Invadosome forms and distributes asymmetrically in differentiated myoblasts. (A) Expression level of different invadosome components in myoblasts upon differentiation. Myoblast lysates derived from different days of DM treatment were immunoblotted with indicated antibodies. Numbers below indicate the fold-change of Dyn2, Tks5, and Cortactin compared with day 0 after normalization with tubulin. (B and C) Colocalization of invadosome components at the myoblast tip of differentiated myoblast. Day 3 differentiated C2C12 myoblasts were immunofluorescence stained to detect endogenous Tks5, Dyn2, MT1-MMP, F-actin, and AP-2. Images were acquired with z-stack confocal microscopy and shown as single focal planes. (D) Matrix degradation ability. Day 3 differentiated myoblasts were seeded onto an FITC-gelatin–coated coverslip and imaged after 24 h. Arrowheads indicate invadosomes. (E–H) Asymmetrical distribution of invadosome in fusing myoblasts. Two close-positioned, day 3 differentiated myoblasts with one cell labeled with Dyn2-GFP or Lifeact-RFP were stained for F-actin (E), Dyn2 and AP-2 (F), Tks5 (G), and tubulin (H). Arrowheads in F indicate the enriched Dyn2 and AP2 at the cell periphery of the receiving cell or the invadosome in attacking cells (G and H). Scale bars (both black and white), 10 µm.