Adhesion dynamics correlates with membrane tension changes during spreading. (A) Cell spreading phases. Red arrows and curve, membrane tension. (B) VASP and actin during spreading. Dashed squares, zooms 1, 2, and 3; yellow arrowheads, VASP in clusters at the back of the lamellipodium; white arrowheads, VASP line at the tip of the leading edge. (C) Sequence of images showing VASP adhesion (green) dynamics relative to actin (magenta) during T. Lamellipodium decreases in size, and VASP adhesions organize as a row near the cell edge. (D) Kymograph of the cell presented in B (dashed line). Yellow arrowheads and arrow, VASP clusters in P1 and P2, respectively; magenta dashed curve, tangential guide to show the change in slope for the cell edge. (E) Row of adhesions positioned during T (increase in membrane tension) matures into focal adhesions later in P2, as indicated by the red outline (representing the adhesion row during T) superimposed with an image of the cell in late P2. (F) Procedure used to quantify adhesion positioning. Left, real frame of the cell; right, reconstructed cell; dashed lines, radius (x); blue dots, original edge position; red lines, aligned edge. (G) Representative mean intensity profiles for P1 and P2, with D (distance between the adhesion clusters and the cell edge) indicated. (H) Mean kymographs for VASP and actin and kymograph plot showing D (arrow in gray region). Magenta and green dashed lines, tangential guides to show the changes in slopes for the cell edge and adhesion clusters, respectively. Changes in slopes occur during T. (I) Variation of D during spreading. D changes from an initial ∼6-µm plateau (P1) to a ∼1-µm plateau (P2). T corresponds to the time necessary to change from one plateau to another (black dashed line, guide to show the change). (J) Mean kymographs for actin with other adhesion proteins. All procedures were repeated for at least 10 different spreading cells, and all showed behaviors similar to those represented in the figure. Bars, 10 µm.