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Cleaning up the mess: cell corpse clearance in Caenorhabditis elegans
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Cleaning up the mess: cell corpse clearance in Caenorhabditis elegans Se´rgio Morgado Pinto1,2 and Michael Otmar Hengartner1 Genetic and cell biology studies have led to the identification in Caenorhabditis elegans of a set of evolutionary conserved cellular mechanisms responsible for the clearance of apoptotic cells. Based on the phenotype of cell corpse clearance mutants, corpse clearance can be divided into three distinct, but linked steps: corpse recognition, corpse internalization, and corpse degradation. Work in recent years has led to a better understanding of the molecular pathways that mediate each of these steps. Here, we review recent developments in our understanding of in vivo cell corpse clearance in this simple but most elegant model organism. Addresses 1 Institute of Molecular Life Sciences, University of Zurich, 8057 Zurich, Switzerland 2 Graduate Program in Areas of Basic and Applied Biology (GABBA), Universidade do Porto, Porto, Portugal Corresponding author: Hengartner, Michael Otmar ([email protected], [email protected]) Current Opinion in Cell Biology 2012, 24:881–888 This review comes from a themed issue on Cell division, growth and death Edited by Julia Promisel Cooper and Richard J Youle For a complete overview see the Issue and the Editorial Available online 1st December 2012 0955-0674/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ceb.2012.11.002
Introduction The ability to safely remove apoptotic cells plays an important role in homeostasis, wound healing and in the development of multicellular organisms [1,2]. Failure to clear corpses can result in secondary necrosis and lead to inflammation and/or autoimmune diseases, such as chronic polyarthritis and systemic lupus erythematosus [3–5]. Programmed cell death in Caenorhabditis elegans occurs in two waves [6]. During development, 131 of the 1090 cells generated in the invariant somatic lineage die soon after their birth and are rapidly removed by their neighboring cells [7]. Later in the adult hermaphrodite gonad, several hundred germ cells die during oogenesis. Germ cells can also induce apoptosis in response to genotoxic stress, DNA damage or bacterial infection [8–10]. The gonadal sheath cells which encase the germ line are responsible for the engulfment and degradation of these cells [9]. www.sciencedirect.com
Both somatic and germ cell corpses can be identified as refractile discs under differential interference contrast (DIC) microscopy (Figure 1). Genetic studies over the past decades led to the identification of a large number of C. elegans ‘cell clearance genes’. As is the case for the apoptotic killing machinery, the process of apoptotic cell clearance is evolutionarily conserved: homologous proteins play similar roles in cell corpse recognition, uptake, and degradation in both worms and humans [11]. Table 1 shows a list of C. elegans proteins and their mammalian homologs involved in cell clearance control. Apoptotic cell clearance in C. elegans can be divided in to three major steps. First, the doomed cells have to be recognized, and then internalized by the phagocytic cell in a process known as engulfment. Finally, internalized corpses undergo a process of phagosome maturation, ultimately forming phagolysosomes in which the ingested corpse is digested.
CED-1, CED-6, CED-7 pathway Genetic studies in the worm identified three partially redundant signaling pathways that mediate cell corpse recognition and internalization [12,13]. The first pathway consists of CED-1 (LRP1/MEGF10), CED-6 (GULP), CED-7 (ABCA1) and DYN-1 (Dynamin). CED-1 is a putative cell corpse receptor composed of a large extracellular domain, a single transmembrane domain and a small intercellular domain responsible for downstream signaling [14]. CED-1 accumulates on the plasma membrane of engulfing cells in the areas that contact apoptotic cells, suggesting that it can recognize molecules or epitopes exposed on the surface of dying cells [14,15,16]. The best characterized apoptosis marker in worms is the phospholipid phosphatidylserine (PS), a common ‘eatme’ signal in mammals [17] that is also selectively exposed on the surface of apoptotic cells in C. elegans [18–20]. Several studies suggest that CED-1 can indirectly bind to PS, using the secreted transthyretinlike protein TTR-52 as a bridging molecule [16,21]. The adapter protein CED-6 is a phosphotyrosine-binding (PTB) domain containing protein which has been shown to interact with CED-1 in a heterologous system [22]. CED-6 is likely to relay CED-1 signals to downstream components regulating corpse engulfment via CED-10 [12] and degradation via the large GTPase DYN-1 (see below) [23] (Figure 2). DYN-1 is enriched transiently on the phagocytic cup where it has been reported to Current Opinion in Cell Biology 2012, 24:881–888
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Figure 1
(a)
promote the fusion of early endosomes with the nascent pseudopodia, allowing the rapid delivery of lipids necessary for the embracement of the dying cell by the membrane extensions of the engulfing cell [24,25].
(b)
(d)
(c)
Current Opinion in Cell Biology
Apoptotic cell corpses in the C. elegans hermaphrodite gonad and embryos. (a, b) Bend region of the hermaphrodite gonad 24 hours postL4/adult molt; (c, d) typical embryos at the 4-fold stage. (a, c) Wild type worms with no visible cell corpses; (b, d) ced-6(n1813) mutant animals with many persistent cell corpses (arrowheads). Scale bar, 10 mm.
Two recent reports suggest that apoptotic cells not only expose PS on their surface, but that they actively transfer PS to the outer surface of the engulfing cells surrounding the corpses [26,27]. This lipid transfer appears to be mediated through the generation of PS-containing and TTR-52-containing lipid particles by apoptotic cells [26]. Loss of the ABC transporter CED-7 or of TTR-52 reduces lipid particle formation and PS transfer. Lipid transfer is also blocked in animals lacking the secreted lipid binding protein NRF-5 [27]. Importantly, mutations that block lipid transfer also prevent CED-1dependent corpse recognition and internalization [21,26,27]. The discovery of PS and TTR-52-containing vesicles raises many interesting questions. Do they act as signaling particles, or is the physical transfer of molecules from apoptotic cell to surrounding cells required for activation of the engulfing cell? Do all dying cells generate lipid
Figure 2
Transmemb. proteins
Transmemb. proteins
PAT-2 PAT-3
UIG-1
CDC-42 ABI-1
UNC-73
MIG-2
MTM-1
CED-5 CED-12
SCRM-1
PS Exposure
PAT-3 INA-1
CED-7
MOM-5 TTR-52 NRF-5
CED-1
SRGP-1
ABL-1
Cytoskeletal rearrangements
CED-10
CED-2 SRC-1 GSK-3 APR-1 CED-6
DYN-1
Phagosome maturation
CED-7 DYING CELL
ENGULFING CELL Current Opinion in Cell Biology
Molecular pathways controlling apoptotic cell recognition and internalization in C. elegans. Arrows indicate direct physical interaction and/or activation. ‘Flat’ arrows indicate inhibitory action. Dashed arrows indicate a proposed interaction and/or activation. PAT-2, UIG-1 and CDC-42 act in muscle cells; INA-1 and CED-1 preferentially act in epithelial cells [40]. Current Opinion in Cell Biology 2012, 24:881–888
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Cell corpse clearance in C. elegans Pinto and Hengartner 883
Table 1 List of C. elegans genes involved in cell clearance and their mammalian homologs. C. elegans
Brief description
Mammalian Homolog
Ref.
PS exposure
SCRM-1
phospholipid scramblase required for PS exposure
Scrm1
[20]
Signal recognition
INA-1 PAT-2 PAT-3 TTR-52 NRF-5 CED-1
integrin a subunit integrin a subunit integrin b subunit promotes PS recognition by the engulfing cell lipid transfer/LPS-binding family protein transmembrane protein possible death cell recognition
Integrin a Integrin a Integrin b sCD14, TTR hLBP family LRP, MEGF10
[39] [40] [39,40] [16] [27] [14,22]
Engulfment
CED-6 CED-7 UNC-73 MIG-2 CED-2 CED-5 CED-12 CED-10 SRGP-1 MOM-5 GSK-3 APR-1 ABL-1 ABI-1 UIG-1 CDC-42 MTM-1
adaptor SH3 and PTB containing protein, relays CED-1 signal downstream ABCA1 lipid transporter, putative creation of lipid subdomains or particles GEF, similar to TRIO which acts on RhoG Ras-related small GTPase, mammalian homolog interacts with CED-12 SH2, SH3 containing adapter protein acts together with CED-12 as a bipartide CED-10 GEF acts together with CED-5 as a bipartide CED-10 GEF Ras-related small GTPase, orchestrates actin cytoskeleton rearrangements CED-10 GTPase activating protein one of the Frizzled homologs glycogen synthase kinase, works downstream of MOM-5 upstream CED-2/5/12 b-catenin binding protein, works downstream of MOM-5 upstream CED-2/5/12 SH2, SH3 containing protein, non-receptor tyrosine kinase interacts with ABL-1contains SH3 domain GEF, specific for CDC-42 Ras-related small GTPase, Cdc42 homolog phosphatidylinositol 3-phosphate 3-phosphatase
GULP, hCED6 ABCA1, ABCA7 TRIO RhoG CrkII Dock180 Elmo Rac1 srGAP1 Frizzled GSK3b APC Abl Abi Clg Cdc42 MTM1
[22,68] [26,69] [28] [28] [34] [30] [33] [12,34] [37] [38] [38] [38] [13] [13] [41] [41] [29,70]
Phagosome maturation
DYN-1 PIKI-1 VPS-34 RAB-5 TBC-2 CCZ-1 SAND-1 RAB-7 VPS-11 VPS-16 VPS-18 VPS-33 VPS-39 VPS-41 UNC-108 RAB-14 BEC-1 EPG-5 ATG-18
Dynamin GTPase ortholog Class II Phosphatidylinositol 3-kinase Class III Phosphatidylinositol 3-kinase Rab5 GTPase ortholog, regulates early steps of phagosome maturation Rab GTPase, promotes RAB-5-GTP to RAB-5-GDP conversion together with SAND-1 regulates RAB-5 to RAB-7 conversion together with CCZ-1 regulates RAB-5 to RAB-7 conversion Rab7 GTPase ortholog, regulates late steps of phagosome maturation HOPS complex subunit, possible GEF activity to RAB-7 HOPS complex subunit, possible GEF activity to RAB-7 HOPS complex subunit, possible GEF activity to RAB-7 HOPS complex subunit, possible GEF activity to RAB-7 HOPS complex subunit, possible GEF activity to RAB-7 HOPS complex subunit, possible GEF activity to RAB-7 Rab2 GTPase homolog, proposed to be required for lysosome recruitment Rab14 GTPase homolog, proposed to be required for lysosome recruitment coiled-coil protein orthologous to yeast and mammalian autophagy proteins protein orthologous to mammalian autophagy protein KIAA1632 WD40 repeat-containing protein ortholog of yeast and mammalian autophagy proteins protein orthologous to yeast and mammalian autophagy proteins
Dynamin Class II PI3K Vps34 Rab5 Armus (TBC1D2) Ccz1 Mon1 Rab-7 Vps11 Vps16 Vps18 Vps33 Vps39 Vps41 Rab2 Rab14 Beclin1 mEPG5 WIPI2
[25,43] [47] [43] [43] [57,58] [55,56] [56] [23,43] [43] [43] [43] [43] [43] [43] [61,62] [63] [71] [72] [71]
MAP-LC3
[71]
LGG-1
particles? Previous studies have for example suggested that CED-7 is not necessary for CED-1 enrichment around apoptotic germ cells [12,19], hinting at different requirements for cell corpse clearance in embryos and adults. Finally, the process described above also shows intriguing similarities to the generation of nascent high density lipoprotein (HDL) particles in humans, in which the close CED-7 homolog ABCA1 transfers lipid molecules from the cell surface onto the lipid-binding protein ApoA-I. Is this similarity coincidental, or indicative of a deeper connection between the two processes? www.sciencedirect.com
CED-2, CED-5, CED-12 pathway In the second pathway, two small RhoGTPases become sequentially activated (Figure 2). First, the guanosine exchange factor (GEF) UNC-73 (Trio) activates the small GTPase MIG-2 (RhoG) [28]. While loss of either gene on its own does not result in any engulfment defect, their role in engulfment becomes quite evident in double mutant combinations with other engulfment genes [28,29]. MIG-2 in turn activates the CED-5 (Dock180)/ CED-12 (Elmo) complex, which acts as a bipartite GEF for the Rac family member CED-10 [30]. Active CED-10 Current Opinion in Cell Biology 2012, 24:881–888
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then drives the cytoskeletal rearrangements necessary for efficient engulfment of the dying cell [12,13]. The human (ELMO1, ELMO2/HCED-12A and BAB14712) and Drosophila (DCED-12) CED-12 homologs contain a lipidbinding Pleckstrin homology (PH) domain driving recruitment to the plasma membrane as well as proline-rich motifs capable of interacting with SRC homology 3 (SH3) domains [31–33]. CED-5/CED-12 can also be activated by the SH2/SH3 domain adapter protein CED-2. Like its mammalian counterpart CrkII, CED-2 is thought to interact with the GEF complex via its C-terminal SH3 domain, further stabilizing it downstream of receptor activation [34,35]. Mutations in ced-2, ced-5, ced-12 or ced-10 not only affect cell corpse engulfment but also result in distal tip cell (DTC) migration defect [33,34], suggesting that this signaling module is also used for cell migration processes.
apoptotic cells by INA-1 requires the activity of the phospholipid scramblase SCRM-1 in the dying cell, probably owing to its stimulation of PS externalization [20,39]. INA-1 promotes engulfment through the CED-5/CED12 GEF complex, via a signaling cascade that also includes the tyrosine kinase SRC-1 (Src) and CED-2 [39]. Finally, a recent report showed that in engulfing muscle cells, an alternative a integrin, PAT-2, is used to recognize cell corpses. Unlike the previously described receptors, PAT-2 signals not via CED-10/Rac but rather through the small GTPase CDC-42 (Cdc42) and its GEF UIG-1 (Clg) [41]. Based on their observation Hsieh et al. also propose that CED-1 and INA-1 use the ‘classical’ pathways (see above) and preferentially act in epithelial cells in cell corpse removal [40]. This report suggests that C. elegans can use different engulfment programs in a tissue dependent manner [40].
Rac activation is negatively regulated by at least two mechanisms. First, the RacGAP protein SRGP-1 can act directly as a GAP for CED-10 [36]. Second, the polyphosphoinositide 3-phosphatase MTM-1 modulates CED-10 activity by controlling the amount of phosphatidylinositol(3,5)P2 available in the plasma membrane, which provides the docking sites for CED-5/CED-12 GEF complex (Figure 2) [29]. Loss of either negative regulator enhances the ability of cells to recognize and take up neighboring cells leading, in extreme cases, even to the internalization and killing of cells that are still viable [29,37].
Clearance of non-apoptotic corpses and of sick cells
ABL-1, ABI-1 pathway A third signaling pathway, which acts in parallel to CED-1/6/7 and CED-2/5/12, is formed by the tyrosine kinase ABL-1 (Abl) and its interacting protein ABI-1 (Abi). ABL-1 negatively regulates engulfment by inhibiting ABI-1, which itself promotes cell clearance, either by regulating CED-10 activity or via an independent pathway (Figure 2) [13].
Cells in C. elegans can also die through non-apoptotic means, including various types of non-apoptotic programmed cell death and necrotic-like death (reviewed in [42]). Intriguingly, the ‘apoptotic’ engulfment machinery can recognize and clear most if not all of types of cell corpses, irrespective of the cause of death. Even more impressive is the ability of this machinery to recognize and clear cells that have been brought to the verge of death through sublethal apoptotic, necrotic or cytotoxic insults [29,37]). Upregulation or downregulation of the engulfment pathways under these conditions can lead to the death of cells that would have been spared, or conversely, the rescue of cells that otherwise would have been ‘eaten alive’ [29,37]. These observations suggest that dead or near-dead cells expose at least one common epitope, which can then be recognized by potential engulfers. Whether this common messenger is PS remains to be determined.
Phagosome maturation ‘Eat-me’ receptors CED-1 is but one of four ‘eat-me’ receptors that have been described in recent years: two of these have been proposed to act via CED-2 rather than CED-6 [38,39] and a third through a novel, CED-10-independent pathway (Figure 2) [40]. 4D-microscopy experiments identified MOM-5 (Frizzled) as a major receptor for cell corpse recognition during early embryonic development, activating an atypical Wnt signaling pathway composed of GSK3 (GSK3b) kinase, APR-1 (APC) and CED-2 (Figure 2) [38]. The CED-1 homologs LRP-5/6 and Arrow act as Frizzled co-receptors in mammals and flies, respectively, suggesting that CED-1 might play a similar role in early embryos [38]. Another ‘eat-me’ receptor is formed by the integrin INA-1 (Integrin a)/PAT-3 (Integrin b). Recognition of Current Opinion in Cell Biology 2012, 24:881–888
In recent years, the clever use of genetic screens and time-lapse imaging techniques has led to the identification of a group of genes with no deficiency in ‘eating’ but rather with problems in ‘digesting’ the dead cells [23,43] (Figure 3). Worms with mutations in these genes show changes in phagosome maturation, a process in which the corpse-containing phagosome undergoes gradual acidification as well as sequential fusion with early endosomes, late endosomes and lysosomes [23,43,44]. Membrane dynamics plays a central role in phagosome maturation. One of the first factors to be recruited to the phagocytic cup is the dynamin DYN-1. DYN-1 acts genetically downstream of CED-1/CED-6 and co-localizes with CED-1 on the phagocytic cup [23,25,43]. However the mechanism of its recruitment is still elusive. Mutations in DYN-1 result in delayed recruitment of Rab www.sciencedirect.com
Cell corpse clearance in C. elegans Pinto and Hengartner 885
Figure 3
LST-4 SNX-1, SNX-6 PIKI-1 DYN-1
Ptdlns(3)P
MTM-1
VPS-34 ATG-18 BEC-1
GEF activity?
CCZ-1 EPG-5
RAB-7
RAB-5
HOPS
CORPSE DEGRADATION
CED-1/CED-6
Ptdlns(3)P dynamic control
SAND-1 TBC-2
UNC-108 RAB-14 Current Opinion in Cell Biology
Molecular pathways controlling phagosome maturation in C. elegans. Arrows indicate direct physical interaction and/or activation. ‘Flat’ arrows indicate inhibitory action. Dashed arrows indicate a proposed interaction and/or activation. HOPS complex: VPS-11, VPS-16, VPS-18, VPS-33, VPS-39 and VPS-41. Shaded area: proteins involved in PtdIns(3)P metabolism.
GTPases to the phagosome membrane and as a consequence a defect in maturation [24].
promote recruitment of RAB-5 (Rab5) in parallel to SNX1/SNX-6 [52,53].
PtdIns(3)P dynamics
Rab GTPases
Another key event in phagosome maturation is the production of phosphatidylinositol 3-phosphate (PtdIns(3)P), a signaling molecule important for many membrane trafficking events [45,46]. A recent report detected two waves of PtdIns(3)P on the phagosome during corpse degradation, controlled by the two phosphoinositol 3-kinases (PI3K) PIKI-1 and VPS-34(Vps34) and by the phosphoinoside 3-phosphatase MTM-1 (Figure 3) [47]. Oscillations in PtdIns(3)P could provide a way of regulating the protein composition of intracellular membranes [48,49], probably via the ability of PtdIns(3)P to recruit protein containing PtdIns(3)P-binding domains, such as the FYVE and PX (Pox homology) domains [50]. Consistent with this hypothesis, at least three PX containing proteins, members of the SNX-BAR subfamily of sorting nexins, are known to function in cell corpse clearance [51–53]. SNX-1 (SNX1) and SNX-6 (SNX6), part of the retromer complex in C. elegans, have been suggested to contribute to apoptotic cell clearance in at least two ways: first, by driving the recycling of CED1 from the phagosome or cytosol to the plasma membrane [51], and second by promoting phagosome maturation [52]. The third sorting nexin LST-4 (SNX9) interacts physically with DYN-1 and cooperates with VPS-34 to
Phagosome maturation involves the sequential recruitment of several Rab GTPases (Figure 3). The first of these, Rab5, might be recruited to the phagocytic cup through direct physical interaction with Dynamin and Vps34 [43]. The mechanism of Rab5 activation on the phagosomal surface is however still unclear. None of the three known RAB-5 GEF orthologs in C. elegans, RABX-5 (Rabex-5), RME-6 (GapEx-5) and TAG-333 (RIN), influences cell clearance, hinting at the existence of a novel protein responsible for this function. When active, RAB-5 promotes homotypic fusion events between membranes containing active RAB-5 (e.g. phagosomes and endosomes) [54]. SAND-1 (Mon1) and its binding partner CCZ-1 (Ccz1) are proposed to act as a complex that promotes the progression from RAB-5 positive stage to RAB-7 (Rab7) positive stage in phagosome maturation [55,56]. In mammals, the complex Mon1a/Ccz1 further controls phagosome maturation by releasing RAB-7 from the inhibitory action of a Rab GDP-dissociation inhibitor (GDI) [54]; whether a similar mechanism acts in C. elegans has yet to be determined. The deactivation of RAB-5 is essential for RAB-7 recruitment and further phagosome maturation; this step is probably mediated by the RabGAP TBC-2 (Armus) [57,58]. In the worm, loss of RAB-7
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886 Cell division, growth and death
results in the arrest of phagosome maturation at a late, acidified stage, probably by impairing the fusion between phagosomes and lysosomes [23,43]. The homotypic fusion and protein sorting (HOPS) complex is formed by six proteins (Vps11, Vps16, Vps18, Vps33, Vps39 and Vps41) first identified in Saccharomyces cerevisiae, where HOPS promotes homotypic vacuole fission (reviewed by [59]). Mutants of this complex in C. elegans have been shown to have phagosome maturation defects and to act downstream of RAB-7 [43,60]. Whether any of its components acts as a direct RAB-7 effector remains to be determined. RAB-14 (Rab14) and UNC-108 (Rab2) also contribute to phagosome maturation [61,62,63]. The localization of these two proteins is rather transient and occurs slightly before RAB-7 recruitment [61]. Loss of function of rab-14 affects several steps of the phagosome maturation including phagosomal acidification and phagolysosome formation [63]. The two proteins are recruited at a similar stage and seem to have a redundant action in the regulation of phagosome maturation. UNC-108 and RAB-14 probably recruit lysosomes while RAB7 is responsible for their fusion with the phagosome [63].
will also provide new hints regarding how this process functions, and can go awry, in us humans.
Acknowledgements We apologize to our colleagues whose work could not be mentioned owing to space constraint. Work in the authors’ laboratory is supported by the Swiss National Science Foundation, the EU FP7 grant PANACEA, the Kanton of Zurich, and a doctoral fellowship from the Portuguese Foundation for Science and Technology (to SMP).
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Autophagy link Recent publications have shown that a series of genes usually associated with autophagy also play an important role in apoptotic cell clearance in C. elegans [64,65,66]. During autophagy, double-membrane structures named autophagosomes envelop cell components and deliver them to lysosomes for destruction, creating a new source of nutrients and limiting cellular damage in moments of metabolic stress [67]. Two independent studies showed that when BEC-1 (Beclin1) is depleted using RNAi, RAB-5 recruitment to phagosomes is compromised [64,65]. Furthermore Li et al., in a recent report identified a group of autophagy proteins (EPG-5, ATG-18 and LGG-1) that act in a sequential way to promote proper phagosome maturation of engulfed C. elegans Q cells [66].
Outlook The past five years have seen a dramatic increase in our understanding of the molecular pathways that mediate cell corpse recognition, internalization, and degradation. With this increased understanding has come the realization that the process is much more complex, but also much more sophisticated than initially imagined. Future work on cell clearance in worms will increasingly need to address this sophistication and complexity, in particular with regards to cell type specificity (both at the level of the cell that dies and the cell that dines). Thanks to continuous advances in light microscopy and molecular genetics, it is likely that studies with C. elegans will continue to generate important insights into how dying cells are cleared in an in vivo setting. As the molecular pathways involved in corpse clearance are largely conserved in evolution, they probably Current Opinion in Cell Biology 2012, 24:881–888
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Cell corpse clearance in C. elegans Pinto and Hengartner 887
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reorganization via a Rho/Rac GTPase signaling pathway. Dev Cell 2001, 1:477-489. 32. Wu YC, Tsai MC, Cheng LC, Chou CJ, Weng NY: C. elegans CED12 acts in the conserved crkII/DOCK180/Rac pathway to control cell migration and cell corpse engulfment. Dev Cell 2001, 1:491-502. 33. Gumienny TL, Brugnera E, Tosello-Trampont AC, Kinchen JM, Haney LB, Nishiwaki K, Walk SF, Nemergut ME, Macara IG, Francis R et al.: CED-12/ELMO, a novel member of the CrkII/ Dock180/Rac pathway, is required for phagocytosis and cell migration. Cell 2001, 107:27-41. 34. Reddien PW, Horvitz HR: CED-2/CrkII and CED-10/Rac control phagocytosis and cell migration in Caenorhabditis elegans. Nat Cell Biol 2000, 2:131-136. 35. Akakura S, Kar B, Singh S, Cho L, Tibrewal N, SanokawaAkakura R, Reichman C, Ravichandran KS, Birge RB: C-terminal SH3 domain of CrkII regulates the assembly and function of the DOCK180/ELMO Rac-GEF. J Cell Physiol 2005, 204:344-351. 36. Neukomm LJ, Kinchen JM: SRGP-1 regulation, targets, and contribution to cell killing in C. elegans. Small Gtpases 2011, 2:177-181. 37. Neukomm LJ, Frei AP, Cabello J, Kinchen JM, Zaidel-Bar R, Ma Z, Haney LB, Hardin J, Ravichandran KS, Moreno S et al.: Loss of the RhoGAP SRGP-1 promotes the clearance of dead and injured cells in Caenorhabditis elegans. Nat Cell Biol 2011, 13:79-86. Demonstrates the ability of the apoptotic clearance machinery to recognize and remove cells that died or were damaged via diverse cell death pathways. 38. Cabello J, Neukomm LJ, Gu¨nesdogan U, Burkart K, Charette SJ, Lochnit G, Hengartner MO, Schnabel R: The Wnt pathway controls cell death engulfment, spindle orientation, and migration through CED-10/Rac. PLoS Biol 2010, 8:e1000297. 39. Hsu TY, Wu YC: Engulfment of apoptotic cells in C. elegans is mediated by integrin alpha/SRC signaling. Curr Biol 2010, 20:477-486. 40. Hsieh HH, Hsu TY, Jiang HS, Wu YC: Integrin a PAT-2/CDC-42 signaling is required for muscle-mediated clearance of apoptotic cells in Caenorhabditis elegans. PLoS Genet 2012, 8:e1002663. 41. Hikita T, Qadota H, Tsuboi D, Taya S, Moerman DG, Kaibuchi K: Identification of a novel Cdc42 GEF that is localized to the PAT-3-mediated adhesive structure. Biochem Biophys Res Commun 2005, 335:139-145. 42. Vlachos M, Tavernarakis N: Non-apoptotic cell death in Caenorhabditis elegans. Dev Dyn 2010, 239:1337-1351. 43. Kinchen JM, Doukoumetzidis K, Almendinger J, Stergiou L, Tosello-Trampont A, Sifri CD, Hengartner MO, Ravichandran KS: A pathway for phagosome maturation during engulfment of apoptotic cells. Nat Cell Biol 2008, 10:556-566. Identifies a conserved genetic pathway controlling phagosome maturation. 44. Kinchen JM, Ravichandran KS: Phagosome maturation: going through the acid test. Nat Rev Mol Cell Biol 2008, 9:781-795. 45. Falasca M, Maffucci T: Rethinking phosphatidylinositol 3monophosphate. Biochim Biophys Acta 2009, 1793:1795-1803. 46. Backer JM: The regulation and function of Class III PI3Ks: novel roles for Vps34. Biochem J 2008, 410:1-17. 47. Lu N, Shen Q, Mahoney TR, Neukomm LJ, Wang Y, Zhou Z: T wo PI 3-kinases and one PI 3-phosphatase together establish the cyclic waves of phagosomal PtdIns(3)P critical for the degradation of apoptotic cells. PLoS Biol 2012, 10:e1001245. Defines the importance of tight regulation of PdtIns(3)P in the membranes of endosomes during phagosome maturation. 48. Velichkova M, Juan J, Kadandale P, Jean S, Ribeiro I, Raman V, Stefan C, Kiger AA: Drosophila Mtm and class II PI3K coregulate a PI(3)P pool with cortical and endolysosomal functions. J Cell Biol 2010, 190:407-425. 49. Noda T, Matsunaga K, Taguchi-Atarashi N, Yoshimori T: Regulation of membrane biogenesis in autophagy via PI3P dynamics. Semin Cell Dev Biol 2010, 21:671-676. Current Opinion in Cell Biology 2012, 24:881–888
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64. Martinez J, Almendinger J, Oberst A, Ness R, Dillon CP, Fitzgerald P, Hengartner MO, Green DR: Microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis is required for the efficient clearance of dead cells. Proc Natl Acad Sci USA 2011, 108:17396-17401. 65. Florey O, Kim SE, Sandoval CP, Haynes CM, Overholtzer M: Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes. Nat Cell Biol 2011, 13:1335-1343. 66. Li W, Zou W, Yang Y, Chai Y, Chen B, Cheng S, Tian D, Wang X, Vale RD, Ou G: Autophagy genes function sequentially to promote apoptotic cell corpse degradation in the engulfing cell. J Cell Biol 2012, 197:27-35. Defines a group of genes necessary for both autophagy and proper phagosome maturation. 67. Sanjuan MA, Green DR: Eating for good health: linking autophagy and phagocytosis in host defense. Autophagy 2008, 4:607-611. 68. Liu QA, Hengartner MO: Candidate adaptor protein CED-6 promotes the engulfment of apoptotic cells in C. elegans. Cell 1998, 93:961-972. 69. Luciani MF, Chimini G: The ATP binding cassette transporter ABC1, is required for the engulfment of corpses generated by apoptotic cell death. EMBO J 1996, 15:226-235.
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Current Opinion in Cell Biology 2012, 24:881–888
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Cleaning up the mess: cell corpse clearance in Caenorhabditis elegans Se´rgio Morgado Pinto1,2 and Michael Otmar Hengartner1 Genetic and cell biology studies have led to the identification in Caenorhabditis elegans of a set of evolutionary conserved cellular mechanisms responsible for the clearance of apoptotic cells. Based on the phenotype of cell corpse clearance mutants, corpse clearance can be divided into three distinct, but linked steps: corpse recognition, corpse internalization, and corpse degradation. Work in recent years has led to a better understanding of the molecular pathways that mediate each of these steps. Here, we review recent developments in our understanding of in vivo cell corpse clearance in this simple but most elegant model organism. Addresses 1 Institute of Molecular Life Sciences, University of Zurich, 8057 Zurich, Switzerland 2 Graduate Program in Areas of Basic and Applied Biology (GABBA), Universidade do Porto, Porto, Portugal Corresponding author: Hengartner, Michael Otmar ([email protected], [email protected]) Current Opinion in Cell Biology 2012, 24:881–888 This review comes from a themed issue on Cell division, growth and death Edited by Julia Promisel Cooper and Richard J Youle For a complete overview see the Issue and the Editorial Available online 1st December 2012 0955-0674/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ceb.2012.11.002
Introduction The ability to safely remove apoptotic cells plays an important role in homeostasis, wound healing and in the development of multicellular organisms [1,2]. Failure to clear corpses can result in secondary necrosis and lead to inflammation and/or autoimmune diseases, such as chronic polyarthritis and systemic lupus erythematosus [3–5]. Programmed cell death in Caenorhabditis elegans occurs in two waves [6]. During development, 131 of the 1090 cells generated in the invariant somatic lineage die soon after their birth and are rapidly removed by their neighboring cells [7]. Later in the adult hermaphrodite gonad, several hundred germ cells die during oogenesis. Germ cells can also induce apoptosis in response to genotoxic stress, DNA damage or bacterial infection [8–10]. The gonadal sheath cells which encase the germ line are responsible for the engulfment and degradation of these cells [9]. www.sciencedirect.com
Both somatic and germ cell corpses can be identified as refractile discs under differential interference contrast (DIC) microscopy (Figure 1). Genetic studies over the past decades led to the identification of a large number of C. elegans ‘cell clearance genes’. As is the case for the apoptotic killing machinery, the process of apoptotic cell clearance is evolutionarily conserved: homologous proteins play similar roles in cell corpse recognition, uptake, and degradation in both worms and humans [11]. Table 1 shows a list of C. elegans proteins and their mammalian homologs involved in cell clearance control. Apoptotic cell clearance in C. elegans can be divided in to three major steps. First, the doomed cells have to be recognized, and then internalized by the phagocytic cell in a process known as engulfment. Finally, internalized corpses undergo a process of phagosome maturation, ultimately forming phagolysosomes in which the ingested corpse is digested.
CED-1, CED-6, CED-7 pathway Genetic studies in the worm identified three partially redundant signaling pathways that mediate cell corpse recognition and internalization [12,13]. The first pathway consists of CED-1 (LRP1/MEGF10), CED-6 (GULP), CED-7 (ABCA1) and DYN-1 (Dynamin). CED-1 is a putative cell corpse receptor composed of a large extracellular domain, a single transmembrane domain and a small intercellular domain responsible for downstream signaling [14]. CED-1 accumulates on the plasma membrane of engulfing cells in the areas that contact apoptotic cells, suggesting that it can recognize molecules or epitopes exposed on the surface of dying cells [14,15,16]. The best characterized apoptosis marker in worms is the phospholipid phosphatidylserine (PS), a common ‘eatme’ signal in mammals [17] that is also selectively exposed on the surface of apoptotic cells in C. elegans [18–20]. Several studies suggest that CED-1 can indirectly bind to PS, using the secreted transthyretinlike protein TTR-52 as a bridging molecule [16,21]. The adapter protein CED-6 is a phosphotyrosine-binding (PTB) domain containing protein which has been shown to interact with CED-1 in a heterologous system [22]. CED-6 is likely to relay CED-1 signals to downstream components regulating corpse engulfment via CED-10 [12] and degradation via the large GTPase DYN-1 (see below) [23] (Figure 2). DYN-1 is enriched transiently on the phagocytic cup where it has been reported to Current Opinion in Cell Biology 2012, 24:881–888
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Figure 1
(a)
promote the fusion of early endosomes with the nascent pseudopodia, allowing the rapid delivery of lipids necessary for the embracement of the dying cell by the membrane extensions of the engulfing cell [24,25].
(b)
(d)
(c)
Current Opinion in Cell Biology
Apoptotic cell corpses in the C. elegans hermaphrodite gonad and embryos. (a, b) Bend region of the hermaphrodite gonad 24 hours postL4/adult molt; (c, d) typical embryos at the 4-fold stage. (a, c) Wild type worms with no visible cell corpses; (b, d) ced-6(n1813) mutant animals with many persistent cell corpses (arrowheads). Scale bar, 10 mm.
Two recent reports suggest that apoptotic cells not only expose PS on their surface, but that they actively transfer PS to the outer surface of the engulfing cells surrounding the corpses [26,27]. This lipid transfer appears to be mediated through the generation of PS-containing and TTR-52-containing lipid particles by apoptotic cells [26]. Loss of the ABC transporter CED-7 or of TTR-52 reduces lipid particle formation and PS transfer. Lipid transfer is also blocked in animals lacking the secreted lipid binding protein NRF-5 [27]. Importantly, mutations that block lipid transfer also prevent CED-1dependent corpse recognition and internalization [21,26,27]. The discovery of PS and TTR-52-containing vesicles raises many interesting questions. Do they act as signaling particles, or is the physical transfer of molecules from apoptotic cell to surrounding cells required for activation of the engulfing cell? Do all dying cells generate lipid
Figure 2
Transmemb. proteins
Transmemb. proteins
PAT-2 PAT-3
UIG-1
CDC-42 ABI-1
UNC-73
MIG-2
MTM-1
CED-5 CED-12
SCRM-1
PS Exposure
PAT-3 INA-1
CED-7
MOM-5 TTR-52 NRF-5
CED-1
SRGP-1
ABL-1
Cytoskeletal rearrangements
CED-10
CED-2 SRC-1 GSK-3 APR-1 CED-6
DYN-1
Phagosome maturation
CED-7 DYING CELL
ENGULFING CELL Current Opinion in Cell Biology
Molecular pathways controlling apoptotic cell recognition and internalization in C. elegans. Arrows indicate direct physical interaction and/or activation. ‘Flat’ arrows indicate inhibitory action. Dashed arrows indicate a proposed interaction and/or activation. PAT-2, UIG-1 and CDC-42 act in muscle cells; INA-1 and CED-1 preferentially act in epithelial cells [40]. Current Opinion in Cell Biology 2012, 24:881–888
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Cell corpse clearance in C. elegans Pinto and Hengartner 883
Table 1 List of C. elegans genes involved in cell clearance and their mammalian homologs. C. elegans
Brief description
Mammalian Homolog
Ref.
PS exposure
SCRM-1
phospholipid scramblase required for PS exposure
Scrm1
[20]
Signal recognition
INA-1 PAT-2 PAT-3 TTR-52 NRF-5 CED-1
integrin a subunit integrin a subunit integrin b subunit promotes PS recognition by the engulfing cell lipid transfer/LPS-binding family protein transmembrane protein possible death cell recognition
Integrin a Integrin a Integrin b sCD14, TTR hLBP family LRP, MEGF10
[39] [40] [39,40] [16] [27] [14,22]
Engulfment
CED-6 CED-7 UNC-73 MIG-2 CED-2 CED-5 CED-12 CED-10 SRGP-1 MOM-5 GSK-3 APR-1 ABL-1 ABI-1 UIG-1 CDC-42 MTM-1
adaptor SH3 and PTB containing protein, relays CED-1 signal downstream ABCA1 lipid transporter, putative creation of lipid subdomains or particles GEF, similar to TRIO which acts on RhoG Ras-related small GTPase, mammalian homolog interacts with CED-12 SH2, SH3 containing adapter protein acts together with CED-12 as a bipartide CED-10 GEF acts together with CED-5 as a bipartide CED-10 GEF Ras-related small GTPase, orchestrates actin cytoskeleton rearrangements CED-10 GTPase activating protein one of the Frizzled homologs glycogen synthase kinase, works downstream of MOM-5 upstream CED-2/5/12 b-catenin binding protein, works downstream of MOM-5 upstream CED-2/5/12 SH2, SH3 containing protein, non-receptor tyrosine kinase interacts with ABL-1contains SH3 domain GEF, specific for CDC-42 Ras-related small GTPase, Cdc42 homolog phosphatidylinositol 3-phosphate 3-phosphatase
GULP, hCED6 ABCA1, ABCA7 TRIO RhoG CrkII Dock180 Elmo Rac1 srGAP1 Frizzled GSK3b APC Abl Abi Clg Cdc42 MTM1
[22,68] [26,69] [28] [28] [34] [30] [33] [12,34] [37] [38] [38] [38] [13] [13] [41] [41] [29,70]
Phagosome maturation
DYN-1 PIKI-1 VPS-34 RAB-5 TBC-2 CCZ-1 SAND-1 RAB-7 VPS-11 VPS-16 VPS-18 VPS-33 VPS-39 VPS-41 UNC-108 RAB-14 BEC-1 EPG-5 ATG-18
Dynamin GTPase ortholog Class II Phosphatidylinositol 3-kinase Class III Phosphatidylinositol 3-kinase Rab5 GTPase ortholog, regulates early steps of phagosome maturation Rab GTPase, promotes RAB-5-GTP to RAB-5-GDP conversion together with SAND-1 regulates RAB-5 to RAB-7 conversion together with CCZ-1 regulates RAB-5 to RAB-7 conversion Rab7 GTPase ortholog, regulates late steps of phagosome maturation HOPS complex subunit, possible GEF activity to RAB-7 HOPS complex subunit, possible GEF activity to RAB-7 HOPS complex subunit, possible GEF activity to RAB-7 HOPS complex subunit, possible GEF activity to RAB-7 HOPS complex subunit, possible GEF activity to RAB-7 HOPS complex subunit, possible GEF activity to RAB-7 Rab2 GTPase homolog, proposed to be required for lysosome recruitment Rab14 GTPase homolog, proposed to be required for lysosome recruitment coiled-coil protein orthologous to yeast and mammalian autophagy proteins protein orthologous to mammalian autophagy protein KIAA1632 WD40 repeat-containing protein ortholog of yeast and mammalian autophagy proteins protein orthologous to yeast and mammalian autophagy proteins
Dynamin Class II PI3K Vps34 Rab5 Armus (TBC1D2) Ccz1 Mon1 Rab-7 Vps11 Vps16 Vps18 Vps33 Vps39 Vps41 Rab2 Rab14 Beclin1 mEPG5 WIPI2
[25,43] [47] [43] [43] [57,58] [55,56] [56] [23,43] [43] [43] [43] [43] [43] [43] [61,62] [63] [71] [72] [71]
MAP-LC3
[71]
LGG-1
particles? Previous studies have for example suggested that CED-7 is not necessary for CED-1 enrichment around apoptotic germ cells [12,19], hinting at different requirements for cell corpse clearance in embryos and adults. Finally, the process described above also shows intriguing similarities to the generation of nascent high density lipoprotein (HDL) particles in humans, in which the close CED-7 homolog ABCA1 transfers lipid molecules from the cell surface onto the lipid-binding protein ApoA-I. Is this similarity coincidental, or indicative of a deeper connection between the two processes? www.sciencedirect.com
CED-2, CED-5, CED-12 pathway In the second pathway, two small RhoGTPases become sequentially activated (Figure 2). First, the guanosine exchange factor (GEF) UNC-73 (Trio) activates the small GTPase MIG-2 (RhoG) [28]. While loss of either gene on its own does not result in any engulfment defect, their role in engulfment becomes quite evident in double mutant combinations with other engulfment genes [28,29]. MIG-2 in turn activates the CED-5 (Dock180)/ CED-12 (Elmo) complex, which acts as a bipartite GEF for the Rac family member CED-10 [30]. Active CED-10 Current Opinion in Cell Biology 2012, 24:881–888
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then drives the cytoskeletal rearrangements necessary for efficient engulfment of the dying cell [12,13]. The human (ELMO1, ELMO2/HCED-12A and BAB14712) and Drosophila (DCED-12) CED-12 homologs contain a lipidbinding Pleckstrin homology (PH) domain driving recruitment to the plasma membrane as well as proline-rich motifs capable of interacting with SRC homology 3 (SH3) domains [31–33]. CED-5/CED-12 can also be activated by the SH2/SH3 domain adapter protein CED-2. Like its mammalian counterpart CrkII, CED-2 is thought to interact with the GEF complex via its C-terminal SH3 domain, further stabilizing it downstream of receptor activation [34,35]. Mutations in ced-2, ced-5, ced-12 or ced-10 not only affect cell corpse engulfment but also result in distal tip cell (DTC) migration defect [33,34], suggesting that this signaling module is also used for cell migration processes.
apoptotic cells by INA-1 requires the activity of the phospholipid scramblase SCRM-1 in the dying cell, probably owing to its stimulation of PS externalization [20,39]. INA-1 promotes engulfment through the CED-5/CED12 GEF complex, via a signaling cascade that also includes the tyrosine kinase SRC-1 (Src) and CED-2 [39]. Finally, a recent report showed that in engulfing muscle cells, an alternative a integrin, PAT-2, is used to recognize cell corpses. Unlike the previously described receptors, PAT-2 signals not via CED-10/Rac but rather through the small GTPase CDC-42 (Cdc42) and its GEF UIG-1 (Clg) [41]. Based on their observation Hsieh et al. also propose that CED-1 and INA-1 use the ‘classical’ pathways (see above) and preferentially act in epithelial cells in cell corpse removal [40]. This report suggests that C. elegans can use different engulfment programs in a tissue dependent manner [40].
Rac activation is negatively regulated by at least two mechanisms. First, the RacGAP protein SRGP-1 can act directly as a GAP for CED-10 [36]. Second, the polyphosphoinositide 3-phosphatase MTM-1 modulates CED-10 activity by controlling the amount of phosphatidylinositol(3,5)P2 available in the plasma membrane, which provides the docking sites for CED-5/CED-12 GEF complex (Figure 2) [29]. Loss of either negative regulator enhances the ability of cells to recognize and take up neighboring cells leading, in extreme cases, even to the internalization and killing of cells that are still viable [29,37].
Clearance of non-apoptotic corpses and of sick cells
ABL-1, ABI-1 pathway A third signaling pathway, which acts in parallel to CED-1/6/7 and CED-2/5/12, is formed by the tyrosine kinase ABL-1 (Abl) and its interacting protein ABI-1 (Abi). ABL-1 negatively regulates engulfment by inhibiting ABI-1, which itself promotes cell clearance, either by regulating CED-10 activity or via an independent pathway (Figure 2) [13].
Cells in C. elegans can also die through non-apoptotic means, including various types of non-apoptotic programmed cell death and necrotic-like death (reviewed in [42]). Intriguingly, the ‘apoptotic’ engulfment machinery can recognize and clear most if not all of types of cell corpses, irrespective of the cause of death. Even more impressive is the ability of this machinery to recognize and clear cells that have been brought to the verge of death through sublethal apoptotic, necrotic or cytotoxic insults [29,37]). Upregulation or downregulation of the engulfment pathways under these conditions can lead to the death of cells that would have been spared, or conversely, the rescue of cells that otherwise would have been ‘eaten alive’ [29,37]. These observations suggest that dead or near-dead cells expose at least one common epitope, which can then be recognized by potential engulfers. Whether this common messenger is PS remains to be determined.
Phagosome maturation ‘Eat-me’ receptors CED-1 is but one of four ‘eat-me’ receptors that have been described in recent years: two of these have been proposed to act via CED-2 rather than CED-6 [38,39] and a third through a novel, CED-10-independent pathway (Figure 2) [40]. 4D-microscopy experiments identified MOM-5 (Frizzled) as a major receptor for cell corpse recognition during early embryonic development, activating an atypical Wnt signaling pathway composed of GSK3 (GSK3b) kinase, APR-1 (APC) and CED-2 (Figure 2) [38]. The CED-1 homologs LRP-5/6 and Arrow act as Frizzled co-receptors in mammals and flies, respectively, suggesting that CED-1 might play a similar role in early embryos [38]. Another ‘eat-me’ receptor is formed by the integrin INA-1 (Integrin a)/PAT-3 (Integrin b). Recognition of Current Opinion in Cell Biology 2012, 24:881–888
In recent years, the clever use of genetic screens and time-lapse imaging techniques has led to the identification of a group of genes with no deficiency in ‘eating’ but rather with problems in ‘digesting’ the dead cells [23,43] (Figure 3). Worms with mutations in these genes show changes in phagosome maturation, a process in which the corpse-containing phagosome undergoes gradual acidification as well as sequential fusion with early endosomes, late endosomes and lysosomes [23,43,44]. Membrane dynamics plays a central role in phagosome maturation. One of the first factors to be recruited to the phagocytic cup is the dynamin DYN-1. DYN-1 acts genetically downstream of CED-1/CED-6 and co-localizes with CED-1 on the phagocytic cup [23,25,43]. However the mechanism of its recruitment is still elusive. Mutations in DYN-1 result in delayed recruitment of Rab www.sciencedirect.com
Cell corpse clearance in C. elegans Pinto and Hengartner 885
Figure 3
LST-4 SNX-1, SNX-6 PIKI-1 DYN-1
Ptdlns(3)P
MTM-1
VPS-34 ATG-18 BEC-1
GEF activity?
CCZ-1 EPG-5
RAB-7
RAB-5
HOPS
CORPSE DEGRADATION
CED-1/CED-6
Ptdlns(3)P dynamic control
SAND-1 TBC-2
UNC-108 RAB-14 Current Opinion in Cell Biology
Molecular pathways controlling phagosome maturation in C. elegans. Arrows indicate direct physical interaction and/or activation. ‘Flat’ arrows indicate inhibitory action. Dashed arrows indicate a proposed interaction and/or activation. HOPS complex: VPS-11, VPS-16, VPS-18, VPS-33, VPS-39 and VPS-41. Shaded area: proteins involved in PtdIns(3)P metabolism.
GTPases to the phagosome membrane and as a consequence a defect in maturation [24].
promote recruitment of RAB-5 (Rab5) in parallel to SNX1/SNX-6 [52,53].
PtdIns(3)P dynamics
Rab GTPases
Another key event in phagosome maturation is the production of phosphatidylinositol 3-phosphate (PtdIns(3)P), a signaling molecule important for many membrane trafficking events [45,46]. A recent report detected two waves of PtdIns(3)P on the phagosome during corpse degradation, controlled by the two phosphoinositol 3-kinases (PI3K) PIKI-1 and VPS-34(Vps34) and by the phosphoinoside 3-phosphatase MTM-1 (Figure 3) [47]. Oscillations in PtdIns(3)P could provide a way of regulating the protein composition of intracellular membranes [48,49], probably via the ability of PtdIns(3)P to recruit protein containing PtdIns(3)P-binding domains, such as the FYVE and PX (Pox homology) domains [50]. Consistent with this hypothesis, at least three PX containing proteins, members of the SNX-BAR subfamily of sorting nexins, are known to function in cell corpse clearance [51–53]. SNX-1 (SNX1) and SNX-6 (SNX6), part of the retromer complex in C. elegans, have been suggested to contribute to apoptotic cell clearance in at least two ways: first, by driving the recycling of CED1 from the phagosome or cytosol to the plasma membrane [51], and second by promoting phagosome maturation [52]. The third sorting nexin LST-4 (SNX9) interacts physically with DYN-1 and cooperates with VPS-34 to
Phagosome maturation involves the sequential recruitment of several Rab GTPases (Figure 3). The first of these, Rab5, might be recruited to the phagocytic cup through direct physical interaction with Dynamin and Vps34 [43]. The mechanism of Rab5 activation on the phagosomal surface is however still unclear. None of the three known RAB-5 GEF orthologs in C. elegans, RABX-5 (Rabex-5), RME-6 (GapEx-5) and TAG-333 (RIN), influences cell clearance, hinting at the existence of a novel protein responsible for this function. When active, RAB-5 promotes homotypic fusion events between membranes containing active RAB-5 (e.g. phagosomes and endosomes) [54]. SAND-1 (Mon1) and its binding partner CCZ-1 (Ccz1) are proposed to act as a complex that promotes the progression from RAB-5 positive stage to RAB-7 (Rab7) positive stage in phagosome maturation [55,56]. In mammals, the complex Mon1a/Ccz1 further controls phagosome maturation by releasing RAB-7 from the inhibitory action of a Rab GDP-dissociation inhibitor (GDI) [54]; whether a similar mechanism acts in C. elegans has yet to be determined. The deactivation of RAB-5 is essential for RAB-7 recruitment and further phagosome maturation; this step is probably mediated by the RabGAP TBC-2 (Armus) [57,58]. In the worm, loss of RAB-7
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Current Opinion in Cell Biology 2012, 24:881–888
886 Cell division, growth and death
results in the arrest of phagosome maturation at a late, acidified stage, probably by impairing the fusion between phagosomes and lysosomes [23,43]. The homotypic fusion and protein sorting (HOPS) complex is formed by six proteins (Vps11, Vps16, Vps18, Vps33, Vps39 and Vps41) first identified in Saccharomyces cerevisiae, where HOPS promotes homotypic vacuole fission (reviewed by [59]). Mutants of this complex in C. elegans have been shown to have phagosome maturation defects and to act downstream of RAB-7 [43,60]. Whether any of its components acts as a direct RAB-7 effector remains to be determined. RAB-14 (Rab14) and UNC-108 (Rab2) also contribute to phagosome maturation [61,62,63]. The localization of these two proteins is rather transient and occurs slightly before RAB-7 recruitment [61]. Loss of function of rab-14 affects several steps of the phagosome maturation including phagosomal acidification and phagolysosome formation [63]. The two proteins are recruited at a similar stage and seem to have a redundant action in the regulation of phagosome maturation. UNC-108 and RAB-14 probably recruit lysosomes while RAB7 is responsible for their fusion with the phagosome [63].
will also provide new hints regarding how this process functions, and can go awry, in us humans.
Acknowledgements We apologize to our colleagues whose work could not be mentioned owing to space constraint. Work in the authors’ laboratory is supported by the Swiss National Science Foundation, the EU FP7 grant PANACEA, the Kanton of Zurich, and a doctoral fellowship from the Portuguese Foundation for Science and Technology (to SMP).
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Autophagy link Recent publications have shown that a series of genes usually associated with autophagy also play an important role in apoptotic cell clearance in C. elegans [64,65,66]. During autophagy, double-membrane structures named autophagosomes envelop cell components and deliver them to lysosomes for destruction, creating a new source of nutrients and limiting cellular damage in moments of metabolic stress [67]. Two independent studies showed that when BEC-1 (Beclin1) is depleted using RNAi, RAB-5 recruitment to phagosomes is compromised [64,65]. Furthermore Li et al., in a recent report identified a group of autophagy proteins (EPG-5, ATG-18 and LGG-1) that act in a sequential way to promote proper phagosome maturation of engulfed C. elegans Q cells [66].
Outlook The past five years have seen a dramatic increase in our understanding of the molecular pathways that mediate cell corpse recognition, internalization, and degradation. With this increased understanding has come the realization that the process is much more complex, but also much more sophisticated than initially imagined. Future work on cell clearance in worms will increasingly need to address this sophistication and complexity, in particular with regards to cell type specificity (both at the level of the cell that dies and the cell that dines). Thanks to continuous advances in light microscopy and molecular genetics, it is likely that studies with C. elegans will continue to generate important insights into how dying cells are cleared in an in vivo setting. As the molecular pathways involved in corpse clearance are largely conserved in evolution, they probably Current Opinion in Cell Biology 2012, 24:881–888
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