Fig. 5. RIAM inhibits the actomyosin-dependent binding of vinculin to talin R1–R2–R3 but not to R11.

a Time lapse showing the recruitment of Vh in disks coated with talin R1–R2–R3 in the absence of actomyosin (top), presence of actomyosin (middle), and presence of actomyosin and RIAM (bottom). This experiment was repeated twice independently with the same results. b, c Kymographs of EGFP-Vh along a cross-section of a disk coated with talin R1–R2–R3 (b) and R11 (c). Conditions: 100 nM EGFP-Vh, 2.4 µM actin, 50 nM myosin, 500 nM (b) or 3 µM (c) mCherry-RIAM, 1 µM talin during the coating step. The images are color coded using the fire LUT of ImageJ. Scale bar in time lapses = 10 µm. In kymographs, horizontal bar = 1000 s, vertical bar = 5 µm. d, e Kinetics of the mean fluorescence of EGFP-Vh corresponding to the conditions described in (b, c). Data are mean ± SD. d n = 54 (−actomyosin) and (+actomyosin), n = 51 disks (+actomyosin + 0.5 µM RIAM). e n = 59 disks. f, g Steady-state binding of Vh (2220 s after sealing the chamber) in disks coated with talin R1–R2–R3 (f) or R11 (g) in the absence and presence of RIAM. f, g Same conditions as in (b, c). Each data point represents the mean fluorescence of Vh in one disk. The bar shows the mean. f n = 54 (+actomyosin), n = 51 disks (+actomyosin + 0.5 µM RIAM). A significant difference was found using a two-tailed t test (P = 3.95 × 10⁻²²). g Left panel: n = 60. No significant difference was found using a two-tailed t test (P = 0.1126). Right panel: n = 59 disks. No significant difference was found using a two-tailed t test (P = 0.3575). ****P < 0.0001 using a two-tailed t test; ns nonsignificant. Source data are provided as a Source data file. See Supplementary Movie 9 and Supplementary Movie 10.