Figure 2. CNM-Bin1 mutants enhance Dyn2... | Rockefeller University Press

Figure 2.

$CNM-Bin1 mutants enhance Dyn2 fission activity. (A) CNM-associated Bin1 mutants used in this study. (B and C) Binding ability between Bin1 mutants and the PRD of Dyn2. GST-PRD was incubated with indicated purified His-tagged Bin1 in the presence of liposome. PRD-bound Bin1 was analyzed with SDS-PAGE and Coomassie Blue staining. The ratio of bound Bin1 was normalized to WT and shown in C (n = 3 experimental replicates). (D) Membrane-binding ability of CNM-Bin1-SH3 mutants. 0.5 µM Bin1 mutants and/or 0.5 µM Dyn2 were incubated with 100 nm liposome for 10 min at 37°C. Liposome-bound proteins (pellet, P) were separated from unbound ones (supernatant, S) through centrifugation sedimentation. (E) Assembly of Bin1 mutants on liposome. 1 µM Bin1 was incubated with liposome and then visualized with negative-stain TEM. Scale bar, 100 nm. Boxed areas were magnified and shown as below. (F) Effects of CNM-Bin1 on Dyn2 fission activity. Dyn2 was incubated with increasing concentrations of Bin1WT, Bin1Q434X, or Bin1K436X in the presence of GTP and SUPER template. The fission activity was determined by sedimentation and fraction of released fluorescent vesicles in the supernatant from total SUPER template (n = 3 experimental replicates). (G and H) Electron micrographs of Dyn2 together with Bin1 mutants assembled onto liposomes. WT Dyn2 and Bin1 mutants were incubated with 100 nm liposomes for 10 min at 37°C, adsorbed to grids, and imaged by negative-stain TEM. Scale bar, 100 nm. Boxed areas were magnified and shown. Averaged tubule diameter was quantified and shown in H (n ≥ 7 liposome tubules). (I) Membrane tubulation ability of Bin1 mutants in cellulo. GFP-tagged Bin1 mutants were overexpressed in C2C12 myoblasts through transfection and imaged under confocal microscopy. Scale bar, 10 µm. (J and K) Morphology of CNM-associated Bin1 mutants coexpressed with Dyn2-mCherry in myoblast. GFP-tagged WT or mutant Bin1 was transfected into C2C12 myoblasts together with Dyn2-mCherry. The Bin1-mediated membrane morphologies were imaged with confocal microscopy. Bottom panels, magnified images from insets in top panels. Scale bar, 10 µm. The Bin1-mediated membrane deformation was categorized into three groups (the examples of cells from each category are shown in top panel of K), and the ratio of each population was quantified and compared with Bin1WT (n ≥ 75 cells from three independent repeats). (L) Time-lapse representative images of CNM-associated Bin1 mutants coexpressed with Dyn2-mCherry in C2C12 myoblast were magnified and shown. White arrowheads indicate the occurrence of membrane fission. Scale bar, 2 µm. Data are shown as average ± SD and analyzed with one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001. Molecular weight is in kD. Source data are available for this figure: SourceData F2.$

CNM-Bin1 mutants enhance Dyn2 fission activity. (A) CNM-associated Bin1 mutants used in this study. (B and C) Binding ability between Bin1 mutants and the PRD of Dyn2. GST-PRD was incubated with indicated purified His-tagged Bin1 in the presence of liposome. PRD-bound Bin1 was analyzed with SDS-PAGE and Coomassie Blue staining. The ratio of bound Bin1 was normalized to WT and shown in C (n = 3 experimental replicates). (D) Membrane-binding ability of CNM-Bin1-SH3 mutants. 0.5 µM Bin1 mutants and/or 0.5 µM Dyn2 were incubated with 100 nm liposome for 10 min at 37°C. Liposome-bound proteins (pellet, P) were separated from unbound ones (supernatant, S) through centrifugation sedimentation. (E) Assembly of Bin1 mutants on liposome. 1 µM Bin1 was incubated with liposome and then visualized with negative-stain TEM. Scale bar, 100 nm. Boxed areas were magnified and shown as below. (F) Effects of CNM-Bin1 on Dyn2 fission activity. Dyn2 was incubated with increasing concentrations of Bin1^WT, Bin1^Q434X, or Bin1^K436X in the presence of GTP and SUPER template. The fission activity was determined by sedimentation and fraction of released fluorescent vesicles in the supernatant from total SUPER template (n = 3 experimental replicates). (G and H) Electron micrographs of Dyn2 together with Bin1 mutants assembled onto liposomes. WT Dyn2 and Bin1 mutants were incubated with 100 nm liposomes for 10 min at 37°C, adsorbed to grids, and imaged by negative-stain TEM. Scale bar, 100 nm. Boxed areas were magnified and shown. Averaged tubule diameter was quantified and shown in H (n ≥ 7 liposome tubules). (I) Membrane tubulation ability of Bin1 mutants in cellulo. GFP-tagged Bin1 mutants were overexpressed in C2C12 myoblasts through transfection and imaged under confocal microscopy. Scale bar, 10 µm. (J and K) Morphology of CNM-associated Bin1 mutants coexpressed with Dyn2-mCherry in myoblast. GFP-tagged WT or mutant Bin1 was transfected into C2C12 myoblasts together with Dyn2-mCherry. The Bin1-mediated membrane morphologies were imaged with confocal microscopy. Bottom panels, magnified images from insets in top panels. Scale bar, 10 µm. The Bin1-mediated membrane deformation was categorized into three groups (the examples of cells from each category are shown in top panel of K), and the ratio of each population was quantified and compared with Bin1^WT (n ≥ 75 cells from three independent repeats). (L) Time-lapse representative images of CNM-associated Bin1 mutants coexpressed with Dyn2-mCherry in C2C12 myoblast were magnified and shown. White arrowheads indicate the occurrence of membrane fission. Scale bar, 2 µm. Data are shown as average ± SD and analyzed with one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001. Molecular weight is in kD. Source data are available for this figure: SourceData F2.