Figure S4.
Active talin constructs form disorganized adhesions when spreading in the absence of intracellular tension. (A) Workflow of image processing for adhesion curvature calculation. The GFP-talin signal was background subtracted and thresholded to generate a binary image, which was then used to generate a skeleton image. The length and curvature of each branch >0.2 µm was used to calculate the curvature of the adhesion. (B) Histograms of adhesion curvature calculated from talinKO cells coexpressing GFP-talinFL and mCh-vinT12 treated in suspension with either DMSO or blebbistatin. (C) FLAP of PAGFP-talinFL in NIH3T3 cells coexpressing mCh-vinT12 after treatment with Y-27632 (50 µM) shows that the interaction between the two proteins is extremely stable (Mf < 95%) in the absence of intracellular tension. Scale bar indicates 5 µm. Error bars represent SEM; n = 30 measurements from five cells. Results are representative of three independent experiments. (D) Still-frame images from movies of vinculinKO MEFs expressing GFP-vin258 or GFP-vin258-A50I treated with Y-27632 (50 µM). Scale bar indicates 10 µm (scale bar in magnified regions indicates 5 µm). The growth and correct maturation of new adhesions requires the interaction between vinculin and talin. Error bars represent SEM; n = 26 (vinT12), 16 (vinT12-A50I), 34 (vin258), and 14 (vin258-A50I) adhesions; **, P < 0.01; ***, P < 0.001, one-way ANOVA with Holm-Sidak’s multiple comparisons test; results are representative of three independent experiments. (E) Histograms of adhesion curvature calculated from talinKO cells expressing GFP-tal∆R2R3 or GFP-talinE1770A, after DMSO or blebbistatin treatment as above. (F) Quantification of the percentage of the cell consisting of adhesions (quantified from the GFP signal) in talinKO cells expressing GFP-tal∆R2R3 or GFP-talinE1770A. Cells were pretreated in suspension with blebbistatin (50 µM) or an equivalent volume of DMSO, for 45 min, before being seeded onto fibronectin-coated glass and fixed after 15 min of spreading. Graphs show the mean and SEM; n = 14 (talinFL), 15 (tal∆R2R3), and 15 (talinE1770A) cells. Results are representative of three independent experiments; **, P < 0.01; ***, P < 0.001, significance against talinFL (one-way ANOVA with Holm-Sidak’s multiple comparisons test).

Active talin constructs form disorganized adhesions when spreading in the absence of intracellular tension. ( A) Workflow of image processing for adhesion curvature calculation. The GFP-talin signal was background subtracted and thresholded to generate a binary image, which was then used to generate a skeleton image. The length and curvature of each branch >0.2 µm was used to calculate the curvature of the adhesion. (B) Histograms of adhesion curvature calculated from talinKO cells coexpressing GFP-talinFL and mCh-vinT12 treated in suspension with either DMSO or blebbistatin. (C) FLAP of PAGFP-talinFL in NIH3T3 cells coexpressing mCh-vinT12 after treatment with Y-27632 (50 µM) shows that the interaction between the two proteins is extremely stable (Mf < 95%) in the absence of intracellular tension. Scale bar indicates 5 µm. Error bars represent SEM; n = 30 measurements from five cells. Results are representative of three independent experiments. (D) Still-frame images from movies of vinculinKO MEFs expressing GFP-vin258 or GFP-vin258-A50I treated with Y-27632 (50 µM). Scale bar indicates 10 µm (scale bar in magnified regions indicates 5 µm). The growth and correct maturation of new adhesions requires the interaction between vinculin and talin. Error bars represent SEM; n = 26 (vinT12), 16 (vinT12-A50I), 34 (vin258), and 14 (vin258-A50I) adhesions; **, P < 0.01; ***, P < 0.001, one-way ANOVA with Holm-Sidak’s multiple comparisons test; results are representative of three independent experiments. (E) Histograms of adhesion curvature calculated from talinKO cells expressing GFP-tal∆R2R3 or GFP-talinE1770A, after DMSO or blebbistatin treatment as above. (F) Quantification of the percentage of the cell consisting of adhesions (quantified from the GFP signal) in talinKO cells expressing GFP-tal∆R2R3 or GFP-talinE1770A. Cells were pretreated in suspension with blebbistatin (50 µM) or an equivalent volume of DMSO, for 45 min, before being seeded onto fibronectin-coated glass and fixed after 15 min of spreading. Graphs show the mean and SEM; n = 14 (talinFL), 15 (tal∆R2R3), and 15 (talinE1770A) cells. Results are representative of three independent experiments; **, P < 0.01; ***, P < 0.001, significance against talinFL (one-way ANOVA with Holm-Sidak’s multiple comparisons test).

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