidly. The platform’s essential features are that: (1) it uses bioluminescence resonance energy transfer (BRET) as the transduction modality; (2) it incorporates multiple classes of biological recognition elements and, within each class, many variants are possible; (3) it works in the fluid phase of a multiplexed microfluidic system. I will describe the technical basis of the technology and illustrate it with applications in food and health diagnostics.

WHOLE GENOME SEQUENCING IDENTIFIES CLINICALLY ACTIONABLE VARIANTS IN FAMILIES WITH CHD

Alakarage D.1, Ip E.1, Szot J.O.1, Munro J.1, Blue G.M.1, 2, 3, Pachter N.4, 6, Chapman G.1, Winlaw D.S.1, 2, 3, Giannoulatou E.1, 5 and Dunwoodie S.L.1, 5 1Victor Chang Cardiac Research Institute. 2Heart Centre for Children, The Children’s Hospital at Westmead. 3University of Sydney. 4Genetic Services of Western Australia, King Edward Memorial Hospital. 5University of New South Wales. 6University of Western Australia.

Congenital heart disease (CHD) affects up to 1% of live births. However, a genetic diagnosis is not made in most cases. The purpose of this study was to assess the outcomes of whole-genome sequencing (WGS) of a heterogeneous cohort of CHD patients. 97 families, with probands born with CHD requiring surgical correction, were recruited for genome sequencing. At minimum, a proband-parents trio was sequenced per family. WGS data were analyzed via a two-tiered method: application of a high-confidence gene screen (hcCHD), and comprehensive analysis. Identified variants were assessed for pathogenicity using the ACMG-AMP guidelines. Clinically relevant genetic variants in known and emerging CHD genes were identified. The hcCHD screen identified a clinically actionable variant in 22% of families. Subsequent comprehensive analysis identified a clinically actionable variant in an additional 9% of families in genes with recent disease associations. Overall, this two-tiered approach provided a clinically relevant variant for 31% of families. Interrogating WGS data using our two-tiered method allowed identification of variants with high clinical utility in a third of our heterogeneous cohort. However, association of emerging genes with CHD etiology, and development of novel technologies for variant assessment and interpretation will increase diagnostic yield during future reassessment of our WGS data.

YEAST 2.0 AND BEYOND: BUILDING THE WORLD’S FIRST SYNTHETIC EUKARYOTE

Paulsen I.T. and The Australian Team For Yeast 2.0. Macquarie University, Sydney Australia.

Yeast 2.0 is an international consortium aiming to build the world’s first synthetic eukaryote by 2017. Systematic genome wide changes in the synthetic yeast include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, transfer RNAs, transposons, and insertion of loxPsym recombination sites. The Australian Yeast 2.0 team is responsible for the design and synthesis of synthetic versions of chromosomes 14 and 16. Construction is essentially complete at Macquarie, with 100% of the synthetic DNA successfully inserted. Troubleshooting and repairing errors identified through genome sequencing is currently ongoing. One of the most interesting features of Yeast 2.0 is the incorporation of the SCRaMbLE system for generating combinatorial genomic diversity through rearrangements at loxPsym recombination sites. This opens up the possibility of harnessing the SCRaMbLE system for adaptive laboratory evolution experiments. We have developed biosensors that respond to a variety of industrially useful metabolites, and are now seeking to use a combination of SCRaMbLE-ing and flow cytometry to identify strains that can produce higher levels of these metabolites.


SymPoSia TUESDay

SYM-48-01 SYM-49-01

SYM-49-02 SYM-49-03

RELIABLE INTERPRETATION OF WATER-USE EFFICIENCY IN CHICKPEA FROM D13C OF LEAF TISSUE

Barbour M.M. and Lockhart E. The University of Sydney, 380 Werombi Road, Brownlow Hill.

The carbon isotope composition of leaf tissue (δ13C) has been widely used for many years as a proxy for both whole plant water-use efficiency (WUE; biomass produced per unit water transpired) and leaf-intrinsic water-use efficiency (WUEi; photosynthetic rate divided by stomatal conductance), and has been successful in many crop species, notably wheat. However, there is conflicting evidence for the utility of δ13C in chickpea as a WUE proxy. Chickpea are somewhat unusual in having high levels of organic acids excreted by leaf hairs, so we tested the hypothesis that surface organic acids are responsible for the inconsistency in δ13C-WUE relationships either directly or indirectly. Across 20 chickpea genotypes, removal of surface acids altered whole leaf tissue δ13C in 3 genotypes, suggesting that acids may contribute to the lack of consistency in δ13C-WUE relationships between different genotypes. Water-soluble CHO samples were 2‰ more enriched, on average, than whole leaf tissue samples. For 2 chickpea genotypes grown in well-watered and droughted conditions, the strongest correlations were found between δ13C of CHO and WUE. Weaker correlations were found for δ13C of CHO and WUEi, and whole leaf δ13C with either WUE or WUEi. Removal of surface acids and extraction of water-soluble CHO is recommended when using leaf δ13C as a proxy for WUE and WUEi in chickpea. Previous studies describing genotypic variability and heritability of δ13C in chickpea may have identified variation in leaf acid presence, or differences in leaf chemical composition, rather than WUE.

MOLECULAR AND EVOLUTIONARY CONSERVATION OF ABSCISIC ACID AND BLUE LIGHT SIGNALLING IN STOMATAL REGULATION

Cai S.1, 2, Chen G.2, Franks P.3 and Chen z.H.2 1School of Science and Health, Western Sydney University, Locked Bag 1797, Penrith, NSW, 2751, Australia. 2College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China. 3School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia.

Evolutionary trajectories of land plants have led to structurally complex and functionally active stomata for terrestrial life. The unique morphology, development and molecular regulation of stomata enable their rapid environmental response. Growing evidence has shown that signals controlling stomatal opening and closure may have evolved in some species of mosses. Here, comparative genomic study showed that many of abscisic acid (ABA) signalling, photoreceptor, membrane transporter, reactive oxygen species (ROS) and nitric oxide (NO) signalling, and protein kinase gene families are conserved over the evolutionary history of green plants (Viridiplantae). Phylogenetic analysis indicated an evolutionarily conserved stomatal response to ABA. Moreover, comparative transcriptomic analysis has identified a suite of ABA responsive differentially expressed genes encoding proteins associated with ABA biosynthesis, transport, reception, transcription, signalling, and ion and sugar transport, which fit the general ABA signalling pathway constructed from Arabidopsis thaliana and Hordeum vulgare. Furthermore, stomatal assays on epidermal peels showed ABA-induced stomatal closure in two fern species (Polystichum proliferum and Nephrolepis exaltata) and blue light-induced stomatal opening of a wide range of fern species in the Orders of Polypodiales, Schizaeales, Psilotales, and Ophioglossales. Stomata of fern species in the Polypodiales and Schizaeales are more responsive to blue light, generating higher ROS production and H+ pumping to the apoplast as compared to other fern species and Arabidopsis. Understanding the evolution stomatal regulation will inform functional manipulation of water use efficiency for plant productivity and will benefit future efforts towards sustainable food production and ecological diversity.

NON-INVASIVE IMAGING OF HYDRAULIC FUNCTION IN LEAVES, STEMS AND ROOTS

Choat B.1, Peters J.M.R.1, Gauthey A.1, Carins-Murphy M.R.2, Rodriguez-Dominguez C.M.2 and Brodribb T.J.2 1Hawkesbury Institute for the Environment, Western Sydney University, Richmond, NSW 2753, Australia. 2School of Biological Sciences, University of Tasmania, Hobart, TAS, 7001, Australia.

Plants have evolved a water transport system that relies on water sustaining a tensile force. Counter intuitively, this means water moves through the plant as a liquid under negative absolute pressures. This mechanism is made possible by the intricate plumbing system that constitutes the xylem tissue of plants. However, water under tension is prone to cavitation, which results in the formation of a gas bubble (embolism). Embolism reduces the capacity of the xylem tissue to deliver water to the canopy, eventually causing dieback and whole plant mortality. Xylem embolism is exacerbated by environmental stresses and is now considered one of the leading causes of plant mortality resulting from drought stress.Non-invasive imaging techniques offer the potential to make direct observations on intact plants at high resolution and in real time. In this presentation, I discuss recent exciting developments in the application of non-invasive imaging technologies such as X-ray Micro Computed Tomography (microCT) and optical imaging to studies of plant vascular function. This includes visualisation of xylem networks during drought stress and recovery in leaves, stems and roots. MicroCT imaging of stems and roots indicated that significant embolism formation occurs at similar time points and levels of water stress in dehydrating plants. This result was observed in herbaceous and woody species, and is surprising given previous hydraulic measurements indicating that, within a plant, roots were more vulnerable to drought-induced embolism than stems. A newly developed optical technique indicates that leaf vasculature is also similar in vulnerability to stems and roots. The overlap in vulnerability suggests that induction of embolism occurs at the same time in different organs or is propagated rapidly through the plant. In examining recovery from drought stress, we saw little evidence of embolism refilling in the xylem of woody plants, except in cases where substantial root pressure is produced. These results suggest that embolism refilling is less widespread than previously thought.

EARLY CAREER RESEARCHER — GRANT WRITING WORKSHOP

Ittner, L.M. University of New South Wales

Grant Writing Workshop


SymPoSia TUESDay

SYM-49-04 SYM-49-05

SYM-50-01 SYM-50-02

PHOSPHORYLATION OF PLANT PLASMA MEMBRANE AQUAPORINS REGULATES ION CHANNEL FUNCTION

McGaughey S.A. 1, Qiu J. 1, Groszmann M.2, Tyerman S.D1. and Byrt C.S1. 1ARC Centre of Excellence in Plant Energy Biology, School of Agriculture, Food and Wine, The University of Adelaide, SA, Australia, 5006. 2ARC Centre of Excellence for Translational Photosynthesis, Australian National Univeristy.

Phosphorylation on the C-terminal domain (CTD) of Arabidopsis plasma membrane aquaporin AtPIP2;1 may be key to its ion channel function. AtPIP2;1 has previously been identified as a Na+ permeable functional water channel. A series of phospho-mimic mutant AtPIP2;1 proteins were expressed in X. laevis oocytes. Phospho-mimic mutations of two CTD residues of AtPIP2;1 had significantly increased Na+ conductivity compared to WT AtPIP2;1. Interestingly, the apparent water permeability of the phospho-mimic AtPIP2;1 mutants were lower than that of WT AtPIP2;1 when PIP expressing X. laevis oocytes were transferred from isotonic to hypotonic solutions that had equivalent ion concentrations. These results suggest that (i) phosphorylation has an important role in the regulation of ion channel function of ion permeable plant aquaporins and (ii) that there may be an inverse relationship between ion permeability and water permeability of these aquaporins. As plant plasma membrane aquaporins are very important for water uptake and transcellular water flow in plant roots, PIPs with ion channel functions may constitute a mechanism by which Na+ enters plant roots in saline conditions. However the exact physiological role of dual water ion plant aquaporins is yet to be elucidated.

ESTIMATING STOMATAL AND BIOCHEMICAL LIMITATIONS DURING PHOTOSYNTHETIC INDUCTION

Deans R.M., Busch F.A. and Farquhar G.D. Research School of Biology 46 Sullivans Creek Rd The Australian National University Acton ACT.

Ross M. Deans, Florian A. Busch and Graham D. Farquhar Research School of Biology 46 Sullivans Creek Road The Australian National University Acton ACT Understanding what limits photosynthesis under different conditions is important for both the targeted improvement of photosynthesis as well as understanding ecological drivers of photosynthesis. Limitation analyses provide such a method to estimate photosynthetic limitations. Despite the use of limitation analyses to calculate biochemical and diffusional limitations at the leaf level being not new, their utility for estimating limitations during photosynthetic induction is still in its infancy. Moreover, few studies have systematically assessed how well different methods estimate limitations. Timecourses of photosynthetic induction provide a rich dataset in which to test the performance of limitation analyses. We compared how well two general ways of estimating limitations predicted both temporal and overall limitations for photosynthetic induction in response to an increase in irradiance. One method sequentially removes the effect of a limitation (elimination), while the other utilised a tangent plane approximation (differential) to estimate limitations. This talk will discuss under what conditions each method works best and why. This last insight has important implications for the general use of limitation analyses beyond photosynthetic induction.

CILIARY SIGNALLING AND THE EXTRACELLULAR MATRIX

McGlashan S.R.1, Leung S.1 and Choi Y.S.2 1University of Auckland. 2University of Western Australia.

Nearly all cells in the body have a primary cilium, a specialised compartment perfectly evolved to be a cellular probe. Primary cilia are microtubule-based organelles (1-15μm long, 200nm wide) that are formed from the centriolar anchor known as the basal body. Primary cilia act as cellular sensors that receive diverse signals from the extracellular environment including light, growth factors and mechanical stimuli in a tissue-specific manner. The cilium is also separated for the main cell body through a ciliary gate known as the transition zone. Our work has focussed on ECM/ cilia interactions in connective tissues such as articular cartilage and the intervertebral disc. Structurally, chondrocyte cilia are structurally associated with the collagen fibres, they express integrins and are mechanically deflected through ECM interactions. We, and others, have shown that chondrocyte primary cilia mechanosensitive, whereby cilia incidence and length are modulated by compressive or tensile forces, and removal of cilia results in in reduced mechanotransduction, and alterations in cartilage ECM gene expression. Current research focuses on chemical, specific mechanical forces such as fluid flow, or gene cues that control cilia, however, few studies have examined how the mechanics of the local microenvironment influences cilia function. We have examined how cilia length and incidence is influenced by different ECM environments in diseases such in osteoarthritis, intervertebral disc disease and, more recently in model gel systems with controlled stiffness. Our data suggest an intricate relationship between substrate stiffness, F-actin and cilia length and more work is essential to understand the critical role of the mechanical microenvironment on ciliary signalling.

COMPRESSIVE FORCES ACTIVATE RHO/ROCK-MEDIATED CELLULAR PROCESSES CHARACTERISTIC OF DISEASE STATES

Boyle S.T.1, Kular J.1, Nobis M.2, Timpson P.2 and Samuel M.S.1 1Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, SA Australia. 2The Kinghorn Cancer Centre & Garvan Institute of Medical Research & St. Vincent’s Clinical School, Victoria St, Darlinghurst, NSW Australia.

Mechanical forces exerted by the extracellular matrix (ECM) upon cells during homeostatic development are counterbalanced by intracellular forces mediated via mechanotransduction signalling pathways that regulate remodelling and tension of the actomyosin cytoskeleton. This phenomenon is termed mechano-reciprocity. The main regulator of these cytoskeletal dynamics is myosin II, and its regulatory subunit myosin regulatory light chain-2 (MLC2) can be directly activated by the RHO/ROCK signalling pathway. Enhanced extracellular matrix tension in diseased states such as cancer enhances mechanotransduction, and so understanding the phenomenon of mechano-reciprocity and its regulation during homeostasis is key to understanding how these processes become corrupted in disease. We have found that acute compressive force applied to cells and epithelial tissues is able to activate the RHO/ROCK signalling pathway, elevating RHOA-GTP levels and increasing regulatory myosin phosphorylation, actomyosin contractility and tension via ROCK. In consequence, cell proliferation was increased, as was the expression of regulators of epithelial-mesenchymal transition. Pharmacological inhibition of ROCK abrogated myosin phosphorylation, whilst inhibition of either RHO or ROCK reversed the physiological effects of compression on cells. Our results strongly suggest that RHO/ROCK-mediated mechanical signalling during cancer, induced by compressive stress from tumour growth within a constricted space, could play a role in tumour progression.


SymPoSia TUESDay

SYM-50-03 SYM-50-04

SYM-50-05 SYM-51-01

A NEW CLASS OF BIOACTIVE NANOPARTICLES FOR CAPABLE OF SPONTANEOUS, LINKER-FREE MULTIFUNCTIONALISATION

Michael P.L.1, Santos M.1, 2, Hung J.1, Lam Y.T.1, 2, Bilek M.M.2 and Wise S.G.1, 2 1The Heart Research Institute, NSW 2042, Australia. 2The University of Sydney, NSW 2006, Australia.

The large number of proposed nanocarriers share limitations in combining fine control of physical properties, low cytoxicity and simple functionalization in a single platform. Multifunctional carbon-based nanoparticles (nanoP3), manufactured in a custom-built plasma chamber, are a new platform that achieves multiple functionalities in a single, simple step enabling delivery of multiple molecular cargos into cells. Size, roughness, surface charge and chemical composition are readily controlled, giving rise to a versatile nanoparticle platform. Unfunctionalized nanoP3 penetrate a diverse range of cell types including cancer lines, fibroblasts, vascular cells and stem cells. nanoP3 accumulate in the cytoplasm, after rapid endosomal escape with no significant effect on viability or morphology, up to 3.1x108 particles per mm2. The presence of long-lived radicals formed during synthesis facilitates direct covalent immobilization of biomolecules by simple incubation. Binding is rapid with >80% coverage within 30 seconds, and has been demonstrated for a range of molecules including drugs, imaging agents, SiRNA, enzymes and antibodies. The addition of multiple functionalities is achieved by co-incubation in solution. To exemplify one possible utility of nanoP3, we delivered functional small interfering RNA targeted to VEGF in HUVECs, impairing the formation of tubules in an established Matrigel assay. To our knowledge this is the first platform to facilitate co-delivery of multiple surface bound cargos into diverse cell systems following one-step co-incubation without chemical pre-functionalization steps. This approach eliminates the trade-off between additional functionality and complexity and could facilitate the upscaling of nanoparticle-based therapies into the clinic.

FIBROBLAST ACTIVATION PROTEIN IN STEATOSIS

zhang H.E.1, Chowdhury S.1, Hamson E.1, Xiang M.1, Lay A.1 and Gorrell M.1, 2 1Centenary Institute. 2The University of Sydney.

Background & Aim: Better understanding of processes of glucose intolerance, insulin resistance and steatosis in diabetes and fatty liver is needed. Fibroblast activation protein (FAP) has a unique post-proline cleaving activity. Normal FAP expression is very low, but is greatly up-regulated in activated liver mesenchymal cells in human liver cirrhosis. Low level of FAP in human plasma strongly associates with lacking liver fibrosis. We aim to understand metabolic outcomes of specific deficiency of FAP activity in diet-induced obesity (DIO). Methods: Wildtype (WT) and FAPgki (gene-knockin; substituting the catalytic serine) mice were in a DIO model. Proteomics/degradomics was performed to identify novel natural substrates of FAP. Results: FAP activity in pancreas was greater in DIO-WT than chow-fed-WT mice. Compared to DIO-WT mice, DIO-FAPgki mice had less glucose intolerance, insulin resistance, insulin secretion, micro-vesicular steatosis, adiposity and circulating cholesterol. We previously found that FAP gene-knockout mice had increased intrahepatic non-esterified free fatty acids, indicative of increased lipolysis and β-oxidation. Concordantly, lipogenic genes (Pparγ, Gck, Acc, Fasn) and hepatic triglyceride and fatty acid uptake genes (Cd36, Apoc3, Ldlr) were downregulated in FAPgki livers. We identified natural substrates of FAP, such as LOXL-1, CSF-1, CCL-2, C1qT6 and FGF21, pointing to roles of FAP in ECM-cell interactions, metabolism and immunoregulation. The starvation hepatokine FGF21 was of particular interest. FGF21 was increased in FAP deficient DIO compared to WT DIO mice. Thus, these metabolic changes depend upon FAP activity and FAP action on FGF-21 is likely involved. Conclusion: This is the first study showing that specific genetic ablation of FAP activity, which mimics a specific potent inhibitor, is protective of DIO-driven glucose intolerance, insulin resistance and liver steatosis in mice.

CLOSER TO NATURE IN VITRO: ORGAN-SPECIFIC EXTRACELLULAR MATRIX-BASED THREE DIMENSIONAL MODELS OF CANCER

Nadort A.1, 2, Iqbal S.1, 2, Parker L.1, 2, Packer N.1, 2, Goldys E.2, 3 and Guller A.2, 3 1Macquarie University, NSW 2109, Australia. 2ARC Centre of Excellence for Nanoscale BioPhotonics, Australia. 3University of New South Wales, NSW, 2032, Australia.

Successful clinical translation of techniques and therapies to detect and treat cancer needs controlled, ethical and practical lab-based tumour models that more accurately represent the biological reality. We repurposed tissue engineering methodology to create a biochemically and structurally authentic environment for in vitro cell culturing and developed an organ-specific three-dimensional (3D) model of cancer closely simulating real tumour tissue. We obtained acellular organ-specific tissue scaffolds with preserved extracellular matrix composition and structure by original decellularization protocols, followed by seeding and culturing the desired cancer cells to obtain tumour tissue engineering constructs (TECs). We focused on aggressive and problematic cancers such as high-grade brain cancer (glioblastoma mulitforme, GBM) and triple negative breast cancer (TNBC) known for its high rate of hepatic metastasis. Following our protocols, we created brain-TECs of GBM cells (human, U87 and U251) and control undifferentiated neurons (rat, PC12), as well as liver-TECs of TNBC cells (human, MDA-MB-231) to mimic hepatic metastasis. We extensively characterized the tumour TECs and compared the hallmarks of tumour progression, such as the growth dynamics, migration behavior, cell morphology, metastatic colonization, angiogenic potential and drug sensitivity, to 2D cultures and control TECs. Our results show a novel biologically accurate, living 3D tumour model, revealing ECM-specific cellular behavior that enables a more realistic study of tumour biology and therapeutic response. Our organ-specific tumour TECs can be used as a tool to improve the detection and treatment of cancer, and represent a sustainable approach to fill the gap between conventional 2D cell cultures, animal studies and clinical trials.

THE BACTERIAL REPLISOME: DESIGN PRINCIPLES FOR A DYNAMIC MOLECULAR MACHINE

Dixon N.E., Spenkelink L.M., Lewis J.S., Xu X.-Q., Jergic S. and van Oijen A.M. Molecular Horizons and School of Chemistry & Molecular Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia.

The E. coli replisome is a complex and dynamic assembly of more than 20 protein subunits that include the multi-protein Pol III chromosomal replicase and the primosome (helicase/primase). The replisome works to achieve simultaneous copying of both strands at a replication fork at rates that approach 1000 bp/s, with near-perfect fidelity. In the textbook view, leading and lagging strand DNA replication are perfectly coordinated processes that are orchestrated to occur deterministically in discrete steps in space and time. However, there is no evolutionary pressure to achieve such elegance, nor do fundamental chemical principles allow it. I will integrate recent structural and single-molecule biophysical studies that are being used to develop a new picture of replisomal function that is messier than the textbook view. In particular, coupled leading and lagging strand replication assays with fully assembled replisomes containing fluorescent proteins enable protein exchange processes at a replication fork to be imaged in real time at the single-molecule level. We have shown, for example, that Pol III replicase complexes undergo frequent exchange at the fork, on a time scale that depends on their concentration in solution [Lewis et al., 2017, eLife, 6, e23932], while single-stranded DNA-binding protein (SSB) can be retained at the fork through multiple cycles of Okazaki fragment synthesis on the lagging strand (Lisanne Spenkelink, unpublished).


SymPoSia TUESDay

SYM-51-02 SYM-51-03

SYM-51-04 SYM-51-05

FANCM SUPPRESSES ALT ACTIVITY BY MODULATING BLM-TOP3A-RMI COMPLEX ACTIVITY AT TELOMERES

Pickett H.A. Children’s Medical Research Institute.

The collapse of stalled replication forks is one of the major drivers of genomic instability, and cells have evolved committed mechanisms to overcome replication stress. These pathways are particularly pertinent at telomeres, which are long terminal repetitive DNA regions that are intrinsically prone to replication fork slowing, stalling and breakage. Cancer cells that use the Alternative Lengthening of Telomeres (ALT) pathway of telomere maintenance display elevated levels of telomere-specific replication stress, and it is thought that ALT cells utilise these degenerate sites as substrates for break-induced telomere synthesis events. FANCM is a multifunctional protein that is central to the Fanconi anaemia (FA) core complex, and can independently bind to the BLM-TOP3A-RMI (BTR) complex. We demonstrate that FANCM depletion provokes excessive ALT activity, evident by rapid induction of extrachromosomal telomeric repeat (ECTR) DNA, and increased firing of break-induced telomere synthesis events. This culminates in an ALT-specific G2/M arrest and loss of cell viability. The MM2 domain of FANCM, which binds the BTR complex, suppresses this response, suggesting that ALT activity is attenuated by FANCM-BTR-mediated replication fork remodelling, and that collapsed replication forks instigate ALT-mediated telomere synthesis events.

PROTECTING THE GENETIC CODE

Richard D.J.1, Adams M.1, Cubeddu L.2, Gamsjaeger R.2, Bolderson E.1, Leong V.1, Burgess J.1, Pacquet N.1 and O’Byrne K.1 1Queensland University of Technology. 2Western Sydney University.

Single-stranded DNA binding (SSB) proteins play a critical role in DNA replication and repair in all three domains of life. SSB proteins all employ an oligonucleotide/oligosacharide binding (OB) fold in order to bind the single-stranded DNA substrate. Human SSB1 (hSSB1, NABP2), has been demonstrated to play key roles in the DNA damage response, especially in the process of double-stranded DNA breaks (DSBs) repair, at stalled DNA replication forks and in response to oxidative damage to DNA. In this presentation, we look at the role hSSB1 plays in the cellular response to oxidative DNA damage. Further, we will present data outlining the molecular mechanism through which hSSB1 recognises the genetic lesion and recruits the correct repair proteins for the processing of the damage. Lastly, we will present data illustrating the critical role hSSB1 plays in cancer cell biology and preliminary data on the first in class inhibitor of hSSB1. In summary, our presentation will highlight the critical role hSSB1 plays in preventing the loss of genetic information within the human genome, a process that drives cancer initiation and progression.

SINGLE MOLECULE SUPER-RESOLUTION MAPPING OF THE SPATIOTEMPORAL ORGANIzATION OF DNA DOUBLE STRAND BREAK REPAIR

Whelan D.R.1, 2, Lee W.T.C.2, Yin Y.2, Fenyo D.2 and Rothenberg E.2 1La Trobe Institute for Molecule Science, La Trobe University, Edwards Road, Flora Hill, Victoria, Australia, 3552. 2Department of Biochemistry and Molecular Pharmacology, Langone Medical Center, New York University, 550 First Ave, New York, NY, USA, 10016.

DNA damage response (DDR) pathways are involved in both the cause and potential treatment of various cancers, auto-immune, and neurodegenerative diseases. Our current understanding of DDR has been elucidated over the course of several decades by combining biochemical and biophysical techniques, however, imaging of damage and repair in vivo has remained challenging. This has predominantly been because of the dense and varied nature of the nuclear environment, and the diffraction limit of light. Here, we have successfully used single molecule localisation super resolution (SR) imaging to circumvent this limit and capture spatially and temporally resolved snapshots of double strand break (DSB) repair in cells. Moreover, we specifically generated individual single-ended DSBs similar to those endogenously created by collapsing replication forks. The resulting repair foci could be visualized in multicolor SR by labelling nascent DNA via modified base incorporation and click chemistry, DSBs via the TUNEL assay or direct ligation, single stranded DNA via BrdU incorporation, and proteins via immunolabelling. The enhanced spatial and temporal resolutions and the singular nature of the DSBs themselves revealed several exciting and novel insights including the dynamic interactions of proteins such as Ku, MRE11 and RAD51 at the DSB, the redundant role of RAD52 in repair, and a critical in vivo BRCA2 dependence on BRCA1. I will present these findings within the context of their importance to the genomic integrity research community, as well as the broader novelty and applicability of the SR assays we have developed.

HISTONE FLIM-FRET MICROSCOPY REVEALS SPATIOTEMPORAL REGULATION OF CHROMATIN ORGANIzATION BY THE DNA DAMAGE RESPONSE

Lou J.1, Scipioni L.2, Gaus K.3, Gratton E.2, Cesare A.4 and Hinde E.1, 3 1Department of Biochemistry, University of Melbourne, Australia. 2Biomedical Engineering, University of California, Irvine, USA. 3EMBL Australia Node in Single Molecule Science, University of New South Wales, Sydney, Australia. 4Children’s Medical Research Institute, University of Sydney, Australia.

Here we describe a biophysical method to measure chromatin organisation in live cells with nucleosome level resolution. The method is based on a localised phasor image correlation analysis (ICS) of FLIM-FRET microscopy data acquired in human cells co-expressing H2B-eGFP and H2B-mCherry. This multiplexed approach produces spatiotemporal maps of nuclear wide chromatin compaction and quantifies the stability, size and spacing between detected chromatin foci. We used this method in cells where double strand breaks (DSBs) were induced by near-infrared laser micro irradiation to assay chromatin dynamics during the DNA damage response (DDR). These experiments revealed that ATM and RNF8 directed rapid local chromatin decompaction at DSBs, coupled with formation of a stable ring of compact chromatin surrounding the repair locus. Based on these data we built a longevity map of sites with high FRET indicating the time scale of large scale compaction events directed by ATM and RNF8. Then by use of a phasor-based ICS analysis we identified the locations where the DDR shapes local and global chromatin dynamics and demonstrate the utility of phasor ICS-FLIM analysis of histone FRET for the study of chromatin biology.


SymPoSia TUESDay

SYM-52-01 SYM-52-02

SYM-52-03 SYM-52-04

USE OF VOLTA PHASE PLATE SINGLE PARTICLE CRYO-ELECTRON MICROSCOPY FOR DETERMINATION OF ACTIVE STATE G PROTEIN-COUPLED RECEPTOR STRUCTURE

Wootten D., Liang L., Khoshouei M., Glukhova A., Draper Joyce C., Christopoulos A. and Sexton P.M. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria.

Cryo-electron microscopy (cryo-EM) has gained prominence as a method of choice for determination of structure for difficult to crystallise membrane proteins, including active state, transducer complexed G protein-coupled receptors (GPCRs). Class GPCRs bind critically important physiological peptides of 30-40 amino acids, and are important targets for major diseases including diabetes, obesity and osteoporosis. Our laboratory has recently applied Volta Phase Plate (VPP) single particle cryo-EM to determine structures of class GPCRs in complex with their canonical transducers, heterotrimeric Gs proteins. This methodology has been applied to minimally modified receptors, including the human calcitonin receptor, the glucagon-like peptide-1 receptor (GLP-1R), and the calcitonin-gene-related peptide receptor (CGRPR), which consists of the calcitonin-related receptor and a single pass transmembrane protein, receptor-activity modifying protein 1 (RAMP1). These studies reveal common macromolecular changes associated with class B GPCR activation and G protein coupling, receptor specific-differences in peptide hormone binding and critical structural insights into GPCR modulation by RAMPs. Moreover, for the GLP-1R we have solved structures bound to different biased peptide agonists that reveal conformational variances within the receptor that can be linked to distinct efficacy for Gs signalling. More recently, we have solved the structure of the class A adenosine A1 receptor bound to its endogenous agonist adenosine and a Gi2 heterotrimeric protein, revealing novel insights regarding GPCR G protein selectivity. Collectively, this work highlights the power of VPP cryo-EM for GPCR structure determination and the similarities and diversities in modes of receptor activation between members of related subfamilies.

STRUCTURAL BASIS OF TIR-DOMAIN ASSEMBLY FORMATION IN MYD88/MAL-DEPENDENT TLR4 SIGNALING

Ve T.1, 2, Vajjhala P.R.1, Hedger A.1, Croll T.3, Dimaio F.4, Horsefield S.1, Landsberg M.J.1, Stacey K.J.1, Egelman E.H.5 and Kobe B.1 1School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD 4072, Australia. 2Institute for Glycomics, Griffith University, Southport, QLD 4222, Australia. 3Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, England. 4Department of Biochemistry, University of Washington, Seattle, Washington, USA. 5Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA.

Toll-like receptor (TLR) signaling represents a key innate immunity response to pathogen products. Recruitment of signaling adapters such as MAL/TIRAP and MyD88 to the receptors requires TIR-domain interactions, which remain structurally elusive. Here we show that MAL TIR domain spontaneously and reversibly forms filaments in vitro, forms a co-filament with TLR4 TIR domain, and induces formation of a MyD88 assembly. A 7 Å resolution cryo-electron microscopy structure reveals a stable MAL proto-filament consisting of two parallel strands of TIR-domain subunits in a BB loop-mediated head-to-tail arrangement. Residues at the interfaces important for the interaction are conserved among different TIR domains. Although large filaments of TLR4, MAL or MyD88 are unlikely to form during signaling in the cell, structure-guided mutagenesis, combined with in vivo interaction assays, demonstrate that the MAL interactions defined within the filament represent a template for a conserved mode of TIR domain interaction involved in both TLR and IL-1R signaling.

INVESTIGATING THE ARCHITECTURE OF A BACTERIOPHAGE USING CRYO-EM, SAXS, AND X-RAY CRYSTALLOGRAPHY

Hardy J.M.1, Dunstan R.1, Grinter R.1, Pickard D.2, Venugopal H.3, Belousoff M.1, Gordon D.2, Lithgow T.1 and Coulibaly F.1 1Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia. 2Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, United Kingdom. 3Ramaciotti Centre for Cryo-Electron Microscopy, Monash University, Clayton, VIC, Australia.

YSD1 is a lytic bacteriophage that infects and kills Salmonella enterica serovar Typhi, the causative agent of typhoid fever. YSD1 is a Siphoviridae homologous with the bacteriophage chi, which uses a flagella-dependent infection mechanism. We have shown by electron microscopy (EM) that YSD1 attaches to flagella through its tail proteins. Currently, there are no high-resolution structures of flagella-dependent bacteriophages. To elucidate the structure of YSD1, purified virions were isolated from cultures of Salmonella typhimurium and imaged by cryo-EM. A 2.8 Å x-ray crystallography structure combined with the 4.7 Å EM map revealed a T=7 icosahedral capsid similar to the HK-97 and T7 phages. However, in contrast to HK-97, which uses crosslinking to reinforce the capsid, YSD1 has an additional cementing protein stabilising the icosahedral shell formed by the major capsid protein. Helical reconstruction of the tail produced a 3.8 Å map which revealed a C6 helical tube related to Type VI secretion systems and the tails of T4 and T5 phages. The YSD1 tail is composed of a central beta-barrel domain decorated by two peripheral domains. A beta-sandwich domain, unique to Chi-like phages, has structural similarity with bacterial adherence factors. The C-terminal domain is flexible and not well resolved in the helical reconstruction. The Ig-like fold and organisation of this domain was determined using modelling and small-angle x-ray scattering (SAXS). The determination of the structure of YSD1 gives us an insight into the assembly and evolution of flagella-dependent bacteriophages.

BIOPHYSICAL APPROACHES TO THE STUDY OF HETEROMERIC AMYLOID FIBRILS INVOLVED IN VIRAL INHIBITION OF NECROPTOSIS

Pham C.L.L.1, Strange M.1, O’Carroll A.2, Shanmugam N.1, Sierecki E.2, Gambin Y.2, Steain M.3 and Sunde M.1 1Discipline of Pharmacology, School of Medical Sciences and Sydney Nano, University of Sydney, NSW 2006, Australia. 2EMBL Australia Node in Single Molecule Sciences, School of Medical Science, University of New South Wales, NSW 2052, Australia. 3Infectious Diseases and Immunology, Central Clinical School, Sydney Medical School, University of Sydney, NSW 2006, Australia.

The large DNA viruses, cytomegalovirus and herpes simplex virus, express proteins that inhibit the host necroptosis programmed cell death pathway. Our recent studies show that the RIP homotypic interaction motif (RHIM) within the viral necroptosis inhibitor proteins renders them amyloidogenic. The viral proteins are therefore functional amyloid structures, in which the biologically active form of the protein is retained or generated in the self-assembled fibrillar form of the protein. We have used a wide range of biophysical techniques, including single molecule fluorescence studies, to investigate the fibrils formed by these proteins. We have demonstrated that these proteins are able to form heteromeric amyloid fibrils through co-assembly with host proteins, RIPK1, RIPK3 and ZBP1/DAI, which each contain RHIMs. Incorporation of viral proteins into host amyloid fibrils alters the structure and properties of the fibrils and renders the component proteins unable to signal for cell death.


SymPoSia WEDNESDay

SYM-53-01 SYM-53-02

SYM-53-03 SYM-53-04

FUNCTIONAL GENOMICS OF SYMBIOTIC NITROGEN FIXATION IN LEGUMES

Udvardi M., Roy S., Liu W., Nova Franco B., Espinoza M., Kang Y., Torres-Jerez I. and Huertas R. Noble Research Institute, Ardmore, OK, USA.

Discovery of the first plant gene required for legume nodule development and symbiotic nitrogen fixation (SNF), LjNIN from Lotus japonicus, occured in 1999 (Schauser et al, 1999). Today, over 150 genes in multiple legume species have been found to be required for nodule development and/or effective nitrogen fixation, via forward-genetics or by genomics-informed reverse-genetics (Roy, Liu, et al., unpublished). These genes have been implicated in signaling between rhizobia and legumes, infection and accommodation of the micro-symbiont in plant cells, nodule organogenesis, and plant metabolism in support of bacterial nitrogen fixation. Although some of these genes appear to be indispensable for SNF in several legumes species, not all are necessary in all species. This may reflect genetic and/or other functional redundancy within species related to either ancient genome duplications or more recent tandem duplications of genes. It also appears to reflect, to some extent, distinct co-evolution of legumes and specific rhizobia. Despite the large number of genes now known to be required for SNF in legumes, our knowledge of the molecular and cellular basis of nodule development and metabolism remains fragmentary. This talk will summarize what is known about the genetics of the various processes that lead to and support SNF, including our work on Medicago truncatula, and highlight some of the gaps in our knowledge in these areas. Finally, we address the question: how can SNF be improved in legumes with so many genes implicated? We are using natural variation in SNF in approximately 200 ecotypes of M. truncatula to identify genes that contribute to effectiveness in this species, via genome-wide association studies, with a view to developing plant breeding strategies to improve SNF in crop legumes. Examples of putative SNF effectiveness genes will be presented.

ALUMINIUM ACTIVATED MALATE TRANSPORTERS FACILITATE GABA TRANSPORT IN PLANTS

Ramesh S.A., Gilliham M. and Tyerman S.D. ARC Centre of Excellence in Plant Energy Biology, School of Agriculture, Food and Wine, Waite Research Institute, University of Adelaide, Glen Osmond, SA 5064 Australia.

Gamma-aminobutyric acid (GABA) is a neurotransmitter regulating membrane potential in nerve cells. GABA rapidly accumulates in plant tissues in response to various stresses, and regulates growth. We identified GABA-binding sites within plant Aluminium-activated Malate Transporter (ALMT) proteins with homology to GABA-binding motifs of mammalian GABAA receptors (GABA-gated anion channels). The ALMTs are currently classified as anion channels but are also regulated by diverse signals leading to a range of physiological responses. We have previously demonstrated that anion flux through ALMTs is negatively regulated by GABA and its analogs with an EC50 in the low micromolar range[1]. We also observed that activation of wheat ALMT (TaALMT1) led to a negative correlation between malate efflux and endogenous GABA concentrations in root tips and heterologous expression systems. Recently we have shown that this negative correlation is a result of GABA efflux facilitated by TaALMT1[2]. Activation of TaALMT1 leads to GABA transport into the cells demonstrated by yeast complementation and 14C[GABA] uptake into Xenopus oocytes. GABA transport into yeast was observed for all ALMTs we examined. Interestingly, mutation of residue (TaALMT1F213C) in the GABA motif prevented GABA influx and efflux in the multiple test systems we used, and abolished the negative correlation between anion efflux and GABA concentration. GABA and malate thus appear to interact with ALMTs in a complex manner to regulate each others transport and this is suggestive of a role for ALMTs in communicating metabolic status of cells. 1. Ramesh, S., et al., GABA signalling modulates plant growth by directly regulating the activity of plant-specific anion transporters. Nature Communications, 2015. 6: p. 7879. 2. Ramesh, S.A., et al., Aluminium-Activated Malate Transporters Can Facilitate GABA Transport. Plant Cell, 2018. 30 (5) 1147-1164; DOI: https://doi.org/10.1105/tpc.17.00864.

ORGANIC ANION EXUDATION FROM ROOTS INFLUENCES MINERAL NUTRITION, ROOT MICROBIOME COMPOSITION AND ROOT ARCHITECTURE

Ryan P.R. CSIRO Agriculture and Food, Canberra.

Plants release an estimated 10-20% of fixed carbon into the soil. Whether released as a consequence of growth or in response to specific triggers and stresses, these exudates modify the chemistry of the rhizosphere which can benefit growth and survival. For example, the release of organic anions from roots enables some plants to cope with the toxic aluminium (Al3+) cations prevalent in acidic soils. Some of the genes controlling the organic anion release have been identified in crop species including wheat, barley, rice and maize. In wheat, the release of malate and citrate anions is controlled by an anion channel encoded by TaALMT1 and a co-transporter encoded by TaMATE1B, respectively. These transporters had only been characterised in young wheat seedlings so more recent experiments examined their physiology in 30 day-old plants with mature root systems. Malate release was detected from the seminal, nodal and lateral roots but citrate release only occurred from seminal and nodal roots. We further showed that citrate release affected the structure and composition of the microbiome near the root apices. These genes therefore provide strategies for manipulating the root microbiome. More surprisingly, the TaMATE1B locus was associated changes in root architecture because when wheat was grown in acid soil or hydroponic solutions with toxic levels of aluminium, lines with the TaMATE1B locus developed 30-50% more nodal roots than near-isogenic lines without this locus. Finally, the constitutive release of malate anions from roots of transgenic rice lines altered the subcellular compartmentation of manganese in leaves. These experiments demonstrate that the release of simple organic anions such as malate and citrate from roots can influence plant growth and physiology in multiple ways.

FROM SMALL RNA TO METABOLITES - HOW EUCALYPTS MEET UP WITH ECTOMYCORRHIzAL FUNGI

Wong J.W.H.1, Lutz A.2, Natera S.2, Wang M.3, Ng V.3, Grigoriev I.3, Martin F.4, Roessner U.2, Anderson I.1 and Plett J.1 1Hawkesbury Institute for the Environment, Western Sydney University, Richmond, NSW 2753, Australia. 2School of BioSciences, University of Melbourne, Parkville, Victoria, 3010, Australia. 3U.S. Department of Energy Joint Genome Institute, Walnut Creek, USA. 4INRA, UMR 1136, INRA-Nancy Universite, Interactions Arbres/Microorganismes, 54280 Champenoux, France.

Ectomycorrhizal (ECM) fungi are often associated with roots of forest trees, bringing benefits to their hosts including enhanced nutrient uptake and increased stress tolerance. How trees communicate with ECM fungi prior to physical interaction, and how this pre-symbiotic exchange of signals differs from plant-pathogen interactions, is largely unexplored. With a focus on this pre-symbiosis stage, we aim to decipher the communication routes between the model tree Eucalyptus grandis and one of its associated ECM fungal species: Pisolithus microcarpus. Considering the roles of metabolites and RNAs in plant-microbial signalling as determined by previous studies, we examined ECM fungi-eucalypt molecular interaction with a combined use of RNA-seq, small RNA-seq and untargeted metabolite profiling.We identified significant changes in both the metabolome and transcriptome (including both small RNAs and RNAs) of eucalypt roots during this pre-symbiotic period with P. microcarpus. These results suggest that eucalypts are able to respond to the presence of ECM fungi prior to physical contact. We then further investigated whether these changes were activated by internal gene regulation of eucalypt roots, or by the trafficking of fungal signals. With our small RNA-seq data, we observed a significant portion of small RNA reads detected in eucalypt roots are originated from P. microcarpus rather than E. grandis. Amongst these fungal reads, we identified one species of fungal small RNAs that putatively targets defense-related R genes of the plant host during pre-symbiosis. Additionally, with the use of isotopic labeling in conjunction to untargeted metabolite profiling, we also identified some ECM fungi-derived metabolite signals in eucalypt roots. Therefore, by dissecting the molecular signals in eucalypt roots using a multi-omics platform, our research suggests that both small RNAs and metabolites are signals used by ECM fungi to prime their host plant during this pre-symbiotic stage of interaction.


SymPoSia WEDNESDay

SYM-53-05 SYM-54-01

SYM-54-02 SYM-54-03

TISSUE-SPECIFICITY OF SODIUM TRANSPORT AND SEQUESTRATION REVEALS THE ROLE OF THE ROOT MERISTEM AS A TENTATIVE SALT SENSOR

Wu H.H.1, 2, Shabala L.1 and Shabala S.1 1Tasmanian Institute for Agriculture, University of Tasmania, Hobart, Tasmania 7001, Australia. 2Department of Botany and Plant Sciences, University of California, Riverside, CA, U.S. 92521.

The progress in plant breeding for salinity stress tolerance is handicapped by the lack of understanding of the specificity of salt stress signalling and adaptation at the cellular and tissue levels. In this study, we used electrophysiological, fluorescence imaging and real-time qPCR tools to elucidate the essentiality of the cytosolic Na+ extrusion in functionally different root zones (elongation; meristem; mature) in a large number of bread and durum wheat accessions. We show that the difference in the root’s ability for vacuolar Na+ sequestration in the mature zone may explain differential salinity stress tolerance between salt sensitive durum and salt tolerant bread wheat species. Bread wheat genotypes also had on average 30% higher capacity for net Na+ efflux from the root elongation zone, providing the first direct evidence for the essentiality of root salt exclusion trait at the cellular level. At the same time, cytosolic Na+ accumulation in the root meristem was significantly higher in bread wheat, leading to suggestion that this tissue may harbor a putative salt sensor. This hypothesis was then tested by investigating patterns of Na+ distribution and relative expression level of several key genes related to Na+ transport in leaves in plants with intact roots and those in which the roots meristems were removed. We show that tampering with this sensing mechanism has resulted in a salt-sensitive phenotype, largely due to compromised plant’s ability to sequester Na+ in mesophyll cell vacuoles. The implications of these findings for plant breeding for salinity stress tolerance are discussed.

DIFFERENTIATION STATES IN THE ORIGIN EVOLUTION AND TREATMENT OF CHILDHOOD LEUKAEMIAEnver T. UCL Cancer Institute, London.

Childhood acute lymphoblastic leukemia is thought in many cases to initiate in utero. The factors that influence the formation of the initiating lesions are not well understood although the nature of these genetic aberrations is well documented and include in approximately a quarter of children the presence of the t12;21 chromosomal translocation that fuses the transcription factors Tel (ETV6) and Aml1 (RUNX1). The Tel-Aml1 fusion gene produces a pre-leukemic clone but in and of itself is insufficient to produce frank leukemic transformation. For this, additional mutations are required and it remains unclear what factors influence their acquisition. These additional mutations tend to arise in loci that are normally involved in cell fate control in the B lineage. Cell fate regulators act in a context dependent manner and thus understanding the nature of the target cell or cells in which the initiating and subsequent mutations arise is of importance. To gain insight with these issues we have been exploring the target genes of TEL-AML1 and associated second hits as well as developing new fetal specific models in which to examine the biological impact of TEL-AML1. In this regard we have identified candidate target cells for TEL-AML1 within the human foetal liver and modelled these using human IPS cells in vitro. This has allowed us to understand that hematopoietic differentiation hierarchies differ in fetal vs adult life and that TEL-AML1 functions primarily as a lineage deregulator. Target gene analysis further reveals how TEL-AML1 alters epigenetic programs and reveals opportunities for therapeutic intervention. Leukemic clones appear to evolve in a branching manner such that at presentation the marrow is replete with multiple variegated subtypes providing a diverse substrate for selection in response to therapy. Beyond genetic heterogeneity, leukemic cells exhibit epigenetic heterogeneity in respect of their immune-phenotypes and functional properties including cell cycle status and niche residence. To obtain a “real-time” longitudinal analysis of sub-clonal dynamics through treatment we have established an in vivo mouse model that allows a patient tumour to be independently exposed to treatment multiple times, enabling us to distinguish between deterministic and stochastic mechanisms of selection during therapy. Our results suggest that epigenetic state - including cycle and differentiation status - rather than genotype dictates resistance to chemotherapy.

ROLE OF ELK1 IN CONGENITAL AND LATE ONSET CARDIAC DISEASE: AT THE HEART OF THE MATTER

Hallab J.C.1, Del-Monte Nieto G.2, Bouveret R.2, Varshney A.1, Huttner I.2, Santiago C.2, Miles L.3, Dworkin S.3, Kikuchi K.2, Hesselson, D.4, Fatkin, D.2, Harvey, R.2 and Ramialison M.1 1Australian Regenerative Medicine Institute, Monash University, Melbourne. 2Victor Chang Cardiac Research Institute, Sydney, Australia. 3Department of Physiology, Anatomy and Microbiology, LaTrobe University, Melbourne. 4Garvan Institute of Medical Research, Sydney, Australia.

Elk1 is an ETS Class I, TCF subfamily transcription factor known as a well- established downstream effector of the MAPK pathway and has been implicated in the causation of a variety of cancers. Recent in vitro evidence places Elk1 in the context of the cardiogenic transcription factor network, although its in vivo role in cardiogenesis remains unexplored. We provide the first in vivo evidence of the role of Elk1 in cardiogenesis using a zebrafish mutant with disrupted DNA binding domain (elk1-543/-543) and cardiac defects including valve displacement and elongation with hypertrophic/hyperplastic changes in the ventricular myocardium. elk1-543/-543 are predisposed to early embryonic death, with high incidence of heart looping defects and accelerated growth among survivors. RNA-sequencing (RNA-seq) at larval stage provides insights into the basis of heart defects, indicating up-regulation of MAPK pathway genes and down-regulation of trim63a, encoding a homeostatic protein involved in reducing muscle mass, dys-regulation of which is associated with Hypertrophic Cardiomyopathy (HCM) in humans. MAPK pathway up-regulation is commonly associated with HCM although the fundamental basis of this relationship is not completely understood. We provide mechanistic insight, suggesting MAPK perturbations could converge via the TCFs at a predicted trim63a enhancer, down-regulating trim63a to mediate HCM. Early developmental RNA-seq indicates loss of Elk1 function is associated with down-regulation of tumor suppressor genes. We hypothesize this promotes embryonic survival via non-optimal pathways, and underlies observed defects. The sum of changes in elk1 -543/-543 mimic a group of congenital syndromes known as “RASopathies” in humans. Our data provides important insights into the time line of molecular events underlying RASopathies/MAPK pathway defects and their relationship to molecules imperative in heart patterning and homeostasis.

CONTROL OF DROSOPHILA MYC TRANSCRIPTION, CELL GROWTH AND DEVELOPMENTAL PATTERNING BY THE SINGLE STRANDED DNA BINDING PROTEIN PSI

zaytseva O.1, 2, Kim N.1, Guo L.1, 2, Mitchell N.C.1, Evers M.1, Marshall O.J.3, Hannan R.D.1, 4, Levens D.L.5 and Quinn L.M.1 1The John Curtin School of Medical Research, ANU, Canberra, Australia. 2The University of Melbourne, Parkville, Australia. 3Menzies Institute for Medical Research, Hobart, Australia. 4Peter MacCallum Cancer Centre, Melbourne, Australia. 5National Cancer Institute, NIH, Bethesda, Maryland, USA.

The transcription factor MYC is upregulated in 70% of cancers and elevated MYC potently drives growth and proliferation. Thus, the capacity of the MYC promoter to integrate developmental signals is essential for correct growth patterning in all multicellular animals. Studies of the MYC promoter identified Far Upstream Binding Protein 1 (FUBP 1), a single stranded DNA binding protein, which acts to activate MYC expression. Consistently with this function, FUBP1 is upregulated in many cancers, including breast, liver, bladder, kidney and lung. Moreover, our recent Drosophila studies revealed that the FUBP1 ortholog (Psi) interacts with the transcriptional Mediator (MED) complex to integrate developmental signals, activate MYC and promote cell and tissue growth in the wing epithelium. Paradoxically, our recent unpublished data demonstrate expansion of the neuroblast stem cell lineage in the Drosophila brain after Psi depletion. In line with tissue-specific functions, FUBP1 knockout mice exhibit hypoproliferation in the embryonic blood lineage, while overgrowth occurs in the brain. Furthermore, in contrast to other tumour types, FUBP1 loss-of-function ranks in the top 10% of predicted driver mutations in oligodendroglioma, the second most common primary brain cancer in adults. The mechanisms behind these striking context-specific roles is unknown; therefore our current studies in Drosophilamodels focus on identification of key Psi transcriptional targets in both wing epithelium and neural stem cells.


SymPoSia WEDNESDay

SYM-54-04 SYM-54-05

SYM-55-01 SYM-55-02

EXPLORING THE FUNCTION OF NKXUS, A NOVEL HEART-ASSOCIATED LNCRNA

Altekoester A.1, 3, Schonrock N.2, Wu J.1, Kesteven S.1 and. Harvey R.P.1 1Victor Chang Cardiac Research Institute, Australia. 2Garvan Institute of Medical Research, Australia. 3University of Cologne, Germany.

A significant large amount of the mammalian genome previously found to not code for proteins and considered “junk”, was found to actually specify a dynamic network of regulatory RNAs, termed long non-coding RNAs (lncRNAs). Here, we identified Nkx2-5UPSTREAM (NkxUS), a heart-associated lncRNA, which lies upstream of the cardiac key transcriptional regulator NKX2-5. In humans, NKX2-5 is essential for proper heart development and mutations are commonly associated with congenital heart disease. We hypothesise that NkxUS either regulates NKX2-5 itself or other processes affecting heart function and development. The aim of my study is to fully characterize NkxUS especially its function. Our studies show that NkxUS is a long, cardiac and nuclear enriched transcript. It is expressed in the heart throughout development and a similar heart specific transcript occurs in humans. We found a heart rate-associated GWAS SNP, located within human NkxUS, which lies within and disrupts a RNA structure conserved between mouse and human. When knocking down the mature transcript, genes involved in heart contraction and calcium signalling are down regulated. Mice lacking the conserved structure exhibit a higher resting heart rate with no differences in ECG parameter intervals, suggesting a possible dysfunction in the sinoatrial node (SAN), the pacemaker of the heart. Currently, we are performing calcium and voltage imaging combined with drug treatments on dissected mouse SANs to identify the basis of the phenotype. Further, we are testing the inducibility of atrial fibrillation (AF) in the same mice as the SNP was also associated with a higher risk of developing AF. Altogether, we present data on a novel cardiac enriched lncRNA and provide a detailed characterisation of this transcript to analyse how it might impact heart function and disease.

NEURAL AND DENDRITIC ACTIVITY DURING SENSORY-BASED BEHAVIOUR

Palmer L.M. Florey Institute of Neuroscience and Mental Health, University of Melbourne.

In the living animal, sensory systems are generally not stimulated in isolation but are instead activated collectively. The task of understanding how neurons receive and transform this sensory input is central to explaining brain function during behaviour. Pyramidal neuron dendrites in the primary somatosensory cortex receive both feedforward input from the thalamus and feedback input from other cortical areas. Since the synaptic location of the different input streams are morphologically and functionally isolated, how sensory input is integrated and computed at the level a single neuron is currently unknown. Here I will present recent results investigating the activity of tuft dendrites of layer 2/3 pyramidal neurons in the primary somatosensory cortex during a sensory-based reward association task. We find that tuft dendrites alter their activity during the behavioural task when presented with multi-sensory input, increasing the occurrence of large Ca2+ events. This modulation of synaptic integration highlights the importance of feedback information in dendritic encoding of sensory-based behaviour.

NEUROPEPTIDE F RECEPTOR ACTS IN THE DROSOPHILA PROTHORACIC GLAND TO REGULATE BODY SIzE AND DEVELOPMENTAL TIMING

Kannangara J.R.1, Henstridge M.A.1, Parsons L.M.1, Kondo S.2, Mirth C.K.1 and Warr C.G.1 1School of Biological Sciences, Monash University, Clayton, Victoria 3800, Australia. 2Invertebrate Genetic Laboratory, National Institute of Genetics, Mishima, Japan.

Drosophila melanogaster is an excellent model organism in which to study the regulation of growth, as many of the major growth factors and signalling pathways are conserved between flies and humans. In Drosophila, growth is primarily regulated by the steroid hormone, ecdysone, which is produced in and secreted from the prothoracic gland in response to various environmental and genetic cues. Regulation of ecdysone biosynthesis in the gland involves a number of neuropeptides, for example the Drosophila insulin-like peptides, which regulate ecdysone production in response to nutrition. Here we identify the receptor for Neuropeptide F (Npf), the fly homologue of mammalian NPY, as a novel regulator of Drosophila growth. Knockdown of Npfrspecifically in the prothoracic gland generates a developmental delay and increased body size. Interestingly, while Npfr mutants also have a developmental delay, they instead have a smaller body size. Likewise, knocking down Npf in Npf-expressing neurons also results in a significant delay to development and smaller body size. These defects can be rescued by feeding larvae ecdysone-enriched food. Furthermore, there is reduced expression of ecdysone biosynthesis genes and reduced ecdysone levels in these animals. This suggests that Npf is involved in regulating ecdysone biosynthesis in the Drosophila prothoracic gland. NPY has been shown to interact with the insulin signalling pathway to regulate energy balance and body size in mammals, thus NPY and Npf may share conserved functions. Studies on Drosophila Npf may therefore further our understanding of human metabolic diseases.

DEVELOPMENTAL DISORDERS OF HISTONE MODIFICATION

Voss A.



SymPoSia WEDNESDay

SYM-55-03 SYM-55-04

SYM-55-05 SYM-56-01

SITAGLIPTIN ALTERS METASTATIC PROGRESSION AND IMMUNE RESPONSE IN AN ID8 OVARIAN CANCER MOUSE MODEL

Wilson A.L.1, 2, Wilson K.L.2, Moffitt L.R.1, 2, Bilandzic M.1, 2, Plebanski M.2, 3 and Stephens A.N.1, 2 1Hudson Institute of Medical Research, VIC, Australia. 2Monash University, VIC, Australia. 3RMIT University, VIC, Australia.

Current methods used to treat epithelial ovarian cancer (EOC) often result in relapse and acquired chemo-resistance; therefore, novel therapies for EOC are urgently needed. Immunotherapy for EOC is gaining traction, and recent studies have suggested that the dipeptidyl peptidase-4 (DPP4) inhibitor sitagliptin can activate the immune system, but this has not been demonstrated in ovarian cancer. Additionally, preclinical models that accurately stage ovarian tumour growth are required for evaluating these therapeutic responses in vivo, as no non-invasive techniques currently exist. We developed a model in which ID8 mouse ovarian cancer cells stably express a near-infrared protein (iRFP) via integration into the ROSA26 genomic region, then used this model to evaluate the anti-tumour effects of sitagliptin in vivo. C57BL/6 mice received an injection of 1x106 pROSA-iRFP720-ID8 EOC cells under the ovarian bursa. Mice were treated with sitagliptin (50mg/kg/body-weight/day) 14-days following implantation until endpoint, and iRFP720 fluorescence was measured weekly to evaluate tumour deposition. T-cell, dendritic cell (DC), macrophage and myeloid-derived suppressor cell (MDSC) populations in the spleen, lymph nodes and blood were assessed by flow cytometry, and in tumour tissue by immunofluorescence. We demonstrated that administration of sitagliptin reduced tumour burden, as indicated by fluorescence and macroscopic tumour observations. Sitagliptin increased tumour-infiltrating T-effector cells, decreased proliferating T-regulatory cells and decreased cytotoxic T-cell apoptosis. In addition, sitagliptin increased circulating DCs and macrophages, and decreased tumour-associated MDSCs. Taken together, these results suggest that administration of sitagliptin decreases tumour burden in an ID8 ovarian cancer model by shifting the balance toward anti-tumour immunity, and it may have therapeutic potential for epithelial ovarian cancer.

THE ROLE OF CDX2 AND HNF4α IN BARRETT’S METAPLASIA

Colleypriest B.J.1, 2, Burke Z.D.1, Griffiths L.P.1, 2, Slack J.M.W.1, 3 and Tosh D.1 1University of Bath, UK. 2Royal United Hospital, Bath, UK. 3Stem Cell Institute, University of Minnesota, USA.

Barrett’s metaplasia is the only known morphological precursor to oesophageal adenocarcinoma and is characterized by replacement of stratified squamous epithelium by columnar epithelium. The cell of origin is uncertain and the molecular mechanisms responsible for the change in cellular phenotype are poorly understood. We therefore explored the role of two key developmental transcription factors, CDX2 and HNF4α in the conversion, using primary organ cultures. Biopsy samples from cases of human Barrett’s metaplasia were analysed for the presence of CDX2 and HNF4α. A new organ culture system for adult murine oesophagus is described. Using this, Cdx2 and HNF4α were ectopically expressed by adenoviral infection. The phenotype following infection was determined by a combination of PCR, immunohistochemical and morphological analyses. In the human biopsy samples we demonstrate the expression of both CDX2 and HNF4α. Our oesophageal organ culture system expressed markers characteristic of the normal SSQE: p63, K14, K4 and loricrin. Ectopic expression of HNF4α, but not of Cdx2, in these cultures induced expression of Tff3, villin, K8 and E-cadherin. So HNF4α is sufficient to induce a columnar-like phenotype in adult mouse oesophageal epithelium and is present in the human condition. These data suggest that induction of HNF4α is a key early step in the formation of Barrett’s metaplasia and are consistent with an origin of Barrett’s metaplasia from the oesophageal epithelium.

MORPHOGENESIS OF THE SEMICIRCULAR CANAL DUCTS OF THE zEBRAFISH INNER EAR

Whitfield T.T. Bateson Centre and Department of Biomedical Science, University of Sheffield, Sheffield, S10 2TN, UK.

The inner ear, the organ of hearing and balance, has a spectacular and complex epithelial morphology. The interlinked chambers and ducts that make up the ear include the three orthogonally-oriented semicircular canals, which function to sense angular acceleration (turning movements) of the head. During embryogenesis, the otic epithelium undergoes fusion and fission events that drive changes in tissue topology, defining the semicircular canal ducts. We are using high-resolution confocal and light-sheet fluorescence microscopy to image and analyse semicircular canal morphogenesis in the live zebrafish embryo. I will present a selection of our imaging studies to illustrate the dynamic cell and tissue movements that accompany semicircular canal formation, both in wild-type embryos, and in mutant lines in which different aspects of semicircular canal development are disrupted. Together, these are giving insight into the molecular and cellular mechanisms underlying semicircular canal formation in the vertebrate embryo.

UNDERSTANDING AND ENGINEERING MICROBIAL SENSORS

Gerth M.L. Victoria University of Wellington, School of Biological Sciences, New Zealand.

Bacterial chemoreceptors are remarkable examples of biological sensors: they can detect chemicals at nanomolar concentrations and discriminate between closely related molecules. They play a central role in chemotaxis, allowing bacteria to detect chemical gradients and bias their swimming behaviour in order to navigate towards favourable environments. There are thousands of putative chemoreceptor genes in bacterial genomes, but for the vast majority, neither what they detect nor how they detect it is understood. These chemoreceptors represent a (largely untapped) source of modular parts for molecular devices. In my laboratory, we are exploring the functional and structural diversity of chemoreceptors. We are also engineering receptors with novel sensing capabilities. Ultimately, our goal is to incorporate these modular domains into handle-held biosensor devices for applications in a variety of industries.


SymPoSia WEDNESDay

SYM-56-02 SYM-56-03

SYM-56-04 SYM-56-05

STRUCTURE AND ASSEMBLY MECHANISM OF THE TYPE III SECRETION SYSTEM NEEDLE TIP COMPLEX

Tuckwell A.J., Xu S., Kyaw W. and Lee L.K. UNSW Sydney.

The type III secretion system (T3SS) is a protein superstructure, consisting of hundreds of subunits, which self-assemble into a molecular syringe that injects virulence factors directly into the host cell. The tip complex caps an extracellular needle filament and is involved in penetrating the host cell membrane. As a surface-exposed antigen, it is also an attractive target for vaccine development. High resolution crystal structures of tip complex subunits have been determined from several species. However, their monomeric conformations appear to be incompatible with EM micrographs. While these also provide information on stoichiometry, the resolution of EM micrographs are at present far too low to determine the arrangement of subunits in an in-tact tip complex. This is in part because the tip complex can only be visualised as part of the entire T3SS superstructure. Here we combine, information from high-resolution crystal structures, solution X-ray scattering and molecular dynamics simulations to obtain insight into the assembly mechanism and structure of an in-tact T3SS tip complex. These structural models are then used to rationally design and construct protein constructs and synthetic structural scaffolds from DNA to artificially stabilise isolated T3SS tip complexes for structural characterisation.

USING EVOLUTION TO GUIDE THE ENGINEERING OF PPR PROTEINS AS CUSTOMIzABLE RNA PROCESSING TOOLS

Bernath-Levin K., Colas Des Francs-Small C., Gutmann B., Honkanen S., McDowell R., Melonek J., Pereira Vincis Sanglard L., Royan S., Sun Y. and Small I.D. ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, University of Western Australia.

Pentatricopeptide repeat (PPR) proteins are modular nucleic acid binding proteins that are highly prevalent in plants, with some species producing thousands of different PPR proteins, each binding a different target sequence. PPR proteins function almost exclusively inside organelles, where they are involved in post-transcriptional steps in gene expression, including RNA cleavage, intron splicing, RNA stabilization, RNA editing and initiation of translation. Their sequence specificity resides in base-specific contacts made by 2-3 amino acids in each repeat that can be described by a simple code, making the target specificity of PPR proteins not only predictable, but programmable. Amongst the many different PPR proteins known, two groups show unusual evolutionary behavior that make them particularly interesting for protein engineering. Restorer-of-fertility factors are a small clade of PPR proteins showing a high degree of inter- and intra-specific variation and strong diversifying selection on the residues that determine RNA binding specificity. We have found that this natural variability makes them ideal subjects for re-engineering and we are currently using them to make targeted knock-downs in mitochondrial gene expression. In a similar vein, but on a much larger scale, PPR RNA editing factors have undergone massive independent expansions in early-diverging land plants such as hornworts, lycophytes and ferns. Inspired by their natural functions, we are attempting to engineer these factors to control gene expression by the targeted creation and removal of start and stop codons using RNA editing.

DIHYDRODIPICOLINATE SYNTHASE IS ABSENT IN FUNGI

Desbois S.1, John U.P.1, 2 and Perugini M.A.1 1Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, VIC 3086, Australia. 2Agriculture Victoria Research, Department of Economic Development, Jobs, Transport and Resources, AgriBio, La Trobe University, VIC 3086, Australia.

The class I aldolase dihydrodipicolinate synthase (DHDPS) catalyses the first committed step of the diaminopimelate (DAP) lysine biosynthesis pathway in bacteria, archaea and plants. Despite the existence in databases of numerous fungal sequences annotated as DHDPS, its presence in fungi has been the subject of contradictory claims. We report the characterisation of DHDPS candidates from fungi. Firstly, the putative DHDPS from Coccidioides immitis (PDB ID: 3QFE) was shown to have negligible DHDPS enzyme activity. Sequence analysis of 3QFE showed that three out of the seven amino acid residues critical for DHDPS activity are absent; however, exact matches to catalytic residues from two other class I aldolases, 2-keto-3-deoxygluconate aldolase (KDGA), and 4-hydroxy-2-oxoglutarate aldolase (HOGA), were identified. The presence of both KDGA and HOGA activity in 3QFE was confirmed in vitro using enzyme assays, the first report of such dual activity. Subsequent analyses of all publically available fungal sequences revealed that no entry contains all seven residues important for DHDPS function. The candidate with the highest number of identities (6 of 7), KIW77228 from Fonsecaea pedrosoi, was shown to have trace DHDPS activity in vitro, partially restored by substitution of the seventh critical residue, and to be incapable of complementing DHDPS-deficient E. colicells. Combined with the presence of all seven sequences for the alternative α-aminoadipate (AAA) lysine biosynthesis pathway in C. immitis and F. pedrosoi, we believe that DHDPS and the DAP pathway are absent in fungi, and further, that robust informed methods for annotating genes need to be implemented.

INTERCHANGEABLE REGULATORY DOMAINS: EXPLORING MODULAR ALLOSTERY EN ROUTE TO CHORISMATEFan Y.1, 2, 5, Cross P.J.2, 5, Jameson G.B.3 and Parker E.J.1, 4 1Ferrier Research Institute, Victoria University of Welington, 6140 Wellington, New Zealand. 2Department of Chemistry, University of Canterbury, 8140 Christchurch, New Zealand. 3Maurice Wilkins Centre, Institute of Fundamental Sciences, Massey University, 4442 Palmerston North, New Zealand. 4Maurice Wilkins Centre, Biomolecular Interaction Centre, University of Canterbury, 8140 Christchurch, New Zealand. 5Biomolecular Interaction Centre, University of Canterbury, 8140 Christchurch, New Zealand.

Engineering desired function into proteins via manipulation of the genes, mimicking natural evolutionary processes, represents a promising approach to synthesizing useful molecular tools. Most proteins comprise two or more domains from a limited suite of protein families. These domains are often rearranged in various combinations through gene fusion events to evolve new protein functions, including the acquisition of protein allostery through the incorporation of regulatory domains. The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of aromatic amino acid biosynthesis and displays a diverse range of allosteric mechanisms. DAH7PSs adopt a common architecture with a shared (β/α)8 catalytic domain which can be attached to an ACT-like or a chorismate mutase regulatory domain that operates via distinct mechanisms. These respective domains confer allosteric regulation by controlling DAH7PS function in response to ligand tyrosine or prephenate. Starting with contemporary DAH7PS proteins, two protein chimeras were created, with interchanged regulatory domains. Both engineered proteins were catalytically active and delivered new functional allostery with switched ligand specificity and allosteric mechanisms delivered by their nonhomologous regulatory domains. This interchangeability of protein domains represents an efficient method not only to engineer allostery in multidomain proteins but to create a new bifunctional enzyme.1 1. Fan, Y., Cross, P.J., Jameson, G.B. and Parker, E.J., 2018. Exploring modular allostery via interchangeable regulatory domains. Proceedings of the National Academy of Sciences, 115(12), pp.3006-3011.


SymPoSia WEDNESDay

SYM-57-01 SYM-57-02

SYM-57-03 SYM-57-04

CROSSTALK BETWEEN ONCOGENIC SIGNALLING PATHWAYS REPROGRAMS LIPID METABOLISM IN CANCER

Brown K.1, 2 1Cancer Metabolism Program and Cancer Therapeutics Program, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia. 2Department of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, VIC, Australia.

Malignant transformation and tumour progression are dependent on reprogramming of cell metabolism to fulfil the unique energetic and biosynthetic needs of cancer cells. A well-recognised, but poorly understood, alteration in metabolism frequently observed in cancer cells is the activation of de novo lipogenesis. Our recent studies have focussed on the oncogenic transcriptional co-activator YAP, a master regulator of organ size and tumourigenesis. Aberrant activation of YAP is widespread in human cancers. There is little knowledge regarding mechanisms by which YAP drives tumourigenesis, in large part because a limited number of YAP target genes and effectors have been identified. We find that YAP overexpression induces de novolipogenesis in vitro and in vivo. Mechanistically, this phenomenon is dependent on the ability of YAP to induce transcriptional upregulation of serum- and glucocorticoid-regulated kinase 1 (SGK1), an effector of the oncogenic phosphoinositide 3-kinase (PI3K) pathway. Downstream of YAP, SGK1 promotes mTORC1 signalling leading to activation of the sterol regulatory element-binding proteins (SREBP1/2), master regulators of lipid metabolism. Importantly, we find that inhibition of key enzymes in the de novo lipogenesis pathway blocks the uncontrolled proliferation associated with YAP-driven transformation in vitro and in vivo. Our data reveal a mechanism of crosstalk between two important oncogenic signalling pathways and reveal a metabolic vulnerability that can be targeted to disrupt oncogenic YAP/PI3K pathway activity.

DYNAMIC ‘OMICS REDEFINES HOW INSULIN SIGNALLING CHOREOGRAPHS GLUCOSE METABOLISM

Krycer J.1, Yugi K.2, 4, Fazakerley D.1, Humphrey S.1, Quek L.1, Hirayama A.3, Soga T.3, Kuroda S.4 and James D.1 1University of Sydney, Sydney, Australia. 2RIKEN IMS, Yokohama, Japan. 3Keio University, Tsuruoka, Japan. 4University of Tokyo, Kashiwa, Japan.

Adipose (fat) tissue plays a crucial role in energy storage and release. Accordingly, adipose metabolism responds to fluctuating nutrient availability and hormonal cues. For instance, after a meal, insulin triggers a phosphorylation cascade to stimulate glucose uptake. This is considered insulin’s primary role, with subsequent energy storage activated by allostery as substrates accumulate. However, our recent phosphoproteomics screen in insulin-treated adipocytes identified hundreds of metabolic proteins - could insulin signalling play a role beyond glucose uptake, coercing glucose down specific metabolic routes? We addressed this by measuring acute, temporal metabolomic changes upon insulin exposure, taking a dynamic approach to traditional steady-state 13C-tracer-experiments. We found three exciting results: (1) Insulin rapidly stimulated glucose uptake (t1/2=4 min), with its kinetics explaining 48% of metabolomic changes in response to insulin. Indeed, glucose was necessary for insulin-stimulated lipogenesis and suppression of fatty-acid oxidation, suggesting glucose facilitates insulin action. (2) Despite this, flux analysis revealed glucose was primarily converted to lactate and favoured NADPH-generating pathways (e.g., pyruvate anaplerosis, pentose phosphate pathway). This is reminiscent of cancer metabolism. We believe this allows glucose to facilitate anabolism beyond being a mere carbon source in (terminally-differentiated) adipocytes. (3) Overlaying metabolomic and phosphoproteomic data (‘transomic analysis’) revealed protein phosphorylation changed rapidly (<5 min), activating anabolism before substrates accumulated. Thus, insulin creates a demand-driven system to ‘drag’ glucose down specific pathways. This complements supply-driven regulation of anabolism by substrate accumulation. Overall, this redefines how signalling coordinates metabolism, with implications for metabolic dysregulation in overactive signalling (cancer) or nutrient oversupply (diabetes).

TRANSCRIPTIONAL REPROGRAMMING OF METABOLISM BY YAP AND C-MYC IN LIVER CANCER

Cox A.G.1, 2 1Peter MacCallum Cancer Centre. 2University of Melbourne.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer and among the most fatal cancer type, as few effective treatments are available. Although the pathophysiology of HCC has not been fully elucidated, the process clearly arises in the context of chronic liver disease brought on by environmental factors. In the lab, we use an combination of transcriptomic, metabolomic and imaging approaches in mutant and transgenic zebrafish to reveal mechanisms by which metabolic reprogramming fuels premalignant hyperplasia and tumourigenesis in vivo. We have recently focused our attention on the role that the oncogenic transcription factors Yap and c-Myc play in reprogramming metabolism. To this end, we have developed a zebrafish model of Yap-driven HCC that recapitulates human HCC at the histological and genetic level. Using this model we found that Yap remodels glucose and glutamine metabolism to enhance the anabolic biosynthesis on nucleotides required for rapid tissue growth. In parallel, we have taken advantage of an inducible zebrafish model of HCC, in which c-Myc is overexpressed specifically in hepatocytes upon exposure to doxycycline. We have used these models to identify oncogenic changes in metabolism and screen potential metabolic interventions for efficacy in suppressing Yap or c Myc driven hyperplasia and HCC. Our long term goal is to identify oncogene-specific metabolic vulnerabilities that can be exploited therapeutically to prevent or combat liver cancer.

NEUTRAL CHOLESTEROL ESTER HYDROLASE 1 (NCEH1) REGULATES EXTRACELLULAR LDL-CHOLESTEROL METABOLISM TO INFLUENCE PROSTATE CANCER PROGRESSIONRaftopulos N.L.1, Washaya T.C.1, Egert A.1,2,3, Aishah A.1, Varney B.1, Nagarajan S.R.1, Butler L.M.4,5, Grewal T.2, and Hoy A.J.1 1The University of Sydney, Charles Perkins Centre, Discipline of Physiology, School of Medical Sciences, Sydney, New South Wales, 2006, Australia. 2Faculty of Pharmacy, The University of Sydney, Sydney, NSW 2006, Australia. 3Universitätsklinikum Hamburg-Eppendorf, 20246 Hamburg, Germany. 4Adelaide Medical School and Freemasons Foundation Centre for Men’s Health, University of Adelaide, Adelaide, South Australia, 5005, Australia. 5South Australian Health and Medical Research Institute, Adelaide, South Australia, 5001, Australia.

Prostate cancer (PCa) remains a leading cause of cancer-related death in Australian men. Advanced PCa is initially treated by depleting androgens thereby removing a critical promotor of tumour growth; however, the low androgen environment exerts selective pressure on remaining PCa cells and the disease evolves into a lethal, aggressive form termed castrate resistant PCa (CRPC). Hypercholesterolemia is a major side effect of androgen deprivation and is linked to more aggressive disease and the development of CRPC. Also, high-grade PCa and metastases exhibit aberrant accumulation of esterified cholesterol. Cholesterol is stored in cytosolic lipid droplets as cholesterol esters (CE) and inhibition of CE formation impairs PCa progression. However, it is now known whether the mobilisation of cholesterol from CE influences CRPC cell proliferation. As expected, C4-2B cell (androgen receptor positive, androgen independent) grew normally in a low androgen environment (charcoal-stripped serum) but had lower CE levels than cells cultured in full serum. CE levels are regulated by Neutral Cholesterol Ester Hydrolase 1 (nCEH1), which hydrolyses CE to free cholesterol and a fatty acid, and inhibition of nCEH1 slowed growth in charcoal-stripped serum, suggesting that cholesterol stored in CE supports cell growth in a low androgen environment. Depletion of lipid-rich lipoproteins (LPDS) from the media reduced C4-2B cell growth and intracellular CE levels, and this was exacerbated in charcoal-stripped LPDS. Supplementation of LPDS with cholesterol-rich LDL fully restored C4-2B cell growth; however, nCEH1 inhibition ameliorated the ability of LDL supplementation to restore C4-2B cell growth in LPDS containing media. These data indicate that LDL-cholesterol promotes C4-2B PCa cell growth via an nCEH1-catalysed metabolism. Future studies aim to further characterise the mechanisms by which CE metabolism influences CRPC cell behaviour. * NLR & TCW contributed equally.


SymPoSia WEDNESDay

SYM-57-05 SYM-58-01

SYM-58-02 SYM-58-03

NON-CANONICAL UBIQUITINATION SITES DIRECT DEGRADATION OF THE SHORTEST KNOWN CHOLESTEROL-DEPENDENT DEGRON

Chua N.K. and Brown A.J. School of Biotechnology and Biomolecular Sciences, UNSW Sydney, NSW 2052, Australia.

Squalene monooxygenase (SM) is the second rate-limiting enzyme in cholesterol synthesis. Excess cholesterol triggers SM degradation via the ubiquitin-proteasome system. The E3 ligase, MARCH6, is responsible for mediating the ubiquitination of SM. Furthermore, this process requires the first 100 amino acids of SM (termed SM N100), which represents the shortest known cholesterol-regulated degron. Ubiquitination is one of the most common post-translational modifications and they often occur on lysine residues, but we have shown that lysine residues are not crucial for the cholesterol-mediated degradation of SM. Despite being an important rate-limiting enzyme in cholesterol synthesis, the precise ubiquitination sites within the cholesterol-regulated degron of SM remain elusive. In this study, we mutated non-canonical ubiquitination residues to alanine. We observed that serine residues are required for the cholesterol-dependent degradation of SM N100. In addition, loss of the key serine residues boosted SM N100 protein levels and MARCH6 knockdown did not further enhance protein levels. Our results reveal non-canonical ubiquitination in the shortest known cholesterol-regulated degron. This finding characterises the mechanism by which SM exerts its role as a rate-limiting enzyme and how cholesterol modulates the stability of SM.

EXPLOITING DIFFERENTIAL METABOLISM IN PARASITE LIFE CYCLES TO LIMIT THE SPREAD OF DRUG RESISTANCE

Goodman C.D., Buchanan H.D. and McFadden G.I. School of BioSciences, University of Melbourne.

Drug resistance is a major factor limiting our ability to control parasitic diseases. Many medically and economically significant parasites undergo immense changes in metabolic activity as they move between their mammalian hosts and invertebrate vectors. Consequently, drug selection for resistance during the vertebrate stage can have dire consequences for parasite fitness during transmission. Using the malaria parasite, Plasmodium, we are investigating anti-malarial drugs whose targets are under markedly reduced selection pressure in the mammalian host. Resistance mutations in these targets tend to drive parasite failure during development in the mosquito vector, thereby inhibiting disease transmission. This approach is particularly effective with drugs targeting the parasite organelles, due to their increased metabolic activity during the mosquito stages and the non-Mendelian inheritance of their genomes. Clinically relevant mutations conferring resistance to the widely used anti-malarial MalaroneTM arise in the mitochondrion-encoded cytochrome b gene. These mutations completely arrest development of infectious sporozoites in the mosquito stages of both P. falciparum and P. berghei, thereby blocking transmission of these resistance genes. The macrolide antibiotic azithromycin kills malarial parasites by inhibiting apicoplast protein synthesis. We generated two azithromycin resistant lines in P. berghei (PbAZMR) harbouring mutations in the apicoplast-encoded rpl4 gene. Such mutations also confer azithromycin resistance in P.falciparum. PbAZMR parasites infect mosquitoes at a lower rate, produce fewer sporozoites, and fail to infect naive mice. Major defects in apicoplast development underly this transmission block. Our results demonstrate that resistance trapping due to differential selection between life stages is a common phenomenon. It can be exploited to develop drugs with greatly reduce levels of resistance transmission; not only in malaria but possibly in other parasites with invertebrate vectors.

IMPROVING ANTI-MALARIAL TREATMENT OF P.VIVAX BY LEVERAGING SHORT AND LONG READ DNA SEQUENCING TECHNOLOGIES

Charnaud S.C.1, 2, Munro J.E.1, Quah Y.W.1, Bahlo M.1, 2 and Mueller I.1, 2, 3 1Walter and Eliza Hall Institute, Parkville, Australia. 2University of Melbourne, Parkville, Australia. 3Institut Pasteur, Paris, France.

The Asia-Pacific region is aiming for malaria elimination by 2030, but despite falling malaria cases Plasmodium vivax persists. The reasons for the greater persistence of P. vivax relate to its unique biology, most importantly its ability to relapse from long-lasting, dormant liver stages (hypnozoites). There are no diagnostic tests for hypnozoites and the only currently available drug to clear hypnozoites is primaquine, which can cause severe haemolysis in people with glucose 6-phosphate dehydrogenase (G6PD) deficiency, and may not work in people with low cytochrome P450 2D6 activity. CYP2D6 is involved in some part in the metabolism of 20-25% of all drugs in clinical use, making it extremely interesting to determine what drugs are likely to work or be contra-indicated in a population. The advent of novel long-read sequencing now makes it possible to sequence complex genes with multiple mutations. We have developed long amplicon barcoded sequencing protocols using PacBio for G6PD and CYP2D6. We will link the genotypes with phenotypes of G6PD deficiency and CYP2D6 activity in populations in PNG and Solomon Islands. This will allow rapid assessment of pharmocogenomic characteristics of malaria endemic populations to tailor antihypnozoite therapy to achieve optimal safety and efficacy in each community. To determine the relatedness of multiple P. vivax infections over time within one person we are also developing a short amplicon based sequencing method. This will allow us to differentiate between (i) recrudescent parasites, indicating drug resistance, (ii) relapse infections from hypnozoite reactivation indicating liver stage treatment failure, and (iii) re-infections indicating high transmission. Together these sequencing methods will allow us to leverage genomics to improve current treatments and to potentially develop new treatments for P.vivax malaria.

IMMUNE RESPONSES IN SCABIES: INSIGHTS FROM A PORCINE MODEL

Mounsey K. School of Health & Sport Sciences, University of the Sunshine Coast, Maroochydore QLD.

Scabies is a skin disease caused by the parasitic mite Sarcoptes scabiei. Critical biological questions surrounding scabies remained unanswered due to the inability to undertake longitudinal study of infestation in humans. The recent development of a porcine model has facilitated in vivo studies on scabies immunopathology. Our research aims to identify factors leading to a dysregulated immune response in crusted scabies, a poorly understood clinical manifestation characterised by an extreme proliferation of mites. We have undertaken several experimental trials, focusing on defining cellular infiltrates and transcription of key cytokines in the skin over the course of infestation. Recent gene expression studies show that scabies is characterised by early immune suppression, followed by dramatic upregulation of pro-inflammatory genes in later weeks of infestation, consistent with the delayed appearance of clinical manifestations. Crusted scabies was associated with a higher number of down regulated genes at all time points, and differential expression of Th17 associated pathways. Transcriptional profiles cluster strongly according to clinical phenotype even prior to the introduction of infection, providing insights into markers of individual susceptibility and resistance to scabies. Outcomes from this research may lead to strategies to protect vulnerable subjects from contracting recurrent crusted scabies, and result in improved skin health for disadvantaged communities where this parasite is endemic.


SymPoSia WEDNESDay

SYM-58-04 SYM-58-05

SYM-59-01 SYM-59-02

RATIONAL DESIGN OF A MULTI-TARGET ANTIMALARIAL COMPOUND WITH IN VIVO ACTIVITY

Drinkwater N.1, Vinh N.2, Malcolm T.1, Charman S.3, De Koning Ward T.4, Avery V.5, Scammells P.2 and McGowan S.1 1Biomedicine Discovery Institute, Department of Microbiology, Monash University, Clayton Melbourne, VIC 3800. 2Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052. 3Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052. 4School of Medicine, Deakin University, Geelong Waurn Ponds Campus, Geelong, VIC 3216. 5Discovery Biology, Griffith Institute for Drug Discovery, Griffith University, Nathan, QLD 4111.

Malaria, particularly that caused by P. falciparum and P. vivax, remains a global health concern. Artemisinin combination therapies, the gold standard of treatment, have played a major role in reducing the malaria burden. However, parasites resistant to artemisinin treatment have emerged, and are spreading rapidly. We implemented an ambitious strategy to use rational drug discovery to develop a single compound capable of inhibiting two antimalarial drug targets, the M1 and M17 aminopeptidases, both key players in the blood stage of malaria infection. This strategy was designed to improve the efficacy of a compound by taking advantage of the synergistic effect achieved by inhibiting multiple targets within the same metabolic pathway, and additionally, to reduce the capacity of parasites to generate resistance, which occurs rapidly when parasites are treated with single-target therapeutics. We discovered potent dual inhibitors of M1 and M17 that show nanomolar in vitro activity against both P. vivax and P. falciparum (including drug resistant strains). Further, in mouse models, our most potent compound is effective against P. berghei infection after oral administration (97% reduction in parasitemia). We have therefore developed a multi-target inhibitor capable of potent activity across multiple Plasmodium species, which represents an exciting lead for further development into a novel antimalarial therapeutic.

DEFINING THE METABOLIC NETWORK OF THE MALARIA PARASITE REVEALS AN ESSENTIAL LIPID REGULATOR

Cobbold S.A.1, Dumont L.1, Marapana D.2, Triglia T.2, Ralph S.A.1, Cowman A.F.2, Tilley L.1 and Mcconville M.J.1 1Bio21 Institute, University of Melbourne, Melbourne, VIC, Australia. 2Walter and Eliza Hall Institute, Melbourne, VIC, Australia.

Asexual development of the malaria parasite is associated with major restructuring of both parasite and host erythrocyte metabolism. Most current antimalarials target metabolic processes - so in the search for novel antimalarials - it is crucial to understand the parasite’s total metabolic capacity and identify which metabolic enzymes are essential. To comprehensively define the metabolic changes that occur in human erythrocytes following malaria infection we utilized an approach that approximates a global stable-isotope labelling strategy. Using untargeted LC-MS we traced the fate of all major carbon sources using 13C-labelled substrates. Differential analyses of all mass/charge features between infected and uninfected cells has led to a draft metabolome of the malaria parasite. The draft metabolome constitutes 43% of the expected metabolome of the parasite (via genomic reconstructions), however 112 observed metabolites did not match to the expected metabolome. To further explore this ‘dark’ metabolome and validate the accuracy of the draft metabolome we targeted potential enzymes with no defined function which could potentially participate in the dark metabolome. CRISPR/Cas9 was used to generate a dual inducible knock-down system for 17 genes encoding uncharacterized metabolic enzymes, including a member of the Haloacid Dehalogense family (HAD5). We demonstrate that HAD5 is essential to normal parasite development and mediates lipid metabolism via dephosphorylating phosphatidate into diacylglycerol species. This lipid regulator was previously unrecognised in the malaria parasite and demonstrates how exploration of the dark metabolome of the parasite could lead to novel antimalarial targets.

UNDERSTANDING THE GENETIC BASIS OF CO2 RESPONSIVENESS UNDER CONTROLLED CONDITIONS: DESIGN CHALLENGES IN THE GENOMICS ERA

Pinkard E.A.1, Brookhouse M.2, Shimono H.3, Bush D.1 and Farquhar G.2 1CSIRO Land and Water. 2Research School of Biology, ANU. 3Faculty of Agriculture Iwate University.

The move towards large genomic experiments, and the substantial phenotyping required to support these experiments, challenges our capacity to conduct reproducible experiments, and correctly identify key traits. If not adequately addressed this can affect the interpretation of results and potentially lead to flawed investment decisions regarding genetic selection for superior performance. In this work, we identify potential problems associated with phenotyping large G x CO2 experiments, and suggest a possible solution. We address the following matters. (1) In fast-growing, young plant material, small differences in environmental conditions or initial plant size can substantially affect outcomes. (2) Though working with clonal material is attractive in that an identical genotypes can be placed in different environments, propagation difficulties and plagiotropic growth can be challenging (3) Size of controlled environment facilities rapidly becomes limiting for large plants, and controlling within-glasshouse spatial variation is an issue, though modern statistical analytical techniques can assist. (4) In genome-wide association experiments, there is often a need to include a large number of genotypes, implying low replication within genotypes. This reduces statistical power for some genetic parameter estimation and requires correction for glasshouse spatial variation (5) The large numbers of plants associated with genome-wide association experiments makes trait assessment difficult to standardise in time, particularly for fast-growing tree species. Many traits are time-consuming to measure and ongoing growth or trait changes due to plant developmental stage during the measurement period can confound results. We discuss the application of pre-screening as a tool in large genome x e[CO2] experiments, to reduce costs and overcome issues of access to suitable CO2-enrichment facilities. In this approach a surrogate to e[CO2] is used to select cultivars for more detailed analysis under e[CO2] conditions. It provides a potential mechanism to screen large numbers of plants at relatively low cost, although to date there has been only limited testing.

AN INTEGRATED SENSING PIPELINE TO MAP THE GENETIC LOCI ASSOCIATED WITH CANOPY RADIATION USE EFFICIENCY IN SORGHUM Barbara Geoge-Jaeggli1,2, Andries Potgieter3, James Watson3, Emma Mace1,2, Colleen Hunt1,2, Adrian Hathorn4, Mark Eldridge1, Kenneth LAWS 1, Scott Chapman4,5, Andrew Borrell 1, David Jordan1 and Graeme Hammer4

1Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Warwick, Queensland, Australia. 2Agri-Science Queensland, Department of Agriculture & Fisheries, Warwick, Queensland, Australia. 3Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Toowoomba, Queensland, Australia. 4Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, Queensland, Australia.5Agriculture & Food, CSIRO, St Lucia, Queensland, Australia.

Cereal yield advances are slowing down and will have to come from biomass production and resource use efficiency, such as improvements in photosynthetic capacity or radiation use efficiency as further improvements in harvest index are becoming more difficult to achieve. Sorghum, which is a C4 crop with more and more re-sequenced genomes and large phenotypic diversity, is an ideal model to study natural variation in traits related to photosynthetic capacity and biomass production. We have developed remote (UAV) and proximal (tractor based) sensing platforms and a data-analysis pipeline which integrates outputs from the various sensors to estimate traits related to dynamic crop growth and canopy photosynthetic capacity, such as canopy radiation use efficiency, for hundreds of field plots. We have used these remote-sensing platforms over the last two field seasons to screen an association panel largely consisting of around 700 sorghum conversion lines, which are short, early-flowering lines developed from a diverse set of exotics through the introgression of dwarfing and photoperiod-insensitivity alleles. Our aim is to map the genetic loci and identify candidate genes associated with photosynthetic capacity in sorghum and potentially other related C4 cereals and use this information to breed lines with greater resource efficiency and yield potential.


SymPoSia WEDNESDay

SYM-59-03 SYM-59-04

SYM-59-05 SYM-60-01

PHENOMICS CAPABILITY DEVELOPMENT AT AGRESEARCH FOR BETTER PASTURE AND FORAGE PRODUCTION

Ghamkhar K. AgResearch, Grasslands Campus, Palmerston North, New Zealand.

New Zealand is recognised for the quality and productivity of its meat and dairy agriculture. Due to the strategic role of forages in securing the continuity of this high quality, accurate trait measurement is extremely important in modern breeding of these forage plants. Major developments have taken place to realise this need in recent years. Beyond automatic systems in contained environments, other promising developments can turn things around for high-throughput characterisation of plants at reasonable costs. Here, I revisit AgResearch’s new approach to the application of sensors and camera technology, describing recent use of non-destructive techniques for accurate phenotyping of a range of traits from yield and growth measurement to weed detection and targeting.

RAIDERS OF THE VAVILOV ARK: SLEUTHING NEW PHOTOSYNTHETIC TRAITS FROM THE PAST

Estavillo G.M.1, Do N.L.1, Blake C.1, Harding C.1, Zwart A.2, Voss-Fels K.P.5, Barrero-Sanchez J.M.1, Hickey L.5, Sharwood R.3, Kramer D.M.4 and Condon A.G.1 1CSIRO Agriculture & Food, Canberra, ACT 2601, Australia. 2CSIRO Data61, Canberra, ACT 2601, Australia. 3The Australian National University, Canberra, ACT 2601, Australia. 4Michigan State University, East Lansing, MI 48824, USA. 5QAAFI, The University of Queensland, St Lucia, QLD 4072, Australia.

Improving photosynthetic capacity is seen as an obvious target for increasing total crop biomass and yield. However the complexity of photosynthesis and lack of high-throughput screening methods hampers the identification of genotypes with improved carbon fixation rates. We have used an affordable, hand-held chlorophyll fluorescence spectrometer (PhotosynQ) to screen for genetic diversity of photosynthetic traits in a set of wheats pre-dating modern breeding (from the Vavilov collection). There was a large range in the rate of linear electron flow (LEF) among 60 selected Vavilov entries representing the whole genetic diversity of the panel. LEF varied from 21 to 80 μmol m-2 s-1 at low light, and from 72 to 167 μmol m-2 s-1 at five times higher irradiance (high light). Additionally, there was large variation in the magnitude of the change in LEF from low to high light, suggesting the presence of plasticity to light acclimation among these lines. There was also considerable variation in how light-derived energy was apportioned between photochemistry and other energy dissipation mechanisms (i.e., heat loss). Concurrent gas exchange, coupled with conventional chlorophyll fluorescence measurements and biochemical analyses were also performed on flag leaves of some selected lines. The results indicated that PhotosynQ could be deployed as a useful screening tool for photosynthetic traits and that screening novel genetic resources could enable the discovery of new traits or enhanced expression of existing traits to improve wheat photosynthesis.

TACKLING THE PHYSIOLOGICAL PHENOTYPING BOTTLENECK WITH LOW-COST, ENHANCED-THROUGHPUT GAS EXCHANGE AND CEPTOMETRY

Salter W.T.1, Gilbert M.G.2, Merchant A.1 and Buckley T.N.2 1School of Life and Environmental Sciences, Sydney Institute of Agriculture, The University of Sydney. 2Department of Plant Sciences, University of California, Davis.

High throughput phenotyping platforms (HTPPs) are increasingly adopted in plant breeding research due to developments in sensor technology, unmanned aeronautics and computing infrastructure. Most of these platforms rely on indirect measurement techniques therefore some physiological traits may be inaccurately estimated whilst others cannot be estimated at all. Unfortunately, existing methods of directly measuring plant physiological traits, such as photosynthetic capacity (Amax), have low throughput and can be prohibitively expensive, creating a bottleneck in the breeding pipeline. We have addressed this issue by developing new low-cost enhanced-throughput phenotyping tools to directly measure physiological traits of wheat (Triticum aestivum). Our eight-chamber multiplexed gas exchange system, OCTOflux, can directly measure Amax with 5-10 times the throughput of conventional instruments, whilst our handmade ceptometers, PARbars, allow us to monitor the canopy light environment of many plots simultaneously and continuously across a diurnal cycle. By custom-building and optimizing these systems for throughput we have kept costs to a minimum, with OCTOflux costing roughly half that of commercially available single-chamber gas exchange systems and PARbars costing approximately 95% less than commercial ceptometers. We recently used these tools to identify variation in the distribution of Amax relative to light availability in 160 diverse wheat genotypes grown in the field. In a two-week measurement campaign we measured Amax in over 1300 leaves with OCTOflux and phenotyped the diurnal light environment of 418 plots using 68 PARbars. These tools could be readily modified for use with any plant functional type and also be useful in validating emerging HTPPs that rely on remotely sensed data to estimate photosynthetic parameters.

IMPACT OF SIGNALLING ACTIVITY ON THE ALLOCATION OF GERM LAYER PROGENITORS: INSIGHTS FROM THE SPATIAL TRANSCRIPTOME OF GASTRULA-STAGE EMBRYO

Tam P.P.L.1, 2 1Embryology Unit, Children’s Medical Research Institute. 2School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney.

Gastrulation is a critical milestone of embryogenesis at which the primary germ layers are formed and the multipotent embryonic cells are allocated to the progenitors of tissue lineages within the germ layers. Analysis of the spatial transcriptome of the epiblast showed that spatially delimited agonistic and antagonistic signalling activity in the epiblast and signalling conditions for the derivation and maintenance of the epiblast stem cells underpin the restriction of the tissue potency of epiblast cells, culminating in the generation of lineage-restricted progenitors for the germ layers and the regionalization of tissue precursors of major body parts in the germ layers during gastrulation.


SymPoSia WEDNESDay

SYM-60-02 SYM-60-03

SYM-60-04 SYM-60-05

EXPLOITING HEPARAN SULFATE PROTEOGLYCANS-GROWTH FACTOR INTERACTIONS TO DIRECT MESENCHYMAL STEM CELL NEUROGENESIS

Yu C., Okolicsanyi R.K., Griffiths L.R. and Haupt L.M. IHBI-QUT, Genomics Research Centre, School of Biomedical Sciences, 60 Musk Avenue, Kelvin Grove, Q 4059.

According to the World Health Organisation, neurological disorders, including trauma, affect 1 in 6 of the world population. With no current effective treatments, much current effort is focussed on the development and improvement of cellular replacement therapies. In particular, the propagation and direction of stem cells into specific neural lineages. Mesenchymal stem cells (MSCs) are capable of neurogenic differentiation but lack high frequency differentiation efficiency, particularly following transplantation. Heparan sulfate proteoglycans (HSPGs), including syndecans (SDCs) and glypicans (GPCs), are ubiquitous within the stem cell microenvironment and essential to numerous cellular activities, including self-renewal, proliferation and differentiation. In this study we examine the role of HSPGs in human MSC (hMSCs) neurogenesis and as potential biomarkers for therapeutic applications. Using two neural differentiation protocols: i.) direct terminal differentiation (TD), and ii.) terminal differentiation via hMSC-induced neurosphere formation (TD via hMSC-INs), hMSC populations (n=3) were directed towards neural lineages with growth factor supplementation for up to 14 days. Brain-derived neurotrophic factor (BDNF) and platelet-derived growth factor (PDGF) bind to HSPGs and have been implicated in neuronal and glial lineage differentiation, respectively. HSPG and neural lineage marker gene expression profiles (focussing on the core protein SDCs and GPCs) were examined under neural specific culture conditions by Q-PCR, WB and ICC. Gene expression analysis identified BDNF-treated cultures to express higher self-renewal (NANOG, POU5F1 and SOX2 (P<0.05)), and oligodendrocyte markers (Olig1 (P<0.05) and Olig2 (P<0.05)) when compared to PDGF-treated cultures. PDGF cultures maintained the highest cell numbers and expressed mature neuronal markers (MAP2 and TUBB3). Analysis of the culture HSPG profiles identified SDCs 1-4 and GPC1, -4, and -6 are likely involved in hMSC neural differentiation. Our data suggests HSPGs, in particular members of the SDC and GPC families, may play key roles during hMSC neurogenesis and are potentially central to controlling human neurogenesis.

ELUCIDATING THE IMPACT OF APP IN DS PATHOGENESIS USING STEM CELL MODELS

Griffiths R.1, Fortuna P.1, Ovchinnikov D.1, Balachandran A.1, Wells C.2, Powell J.3, Mar J.1 and Wolvetang E.1 11Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, QLD 4072, Australia. 2Centre for Stem Cell Systems, MDHS, University of Melbourne, Melbourne, VIC 3010, Australia. 3Garvan Institute of Medical Research, Sydney, Australia.

Down syndrome (DS) is due to trisomy of HSA21. Which genes on chromosome 21 are responsible for the pronounced Alzheimer’s disease and neurocristopathy features of Down syndrome remains largely unclear. Here we use CRISPR-assisted genome manipulation to normalize the copynumber of APP or DYRK1A in DS induced pluripotent stem cells. Comparison of cellular phenotypes and transcriptomes of these isogenic euploid, DS and genome edited DS IPSC following differentiation into cortical neurons in 2D or cerebral organoid setings or analysis neural crest cells reveals important roles for APP in amyloidigenic but not tau-pathology of Alzheimers disease and the strong impact of DYRK1A dosage on neural crest cell generation, survivial and migration. We further show that these in vitro models can be used to test the efficiacy of therapeutic compounds.

FROM SERVANT TO MASTER: THE RIBOSOME’S INSTRUCTIVE ROLE IN HEMATOPOIETIC CELL FATE DETERMINATION

Chahal A.S.1, 2, Mitchell N.1, Zaytseva O.1, Geroge A.1, Hannan R.D.1 and Quinn L.M.1 1JCSMR, Australian National University. 2University of Melbourne.

Transcriptional control of gene expression is viewed as the key instructor of stem cell fate. In contrast, dogma has relegated ribosome biogenesis and mRNA translation as a necessary, but passive, contributor to cell and tissue specification during development. Intuitively, impaired ribosome function should (and usually does) cause reduced tissue growth and stunted development. Inexplicably, cancer arises more frequently in patients with ribosomal protein (RP) loss, particularly in the blood lineage. Our recently developed Drosophila hematopoietic models may provide a rationale for the prevalence of leukemia in patients with reduced RP levels. Following tissue specific depletion of the RPs (s19 and s24) most frequently mutated in the ribosomopathy Diamond Blackfan Anemia (DBA), we observe stem cell fate defects, overproliferation and massive overgrowth of the blood compartment. Interestingly, knockdown of RPs19 and RPs24 differentially altered stem and progenitor cell differentiation, which suggests depletion of particular RPs from ribosomes might actively disrupt cell fate by modulating the classes of transcripts translated. Indeed, Mass Spec data from the RP deficient lymph glands revealed increased protein abundance for several factors previously implicated in driving developmental programs of growth and differentiation, including master transcriptional regulators and chromatin remodeling machinery. As RNA-Seq revealed mRNA transcript levels for these factors were unchanged, we hypothesise that their defective translation underlies the stem cell fate defects and blood compartment overgrowth. A combination of TEM and Bi-FC analysis revealed that RP depletion did not significantly reduce mature ribosomes in the cytoplasm, suggesting that altered protein abundance is not due to competition for ribosomes. We are currently using mass spec to determine if RPs19 or s24 depletion alters ribosomal composition in Drosophila hematopoietic cell lines, and whether mRNA species encoding stem cell fate proteins are differentially translated using RNA-seq of polysome fractions.

INNER EAR ORGANOIDS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS: COMPARISONS TO HUMAN FOETAL INNER EAR

Mattei C.1, 2, Lim R.3, Drury H.R.3, Tadros M.A.3,Nasr B.1, 4, Chatterton R.1, 2, Kulkarni T.1, 2, Jamshidi P.1, 2, D’Abaco G.2, Nayagam B.A.5 and Dottori M.1, 2, 6 1Centre for Neural Engineering, Melbourne School of Engineering, University of Melbourne, Victoria, Australia. 2Department of Biomedical Engineering, Melbourne School of Engineering, University of Melbourne, Victoria, Australia. 3School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle, NSW, Australia. 4Department of Electrical and Electronic Engineering, Melbourne School of Engineering, University of Melbourne, Victoria, Australia. 5Departments of Audiology and Speech, Pathology & Ophthalmology, University of Melbourne, Victoria, Australia. 6Illawarra Health and Medical Research Institute, University of Wollongong, NSW, Australia.

Introduction: In mammals, sensory hair cells do not regenerate. Consequently, the derivation of inner ear tissue from human pluripotent stem cells (hPSC) offers an opportunity to study human inner ear development and provides a platform for drug screening and disease modelling. Methods: A dynamic three dimensional rotary cell culture system was used to derive inner ear organoids from human PSCs for 16 weeks in-vitro. Differentiation and mechanosensitiviy of hPSCs-derived organoids were examined using a combination of qPCR, immunofluorescent labelling, and AM142 staining. Helium microscopy and electrophysiology compared the anatomical and physiological characteristics of inner ear organoids to foetal human inner ear. Results: Inner ear organoids show temporal expression of key developmental hair cell markers including Pax2, Atoh1, MyosinVIIa, and CtBP2 by immunofluorescence and qPCR. AM143 fluorescence in organoid cells is indicative of mechanosensitivity. Cells have outward currents (350 pA to 5 nA) consistent with developing human type II vestibular hair cells (12-16 weeks gestation). A subset of oganoid cells also have sodium currents. Striking morphological similarities were detected between inner ear organoids and developing inner ear using helium microscopy. Conclusion: We describe a novel three dimensional system for modelling human inner ear development using rotary cell culture. Preliminary data suggests this system is capable of generating a population of inner ear hair cells which resemble an early vestibular phenotype.


SymPoSia WEDNESDay

SYM-61-01 SYM-61-02

SYM-61-03 SYM-61-04

THE COMPANION OF CELLULOSE SYNTHASE 1 CONTROLS MICROTUBULE DYNAMICS THROUGH A TAU-LIKE MECHANISM TO CONFER SALT TOLERANCE IN PLANTS

Kesten C.1, Wallmann A.2, Oschkinat H.2 and Persson S.1 1School of Biosciences, University of Melbourne, Parkville 3010 VIC, Melbourne, Australia. 2Leibniz-Forschungsinstitut fur Molekulare Pharmakologie (FMP), NMR-supported Structural Biology, Robert-Rossle-Str. 10, 13125, Berlin, Germany.

Microtubules are filamentous structures necessary for cell division, motility and morphology. Microtubule dynamics are critically regulated by microtubule-associated proteins (MAPs). We outline the molecular mechanism by which the MAP, COMPANION OF CELLULOSE SYNTHASE1 (CC1), controls microtubule-bundling and dynamics in plants under salt stress conditions. CC1 contains an intrinsically disordered N-terminus that joins microtubules through conserved hydrophobic regions at evenly distributed foci. Structural data on the microtubule-bound CC1 N-terminus and mutation studies revealed the regions, and specific amino acids, that contribute to microtubule-binding. The importance of these regions and amino acids was confirmed through in vivo live cell imaging, which also explains how CC1 maintains cellulose synthesis during salt exposure. Surprisingly, the microtubule-binding mechanism of CC1 is remarkably similar to that of the prominent neuropathology-related protein Tau. Hence, we outline how MAP functions have converged during evolution across animal and plant cells.

DEVELOPMENT OF THREE-DIMENSIONAL CULTURE MODELS TO STUDY CANCER DEVELOPMENT AND METASTASIS

Bray L.J.1, Murekatete B.1, Jaeschke A.1, Koch M.1, Tsurkan M.V.2, Risbridger G.3, Werner C.2 and Hutmacher D.W.1 1Centre for Regenerative Medicine, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia. 2Leibniz Institute for Polymer Research, Max Bergmann Center of Biomaterials, Dresden, Saxony, Germany. 3Monash University, Clayton, Victoria, Australia.

The culture of cells on rigid 2D substrates, such as tissue culture plastic, does not recreate the dynamic and highly complex tissue microenvironment, but rather distorts cell-integrin and cell-cell interactions, affecting gene expression, signal transduction, cell proliferation and differentiation, and thus is physiologically irrelevant. Naturally, cells are embedded in an extracellular matrix (ECM) that provides not only architectural support, but also chemical and mechanical signals to cells in vivo. Hydrogels prepared from star-shaped poly(ethylene glycol) (PEG) and maleimide-functionalised heparin provide a potential matrix for use in developing three dimensional (3D) models. We have previously demonstrated that these hydrogels support tri-cultures of human umbilical vein endothelial cells (HUVECs) with mesenchymal stromal cells (MSCs) and breast or prostate cancer epithelial cell lines. We extended this body of work to study the effects of cancer associated fibroblasts (CAFs) on tumour angiogenesis. Also, we investigated the ability to produce a tri-culture mimicking tumour angiogenesis with epithelial cells, and tissue-specific microvascular endothelial cells and fibroblasts. Cultures were analysed via immunostaining and observed using confocal microscopy. We demonstrated the ability of starPEG-heparin hydrogels to support co- and tri-cultures of primary patient-derived prostate or breast cells, with capillary formation by the endothelial cells. Interactions were visualised between all cell types via confocal microscopy. Our results confirm the suitability of hydrogels constructed from starPEG-heparin for the co-cultivation of primary patient-derived tissue specific cells to study cell-cell and cell-matrix interactions in a 3D microenvironment. This represents a step forward in the development of 3D culture models to study the pathomechanisms of various cancer types.

AN UNBIASED CHEMICAL SCREEN OF ACTIN ORGANISATION ENABLES DRUG DISCOVERY AND MECHANISTIC ANALYSES OF THE CYTOSKELETON

Bryce N.S., Lock J.G., Stehn J., Hardeman E.C. and Gunning P.W. School of Medical Sciences, UNSW Australia, Sydney, NSW, Australia.

The actin cytoskeleton is a highly organised and dynamic system that processes both external and internal signalling cues into changes in the structure of cellular architecture. Dysregulation of actin organisation is seen in many tumour types and disease processes, however actin itself is not a clinical drug target, due to cardiotoxic side-effects. Actin binding proteins are a diverse group of proteins that have distinct functions and tissue and cellular expression profiles, and as such present as more attractive therapeutic targets. This project aims to identify compounds that impact on the organisation of the actin cytoskeleton either through signalling pathways or direct actin binding proteins. We screened 114,400 diverse compounds in an image-based screen using F-actin organisation as a readout, quantifying 75 features per condition and defining >2600 unknown compounds impacting actin organisation. Unbiased clustering of responses delineated 27 distinct actin phenotypes. This suggests that there are a discrete number of ways that polymeric actin can be reorganised in a mammalian cell. We hypothesized that similar actin phenotypes may have similar drug targets, establishing 3 strategies to accelerate mechanism discovery: 1) Comparison with known actin compounds used as positive controls in the screen (>260 compounds identified); 2) Comparison with known genetic changes (3 talin-binding compounds defined), and; 3) Retrospective integration (RetInt) of additional positive controls into the original screen data (2 ROCK inhibitors defined). The RetInt strategy may enhance accelerated drug discovery by providing near-unlimited capacity to extend mechanistic analyses in this and other phenotypic screens.

ACTIN POLYMERIzATION ALTERS NUCLEAR ARCHITECTURE IN RESPONSE TO DNA REPLICATION STRESS TO MAINTAIN GENOME STABILITY

Lamm-Shalem N.1, Masamsetti V.P.1, Biro M.2 and Cesare A.J.1 1Genome Integrity Unit, Children’s Medical Research Institute, University of Sydney, Westmead, NSW 2145, Australia. 2EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia.

Impediments that that slow the rate of DNA replication are collectively referred to as replicaiton stress. Replication stress is the main driver of genome instability in early cancer development and is recognized as a hallmark of cancer. Actin is a cytoskeletal protein that forms filaments to provide cells with mechanical support and driving force for movement. While actin is traditionally considered a cytoplasmic protein, recent evidence indicates actin polymerization can also occur inside the nucleus. However, the role for nuclear actin fibres, the mechanism(s) triggering their polymerization, and the impact of nuclear actin on the genome remains unclear. Using live-cell and super-resolution imaging, chromatin fibre analysis, biochemistry, cell and molecular biology, we discovered that actin polymerization plays a prominent role in the replication stress response. Consistent with induced DNA replication stress, pharmacological inhibition of actin polymerization in human cells resulted in S-phase elongation, reduced DNA replication rate, shortened distance between replication origins, and increased occurrence of micronuclei and anaphase abnormalities. Pharmacological replication stress induced ATR and mTOR-dependent nuclear actin polymerization, which altered the nuclear architecture through the expansion of nuclear volume and directed migration of stalled replication forks along nuclear actin fibres. Inhibiting ATR, mTOR or actin polymerization, suppressed replication stress-dependent nuclear alteration, prevented fork migration, and prevented the restart of stalled replication forks. Preliminary data indicate co-localization of stalled replication forks with certain repair factors is compartmentalized to the nuclear periphery, suggesting that actin polymerization facilitates movement of stalled forks to the nuclear periphery for repair. Cumulatively, these data reveal a novel pathway where actin dependent forces shape the nucleus in response to replication stress to maintain genome health. These findings suggest nuclear actin polymerization may have additional nuclear roles that that impact genome function.


SymPoSia WEDNESDay

SYM-61-05 SYM-62-01

SYM-62-02 SYM-62-03

ASYMMETRICAL DISASSEMBLY OF APOPTOTIC CELLS AND THE MECHANISMS UNDERPINNING THIS PROCESS

Jiang L., Tixeira R., Atkin-Smith G., Caruso S., Phan K., Baxter A., Hulett M. and Poon I. La Trobe Institute for Molecular Science, La Trobe University.

Apoptotic bodies (ApoBD) generated from apoptotic cells is considered a major class of extracellular vesicles that could facilitate cell clearance and intercellular communication. Efficient disassembly and clearance of dying cells is significant in avoiding accumulation of cellular debris, also the release of pro-inflammatory intracellular contents. Thus, the disassembly of apoptotic cells may also play a key role in preventing autoimmune diseases such as systemic lupus erythematosus. However, the mechanisms underpinning this process is not entirely clear yet. When observing T cells undergoing apoptosis by time-lapse microscopy, we discovered the establishment of asymmetric form “octopus structure” during apoptosis. Moreover, by using fluorescent microscope, we discovered that cellular organelle (e.g. nucleus, mitochondria, Golgi apparatus and lysosomes) and cell surface markers are also distributed asymmetrically as well during apoptotic membrane blebbing, which will then lead to asymmetrical disassembly and the generation of distinct apoptotic bodies with different contents and morphological characteristics. Additionally, we discovered similar morphology in different cell lines during apoptosis, indicating a unified cellular content sorting mechanism during apoptotic cell disassembly. Furthermore, we found that dynamic blebbing is essential for asymmetrical distribution of nuclear content and ROCK1 inhibitor GSK can inhibit this process efficiently. Overall, these data uncover a novel characteristic of apoptotic cell disassembly and molecular regulators involved in this process.

THE ARCHITECTURE OF THE MEMBRANE ASSOCIATED RETROMER-SORTING NEXIN COMPLEX REVEALED BY CRYO-ELECTRON TOMOGRAPHY

Leneva N.1, Kovtun O.2, Ariotti N.3, Teasdale R.4, Owen D.1, Briggs J.2 and Collins B.4 1CIMR, Cambridge. 2MRC-LMB, Cambridge. 3UNSW, Sydney. 4UQ, Brisbane.

Compartmentalisation is a defining feature of all eukaryotic cells, and we have evolved highly sophisticated protein machineries to control the flow of transmembrane molecules and membrane lipids between different organelles. The retromer protein complex is required for generating cargo-selective tubulovesicular carriers from endosomal membranes (1,2). Retromer-mediated trafficking is an essential process in all eukaryotes, controlling the cellular localisation and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders (3). However, how retromer is assembled and how it is recruited to form membrane tubules remains unknown. Here we describe the structure of the Chaetomium thermophilum trimeric retromer complex (Vps26-Vps29-Vps35) assembled on membrane tubules with the sorting nexin protein Vps5, using X-ray crystallography, molecular modelling and cryo-electron tomography with sub-tomogram averaging at sub-nanometre resolution. The structure reveals two interwoven layers where Vps5 forms a membrane-associated lattice, while the outer lattice consists of arches of retromer that extend away from the membrane surface. Vps35 forms the legs of these arches, Vps29 sits at the apex where it is free to interact with regulatory factors, and the feet of the arches connect to each other through Vps26. Vps26 also forms the primary interface with Vps5 via the same site previously shown to mediate Snx3 and cargo interactions (4), suggesting the existence of distinct retromer-sorting nexin complexes. The architecture of the tubulovesicular retromer coat revealed here provides key insights into the conserved mechanisms of retromer assembly and retromer-mediated endosomal trafficking.

STRUCTURAL INSIGHTS INTO THE ARCHITECTURE AND MEMBRANE INTERACTIONS OF THE CONSERVED COMMD PROTEINS

Healy M.1, Hospenthal M.2, Hall R.1, Chilton M.3, Chandra M.1, Chen K.1, Cullen P.3, Lott S.2, Collins B.1 and Ghai R.1 1Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, 4072, Australia. 2School of Biological Sciences, The University of Auckland, Auckland 1142, New Zealand. 3School of Biochemistry, Biomedical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.

The COMMD proteins are a conserved family of proteins with central roles in intracellular membrane trafficking and transcription. They form oligomeric complexes with each other and act as components of a larger assembly called the CCC complex, which is localized to endosomal compartments and mediates the transport of several transmembrane cargos. How these complexes are formed however is completely unknown. Here, we have systematically characterised the interactions between several human COMMD proteins, and determined structures of COMMD proteins using X-ray crystallography and X-ray scattering to define the core principles of their homo- and heteromeric assembly. All COMMD proteins possess an α-helical N-terminal domain, and a highly conserved C terminal domain that forms a tightly interlocked dimeric structure responsible for COMMD-COMMD interactions. The COMM domains also bind directly to components of CCC and mediate non-specific membrane association. Overall these studies show that COMMD proteins function as obligatory dimers with conserved domain architectures.

T CELL RECEPTOR CLUSTERING: A MECHANISM OF SIGNAL TRANSDUCTION

Gaus K.1, 2 1EMBL Australia Node in Single Molecule Science. 2ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney, Australia.

Antigen recognition by the T cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. We develop single-molecule localization microscopy (SMLM) approaches and novel analysis to determine how spatial organization regulates signal initiation and propagation. For example, we used SMLM data to map the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggered and non-triggered receptor copies. We found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. This lead us to propose a model in which antigen recognition is first translated into receptor clustering and then the density of receptor nanoclusters is translated into signaling. This model may explain how T cells can respond to both the affinity and dose of pMHC molecules with a common signal transduction mechanism (Pageon et al. PNAS 2016). We also developed novel FRET sensors to monitor the rate of receptor clustering (Maet al. Nat Commun 2017) and a sensor that reports membrane charges (Ma et al. Nat Biotech 2017) to understand how biophysical properties of the plasma membrane contribute to TCR signaling.


SymPoSia WEDNESDay

SYM-62-04 SYM-62-05

SYM-63-01 SYM-63-02

DEFINING ALLOSTERIC BINDING SITES AND BIASED AGONISM OF CLASS C G PROTEIN-COUPLED RECEPTORS

Gregory K.J. Drug Discovery Biology and Dept. of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University.

Class C G protein-coupled receptors (GPCRs) are cell surface transmembrane proteins that are attractive therapeutic targets for multiple disorders. Our work focuses on two well-characterised members, metabotropic glutamate receptor 5 (mGlu5) and the Calcium-sensing receptor (CaSR), which respond to glutamate and extracellular Ca2+ respectively. Allosteric modulators that generally bind to sites within the 7 transmembrane-spanning domains, which are distinct from the endogenous agonists, are of significant interest due to their ability to fine-tune GPCR activity. Positive allosteric modulators (PAMs) enhance, whereas negative allosteric modulators (NAMs) inhibit the orthosteric agonist response. Allosteric modulators offer greater subtype selectivity and the potential to fine-tune GPCR activity. Indeed, cincalcet, a CaSR PAM, was the first GPCR allosteric modulator to enter the clinic. Discovery programs commonly rely on potency determinations when screening for allosteric modulators, however, we have shown this approach lacks sufficient rigor and can result in misinterpretation of activity. We apply rigorous analytical methods and investigate multiple measures of activation to dissect the structural basis and functional consequences of class C GPCR allosteric modulation. We have found that Class C GPCR allosteric modulators can differentially activate and/or modulate distinct signalling pathways, referred to as biased agonism and biased modulation, respectively. Distinct bias profiles can be linked to in vivo efficacy and may be predictive of adverse effect liability. Moreover, previously unappreciated biased modulation has revealed that ligands originally classified as selective ligands at other class C GPCRs have off-target activity at mGlu5. Through structure-function analyses we are defining the key ligand-receptor interactions that govern these effects. Ultimately, our work will provide a better understanding of the mechanisms driving on-target therapeutic versus adverse effects and provide a framework for future rational discovery campaigns for biased modulators that can fine-tune receptor activity at the pathway level.

CRYO-EM STRUCTURE OF THE HUMAN ADENOSINE A1 RECEPTOR-GI2-PROTEIN COMPLEX BOUND TO ITS ENDOGENOUS AGONIST

Draper-Joyce C.J.1, Khoshouei M.2, 3, Thal D.M.1, Liang Y.L.1, Furness S.G.B.1, May L.T.1, Wootten D.1, Sexton P.M.1, Glukhova A.1 and Christopoulos A.1 1Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia. 2Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany. 3Norvatis Institutes for Biomedical Research, Norvatis Pharma AG, 4002 Basel, Switzerland.

G protein-coupled (GPCRs) are responsible for the majority of cellular responses to hormones, neurotransmitters, and a variety of other small molecules. In recent years there has been an exponential growth in the amount of determined inactive GPCR structures, however, there still remains a dearth of active-state, G-protein-bound, GPCRs. To date, all solved active-GPCRs have been coupled to the stimulatory Gs protein. The class A adenosine A1 receptor (A1R) is a GPCR that preferentially couples to the inhibitory Gi/o family of heterotrimeric G-proteins, has been implicated in numerous diseases, yet remains poorly targeted. We have recently solved the 3.6 Å structure of the human A1R in complex with adenosine and heterotrimeric Gi2-protein determined by Volta phase plate cryo-electron microscopy. Compared to inactive A1R, there is contraction at the extracellular surface in the wide orthosteric binding site that is mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G-protein engages the A1R primarily via amino acids in the C-terminus of the Gαi α5 helix, concomitant with a 10.5 Å outward movement of A1R transmembrane domain 6. Comparison with the agonist-bound β2adrenergic receptor-Gs-protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active A1R structure provides novel molecular insights into receptor/G-protein selectivity.

NEW PATHWAYS IN HEPATIC STEATOSIS, INSULIN RESISTANCE, AND TYPE 2 DIABETES

Koay Y.C.1, 2, Yang P.3, Chen D.L.4, Jenkins A.B.4, Wood C.5, Li M.3, Greenfield J.R.4, 6, 7, Samocha-Bonet D.4, 6 and O’Sullivan J.F.1, 2, 8 1Sydney Medical School, The University of Sydney, Australia. 2Heart Research Institute, Sydney, Australia. 3School of Mathematics and Statistics, University of Sydney, Australia. 4Diabetes and Metabolism Division, Garvan Institute of Medical Research, Sydney, Australia. 5Talented Scientists Progam, School of Biomedical Science, The University of Sydney, Australia. 6Faculty of Medicine, UNSW, Australia. 7Department of Endocrinology and Diabetes Centre, St Vincent’s Hospital, Sydney, Australia. 8Royal Prince Alfred Hospital, Department of Cardiology, Sydney, Australia.

The global obesity epidemic, leading to non-alcoholic fatty liver disease (NAFLD) and Type 2 Diabetes (T2D), continues unabated despite sustained efforts to combat it, and is our greatest modern healthcare challenge. NAFLD is the commonest form of liver disease in the Western world, affecting one in three people in the general population, 90% of obese patients with type 2 diabetes (T2D), and 5.5 of 6 million Australians with liver disease – accounting for much of the $51 billion annual cost to our healthcare system. Although exact mechanisms remain unclear, accumulation of liver fat is a principal cause of T2D. We have recently discovered a new pathway in hepatic steatosis that independently predicts type 2 diabetes over 12 years in advance. Our subsequent work has revealed novel lipid biomarkers for NAFLD that can distinguish between liver insulin resistance and muscle insulin resistance. Three representative lipids alone can accurately classify patients according to the organ driving insulin resistance with >99% accuracy. We have also recently revealed how a common SNP (MAF.

EVALUATION OF LIVER SECRETED PROTEINS IDENTIFIES NOVEL REGULATORS OF GLUCOSE METABOLISM

Watt M.J. and Montgomery M.K. Department of Physiology, University of Melbourne, Melbourne, 3010.

Obesity is a risk factor for the development of inter-related complications including dyslipidemia, non-alcoholic fatty liver disease (NALFD), cardiovascular disease and type 2 diabetes. An accumulation of lipid in the liver, which is clinically known as hepatic steatosis, is a pathologic abnormality that is common in obese and type 2 diabetes patients. Hepatic steatosis occurs when fatty acid supply outweighs fatty acid demand and occurs in a time-course that usually precedes the induction insulin resistance and type 2 diabetes. This presentation describes our laboratories use of proteomics to evaluate how ‘heptokine’ secretion is altered in murine models of NAFLD and non-alcoholic steatohepatitis. We report on the pre-clinical validation of several liver secreted factors that either cause insulin resistance and disturbances in systemic metabolic homeostasis or enhance glycemic control in diabetes. These studies have unravelled previously appreciated biology and pave the way for potential therapeutic intervention for type 2 diabetes.


SymPoSia WEDNESDay

SYM-63-03 SYM-63-04

SYM-63-05 SYM-64-01

PRECLINICAL CHARACTERISATION OF JAK1/JAK2 INHIBITORS FOR TREATMENT OF TYPE 1 DIABETES

Trivedi P.M.1, Scott N.A.1, Jenkins M.R.2, Brodnicki T.C.1, Kay T.W.1 and Thomas H.E.1 1St Vincents Institute, Fitzroy, Victoria, Australia. 2Walter and Eliza Hall Institute, Parkville, Victoria, Australia.

There is an opportunity to repurpose therapeutics from other diseases to type 1 diabetes, especially when there is evidence for overlapping mechanisms. We generated directed networks based on the phenotypes of gene knockout non-obese diabetic (NOD) mice, a mouse model of type 1 diabetes. The JAK-STAT pathway was important in T cells and pancreatic beta cells, the target of T cell-mediated destruction. We tested the hypothesis that blocking the response to cytokines that activate the JAK-STAT pathway would prevent autoimmune diabetes and tested the effects of drugs that selectively inhibit JAK1/JAK2. JAK inhibitors successfully blocked IFNγ-induced phosphorylation of STAT1 and IFNγ-induced MHC class-I upregulation in mouse and human islets. Mouse islets were protected from CTL-mediated killing in vitro by JAK inhibitors. Time-lapse microscopy shows the inhibitor prevented the direct interaction between CTLs and beta cells, reducing calcium flux in the CTL and synapse duration. JAK inhibitors blocked the effect of cytokines on beta cells in vivo by inhibiting MHC class I upregulation, and reduced infiltration of immune cells into islets. Furthermore, NOD mice treated with JAK inhibitors were protected from autoimmune diabetes, and diabetes was reversed in newly diagnosed NOD mice. Interestingly, deficiency of receptors for all three interferons (IFNγ, IFNα, IFNλ) did not prevent diabetes in NOD mice, suggesting that JAK inhibitors block cytokine signals other than interferons that are crucial for diabetes development. Future work will study this. Our work provides mechanistic groundwork for re-purposing clinically approved JAK inhibitors for type 1 diabetes, and provides a platform for testing many of the >150 kinase inhibitors under development for other diseases for efficacy in autoimmune diabetes.

THE ROLE OF TRAFFICKING REGULATOR OF GLUT4 1 (TRARG1) IN GLUT4 TRAFFICKING

Duan X.1, Krycer J.R.1, Cooke K.C.1, Yang G.1, James D.E.1, 2 and Fazakerley D.J.1 1Charles Perkins Centre, School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia. 2Sydney Medical School, The University of Sydney, Sydney, NSW 2006, Australia.

Insulin lowers blood glucose, in part, by enhancing glucose transport into adipose and muscle tissues through the redistribution of glucose transporter GLUT4 from specialised intracellular GLUT4 storage vesicles (GSVs) to the plasma membrane. This process is impaired in insulin resistance, which is a precursor to numerous metabolic disorders including Type 2 diabetes. Trafficking regulator of GLUT4 1 (TRARG1) localises to GSVs and positively regulates GLUT4 trafficking in response to insulin. Understanding how TRARG1 regulates GLUT4 is of interest since lower TRARG1 expression may contribute to impaired GLUT4 trafficking in insulin resistance. To begin to address this we have used a combination of bioinformatics prediction tools and biochemical assays to define the membrane topology of the 173-amino acid mouse TRARG1. This revealed that TRARG1 contains a single transmembrane domain at its C-terminus with a cytosolic N-terminus and extracellular/luminal C-terminus. Interestingly, our studies also revealed a re-entrant loop that also confers TRARG1 membrane association. Based on this model of TRARG1 membrane topology, our current studies are focussed on characterising TRARG1, including identifying the signals that determine its localisation to GSVs, and determining how TRARG1 regulates GLUT4 trafficking.

TREATMENT WITH NAD+ PRECURSORS IMPROVE WOUND HEALING IN DIABETIC AND OLD MICE

Das A.1, 2, Giatsidis G.2, Garg N.2, Wu L.1, Orgill D.2 and Sinclair D.1, 2 1University of New South Wales, Sydney. 2Harvard Medical School, USA.

Delayed wound healing is one of the major complications in diabetes and ageing, which may result in limb amputation. Recent reports suggest that Sirtuins, a family of NAD+-dependent protein deacylases, play an important role in skin wound healing in both diabetes and ageing. Transgenic mice systemically overexpressing SIRT1, the mammalian sirtuin orthologue, showed improved skin wound healing in old and diabetic mice. The activity of SIRT1 is dependent on the levels of NAD+ inside the cell. The NAD+levels however decrease in different tissues with ageing and diabetes, resulting in declining SIRT1 activity. Boosting the levels of NAD+ using NAD+ boosters is emerging to be an effective strategy to activate and reproduce the effects of Sirtuins in ageing and diabetes. Here, we tested the efficacy of two NAD precursors – Nicotinamide riboside (NR) and Nicotinamide Mononucleotide (NMN) – in wound healing in old and diabetic mice. Topical administration of both of these compounds resulted in enhanced wound healing, increased wound vascularity, and significant improvement in the ischemic neovascular response. These findings have implications to significantly improve quality of life of diabetic patients and ageing population who have severely impaired wound-healing capabilities after injuries.

A-TO-I RNA EDITING - NOT ALL EVENTS ARE CREATED EQUAL

Heraud-Farlow J.E. St. Vincent’s Institute of Medical Research, Fitzroy, Vic, 3065 Australia.

Adenosine-to-inosine (A-to-I) editing of double-stranded RNA (dsRNA) by ADAR proteins is a highly prevalent form of RNA base modification in higher eukaryotes. Tens of thousands of A-to-I editing events are defined in the mouse, yet the functional impact of most is unknown. Inosine is interpreted as guanosine by the ribosome and other cellular machinery and therefore editing has the ability to change the amino acid sequence of encoded proteins, splice sites, the binding sites of RNA-binding proteins as well as modify the RNA secondary structure. Editing causing protein recoding is the essential function of ADAR2, but an essential role for recoding by ADAR1 has not been demonstrated. Additionally, ADAR1 has been proposed to have editing-dependent and editing-independent functions however, the relative contribution of these in vivo has not been clearly defined. A critical function of ADAR1 is editing of endogenous RNA to prevent activation of innate immune pathways via the dsRNA sensor MDA5 (Ifih1). Concurrent deletion of MDA5 rescues the embryonic lethality of Adar1 editing deficient mice (Adar1E861A/E861A). Outside of this role however, it is uncertain how ADAR1 editing contributes to normal development and homeostasis. To address this, we analysed the consequences of ADAR1 editing deficiency on murine homeostasis in the absence of MDA5. Our findings indicate that outside of the editing of MDA5-targeted self dsRNA, editing by ADAR1 is dispensable for normal murine development.


SymPoSia WEDNESDay

SYM-64-02 SYM-64-03

SYM-64-04 SYM-64-05

SINGLE MOLECULE INVESTIGATION OF LESION HAND-OFF DURING TRANSCRIPTION COUPLED REPAIR

Ho H.N.1, 2, van Oijen A.M.1, 2 and Ghodke H.1, 2 1Molecular Horizons Institute and School of Chemistry, University of Wollongong, Wollongong, Australia. 2Illawarra Health and Medical Research Institute, Wollongong, Australia.

During transcription elongation, bacterial RNA polymerase (RNAP) can pause, backtrack or stall on template DNA. Stalled transcription elongation complexes (TECs) at sites of lesions can be rescued by the transcription terminator Mfd. In this transcription-coupled repair (TCR) reaction, Mfd recognizes stalled TECs and recruits the nucleotide excision repair machinery (NER) to the site. Extensive biochemical, structural, genetic and single-molecule investigations have shed insight into various aspects of TCR. Despite these studies, the molecular mechanisms of stalled RNAP recognition by Mfd and recruitment of the NER machinery in vivo largely remain unknown. Single-molecule live-cell imaging of fluorescently labelled Mfd revealed that Mfd associates with TECs even in the absence of exogenous genotoxic stresses. This interaction requires an intact RNAP-interacting domain of Mfd, and is stabilized under conditions in which RNAP is stalled on template DNA. Binding of Mfd is highly regulated in cells, and mutations in its ATPase domain or the UvrB-homology module result in deregulated and non-specific DNA binding. Using an interval imaging approach on live-cells, we determined the lifetime of Mfd to be 18s during TCR in wild-type cells. We found that this lifetime is influenced by the presence of the NER machinery components UvrA and UvrB. Whereas absence of UvrA led to a longer lifetime of 30s, cells lacking functional UvrB exhibited Mfd foci that were arrested on DNA for 60-80s. Our findings shed insight into the recruitment and regulation of the transcription-repair coupling factor, and the execution of the TCR in cells.

MECHANISTIC DIFFERENCES IN DNA BINDING BEHAVIOUR OF ISL1/LHX3 HOMEODOMAINS IN TRANSCRIPTIONAL COMPLEXES

Smith N.C., Stokes P.H. and Matthews J.M. University of Sydney, Sydney, NSW, Australia.

LIM-homeodomain (LIM-HD) transcription factors act as key developmental regulators, both through their ability to bind DNA through homeodomain:DNA interactions, and through their ability to form higher order complexes through protein:protein interactions. The LIM-HD proteins Islet-1 (Isl1) and LIM homeobox protein 3 (Lhx3) have been implicated in the development of a broad range of tissues, most comprehensively in cell fate determination in the developing central nervous system. These proteins, along with the protein cofactor LIM-domain binding protein 1 (Ldb1) interact to form cell-specific transcriptional complexes. The presence or absence of Isl1 in complex with Lhx3 and Ldb1 is sufficient to redirect the complex to target different genes. This then directs the differentiaton of neural precursors into motor neurons or interneurons. Recapitulating this system in vitro, we have shown that Isl1 and Lhx3 bind differently in isolation than they do in combination, displaying a change in specificity that is not explained by simple cooperative binding of the homeodomains. Through the use of comparative electrophoretic mobility shift assays (EMSAs), multi angle laser light scattering (MALLS) and small angle X-ray scattering (SAXS) experiments, we we have studied the binding behaviours of these homeodomains in different combinations with each other and with or without additional protein:protein interaction domains. Through this, we have gained new understanding of the mechanisms by which these complexes can discern targets in the cell. Our results suggest that while the stoichiometry of binding is as expected, only one homeodomain is responsible for the affinity of binding to different targets, while the other appears to be important in stabilising binding to specific target sequences. Taken together, these data suggest a novel molecular mechanism for the combinatorial action of these transcription factors that act to influence cell fate during development.

TRANSPOSON-DERIVED TRANSCRIPTION FACTOR BINDING SITES AND PROMOTER ACTIVITIES IN BREAST CANCER

Jiang J. and Upton K.R. The University of Queensland, The School of Chemistry and Molecular Biosciences.

Transposons, a type of repetitive DNA, are pervasive in the human genome, occupying approximately 45% of the total genomic sequence. Since their initial discovery by Barbara McClintock, transposons were predominantly viewed as junk or selfish DNA. However, recent progress has revealed the extensive co-option of transposons for the transcriptional regulation of host genes. While generally inactivated in normal somatic tissues, transposons can become transcriptionally active in epithelial cancers and may even act as promoters or enhancers that subsequently activate oncogenes and contribute to tumorigenesis. Currently, the regulatory impact of transposons in breast cancer remains largely unknown. To address this gap in knowledge, we investigated the landscape of transposon-derived oncogenic transcription factor (TF) binding in breast cancer, predicted transposon-derived promoters, and validated these predictions. Our results demonstrated that transposons were an abundant source of TF binding sites in breast cancer, where ~38% of all binding sites of MYC, E2F1 and C/EBPβ were harboured by transposons. We identified 399 transposon subfamilies as significantly overrepresented in the oncogenic TF binding sites, suggesting a widespread role of these subfamilies in cancer transcriptional networks. TF-bound transposons were also associated with active histone modifications, further supporting the regulatory role of transposons in breast cancer. Finally, we validated the promoter activity of individual transposons in triple negative breast cancer cell lines. Luciferase assay results revealed that the promoter activities of SYT1, UCA1, AK4 and PSAT1 oncogenes were significantly reduced, and in some cases, almost completely abolished following transposon deletion. Transposons with strong promoter activity were also found to be epigenetically deregulated in breast cancer, characterised by hypomethylation and/or increased DNAse sensitivity. Our results provide an insight into the contribution of transposons to breast cancer transcriptional regulation, and will facilitate the development of novel diagnostic and prognostic biomarkers for breast cancer.

MODULATION OF EPIGENETIC PLASTICITY IN CANCER CELLS: FROM SIGNALLING PATHWAYS TO ENGINEERED PROTEINS

Blancafort, P.



SymPoSia WEDNESDay

SYM-65-01 SYM-65-02

SYM-65-03 SYM-65-04

FARMING MALLEE EUCALYPTS FOR VALUABLE NATURAL PRODUCTS

Woodrow I.E.1, Fernando S.2, Humphries J.2 and Goodger J.Q.D.2 1School of Ecosystem and Forest Sciences, The University of Melbourne, VIC 3010, Australia. 2School of BioSciences, The University of Melbourne, VIC 3010, Australia.

Mallee eucalypts have several properties that are advantageous for producing natural products on a large scale. These include the capacity to grow well on marginal land where agricultural cropping is unprofitable and an ability to be harvested mechanically in a short rotation coppice cycle. The leaves of some mallee species contain exceptionally high levels of natural products, which are stored in the extracellular lumina of embedded secretory cavities, commonly known as oil glands. These glands can comprise up to 15% of total leaf volume. Two examples of natural product production are explored in this presentation. In the first, the research underpinning the development of profitable blue mallee (Eucalyptus polybractea) plantations for pharmaceutical eucalyptus oil production is described. Specifically, the biochemical mechanism for oil synthesis and storage is outlined, and the development of exceptionally high yielding plantations through a clonal forestry approach is described. In the second example, the ability of mallee eucalypts to produce highly valuable flavonoids is discussed. Importantly, some of these compounds are already in the latter stages of testing for their ability to diminish the symptoms of several diseases of the central nervous system, including Alzheimer’s and Parkinson’s diseases and ischemic stroke. The potential to establish a mallee based industry for natural flavonoid production is discussed.

PRECISION LANDSCAPE REGENERATION FOR FOOD, ECOSYSTEM AND CLIMATE SECURITY

Borevitz J.O. Australian National University.

Global trends project a convergence of crisis in the next decades as demand for land exceeds planetary biocapacity. Agriculture depends on soil and water supplied by ecosystems which are alsol holding run away climate change. Regenerative agro-ecosystems grow food, are water resilient and build soil using perennial cropping, silvapasture and agroforestry approaches with biochar and enhanced rock weathering. When deployed at scale (1B hectares), regenerative landscapes can also stabilize the global climate by drawing down 10GtCO2e/y. I will outline a high tech (Genome2Phenome2Environment) experimental, modeling and social/economic research program to scale regenerative agriculture nationally and globally by 2030. Australia’s diverse landscape with synergistic primary industry capacity (agriculture and mafic rock mining) can contribute 10% of this solution valued at $1T over the decade.

APPLYING GENOMICS TO CONSERVATION: INVESTIGATING CLIMATE ADAPTATION IN EUCALYPTUS MICROCARPA AND IMPLICATIONS FOR RESTORATION

Jordan R.1, 2, Prober S.3, Dillon S.4 and Hoffmann A.1 1Bio21 Institute, School of BioSciences, University of Melbourne, 30 Flemington Rd, Parkville Vic 3010, Australia. 2CSIRO Land & Water, 15 College Rd, Sandy Bay Tas 7005, Australia. 3CSIRO Land & Water, 147 Underwood Ave, Floreat WA 6014, Australia. 4CSIRO Agriculture, Clunies Ross Street, Black Mountain ACT 2601, Australia.

Under continued environmental change, the simple presence of plant species within the landscape does not necessarily equate to their long-term survival. This is especially true in highly fragmented regions where reductions in population size and connectivity may decrease the potential for future adaptation. To ensure evolutionary resilience under changing conditions, conservation and restoration relies on capturing genetic diversity and thus adaptive potential. Genomics offers a powerful new approach for investigating genetic diversity and adaptation across a species’ range. Using the test case of Eucalyptus microcarpa (Grey Box), we present an example of how genomic approaches can be applied to conservation, in particular restoration of tree species under climate change. Employing DArTseq, a reduced-representation genomics approach, we firstly compared genomic diversity between natural and revegetated sites to assess how well current revegetation strategies capture genomic diversity and thus adaptive potential. Secondly, we investigated adaptation at the genomic-level, aiming to identify genomic regions important for climate adaptation as well as environmental factors potentially driving adaptive diversity in this species. Through this case-study, we demonstrate how genomics can provide deeper insight for restoration under climate change; moving beyond general genetic diversity towards knowledge of genomic signatures of climate adaptation and thus potential adaptive diversity. We show the power of genomics to provide in-depth knowledge that may assist in improving seed sourcing strategies and evolutionary-resilience of future revegetation efforts.

RESPONSES OF ISOTOPE DISCRIMINATION AND INTERCELLULAR RELATIVE HUMIDITY TO VAPOUR PRESSURE DEFICIT IN WILDTYPE AND ABSCISIC ACID INSENSITIVE POPULUS X CANESCENS

Cernusak L.A.1, Goldsmith G.2, Arend M.3 and Siegwolf R.4 1James Cook University, Cairns, Australia. 2Chapman University, Orange, California, USA. 3Basel University, Basel, Switzerland. 4Paul Scherrer Institute, Villigen, Switzerland.

We investigated responses to vapour pressure deficit (D) in abscisic acid insensitive (abi) and wildtype (WT) Populus x canescens, in order to better understand the crucial role of stomata in modulating leaf gas exchange and stable isotope discrimination, and to test for unsaturation of intercellular relative humidity (hi). Transpiration rate increased linearly with D in abi plants up to D of about 2 kPa, beyond which it declined precipitously; this was followed by leaf death within hours of removing the leaf from the gas exchange cuvette. On the other hand, WT leaves showed a steady or slightly declining transpiration rate up to D of nearly 7 kPa, and fully recovered photosynthetic function when measured the next day. Discrimination against 13CO2 (Δ

13C) declined in response to increasing D in WT plants, consistent with declining intercellular CO2 concentrations. For the D range over which abi plants were measured, Δ13C was higher in abi plants than in WT plants by about 6 permil on average, and did not decrease in response to increasing D. Discrimination against C18OO (Δ18O) differed markedly between WT and abi plants. In WT plants, Δ18O increased with increasing D by about 2 permil kPa-1, whereas in abi plants the rate of increase was about 66 permil kPa-1. This reflected progressive stomatal closure in the WT plants with increasing D. In abi plants, in contrast, stomata remained open in response to increasing D allowing CO2 to readily diffuse into the leaf interior, exchange oxygen atoms with the 18O enriched leaf water, and then diffuse back into the air exiting the gas exchange cuvette. The coupled measurements of Δ18O and gas exchange allowed us to estimate hi. In WT leaves we saw no evidence of unsaturation of hi, even at D above 6 kPa. However, in abi leaves, hi decreased at a rate of about -0.25 kPa-1, thus declining to values approaching 0.6 before the precipitous decline in transpiration rate and subsequent leaf death.


SymPoSia WEDNESDay

SYM-65-05 SYM-66-01

SYM-66-02 SYM-66-03

REGULATING TREE VIGOUR TO OPTIMISE THE LONG-TERM PRODUCTIVITY OF WALNUT (JUGLANS REGIA L.) ORCHARDS

Simpson J.E.1, Lang M.D.2 and Lewis J.3 1NSW Department of Primary Industries, Orange Agricultural Institute, 1447 Forest Rd, Orange, NSW. 2800. 2Webster Limited, 148 Colinroobie Road, Leeton, NSW 2725. 3NSW Department of Primary Industries, Yanco Agricultural Institute, Yanco, NSW, 2703.

Walnut orchards in Australia are typically high-density plantings comprising one or more cultivars of Juglans regia L. An important criterion for cultivar selection is tree vigour, with greater vigour improving the profitability of orchards during the early years of tree production which increases profitability. As trees mature unregulated growth leads to shading of fruiting points and ultimately reduces tree productivity. This study aimed to manage tree vigour with an exogenously applied plant growth regulator (PGR), and to determine the impact of reduced vigour on potential yield and nut quality in young (2-4 years) and mature (8-10 years) walnut trees. The triazole-type PGR, uniconazole-P, reduces the internode length by inhibiting gibberellin biosynthesis ergo is a potential means of managing tree vigour. Sunny PGR (active ingredient: 50 g/L uniconazole-P), was applied to the root zone of mature walnut trees at four different rates (0, 0.75, 1.5, 3.0 L ha-1) and at two different timings (autumn and spring) in three cultivars: Serr, Vina and Chandler. Young trees were treated in the same manner as mature trees, but in cultivar Chandler only. All trials were randomised complete block designs with five-replicates, of four- and single-tree-plots in mature and young trees respectively. General linear modelling will be used to describe the relationship between tree growth (internode length), crop yield and nut quality with PGR treatment (rate, timing) and cultivar in mature trees, and between tree growth and PGR treatment in young trees.

SEPARATING THE FGF SIGNALING REQUIREMENTS OF CLOCK OSCILLATION FROM WAVEFRONT ACTIVITY DURING SOMITOGENESIS

Lewandoski M.1, Kageyama R.2 and Anderson M.1 1National Cancer Institute, NIH, USA. 2Kyoto University, Kyoto, Japan.

The vertebrate axial skeleton is comprised of segmented vertebrae, differing in size and shape, but with a pattern nearly invariant between individuals. Vertebrae form from somites, which segment from the posterior presomitic mesoderm (PSM). Somite segmentation is controlled by two classical activities, known as the “clock” and “wavefront”. Wavefront activity maintains the posterior PSM in an undifferentiated state; we previously showed that Fgf4 and Fgf8 each encode this activity. The clock is governed by oscillating Notch signals that segment new somites at the anterior wavefront edge. Loss of only Fgf4 in the PSM causes misshaped vertebrae specifically in cervical and thoracic regions. We have characterized these mutants with Hairpin Chain Reaction mRNA in situ hybridization, allowing for direct quantification of up to four different mRNA sequences per embryo. We found that wavefront gene expression is unaffected in Fgf4 mutants; however, clock gene oscillations are highly abnormal. Hes7, a transcriptional repressor of Notch signaling, is reduced in Fgf4 mutant PSM, but only during the developmental window when future malformed vertebrae are generated. Due to the central role of Hes7 in regulating clock oscillation, we hypothesize that this reduction is the cause of Fgf4 segmentation defects. We also find that defects are more extreme when one copy of Fgf8 is additionally removed and are rescued when Fgf8 is overexpressed. We conclude that correct clock oscillations require an FGF signal that is genetically separable from the FGF wavefront requirement. The FGF signal appears to be permissive and provides a robustness factor for somite segmentation, buffering the system to changes in Hes7 expression dynamics.

TREATMENT OF SOLID TUMOURS BY CO-TARGETING BCL-2 PRO-SURVIVAL PROTEINS

Lee E.F.1, Harris T.2, Tran S.2, Evangelista M.2, Herold M.J.3 and Fairlie W.D.2 1LIMS, La Trobe University. 2Olivia Newton-John Cancer Research Institute. 3Walter and Eliza Hall Institute.

Defective apoptosis signalling is a hallmark of most, if not all, cancers. Typically this involves abnormally high expression of the pro-survival members of the Bcl-2 family of proteins. This enables damaged cell to survive when they should otherwise be removed. Over the last decade, a new class of drugs (“BH3-mimetics”) has been developed to target the Bcl-2 proteins and there are now molecules that enable most of them to be selectively antagonised. In this presentation, I will discuss our recent data using a panel of these drugs on different types of highly chemoresistant solid tumours. These data combined with results from experiments using protein-based ligands we have engineered, as well as genetically engineered cell lines, have enabled us to dissect the critical “survival” factors in these tumours. Critically, we show a consistent pattern of proteins that must be antagonised to provide potent and synergistic tumour cell killing Interestingly, the same pattern is also starting to emerge for other cancers types, suggesting a potential standardised treatment strategy for many cancers using these drugs.

MEF2C PHOSPHORYLATION IS REQUIRED FOR CHEMOTHERAPY RESISTANCE IN ACUTE MYELOID LEUKEMIA

Brown F.C.1, 2 and Kentsis A.1 1Memorial Sloan Kettering Cancer Center, NY, USA. 2Monash University, Melbourne, Australia.

In acute myeloid leukemia, chemotherapy resistance remains highly prevalent, representing the major barrier to cure in children and adults alike. We sought to investigate molecular mechanisms that may explain primary chemotherapy resistance in AML using targeted genomic sequencing and high-resolution mass spectrometry proteomics. This analysis identified aberrant phosphorylation of MEF2C S222 in primary chemoresistant human AML specimens, and using an affinity-purified antibody, established its prevalence and prognostic significance in a cohort of 47 patients, spanning the major biologic subtypes of human AML. MEF2C is a transcription factor required for hematopoietic cell fate determination. We found that Mef2cS222A/S222A knock-in mutant mice engineered to block MEF2C phosphorylation exhibited normal hematopoiesis, but were resistant to leukemogenesis induced by MLL-AF9. MEF2C phosphorylation was required for leukemia stem cell maintenance in both MLL-rearranged and non-MLL-rearranged mouse AML in vivo, and chemotherapy resistance and leukemogenicity of human AML cells. Transcriptome and chromatin analysis of gene expression changes and composition of MEF2C transcriptional complexes showed assembly of an active MEF2C transactivation complex specifically induced by MEF2C phosphorylation in leukemia cells. MARK3 activation was sufficient to cause MEF2C phosphorylation, leading to enhanced transcription of MEF2 response elements in cells. Notably, treatment with the selective MARK inhibitor MRT199665 caused apoptosis and conferred enhanced sensitivity to cytarabine of MEF2C-activated human AML cell lines and primary patient specimens, but not those lacking MEF2C phosphorylation. These findings identify kinase-dependent dysregulation of transcription factor control as a determinant of therapy response in AML, with immediate potential for improved diagnosis and therapy for this disease.


SymPoSia WEDNESDay

SYM-66-04 SYM-66-05

SYM-67-01 SYM-67-02

EXPLOITING TUMOUR ACIDITY FOR POLYMER BASED DRUG DELIVERY

Whitty E.G.1, 2, 3, Castignolles P.2, Gaborieau M.2, 3 and Callaghan R.1 1Department of Biomedical Science and Biochemistry, Research School of Biology, The Australian National University, Canberra, ACT, 0200, Australia. 2Australian Centre for Research on Separation Science (ACROSS), School of Science and Health, Western Sydney University, Parramatta, NSW, 2150, Australia. 3Medical Sciences Research Group, School of Science and Health, Western Sydney University, Parramatta, NSW, 2150, Australia.

An acidic micro-environment is a hallmark feature of solid tumours generated by metabolic adaptations including an increased reliance on glycolysis and its concomitant production of lactic acid. The acidic environment has deleterious effects on many anticancer drugs; however, several novel therapeutic strategies are beginning to exploit this property. Our consortium has developed three configurations of poly(sodium acrylate) (PNaA) as a pH-sensitive drug delivery system. Initial efforts have focussed on the anticancer drug cisplatin, which is beset with poor stability, toxicity and pharmacokinetic properties. Loading cisplatin onto a polymer may circumvent many of these issues by providing targeted delivery and increased stability. PNaA is loaded with cisplatin at pH9 and the attachment is stable, reversible and displays a pH-dependent release process. The three configurations of PNaA differ in their branching structure and thereby offer distinct loading capacities. In the present investigation we have undertaken preliminary characterisation of PNaA in a monolayer cell culture system. Unloaded polymers did not alter cell cycle progression nor where they associated with significant levels of apoptosis. Cisplatin loaded polymers displayed a greater potency than observed with free drug. The increase in drug potency demonstrates the viability of this polymer as a drug delivery system.

DUAL SPHINGOSINE KINASE AND BCL-2 INHIBITION EXHIBITS SYNERGISTIC CELL DEATH IN ACUTE MYELOID LEUKEMIA

Lewis A.C.1, Tea M.1, Wallington-Beddoe C.T.1, Anderson D.2, Creek D.2, Powell J.A.1 and Pitson S.M.1 1Centre for Cancer Biology, University of South Australia, CRI Building, North Terrance, Adelaide, SA 5001, Australia. 2Department of Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

Pro-survival Bcl-2 family proteins such as Mcl-1 and Bcl-2 have garnered significant interest as therapeutic targets due to their up-regulation in many cancers, including acute myeloid leukaemia (AML), leading to enhanced cancer cell survival. Small molecule inhibitors such as the selective Bcl-2 inhibitor, Venetoclax, are very effective in some cancers that are highly on Bcl-2, but have demonstrated poor single agent efficacy in AML due to these cells being highly dependent on Mcl-1, which is not targeted by this agent. Sphingosine kinase 1 (SK1) is a signalling enzyme with established roles in oncogenesis and has recently emerged as a potential therapeutic target in leukaemia. We recently demonstrated that the selective SK1 inhibitor, MP-A08 exhibits anti-leukemic activity in vitro and in vivo using patient-derived AML xenograft models. MP-A08-mediated cytotoxicity in AML cells correlated with a reduction in Mcl-1 levels, as well as upregulation of BH3 only proteins. Here, we found that combinational therapies with MP-A08 and Venetoclax induced synergistic cell death in AML cell lines and patient samples. Mechanistically, MP-A08 induces transcriptional upregulation of BH3-only protein, Noxa and formation of Noxa/Mcl-1 complexes. MP-A08 appears to exert its cytotoxicity in AML cells through loss of Mcl-1 as a consequence of Noxa binding. Using patient-derived xenografts, we demonstrate that MP-A08 and Venetoclax treatment exhibits anti-leukemic activity in vivo providing pre-clinical evidence to target SK1 and Bcl-2 in AML.

MCL-1 INHIBITION PROVIDES A NEW WAY TO SUPPRESS BREAST CANCER METASTASIS AND INCREASE SENSITIVITY TO DASATINIBYoung A.I.J.1, Castillo L.1, Law A.M.K.1, Brummer T.2, 3, Lee E.F.4, 5, 6, Fairlie E.F.4, 5, 6, Timpson P.1, 7, Gallego-Ortega D.1, 7, Ormandy C.J.1, 7 and Oakes S.R.1, 7 1Cancer Research Division, Garvan Institute of Medical Research and the Kinghorn Cancer Centre, 384 Victoria Street, Darlinghurst, NSW, 2010, Australia. 2Centre for Biological Systems Analysis (ZBSA) and Centre for Biological Signallling Studies, Albert-Ludwigs-University, Stefan-Meier-Strasse 17, 79104, Freiburg, Germany. 3Spemann Graduate School for Biology and Medicine and Faculty of Biology, Albert-Ludwigs-University, Stefan-Meier-Strasse 17, 79104, Freiburg, Germany. 4Olivia Newton-John Cancer Research Institute, 145 Studley Rd, Heidelberg, Victoria, 3084, Australia. 5School of Cancer Medicine and Department of Chemistry and Physics, La Trobe University, Melbourne, Victoria, 3086, Australia. 6The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria, 3052, Australia. 7St. Vincent’s Clinical School, UNSW Medicine, Victoria Street, Darlinghurst, NSW, 2052, Australia.

Metastatic disease is largely resistant to therapy and accounts for almost all cancer deaths. Myeloid cell leukemia-1 (MCL-1) is an important regulator of cell survival and chemo-resistance in a wide range of malignancies, and thus its inhibition may prove to be therapeutically useful. To examine whether targeting MCL-1 may provide an effective treatment for breast cancer, we constructed inducible models of BIMs2A expression (a specific MCL-1 inhibitor) in MDA-MB-468 (MDA-MB-468-2A) and MDA-MB-231 (MDA-MB-231-2A) cells. MCL-1 inhibition caused apoptosis of basal-like MDA-MB-468-2A cells grown as monolayers, and sensitized them to the BCL-2/BCL-XL inhibitor ABT-263, demonstrating that MCL-1 regulated cell survival. In MDA-MB-231-2A cells, grown in an organotypic model, induction of BIMs2A produced an almost complete suppression of invasion. Apoptosis was induced in such a small proportion of these cells that it could not account for the large decrease in invasion, suggesting that MCL-1 was operating via a previously undetected mechanism. MCL-1 antagonism also suppressed local invasion and distant metastasis to the lung in mouse mammary intraductal xenografts. Kinomic profiling revealed that MCL-1 antagonism modulated Src family kinases and their targets, which suggested that MCL-1 might act as an upstream modulator of invasion via this pathway. Inhibition of MCL-1 in combination with dasatinib suppressed invasion in 3D models of invasion and inhibited the establishment of tumors in vivo. These data provide the first evidence that MCL-1 drives breast cancer cell invasion and suggests that MCL-1 antagonists could be used alone or in combination with drugs targeting Src kinases such as dasatinib to suppress metastasis.

EXPLORING BACTERIAL RESISTANCE WITH ANTIBIOTIC-DERIVED FLUORESCENT PROBES

Blaskovich M.A.T. Centre for Superbug Solutions, Institute for Molecular Bioscience, The University of Queensland.

Bacteria are becoming resistant to every antibiotic, leading to multi-drug resistant ‘superbugs’ that will kill millions of people each year. New antibiotics are urgently needed, but few new drugs are available or in clinical development. It is imperative to discover and develop new antibiotics to fight these superbugs, but for this to occur we require an improved understanding of how antibiotics work and how bacteria function and develop resistance. This requires new tools and techniques to advance our knowledge of bacterial metabolism, efflux pumps and other responses to antibiotics, allowing for analysis of key aspects of bacterial growth, division, metabolism and resistance. In order to create such tools, we have been systematically converting representatives of the major classes of antibiotics into mechanism-specific fluorescent probes that retain the biological profile of the parent compound. This talk will present examples of how these probes are able to provide information about bacterial resistance mechanisms by assessing membrane permeability and efflux pump activity. We will also show how they have helped to decipher differences in the mode of action of apparently similar antibiotics possessing strikingly different activity against resistant bacteria, and how they may also be useful in assessing antibiotic toxicity to human cells.


SymPoSia WEDNESDay

SYM-67-03 SYM-67-04

SYM-67-05 SYM-68-01

TARGETING MULTIFACTORIAL RESISTANCE MECHANISMS ASSOCIATED WITH MICROTUBULE PROTEINS IN CANCER CELLS

Kavallaris M.1, 2, 3 1Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Sydney. 2ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, UNSW Sydney. 3Australian Centre for NanoMedicine, UNSW Sydney.

Cancer is one of the major causes of morbidity and mortality in the world. A major cause of cancer treatment failure is poor access of chemotherapeutic drugs to tumour sites and resistance to therapy. Microtubules and other mitotic proteins are often deregulated in cancer and are responsible for diverse effects including drug resistance, tumour formation and metastasis. In particular, aberrant expression of specific microtubule proteins in epithelial cancers is a poor prognostic indicator and is associated with clinical resistance. Recent data on understanding the mechanisms mediating these multifactorial resistance effects and strategies to overcome resistance will be presented.

DICHLOROACETATE AT CLINICALLY ACHIEVABLE CONCENTRATIONS CAN REDUCE PPDH AND REVERSE THE GLYCOLYTIC PHENOTYPE IN MULTIPLE MYELOMA CELLS

Tian D.D. and Blackburn A.C. ACRF Dept Cancer Biology and Therapeutics, John Curtin School of Medical Research, Australian National University, Acton, ACT, Australia.

Background: Multiple myeloma (MM) is a B-cell malignancy with a glycolytic phenotype. Dichloroacetate (DCA) is a pyruvate dehydrogenase (PDH) kinase (PDK) inhibitor that can reverse the glycolytic phenotype. In vitro, DCA (≥10mM) inhibited MM proliferation and induced apoptosis, but these concentrations are above those clinically achievable and are likely acting off-target. We examined whether DCA at clinically achievable levels can act on target, inhibit MM cell growth and enhance effects of other drugs. Methods: The response of five human MM cell lines to DCA alone or combined with nutrient deprivation, hypoxia, 2-deoxyglucose, dexamethasone or orlistat was measured using neutral red viability assay. pPDH/tPDH was measured by western blotting. PDK expression was measured by rt-PCR and western blotting. Cell proliferation (CFSE), cell cycle (BrdU/PI) and apoptosis (Annexin V/7AAD) were measured by FACS. Results: DCA (1-5mM) decreased cell proliferation without increasing apoptosis or inducing cell cycle arrest. This was accompanied by decreased pPDH and lactate which were dependent on the presence of glucose. DCA treatment as low as 0.1-0.3mM decreased pPDH/tPDH within 5-24hr. These concentrations are based on DCA serum peak and trough levels in MM patients in our DiCAM clinical trial (1). DCA had a greater effect on growth inhibition under hypoxia, corresponding with induction of the target PDKs. DCA combined with dexamethasone (inhibits glycolysis) or orlistat (fatty acid synthase inhibitor) had additive effects in reducing viable cells, and were accompanied by greater changes in metabolism. Conclusion: DCA can act on target in MM cells at clinically achievable levels and may be a useful therapy in combination with other low toxicity metabolism modifying drugs.

FUNCTIONAL ANALYSIS OF A CAMPYLOBACTER JEJUNI NUTRIENT TRANSPORT PROTEIN USING PROTEOMICS AND METABOLOMICS

Man L.1, 2, Cain J.A.1, 2, Solis N.2, Klare W.1, 2, Niewold P.1, 2, 3, Sumer-Bayraktar Z.1, 2 and Cordwell S.J.1, 2, 3 1School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, Australia. 2Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia. 3Discipline of Pathology, School of Medical Sciences, The University of Sydney, Sydney, NSW, Australia.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis in the developed world. Infection occurs predominantly through the consumption of undercooked/poorly prepared commercial chicken products. Importantly, C. jejuni exists mainly as a commensal organism within the intestines of chickens, but is pathogenic in humans. While the exact mechanism of this difference in pathogenicity is unknown, factors such as motility and nutrient uptake are thought to be significant. C. jejuni is generally considered assaccharolytic and primarily utilizes amino and organic acids as carbon sources. We conducted proteomic analysis of C. jejuni to identify proteins associated with growth in deoxycholate, mimicking gut bile salts encountered during human infection. We quantified 1561 proteins, and the most significantly induced protein was the product of the cj0025 gene, which has been previously annotated as a ‘putative C4-dicarboxylate transport protein’. To determine the function of Cj0025, metabolomic profiles of media inoculated with C. jejuni wild-type or a Δcj0025 deletion strain were compared. Our results showed that the mutant was not impaired in the uptake of amino acids or organic acids, but was for cystine, a cysteine dimer. This was reflected both in the mutant proteome, which showed a downregulation of sulfur-related proteins, and a growth-inhibition assay utilising a toxic analogue of cystine.

THE ROLES OF H3.3 AND ATRX ABNORMALITIES IN DRIVING ALTERNATIVE LENGTHENING OF TELOMERES AND CHROMATIN ABERRATIONS IN CANCERS

Udugama M., Voon H.P., Hii L. and Wong L.H. Biomedicine Discovery Institute, Monash University.

One striking finding in the area of cancer epigenetics has been the identification of mutated histone genes (oncohistones) in paediatric glioblastomas (pGBMs). Two H3.3 mutations are found. The first mutation replaces lysine 27 with a methionine. The second one replaces glycine 34 by an arginine (G34R). H3.3G34R mutations always overlap with ATRX and p53 mutations, and these pGBMs are activated in the Alternative Lengthening of Telomeres (ALT) pathway, suggesting that H3.3G34R/ATRX/p53 mutations cooperate to drive ALT and GBM development. We have created cell models carrying H3.3G34R/ATRX/p53 mutations to recapitulate the initial driver epigenetic events that promote ALT. These mutants are compromised in heterochromatin formation at the telomeres, accompanied with telomere damage (and aberrant telomere protein binding) and formation of aberrant PML nuclear bodies (PML-NBs) that are irregular in size, shape and number. KDM4 proteins are demethylases or epigenetic erasers that remove the methyl group from trimethylated H3K9 and H3K36. We find that the H3.3G34R inhibits KDM4 catalytic function of and drives its aberrant distribution. As a result, it induces aberrant histone methylation pattern and affects telomere chromatin maintenance. IDH1 (which is also mutated in GBM) is also known to inhibit KDM4 function. We propose KDM4 chromatin network as a major driver that promotes ALT and the oncogenic process in GBMs. In the H3.3G34R/ATRX mutants, we also detect DNA copy loss at ATRX-bound ribosomal repeats, accompanied with severely reduced rRNA synthesis. ALT positive human sarcoma tumour samples are substantially reduced in rDNA copy. Moreover, ALT cancer cells show increased sensitivity to RNA polymerase I transcription inhibitor, suggesting the therapeutic potential of targeting Pol I transcription in ALT cancers. Our study provides insights into chromatin defects associated with ATRX/H3.3 mutations and development of ALT cancers.


SymPoSia WEDNESDay

SYM-68-02 SYM-68-03

SYM-68-04 SYM-68-05

INVESTIGATING THE ROLE OF DE NOVO DNA METHYLATION IN REGULATING LIVER METABOLISM

Youngson N.A.1, Prates K.V.1, Yao S.1, McRae A.F.2 and Morris M.J.1 1School of Medical Sciences, UNSW Sydney. 2Institute for Molecular Bioscience, The University of Queensland.

The de novo DNA methyltransferases are required for development as their constitutive deletion in mice results in lethality either in the late embryonic period (Dnmt3a) or in the first weeks after birth (Dnmt3b). Beyond development, post-natal DNA methylation changes have been identified that associate with disease states or particular diets. However, despite hundreds of studies that associate de novo methylation changes in mammals, a functional requirement for those DNA methylation changes is still not proven. Uncovering the true role of DNA methylation in disease is vital for understanding pathogenesis, and in directing the development of therapies. If DNA methylation is just a consequence of, or a minor player in, disease associated transcriptional change, then future epigenetic studies in this area should focus on other molecules such as histone tail modifications. To investigate this issue we have generated mice that are homozygous mutants for both Dnmt3a and Dnmt3b in post-natal liver. These mice are viable and fertile. Here we present data on the requirement for liver de novo methylation for systemic glucose metabolism, in control and obese mice. Our model also sheds light on how in vivo inhibition of de novo DNA methylation in liver affects other epigenetic regulatory systems, and systematic physiology and behaviour.

SINGLE-CELL EPIGENOMICS FOR ANALYSIS OF HETEROGENEOUS AND RARE CELL POPULATIONS

Lee H.J. The University of Newcastle.

Single-cell sequencing technologies are revolutionising our understanding of heterogeneous cell populations in development and disease. Incorporation of epigenetic information with single-cell transcriptomic and genomic analyses will provide valuable insights into the molecular mechanisms of gene regulation (Clark, Lee, Smallwood et al. 2016 Genome Biol 17:72). DNA methylation occurs on cytosine residues of CpG dinucleotides in mammalian cells. This epigenetic modification is dynamically regulated during development and is globally dysregulated in many cancer types. We developed single-cell bisulphite sequencing (scBS-seq) (Smallwood, Lee, et al. 2014 Nat Methods 11:817-202), which provides quantitative, single-nucleotide information on DNA methylation for up to 50% of cytosines across the genome. Extending on this work, we recently reported parallel single-cell methylome and transcriptome sequencing (scM&T-seq) (Angermueller, Clark, Lee, Macaulay, et al. 2016 Nat Methods 13:229-32), which allows both DNA methylation and gene expression to be assayed from the same single cell. To illustrate the power of integrated single-cell multi-omics, biological insights gained from scM&T-seq analysis of mouse embryonic stem cells will be presented.

MIRNA:MIRNA INTERACTIONS IN HEAD AND NECK SQUAMOUS CELL CARCINOMA

Hill M.1 and Tran N.1, 2 1School of Biomedical Engineering, Faculty of Engineering and IT, University of Technology Sydney, NSW, Australia. 2The Sydney Head and Neck Cancer Institute, Sydney Cancer Centre, Royal Prince Alfred Hospital, NSW, Australia.

Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common form of cancer. It is commonly caused by smoking, alcohol, and Human Papilloma Virus (HPV). Due to its recent increase in incidence worldwide, it is important to investigate the molecular mechanisms responsible for HNSCC. MicroRNAs (miRNAs) control messenger RNA (mRNA) and have an important role in disease development. Normally, miRNAs perform their regulatory function by binding to the 3’ untranslated region (UTR) of mRNA. However, several studies have demonstrated that miRNAs are involved in the regulation of other miRNAs, known as a miRNA:miRNA interaction. This mode of regulation is unexplored in HNSCC. Our lab has identified miR-21 and miR-499 as important regulators in HNSCC. The overexpression of miR-21 in HNSCC cells reduced the levels of mature miR-499. However, miR-499 did not alter miR-21 levels. To determine if miR-21 overexpression has any global effect, we measured the expression of 750 miRNAs. From this analysis, both miR-21 and miR-499 overexpression could regulate specific miRNAs such as miR-148, miR-100, and miR-31. This data was then used to create an interactome to visualise the identified miRNA:miRNA interactions and their connections to genes. Furthermore, miR-21 overexpression dysregulated the RNA levels of the biogenesis components, Ago2, Ago3, and Dicer. This has implications on mature miRNA levels, and may affect the regulation of HNSCC related genes. In summary, our study provides the first characterisation of miRNA:miRNA interactions in HNSCC cells, and demonstrates that miR-21 can alter the levels of specific miRNAs and affect expression of the biogenesis machinery.

UNLOCKING FEMALE ES CELLS FOR RESEARCH

Keniry A.J., Jansz N. and Blewitt M.E. Walter and Eliza Hall Institute.

Female mouse embryonic stem cells (mESCs) are significantly harder to grow and expand compared to their male counterparts. This discrepancy has resulted in the majority of mESC studies being performed in male cells, and therefore our appreciation of characteristics particular to female mESCs is severely lacking. This is worrying as in addition to being harder to maintain, female mESCs have also been found to be less karyotypically and epigenetically stable and display slower differentiation kinetics. Clearly female mESCs are very different to their male equivalents and demand significantly more research attention. To achieve this however, female mESCs must become more experimentally tractable through development of robust methods to both derive and maintain them. It is not fully understood what makes female mESCs less robust in culture, however there is some evidence that the second X chromosome is to blame as female XO mESCs behave similarly to male XY cells. Interestingly, female mESCs are the only female cell type that has not undergone the process of X chromosome inactivation, and thus are the only cells to express from both X chromosomes, suggesting that it may be the activity of the second X, as opposed to the second chromosome per se, that makes female mESCs less amenable to culture. In order to study female mESCs we have tagged each X chromosome with a different fluorescent reporter, such that we can monitor activity from each X rapidly and accurately by FACs. The system allows for rapid monitoring of cell fitness, karyotype and X inactivation status and has enabled us to establish robust methods for the derivation, culture and manipulation of female mESCs. We have also experimentally proven the utility of these cells for the study of pluripotency, differentiation, induced pluripotency and X inactivation. Moreover, we have used the cells to perform screens for epigenetic regulators of X inactivation. These screens have revealed genes from completely unexpected pathways and suggest unappreciated mechanisms are required for X chromosome inactivation and indeed gene silencing more broadly. We are now moving on to validating and characterising these discoveries; first in XCI and then in other epigenetic processes.


SymPoSia WEDNESDay

SYM-69-01 SYM-69-02

SYM-69-03 SYM-69-04

DEVELOPING THE NEXT-GEN THERAPEUTIC MONOCLONAL ANTIBODIES: NEW INSIGHTS BY TARGETING AND MANIPULATING HUMAN FCR: ANTIBODY DEPENDANT FUNCTIONS

Hogarth P.M.1, 2, 3, Chenoweth A.M.1, Wines B.D.1, Trist H.M.1 and Esparon S.E.1 1Immune Therapies Group, Burnet Institute, Melbourne. VIC. 2Clinical Pathology, University of Melbourne, Parkville VIC. 3Immunology and Pathology Monash University, Melbourne.

The potency of many therapeutic antibodies, particularly in cancer and immune agonism, requires antibody interaction with human Fc receptors. The FcR are specific, cell surface receptors for immunoglobulins which provide a potent, system of activation and regulation of cell-based effector responses used by therapeutic antibodies. These Fc-dependant reposes include antibody dependent cell mediated cytotoxicity, phagocytosis, potent degranulation and mediator release. In addition to therapeutic potency, the Fc:FcR interaction of antibodies or Fc fusions can also induce unwanted adverse reactions. Manipulation of the antibody Fc will provide the next generation therapeutic mabs and Fc fusions with more potent and specific actions i.e. cell or virus killing in anti-cancer mAbs or inhibitory anti-inflammatory modulation in autoimmunity and allergy or the ablation of FcR-dependant function to avoid adverse effects. Our therapeutic biologicals program uses our pioneering studies of antibody and Fc receptor structure and function of human, and non-human primates, as the basis for the engineering of next generation therapeutic antibodies for profoundly altered function and specificity. This will include our work on (i) Fc receptors forms and roles in antibody effector function. (ii)Manipulation of mAbs and Fc receptors for antibody therapy (iii)Novel methods of evaluation of antibody:FcR effector functions in immune therapy. (iv) Challenges in using non-human primates as models of human antibody function. WWW http://www.burnet.edu.au/staff_members/182_mark_hogarth.

GERMINAL CENTER ANTIBODY MUTATION TRAJECTORIES ARE DETERMINED BY RAPID SELF/FOREIGN DISCRIMINATION

Burnett D.L., Langley D.B., Schofield P., Hermes J., Chan T.D., Jackson J., Bourne K., Brink R., Christ D. and Goodnow C. Garvan Institute of Medical Research.

Antibodies have exquisite specificity to differentiate foreign antigens that mimic “self”, but it remains unclear how such specificity is acquired. We generated B-cells displaying an antibody that cross-reacts with two related protein antigens expressed on self versus foreign cells. B-cell anergy was imposed by self antigen but reversed upon challenge with high-density foreign antigen, leading to germinal center recruitment and antibody gene hypermutation. Single-cell analysis revealed rapid selection for mutations that decrease self affinity and slower selection for epistatic mutations that specifically increase foreign affinity. Crystal structures revealed the mutations exploited subtle topological differences to achieve 5,000-fold preferential binding to foreign targets over self epitopes. Strikingly, resolution of antigenic mimicry drove the optimal affinity maturation trajectory, highlighting the value of retaining self-reactive clones as substrates for protective antibody responses.

INTERROGATING HUMAN B CELL IMMUNITY TO INFORM UNIVERSAL INFLUENzA VACCINE DESIGN

Wheatley A.K. Department of Microbiology and Immunology, University of Melbourne.

Influenza inflicts significant global mortality and morbidity that can be combated by effective immunisation. However, the protective efficacy of current vaccines is limited by the variability and rapid mutagenesis of circulating strains. There is tremendous interest in developing universal influenza vaccines for life-long protection. Here, we use recombinant analogues of the viral hemagglutinin protein and flow cytometry to interrogate human B cell and antibody responses to influenza. Our approach enables the enumeration and phenotypic characterisation of B cell responses to seasonal influenza vaccines. In addition, we can pan the B cell repertoire for rare specificities allowing near universal influenza recognition and in some cases protection. Theses approaches allow the identification of potentially protective epitopes and may inform the design of next-generation influenza vaccines. In addition, we can recover human monoclonal antibodies with potential prophylactic, therapeutic, or diagnostic applications for eventual clinical use.

PATHOGEN SENSING BY THE INTRACELLULAR ANTIBODY RECEPTOR, TRIM21, IS REGULATED BY B-BOX AUTOINHIBITION AND RING PHOSPHORYLATION

Dickson C.F.1, 2, Fletcher A.J.1, Vaysburd M.1, Yang J.C.1, Mallery D.L.1, Mclaughlin S.H.1, Chin J.W.1, Neuhaus D.1 and James L.C.1 1MRC Laboratory of Molecular Biology, Cambridge, UK. 2SMS, School of Medical Sciences, UNSW, Australia.

TRIM21 is a cytosolic antibody receptor that performs pathogen sensing and antibody-dependant intracellular neutralisation of viruses. Both signalling and effector functions require E3 ubiquitin ligase activity, which is provided by an N-terminal RING domain. TRIM21 induces a potent antiviral immune response, however, it does not constitutively signal, suggesting that the enzymatic activity of the RING is tightly regulated. We have used structural studies together with in vitro ubiquitination assays to show that a domain of unknown function, the B-box domain, represses TRIM21 activity by competing with E2 ubiquitin conjugating enzymes for binding to the RING. We further demonstrated that cellular TRIM21 is phosphorylated by IKKβ and TBK1 at serine 80, located at the RING-B-box interface. By introducing an phosphomimetic or a phosphoserine at position 80 in a recombinant RING-B-box protein we showed that phosphorylation of the interface relieves B-box mediated autoinhibition of the RING by promoting E2 binding and restoring enzymatic activity to the uninhibited state. Cells expressing TRIM21 carrying an S80E mutation constitutively activate NF-κB and produce a more robust cytokine response upon infection with DNA or RNA viruses, while an S80A mutation attenuated cytokine production. In contrast, neutralisation activity was unaffected by mutation of S80, consistent with a different threshold for these two activities. This work identifies several layers of regulation that fine tune the signalling activity of TRIM21 and highlights the importance of controlling inflammation, which unchecked has the potential to cause tissue damage and autoimmune disease.

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SYMPOSIA - ComBio · repressed and male differentiation was not promoted similar to the case of NANOS2-null. On the other hand, some NANOS2-null properties, meiosis initiation and