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Figure 5


Fig. 5. Paxillin phosphorylation during focal adhesion development. (A) First two rows of the time-lapse recording show localization of CFP-paxillin and YFP-dSH2 along the lamellipodium of NIH 3T3 fibroblast; for better visualization black and white inverted-contrast images are shown. The third row shows calculated FI between CFP-paxillin and YFP-dSH2. Note that during the development of focal adhesions, paxillin accumulation precedes the increase in PY levels (compare encircled areas in rows one and two). FRET values of focal complexes in the encircled area increase steadily until reaching a level of FI>20, as in mature focal adhesions. High FRET values in mature focal adhesions (ellipse) remained at FI>20 during the entire period of time-lapse recording. (B) Measurements of fluorescence intensity maxima during transition of focal complexes to focal adhesions (black lines; encircled area in A) compared with those in focal adhesions (grey lines; ellipse area in A) during the period of observation. Note that during the transition of focal complexes to focal adhesions, acceptor fluorescence intensity, although initially below donor intensity, rises above the donor intensity at 8 minutes. In focal adhesions, acceptor intensity remains in excess of the donor intensity.





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