The talin R3 domain is a critical regulator of vinculin binding and talin activation in response to disruption of talin autoinhibition. (A) Schematic showing talin in a closed compact conformation where the R9 rod domain interacts with F3 of the FERM domain and an open talin conformation. Blue helices indicate VBSs. (B) GFP-talinFL^E1770A (autoinhibition relieved) is recruited to mCh-vinFL-cBAK when coexpressed in NIH3T3 cells. Scale bar indicates 10 µm. (C) FLAP experiments in NIH3T3 cells shows there is minimal loss of fluorescence over time of photoactivated PAGFP-talinFL^E1770A when bound to mCh-vinFL-cBAK at mitochondria. Scale bar indicates 5 µm. Error bars show the SEM; n = 7 mitochondria from five cells. Results are representative of three independent experiments. (D) GFP-talFL^I805S (an R3-destabilizing mutant) is even recruited to wild-type mCh-vinFL-cBAK at mitochondria. (E) A GFP-talin construct in which F3-R9 autoinhibition is relieved but R3 is stabilized (GFP-tal^IVVI+E1770A) is not recruited to wild-type mCh-vinFL-cBAK at mitochondria. (F) GFP-tal^IVVI (R3 rod domain–stabilizing mutant) is capable of binding to constitutively active mCh-vinT12-cBAK. Experiments in D–F were all performed in NIH3T3 cells, and scale bars indicate 10 µm. (G) Model explaining how relief of talin autoinhibition regulates the potential for vinculin to bind to the R3 rod domain of talin. In the closed, autoinhibited talin conformation, only active vinculin is capable of binding to talin R3. When the F3-R9 autoinhibition is relieved, the R3 domain undergoes a conformational change, allowing wild-type vinculin to bind.