BAR scaffolds drive membrane fission by crowding disordered domains

Wilton T. Snead; Wade F. Zeno; Grace Kago; Ryan W. Perkins; J Blair Richter; Chi Zhao; Eileen M. Lafer; Jeanne C. Stachowiak

Using this assay, we found that Amph-FL in the concentration range of 50-150 nM transformed vesicles with an average initial diameter of 200 nm (Fig. 2B ,C) to a population of high curvature fission products (Fig. 2B ,D) with a median diameter of 22 nm at 150 nM, in agreement with results from TEM ( Fig. 1D ,E). The proportion of vesicles that fell within the high curvature group (diameters below approximately 45 nm) increased with increasing protein concentration, from less than 1% at 50 nM to approximately 38% at 150 nM (Fig. 2F ). In contrast, the N-BAR domain did not drive fission, even at higher protein concentrations (Fig. 2B , E, and F). While lipid tubules were not apparent in tethered vesicle experiments with N-BAR (Fig. 2B ), tubules were observed at micromolar N-BAR concentrations (Fig. S1E) , consistent with the tubules formed in TEM experiments (Fig. 1C) . Notably, higher concentrations of Amph-FL were required to observe fission in TEM experiments compared to tethered vesicle experiments. This increase is due to the high lipid concentration used in TEM experiments (approximately 100-fold greater than tethered vesicle experiments), which is necessary to achieve an adequate density of lipid structures for TEM (see methods). Taken together, our results from electron microscopy and tethered vesicle experiments confirm that Amph-FL is a potent driver of membrane fission, while the isolated N-BAR domain is only capable of forming membrane tubules. Full-length amphiphysin produces highly curved fission products. Tethered vesicle composition: 76 mol% DOPC, 5 mol% PtdIns(4,5)P 2 , 15 mol% DOPS, 2 mol% Oregon Green 488-DHPE, 2 mol% DP-EG10-biotin. (A) Schematic of tethered vesicle fission experiment. (B) Representative spinning disc confocal micrographs of tethered vesicles before exposure to protein (top), after exposure to 150 nM Amph-FL (middle), and after exposure to 300 nM N-BAR (bottom). Contrast settings in top and bottom images are the same while contrast in middle image is adjusted to clearly show vesicle puncta. Dashed yellow boxes indicate puncta intensity profiles on right, where bar heights are all scaled between 90 and 6,000 brightness units while each color map corresponds to specified intensity range. (C-E) Distributions of All rights reserved. No reuse allowed without permission.