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First published online 14 February 2006
doi: 10.1242/jcs.02794

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Molecular mapping of tyrosine-phosphorylated proteins in focal adhesions using fluorescence resonance energy transfer

Department of Molecular Cell Biology, The Weizmann Institute of Science, 76100 Rehovot, Israel

View larger version (31K): [in a new window]   Fig. 1. Tyrosine phosphorylation of focal adhesion components. A construct consisting of two consecutive SH2 domains (dSH2) fused to CFP or YFP was used as a reporter for PY residues. For FRET measurements between PY and vinculin or paxillin, dSH2 (fused to CFP or YFP) was co-transfected with paxillin or vinculin, fused to the complementary fluorophore. (A) Single-color images, ratio images and FRET images are displayed for the indicated pairs. Note high CFP-paxillin/YFP-dSH2 (pax-dSH2) and low FRET for CFP-vinculin/YFP-dSH2 (vin-dSH2). Bar, 2 µm. (B) Quantification of FI measurements with the indicated FRET pairs. The data are presented as mean ± standard error of FI in focal adhesions of 15 to 32 cells. (C) Western blots showing, on the left, immunoprecipitation of YFP and YFP-dSH2 from cells expressing these constructs. The blot on the upper right shows that a large number of tyrosine phosphorylated proteins co-immunoprecipitate with the YFP-dSH2 domain expressed in NIH 3T3 cells. One of the proteins that co-precipitated is paxillin (lower right blot). No tyrosine-phosphorylated proteins co-precipitated from control lysates from NIH 3T3 cells expressing YFP only. (D) Quantification of FRET measurements with the indicated FRET pairs. Note that FRET between dSH2 and a truncated form of paxillin devoid of major tyrosine phosphorylation sites is reduced by half compared with FI values between dSH2 and full-length paxillin, but still significantly higher than between dSH2 and vinculin.

View larger version (36K): [in a new window]   Fig. 2. Paxillin proximity to PY sites, as detected by FRET measurements via acceptor photobleaching. (A) Cells expressing paxillin-CFP and dSH2-YFP were placed on an inverted microscope. For FRET measurements CFP and YFP images were captured before and after YFP photobleach. FRET efficiency (E%) was calculated pixel by pixel: 1-CFP before photobleaching and CFP after photobleaching. Note high FI values are seen in `hotspots' of the total focal adhesion area. Bar, 4 µm. (B) Left, focal adhesion donor (CFP) image before (red) and after (green) acceptor photobleaching. Right, a line scan through the focal adhesion images showing the increase in donor fluorescence as a result of the acceptor bleaching.

View larger version (61K): [in a new window]   Fig. 3. FAK and CAS are in FRET proximity to PY-containing sites. (A) Single-color images, ratio images and FRET images are displayed for the indicated pairs. High FI values were detected for both dSH2-FAK and dSH2-CAS donor-acceptor pairs. Bar, 2 µm. (B) Quantification of FRET measurements with the indicated FRET pairs. The data are presented as mean ± s.e. of three experiments. (C) Immunoprecipitation of CAS from lysates of 293 cells expressing YFP-CAS together with CFP-dSH2 leads to the co-precipitation of CFP-dSH2 (left). CFP-dSH2 co-precipitates with FAK in immunoprecipitation experiments from lysates of 293 cells expressing YFP-FAK and CFP-dSH2 (right).

View larger version (133K): [in a new window]   Fig. 4. Paxillin, CAS, and FAK phosphorylation in focal complexes and focal adhesions. NIH 3T3 cells were fixed and labeled for paxillin (A,D,G); phosphospecific antibody to paxillin PY118 (B); phosphospecific antibody against the FAK autophosphorylation site PY397 (E); phosphospecific antibody directed against CAS PY165 (H). Merged images are shown in the right-hand column (C,F,I). Note that paxillin, FAK and CAS are phosphorylated on the specific PY sites in focal complexes and focal adhesions. Bars, 5 µm.

View larger version (79K): [in a new window]   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.

View larger version (56K): [in a new window]   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.

View larger version (67K): [in a new window]   Fig. 7. FRET for FAK together with either paxillin or CAS. NIH 3T3 cells were transfected with cDNA encoding pairs of CFP- and YFP-fusion proteins as indicated. (A) Images are displayed in spectral scale for the CFP and YFP fusion proteins, for their fluorescence ratio, and for the calculated FRET between the co-expressed molecules. CFP-paxillin/YFP-FAK (pax-FAK) and CFP-FAK/YFP-CAS (FAK-CAS) pairs display relatively high FRET values. (B) FRET of indicated donor/acceptor pairs in focal complexes. First image displays the localization of the acceptor in inverted grey levels (C) Quantitative FRET analysis of the indicated pairs. The data are presented as mean ± s.e. of FI in focal adhesions measured in 8 to 15 cells. Bar, 2 µm.

View larger version (78K): [in a new window]   Fig. 8. Dynamic changes of fluorescence intensity ratio and of FRET of paxillin-dSH2 and FAK-dSH2 pairs. The fluorescence measurements were taken at 1-minute intervals. The color scales on the right indicate the fluorescence ratio or FI values. Note that the temporal variation in the ratio of both donor/acceptor pairs is relatively stable. FI values for the FAK-SH2 pair showed some local fluctuations, whereas high values for paxillin-dSH2, mostly restricted to areas with high PY remained stable over time. Bar, 2 µm.

View larger version (16K): [in a new window]   Fig. 9. A highly tyrosine phosphorylated focal adhesion at the retracting edge of a cell displays a dynamic reshaping, including a disconnected region (area indicated by asterisk) that moves towards the cell center.

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