Recruitment of t alin to truncated vinculin constructs. (A) Coexpression of GFP-talinFL and mCh-vinFL-cBAK in NIH3T3 cells. Imaging of the mitochondria plane (top panel) or the FA plane (bottom plane) shows that, while the constructs do not colocalize at mitochondria, both constructs are present at FAs (red arrows). Scale bar indicates 5 µm. (B) Coexpression of GFP-talinFL with either mCh-vin880-cBAK (lacking the C-terminal vinculin tail) or mCh-vin258-cBAK (the D1 domain of vinculin only) in NIH3T3 cells shows that both cBAK constructs can recruit talin. Scale bar indicates 10 µm. (C) FLAP curve and images of PAGFP-cBAK at mitochondria marked using MitoTracker Deep Red FM in NIH3T3 cells reveals that this construct stably integrates into the outer mitochondrial membrane. Scale bar indicates 5 µm; error bars represent SEM, n = 15 mitochondria from five cells. Results are representative of three independent repeats. (D) FLAP curves of PAGFP-talinFL at FAs coexpressed with either mCh-vinFL or mCh-vinT12. Note the reduced turnover of talin at FAs when coexpressed with vinT12. Error bars represent SEM; n = 92 (vinFL) or 68 (vinT12) FAs, from 10–15 cells. Data are pooled from three independent experiments.