Tks5 interacts with Dyn2 and regulates its assembly around actin. (A) Domain structure of different Tks5 constructs used in this study. (B) Direct interaction between Dyn2 and Tks5. GST pulldown assay was performed using purified full-length Dyn2 and three GST-tagged Tks5 fragments, GST-PX, GST-PX3A, and PX3C. Bound His-Dyn2 was detected with immunoblotting, and GST-tagged proteins were shown with Coomassie Blue stain. (C and D) F-actin bundling assay. Prepolymerized filamentous actin was incubated with purified Dyn2 or Tks5 as indicated. After 30 min of incubation, bundled actin was sedimented into pellet (P), and the ratio of bundled actin (pellet/total) was quantified with SDS-PAGE, Coomassie Blue staining, and ImageJ (D). (E and G) Electron micrographs of Dyn2-mediated actin bundling with or without Tks5. Single Dyn2 spiral was indicated by red braces in the magnified panels, and the diameters of actin bundles or Dyn2 spirals in the presence or absence of Tks5 were quantified with ImageJ (F and G). Black bar, 100 nm. White bars, 50 nm. *, P < 0.05; ***, P < 0.001.