Abstract
Clearance of apoptotic cells is of key importance during development, tissue homeostasis and wound healing in multi-cellular animals. Genetic studies in the nematode Caenorhabditis elegans have identified a set of genes involved in the early steps of cell clearance, in particular the recognition and internalization of apoptotic cells. A pathway that orchestrates the maturation of phagosomes containing ingested apoptotic cells in the worm has recently been described. However, many steps in this pathway remain elusive. Here we show that the C. elegans SNX9-family member LST-4 (lateral signaling target) and its closest mammalian orthologue SNX33 play an evolutionary conserved role during apoptotic cell corpse clearance. In lst-4 deficient worms, internalized apoptotic cells accumulated within non-acidified, DYN-1-positive but RAB-5-negative phagosomes. Genetically, we show that LST-4 functions at the same step as DYN-1 during corpse removal, upstream of the GTPase RAB-5. We further show that mammalian SNX33 rescue C. elegans lst-4 mutants and that overexpression of truncated SNX33 fragments interfered with phagosome maturation in a mammalian cell system. Taken together, our genetic and cell biological analyses suggest that LST-4 is recruited through a combined activity of DYN-1 and VPS-34 to the early phagosome membrane, where it cooperates with DYN-1 to promote recruitment/retention of RAB-5 on the early phagosomal membrane during cell corpse clearance. The functional conservation between LST-4 and SNX33 indicate that these early steps of apoptotic phagosome maturation are likely conserved through evolution.
Highlights
Apoptotic cell clearance is an important process during development, tissue homeostasis and wound healing [1,2]
To confirm our RNAi results, we analyzed cell corpse clearance in animals carrying the lst4(tm2423) mutation, a 212 bp deletion predicted to completely inactivate the gene (Fig. S2B). lst-4(tm2423) mutant worms showed a strong accumulation of non-acidified, AO-negative cell corpses in the hermaphrodite germline, a similar phenotype observed in dyn-1(ky51) mutants [10] (Fig. 1 A–D9, S1), suggesting a defect in cell corpse clearance upstream of phagosome acidification
LST-4 co-localized extensively with actin and DYN-1 around apoptotic cells (Fig. 2H– K, R, S) but only minimally with RAB-5 (Fig. 2R, S) and not at all with RAB-7 (Fig. 2L–O, R, S). These results indicate that engulfment signaling is required for LST-4 recruitment and suggest that both LST-4 and DYN-1 act at an early step of phagosome maturation (Fig. 2I–K) [10]
Summary
Apoptotic cell clearance is an important process during development, tissue homeostasis and wound healing [1,2]. During clearance of apoptotic cells, two partially redundant pathways comprised of CED-1/MEGF10/ LRP1, CED-6/GULP and CED-7/ABCA1, and of MIG-2/ RhoG, UNC-73/Trio, CED-2/CrkII, CED-5/DOCK180 and CED-12/ELMO respectively, regulate the small GTPase CED10/Rac, which in turn directs the actin polymerization and membrane extension required to engulf the dying cell [4]. The two small GTPases RAB-5 and RAB-7, together with many additional factors, sequentially control the maturation of the nascent phagosome, leading to lysosome fusion and corpse degradation [8,9,10,11,12,13,14,15] for review see add [16,17]. We previously described an evolutionary conserved mechanism for the recruitment of RAB-5 to the early phagosome involving the large GTPase DYN-1/Dynamin and the phophatidylinositol-3 kinase VPS-34 [18,10]. Zhou and colleagues recently extended these observations by demonstrating that the sorting nexin LST-4 functions at this step, interacting physically with DYN-1 and promoting the fusion of the maturing phagosomes with endosomes and lysosomes [19]
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