The small GTPase RAB-35 defines a third pathway that is required for the recognition and degradation of apoptotic cells

Abstract

In metazoans, apoptotic cells are swiftly engulfed by phagocytes and degraded inside phagosomes. Multiple small GTPases in the Rab family are known to function in phagosome maturation by regulating vesicle trafficking. We discovered rab-35 as a new gene important for apoptotic cell clearance from a genetic screen targeting putative Rab GTPases in Caenorhabditis elegans. We further identified TBC-10 as a putative GTPase-activating protein (GAP), and FLCN-1 and RME-4 as two putative Guanine Nucleotide Exchange Factors (GEFs), for RAB-35. We found that RAB-35 was required for the efficient incorporation of early endosomes to phagosomes and for the timely degradation of apoptotic cell corpses. More specifically, RAB-35 promotes two essential events that initiate phagosome maturation: the switch of phagosomal membrane phosphatidylinositol species from PtdIns(4,5)P ₂ to PtdIns(3)P, and the recruitment of the small GTPase RAB-5 to phagosomal surfaces. These functions of RAB-35 were previously unknown. Remarkably, although the phagocytic receptor CED-1 regulates these same events, RAB-35 and CED-1 appear to function independently. Upstream of degradation, RAB-35 also facilitates the recognition of apoptotic cells independently of the known CED-1 and CED-5 pathways. RAB-35 localizes to extending pseudopods and is further enriched on nascent phagosomes, consistent with its dual roles in regulating apoptotic cell-recognition and phagosome maturation. Epistasis analyses indicate that rab-35 acts in parallel to both of the canonical ced-1/6/7 and ced-2/5/10/12 clearance pathways. We propose that RAB-35 acts as a robustness factor, defining a novel pathway that aids these canonical pathways in both the recognition and degradation of apoptotic cells.

Author summary

After apoptosis, cell corpses must be promptly recognized and phagocytosed by engulfing cells. These nascent phagosomes then undergo a maturation process that results in the degradation of the apoptotic cell corpse. Phagosome maturation is enabled and coordinated through Rabs, small GTPases that recruit specific sets of effectors to the phagosome. The ced-1/6/7/dyn-1 and ced-2/5/10/12 pathways represent the two major canonical pathways in apoptotic cell clearance. However, substantial apoptotic cell clearance persists even when both of these pathways are inactivated. We show that the small GTPase RAB-35 promotes the recognition of cell corpses necessary for engulfment, as well as the initiation of phagosome maturation after engulfment. Specifically, RAB-35 localizes to nascent phagosomes and promotes simultaneous PtdIns(4,5)P ₂ turnover and PtdIns(3)P production, a recently discovered process that we refer to as the PtdIns(4,5)P ₂-to-PtdIns(3)P switch. We also found that RAB-35 aids in the recruitment of RAB-5 and early endosomes to the nascent phagosome. Although CED-1 also performs many of these same functions, we found that rab-35 acts in parallel to both the ced-1/6/7/dyn-1 and ced-2/5/10/12 pathways. Therefore, we claim that RAB-35 defines a third pathway that functions throughout apoptotic cell clearance in order to make it more robust.