Vinculin mediates F-actin flow engagement in maturing FA and high ECM traction. (A) Alexa Fluor 488–phalloidin staining of F-actin (green), paxillin (red), and myosin light chain-2 (MLC-2; blue; asterisk) immunofluorescence staining of a primary control MEF. Bars, 5 µm. (B) qFSM of control and Vcl-KO MEF microinjected with X-rhodamine actin and EGFP-paxillin cDNA. (left to right) SDC-FSM images of F-actin (Bar, 5 µm); F-actin flow maps (Bar, 2 μm/min); F-actin speed maps (μm/min); and EGFP-paxillin image showing FA location. Note a wider band of fast retrograde F-actin flow in Vcl-KO (arrowhead) compared to control (arrow) MEF. 10-s frame rate. (C) Box and whisker plot of mean F-actin flow velocities in lamellipodium (LP) and lamellum (LM) of control and Vcl-KO MEF, calculated from qFSM F-actin speed maps. n = 30 (control) and n = 40 (Vcl-KO) time points during protrusion (6–8 cells/condition); means indicated; *, P < 0.01, Mann-Whitney U test. (D) Overlays of EGFP-paxillin (left) and F-actin flow maps (right) of control and Vcl-KO MEF. Bars: (left) 2 µm; (right, flow), 2 μm/min. (E) Box and whisker plot of F-actin flow velocities within and outside of segmented FA during protrusion phases (6–8 cells per condition, qFSM, X-rhodamine actin/EGFP-paxillin); n = 1,000 per group (uniformly sampled among all segmented FA of each group, or among all pixels outside of segmented FA in a given region); means indicated; *, P < 0.0001, Mann-Whitney U test. (F) High resolution TFM of EGFP-paxillin expressing control and Vcl-KO MEF on FN-coated polyacrylamide substrates. (left to right) EGFP-paxillin SDC images and ECM traction maps (kPa). Arrows (control) and arrowheads (Vcl-KO) denote individual FA. Bar, 5 µm. (G) Box and whisker plot of ECM traction stresses of n = 48 (control) and n = 20 (Vcl-KO) segmented FA (eight control and six Vcl-KO MEF); means indicated; *, P < 0.01, Student’s t test.