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

Spinning disc confocal z-stacks of BFP-CLC and the mCherry fusion protein were collected with a z-step of 0.25 µm. Z-stacks were analyzed for the number of tubes per cell and tube length. Image analysis was performed using ImageJ software. The plasma membrane frame was chosen by identifying the BFP-CLC frame in which the clathrincoated structures were best in focus. The plasma membrane expression level of the mCherry fusion protein was then quantified by measuring the mean brightness on a region of the plasma membrane, away from the nucleus and bright structures. Membrane tubes were counted at one frame above the plasma membrane frame. Tube lengths were quantified as the end-to-end distances of the tubes. Fig. S4A shows an image of a cell stained with CellMask Green plasma membrane stain (Thermo Fisher). Before imaging, the cells were incubated for 5 min at 37 °C in a solution of the CellMask Green stain diluted 1000-fold in sterile PBS. The solution was removed and the cells were washed three times with media before imaging.