Force-independent interactions between activated vinculin and talin, or vinculin and activated talin, are direct. ( A and B) Coexpression in NIH3T3 cells of mCh-talinFL-cBAK with active forms of vinculin bearing a point mutation in the canonical talin binding site in the vinculin head domain (D1) that blocks the interaction with talin: (A) GFP-vinT12-A50I or (B) YFP-vin258-A50I. (C) An activated talin deletion construct (Atherton et al., 2015) targeted to mitochondria (mCh-tal∆R2R3-cBAK) recruits endogenous vinculin (hVin1 antibody staining) in NIH3T3 cells. (D) Mutating the canonical talin binding site within the D1 domain of full-length vinculin (GFP-vin-A50I) blocks the recruitment of vinculin to active talin at mitochondria (mCh-tal∆R2R3-cBAK). Scale bars in A–D indicate 10 µm. (E) FLAP experiments in NIH3T3 cells coexpressing mCh-vinT12-cBAK and PAGFP-talinFL show that there is minimal loss of fluorescence over time after activation in the presence of either blebbistatin (50 µM) or cytochalasin D (2.5 µg/ml). Scale bar indicates 2 µm. Error bars represent SEM; n = 21 (DMSO), 24 (Blebbistatin), and 27 (Cytochalasin D) mitochondria per cell; N = 5 (DMSO), 7 (Blebbistatin), and 5 (Cytochalasin D) cells; results are representative of three independent repeats.