Figure S4.
Active vinculin can rescue tension-independent adhesion formation, but requires the vinculin-talin interaction. (A) Representative images of vinculin-null cells (vinKO MEFs) co-expressing GFP-tensin3-FL or GFP-tensin3-FL∆H1 together with either mCh-vinFL or mCh-vinT12, treated in suspension with blebbistatin (50 µM) or an equivalent volume of DMSO, for 60 min. Cells were fixed after spreading on fibronectin-coated glass for 60 min. Quantification of mean adhesion size per cell using the GFP signal shows that co-expression of active vinculin (mCh-vinT12) can rescue formation of GFP-tensin3 positive adhesions under tension-free (blebbistatin-treated) conditions; this phenotype is not seen in cells expressing GFP-tensin3-FL∆H1. Error bars are SEM; n = 19–24 cells, *** indicates P < 0.001 (ANOVA), n.s. indicates not significant. (B) Representative images of vinculin-null cells (vinKO MEFs) co-expressing GFP-tensin3-FL together with either mCh-vinT12 or mCh-vinT12-A50I, treated in suspension with blebbistatin (50 µM) or an equivalent volume of DMSO, for 60 min. Cells were fixed after spreading on fibronectin-coated glass for 60 min. Quantification of mean adhesion size per cell using the GFP signal shows that inhibiting the vinculin-talin interaction (vinT12-A50I expression) blocks the formation of large GFP-tensin3-FL positive adhesions in tension-free conditions. Error bars are SEM; n = 19–21 cells, ** indicates P < 0.001 (Kruskal-Wallis test with Dunn’s multiple comparison test). Scale bars in A and B indicate 10 µm.

Active vinculin can rescue tension-independent adhesion formation, but requires the vinculin-talin interaction. (A) Representative images of vinculin-null cells (vinKO MEFs) co-expressing GFP-tensin3-FL or GFP-tensin3-FL∆H1 together with either mCh-vinFL or mCh-vinT12, treated in suspension with blebbistatin (50 µM) or an equivalent volume of DMSO, for 60 min. Cells were fixed after spreading on fibronectin-coated glass for 60 min. Quantification of mean adhesion size per cell using the GFP signal shows that co-expression of active vinculin (mCh-vinT12) can rescue formation of GFP-tensin3 positive adhesions under tension-free (blebbistatin-treated) conditions; this phenotype is not seen in cells expressing GFP-tensin3-FL∆H1. Error bars are SEM; n = 19–24 cells, *** indicates P < 0.001 (ANOVA), n.s. indicates not significant. (B) Representative images of vinculin-null cells (vinKO MEFs) co-expressing GFP-tensin3-FL together with either mCh-vinT12 or mCh-vinT12-A50I, treated in suspension with blebbistatin (50 µM) or an equivalent volume of DMSO, for 60 min. Cells were fixed after spreading on fibronectin-coated glass for 60 min. Quantification of mean adhesion size per cell using the GFP signal shows that inhibiting the vinculin-talin interaction (vinT12-A50I expression) blocks the formation of large GFP-tensin3-FL positive adhesions in tension-free conditions. Error bars are SEM; n = 19–21 cells, ** indicates P < 0.001 (Kruskal-Wallis test with Dunn’s multiple comparison test). Scale bars in A and B indicate 10 µm.

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