BAR scaffolds drive membrane fission by crowding disordered domains

Abstract

Cellular membranes are continuously remodeled. The crescent-shaped bin-amphiphysin-rvs (BAR) domains remodel membranes in multiple cellular pathways. Based on studies of isolated BAR domains in vitro, the current paradigm is that BAR domain-containing proteins polymerize into cylindrical scaffolds that stabilize lipid tubules. But in nature, proteins that contain BAR domains often also contain large intrinsically disordered regions. Using in vitro and live cell assays, here we show that full-length BAR domain-containing proteins, rather than stabilizing membrane tubules, are instead surprisingly potent drivers of membrane fission. Specifically, when BAR scaffolds assemble at membrane surfaces, their bulky disordered domains become crowded, generating steric pressure that destabilizes lipid tubules. More broadly, we observe this behavior with BAR domains that have a range of curvatures. These data suggest that the ability to concentrate disordered domains is a key driver of membrane remodeling and fission by BAR domain-containing proteins.

Figure 1.

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.

Figure 2.

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.

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.

Figure 4.

Disordered domains disrupt N-BAR mediated membrane tubulation in live cells. (A) Schematic of mCherry (PDB 2H5Q) fusion constructs expressed in cells. (B) Confocal images of RPE cells expressing N-BAR (top), Amph-FL (middle), and N-BAR-NfM CTD (bottom). Yellow dashed boxes indicate zoomed regions to the right. White arrows indicate tubules. All cells are within the same range of protein expression level used for quantification in D and E. (C) Number of tubes per cell as a function of protein expression level, quantified as the background-subtracted protein intensity at the plasma membrane (see Materials and methods). Lines indicate linear regression with y-intercept set to 0. Shaded regions indicate 99% CI. Line color matches the respective marker color. n > 90 cells per condition from two independent transfections. (D) Number of tubes per cell within the expression level range of 200–400 brightness units. Bars indicate mean ± SEM; n > 20 cells per condition. (E) Length of tubes in cells within the expression level range of 200–400 brightness units. Points indicate data; black lines indicate means. n > 80 tubes per condition. (F) Lifetime of tubes in cells measured from time-lapse total internal reflection fluorescence (TIRF) microscopy videos (see Materials and methods). Points indicate data; black lines indicate means. n > 40 tubes per condition. All P values: two-tailed, unpaired Student’s t tests. (B) Bars, 10 µm; insets, 5 µm. See also Fig. S4 and Video 6.

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.