Load across vinculin is strongly affected by the ability of vinculin to bind actin, but not talin. (A) TSMod was inserted into vinculin variants. (Left to right) Shown here is WT VinTS, mutation disrupting the vinculin binding to talin (A50I), and mutation disrupting the vinculin binding to actin (I997A). (B) Representative acceptor (top row) and masked FRET efficiency (bottom row) images are given of single Vinc −/− MEFs expressing each of the VinTS constructs. Scale bars, 30 μm. (C) Zoomed-in view is given of the regions indicated in (B). (D) A box-whisker plot is given of cell-averaged FRET efficiency (n = 150, 166, and 79 cells, respectively, from seven independent experiments) compared to previously established zero-load (dotted line). Differences between groups were detected using the Steel-Dwass test (∗∗∗p < 0.001); p values for all comparisons can be found in Table S1. (E) Line scans are shown of vinculin distribution and FRET efficiency across peripheral FAs from distal to proximal tip. Thin lines represent each individual adhesion (n = 27 FAs from three independent experiments); thick lines represent a smoothing spline fit to the collective FA data. To see this figure in color, go online.