Tensin3 regulates integrin activity through its interaction with talin and integrin. (A) Representative images of TLNKO cells co-expressing GFP-TLN1 with mCh-vector or mCh-TNS3, respectively. Active β1 integrins were stained with 9EG7 antibody. (B) Quantification of the fold change in 9EG7-positive adhesion counts in A. The fold change was calculated by normalizing the values for cells expressing mCh-TNS3 to the mean value for cells expressing mCh-vector. (C) Representative integrin activation (β1, 9EG7 antibody) profiles of TLNKO cells co-expressing GFP-TLN1 with mCh-vector (light gray) or mCh-TNS3 (dark gray) as measured by flow cytometry. (D) Integrin (β1) activation index (normalized to cells expressing GFP-TLN1 and mCh-vector) calculated from triplicate experiments of C. (E) Schematic of the tensin3 deletion and mutation constructs used, with the TBS motif (red) and the integrin-binding PTB domain indicated by orange dashed lines. All constructs are N-terminally labeled with GFP or mCherry. (F) Images of U2OS TNS3KO cells expressing the mCh-TNS3 construct shown in E, or a vector control. Cells were plated overnight on FN-coated glass-bottom dishes and stained for active β1 integrin (9EG7). (G) Quantification of β1 integrin–positive adhesions in F. n = 66 (vector), 70 (WT), 58 (L702E), 55 (ΔPTB), and 58 (L702E+ΔPTB) cells. (H) Integrin activation index (normalized to cells expressing GFP-TLN1 and mCh-TNS3-WT) calculated from triplicate flow cytometry experiments of Fig. S3, F and G. (I) Schematic of the tensin3 C-terminal deletion construct used. Note that this construct is N-terminally labeled with GFP. (J) U2OS TNS3KO cells expressing GFP-TNS3 constructs shown in E and I were cultured overnight before treatment with blebbistatin (50 μM) or an equivalent volume of DMSO (shown in Fig. S3 H) for 60 min. (K) Quantification of β1 integrin–positive adhesion in J. n = 53 (WT), 43 (L702E), 66 (ΔPTB), and 53 (Cterm) cells. All data are collected from three independent experiments. Scale bars: 10 μm (A and J); 20 μm (F). Error bars are the SEM; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001 (B, D: unpaired t test; G and K: Kruskal–Wallis test with Dunn’s multiple comparisons test; H: ordinary one-way ANOVA with Turkey’s multiple comparisons).
Figure 5.

Tensin3 regulates integrin activity through its interaction with talin and integrin. (A) Representative images of TLNKO cells co-expressing GFP-TLN1 with mCh-vector or mCh-TNS3, respectively. Active β1 integrins were stained with 9EG7 antibody. (B) Quantification of the fold change in 9EG7-positive adhesion counts in A. The fold change was calculated by normalizing the values for cells expressing mCh-TNS3 to the mean value for cells expressing mCh-vector. (C) Representative integrin activation (β1, 9EG7 antibody) profiles of TLNKO cells co-expressing GFP-TLN1 with mCh-vector (light gray) or mCh-TNS3 (dark gray) as measured by flow cytometry. (D) Integrin (β1) activation index (normalized to cells expressing GFP-TLN1 and mCh-vector) calculated from triplicate experiments of C. (E) Schematic of the tensin3 deletion and mutation constructs used, with the TBS motif (red) and the integrin-binding PTB domain indicated by orange dashed lines. All constructs are N-terminally labeled with GFP or mCherry. (F) Images of U2OS TNS3KO cells expressing the mCh-TNS3 construct shown in E, or a vector control. Cells were plated overnight on FN-coated glass-bottom dishes and stained for active β1 integrin (9EG7). (G) Quantification of β1 integrin–positive adhesions in F. n = 66 (vector), 70 (WT), 58 (L702E), 55 (ΔPTB), and 58 (L702E+ΔPTB) cells. (H) Integrin activation index (normalized to cells expressing GFP-TLN1 and mCh-TNS3-WT) calculated from triplicate flow cytometry experiments of Fig. S3, F and G. (I) Schematic of the tensin3 C-terminal deletion construct used. Note that this construct is N-terminally labeled with GFP. (J) U2OS TNS3KO cells expressing GFP-TNS3 constructs shown in E and I were cultured overnight before treatment with blebbistatin (50 μM) or an equivalent volume of DMSO (shown in Fig. S3 H) for 60 min. (K) Quantification of β1 integrin–positive adhesion in J. n = 53 (WT), 43 (L702E), 66 (ΔPTB), and 53 (Cterm) cells. All data are collected from three independent experiments. Scale bars: 10 μm (A and J); 20 μm (F). Error bars are the SEM; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001 (B, D: unpaired t test; G and K: Kruskal–Wallis test with Dunn’s multiple comparisons test; H: ordinary one-way ANOVA with Turkey’s multiple comparisons).

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