Figure 5.
Figure 5. Disordered domain crowding opposes inverted membrane bending by I-BARs and promotes membrane fission by F-BARs. GUV membrane composition: 79.5 mol% DOPC, 5 mol% PtdIns(4,5)P2, 15 mol% DOPS, and 0.5 mol% Oregon Green 488–DHPE. SUPER template membrane composition: 79 mol% DOPC, 5 mol% PtdIns(4,5)P2, 15 mol% DOPS, and 1 mol% Texas Red–DHPE. Tethered vesicle membrane 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. Membrane composition for vesicles in TEM: 80 mol% DOPC, 5 mol% PtdIns(4,5)P2, and 15 mol% DOPS. (A) Schematic of I-BAR-AP180 CTD chimera dimer. IRSp53 I-BAR domain: PDB 1Y2O. (B) Two representative confocal micrographs of GUVs after exposure to I-BAR or I-BAR-AP180 CTD. Asterisks indicate direction of membrane bending (magenta: inward; cyan: outward). Fluorescence signal comes from Atto 594–labeled protein. (C) SUPER template membrane release comparing I-BAR and I-BAR-AP180 CTD. Dots indicate data and lines indicate mean; n = 3 independent experiments. P value: one-tailed, unpaired Student’s t test. (D) Tethered vesicle fission experiments reveal that 5 µM I-BAR-AP180 CTD generates highly curved fission vesicles. (E) Schematic of FCHo1-FL dimer. F-BAR domain: PDB 2V0O. µ-Homology domain: PDB 5JP2, chain A. (F–H) Negative stain TEM micrographs of 200 nm extruded vesicles before exposure to protein (F), after exposure to 33 µM F-BAR (G), and after exposure to 2 µM FCHo1-FL (H). Dashed boxes indicate zoomed regions to the right. Black arrows indicate membrane tubules; red arrowheads indicate fission vesicles. (I) Tethered vesicle fission experiments reveal FCHo1-FL generates highly curved fission products over the concentration range of 10–250 nM. (J) F-BAR does not drive fission in tethered vesicle fission experiments, even at concentrations up to 2,000 nM. (K) Summary of tethered vesicle and TEM experiments, expressed as the proportion of vesicle diameters within the high curvature group of 45 nm or smaller (compare to Fig. 2 F). Markers for tethered vesicle data represent mean ± first SD; n = 3 independent experiments. TEM data from Fig. S5 D. (B) Bars, 5 µm. (F–H) Bars, including insets, 200 nm. See also Fig. S5 and Videos 7 and 8.

Disordered domain crowding opposes inverted membrane bending by I-BARs and promotes membrane fission by F-BARs. GUV membrane composition: 79.5 mol% DOPC, 5 mol% PtdIns(4,5)P2, 15 mol% DOPS, and 0.5 mol% Oregon Green 488–DHPE. SUPER template membrane composition: 79 mol% DOPC, 5 mol% PtdIns(4,5)P2, 15 mol% DOPS, and 1 mol% Texas Red–DHPE. Tethered vesicle membrane 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. Membrane composition for vesicles in TEM: 80 mol% DOPC, 5 mol% PtdIns(4,5)P2, and 15 mol% DOPS. (A) Schematic of I-BAR-AP180 CTD chimera dimer. IRSp53 I-BAR domain: PDB 1Y2O. (B) Two representative confocal micrographs of GUVs after exposure to I-BAR or I-BAR-AP180 CTD. Asterisks indicate direction of membrane bending (magenta: inward; cyan: outward). Fluorescence signal comes from Atto 594–labeled protein. (C) SUPER template membrane release comparing I-BAR and I-BAR-AP180 CTD. Dots indicate data and lines indicate mean; n = 3 independent experiments. P value: one-tailed, unpaired Student’s t test. (D) Tethered vesicle fission experiments reveal that 5 µM I-BAR-AP180 CTD generates highly curved fission vesicles. (E) Schematic of FCHo1-FL dimer. F-BAR domain: PDB 2V0O. µ-Homology domain: PDB 5JP2, chain A. (F–H) Negative stain TEM micrographs of 200 nm extruded vesicles before exposure to protein (F), after exposure to 33 µM F-BAR (G), and after exposure to 2 µM FCHo1-FL (H). Dashed boxes indicate zoomed regions to the right. Black arrows indicate membrane tubules; red arrowheads indicate fission vesicles. (I) Tethered vesicle fission experiments reveal FCHo1-FL generates highly curved fission products over the concentration range of 10–250 nM. (J) F-BAR does not drive fission in tethered vesicle fission experiments, even at concentrations up to 2,000 nM. (K) Summary of tethered vesicle and TEM experiments, expressed as the proportion of vesicle diameters within the high curvature group of 45 nm or smaller (compare to Fig. 2 F). Markers for tethered vesicle data represent mean ± first SD; n = 3 independent experiments. TEM data from Fig. S5 D. (B) Bars, 5 µm. (F–H) Bars, including insets, 200 nm. See also Fig. S5 and Videos 7 and 8.

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