Paxillin can bind both talin and vinculin when either is in an inactive conformation. (A) Coexpression of GFP-paxillin with either mCh-vinFL-cBAK or mCh-talinFL-cBAK in NIH3T3 cells. The colocalization suggests that paxillin can bind to inactive vinculin or talin independently of forces. (B) TalinKO cells expressing GFP-paxillin and mCh-vinFL fixed and stained with 647-Phalloidin after Mn²⁺ (5 mM)-induced spreading on fibronectin. Note the colocalization of both in FAs. Scale bar in magnified region indicates 5 µm. (C) TalinKO cells expressing GFP-paxillin and mCh-vinFL-cBAK or mCh-vin258-cBAK spread on fibronectin in the presence of Mn²⁺ (5 mM). Note that mCh-vin258-cBAK does not recruit paxillin. (D) TalinKO cells expressing mCh-vin258-cBAK spread on fibronectin in the presence of Mn²⁺ (5 mM) were fixed after 1 h of spreading and stained for paxillin. Note the localization of mCh-vin258 to adhesions in these cells. Scale bars in A–D indicate 10 µm.