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

Vesicles in experiments with full-length amphiphysin and N-BAR were composed of 76 mol% DOPC, 15 mol% DOPS, 5 mol% PtdIns(4,5)P 2 , 2 mol% DP-EG10-biotin, and 2 mol% Oregon Green 488-DHPE. DOPS and PtdIns(4,5)P 2 were replaced with 20 mol% DOGS-NTA-Ni in experiments with Amph CTD ∆SH3. Dried lipid films were hydrated in 20 mM MOPS pH 7.35, 150 mM NaCl buffer (0.5 mM EGTA and EDTA was included in experiments with PtdIns(4,5)P 2 ) and extruded to 30 or 200 nm. Imaging wells were prepared as described above. Vesicles were diluted to 5 µM in the wells and allowed to tether for 10 min. Untethered vesicles were removed by thorough washing with MOPS buffer. After tethering, Atto 594-labeled protein was added to the specified concentration, and multiple spinning disc confocal z-stacks of lipid and protein fluorescence were acquired, with a z-step of 0.1 µm. Images were collected after approximately 15 min incubation of protein with vesicles. The same laser power and camera gain settings were used for all experiments. Fig. S2A shows images of tethered vesicles with 10 and 25 nM All rights reserved. No reuse allowed without permission.