Chemically induced vesiculation as a platform for studying TMEM16F activity

Tina W Han,Wenlei Ye,Neville P Bethel,Mario Zubia,Andrew Kim,Kathy H Li,Alma L Burlingame,Michael Grabe,Yuh Nung Jan,Lily Y Jan

doi:10.1073/pnas.1817498116

Abstract

Calcium-activated phospholipid scramblase mediates the 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-dependent lipid scrambling activity are deficient in generation of extracellular vesicles (EVs) that shed from the plasma membrane in a Ca2+-dependent manner, namely microvesicles. We have adapted chemical induction of giant plasma membrane vesicles (GPMVs), which require both TMEM16F-dependent phospholipid scrambling and calcium influx, as a kinetic assay to investigate the mechanism of TMEM16F activity. Using the GPMV assay, we identify and characterize both inactivating and activating mutants that elucidate the mechanism for TMEM16F activation and facilitate further investigation of TMEM16F-mediated lipid translocation and its role in extracellular vesiculation.

Highlights

Calcium-activated phospholipid scramblase mediates the energyindependent bidirectional translocation of lipids across the bilayer, leading to transient or, in the case of apoptotic scrambling, sustained collapse of membrane asymmetry
To assess whether the giant plasma membrane vesicles (GPMVs) assay could be used as a tool for studying the role of TMEM16F in vesiculation, we first asked whether production of these giant vesicles was dependent on expression of the TMEM16F protein
We have developed a robust and quantitative assay for assessing TMEM16F activity by using GPMVs, chemically induced giant vesicles that bud from the plasma membrane

Summary

CELL BIOLOGY

Cells lacking TMEM16F-dependent lipid scrambling activity are deficient in generation of extracellular vesicles (EVs) that shed from the plasma membrane in a Ca2+-dependent manner, namely microvesicles. We have adapted chemical induction of giant plasma membrane vesicles (GPMVs), which require both TMEM16F-dependent phospholipid scrambling and calcium influx, as a kinetic assay to investigate the mechanism of TMEM16F activity. We use chemically induced giant plasma membrane vesicles (GPMVs) to investigate the role of TMEM16F phospholipid scrambling activity in extracellular vesiculation. Due to their enlarged dimensions, GPMVs can be monitored in real time by conventional light microscopy, revealing that vesiculation depends on TMEM16F-mediated lipid scrambling and vesicle formation begins after the initiation of PS exposure. Using the GPMV assay, we identify and characterize both inactivating and activating TMEM16F mutants in kinetic analyses that facilitate further investigation of TMEM16F-mediated lipid translocation and extracellular vesicle formation

Results

Discussion

Materials and Methods