Phosphatidylserine (PS) Externalization Facilitates Membrane Vesiculation through Decreasing Membrane Stiffness

Abstract

Extracellular vesicles (EVs) including exosomes and microvesicles play critical roles in intercellular communication by exchanging proteins, lipids, and genetic materials between cells. However, the origin and biogenesis of these vesicles remain unclear. It has been noted that almost all EVs present the anionic membrane lipid phosphatidylserine (PS) on their outer leaflet, in opposition to their cells of origin, which almost exclusively retain PS in the cytoplasmic leaflet. The "scrambling” of PS from the inner to the outer leaflet of the bilayer is facilitated by the lipid channel Ano6. This process of lipid scrambling has been implicated in a variety of physiological contexts, including apoptotic cell removal, bone mineralization, viral infection and blood coagulation. However, the biophysical role of PS externalization in the formation of extracellular vesicles is still a mystery. Using micrometer-sized giant plasma membrane vesicles (GPMVs) that are produced by membrane blebbing from mouse B-cells (BaF3), we studied the role of PS externalization in membrane vesiculation. We first found that a scramblase (i.e. Ano6) knockout BaF3 cell line was deficient in producing large vesicles compared with the wild type cell line, indicating that PS externalization is necessary for membrane budding. Importantly, through fluorescence lifetime imaging microscopy (FLIM) using a reporter of lipid packing (Di4), we found that PS exposure lead to a decrease of plasma membrane packing, potentially make the membrane softer for bending and thus facilitating membrane budding. To confirm this assumption, we artificially decreased the lipid order on the plasma membrane by increasing the incubation temperature or treating the cells with methyl-β-cyclodextrin (MβCD). Under all conditions which reduced membrane stiffness, large vesicles were efficiently produced from the PS externalization-deficient Ano6 knockout cell line, confirming the crucial role of membrane stiffness in cell vesiculation.