Figure 5.
Activated vinculin or talin support adhesion formation under tension-release conditions. (A) TalinKO cells coexpressing GFP-talinFL with either autoinhibited mCh-vinFL or activated mCh-vinT12 were pretreated in suspension with blebbistatin (50 µM) or an equivalent volume of DMSO for 45 min. Cells were fixed after spreading on fibronectin-coated glass for 15 min. The percentage of the cell consisting of adhesions was quantified from the GFP signal. Graphs show the mean and SEM; n = 15 (talinFL + vinFL DMSO), 14 (talinFL + vinFL Blebbistatin), 16 (talinFL + vinT12 DMSO), and 16 (talinFL + vinT12 Blebbistatin) cells; results are representative of three independent experiments. *, P < 0.05; ***, P < 0.001; one-way ANOVA with Holm-Sidak’s multiple comparison test. (B) Representative images of talinKO cells as described above. Scale bar indicates 10 µm (scale bar in the magnified region indicates 2 µm). Line profile shows that mCh-vinFL and GFP-talinFL colocalize at peripheral adhesion structures, whereas mCh-vinT12 and GFP-talinFL colocalize at all adhesions throughout the cell. (C) Quantification of the curvature of talin-positive structures in these cells. Graphs show the mean and SEM; n = 1,817 structures analyzed from 16 (DMSO) and 1,171 structures analyzed from 15 (blebbistatin) cells. ***, P < 0.001, unpaired two-tailed t test. Results are representative of three independent experiments. (D) Still frame images from a video of a vinculinKO MEF expressing GFP-vinT12 (green) and RFP-LifeAct (magenta) before and during treatment with Y-27632 (50 µM). Note the buckling and bending of the adhesions (arrowheads). Scale bar indicates 5 µm. (E) VinculinKO MEF expressing GFP-vinT12 together with RFP-LifeAct treated with Y-27632 (50 µM) and imaged every minute for 60 min. Active vinculin supports the formation and growth of new adhesions (upper panels, orange arrowheads), with actin stress fibers remaining bundled at the center of the cell (lower panels, cyan arrowheads). Scale bar indicates 10 µm (scale bars in magnified regions indicate 5 µm). (F) TalinKO cells expressing GFP-talΔR2R3 or GFP-talinE1770A, after DMSO or blebbistatin treatment (as described above in A), with phalloidin staining shows GFP-positive structures form during cell spreading without intracellular tension. Scale bar indicates 10 µm. (G) Quantification of the curvature of talin-positive structures in these cells. Graph shows the mean and SEM; n = 383 (talΔR2R3 DMSO), 605 (talΔR2R3 blebbistatin), 428 (talinE1770A DMSO), and 806 (talinE1770A Blebbistatin) structures analyzed from 15 cells for each condition. ***, P < 0.001, one-way ANOVA with Holm-Sidak’s multiple comparison test. Results are representative of three independent experiments.

Activated vinculin or talin support adhesion formation under tension-release conditions. (A) TalinKO cells coexpressing GFP-talinFL with either autoinhibited mCh-vinFL or activated mCh-vinT12 were pretreated in suspension with blebbistatin (50 µM) or an equivalent volume of DMSO for 45 min. Cells were fixed after spreading on fibronectin-coated glass for 15 min. The percentage of the cell consisting of adhesions was quantified from the GFP signal. Graphs show the mean and SEM; n = 15 (talinFL + vinFL DMSO), 14 (talinFL + vinFL Blebbistatin), 16 (talinFL + vinT12 DMSO), and 16 (talinFL + vinT12 Blebbistatin) cells; results are representative of three independent experiments. *, P < 0.05; ***, P < 0.001; one-way ANOVA with Holm-Sidak’s multiple comparison test. (B) Representative images of talinKO cells as described above. Scale bar indicates 10 µm (scale bar in the magnified region indicates 2 µm). Line profile shows that mCh-vinFL and GFP-talinFL colocalize at peripheral adhesion structures, whereas mCh-vinT12 and GFP-talinFL colocalize at all adhesions throughout the cell. (C) Quantification of the curvature of talin-positive structures in these cells. Graphs show the mean and SEM; n = 1,817 structures analyzed from 16 (DMSO) and 1,171 structures analyzed from 15 (blebbistatin) cells. ***, P < 0.001, unpaired two-tailed t test. Results are representative of three independent experiments. (D) Still frame images from a video of a vinculinKO MEF expressing GFP-vinT12 (green) and RFP-LifeAct (magenta) before and during treatment with Y-27632 (50 µM). Note the buckling and bending of the adhesions (arrowheads). Scale bar indicates 5 µm. (E) VinculinKO MEF expressing GFP-vinT12 together with RFP-LifeAct treated with Y-27632 (50 µM) and imaged every minute for 60 min. Active vinculin supports the formation and growth of new adhesions (upper panels, orange arrowheads), with actin stress fibers remaining bundled at the center of the cell (lower panels, cyan arrowheads). Scale bar indicates 10 µm (scale bars in magnified regions indicate 5 µm). (F) TalinKO cells expressing GFP-talΔR2R3 or GFP-talinE1770A, after DMSO or blebbistatin treatment (as described above in A), with phalloidin staining shows GFP-positive structures form during cell spreading without intracellular tension. Scale bar indicates 10 µm. (G) Quantification of the curvature of talin-positive structures in these cells. Graph shows the mean and SEM; n = 383 (talΔR2R3 DMSO), 605 (talΔR2R3 blebbistatin), 428 (talinE1770A DMSO), and 806 (talinE1770A Blebbistatin) structures analyzed from 15 cells for each condition. ***, P < 0.001, one-way ANOVA with Holm-Sidak’s multiple comparison test. Results are representative of three independent experiments.

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