Tensin3–talin interaction controls FB formation and FN fibrillogenesis. (A) Representative images of U2OS TNS3KO cells expressing mCh-TNS3-WT or -L702E spread overnight on FN-coated glass-bottom dishes, before being stained for α5 integrins (SNAKA51). (B) Left panel: Quantification of the mean number of α5 integrin-positive adhesions in A; n = 37 (WT) and 47 (L702E) cells. Right panel: Histograms and associated Gaussian curve fits for the frequency of the normalized distance (percentage of maximum) to the cell edge of each α5 integrin–positive adhesion; n = 3,335 (WT, 37 cells) and 1,860 (L702E, 47 cells) adhesions, respectively. (C) Images (background-subtracted) of cell-derived FN fibrils (labeled with IST9 antibody) produced by TNS3KO cells expressing GFP-TNS3-WT or GFP-TNS3-L702E, spread overnight on FN-coated glass. (D) Left panel: Quantification of FN fibrils in 40 × 40 μm square areas applied to each image; n = 56 (WT) and 54 (L702E) cells. Right panel: Fold change in FN-covered area (normalized to WT). (E) Images of U2OS TNS3KO cells expressing mCh-TNS3-WT or mCh-TNS3-L702E spread overnight in serum-free medium on VN-coated glass-bottom dishes, before being fixed and stained for the FA marker vinculin. (F) Quantification of the mean number of vinculin-positive adhesions in E; n = 74 (WT) and 51 (L702E) cells. (G) Images of TLNKO cells co-expressing GFP-TLN1 constructs (WT, R8m, R11m, or R8m+R11m) with mCh-TNS3-WT. Cells co-expressing GFP-TLN1-WT and mCh-TNS3-L702E (in the red box) were used as a negative control as the complete disruption of the talin–tensin3 interaction. (H) Left panel: Quantification of TNS3-positive adhesions in G. n = 29 (WT), 27 (R8m), 29 (R11m), 37 (R8m+R11m), and 35 (L702E) cells. Middle and right panels: Gaussian curve fits and means for the normalized distance (percentage of maximum) to the cell edge of each TNS3-positive adhesion in G; n = 3,528 (WT), 1,720 (R8m), 1,433 (R11m), 1,030 (R8m+R11m), and 790 (L702E) adhesions from 29, 27, 29, 37, and 35 cells, respectively. ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001 (B and D: Mann–Whitney test; F: unpaired t test; H left panel: Ordinary one-way ANOVA test with Tukey’s multiple comparisons test; H right panel: Kruskal–Wallis test with Dunn’s multiple comparisons test). All results are collected from three independent experiments. Scale bars: 10 μm (A and E), 20 μm (G), and 5 μm (C). Error bars are the SEM.
Figure 4.

Tensin3–talin interaction controls FB formation and FN fibrillogenesis. (A) Representative images of U2OS TNS3KO cells expressing mCh-TNS3-WT or -L702E spread overnight on FN-coated glass-bottom dishes, before being stained for α5 integrins (SNAKA51). (B) Left panel: Quantification of the mean number of α5 integrin-positive adhesions in A; n = 37 (WT) and 47 (L702E) cells. Right panel: Histograms and associated Gaussian curve fits for the frequency of the normalized distance (percentage of maximum) to the cell edge of each α5 integrin–positive adhesion; n = 3,335 (WT, 37 cells) and 1,860 (L702E, 47 cells) adhesions, respectively. (C) Images (background-subtracted) of cell-derived FN fibrils (labeled with IST9 antibody) produced by TNS3KO cells expressing GFP-TNS3-WT or GFP-TNS3-L702E, spread overnight on FN-coated glass. (D) Left panel: Quantification of FN fibrils in 40 × 40 μm square areas applied to each image; n = 56 (WT) and 54 (L702E) cells. Right panel: Fold change in FN-covered area (normalized to WT). (E) Images of U2OS TNS3KO cells expressing mCh-TNS3-WT or mCh-TNS3-L702E spread overnight in serum-free medium on VN-coated glass-bottom dishes, before being fixed and stained for the FA marker vinculin. (F) Quantification of the mean number of vinculin-positive adhesions in E; n = 74 (WT) and 51 (L702E) cells. (G) Images of TLNKO cells co-expressing GFP-TLN1 constructs (WT, R8m, R11m, or R8m+R11m) with mCh-TNS3-WT. Cells co-expressing GFP-TLN1-WT and mCh-TNS3-L702E (in the red box) were used as a negative control as the complete disruption of the talin–tensin3 interaction. (H) Left panel: Quantification of TNS3-positive adhesions in G. n = 29 (WT), 27 (R8m), 29 (R11m), 37 (R8m+R11m), and 35 (L702E) cells. Middle and right panels: Gaussian curve fits and means for the normalized distance (percentage of maximum) to the cell edge of each TNS3-positive adhesion in G; n = 3,528 (WT), 1,720 (R8m), 1,433 (R11m), 1,030 (R8m+R11m), and 790 (L702E) adhesions from 29, 27, 29, 37, and 35 cells, respectively. ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001 (B and D: Mann–Whitney test; F: unpaired t test; H left panel: Ordinary one-way ANOVA test with Tukey’s multiple comparisons test; H right panel: Kruskal–Wallis test with Dunn’s multiple comparisons test). All results are collected from three independent experiments. Scale bars: 10 μm (A and E), 20 μm (G), and 5 μm (C). Error bars are the SEM.

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