Amph-FL produces highly curved fission products. Tethered vesicle composition: 76 mol% DOPC, 5 mol% PtdIns(4,5)P2, 15 mol% DOPS, 2 mol% Oregon Green 488–DHPE, and 2 mol% DP-EG10-biotin. (A) Schematic of tethered vesicle fission experiment. (B) Representative spinning disc confocal micrographs of tethered vesicles before exposure to protein (top), after exposure to 150 nM Amph-FL (middle), and after exposure to 300 nM N-BAR (bottom). Contrast settings in top and bottom images are the same while contrast in middle image is adjusted to clearly show vesicle puncta. Dashed yellow boxes indicate puncta intensity profiles on the right, where bar heights are all scaled between 90 and 6,000 brightness units while each color map corresponds to the specified intensity range. (C–E) Distributions of vesicle diameter measured by tethered vesicle assay before exposure to protein (C), after exposure to Amph-FL at the specified concentrations (D), and after exposure to N-BAR at the specified concentrations (E). (F) Summary of tethered vesicle and TEM experiments, expressed as the proportion of vesicle diameters within the high curvature group of 45 nm or smaller. Markers for tethered vesicle data represent mean ± first SD; n = 3 independent experiments. TEM data from Fig. 1 E. (G) Number of membrane-bound proteins per 1,000 nm2 of membrane surface area versus concentration of N-BAR or Amph-FL. (H) Data in G plotted as the coverage of the membrane surface by proteins as a function of protein concentration. Error bars in G and H represent 95% CI; n > 1,700 vesicles for each condition. Amph-FL and N-BAR data collected using 30-nm–extruded vesicles and sonicated vesicles, respectively (see Materials and methods). (B) Bars, 2 µm. See also Figs. S1, S2, and S3.
