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

Membrane binding experiments with fluorescently-labeled Amph CTD ∆SH3 showed that when 10 µM of protein was present in solution, the protein covered approximately 40% of the membrane surface . At this coverage, steric pressure from protein crowding is expected to be sufficient to overcome the energetic barrier to membrane fission (Snead et al., 2017) . Therefore, the requirement for a high solution concentration of Amph CTD ∆SH3 reflects the conditions necessary to promote crowded binding to the membrane surface. In contrast, the ability of Amph-FL to drive fission at much lower concentrations likely arises from polymerization of the BAR domain scaffold, which enables multivalent membrane binding (Simunovic et al., 2016; Sorre et al., 2012) . In line with this thinking, we found that 100 nM Amph-FL, a concentration which drove potent fission (Fig. 2D ,F), covered greater than 70% of the membrane surface (Fig. S2F ). This coverage is significantly higher than has been observed for non-assembling proteins (Feder, 1980; Snead et al., 2017) . Collectively, these results suggest that the ability of amphiphysin to drive efficient membrane fission arises from a collaboration between its N-BAR domain and disordered domain. respectively) as well as hydrodynamic radius . Tethered vesicle fission experiments revealed that N-BAR-epsin CTD generated highly curved fission products from vesicles with an initial average diameter of 200 nm within a similar range of protein concentrations to Amph-FL (Fig. 3F , compare to Fig. 2D ). Further, both N-BAR-epsin CTD and Amph-FL produced a very similar fraction of fission products with diameters below 45 nm at equivalent concentrations in solution (Fig. 3G) . Finally, SUPER template membrane shedding experiments revealed that in the concentration range of 50-1,000 nM, N-BAR-epsin CTD drove greater membrane release compared to the isolated N-BAR domain (Fig. 3H) , similar to the results of SUPER template experiments comparing N-BAR and Amph-FL (Fig. 1F ). These findings illustrate the ability of N-BAR scaffolds to promote membrane fission by crowding arbitrary disordered domains at membrane surfaces.