Active talin can bind to vinculin independently of force. (A) Schematics of the talin constructs used. Blue indicates VBSs within the four-and five-helix bundles that make up the talin rod (R1–R13), and the green halo indicates the rod domains involved in binding F-actin. The position of the talin rod domains deleted in talΔR2R3 and talΔR4-R10 are indicated by brackets. (B and C) GFP-talΔR4-R10 and GFP-talΔR2R3 are both recruited to either constitutively active mCh-vinT12-cBAK (B) or wild-type mCh-vinFL-cBAK (C) when coexpressed in NIH3T3 cells. Scale bars indicate 10 µm. (D) FLAP experiments in NIH3T3 cells show that there is minimal loss of fluorescence over time, indicating that the interaction between vinFL-cBAK and talΔR4-R10 (upper panel) and talΔR2R3 (lower panel) is very stable. Error bars represent SEM; n = 8 (PAGFP-talΔR2R3) or 6 (PAGFP-talΔR4-R10) mitochondria from five cells. Results are representative of three independent experiments. Scale bar indicates 5 µm.