![The tensin3 talin binding site regulates α5 integrin localization. (A) Representative images of wild-type (WT) or CRISPR-mediated tensin3 knock-down (TNS3 KO) U2OS cells spread overnight on fibronectin-coated glass before fixation and immunostaining for α5 integrin. Quantification shows that tensin3 KO cells (clone #2) have reduced α5 integrin-positive adhesions; error bars are SEM, n = 15 (WT) and 19 (KO) cells; *** indicates P < 0.001 (t test). (B) Histograms and accompanying gaussian curve fit for the normalized distance (percent of maximum) of each a5-integrin positive adhesion structure from the cell edge to the cell center; bar graphs show the mean and SEM of the normalized distance of all the adhesions; n = 836 (WT) and 662 (KO) adhesions from 15 to 19 cells, respectively; **** indicates P < 0.0001 (Mann-Whitney t test). Note that TNS3 KO cells have a significantly reduced number of centrally located adhesions. (C and D) Representative images of TNS3 KO cells expressing the indicated GFP-tagged tensin3 construct fixed and stained for α5 integrin. Number of adhesions (n = 13 [GFP-tensin3-FL] and 15 [GFP-tensin3-FL∆TBS] cells) and the distance from the cell periphery was quantified as above, revealing that expression of the GFP-tensin3-FL∆TBS construct is unable to rescue the formation of centrally located α5 integrin-positive adhesion structures. Note that while rescue with GFP-tensin3-FL resembles the WT U2OS phenotype, rescue with GFP-tensin3-FL∆TBS resembles the TNS3 KO phenotype. Error bars are SEM, n = 1,836 (GFP-tensin3-FL), 953 (GFP-tensin3-FL∆TBS) adhesions from 13 to 15 cells, respectively; *** indicates P < 0.001 (Mann-Whitney t test). (E) Representative TIRF images of talin1/2 KO cells co-expressing GFP-talin together with either mCh-tensin3-FL or mCh-tensin3-FL∆TBS. Quantification of the number of GFP-talin positive adhesions reveals fewer adhesion structures in cells expressing mCh-tensin3-FL∆TBS; n = 30 (mCh-tensin3-FL) and 29 (mCh-tensin3-FL∆TBS) cells; *** indicates P = 0.0002 (t test). (F) Histograms and accompanying gaussian curve fit for the normalized distance (percent of maximum) of each GFP-talin positive adhesion structure from the cell edge to the cell center; bar graphs show the mean and SEM of the normalized distance of all adhesions; n = 1,650 (WT) and 991 (KO) adhesions from 30 to 29 cells, respectively; **** indicates P < 0.0001 (Mann-Whitney t test). All results shown in A–F are representative of three independent repeats; scale bars indicate 10 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/221/10/10.1083_jcb.202107022/2/jcb_202107022_fig6.png?Expires=1771108062&Signature=g4B2ZsLvmJIgNoeuD2nkQ9fqAhlo4wovQ8M-3YEYXei7m2YKCsqFPKKnRxgASPt283yo~GEwHaRvbiXiVlEeRC3ZtYVdyV2zGfnWvbgPqSYDzsIfw0XfuVpM6XdhxceW~LBNW2Sq5FPyWR2Bvj4gqXI-Z-rrjrW0Z6FDOaahnBO0qFrGHlKjuCdjLSVus-uN6XlD-ftDNAv9~WNlGNvZ3XcE72BJ5FnQgo-ZIe3km8iH96qynjEUodMgh87zHBbuqSaFMIIxcU04AkUk3GReuv~QbPWD8yWXi~ZE-oB2wJUl3CAUK1f1Ax-uEpa~Xu0r7fdrA7vUvReXKbfZNqhn8g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The tensin3 talin binding site regulates α5 integrin locali z ation. (A) Representative images of wild-type (WT) or CRISPR-mediated tensin3 knock-down (TNS3 KO) U2OS cells spread overnight on fibronectin-coated glass before fixation and immunostaining for α5 integrin. Quantification shows that tensin3 KO cells (clone #2) have reduced α5 integrin-positive adhesions; error bars are SEM, n = 15 (WT) and 19 (KO) cells; *** indicates P < 0.001 (t test). (B) Histograms and accompanying gaussian curve fit for the normalized distance (percent of maximum) of each a5-integrin positive adhesion structure from the cell edge to the cell center; bar graphs show the mean and SEM of the normalized distance of all the adhesions; n = 836 (WT) and 662 (KO) adhesions from 15 to 19 cells, respectively; **** indicates P < 0.0001 (Mann-Whitney t test). Note that TNS3 KO cells have a significantly reduced number of centrally located adhesions. (C and D) Representative images of TNS3 KO cells expressing the indicated GFP-tagged tensin3 construct fixed and stained for α5 integrin. Number of adhesions (n = 13 [GFP-tensin3-FL] and 15 [GFP-tensin3-FL∆TBS] cells) and the distance from the cell periphery was quantified as above, revealing that expression of the GFP-tensin3-FL∆TBS construct is unable to rescue the formation of centrally located α5 integrin-positive adhesion structures. Note that while rescue with GFP-tensin3-FL resembles the WT U2OS phenotype, rescue with GFP-tensin3-FL∆TBS resembles the TNS3 KO phenotype. Error bars are SEM, n = 1,836 (GFP-tensin3-FL), 953 (GFP-tensin3-FL∆TBS) adhesions from 13 to 15 cells, respectively; *** indicates P < 0.001 (Mann-Whitney t test). (E) Representative TIRF images of talin1/2 KO cells co-expressing GFP-talin together with either mCh-tensin3-FL or mCh-tensin3-FL∆TBS. Quantification of the number of GFP-talin positive adhesions reveals fewer adhesion structures in cells expressing mCh-tensin3-FL∆TBS; n = 30 (mCh-tensin3-FL) and 29 (mCh-tensin3-FL∆TBS) cells; *** indicates P = 0.0002 (t test). (F) Histograms and accompanying gaussian curve fit for the normalized distance (percent of maximum) of each GFP-talin positive adhesion structure from the cell edge to the cell center; bar graphs show the mean and SEM of the normalized distance of all adhesions; n = 1,650 (WT) and 991 (KO) adhesions from 30 to 29 cells, respectively; **** indicates P < 0.0001 (Mann-Whitney t test). All results shown in A–F are representative of three independent repeats; scale bars indicate 10 µm.
