Figure 7.
Fibronectin fibrillogenesis is dependent on the tensin3–talin interaction. (A and B) Representative (background subtracted) images of fibronectin fibrils (using an antibody against cell-derived fibronectin (Serini et al., 1998) produced by (A) WT or TNS3 KO U2OS cells or (B) TNS3 KO U2OS cells expressing GFP-tensin3-FL or GFP-tensin3-FL∆TBS, spread overnight on fibronectin-coated glass. Fibril formation was quantified in 70 × 70 µm squares applied to each image and is normalized to U2OS WT (A) or U2OS TNS3 KO expressing GFP-tensin3-FL (B) to calculate the fold-change in the area covered by fibronectin fibrils. Quantification shows 50% reduction in fibronectin fibrils produced by TNS3 KO (A) and GFP-tensin3-FL∆TBS (B) cells. Error bars are SEM, n = 57 (WT); 60 (KO); 76 (GFP-tensin3-FL); 69 (GFP-tensin3-FL∆TBS) squares from 30 to 35 images, pooled from three independent experiments; **** indiciates P < 0.0001 (Mann-Whitney t test). (C) Representative images (background subtracted) of fibronectin fibrils produced by TNS3 KO U2OS cells expressing GFP-tensin3-FL or GFP-tensin3-FL∆TBS, spread overnight on fibronectin-coated soft (5 kPa) or stiff (50 kPa) polyacrylamide gels (quantified as Fig. 7, A and B) normalized to GFP-tensin3-FL on 5 kPa polyacrylamide gels. Note the reduced fibronectin fibrils produced by GFP-tensin3-FL∆TBS expressing cells on both substrates. Error bars are SEM, n = 75 (GFP-tensin3-FL, 5 kPa); 48 (GFP-tensin3FL, 50 kPa); 58 (GFP-tensin3-FL∆TBS, 5 kPa); 41 (GFP-tensin3-FL∆TBS, 50 kPa) squares from 30 to 35 images, pooled from three independent experiments; **** indicates P < 0.0001 (Kruskal-Wallis ANOVA with Dunn’s multiple comparison test). (D) Live-cell imaging of vinKO MEFs expressing GFP-tensin3-FL with or without mCh-vinFL co-expression; temporal color maps of adhesion movement obtained from the GFP signal show that tensin3-positive adhesions in cells without vinculin are more dynamic. Images were acquired every 5 min for 2 h. Quantification of mean adhesion speed and lifetime from time-lapse movies of vinKO MEFs expressing GFP-tensin3-FL with or without mCh-vinFL co-expression. Error bars are SEM, n numbers are pooled from 6 (vinKO) or 10 (+vinFL) cells; *** indicates P < 0.001 (t test). (E) Representative images of vinKO MEFs expressing GFP-tensin3-FL with or without mCh-vinFL. Alexa Fluor 647-labeled fibronectin (647-FN) was added to the culture medium for 2 h before fixation. Quantification of the mean distance of 647-FN fibrils from the cell periphery shows that cells without vinculin have significantly fewer centrally located FN fibrils compared to cells expressing mCh-vinFL. Error bars are SEM; n = 1,318 (vinKO) or 1,875 (+vinFL) fibres from 24 (vinKO) or 22 (+vinFL) cells (see Fig. S5 I for accompanying histograms); **** indicates P < 0.0001 (t test). Data are representative of two independent experiments. Scale bars in A–D indicate 10 µm.

Fibronectin fibrillogenesis is dependent on the tensin3–talin interaction. (A and B) Representative (background subtracted) images of fibronectin fibrils (using an antibody against cell-derived fibronectin (Serini et al., 1998) produced by (A) WT or TNS3 KO U2OS cells or (B) TNS3 KO U2OS cells expressing GFP-tensin3-FL or GFP-tensin3-FL∆TBS, spread overnight on fibronectin-coated glass. Fibril formation was quantified in 70 × 70 µm squares applied to each image and is normalized to U2OS WT (A) or U2OS TNS3 KO expressing GFP-tensin3-FL (B) to calculate the fold-change in the area covered by fibronectin fibrils. Quantification shows 50% reduction in fibronectin fibrils produced by TNS3 KO (A) and GFP-tensin3-FL∆TBS (B) cells. Error bars are SEM, n = 57 (WT); 60 (KO); 76 (GFP-tensin3-FL); 69 (GFP-tensin3-FL∆TBS) squares from 30 to 35 images, pooled from three independent experiments; **** indiciates P < 0.0001 (Mann-Whitney t test). (C) Representative images (background subtracted) of fibronectin fibrils produced by TNS3 KO U2OS cells expressing GFP-tensin3-FL or GFP-tensin3-FL∆TBS, spread overnight on fibronectin-coated soft (5 kPa) or stiff (50 kPa) polyacrylamide gels (quantified as Fig. 7, A and B) normalized to GFP-tensin3-FL on 5 kPa polyacrylamide gels. Note the reduced fibronectin fibrils produced by GFP-tensin3-FL∆TBS expressing cells on both substrates. Error bars are SEM, n = 75 (GFP-tensin3-FL, 5 kPa); 48 (GFP-tensin3FL, 50 kPa); 58 (GFP-tensin3-FL∆TBS, 5 kPa); 41 (GFP-tensin3-FL∆TBS, 50 kPa) squares from 30 to 35 images, pooled from three independent experiments; **** indicates P < 0.0001 (Kruskal-Wallis ANOVA with Dunn’s multiple comparison test). (D) Live-cell imaging of vinKO MEFs expressing GFP-tensin3-FL with or without mCh-vinFL co-expression; temporal color maps of adhesion movement obtained from the GFP signal show that tensin3-positive adhesions in cells without vinculin are more dynamic. Images were acquired every 5 min for 2 h. Quantification of mean adhesion speed and lifetime from time-lapse movies of vinKO MEFs expressing GFP-tensin3-FL with or without mCh-vinFL co-expression. Error bars are SEM, n numbers are pooled from 6 (vinKO) or 10 (+vinFL) cells; *** indicates P < 0.001 (t test). (E) Representative images of vinKO MEFs expressing GFP-tensin3-FL with or without mCh-vinFL. Alexa Fluor 647-labeled fibronectin (647-FN) was added to the culture medium for 2 h before fixation. Quantification of the mean distance of 647-FN fibrils from the cell periphery shows that cells without vinculin have significantly fewer centrally located FN fibrils compared to cells expressing mCh-vinFL. Error bars are SEM; n = 1,318 (vinKO) or 1,875 (+vinFL) fibres from 24 (vinKO) or 22 (+vinFL) cells (see Fig. S5 I for accompanying histograms); **** indicates P < 0.0001 (t test). Data are representative of two independent experiments. Scale bars in A–D indicate 10 µm.

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