Membrane tension increase induces adhesion row positioning. Paxillin and actin behaviors followed in four conditions: control (A–C), hypo-OS (D–G), 10 µM BBI (H–J), and 10 µM BBI plus hypo-OS (K–N). (A, H, D, and K) Representative images in P1, T, or hypo-OS and P2 for actin and paxillin in all four conditions. Arrowheads, paxillin-containing adhesion clusters. (B, I, E, and L) Merged kymographs of paxillin (green) and actin (magenta) plotted from the dashed lines in A, D, H, and K. Yellow arrows, strips of paxillin-containing adhesions positioned during T (B and I) and hypo-OS (E and L); white arrows, strips of paxillin-containing adhesions positioned in P2 (after T or hypo-OS). (C, J, F, and M) Variations of D (in micrometers) during spreading for each experimental condition. Red dashed lines, beginning and end of T or hypo-OS. (G and N) Adhesion row positioned during hypo-OS (increase in membrane tension) matures into focal adhesions later in P2 in control case G but not in myosin II–inhibited cells in N, as indicated by the red outline (representing the adhesion row positioned during hypo-OS) superimposed with an image of the cell in late P2. All procedures were repeated for five different spreading cells of each experimental condition. All cells showed behaviors similar to those represented. Bars, 10 µm.