ICA of vin880 with other FA proteins. (A) Sections of NIH3T3 cells expressing CFP- and YFP-tagged proteins as indicated. Fluorescence intensity profiles depict the area of the line drawn in image overlays. Potential direct interacting proteins talin and α-actinin show different correlations with vin880. In contrast to talin, which colocalizes identically with vin880, α-actinin colocalizes only poorly with pixels positive for vin880. vin880 colocalization with potentially indirectly associating proteins paxillin and FAK and correlation with phosphotyrosine in FA is shown. Paxillin colocalizes identically with vin880 in oversized FAs, whereas pixels positive for FAK and a probe that recognizes phosphotyrosine in FA (dSH2) correlate less well with vin880 localization. Bar, 2 μm. (B) Quantification of colocalization by ICA based on the Pearson's correlation coefficient (a perfect linear correlation is shown at +1; see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200703036/DC1). Note the high correlation of vin880 with talin and paxillin but lower correlation with α-actinin, FAK, and dSH2. Red asterisks indicate significant differences of correlation values compared with pax-pax control (P < 0.0001, HSD test). Blue asterisks indicate no statistical differences. (C) Quantification of colocalization in FAs by ICA based on the Pearson's correlation coefficient of GFP-vinT12/RFP-paxillin, CFP-paxillin/YFP-vin258, GFP-vinT12/RFP-talin, and YFP-vin258/RFP-talin. The correlation between indicated pairs was as equally high as CFP-paxillin/YFP-paxillin, demonstrating identical colocalization. Error bars indicate ± SEM.

YFP-vin880 is enriched in FN– integrin complexes and coimmunoprecipitates talin and integrin. (A) FN–bead bound complexes isolated from NIH3T3 cells expressing either YFP-vin880 or -vinFL and immunoblotted for GFP, α5 integrin, or talin. (B) Quantification of A expressed as a ratio of vin880/vinFL after immunoblot band signal intensity normalization to the respective α5 integrin signal intensity. Open bars represent ratios of indicated proteins from pull-down experiments with pFN-coated beads; shaded bars represent protein ratios detected in total cell lysate (TCL) of cells expressing YFP-vin880 or -vinFL. (C) Immunoprecipitations using anti-GFP or control mouse IgG (MuIgG) from NIH3T3 cells expressing either YFP-vin880 or -vinFL. (D and E) Immunoprecipitations using anti-GFP or control MuIgG from NIH3T3 cells expressing YFP-vin880 after treatment with a chemical cross-linker (D) or from cells in suspension for 15 min (E) versus cells left attached to tissue culture dishes. All blots are representative of more than two independent transfections. Mrks denotes the position of molecular mass standards (250 kD for talin blots and 150 kD for GFP and α5 integrin blots), which are visible by Western blotting using the infrared imaging system.

Vinculin-induced FA hypertrophy and paxillin recruitment to FAs is not due to the potential dimerization of tailless vinculin constructs with endogenous vinculin. (A) CFP-paxillin was coexpressed with YFP-vinFL or -vin258 in vin−/− MEF cells. Overlay images are from insert areas outlined in the vinFL and vin258 labeled images. The line profiles taken from the lines indicated on the left outline the nearly identical localization of the respective coexpressed proteins in FAs. Bar, 8 μm. (B) Quantification of FA sizes in vin−/− MEF cells expressing the indicated YFP fusion proteins. Area of FAs was calculated as a percentage of the total cell area. Red asterisks indicate significant differences of correlation values compared with vinFL (P < 0.0001, HSD test). Blue asterisks indicate no statistical differences to vinFL. (C) YFP-vin880 t_1/2 of recovery is increased compared to YFP-vinFL when expressed in vin−/− MEF cells. Asterisk indicates statistical significance (P < 0.001, t test).

Vin880 stabilizes adhesion sites in cells despite the inhibition of actomyosin function or disruption of actin filaments. (A, top) NIH3T3 cells expressing YFP-vinFL or -vin880 treated with 100 μM Y-27632 for 60 min. Although only small dotlike adhesions (focal complexes) can be seen at the cell periphery in vinFL-expressing cells, a large number of adhesion sites are still apparent in cells expressing vin880, despite the absence of stress fibers. (bottom) Cells treated with 1 μM cytochalasin D for 30 min. Although essentially no adhesions can be seen in YFP-vinFL–expressing cells, many adhesion sites are still visible in YFP-vin880–expressing cells. Fluorescence intensity profiles on the right are from the area of the line drawn in image overlays. Note that there is little if any correlation of high vinFL or vin880 intensity peaks with those of actin. Bar, 12 μm. (B) Quantification of FA area. FA area was calculated as a percentage of the total cell area. Note the significant loss in FA area in vinFL-expressing cells when treated with cytochalasin D (cyto D) or Y-27632. No loss of adhesion area was found in vin880 cells treated with actin-perturbing reagents. Asterisks indicate significant differences of values compared with nontreated vinFL (P < 0.001, HSD test). The apparent slight increase of vin880 FA area in vin880-expressing cells treated with cytochalasin D or Y-27632 compared to nontreated cells was caused by a decrease in total cell area (in the case of cytochalasin D) or increased de novo formation of adhesions at the cell periphery (in the case of Y-27632).

Effect of wild-type and mutant forms of vinculin on FA growth. (A) Vinculin constructs that were expressed as fusion constructs to GFP derivatives in NIH3T3 cells: vinFL; vinculin comprising head and neck domains, amino acids 1–880 (vin880); vinculin head domain 1, amino acids 1–258 (vin 258); vinT, amino acids 880–1066; vinT12 (Cohen et al., 2005), with point mutations that render the molecule constitutively active; and vinLD (Chandrasekar et al., 2005), with point mutations that inhibit PIP2 binding. (B) NIH3T3 cells expressing indicated GFP-tagged constructs. Note the dramatic increase of FAs expressing vinT12, vin880, and vin258 compared to cells expressing vinFL or vinLD. vinT localizes to FAs and filamentous structures. (C) Quantification of FA size and number. FAs were masked, and their sizes and numbers where calculated. Asterisks indicate statistical significance (P < 0.001, t test). (D) Quantification of FA areas. FA area was calculated as a percentage of the total cell area. Asterisks indicate significant differences of values compared with vinFL (P < 0.0001, HSD test). There was no statistical difference in FA values between vinFL and vinLD. Error bars indicate ± SEM. Bars, 10 μm.

Vinculin regulates integrin clustering and dynamics. (A) HeLa cells overexpressing YFP-vin880 were costained with an antibody recognizing active β1 (9EG7; Bazzoni et al., 1995) and the total pool of β1 integrin (TS2/16). FAs positive for YFP-vin880 are also positive for β1 integrins. The intensity profiles in red and green outline the high correlation of YFP-vin880 and active β1 integrins. The blue intensity profile shows the high levels of β1 integrins in the cell membrane and FAs. Fluorescence intensity profiles depict the area of the lines drawn in image overlays. (B) FAs induced by YFP-vin880 (left) are readily visible by interference reflection microscopy (middle), indicating that they participate in adhesion to ECM proteins. Overlay fluorescence and interference are shown on the right. (C) Images and line profiles drawn over the area of NIH3T3 cells expressing indicated constructs demonstrate that the cytoplasmic pool of GFP-vinFL is higher than that of GFP-vinT12. (D) To assess the turnover of indicated proteins, circular areas of 1.5-μm diameter were bleached, and recovery was measured. Note the slower fluorescence recovery of bleached areas in YFP-vin880–expressing cells compared to areas in YFP-vinFL–expressing cells. (E) Normalized recovery of vinFL, vin880, and vinT12 in FAs, with lines indicating the single exponential fit of the data. MF (F) and t_1/2 of recovery (G) of indicated YFP-fusion proteins in FAs of NIH3T3 cells. Red asterisks indicate significant differences compared to vinFL (P < 0.005, HSD test). Blue asterisks indicate no statistical differences. (H) Talin t_1/2 of recovery is longer when coexpressed with vin880 in comparison with vinFL. Asterisk indicates statistical significance (P < 0.05, t test). Comparison of MFs (I) and t_1/2 (J) of YFP-vinFL, -vin880, and −α5 integrin in FA. Note the striking similarity of vin880 and α5 integrin dynamics. Red asterisks indicate significant differences compared to vinFL (P < 0.001, HSD test). (K) Coexpression of vinFL or vin880 leads to a longer t _1/2 of recovery of YFP–α5 integrin in FAs of vin−/− MEF. Red asterisks indicate significant differences between the samples (P < 0.01, HSD test). (L and M) Expression of vin880 changes neither the MF (L) nor the t_1/2 of recovery (M) of paxillin in FAs. Error bars indicate ± SEM. Bars: (A) 2 μm; (B and C) 10 μm; (D) 2 μm.

FA size is regulated by vinculin interaction with talin. (A) Paxillin was knocked-down using small hairpin RNA in B16 melanoma cells expressing vin880. Despite the absence of paxillin, vin880 still induced FA growth. (B) Quantification of FA number, size, and area fraction of indicated constructs in B16 cells with or without (sh pax) paxillin. (C) Immunoprecipitations from cell lysates of HeLa cells expressing either YFP-vin880, -vin880 (A50I), -vinT12, or -vinT12 (A50I) with anti-GFP antibodies. Note talin coimmunoprecipitates with vin880 and vinT12 but not with A50I mutants. (D) NIH3T3 cells expressing YFP-vinFL and -vin258 (A50I) show no significant differences in FA size in contrast to cells expressing YFP-vin258. (E) Quantification of FA size of cells expressing indicated constructs. Area of FAs was calculated as a percentage of the total cell area. Red asterisks indicate significant differences compared with vinFL (P < 0.0001, HSD test). Blue asterisks indicate no statistical differences. Black asterisk indicates statistical difference with vinFL (P < 0.001, t test). Error bars indicate ± SEM. Bars: (A) 10 μm; (D) 10 μm.

vinT colocalization correlates with a subset of actin but not paxillin. (A) Cells expressing vinT were either labeled for actin or cotransfected with CFP–α-actinin (bottom). The intensity profiles on the right are from the area covered by the line in the overlay images. Note the high correlation of actin stress fibres with YFP-vinT. However, protrusive areas (white arrowheads) positive for α-actinin remain free of YFP-vinT. Bar, 10 μm. (B) In vin−/− MEF cells, there is little YFP-vinT in protruding lamellipodia when compared to F-actin labeled with rhodamine-phalloidine. Note the strong correlation of the intensity profiles (right) on the left side and the lack of correlation on the right side (lamellipodium). (C) NIH3T3 cell expressing CFP-paxillin and YFP-vinT. The protruding area of cell shows many FAs in the protruding area positive for paxillin but devoid of vinT. To the right are line profiles of fluorescence intensities of the line in the overlay image (compare with Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200703036/DC1). (D) NIH3T3 cell expressing YFP-vin880 and colabeled for actin. (a) FAs at the cell periphery with similar intensity profiles, indicating that these FAs are linked to actin stress fibers. (b) FAs away from the periphery of the cell. Intensity profiles show that intensity peaks of vin880 do not correlate with the actin intensities. (E) NIH3T3 cell expressing vinT12 and colabeled for actin. Intensity profiles taken from lines in inserts of peripheral and inner FA demonstrate a significant overlap of vinT12 and actin fluorescence intensity peaks, suggesting a link of these FAs with the actin cytoskeleton. (F) Kymograph analysis of NIH3T3 cells expressing YFP-vin880, GFP-vinT12, and YFP-vinT. Kymograph analysis shown in a spectral fluorescence intensity scale derived from a one-pixel line perpendicular to the cell edge of time-lapse recordings presented in Videos 2–4. Note that high intensity areas in vin880 cells remain stable with time and therefore are visualized as straight lines in this type of analysis, whereas those of vinT12 and vinT follow a retrograde flow. Bars (A–C) 10 μm; (D and E) 8 μm.

Model of vinculin action in cells. See text in Discussion.