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

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/276147 doi: bioRxiv preprint Figure S3 . Amph-FL drives fission of membranes that contain high concentrations of negatively charged lipids. Vesicle composition: 68 mol% DOPS, 23 mol% DOPE, 5 mol% cholesterol, 2 mol% DP-EG10-biotin, and 2 mol% Oregon Green 488-DHPE, extruded to 200 nm. Composition based on Mim et al, Cell 2012 . (A) Representative spinning disc confocal micrographs of vesicles (top) before protein exposure, (middle) after exposure to 500 nM Amph-FL, and (bottom) after exposure to 500 nM N-BAR. 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 3,050 brightness units while each color map corresponds to specified intensity range. Scale bars: 2 µm. (B-D) Distributions of vesicle diameter measured by tethered vesicle assay (B) before exposure to protein, (C) after exposure to Amph-FL at 150, 350, and 500 nM, and (D) after exposure to N-BAR at 500 nM. (E) Summary of tethered vesicle fission data, expressed as the ratio of the distribution area below 45 nm diameter to the total distribution area. Compare to Fig. 2F . Markers represent mean ± first s.d., n = 3 independent experiments. All rights reserved. No reuse allowed without permission.