Visualizing Phagocytic Cargo In Vivo from Engulfment to Resolution in Caenorhabditis elegans.

The nematode Caenorhabditis elegans offers many experimental advantages to study conserved mechanisms of phagocytosis and phagocytic clearance. These include the stereotyped timing of phagocytic events in vivo for time-lapse imaging, the availability of transgenic reporters labeling molecules involved in different steps of phagocytosis, and the transparency of the animal for fluorescence imaging. Further, the ease of forward and reverse genetics in C. elegans has enabled many of the initial discoveries of proteins involved in phagocytic clearance. In this chapter, we focus on phagocytosis by the large undifferentiated blastomeres of C. elegans embryos, which engulf and eliminate diverse phagocytic cargo from the corpse of the second polar body to cytokinetic midbody remnants. We describe the use of fluorescent time-lapse imaging to observe the distinct steps of phagocytic clearance and methods to normalize this process to distinguish defects in mutant strains. These approaches have enabled us to reveal new insights from the initial signaling to induce phagocytosis up until the final resolution of phagocytic cargo in phagolysosomes.

The ATPase activity of the phosphatidylethanolamine flippase TAT-5 inhibits extracellular vesicle budding from the plasma membrane.

Cells release extracellular vesicles (EVs) from their surface, but the mechanisms that govern EV release by plasma membrane budding are poorly understood. The lipid flippase TAT-5 inhibits EV release from the plasma membrane in C. elegans , but how the level of flippase activity regulates EV release was unknown. We generated point mutations in the DGET motif of TAT-5 predicted to lead to a partial or complete loss of ATPase activity. We discovered that tat-5(E246Q) mutants were sterile, while tat-5(D244T) mutants produced embryos that arrested during development. Using degron-based reporters, we found that EV release was increased in tat-5(D244T) mutant embryos and that phagocytosis was also disrupted. These data suggest that a low level of flippase activity can promote fertility, while a higher level of flippase activity is required to inhibit EV release, allow phagocytosis, and carry out embryonic development.