The mitochondrial targeting system confirms tensin interactions with other FA proteins. (A) U2OS cells stably expressing BirA-control or BirA-tensin3 were incubated with biotin for 16 h before being fixed and stained for myc and paxillin and biotinylated proteins (using fluorophore [Dylight 488]-conjugated streptavidin). Scale bar indicates 10 µm. (B) The C-terminus of tensin1, 2, or 3 was fused to the short mitochondrial targeting sequence from the outer mitochondrial membrane protein BAK (cBAK), with an N-terminal mCherry (mCh) tag. When expressed in NIH3T3 cells each of these constructs co-localize with the mitochondria-specific dye MitoTracker. (C) Representative images of NIH3T3 cells co-expressing the indicated GFP-tagged cell-matrix adhesion protein together with the indicated mCh-tensin-cBAK construct. (D) Representative images of NIH3T3 cells expressing mCh-tensin-cBAK constructs immunostained for endogenous talin, vinculin or paxillin. (E) Representative images of NIH3T3 cells expressing mCh-tensin1-cBAK or mCh-tensin3-cBAK immunostained for active β1 integrin (9EG7 stain) reveals the absence of active integrins. Scale bars in B–E indicate 5 µm.