Figure 3.
Figure 3. The disordered domain of amphiphysin alone drives membrane fission, but the N-BAR scaffold substantially enhances fission efficiency. Membrane composition in Amph CTD ΔSH3 tethered vesicle experiments: 76 mol% DOPC, 20 mol% DOGS-NTA-Ni, 2 mol% Oregon Green 488–DHPE, and 2 mol% DP-EG10-biotin. In tethered vesicle experiments with N-BAR-epsin CTD, DOGS-NTA-Ni was replaced with 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 CTD ΔSH3. (B) Tethered vesicle fission experiments show that Amph CTD ΔSH3 forms highly curved fission products. (C) Summary of data from tethered vesicle fission experiments with Amph CTD ΔSH3 expressed as the ratio of the distribution area below 45 nm to the total distribution area (compare to Fig. 2 F). (D) Coverage of the membrane surface by Amph CTD ΔSH3 and Amph-FL as a function of protein concentration. Amph-FL data from Fig. 2 H. (E) Fraction of vesicle diameters below 45 nm generated by Amph CTD ΔSH3 and Amph-FL versus coverage of the membrane surface by proteins. Amph-FL fission data from Figs. 2 F and S2 M. Amph CTD ΔSH3 fission data from Fig. 3 C. (F) Schematic of N-BAR-epsin CTD chimera dimer. (G) Tethered vesicle fission measurements show that N-BAR-epsin CTD generates highly curved fission vesicle populations over the concentration range of 10–150 nM, similar to Amph-FL (compare to Fig. 2 D). (H) Summary of data from tethered vesicle fission experiments with N-BAR-epsin CTD, expressed as the ratio of the distribution area below 45 nm to the total distribution area. Amph-FL and N-BAR data from Fig. 2 F. (I) SUPER template membrane shedding experiments show that N-BAR-epsin CTD drives greater membrane release compared with N-BAR (compare to Fig. 1 F). Dots indicate data and lines indicate mean; n = 3 independent experiments. P value: one-tailed, unpaired Student’s t test. Amph CTD ΔSH3 markers in C and D and all markers in H represent mean ± first SD; n = 3 independent experiments. (J) Schematic of the N-BAR scaffold (EMDB 3192; Adam et al., 2015) with attachment points of some of the disordered domains marked (two per N-BAR dimer). Dashed circles indicate approximate volumes occupied by undeformed disordered domains. See also Figs. S2 and S3.

The disordered domain of amphiphysin alone drives membrane fission, but the N-BAR scaffold substantially enhances fission efficiency. Membrane composition in Amph CTD ΔSH3 tethered vesicle experiments: 76 mol% DOPC, 20 mol% DOGS-NTA-Ni, 2 mol% Oregon Green 488–DHPE, and 2 mol% DP-EG10-biotin. In tethered vesicle experiments with N-BAR-epsin CTD, DOGS-NTA-Ni was replaced with 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 CTD ΔSH3. (B) Tethered vesicle fission experiments show that Amph CTD ΔSH3 forms highly curved fission products. (C) Summary of data from tethered vesicle fission experiments with Amph CTD ΔSH3 expressed as the ratio of the distribution area below 45 nm to the total distribution area (compare to Fig. 2 F). (D) Coverage of the membrane surface by Amph CTD ΔSH3 and Amph-FL as a function of protein concentration. Amph-FL data from Fig. 2 H. (E) Fraction of vesicle diameters below 45 nm generated by Amph CTD ΔSH3 and Amph-FL versus coverage of the membrane surface by proteins. Amph-FL fission data from Figs. 2 F and S2 M. Amph CTD ΔSH3 fission data from Fig. 3 C. (F) Schematic of N-BAR-epsin CTD chimera dimer. (G) Tethered vesicle fission measurements show that N-BAR-epsin CTD generates highly curved fission vesicle populations over the concentration range of 10–150 nM, similar to Amph-FL (compare to Fig. 2 D). (H) Summary of data from tethered vesicle fission experiments with N-BAR-epsin CTD, expressed as the ratio of the distribution area below 45 nm to the total distribution area. Amph-FL and N-BAR data from Fig. 2 F. (I) SUPER template membrane shedding experiments show that N-BAR-epsin CTD drives greater membrane release compared with N-BAR (compare to Fig. 1 F). Dots indicate data and lines indicate mean; n = 3 independent experiments. P value: one-tailed, unpaired Student’s t test. Amph CTD ΔSH3 markers in C and D and all markers in H represent mean ± first SD; n = 3 independent experiments. (J) Schematic of the N-BAR scaffold (EMDB 3192; Adam et al., 2015) with attachment points of some of the disordered domains marked (two per N-BAR dimer). Dashed circles indicate approximate volumes occupied by undeformed disordered domains. See also Figs. S2 and S3.

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