Journal: bioRxiv

Article Title: BAR scaffolds drive membrane fission by crowding disordered domains

doi: 10.1101/276147

Figure Lengend Snippet: 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)P 2 , 15 mol% DOPS. SUPER template membrane composition: 79 mol% DOPC, 5 mol% PtdIns(4,5)P 2 , 15 mol% DOPS, 1 mol% Texas Red-DHPE. ( A ) Schematic of full-length amphiphysin (Amph-FL) dimer. BAR domain: PDB 4ATM. SH3 domain: PDB 1BB9. ( B-D ) Negative stain TEM micrographs of 200 nm extruded vesicles ( B ) before exposure to protein, ( C ) after exposure to 26 μM N-BAR, and ( D ) after exposure to 5 μM Amph-FL. Dashed boxes indicate zoomed regions to the right. Black arrows indicate membrane tubules, red arrowheads indicate fission vesicles. ( E ) Histograms of vesicle diameters measured from electron micrographs. Vesicles alone: n = 1,302 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. Scale bars in (B-D): 500 nm. Zoomed region scale bars: 200 nm. See also and Movies S1-S4.

Article Snippet: The pCAGEN mammalian expression vector containing the N-BAR domain of human amphiphysin (residues 1-256), tagged at the C-terminus with mCherry, was a gift from Tobias Meyer (Addgene plasmid # 85130).

Techniques: Staining, Sedimentation, One-tailed Test

Journal: bioRxiv

Article Title: BAR scaffolds drive membrane fission by crowding disordered domains

doi: 10.1101/276147

Figure Lengend Snippet: Full-length amphiphysin produces highly curved fission products. Tethered vesicle composition: 76 mol% DOPC, 5 mol% PtdIns(4,5)P 2 , 15 mol% DOPS, 2 mol% Oregon Green 488-DHPE, 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 right, where bar heights are all scaled between 90 and 6,000 brightness units while each color map corresponds to specified intensity range. ( C-E ) Distributions of vesicle diameter measured by tethered vesicle assay ( C ) before exposure to protein, ( D ) after exposure to Amph-FL at 50, 75, 100, and 150 nM, and ( E ) after exposure to N-BAR at 150 and 300 nM. ( F ) Summary of tethered vesicle fission data, expressed as the ratio of the distribution area below 45 nm diameter to the total distribution area. Markers represent mean ± first s.d., n = 3 independent experiments. Scale bars in (B): 2 μm. See also - .

Article Snippet: The pCAGEN mammalian expression vector containing the N-BAR domain of human amphiphysin (residues 1-256), tagged at the C-terminus with mCherry, was a gift from Tobias Meyer (Addgene plasmid # 85130).

Techniques: Vesicle Assay

Journal: bioRxiv

Article Title: BAR scaffolds drive membrane fission by crowding disordered domains

doi: 10.1101/276147

Figure Lengend Snippet: The disordered domain of amphiphysin alone drives membrane fission, but the N-BAR scaffold substantially enhances fission efficiency. Membrane composition in Amph CTD ∆SH3 tethered vesicle experiments: 76 mol% DOPC, 20 mol% DOGS-NTA-Ni, 2 mol% Oregon Green 488-DHPE, 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)P 2 and 15 mol% DOPS. SUPER template membrane composition: 79 mol% DOPC, 5 mol% PtdIns(4,5)P 2 , 15 mol% DOPS, 1 mol% Texas Red-DHPE. ( A ) Schematic of Amph CTD ∆SH3. ( B ) Tethered vesicle fission experiments show that Amph CTD ∆SH3 forms highly curved fission products. ( C ) Summary of data from tethered vesicle fission experiments with Amph CTD ∆SH3 expressed as the ratio of the distribution area below 45 nm to the total distribution area (compare to ). ( D ) Coverage of the membrane surface by Amph CTD ∆SH3 for each concentration at which fission was measured. ( E ) Schematic of N-BAR-epsin CTD chimera dimer. ( F ) 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 ). ( G ) 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 . ( H ) SUPER template membrane shedding experiments show that N-BAR-epsin CTD drives greater membrane release compared to N-BAR (compare to ). Dots indicate data and lines indicate mean, n = 3 independent experiments. P-value: one-tailed, unpaired Student’s t-test. Markers in (C), (D), and (G) represent mean ± first s.d., n = 3 independent experiments. ( I ) Schematic of the N-BAR scaffold (EMDB 3192) 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 and .

Article Snippet: The pCAGEN mammalian expression vector containing the N-BAR domain of human amphiphysin (residues 1-256), tagged at the C-terminus with mCherry, was a gift from Tobias Meyer (Addgene plasmid # 85130).

Techniques: Concentration Assay, One-tailed Test

Journal: bioRxiv

Article Title: BAR scaffolds drive membrane fission by crowding disordered domains

doi: 10.1101/276147

Figure Lengend Snippet: Imaging tubules generated in live cells by N-BAR, Amph-FL, and N-BAR-NfM CTD. ( A ) Spinning disc confocal image of a live RPE cell overexpressing the N-BAR domain of amphiphysin tagged with mCherry and stained with CellMask Green plasma membrane stain. Yellow dashed box indicates the zoomed region below, where the individual channels are shown. White arrows indicate lipid tubes. The co-localization of the plasma membrane stain with N-BAR-coated tubes indicates that the tubes are derived from the plasma membrane. Scale bar in top image: 10 μm. Scale bar in zoomed region: 5 μm. ( B ) Representative tubes in RPE cells overexpressing the indicated proteins tagged with mCherry. Images were acquired in TIRF at 37 °C. Tubules are from cells with similar protein expression level, and all images are displayed with equal contrast settings. White arrows indicate tubes. N-BAR shows greater enrichment on the tube relative to the local background compared to Amph-FL and N-BAR-NfM CTD. Scale bars: 1 μm. ( C ) Protein intensity on membrane tubes in live cells, quantified as the ratio of the tube intensity to the local background intensity. Points indicate individual tubes, and black lines indicate means. Data were quantified from TIRF movies that were taken under identical imaging settings. n = 100, 50, and 91 tubes for N-BAR, Amph-FL, and N-BAR-NfM CTD, respectively. P-values: unpaired, two-tailed Student’s t-tests. These data indicate the disordered domains of Amph-FL and N-BAR-NfM CTD did not promote fission of lipid tubules in live cells by enhancing protein binding to the membrane surface.

Article Snippet: The pCAGEN mammalian expression vector containing the N-BAR domain of human amphiphysin (residues 1-256), tagged at the C-terminus with mCherry, was a gift from Tobias Meyer (Addgene plasmid # 85130).

Techniques: Imaging, Generated, Staining, Derivative Assay, Expressing, Two Tailed Test, Protein Binding