Veit, S.; Dearden, G. I.; Menon, K. M.; Noor, F.; Menon, A. K.; Pomorski, T. G.

Scramblases play important roles in physiology by translocating phospholipids bidirectionally across cell membranes. For example, scrambling facilitated by VDAC1 dimers is the primary mechanism by which endoplasmic reticulum-derived phospholipids cross the outer membrane to enter mitochondria. Precise quantification of lipid scrambling, while critical for mechanistic understanding, cannot be obtained from ensemble averaged measurements of the activity of reconstituted scramblases. Here, we describe a microscopy platform for high-throughput imaging of single vesicles reconstituted with fluorescently-labeled phospholipids and heterogeneously crosslinked VDAC1 dimers. For each vesicle, we quantify size, protein occupancy and scrambling rate. Notably, we find that individual VDAC1 dimers have different activities, ranging from <100 to >10,000 lipids per second. This kinetic heterogeneity, masked in ensemble measurements, reveals that only some dimer interfaces are capable of promoting rapid scrambling, as suggested by molecular dynamics simulations. Our platform provides a versatile framework for quantifying scramblases and exploring their regulation.