Amphiphysin drives membrane fission, while the N-BAR domain stabilizes membrane tubules. Membrane composition for vesicles in TEM: 80 mol% DOPC, 5 mol% PtdIns(4,5)P2, and 15 mol% DOPS. SUPER template membrane composition: 79 mol% DOPC, 5 mol% PtdIns(4,5)P2, 15 mol% DOPS, and 1 mol% Texas Red–DHPE. (A) Schematic of Amph-FL dimer. BAR domain: PDB 4ATM. SH3 domain: PDB 1BB9. (B–D) Negative stain TEM micrographs of 200 nm extruded vesicles before exposure to protein (B), after exposure to 26 µM N-BAR (C), and after exposure to 5 µM Amph-FL (D). Dashed boxes indicate zoomed regions to the right. Black arrows indicate membrane tubules; red arrowheads indicate fission vesicles. Yellow asterisks indicate small vesicles that are present in the vesicle population before protein exposure. (E) Histograms of vesicle diameters measured from electron micrographs. Vesicles alone: n = 1,302 vesicles. 26 µM N-BAR: n = 462 vesicles. 5 µM Amph-FL: n = 1,071 vesicles. (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. (B–D) Bars, 500 nm; insets, 200 nm. See also Fig. S1 and Videos 1, 2, 3, 4, and 5.
