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 directly visualize the morphology of membranes at the end of remodeling, we utilized negative stain transmission electron microscopy (TEM) to resolve membrane structures below the optical diffraction limit. As expected from previous findings (Gallop et al., 2006; Peter et al., 2004) , the N-BAR domain transformed vesicles that had an average initial diameter of 200 nm into long tubules with average outer diameter 44±6 nm s.d. (Fig. 1B ,C and S1B,C). In contrast, Amph-FL did not drive appreciable membrane tubule formation in TEM experiments. Rather, Amph-FL divided the vesicles of initially 200 nm diameter into a population of highly curved vesicles with a peak diameter centered near 22 nm (Fig. 1D,E and S1D). This result suggests that Amph-FL is capable of driving membrane fission, a more energetically demanding process than membrane tubule formation (Campelo et al., 2012) . (F) Membrane release from SUPER templates, measured as Texas Red signal present in the supernatant after sedimentation of the SUPER templates. Membrane release in the absence of protein was measured and subtracted as background. Dots indicate data and lines indicate mean, n = 3 independent experiments. P-value: one-tailed, unpaired Student's t-test. Scale bars in (B-D): 500 nm. Zoomed region scale bars: 200 nm. See also Fig. S1 and Movies S1-S4.