Fig 4. Nonphosphorylatable Dyn1 mutant mimics GSK3β effects and can partially substitute for Dyn2.

CCP initiation densities (A), median lifetimes (B), and the lifetime distribution (C) of bona fide CCPs analyzed in H1299 Dyn1^KO cells reconstituted with Dyn1^WT- or Dyn1^S774/8A-eGFP, determined as described in Fig 2. (D) Representative TIRFM images of overexpressed Dyn1^WT-eGFP or Dyn1^S774/8A-eGFP and mRuby2-CLCa and (E) quantification of their average recruitment to CCPs with lifetimes between 40 and 60 s. (F) Maximum intensities of Dyn1^WT-eGFP or Dyn1^S774/8A-eGFP averaged among individual bona fide CCP tracks. (G) Effect of siRNA knockdown of Dyn2 on TfnR endocytosis in parental and Dyn1^KO H1299 cells and Dyn1^KO cells reconstituted with either Dyn1a^WT-eGFP or Dyn1a^S774/8A-eGFP. (H) Representative TIRFM images of Dyn2 siRNA-treated Dyn1^KO cells overexpressing Dyn1a^WT-eGFP and mRuby2-CLCa treated or not with GSK3β inhibitor and (I) quantification of the average recruitment of Dyn1^WT-eGFP to CCPs with lifetimes between 40 and 60 s in Dyn2 knockdown cells treated or not with GSK3β inhibitor. The underlying data of panels A–C, F, and G can be found in S1 Data. CCP, clathrin-coated pit; CLCa, clathrin light chain a; Dyn1, dynamin-1; GSK3β, glycogen synthase kinase-3 beta; siRNA, small interfering RNA; TfnR, transferrin receptor; TIRFM, total internal reflection fluorescence microscopy.