Introduction

A large number of proteins interact with the actin cytoskele-ton and play important roles in regulating cell motility, celldifferentiation and cell shape.1–4 The analysis of mutationsthat impair flight abilities in Drosophila melanogaster hasled to significant advances in characterizing the genesinvolved in the regulation of myogenesis, myofibrillarassembly and the mechanics of muscle contraction. Thisapproach can also be used to understand actin cytoskeletalactivity in non-muscle cells, in part because of similarities inmuscle sarcomeres and non-muscle actin stress fibres. It haspreviously been shown that several mutations that causeflightlessness in Drosophila melanogaster occur in theflightless I (fliI ) gene, which is located in subdivision 19F onthe X chromosome.5,6 The fliI gene encodes a 1256 aminoacid protein with a predicted molecular weight of143 672 Da. The fliI protein is characterized by a leucine-rich repeat (LRR) region at the N-terminus, a motif knownto be involved in protein–protein or protein–lipid interac-tions.7–9 The C-terminus has significant sequence homologyto the gelsolin-like family of actin-binding proteins.10,11 Thepresence of these two domains in a single protein has led tospeculation that the fliI protein is involved in augmenting the versatility of actin filament structures and contributing

to the dynamics of filament rearrangements. The fliI genehas been identified in Caenorhabditis elegans,10 mouse12 andhuman10 and northern analysis of human FLII mRNA expres-sion patterns have shown that the highest level is in skeletalmuscle. Drosophila embryos with weak mutations in the fliIlocus undergo apparently normal embryonic development,but exhibit abnormal flight muscle morphology.5,13 Severedisruptions to the fliI locus result in lethality, indicating theessential requirement of the fliI protein for the survival of theorganism to adulthood.14 All of these findings are consistentwith a role for flightless I in providing a direct link betweenmolecules involved in signal transduction pathways and theactin cytoskeleton. It has recently been shown that expressedforms of the human and C. elegans fliI are able to bind actinin vitro;15,16 however, evidence of the endogenous fliI proteininteracting with actin monomers or filaments remains to beshown. We have developed rabbit antibodies specific for thefliI protein and have investigated its localization in relation-ship to actin during developmental stages in D. melanogaster,mouse and chick embryos. We show that the fliI proteinlocalizes to actin-rich regions in parasympathetic neuronsfrom chick embryos, early stages of mouse development andcellularization structures in D. melanogaster embryos.

Materials and Methods

Immunization with flightless I-specific peptides

The cDNA sequences of flightless I (fliI) and homologues have been determined (D. melanogaster GenBank accession no. U01182, C. elegans GenBank U01183 and Homo sapiens GenBankU01184).10 A peptide corresponding to sequences within the

Immunology and Cell Biology (2000) 78, 423–429

Curtin Conference

The f lightless I protein localizes to actin-based structures duringembryonic development

DEBORAH A DAVY, 1 ELDON E BALL, 2 KLAUS I MATTHAEI, 3 HUGH D CAMPBELL 2

and MICHAEL F CROUCH 1

1Molecular Signalling Group, Division of Neuroscience, 3Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research and 2Molecular Genetics and Evolution Group and Centre for MolecularStructure and Function, Research School of Biological Sciences, Australian National University, Canberra,Australian Capital Territory, Australia

Summary The product of the flightless I gene is predicted to provide a link between molecules of an as yetunidentified signal transduction pathway and the actin cytoskeleton. Previous work has shown that weak and severemutations of the flightless I locus in Drosophila melanogaster cause disruption in the indirect flight muscles andin embryonic cellularization events, respectively, indicative of a regulatory role for the flightless I protein incytoskeletal rearrangements. A C-terminal domain within flightless I with significant homology to the gelsolin-like family of actin-binding proteins has been identified, but evidence of a direct interaction between endogenousflightless I and actin remains to be shown. In the present study, chick, mouse and Drosophila melanogaster embryoshave been examined and the localization of flightless I investigated in relation to the actin cytoskeleton. It is shownthat flightless I localization is coincident with actin-rich regions in parasympathetic neurons harvested from chicks,in mouse blastocysts and in structures associated with cellularization in Drosophila melanogaster.

Key words: actin, cytoskeleton, development, Drosophila melanogaster, flightless I, neuron.

Correspondence: Deborah A Davy and Michael F Crouch, JohnCurtin School of Medical Research, GPO Box 334, Canberra, ACT2601, Australia. Email: Deb.Davy@anu.edu.au or Michael.Crouch@anu.edu.au

Received 11 April 2000; accepted 11 April 2000.

gelsolin-like domain of human fliI was synthesized (BiomolecularResource Facility, JCSMR) for subsequent production of antipeptideantibodies. The amino acid sequence corresponding to the peptide isidentical in human, C. elegans, D. melanogaster and mouse.12 Thepeptide (CSHFKRKFIIH, amino acids 1032–1041) was conjugated tokeyhole limpet haemocyanin (Sigma Chemical Co., St Louis, MO,USA), using the protocol described by Goldsmith and coworkers.17

The N-terminal cysteine was added to the peptide for coupling purposes. A New Zealand white rabbit was injected, boosted andserum collected following clot retraction and stored at – 70°C. Therabbit was further boosted at regular intervals and further sampleswere collected.

The flightless I antipeptide antibody preparation

Antibodies were affinity-purified using 1 mL N-hydroxysuccinimide(NHS)-activated HiTrap affinity columns (Pharmacia, Uppsala,Sweden) coupled with the peptide, according to the manufacturer’sinstructions.

Mammalian cell culture and harvest of Swiss 3T3 fibroblasts

Swiss 3T3 fibroblasts (Commonwealth Serum Laboratories,Parkville, Vic., Australia) were cultured and harvested as describedby Crouch and Simson.18

Fractionation of Swiss 3T3 fibroblasts

Lysates were fractionated as previously described by Franze-Fernandez and Pogo19 into the nuclear (N), cytoskeletal (C) or membrane/cytosol (M) fractions. The resulting fractionated lysateswere used for immunoprecipitation procedures.

Immunoprecipitation of fractionated Swiss 3T3 fibroblasts

The antipeptide flightless I antibody was added to lysed and frac-tionated Swiss 3T3 fibroblasts and incubated overnight on a rotatingmixer. Protein A-Sepharose beads (Pharmacia) were added andsamples mixed for 1 h. For peptide blocking, 50 µg of peptide wasincubated with 50 µL of the antibody for 1 h at room temperatureprior to use. This preparation was then used in the same manner as antibodies that were not blocked. Beads were pelleted by centrifu-gation for 45 s at 17 400 g and the supernatant discarded. Protein A-Sepharose-antibody-antigen pellets were rinsed three times with1 mL Tris-buffered saline (TBS; 50 mmol/L Tris/HCl pH 7.4,0.2 mol/L NaCl). Following the final centrifugation, SDS–PAGEsample buffer containing 15 mg/mL dithiothreitol was added to theimmunoprecipitates and the samples were stored at – 20°C. Prior toSDS–PAGE analysis, samples were heated for 5 min at 100°C.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

Proteins were electrophoresed on 7% SDS-polyacrylamide gels andwestern transferred to nitrocellulose membrane (Schleicher andSchuell, Dassel, Germany). The blot was incubated with the antibodyovernight at 4°C. Detection of the antigens after the primary anti-body incubation was performed using horseradish peroxidase-labelled secondary antibodies followed by chemiluminescentdetection (ECL; Amersham, Buckinghamshire, UK).

Preparation and immunohistochemistry of Drosophilamelanogaster embryos

Drosophila eggs of various stages were collected, dechorionated andfixed in 3.7% formaldehyde following standard protocols.20 Theywere then dehydrated through a methanol series and stored inabsolute methanol at – 20°C until needed, at which time they weregradually rehydrated through a methanol/PBS series. After severalrinses in PBS/0.2% Triton X-100, totalling greater than 30 min,embryos were ready for antibody immunohistochemistry. Antibodystaining followed standard techniques for Drosophila melano-gaster.21,22 For double-labelled preparations, best results wereobtained with sequential staining using antibodies at the followingconcentrations: rabbit antifliI, 1:150, donkey antirabbit FITC(Jackson Immunoresearch, West Grove, PA, USA), 1:100; mouseantiactin (Mab JLA-2023), 1:15; goat antimouse Texas Red (JacksonImmunoresearch), 1:125. Embryos were cleared through a glycerolseries and mounted and visualized using confocal microscopy in90% glycerol. The JLA-20 mAb developed by JJ-C Lin was obtainedfrom the Developmental Studies Hybridoma Bank developed underthe auspices of the National Institute of Child Health and HumanDevelopment (NICHD) and maintained by the University of Iowa,Department of Biological Sciences, Iowa City, IA, USA.

Dissociation and culture of embryonic chick ciliaryneurons

Ciliary ganglia were removed from embryonic day 8 chick embryosas previously described.24,25 Briefly, ganglia were incubated inCa2+/Mg2+-free Hanks’ solution containing trypsin (0.8 mg/mL) for20 min at 37°C, then washed 3 times with DMEM/10% horse serumand finally resuspended in DMEM/10% horse serum. Ganglia werethen triturated using a Pasteur pipette to disperse the individual cells.Neurons were cultured in DMEM in the presence of insulin(16 µmol/L) and phorbol ester (752 nmol/L) and incubated at 37°C(5% CO

2in air) for 24 h. This stimulation protocol has been shown

to cause maximal survival and neurite growth.26 Neurons were subsequently fixed with 2% paraformaldehyde as described below.

Harvesting and culturing of BALB/c mouse embryos

BALB/c mice were mated, and pregnant females were humanelykilled by cervical dislocation 3.5 days postfertilization. The femalereproductive organs were removed via an abdominal incision and theuterus resected by cutting below the junction of the oviduct.Embryos were flushed with DMEM and were incubated (10% CO

2

in air at 37°C) for approximately 2 days, until the blastocyst hatchedfrom the surrounding zona and attached. Embryos were subsequentlyfixed with 2% paraformaldehyde as described later.

Immunohistochemistry

Mouse embryos or chick neurons were cultured as described andfixed (2% paraformaldehyde, 0.1 mol/L phosphate buffer pH 7.5) for15–20 min at room temperature and then washed 5 times with coldPBS. Neurons were permeabilized with PBS/1.0% BSA/0.1% SDSfor 15 min and non-specific sites were blocked by treatment withPBS/1.0% BSA for 1 h at room temperature. The PBS/0.1% BSAwith the antibody was added to the well and incubation continuedovernight at 4°C. Wells were rinsed 5 times with cold PBS then incu-bated with FITC-labelled secondary antibodies (Jackson Immuno-research) for 1 h at room temperature. In some cases (as noted infigure legend) specimens were also labelled with Texas Red phal-loidin (Molecular Probes, Eugene, OR, USA). Phalloidin was dried

DA Davy et al.424

under nitrogen and redissolved in PBS/1.0% BSA. Phalloidin (25 U/mL) was added to each well and the same protocol as for secondary antibodies was followed. There was no crossover of fluorescence between FITC and Texas Red wavelengths. Cellular fluorescence was visualized and recorded with a Leica (Bensheim,Germany) confocal microscope.

Results

The flightless I antibody recognized a 145 kDa protein inSwiss 3T3 fibroblasts

Cultured Swiss 3T3 fibroblasts were harvested, fractionatedand subsequently incubated with the antipeptide flightless I(fliI) antibody. The antigen–antibody complex was resolvedusing SDS–PAGE analysis and western transfer. Figure 1shows that a single, 145 kDa protein was identified in thenuclear, membrane/cytosol and cytoskeletal fractions. ThefliI protein was present in the nuclear fraction in unstimulatedcells (lane 1), but the amount of protein present in the nuclearfraction was greatly reduced following stimulation with 10%fetal calf serum for 18 h (lane 2). Activation with seruminduced a corresponding increase in the amount of fliIprotein in the membrane/cytosol fraction (lanes 3 and 4), butthere was little change in the amount of protein found in thecytoskeletal fraction following stimulation with serum (lanes5 and 6). This protein was absent when the antibody wasblocked with peptide prior to use (not shown). The fliI-specific antibody also recognized fliI protein expressed usingan in vitro translation/transcription kit from Promega(Madison, WI, USA; data not shown). This result shows thatthe antipeptide fliI antibody specifically recognizes a singleprotein with a molecular weight equal to that predicted forfliI in Swiss 3T3 fibroblasts.

The flightless I protein localizes to actin-rich regions inparasympathetic neurons

Permeabilized cultured neurons were labelled with the fliIantibody and examined using confocal microscopy. The fliIprotein localized to punctate regions along neurites (Fig. 2a)extending from the cell body. The fliI protein was abundantin the main body of growth cones and also in discrete accumulations at the tip of filopodia extending from theleading edge of the growth cone (Fig. 2b, arrows). The local-ization of fliI correlated with the actin filaments localized to filopodia when neurons were labelled with phalloidin (not presented).

The flightless I protein localizes to phalloidin-definedregions in mouse embryos

Mouse embryos, 3.5 days postfertilization, were harvestedand cultured in DMEM for approximately 2 days afterremoval from the mother, until the blastocyst hatched fromthe surrounding zona and attached. Embryos were subse-quently fixed, permeabilized and labelled with antifliI andphalloidin. The views presented in Fig. 3 are of whole pre-implantation embryos, the major feature being the inner cellmass. Fluorescence was visualized by confocal microscopy.Visualizing a clear focal plane was difficult due to the large

size of the specimen. Individual cells are rich in actin(Fig. 3a) and it appeared that actin localized to the outerregions of each cell in the mouse embryo. When the rabbitfliI antibody distribution was visualized, it was found that fliIlocalized similarly to the outer regions of individual cells(arrows, Fig. 3b).

The flightless I protein localizes to cellularization structures in syncytial Drosophila melanogaster embryos

Embryos of varying developmental stages were permeabi-lized and fixed. The embryos were exposed to the primaryantibody specific for the fliI protein and also subsequentlylabelled with an antiactin antibody. The fliI protein clearlylocalized with the actomyosin hexagonal array that prefiguresthe sites of membrane invaginations prior to cellularization(Fig. 4a,b). Optical sections of a cellularizing blastodermrevealed that actin is localized predominantly to the eggcortex and the early stages of membrane invagination furrows(Fig. 4c). Similarly, when fliI localization was examined, thestaining was clearly coincident with the actin-based invagi-nation furrows (Fig. 4d). There was also a concentration offliI protein localizing to the leading edge of the invaginatingfurrows (arrows, Fig. 4d). Double labelling with the actin(Fig. 4e) and fliI (Fig. 4f) antibodies in Drosophila embryosthat had almost completed cellularization, showed that actinand fliI localize to invaginating membrane furrows.

The flightless I protein and actin 425

Figure 1 Detection of the mouse flightless I protein on westernblots of Swiss 3T3 fibroblast extracts using antiflightless Iantipeptide antibody. Swiss 3T3 fibroblasts were starvedovernight in 0% FCS and either left unstimulated (0) or activatedwith 10% FCS for 18 h (S). Nuclear (N), membrane/cytosol (M)and cytoskeletal (C) fractions are indicated. The fractions wereimmunoprecipitated with antiflightless I antipeptide antibodyprior to gel electrophoresis. A rabbit antipeptide flightless I anti-body was then used for detection after western transfer. The position of the rabbit antibody heavy chain (h.c.) is indicated. Asingle protein of approximately 145 kDa was precipitated andimmunoblotted by the flightless I antibody. Molecular weight(Mol. wt.) marker sizes are shown in kDa.

Discussion

The polymerization and depolymerization of actin plays anactive role in a large number of cellular functions, includingreorganization of cell shape and cell motility.3,27,28 Thecontrol of G- and F-actin pools is under the influence ofactin-binding proteins, some of which can sequester G-actin,making it unavailable for incorporation into filaments.29,30

Other actin-binding proteins are able to bind actin monomers,creating nucleation sites for the initiation of actin polymer-ization, or can bind, cap and/or sever actin filaments, result-ing in the reorganization of actin filaments.31,32 Manydifferent actin-binding proteins are required to work inconcert to regulate the actin cytoskeletal activities in cells.One of the proteins possibly responsible for interacting withand modifying actin filaments is the fliI protein, a protein

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Figure 2 Localization of antiflightless I immunoreactive material in cultured neurons from ciliary ganglia from 8-day-old chickembryos. (a) Low magnification view indicating that the flightless I protein is spread throughout the cell, including the cell body, neurites and growth cones, as indicated. Punctate regions of flightless I are indicated with arrows. (b) High magnification view of a growthcone. The growth cone extends filopodia from the cell periphery, but definition of these structures is difficult to see in this image. Thetips of each filopodia are strongly immunoreactive to the antiflightless I antibody (arrows).

Figure 3 Immunolocalization of flightless I protein (b) and actin (a) in early mouse embryos. BALB/c mouse embryos were harvested,fixed in 2% paraformaldehyde and labelled with Texas Red phalloidin (a) and flightless I antibody (b), which was visualized with FITCantirabbit secondary antibodies. Arrows mark regions of colocalization between actin and flightless I at the plasma membrane of individual cells.

with significant homology to the gelsolin-family of actin-binding proteins.10

The functional organization of the adult nervous systemdepends on the connections formed during development,when axons grow out from cell bodies and extend alongdefined pathways to connect with their targets. This directedaxonal elongation is due to motile structures at the axon tips,the growth cones, which establish the direction of axonalelongation by detecting and responding to guidance cues.Growth cone behaviours, including protrusion, retraction and turning, depend on the dynamic reorganization of thecytoskeleton.33 Growth cones navigate by constantly extend-ing and retracting sensory protrusions, in the form of broadlamellipodia and finger-like projections called filopodia,from the leading edge. It has been suggested that actinrearrangements at the tips of filopodia control their extensionand retraction.34 Actin-binding proteins influence the behav-iour of the cytoskeleton, which in turn influences the protru-sive force generated at the leading edge. The fliI proteinlocalizes in a punctate distribution along neurites in culturedchick ciliary neurons. These sites correspond to varicosities,35

sites that form areas of close membrane apposition with thetarget cell membrane. These areas are similar to the localvariations in fluorescence observed when the actin distribu-tion is visualized in neurites,36 also identified as varicosities.The presence of fliI protein within migratory structures suchas neurites, growth cones and sites of dynamic actinrearrangements, such as tips of filopodia, is consistent witha role for fliI in actin rearrangements. The process by whichfilopodia extend is believed to involve the addition of actinmonomers and/or short actin fragments to the barbed end ofthe filament.34 This process of treadmilling involves thedepolymerization of actin from the minus end of the filamentand the incorporation of actin monomers and/or short actinfilaments at the barbed, or plus, end of the growing filament.Recent evidence has shown that the expressed form of C.elegans fliI is able to sever actin16 and may account for thepresence of fliI at the tips of filopodia extending frommigrating growth cones.

Fertilization of a mouse oocyte triggers cell division and movement of the blastocyst along the oviduct to theuterus. The migrating blastocyst continues to divide and

The flightless I protein and actin 427

Figure 4 Immunolocalization of the flightless I protein and actin in double-labelled Drosophila melanogaster embryos. Each of the pairs(a,b), (c,d) and (e,f) represent individual embryos viewed first with FITC to localize actin (a,c,e) and then with Texas Red to localize flight-less I (b,d,f). (a,b) Actin and flightless I colocalize to the hexagonal pattern of actomyosin that prefigures the sites of membrane invagi-nations prior to cellularization during early stages of cellularization. (c,d) Colocalization of actin and flightless I to membrane invaginationfurrows as cellularization advances. (e,f) Flightless I and actin colocalize to membranes and membrane furrows when cellularization isalmost complete.

differentiate, forming an embryo consisting of two differentcell types, the trophectoderm, which forms the basis of theplacenta, and the inner cell mass, from which the fetus willdevelop. During the fifth day of development, the blastocysthatches from the surrounding zona and is ready for implan-tation into the walls of the uterus. Division and differentiationare processes that require an intact cytoskeleton, and thepresence of this network in developing embryos can be visu-alized when embryos are labelled with phalloidin. The local-ization of the fliI protein in mouse embryos is coincident withthese actin-rich regions. In Drosophila, 13 synchronousmitotic divisions without cytokinesis produce a syncytialblastoderm characterized by a uniform monolayer of corticalnuclei. Immediately following the alignment of the nuclei,the cell membrane begins to synchronously invaginatebetween adjacent nuclei, partitioning them into individualcells. This stage, known as cellularization, is a specializedform of cytokinesis37 and as such requires an intact, func-tional actin cytoskeleton under the control of other proteins38

to occur. Just prior to cellularization, actin is concentrated incap-like structures between the egg cortex and the underlyingnuclei. These caps expand and, as their edges reach eachother, a hexagonal network of actin filaments is formed thatprefigures the sites of membrane invaginations. Actinremains associated with the newly formed cell membranesand the advancing cleavage furrows, while different actin-binding proteins associate with various domains of the sub-cortical actin network. Germ line clone analysis of embryoswith severe disruptions of the fliI transcription unit, resultingin the depletion of maternally supplied protein, has revealedthat they fail to undergo cellularization. Although nuclei suc-cessfully align at the egg cortex, they lose their position at theperiphery before cellularization is complete. Abnormal meso-dermal invagination ensues, followed by defective gastrula-tion and finally death, indicating an absolute requirement forthe fliI gene product at this stage of cellularization. Thematernally synthesized fliI protein appears to be required forthe proper distribution of actin along the membrane networkand for cellularization to proceed correctly. It apparently reg-ulates the state of the actin cytoskeleton, rather than onlybeing involved in attaching or moving proteins along theactin filaments.14 When cellularizing embryos are labelledwith the fliI antibody, this protein is seen localizing to actin-positive invaginating membrane, consistent with a regulatoryrole in this process. The observation that fliI appears toprecede the invagination of the actin-based furrows suggeststhat fliI may predefine or prepare material for the oncominginvaginating membranes. The irregularity of actin filamentsin the mutant may arise from the network being expanded bythe contractile apparatus without new actin being inte-grated.14 Vesicles observed near the invaginating membranesof fliI mutant embryos are believed to contain material thathas failed to be incorporated, as seen in embryos mutant inother genes that affect cellularization.39

The results presented here show that the fliI protein,which a number of lines of evidence indicates may mediatesignal transduction regulation of the actin cytoskeleton, local-izes to actin-defined structures during development. We showthat the fliI protein localizes to regions involved in cellular-ization in the Drosophila embryo, to migratory structures inthe growth cones of chick ciliary neurons and to the outer

regions of individual cells of mouse blastocysts. The actionsof fliI at these sites remain unclear, but it has recently beenshown that the C. elegans form of fliI is able to sever actin.This may explain the presence of fliI, and the function forfliI, within neuronal migratory structures, cell membranesand cellularization structures in D. melanogaster.

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