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

Images were cropped and individual vesicle puncta were detected using cmeAnalysis software (Aguet et al., 2013) , following a similar approach described in the previous section. Here we only accepted puncta that persisted at the same location through three consecutive imaging frames. The algorithm also searched for fluorescent puncta in the protein channel using the centroids of the detected fluorescent puncta in the master lipid channel. The search region in the protein channel was three times the standard deviation of the Gaussian fit to the point spread function of our microscope. We estimated vesicle diameters from lipid fluorescence brightnesses by calibrating against dynamic light scattering, as described in the previous section. We estimated the number of bound proteins on each vesicle by comparing brightness values in the protein channel to the brightness of a single molecule of Atto 594-labeled protein. Images of single molecules of Atto 594-labeled proteins were obtained by adding a dilute concentration of protein to an imaging well on an ultraclean coverslip, and imaging single proteins adhered to the coverslip surface in a similar manner as described for the tethered vesicles. A linear correction for camera exposure time was applied to the single molecule brightness, as longer exposure times were required to image single molecules compared to membranebound protein. Fig. S2B shows a plot of the raw protein intensity values as a function of vesicle intensity for 10 and 25 nM Amph-FL. The 25 nM data shows a higher slope than 10 nM, indicating greater membrane coverage. Fig. S2C shows this same data after processing, plotted as the number of membrane-bound proteins as a function of vesicle diameter.