Fig. 3: Migrating myotubes form stable cell–matrix adhesions at their free edge and adherens junctions at their cell–cell edge.

a Wild-type and b arp3 knockdown myotubes. Scale bar, 10 µm. c Quantification of filopodia number per cell edge length. d Still images of a front-row myotube expressing Arp3-EGFP. Arrowheads mark positions of Arp3 at filopodial branch points. Scale bar, 10 µm. e Still images of a front-row myotube coexpressing Arp3-EGFP and Myr-RFP. The arrowhead marks a position of Arp3. Scale bar, 10 µm. f, g Close-up of myotubes at the front edge of the migrating sheet, 6 min in ex vivo culture. Cell–matrix adhesions (green) are found in a beaded string-like manner along filopodia. Scale bar: 10 µm. g Some filopodia build branches. UAS-FAT-EGFP, scale bar: 10 µm. h Dashed lines represent the area magnified in h’ and h”. h’ Matrix adhesions are found at filopodia tips (white arrowheads). Actin cables are marked by yellow arrowheads. h” Cell–matrix adhesions colocalize with actin fibers (white arrowheads). h’, h” Scale bar, 10 µm. i–k. Boxplot center: median. Bounds: 25th and 75th percentiles. Whiskers: minimum/maximum values. *P ≤ 0.05, **P  ≤ 0.01, ***P  ≤ 0.001, ****P  ≤ 0.0001. Quantification revealed i a significant bias in directionality of cell–matrix adhesions, and j an increased number of cell–matrix adhesions at the cell front, N = 20 cells. Mann–Whitney test (two tailed) and k an increased number of cell–matrix adhesions at the free edge compared to cell–cell edges. N = 20 cells. Kruskal–Wallis test. l, m Cell–matrix adhesions during migration. Quantified adhesions at the free edge are depicted in red and at the cell–cell edge in green. Scale bar: 10 µm. l Front edge of the migrating sheet. l’ Magnification at 0 min. j” Magnification at 40 min. m Following cells in the same sheet as in H. m’ Magnification at 0 min. m” Magnification at 40 min. n Quantification revealed that cell–matrix adhesions longevity is significantly higher at the free edge” compared to the cell–cell-edge”. N = 980 matrix adhesions in three testes. Mann–Whitney test (two tailed). o Cell–matrix adhesions before and after laser ablation. After laser ablation, new cell–matrix adhesion formed along the free edge (arrowhead). Scale bar: 10 µm.