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pp60^c-src and related tyrosine kinases: a role in the assembly and reorganization of matrix adhesions

Tova Volberg¹, Lewis Romer^2,*, Eli Zamir¹ and Benjamin Geiger^1,

¹ Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel
² Departments of Pediatrics, Cell Biology and Anatomy, and Anesthesiology, University of North Carolina at Chapel Hill, NC 27599-7220, USA
^* Present address: Departments of Anesthesiology and Cell Biology, Johns Hopkins University, Blalock 904, Baltimore, MD 21215-4904, USA

View larger version (59K): [in a new window]   Fig. 1. Quantitative immunofluorescence labeling of wild-type, Src-/-, wt6 and SYF cells for phosphotyrosine. The cells were plated on fibronectin-coated cover glasses for 15 minutes or 24 hours, permeabilized, fixed and immunolabeled with anti-phosphotyrosine antibodies. (A) Immunofluorescence patterns of labeling, presented by a linear color look-up table. (B) Scattergrams presenting the fluorescence labeling intensity, in arbitrary units (AU), and area, in µm2, of phosphotyrosine-containing focal contacts in the different cell types. Each scattergram shows all the focal contacts identified in 10 representative cells, each of which contains 50-150 focal contacts. In each scattergram, the average number of relatively large and/or intensely labeled adhesions per cell is indicated. The size and intensity threshold levels were selected so that only <10% of the adhesion sites of SYF cells were in the ‘large or intense’ category. Note the low labeling intensity of phosphotyrosine in Src-/- and SYF cells compared with control wild-type and wt6 cells.

View larger version (36K): [in a new window]   Fig. 2. (A) Phosphotyrosine-containing proteins in wild-type, Src-/- and wt6 cells. The cells were plated on fibronectin-covered tissue culture dishes, incubated for the indicated times, then extracted in Laemmli sample buffer, subjected to 5-15% gradient SDS-PAGE, immunoblotted and labeled with anti-phosphotyrosine antibodies. The immunoreactive bands were detected using ECL. Note the progressive increase in phosphorylation of the 125 kDa (FAK) and 68 kDa (paxillin) bands in wild-type and wt6 cells. FAK phosphorylation gradually increased in wt6 cells. In Src-/- cells, no increase in FAK phosphorylation above basal level was detected and only a small increase in paxillin phosphorylation was observed at 24 hours. (B) Western blot analysis of tyrosine phosphorylation of tensin in Src-/- and wild-type cells. The two cell types were extracted 24 hours after plating and equal amounts of protein were subjected to immunoprecipitation with tensin antibodies and immunoblotting with both tensin and phosphotyrosine antibodies. Note the comparable amounts of tensin in both cell lines and the marked decrease in tensin phosphorylation in the Src-/- cells. (C) Western blot analysis of FAK and paxillin in wild-type and Src-/- cells. The blots reveal that the levels of FAK and paxillin in the two cell lines are essentially identical (tubulin is used as an internal control for equal protein loading).

View larger version (63K): [in a new window]   Fig. 3. Quantitative immunofluorescence labeling of wild-type, Src-/-, wt6 and SYF cells for tensin. The cells were plated on fibronectin-coated cover-glasses for 15 minutes or 24 hours, permeabilized, fixed and immunolabeled with anti-tensin antibodies. (A) Immunofluorescence patterns of labeling, presented by a linear color look-up table. (B) Scattergrams presenting the fluorescence labeling intensity, in arbitrary units (AU), and area, in µm2, of tensin-containing matrix adhesions in the different cell types. The quantitative analysis of tensin-containing matrix adhesions was performed as described for Fig. 1. The size and intensity-threshold levels were selected so that only <5% of the adhesion sites of wild-type or wt6 cells were in the ‘large or intense’ category. Note the intense labeling for tensin in the focal contacts of Src-/- and SYF cells, compared with control wild-type and wt6 cells.

View larger version (62K): [in a new window]   Fig. 4. Fluorescence ratio imaging (tensin/vinculin) showing accumulation of tensin in focal contacts of Src-/- and SYF cells. The cells were plated on fibronectin-coated cover-glasses for 15 minutes or 24 hours, permeabilized, fixed and double-immunolabeled with antibodies against tensin and vinculin. The tensin/vinculin ratios per pixel were calculated and presented by a logarithmic color look-up table, which enabled the presentation of ratio value variations spanning two orders of magnitude (from 0.1 to 10). Note the relatively low (blue) content of tensin in peripheral focal contacts in wild-type and wt6 cells compared with Src-/- and SYF cells, where extensive colocalization with vinculin (yellow) is apparent.

View larger version (49K): [in a new window]   Fig. 5. Quantitative immunofluorescence labeling of wild-type, Src-/- and wt6 cells for FAK. The cells were plated on fibronectin-coated cover-glasses for 15 minutes or 24 hours, permeabilized, fixed and immunolabeled with anti-FAK antibodies. (A) Immunofluorescence patterns of labeling, presented by a linear color look-up table. (B) Scattergrams presenting the fluorescence labeling intensity, in arbitrary units (AU), and area, in µm2, of FAK-containing focal contacts in the different cell types. The quantitative analysis of FAK-containing matrix adhesions was performed as described for Fig. 1. Note the high FAK labeling in Src-/- cells at 15 minutes of incubation.

View larger version (63K): [in a new window]   Fig. 6. Inhibition of FAK and Src activity by AG1007. (A) pp125FAK that was immunoprecipitated from cultured swiss3T3 cells extracts by antibodies to FAK, was equally divided to two samples. One was used as a control (NT) and the other was incubated with 100 µM AG1007 for 15-20 minutes at room temperature. Both samples were incubated for additional 20 minutes with 6 µCi per sample, 32P-labeled ATP. The samples were subjected to 10% SDS-PAGE and the radioactive bands detected using a phosphoimager. Note that FAK activity was inhibited up to 60% by AG1007. (B) GST-Src was assayed for its activity on poly Glu-Tyr ELISA assay in the absence or presence of 50 µM AG1007. Note that AG1007 inhibited Src activity by 80%.

View larger version (96K): [in a new window]   Fig. 7. Reorganization of tensin in wild-type cells After treatment with AG1007. Wild-type cells were cultured for 24 hours, treated with 100 µM AG1007 for 2 hours and then trypsinized and re-plated on fibronectin-coated cover glasses in the presence of 100 µM AG1007, for additional 5 hours. The cells were fixed and double immunolabeled for tensin and phosphotyrosine. The treatment induced tensin accumulation in large peripheral focal contacts (upper panel) and reduction in phosphotyrosine levels (middle panel and super-imposed image, bottom panel). Phosphotyrosine was associated with focal contacts in both treated and untreated cells.

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