Draft - University of Toronto T-SpaceDraft 1 1 Phagocytosis: What’s on the menu? 2 3 Charlene E. Lancaster1,2, Cheuk Y. Ho1, Victoria E.B. Hipolito3,4, Roberto J. Botelho3,4, 4 Mauricio

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Phagocytosis: What’s on the menu?

Journal: Biochemistry and Cell Biology

Manuscript ID bcb-2018-0008.R1

Manuscript Type: Mini Review

Date Submitted by the Author: 02-May-2018

Complete List of Authors: Lancaster, Charlene E.; University of Toronto at Scarborough Department of Biological Sciences; University of Toronto at Scarborough Department of Biological Sciences Ho, Cheuk Y.; University of Toronto at Scarborough Department of Biological Sciences Hipolito, Victoria E.B.; Ryerson University, Molecular Science Graduate Program; Ryerson University, Chemistry and Biology Botelho, Roberto J.; Ryerson University, Molecular Science Graduate Program; Ryerson University, Chemistry and Biology Terebiznik, Mauricio R.; University of Toronto at Scarborough Department of Biological Sciences; University of Toronto Department of Cell and

Systems Biology

Keyword: Phagocytosis, Target morphology, Phagocytic plasticity, Phagosomal maturation, Phagocytic cup

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Biochemistry and Cell Biology

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Phagocytosis: What’s on the menu? 1 2

Charlene E. Lancaster1,2, Cheuk Y. Ho1, Victoria E.B. Hipolito3,4, Roberto J. Botelho3,4, 3

Mauricio R. Terebiznik1,2† 4

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1Department of Biological Sciences and 2Department of Cell and System Biology, University of 6

Toronto at Scarborough, Toronto, Canada 7

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3Molecular Science Graduate Program and 4Department of Chemistry and Biology, Ryerson 9

University, Toronto, Canada 10

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†To whom correspondence should be addressed: 12

Mauricio R. Terebiznik 13

E-mail: [email protected] 14

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Abstract 15

Phagocytosis is an evolutionarily conserved process. In Protozoa, phagocytosis fulfills a 16

feeding mechanism, while in Metazoa, phagocytosis diversified to play multiple organismal 17

roles, including immune defence, tissue homeostasis and remodeling. Accordingly, phagocytes 18

display a high level of plasticity in their capacity to recognize, engulf and process targets that 19

differ in composition and morphology. Here, we review how phagocytosis adapts to its multiple 20

roles and discuss in particular the effect of target morphology in phagocytic uptake and 21

phagosome maturation. 22

23

Keywords: Phagocytosis, Target morphology, Phagocytic plasticity, Phagosomal maturation, 24

Phagocytic cup 25

26

Overview of Phagocytosis and Phagosome Maturation 27

Protozoa and certain cells within Metazoa have the ability to selectively internalize 28

particles and degrade them intracellularly through a process known as phagocytosis. In this 29

process, plasma membrane remodeling promotes the engulfment of particles greater than 0.5 µm 30

in diameter into enclosed vacuoles called phagosomes, where the targets are degraded. 31

Phagocytic cells express receptors, like lectins and scavenger receptors, that can recognize 32

distinct molecular signatures endogenous to the target (Naik and Harrison 2013, Brubaker et al. 33

2015). Alternatively, phagocytes also express opsonic receptors that bind to opsonins deposited 34

on the target, like complement and Fcγ receptors that bind to complement fragments and 35

immunoglobulin G (IgG), respectively (Naik and Harrison 2013, Brubaker et al. 2015). 36

Receptor-ligand clustering triggers changes in the composition of plasma membrane 37

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phosphoinositide lipids and the activation of small Rho GTPases, like RhoA, Rac1/2 and Cdc42, 38

to stimulate the formation of actin-rich, membrane pseudopods, which surround the target, 39

thereby forming a phagocytic cup (Swanson et al. 1999, Herre et al. 2004, Tomasevic et al. 40

2007). 41

The closure of the phagocytic cup results in the formation of a new phagosome, which 42

undergoes maturation through a stepwise process that promotes the degradation of the 43

internalized particle (Figure 1A). The internal environment of the phagosome changes from a 44

neutral to an acidic pH and acquires hydrolases through the fusion with the endolysosomal 45

compartments (early endosomes, late endosomes and lysosomes). In the initial stage of 46

maturation, the nascent phagosome acquires the small GTPase, Rab5, whose recruitment and 47

activity is presumed (based on endosomal maturation) to be promoted through its cognate GEF, 48

Rabex-5, as well as through a positive feedback loop established by the Rab5/Rabaptin-5 49

complex (Horiuchi et al. 1997, Lippé et al. 2001). Rab5 activity promotes early endosomal 50

fusion through the recruitment of classical early endosome marker Early Endosome Autoantigen 51

1 (EEA1) and the p150/Vps34 complex (Mu et al. 1995, Christoforidis et al. 1999b, Vieira et al. 52

2002). In turn, the acquisition of Vps34 increases the localized synthesis of phosphatidylinositol-53

3-phosphate [PtdIns(3)P], which then further enhances EEA1 recruitment (Christoforidis et al. 54

1999b). EEA1 acts like a tether to coordinate and promote fusion between Rab5-positive 55

organelles, like early phagosomes and early endosomes (Christoforidis et al. 1999a). 56

Following endosomal fusion, the early phagosome transitions into the phagolysosome. 57

The Mon1-Ccz1 complex facilitates the loss of Rab5 and subsequent acquisition of Rab7, by 58

displacing Rabex-5 and Rabaptin-5 (Kinchen and Ravichandran 2010, Poteryaev et al. 2010). 59

The recruitment of active Rab7 primes the phagosome for fusion with the late endosomes and 60

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lysosomes through yet unclear mechanisms. It is known that Rab7-interacting lysosomal protein 61

(RILP) and oxysterol-binding protein-related protein 1L (ORP1L) work together to mediate 62

microtubule-dependent trafficking of the phagosome towards the lysosome via the dynein-63

dynactin complex (Harrison et al. 2003, Johansson et al. 2007). Moreover, tubular phagosome 64

extensions are formed in a RILP-dependent manner and allow the phagosome to fuse with the 65

late endosomes and lysosomes (Harrison et al. 2003). The homotypic fusion and protein sorting 66

(HOPS) complex promotes fusion through two Rab7-interacting subunits, Vps39 and Vps41, 67

which tether two Rab7-positive compartments together to initiate and facilitate fusion via trans-68

SNARE complexes (Kramer and Ungermann 2011, Balderhaar and Ungermann 2013). The 69

GTPase, ADP Ribosylation Factor Like GTPase 8B (Arl8B), also appears to coordinate HOPS 70

complex association via kinesin-1 (Garg et al. 2011). In addition, the lysosomal Ca2+ channel, 71

transient receptor potential mucolipin 1 (TRPML1/MCOLN1), and phosphatidylinositol-3,5-72

bisphosphate [PtdIns(3,5)P2] are also necessary for maturation (Kim et al. 2014, Dayam et al. 73

2015, 2017). The synthesis of PtdIns(3,5)P2 by the lipid kinase PIKfyve helps to deplete 74

PtdIns(3)P from the early phagosome and promote its conversion to a late-stage phagosome 75

(Hazeki et al. 2012, Kim et al. 2014). Moreover, PtdIns(3,5)P2 is thought to activate 76

TRPML1/MCOLN1, thus releasing Ca2+ from lysosomes and facilitating phagosome-lysosome 77

fusion (Dayam et al. 2015). PIKfyve may also play a role in recovering nutrients during the 78

degradation of phagosomal cargo, as was shown for phagosomes containing apoptotic bodies 79

(Krishna et al. 2016). 80

As a consequence of the maturation process, the luminal content of the phagosome 81

changes profoundly. The acquisition of vacuolar-ATPases establishes an acidic pH (4-4.5), 82

which is optimal for the action of a number of hydrolytic enzymes, such as proteases, peptidases, 83

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phosphatases, nucleases, glycosidases, sulfatases and lipases (Appelqvist et al. 2013). The 84

ultimate function of these hydrolases is to degrade the target particle, which will provide 85

metabolites to the cell and produce antigen to prime the adaptive immune response. 86

87

Phagocytic Branches on the Tree of Life 88

In its origins, phagocytosis was limited to a nutritional role and can be hypothetically 89

traced back to predatory, phagotrophic archaeans. According to the endosymbiotic theory, these 90

archaeans originated the eukaryotic lineage upon the ingestion of α-proteobacteria from which 91

ancestral mitochondria developed (Yutin et al. 2009, Davidov and Jurkevitch 2009, Koonin 92

2015, Martin et al. 2017). Interestingly, comparative genomic analysis identified actin and actin-93

associated protein homologues in Archaea, indicating that these organisms could have primordial 94

actin cytoskeletons and the capacity to modulate them. This thereby raises the possibility that 95

archaeans can perform a primitive form of phagocytosis (Hara et al. 2007, Ettema et al. 2011, 96

Lindås et al. 2014, 2017, Izoré et al. 2016). 97

Phagocytosis is the feeding mechanism utilized by many species of Protozoa. This has 98

been particularly well studied for the amoebae, Entamoeba histolytica, Amoeba proteus and 99

Dictyostelium discoideum, among others (Cosson and Soldati 2008, Sobczak et al. 2008, Clarke 100

et al. 2010). Amoebae thrive within aquatic and soil environments by phagocytosing bacteria and 101

other protozoans. The phagocytosed prey is trafficked into an internal compartment known as 102

food vacuole, where it is later digested by host enzymes (Müller et al. 2005, King et al. 2013, 103

Leiba et al. 2017). Phagocytosis is also involved in the feeding of bacteriotropic ciliates and 104

flagellate protozoans. The bacteriotropic ciliates, like Paramecium and Tetrahymena, utilize cilia 105

from their extensive ciliary membranelle structures to push bacteria and other food particles into 106

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a small buccal cavity (Allen and Fokt 2000, Grønlien et al. 2002, Guerrier et al. 2017). 107

Choanoflagellates instead draw bacteria into a collar of actin-filled microvilli through an apical 108

flagellum (Leadbeater et al. 2009, Nielsen et al. 2017). Once the bacterium is positioned at the 109

base of either the buccal cavity or collar of microvilli, the bacterium is engulfed into a food 110

vacuole (essentially a phagosome) through phagocytosis. As the vacuole is trafficked away from 111

the site of phagocytosis via a microtubule-dependent transport system, the food vacuole undergo 112

maturation, during which it acquires hydrolytic enzymes from lysosomes (Kovács and Csaba 113

2006, Dayel and King 2014, Nielsen et al. 2017). The degraded materials supply the nutritional 114

requirements for the phagotrophic protozoans, while the unwanted waste material is expelled out 115

into the surrounding environment (Guerrier et al. 2017). 116

The aquatic sponges within the phylum Porifera are sessile organisms that feed by 117

filtering large quantities of water. Sponges bear different arrays of phagocytic cells that can 118

capture food particles through phagocytosis, including choanocytes, which resemble the 119

protozoan choanoflagellates due to their similar collar of microvilli (Leys and Eerkes-Medrano 120

2006, Wehrl et al. 2007). In addition to a feeding mechanism, Porifera phagocytes capture and 121

eliminate pathogens, thereby indicating that the function of phagocytosis in these organisms had 122

diversified into an immune defence role (Müller and Müller 2003, Vacelet and Duport 2004, 123

Mukherjee et al. 2015, Neely et al. 2016). 124

Élie Metchnikoff’s seminal work in cellular immunity, where he described macrophages 125

and coined the term phagocytosis, was completed in starfish larvae (Gordon 2016). The immune 126

system of echinoderms consists of multiple types of phagocytes found within their coelomic 127

fluid, which are important for the removal of bacteria, xenogeneic cells and foreign material 128

(Smith et al. 2010). Furthermore, echinoderms have a basic complement system, where 129

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coelomocytes express the opsonic protein SpC3, which is homologous to the mammalian C3 130

protein, and phagocytes contain receptors that can bind to C3-like proteins (Al-Sharif et al. 1998, 131

Smith et al. 2010). 132

Within invertebrates in the phyla Nematoda and Arthropoda, phagocytosis is further 133

incorporated into a variety of cellular functions, including tissue homeostasis, remodelling, 134

development and immunity. Surface expression of phosphatidylserine on apoptotic cells in 135

Caenorhabditis elegans triggers their removal by neighbouring cells through phagocytic 136

mechanisms, while endoderm cells are known to internalize primordial germ cells during cellular 137

development (Hsieh et al. 2012, Mapes et al. 2012, Abdu et al. 2016). Haemocytes within 138

insects, like Drosophila melanogaster, internalize pathogens and virus-infected cells, as part of 139

their immune responses (Brock et al. 2008, Moreira et al. 2011, Honti et al. 2014, Nainu et al. 140

2015). The Drosophila integrin βv on haemocytes recognizes and initiates phagocytosis of 141

apoptotic cells, while it also works in conjunction with the receptor Draper to recognize and bind 142

to peptidoglycan and lipoteichoic acid during the phagocytosis of Staphylococcus aureus 143

(Shiratsuchi et al. 2012). While during neuronal development and degeneration, clearance of 144

neurites by phagocytosis is achieved by neighbouring epidermal cells rather than haemocytes 145

(Han et al. 2014). 146

The adaptive immune system first appeared in jawless fish, as established in lampreys 147

through the discovery of B and T cells and antigen receptors, called variable lymphocyte 148

receptors (Flajnik 2018). However, many of the features of the mammalian adaptive immune 149

system emerged in cartilaginous fish, including immunoglobulins and major histocompatibility 150

complex (MHC) class I and II molecules (Flajnik 2018). Thus, phagocytosis plays a new role in 151

vertebrates by providing a link between the innate and adaptive immune response. In this 152

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context, the phagosomal degradation of the target, which was engulfed by phagocytes, generates 153

antigens that are presented by MHC class II molecules to cells of the adaptive immune system, 154

thereby leading to the production of antibodies against the target. 155

Phagocytosis within vertebrates can be performed by cells originating from different 156

lineages and these cells can be classified as either professional or non-professional phagocytes 157

(Rabinovitch 1995, Han et al. 2016). Epithelial cells and fibroblasts retained the ability to 158

perform phagocytosis and thus can participate in functions like wound healing or neuronal repair 159

(Rasmussen et al. 2015, DeBerge et al. 2017). However, macrophages, dendritic cells and 160

neutrophils are able to more efficiently internalize targets and therefore are called professional 161

phagocytes. Dendritic cells are highly specialized antigen presenting cells that present exogenous 162

peptides derived from degraded microorganisms on MHC class II molecules. These antigens 163

trigger the proliferation of CD4+ T lymphocytes, thereby activating the adaptive immune system 164

(Desjardins et al. 2005, Hoffmann et al. 2012). Like dendritic cells, macrophages can present 165

antigens to T and B lymphocytes. However, these cells also play a number of regulatory and 166

homeostatic roles, including killing microbes and clearing apoptotic cells. Neutrophils specialize 167

in the removal of microorganisms from the body and are considered the most efficient phagocyte 168

in regards to both speed of internalization and quantity of targets phagocytosed (Lim et al. 2017). 169

Besides phagocytosis, neutrophils have mechanisms to extracellularly kill pathogens, as 170

discussed later in this review (Lim et al. 2017). 171

Macrophages are located within most tissues of the body, including the lungs, central 172

nervous system and spleen, where the tissue-resident macrophages serve as the first line of 173

defence against an infection. These phagocytes can be derived from circulating monocytes, 174

where they are replenished by new monocytes from within the blood, or can be derived early in 175

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development from embryonic sources, where they self-replenish locally within the tissue 176

(Hashimoto et al. 2013, Ogle et al. 2016). Despite their similarity in the mechanisms they 177

employ, macrophages are a very heterogeneous group of cells, whose function depends not only 178

on the tissue in which they reside, but also the microanatomical niche (Davies et al. 2013). 179

Moreover, macrophages are highly plastic cells that can adopt different states of activation, such 180

as the inflammatory M1 macrophages and anti-inflammatory M2 macrophages (Ogle et al. 181

2016). Evidently, phagocytosis is a highly conserved mechanism that has coevolved with 182

eukaryotes into a more diverse and sophisticated process, in order to maintain complex tissue 183

homeostasis and combat the ever-changing infectious pathogens. 184

185

The War between Predators and Prey 186

Phagocytes and their targets are engaged in a deadly predator-prey war, which can be 187

traced back to the co-evolution of phagotrophic protozoans and bacteria (Brüssow 2007). In 188

order to recognize their prey, Protozoa evolved receptors. The majority of receptors identified so 189

far fall under two main categories: integrins and lectins (Roberts et al. 2011). The protein, SibA 190

found in Dictyostelium discoideum is both structurally and functionally similar to the mammalian 191

integrin β chains, where it is critical for attaching bacteria (Cornillon et al. 2006). Whereas the 192

parasitic protozoan, Entamoeba histolytica, uses a galactose/N–acetylgalactosamine 193

(Gal/GalNAc) lectin to recognize and bind to its prey (Okada et al. 2005). Although these 194

receptors are critical for protozoan phagocytosis, some parasitic bacteria have evolved to utilize 195

these receptors as a means of entry into their host. Legionella pneumophila, which is an 196

intracellular parasite in Protozoa and an intracellular pathogen in humans, attaches to and 197

invades the Protozoa, Hartmannella vermiformis, by taking advantage of a Gal/GalNAc lectin 198

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Figure 1. The phagocytosis of long filamentous targets is dependent on the orientation of the target in relation to the cell. Schematics showing how the morphology of the target can impact internalization. (A) The internalization of short bacillary targets occurs independently of the orientation of the target. After the

phagocytic cup closes (i), the phagosome undergoes fusion with the endolysosomal compartments (ii), including the early endosomes (green), late endosomes (blue) and lysosomes (red), in order to gain the characteristics required to degrade the target (iii). (B) In opposition, the phagocytosis of long filamentous targets is highly dependent on the orientation of the target. Phagocytes that encounter the long axis of the target are not able to proceed with phagocytosis until the filament is reoriented, such that the cell can

capture one of its poles (iv). Once the phagocyte has oriented the target correctly, internalization proceeds through a tubular phagocytic cup (v), which undergoes remodelling through its fusion with the

endolysosomal compartments (vi). Although the tubular phagocytic cup acquires lysosomal markers, the compartment does not become degradative until the cup seals (vii).

100x64mm (300 x 300 DPI)

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Figure 2. The maturation of the tubular phagocytic cup. During the uptake of filamentous targets, macrophages extend actin-driven pseudopodia around the target, thereby forming a tubular phagocytic cup (i). The tubular phagocytic cup first recruits early endosome markers, Rab5 and EEA1, as well as the class

III PI3K, Vps34, which synthesizes PtdIns(3)P (ii). Subsequently, the early endosome markers disassociate from the tubular phagocytic cup, with the exception of Vps34 and PtdIns(3)P, and endolysosomal

components, like LAMP1 and Rab7, are acquired (iii). During stages i-iii, a F-actin jacket is formed at the neck of the tubular phagocytic cup, which generates forces that constrict the target and favours the

formation of an integrin-dependent diffusion barrier and membrane fences. Despite the diffusion barrier, small molecules within the tubular phagocytic cup, like hydrolytic enzymes and H+, are able to leak out.

Tubular phagocytic cups that are longer than 20 µm will begin to acidify and will be able to partially retain ROS (iv). Acidification results in the disassociation of Vps34, thereby halting the production of PtdIns(3)P. The remaining PtdIns(3)P on the luminal region of the tubular phagocytic cup is thus depleted, when it is

consumed by PIKfyve. Once the target is fully enclosed, the tubular phagocytic cup seals to form a phagosome, which completely acidifies and accumulates hydrolytic enzymes (v). This microbicidal/hydrolytic

environment allows for the complete degradation of the phagocytic target (vi). Red arrows indicate the pressure exerted by actin, while black dotted arrows show the endolysosomal content escaping from the

tubular phagocytic cup.

204x157mm (300 x 300 DPI)

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Documents

Draft - University of Toronto T-SpaceDraft 1 1 Phagocytosis: What’s on the menu? 2 3 Charlene E. Lancaster1,2, Cheuk Y. Ho1, Victoria E.B. Hipolito3,4, Roberto J. Botelho3,4, 4 Mauricio