A single-vesicle fluorescence microscopy platform to quantify phospholipid scrambling

Scramblases are physiologically important proteins that translocate phospholipids bidirectionally across cell membranes. For example, scrambling facilitated by dimers of the voltage-dependent anion channel 1 (VDAC1) enables endoplasmic reticulum-derived phospholipids to cross the outer membrane to enter mitochondria. Here we describe a protocol to obtain lipid translocation rates at a single-protein level, allowing for mechanistic understanding of scramblases. We reconstituted vesicles with fluorescent phospholipids and VDAC1 dimers and use high-throughput imaging to quantify their size and dimer content. We measure scrambling in each vesicle using a new assay and find that individual human VDAC1 dimers scramble lipids at rates ranging from under 100 to over 10,000 per second. This kinetic heterogeneity, masked in ensemble measurements, revealed that rapid scrambling is facilitated by specific VDAC1 dimers. Extending our analyses to bovine opsin, a monomeric G-protein-coupled receptor scramblase, we demonstrate the versatility of our platform for quantifying lipid scrambling and exploring its regulation.