Tks5 regulates the physical properties of Dyn2-actin bundles. (A–D) AFM topography and stiffness map (Young’s modulus) of Dyn2-actin bundles. Height (A) and corresponding stiffness (C) of Dyn2-actin bundles with or without GST-PX3A addition were recorded and quantified with Peakforce QNM. Quantification results are shown in C and D. n ≥ 10. Scale bars, 800 µm. ***, P < 0.001. (E and F) Dyn2 knockdown in c-SrcY527F–transformed NIH3T3 cells. Dyn2 was depleted by lentiviral shRNA and selected with puromycin for 3 d. Cells were processed for immunoblotting (E) or immunostaining (F). Scale bar, 10 µm. (G and H) AFM stiffness map (Young’s modulus) of podosome rosette. Podosome rosettes in control or Dyn2-depleted c-SrcY527F–transformed NIH3T3 cells with diameter ∼5 µm were analyzed with AFM probe equipped with a 5-µm bead in medium (G). Approximately 40 podosome rosettes in >10 cells of each condition were measured and quantified in H. ^#, P = 0.079. (I) Tks5 mediates invadosome maturation through regulating dynamin-actin organization to drive myoblast fusion. Upon myoblast differentiation, Tks5 is up-regulated and undergoes isoform switching to the long isoform Tks5α that encodes a membrane-interacting PX domain. Tks5α promotes the formation and maturation of an invadosome by dictating Dyn2 assembly, strengthening the protrusive actin-rich structure and perhaps coupling these force-generating filaments to the membrane. The invadosome thus functions as a molecular drill to propel both the first- and second-phase myoblast fusion.