Paxillin can recruit inactive vinculin and talin independently of force. ( A) Staining for endogenous paxillin in NIH3T3 cells expressing either mCh-vinFL-cBAK or mCh-talinFL-cBAK shows paxillin is recruited to both inactive vinFL-cBAK and talinFL-cBAK. (B) Colocalization of YFP-paxillin168-557 (upper panel) or YFP-paxillin314-557 (lower panel) with mCh-vinFL-cBAK shows a stretch of paxillin containing the third, fourth, and fifth LD motifs are required for the interaction between paxillin and mCh-vinFL-cBAK. (C) Staining for phosphotyrosine in NIH3T3 cells expressing mCh-vinT12-cBAK reveals that no tyrosine phosphorylation is present at these mitochondria. (D) Coexpression of mCh-paxillin-cBAK with either GFP-vinFL or GFP-talinFL in NIH3T3 cells shows mCh-paxillin-cBAK is able to recruit either GFP-vinFL or GFP-talinFL to mitochondria. Scale bars in A–D indicate 10 µm. (E) Cell lysates from talinKO cells transfected with siRNA targeting paxillin were separated by SDS-PAGE and stained for paxillin and tubulin. Either siRNA sequence reduced paxillin levels by ∼60%. (F) TalinKO cells transfected with two separate siRNA sequences targeting paxillin were plated on fibronectin in the presence of Mn²⁺ (5 mM) and fixed after 1 h. Cell area was measured from 10 images (n = 251 [Scrambled], 295 [Paxillin siRNA #1], and 262 [Paxillin siRNA #2] cells) and is presented as a histogram showing the percentage of cells with different areas with bins of 25 µm. Results are representative of three independent experiments; ***, P < 0.001 (one-way ANOVA with Dunnett’s multiple comparison test). Note that paxillin knockdown strongly reduces the number of spread cells; cells still containing residual paxillin are able to spread (yellow arrowhead), unlike those lacking paxillin (red arrowheads). Scale bar indicates 20 µm.