Figure 1. Bin1 inhibits Dyn2 fission... | Rockefeller University Press

Figure 1.

$Bin1 inhibits Dyn2 fission activity via SH3 domain. (A) Domain structure of dynamin and Bin1 constructs used in this study. Pleckstrin homology domain and GTPase effector domain of dynamin are abbreviated to PH and GED, respectively. (B) In vitro fission assay of Dyn2. Purified Dyn2 was incubated with increasing concentrations of Bin1 or Bin1ΔSH3 in the presence of GTP and SUPER template. The fission activity was measured as the fraction of vesicle released from total SUPER template (n = 3 experimental replicates). (C–E) Effect of Bin1 on the fission activity of dynamins. Purified Dyn1 (C) or Dyn2 CNM mutants (D) is incubated with Bin1, Bin1ΔSH3, or increasing concentrations of Bin1 (E; n = 3 experimental replicates). (F and G) Liposome binding ability. Dyn2 or Bin1 were incubated with 100 nm liposome for 10 min at 37°C. Liposome-bound proteins (pellet, P) were separated from unbound ones (supernatant, S) with centrifugation sedimentation. The fraction of liposome-bound proteins was quantified and shown in G (n = 3 experimental replicates). Molecular weight is in kD. (H) Assembly of Dyn2 or Bin1 on liposome. Dyn2 and/or Bin1 were incubated with liposomes and then visualized with negative-stain TEM. Scale bar, 100 nm. Boxed areas were magnified and shown as below. (I) Liposome-stimulated GTPase activity of Dyn2. Dyn2 was incubated alone or with Bin1 or endophilin in the presence of liposome and GTP at 37°C. The rate of GTP hydrolysis was measured using a colorimetric malachite green assay (n = 3 experimental replicates). Data are shown as average ± SD and analyzed with one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData F1.$

Bin1 inhibits Dyn2 fission activity via SH3 domain. (A) Domain structure of dynamin and Bin1 constructs used in this study. Pleckstrin homology domain and GTPase effector domain of dynamin are abbreviated to PH and GED, respectively. (B) In vitro fission assay of Dyn2. Purified Dyn2 was incubated with increasing concentrations of Bin1 or Bin1ΔSH3 in the presence of GTP and SUPER template. The fission activity was measured as the fraction of vesicle released from total SUPER template (n = 3 experimental replicates). (C–E) Effect of Bin1 on the fission activity of dynamins. Purified Dyn1 (C) or Dyn2 CNM mutants (D) is incubated with Bin1, Bin1ΔSH3, or increasing concentrations of Bin1 (E; n = 3 experimental replicates). (F and G) Liposome binding ability. Dyn2 or Bin1 were incubated with 100 nm liposome for 10 min at 37°C. Liposome-bound proteins (pellet, P) were separated from unbound ones (supernatant, S) with centrifugation sedimentation. The fraction of liposome-bound proteins was quantified and shown in G (n = 3 experimental replicates). Molecular weight is in kD. (H) Assembly of Dyn2 or Bin1 on liposome. Dyn2 and/or Bin1 were incubated with liposomes and then visualized with negative-stain TEM. Scale bar, 100 nm. Boxed areas were magnified and shown as below. (I) Liposome-stimulated GTPase activity of Dyn2. Dyn2 was incubated alone or with Bin1 or endophilin in the presence of liposome and GTP at 37°C. The rate of GTP hydrolysis was measured using a colorimetric malachite green assay (n = 3 experimental replicates). Data are shown as average ± SD and analyzed with one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData F1.