Small GTPases 1:3, 170-173; November/December 2010; © 2010 Landes Bioscience

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170 Small GTPases volume 1 issue 3

Key words: Rab GTPase, RAB-14, apop-totic cell degradation, phagosome matu-ration, phagolysosome, C. elegans

Submitted: 12/03/10

Accepted: 12/17/10

Previously published online: www.landesbioscience.com/journals/smallgtpases/article/14511

DOI: 10.4161/sgtp.1.3.14511

*Correspondence to: Xiaochen Wang; Email: wangxiaochen@nibs.ac.cn

during apoptosis, apoptotic cells are recognized and quickly engulfed by

phagocytes. the internalized cell corpses are enclosed within membrane-bound vesicles called phagosomes. cell corpse degradation depends on the phagosomes undergoing a maturation process, but regulation of phagosomal maturation is not well understood. recently, we identi-fied C. elegans rab Gtpase 14 as a novel regulator of apoptotic cell degradation. loss of rab-14 function affects several steps of phagosome maturation, causing accumulation of persistent cell corpses. rab-14 and unc-108 (rab Gtpase 2) function redundantly to regulate phagosome maturation. three rabs, rab-14, unc-108/rab2 and rab-7, act cooperatively to control phagolyso-some formation. rab-14 and unc-108 recruit lysosomes, while rab-7 mediates fusion of lysosomes to phagosomes. our data thus reveal the sequential action of rab Gtpases in regulating tether-ing, docking and fusion of lysosomes to phagosomes.

The phagocytosis of apoptotic cells requires recognition of cell corpses followed by internalization. The resulting apoptotic cell-containing phagosomes undergo a maturation process involving fusion with early endosomes, late endosomes and lyso-somes, culminating in the formation of phagolysosomes in which cell corpses are degraded. Rab GTPases are central players in almost all membrane trafficking events and have important roles in the matura-tion of apoptotic cell-containing phago-somes.1-3 For example, three Rabs, RAB-5, UNC-108/RAB2 and RAB-7, have been reported to regulate apoptotic cell degra-dation through phagosome maturation.4-7

rab Gtpases act in sequential steps to regulate phagolysosome formation

Pengfei Guo and Xiaochen Wang*National Institute of Biological Sciences; Beijing, China

However, many aspects of this process are poorly understood, including how these Rab GTPases act at a specific maturation step, whether other Rabs are also needed, and how the functions of different Rabs are coordinated.

In an effort to identify additional regu-lators involved in apoptotic cell removal by a forward genetic approach, we isolated a rab-14 mutation and found it caused accumulation of persistent cell corpses owing to defective cell corpse clearance.8 Our results indicate that RAB-14 acts in engulfing cells and transiently associates with apoptotic cell-containing phago-somes, suggesting that it may regulate phagosome maturation. Previously it was found that C. elegans RAB-5 and RAB-7 act at early and late stages of phagosome maturation, respectively, while UNC-108/RAB2 may affect multiple steps.4-7 We found that RAB-14 is recruited to phagosomes at a similar stage as UNC-108 and RAB-7, which is preceded by recruitment of RAB-5 and the onset of PtdIns(3)P synthesis. The association of RAB-14, UNC-108 or RAB-7 with phagosomes all requires RAB-5 activity but not that of other Rabs, whereas RAB-5 recruitment is not affected when RAB-14, UNC-108 or RAB-7 is lost. This indicates that RAB-14, UNC-108 and RAB-7 may act at closely related steps downstream of RAB-5 activation.

One of the key events occurring down-stream of RAB-5 activation is the gradual acidification of the phagosomal lumen, which functions as a critical determinant of the degradative activity of phagosomes and also coordinates membrane fusion events during phagosomal maturation.3,9,10 We found that UNC-108 and RAB-14, but not RAB-7, are required for the initial

Extra View to: Guo P, Hu T, Zhang J, Jiang S, Wang X. Sequential action of Caenorhabditis elegans Rab GTPases regulates phagolysosome formation during apoptotic cell degradation. Proc Natl Acad Sci USA 2010; 107:18016–21; PMID: 20921409; DOI: 10.1073/pnas.1008946107.

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regulate endocytic transport through early endosomes in different cell types.6,17 Given that RAB-14 and UNC-108 mainly local-ize to early endosomes and the TGN in engulfing cells, and that they transiently associate with apoptotic cell-containing phagosomes, these two Rabs may regu-late phagosome acidification by direct-ing the tethering, docking and fusion of early endosomes to phagosomes, thereby allowing rapid acquisition of V-ATPase (fig. 1a). We are currently testing this hypothesis by examining the association of V-ATPase with apoptotic cell-contain-ing phagosomes and its involvement in apoptotic cell degradation.

with this, significantly higher numbers of cell corpses persist in unc-108;rab-14 double mutants than in either of the single mutants. Organellar acidification, including that of phagosomes, is thought to be regulated by vacuolar-type ATPase complex (V-ATPase), a proton pump that couples ATP hydrolysis and proton trans-location.12,13 Phagosomes may acquire V-ATPase via fusion with early endo-somes.10 In mammalian cells, RAB2 and RAB14 have been implicated in direct-ing ER-Golgi transport and trafficking between the trans-Golgi network (TGN) and early endosomes, respectively.14-16 In C. elegans, UNC-108/RAB2 is reported to

acidification of phagosomes. This is sup-ported by the observation that most cell corpses persisting in rab-7(lf), but not unc-108(lf) or rab-14(lf), worms are stained by Lysosensor Green. This result differs from that obtained in macrophages, in which RAB7 is required for optimal acidification of latex bead-containing phagosomes.11 Thus, different regulatory mechanisms might be employed in phagosomes car-rying distinct cargoes. Interestingly, we observed that loss of both unc-108 and rab-14, but not either of them alone, com-pletely abolishes phagosome acidifica-tion, indicating that these genes function redundantly in this process. In agreement

Figure 1. Multiple rabs cooperate to regulate phagosome acidification and phagolysosome formation for apoptotic cell degradation in C. elegans. (a) raB-14 and UNC-108/raB2 may act sequentially to deliver v-type aTPase to apoptotic cell-containing phagosomes. UNC-108 mainly associates with vesicles derived from the trans-Golgi network (TGN) and promotes the transport of v-type aTPase to early endosomes. raB-14 functions in the follow-ing step to deliver v-type aTPase to phagosomes by mediating the tethering, docking and fusion of early endosomes to phagosomes through the re-cruitment of tethering effectors. The acquisition of v-type aTPase initiates phagosomal acidification. (B) raB-14, UNC-108 and raB-7 act in sequential steps to regulate phagolysosome formation. raB-14 and UNC-108 recruit lysosome-targeting tethers as effectors to bring lysosomes in close contact with phagosomes. raB-7 acts in the next step to mediate lysosome docking and fusion by promoting SNarE paring and complex formation. The HOPS complex may serve as the effector for both UNC-108 (raB-14) and raB-7 to coordinate the tethering and fusion of lysosomes to phagosomes.

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downstream Rab effectors, are needed to reveal how multiple Rab GTPases coordi-nate distinct membrane trafficking events to control phagolysosome formation.

acknowledgements

This work was supported by the National High Technology Project 863 to X.W.

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8. Guo P, Hu T, Zhang J, Jiang S, Wang X. Sequential action of Caenorhabditis elegans Rab GTPases reg-ulates phagolysosome formation during apoptotic cell degradation. Proc Natl Acad Sci USA 2010; 107:18016-21.

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We identified redundant functions of RAB-14 and UNC-108/RAB2 in regu-lating both phagosomal acidification and phagolysosome formation. This has not been reported in other membrane traffick-ing processes. The redundancy is not sim-ply due to overlapping functions because overexpression of GTP-bound RAB-14 fails to bypass the functional requirement for UNC-108 and vice versa. As UNC-108 mainly localizes to the TGN while RAB-14 is found mostly on early endosomes in engulfing cells, we suspect that they may act sequentially to deliver V-ATPase to phagosomes (fig. 1a). On the other hand, the redundant roles of RAB-14 and UNC-108 in phagolysosome formation may be accomplished by recruiting differ-ent tethering factors (fig. 1b). Emerging evidence suggests that luminal acidifica-tion may play direct roles in coordinating membrane fission/fusion events, thereby promoting endocytic transport and phagosome maturation.10,12,21 As loss of rab-14 and unc-108 function affects both phagosomal acidification and lysosome recruitment, it is possible that pH-sensi-tive mechanisms are involved in targeting lysosomes to phagosomes. Further identi-fication of RAB-14 and UNC-108 effec-tors acting in both processes is needed to address these questions.

The coordination of diverse mem-brane trafficking events by Rab GTPases requires crosstalk among different Rabs. For example, Rab cascades, in which one activated Rab recruits the GEF for another Rab acting in the next trafficking step, have been described in both secre-tory and endocytic pathways.22 Regulated RAB5-RAB7 conversion is important for the maturation of phagosomes containing pathogenic bacterial or apoptotic cells.23,24 Moreover, some Rab effectors appear to contain separate binding sites for mul-tiple Rabs, thereby coordinating their functions.18 Our findings provide another example of coordinated Rab function in which three Rabs act in sequential steps to regulate tethering, docking and fusion of lysosomes to phagosomes. This regulatory mechanism is likely conserved in mam-mals as mouse RAB2 and RAB14 can substitute for C. elegans UNC-108 and RAB-14 in removing cell corpses. Further studies, especially the identification of

The progressive maturation of phago-somes culminates in the formation of pha-golysosomes, in which apoptotic cells are degraded. Very little is known about how this final maturation step is regulated. We found that the three Rabs, RAB-14, UNC-108/RAB2 and RAB-7 are all required for phagolysosome formation, but appear to act at different steps. For example, loss of rab-7 function affects the incorporation but not the attachment of lysosomes to phagosomes. Loss of unc-108 or rab-14 function partially blocks the recruitment of lysosomes to phagosomes, which is completely abolished in unc-108;rab-14 double mutants. Therefore, RAB-14, UNC-108 and RAB-7 appear to act sequentially to regulate phagolyso-some formation. RAB-14 and UNC-108 function redundantly to recruit lyso-somes, while RAB-7 mediates the fusion of lysosomes to phagosomes. Vesicle teth-ering, docking and fusion are mediated by tethering factors and SNAREs that act in a regulated cascade.18 Acting as molecular switches on distinct intracellular mem-branes, Rab GTPases coordinate these events through the recruitment of effec-tor proteins. For example, several known tethers including exocyst, COG and HOPS are found to be effectors of Rabs.19 In addition, Rab effectors, including some tethering factors, can directly inter-act with SNAREs to promote SNARE-mediated membrane fusion.18,19 In the case of phagolysosome formation, lyso-some-targeting tethers may be recruited by RAB-14 and UNC-108 as effectors to bring the lysosomes in closer contact with phagosomes, which subsequently allows RAB-7 to mediate lysosome docking and fusion by promoting SNARE pairing and complex formation (fig. 1b). The HOPS complex has been shown to act as both effector and GEF of Ypt7p, the yeast homolog of RAB7, to coordinate vesicle tethering, docking and fusion with vacu-oles (yeast lysosomes).19 Notably, compo-nents of the HOPS complex were found to function at late maturation steps to remove cell corpses.7,20 It will therefore be interesting to determine whether the HOPS complex can serve as the effector for multiple Rabs to coordinate the teth-ering, docking and fusion of lysosomes to phagosomes.

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23. Kinchen JM, Ravichandran KS. Identification of two evolutionarily conserved genes regulating processing of engulfed apoptotic cells. Nature 2010; 464:778-82.

24. Roberts EA, Chua J, Kyei GB, Deretic V. Higher order Rab programming in phagolysosome biogen-esis. J Cell Biol 2006; 174:923-9.

21. Steinberg BE, Huynh KK, Grinstein S. Phagosomal acidification: measurement, manipulation and functional consequences. Biochem Soc Trans 2007; 35:1083-7.

22. Markgraf DF, Peplowska K, Ungermann C. Rab cascades and tethering factors in the endomembrane system. FEBS Lett 2007; 581:2125-30.

18. Stenmark H. Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 2009; 10:513-25.

19. Cai H, Reinisch K, Ferro-Novick S. Coats, tethers, Rabs and SNAREs work together to mediate the intracellular destination of a transport vesicle. Dev Cell 2007; 12:671-82.

20. Xiao H, Chen D, Fang Z, Xu J, Sun X, Song S, et al. Lysosome biogenesis mediated by vps-18 affects apoptotic cell degradation in Caenorhabditis elegans. Mol Biol Cell 2009; 20:21-32.