Phagolysosomes break down the membrane of a non-apoptotic corpse independent of macroautophagy

Cell corpses must be cleared in an efficient manner to maintain tissue homeostasis and regulate immune responses. Ubiquitin-like Atg8/LC3 family proteins promote the degradation of membranes and internal cargo during both macroautophagy and corpse clearance, raising the question how macroautophagy contributes to corpse clearance. Studying the clearance of non-apoptotic dying polar bodies in Caenorhabditis elegans embryos, we show that the LC3 ortholog LGG-2 is enriched inside the polar body phagolysosome independent of autophagosome formation. We demonstrate that ATG-16.1 and ATG-16.2, which promote membrane association of lipidated Atg8/LC3 proteins, redundantly promote polar body membrane breakdown in phagolysosomes independent of their role in macroautophagy. We also show that the lipid scramblase ATG-9 is needed for autophagosome formation in early embryos but is dispensable for timely polar body membrane breakdown or protein cargo degradation. These findings demonstrate that macroautophagy is not required to promote polar body degradation, in contrast to recent findings with apoptotic corpse clearance in C. elegans embryos. Determining how factors regulating Atg8/LC3 promote the breakdown of different types of cell corpses in distinct cell types or metabolic states is likely to give insights into the mechanisms of immunoregulation during normal development, physiology, and disease.

Methods
Gravid C. elegans hermaphrodites were dissected in egg salts (94 mM NaCl, 32 mM KCl, 2.8 mM MgCl2, 2.8 mM CaCl2, 2 mM HEPES, pH 7.5) to isolate embryos and mounted on a 4% agarose pad for live imaging. Z-stacks were collected using a Zeiss Axio Observer 7 inverted microscope with Plan-APO 40X 1.4 NA oil objective with Excelitas Technologies X-Cite 120LED Boost illumination, and Hamamatsu ORCA-Fusion sCMOS camera controlled by 3i SlideBook6 software. Time-lapse imaging was acquired sequentially for mCherry and GFP every minute. Data is provided in the SlideBook format (.sld).