Cycles of actin buckling are correlated with adhesion positioning. (A) Tirf kymographs for VASP, actin, and their merge during five buckling cycles in P2. Arrowheads, adhesion rows. (B) DIC, epi (actin), and tirf (actin and paxillin) kymographs of a cell in P2 showing six buckling cycles. Dashed lines in zooms separate each buckling cycle. (C) Actin epi, actin tirf, and their merge showing the actin buckling at the leading edge of a spreading cell during P2. Top right color image is the same kymograph as in B, with actin epi (blue) and actin tirf (red). (D) Fluorescence intensity heat map of actin in tirf and epi for the same kymograph as in B. Fluorescence intensity levels (a.u.) are indicated as color bars. (E) Schematic top and side representations of actin buckling during P2, observed with tirf (red) and epi (blue). The experiment was repeated for 10 different spreading cells, and all show behaviors similar to those represented in the figure. Bar, 5 µm.