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

Imaging wells for fluorescence correlation spectroscopy (FCS) utilized supported lipid bilayers (SLBs) to passivate the glass surface and prevent protein adsorption. Briefly, a well was created with a silicone gasket on an ultraclean coverslip, and a solution of sonicated DOPC vesicles at 1 mM lipid was added. The SLB was formed for 10 min and thoroughly washed in experiment buffer of 50 mM Tris pH 8.0, 10 mM CaCl 2 , 150 mm NaCl, 15 mM EGTA, 5 mM EDTA, 5 mM TCEP. Atto 488-labeled proteins were diluted in experiment buffer and added to the imaging well such that the concentration of Atto 488 dye was approximately 1 nM. FCS measurements were acquired on a custom-built timecorrelated single photon counting confocal microscope using a 486 nm picosecond pulsed diode laser. The laser was focused in solution approximately 3 µm above the bilayer passivation surface, and fluorescence signal was collected as proteins diffused through the focused laser volume. The signal was split onto separate GaAsP photomultiplier tubes (Hamamatsu) for cross-correlation using Becker and Hickl software. FCS traces were collected for 120 s. The number of FCS traces acquired for Amph CTD ∆SH3, AP180 CTD, and transferrin were: 10, 5, and 3, respectively. Each FCS trace was fit with the 2D autocorrelation function: All rights reserved. No reuse allowed without permission.