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

SUPER templates were prepared according to the protocol of Neumann et al . A lipid mixture of 5 mol% PtdIns(4,5)P 2 , 15 mol% DOPS, 1 mol% Texas Red DHPE, and 79 mol% DOPC was mixed in a clean glass test tube, the solvent was evaporated, and the lipid film was further dried under vacuum. The lipid film was hydrated in Milli-Q water, subjected to three freeze-thaw cycles in liquid nitrogen, and extruded through a 100 nm pore filter (Whatman). SUPER templates were made by creating a 100 µL mixture consisting of 200 µM liposomes, 1 M NaCl, and 5 x 10 6 of 2.5 µm m-type silica beads (Corpuscular; Cold Spring, NY, USA) in a low-adhesion microcentrifuge tube. The mixture was incubated for 30 min at room temperature and gently agitated periodically. The mixture was washed by adding 1 mL Milli-Q water, gently mixing, and spinning at 300 g for 2 min in a swinging bucket rotor to pellet the SUPER templates. 1 mL of supernatant was removed, SUPER templates were resuspended in the remaining 100 µL, and washing was repeated a total of four times. SUPER templates were kept on ice and used within 4 h. All rights reserved. No reuse allowed without permission.