The phosphorylation of Dyn2-S848 relieves Bin1 inhibition. (A) The amino acid sequence of Dyn2-PRD. Two potential phosphorylated serine, S848 and S856, are boxed and highlighted in yellow. The SH3-binding pockets were highlighted in light blue. (B and C) GST pulldown assay. GST or GST-tagged Bin1 SH3 domain was incubated with indicated Dyn2 mutants. The ratio of bound Dyn2 was detected with Coomassie Blue staining, quantified with ImageJ, and normalized to WT as shown in C (n = 3 experimental replicates). Molecular weight is in kD. (D) Fission activity of Dyn2 mutants in the presence of Bin1. Indicated Dyn2 proteins were incubated with increasing concentrations of Bin1 in the presence of GTP and SUPER templates for fission activity analysis. The data was shown as fold change relative to the Dyn2 fission activity in the absence of Bin1 (n = 3 experimental replicates). (E and F) Effect of Dyn2 mutants on GFP-Bin1 tubules in myoblasts. WT or indicated Dyn2-mCherry mutants were transfected into myoblasts together with Bin1-GFP. Scale bar, 10 µm. The Bin1-induced membrane morphologies were analyzed as in Fig. 2 K and shown in F (n ≥ 100 cells from three independent repeats). (G) Assembly of Dyn2 mutants on liposome. Indicated Dyn2 proteins were incubated with liposome alone (top) or together with Bin1 (bottom) and then visualized with negative-stain TEM. Scale bar, 100 nm. Data are shown as average ± SD and analyzed with one-way ANOVA. #P ≤ 0.06; *P < 0.05; **P < 0.01. Source data are available for this figure: SourceData F3.