Multiple Lipid Scramblases Regulate the Quantity of Extracellular Vesicle Production From the Plasma Membrane

Extracellular vesicle (EV) biogenesis is linked to the disruption of lipid bilayer asymmetry during diverse processes from coagulation to programmed cell death. However, the specific contributions of lipid scramblase proteins to the regulation of EV production and the potential redundancy of diverse families of scramblases are not well understood. We developed a semi-automated image analysis pipeline that accurately quantifies fluorescently labelled EVs on the cell surface of live Caenorhabditis elegans embryos. Using this method in a sensitized background with enhanced EV production, we performed an RNA interference (RNAi) screen targeting a subset of plasma membrane and intracellular scramblases. We found that knockdown of most scramblase genes we tested resulted in a minor but significant reduction in the number of EVs. We confirmed these significant decreases in EV abundance with anoh-2 and atg-9 scramblase mutants. However, loss-of-function egli-1 or scrm-3 scramblase mutants displayed no significant change in EV production, unlike egli-1 or scrm-3 knockdown. We also observed no effect on EV production in ced-7 ABC transporter mutants. The discrepancy between the phenotypes of acute scramblase knockdown and chronic loss suggested the presence of compensatory mechanisms that maintain EV homeostasis in response to the permanent absence of a scramblase. RNA sequencing revealed changes in expression levels for conserved sets of genes in egli-1 and scrm-3 mutants. Together, our data suggest that multiple scramblases involved in diverse pathways regulate EV budding, potentially responding to distinct physiological or metabolic cues.