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Efficient degradation of phagocytic cargo in lysosomes is crucial to maintain cellular homeostasis and defending cells against pathogens. However, the mechanisms underlying the degradation and recycling of macromolecular cargo within the phagolysosome remain incompletely understood. We previously reported that the phagolysosome containing the corpse of the polar body in C. elegans tubulates into small vesicles to facilitate corpse clearance, a process that requires cargo protein degradation and amino acid export. Here we show that degradation of hexosylceramides by the prosaposin ortholog SPP-10 and glucosylceramidases is required for timely corpse clearance. We observed accumulation of membranous structures inside endolysosomes of spp-10-deficient worms, which are likely caused by increased hexosylceramide species. spp-10 deficiency also caused alteration of additional sphingolipid subclasses, like dihydroceramides, 2-OH-ceramides, and dihydrosphingomyelins. While corpse engulfment, initial breakdown of corpse membrane inside the phagolysosome and lumen acidification proceeded normally in spp-10-deficient worms, formation of the cargo-containing vesicles from the corpse phagolysosome was reduced, resulting in delayed cargo degradation and phagolysosome resolution. Thus, by combining ultrastructural studies and sphingolipidomic analysis with observing single phagolysosomes over time, we identified a role of prosaposin/SPP-10 in maintaining phagolysosomal structure, which promotes efficient resolution of phagocytic cargos.

Summary: The cuticle of C. elegans is impermeable to chemicals, toxins, and pathogens. However, increased permeability is a desirable phenotype because it facilitates chemical uptake. Surface lipids contribute to the permeability barrier. Here, we identify the lipid transfer protein GMAP-1 as a critical element setting the permeability of the C. elegans cuticle. A gmap-1 deletion mutant increases cuticular permeability to sodium azide, levamisole, Hoechst, and DiI. Expressing GMAP-1 in the hypodermis or transiently in the adults is sufficient to rescue this gmap-1 permeability phenotype. GMAP-1 protein is secreted from the hypodermis to the aqueous fluid filling the space between collagen fibers of the cuticle. In vitro, GMAP-1 protein binds phosphatidylserine and phosphatidylcholine while in vivo, GMAP-1 sets the surface lipid composition and organization. Altogether, our results suggest GMAP-1 secreted by hypodermis shuttles lipids to the surface to form the permeability barrier of C. elegans.