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First published online 23 January 2003
doi: 10.1242/jcs.00284

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Live-cell monitoring of tyrosine phosphorylation in focal adhesions following microtubule disruption

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

View larger version (41K): [in a new window]   Fig. 1. Double immunostaining of serum-starved SV80 cells for vinculin (left column) and phosphotyrosine (middle column) following treatment with nocodazole for up to 60 minutes. The right column shows the ratio between vinculin and PY intensities. All images are presented in a spectrum scale as indicated in the lookup table for each column. Although focal complexes are most prominent at time=0, FAs are apparent at 3 minutes, and constitute the majority of adhesive structures from 10 minutes onwards. FAs reach their maximal size at 30 minutes. Vinculin intensity increases from 0 to 30 minutes. Note the `centripetal shift' of vinculin relative to PY at 0 and 3 minutes, which can still be seen in some FAs after 10 minutes (arrowheads). Bar, 1 µm.

View larger version (49K): [in a new window]   Fig. 2. Double immunostaining of paxillin and phosphotyrosine. All images were acquired and produced as in Fig. 1. Whereas paxillin levels are continuously increasing from 0 to 30 minutes, PY levels increase significantly only later, between 3 and 10 minutes, and then reach a plateau. Note the overall extensive overlap between paxillin and PY labeling. Main differences are detected along the paxillin-rich and PY-poor peripheral margins of FAs at 30 minutes (arrowheads). Bar, 1 µm.

View larger version (43K): [in a new window]   Fig. 3. Double immunostaining of FAK and phosphotyrosine. All images were acquired and processed as in Fig. 1. Note that FAK levels in small adhesions have already increased at 3 minutes, whereas PY levels remain almost unchanged. Both FAK and PY reach their maximal levels at 10 minutes, remain high for up to 30 minutes, and return to control levels by 60 minutes. Bar, 1 µm.

View larger version (34K): [in a new window]   Fig. 4. Quantitative analysis of the immunostaining data shown in Figs 1,2,3, depicting the percent increase in average intensity and area compared with control, untreated cells. Vinculin, paxillin and FAK levels are already elevated after 3 minutes of treatment, whereas PY levels remain essentially unchanged. Vinculin and paxillin levels continue to increase until 30 minutes and decrease only slightly afterwards, whereas FAK and PY levels reach a plateau after 10 minutes and decrease to control levels between 30 and 60 minutes. FA growth starts immediately and continues for 30 minutes.

View larger version (81K): [in a new window]   Fig. 5. (A) Domain structure of pp60Src and amino acid sequence of the SH2 domain (from mouse neuronal Src). (B) Schematic representation of the YFP-SH2 and YFP-dSH2 constructs. (C) Live-cell recording of SV80 cells transfected with YFP-SH2. The fusion protein localizes to FAs only weakly and shows a high cytoplasmic background. (D) Live-cell recording of SV80 cells transfected with YFP-dSH2. Note the distinct FA staining and relatively low cytoplasmic background. (E) Fixed cell transfected with YFP-SH2, showing loss of FA localization. (F) Fixed, YFP-dSH2 transfected cell, showing retention of FA labeling after fixation. Bar, 10 µm.

View larger version (53K): [in a new window]   Fig. 6. (A,B) Two fields of YFP fluorescence of SV80 cells transfected with YFP-dSH2. (C,D) Anti-PY staining of the corresponding fields. Images A-D show the fluorescence intensities represented in a spectrum scale, as indicated in the calibration bars. (E,F) Ratio images of the cells shown in (A,C) and (B,D), respectively, depicting the relative fluorescence of PY and YFP-dSH2. The cells shown are numbered consecutively from 1-6. Note the uniform ratio between the two labels in YFP-dSH2 expressing cells (1, 4 and 5), indicating strong correlation between YFP-dSH2 and PY. The cells (2, 3 and 6) showing red FAs (i.e. excess PY) are not expressing detectable levels of YFP-dSH2. (G) Plot of intensity of PY staining against the intensity of labeling in YFP-dSH2-overexpressing cells 1 and 4. (H) Similar plot, as in G, for nonexpressing cells 2, 3, 6, and YFP-dSH2 expressing cell 5. FAs from field 1 are represented in red, whereas FAs from field 2 are represented in black. Notice the linear correlation between the two labels, the increase in PY labeling upon expression of particularly high levels of YFP-dSH2, and the normal PY levels in the moderately transfected cell. Bar, 10 µm.

View larger version (61K): [in a new window]   Fig. 7. Frames from a two-color movie showing a serum-starved SV80-cell transfected with CFP-vinculin and YFP-dSH2 and treated with nocodazole. Row 1 shows the CFP-vinculin staining at 4 different time points, and the images in row 2 are time ratio (FRIT) images comparing consecutive time points. Row 3 and 4 show the corresponding YFP-dSH2 images. Row 5 shows the vin/dSH2 ratio at all time points. Note the strong initial increase in vinculin after 2 minutes, in comparison with the low and inconsistent changes in PY levels. The adhesions used for the quantification presented in Fig. 8 are marked in the 0' frame of YFP-dSH2. The whole movie (2 colors and ratio) can be found in the online supplemental material (http://jcs.biologists.org/supplemental). Bar, 10 µm.

View larger version (15K): [in a new window]   Fig. 8. Quantitative analysis of the three FAs marked in Fig. 7. The temporal changes of the integrated intensities (total label present in each adhesion) of CFP-vinculin and YFP-dSH2 are presented. Note that vinculin intensity increases sharply during the first two minutes in all three cases, whereas there is almost no increase in PY at the same time. The fourth panel shows the average values of all FA-associated CFP-vinculin and YFP-dSH2 in the tested cell, which is consistent with the data obtained for the fixed cells (see Fig. 4).

View larger version (27K): [in a new window]   Fig. 9. (A) FRIT images (fluorescence ratio of images taken at different time points) of a movie showing an SV80 cell transfected with YFP-dSH2, serum starved, and treated with nocodazole for 30 minutes. The four spectrum scale images (see lookup table) are temporal ratios of two consecutive frames. Structures that appear only at the later image are shown in red, whereas structures that appear only at the earlier of the two time points are blue. Unchanged structures are represented in yellow. The numbers inserted into the 6'/2' image mark FAs that were further subjected to quantitative analysis (see below). The arrow points to a FA that is not present at 6' but appears at 10', whereas the arrowhead points to a FA that is present at 18' but has disappeared at 30'. The whole movie can be found in the online supplemental material (http://jcs.biologists.org/supplemental). Bar, 10 µm. (B) Quantitative analysis of the four FAs that were marked above (I-IV). The temporal changes of their average intensities and sizes are depicted. Often, neighboring FAs show different PY dynamics (compare FA I and II), and the changes in PY intensity are not always correlated in time with the growth of the FA. Note that the changes in PY level and FA size may be correlated in some cases (e.g. adhesions II and IV), or may be completely different (e.g. adhesions 1 and 3).

View larger version (11K): [in a new window]   Fig. 10. A scheme summarizing the proposed sequence of dynamic molecular events during nocodazole-induced FA growth (see text for details). The time is shown on a logarithmic scale.

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