Vinculin–F-actin binding restricts lamellipodium width and limits F-actin flow velocity in lamellipodium and mature FA. (A) qFSM of Vcl-KO MEF expressing mApple-actin and the indicated EGFP-vinculin cDNA. (left to right) SDC-FSM images of F-actin (Bar, 5 µm); F-actin flow maps (Bar, 2 μm/min); and F-actin speed maps (μm/min). 5-s frame rate. (B) Cortactin immunofluorescence (purple) staining of Vcl-KO MEF expressing the indicated EGFP-vinculin cDNA. Note distinct, narrow cortactin band in WT- and PA-vinculin (arrows) expressing Vcl-KO MEF and wider, diffuse cortactin band in nontransfected, ΔAB-, and PA-ΔAB-vinculin (arrowheads) expressing Vcl-KO MEF. Bars, 2 µm. (C) Box and whisker plot of mean F-actin flow velocities in protruding lamellipodia of Vcl-KO MEF expressing the indicated EGFP-vinculin cDNA, data calculated from qFSM F-actin speed maps; n = 70 (nontransfected), n = 120 (WT), n = 35 (PA), n = 110 (ΔAB), and n = 45 (PA-ΔAB) time points during protrusion (7–22 cells/condition; means indicated; *, P < 0.01, Mann-Whitney U test). (D) Box and whisker plot of local F-actin flow velocity within maturing FA in Vcl-KO MEF expressing the indicated EGFP-vinculin cDNA; n = 41 (nontransfected), n = 32 (WT), n = 22 (PA), n = 30 (ΔAB), and n = 22 (PA-ΔAB) FA of 7–16 cells/condition. EGFP-vinculin mutants were used as FA markers (EGFP-paxillin, nontransfected) and F-actin flow was measured on kymographs; means indicated; *, P < 0.02, Student’s t test. (E) Box and whisker plot of lamellipodium (LP) width at I_max/2 of cortactin line scans, placed through the lamellipodia at regular intervals; n = 145 (nontransfected), 160 (WT), 150 (PA), 195 (ΔAB), and 195 (PA-ΔAB) scans from 29–39 cells/condition; means indicated; *, P < 0.001, Mann-Whitney U test.