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

To better understand the ability of amphiphysin to drive membrane fission, we compared N-BAR and Amph-FL in two additional assays of membrane fission. In the first of these experiments, we used supported bilayers with extra membrane reservoir (SUPER templates), which are glass beads surrounded by a low-tension membrane. Exposure of SUPER templates to fission-driving proteins results in measurable membrane release from the beads Pucadyil and Schmid, 2008) . SUPER template experiments revealed that while both N-BAR and Amph-FL drove membrane release in the concentration range of 50-1,000 nM, Amph-FL drove more than twice as much (2.6 to 4.7-fold greater) membrane release at each concentration (Fig. 1F) . Notably, membrane release does not directly imply efficient membrane fission, as both vesicles and lipid tubules can be shed from SUPER templates. Therefore, we next employed a tethered vesicle assay to quantify the distributions of fission vesicle diameters over a range of protein concentrations (Snead et al., 2017) . Specifically, we tethered fluorescent vesicles to a coverslip passivated with PEG and PEG-biotin ( Fig. 2A) . Vesicles in these experiments contained a biotinylated lipid, which facilitated tethering to the substrate via binding to neutravidin. Vesicles also contained the fluorescent lipid Oregon Green 488-DHPE, which we used to quantify the brightness of each vesicle after imaging in confocal fluorescence microscopy (Aguet et al., 2013) (Fig. 2B ). We then converted the resulting distributions of vesicle brightness to approximate distributions of vesicle diameter by calibrating against the initial vesicle diameter distribution measured using dynamic light scattering (see methods).