TMEM16F is a Calcium-Activated Phospholipid Scramblase Required for Chemically-Induced Giant Plasma Membrane Vesicles

Abstract

Cellular membranes are composed of heterogeneously partitioned phospholipids such that certain lipid species are sequestered to the inner leaflet while others are exposed on the cell surface. Lipid asymmetry is established and maintained by ATP-dependent unidirectional phospholipid translocases which are colloquially referred to as “flippases” and “floppases”. TMEM16F belongs to a third class of proteins aptly named “scramblases” which mediate Ca2+-activated, energy-independent bidirectional translocation of lipids across the bilayer, leading to transient or, in the case of apoptotic scrambling, sustained collapse of membrane asymmetry. Cells lacking TMEM16F-mediated lipid scrambling activity are also deficient in generation of a subtype of extracellular vesicle (EV) called “microvesicles” or “ectosomes” though the relationship between scrambling and vesiculation is not well understood. Microvesicles, which range between 100 nm to 1 μm in diameter depending on the cell type of origin, shed distinctively from the plasma membrane in a Ca2+-dependent manner and are thought to carry bioactive cargo in the form of RNAs, proteins, and lipids which can elicit biological activity in neighboring cells. Quantification of EVs using conventional techniques is challenging due to the inherent difficulties of resolving particles on such a diminutive scale. We have adapted the use of chemically-induced giant plasma membrane vesicles (GPMVs) generation which can be monitored in real time by conventional light microscopy to investigate the role of TMEM16F phospholipid scrambling activity in extracellular vesiculation. Using the GPMV assay, we identify and characterize both inactivating and activating mutants that emphasize residues critical for TMEM16F function and that allow us to further examine the mechanism of lipid translocation through TMEM16F.