RAB-35 aids apoptotic cell clearance by regulating cell corpse recognition and phagosome maturation

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 using an RNAi 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. RAB-35 function was found to be required for the incorporation of early endosomes to phagosomes and for the timely degradation of apoptotic cell corpses. More specifically, RAB-35 facilitates the switch of phagosomal membrane phosphatidylinositol species from PtdIns(4,5)P2 to PtdIns(3)P and promotes the recruitment of the small GTPase RAB-5 to phagosomal surfaces, processes that are essential for phagosome maturation. Interestingly, we observed that CED-1 performs these same functions, and to a much larger extent than RAB-35. Remarkably, in addition to cell corpse degradation, RAB-35 also facilitates the recognition of cell corpses independently of the 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 cell corpse-recognition and phagosome maturation. Epistasis analyses indicate that rab-35 represents a novel third genetic pathway that acts in parallel to both of the canonical ced-1/6/7 and ced-2/5/10/12 engulfment pathways. We propose that RAB-35 acts as a robustness factor, leading a pathway that aids the canonical pathways for the engulfment and degradation of apoptotic cells.