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Article Snippet: Rabbit polyclonal anti-CEBPβ , Bethyl , Cat#A302-738A; RRID: AB_10627809.

Techniques: Recombinant, Cell Isolation, Activation Assay, Staining, Flow Cytometry, Expressing, Transfection, TA Cloning, Plasmid Preparation, Methylation, Immunoprecipitation, Purification, Sequencing, Methylation Sequencing, Amplification, Software

Journal: The Journal of cell biology

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

doi: 10.1083/jcb.201807119

Figure Lengend Snippet: Figure 1. Amphiphysin drives membrane fission, while the N-BAR domain stabilizes membrane tubules. Mem- brane 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 indi- cate membrane tubules; red arrowheads indicate fission vesi- cles. Yellow asterisks indicate small vesicles that are present in the vesicle population before protein exposure. (E) Histograms of vesicle diameters measured from electron micrographs. Ves- icles 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 indi- cate 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.

Article Snippet: The pCAG EN 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 T. Meyer, Stanford University, Stanford, CA (Addgene plasmid 85130).

Techniques: Membrane, Staining, Sedimentation, One-tailed Test

Journal: The Journal of cell biology

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

doi: 10.1083/jcb.201807119

Figure Lengend Snippet: 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 exper- iments with Amph CTD ΔSH3 expressed as the ratio of the distribution area below 45 nm to the total distri- bution 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 dis- tribution area below 45 nm to the total distribution area. Amph-FL and N-BAR data from Fig. 2 F. (I) SUP ER 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 inde- pendent 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 indi- cate approximate volumes occupied by undeformed disordered domains. See also Figs. S2 and S3.

Article Snippet: The pCAG EN 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 T. Meyer, Stanford University, Stanford, CA (Addgene plasmid 85130).

Techniques: Membrane, Protein Concentration, Generated, Concentration Assay, One-tailed Test