Fig. 6. Paxillin phosphorylation in focal complexes and focal adhesions. (A) Single-color images, ratio and FRET image of CFP-paxillin and YFP-dSH2 in NIH 3T3 cells treated with the ROCK inhibitor Y27632 to induce accumulation of focal complexes. Note the high FI in focal complexes between CFP-paxillin and YFP-dSH2 indicating high tyrosine phosphorylation of paxillin in these structures. (B) A cell expressing a dominant-active form of Rac in addition to the donor-acceptor pair paxillin-dSH2. Note the coexistence of focal complexes at the cell periphery and the larger focal adhesions behind the cell edge. Single-color images of CFP-paxillin and YFP-dSH2 are presented as inverted black and white images for better visualization. High FI values in focal adhesions and focal complexes suggest that a large and comparable fraction of paxillin is tyrosine phosphorylated in both structures. (C) Quantification of FRET between the indicated donor-acceptor pairs in focal adhesions, in focal complexes of Y27632-treated cells, in focal adhesions plus focal complexes in cells expressing dominant-active Rac1 (daRac) and in focal adhesions of cells expressing active RhoA (daRho). The data are presented as mean ± s.e. of FI in focal adhesions or focal complexes measured in five to ten cells.