Figure 4. A helical motif in the tensin3... | Rockefeller University Press

Figure 4.

$A helical motif in the tensin3 IDR mediates the association with talin and vinculin. (A) Secondary structure prediction of the tensin3 IDR reveals two stretches of amino acids predicted to form a helix, termed “H1” and “H2.” (B) Co-expression of mCh-vinFL-cBAK together with a tensin3-IDR construct lacking either H1 (GFP-tensin3-IDR∆H1) or H2 (GFP-tensin3-IDR∆H2) in NIH3T3 cells shows that the H1 motif is responsible for the interaction between the tensin3 IDR and vinculin. (C) Immunostaining of NIH3T3 cells expressing mCh-tensin3-∆H1-cBAK shows that the H1 motif is responsible for the recruitment of endogenous vinculin and talin to mitochondria. (D) Tensin3 lacking the H1 motif (GFP-tensin3-FL∆H1) expressed in NIH3T3 cells localization to cell-matrix adhesions. Line profiles show that this construct has a higher cytoplasmic fraction compared to GFP-tensin3-FL. (E) FRAP experiments in NIH3T3 cells show the H1 motif regulates the turnover of tensin3 within cell-matrix adhesion sites; mobile fraction 36.49% ± 1.62 and 56.51% ± 1.34. Error bars are SEM, n = 74 (GFP-tensin3-FL) or 99 (GFP-tensin3-FL∆H1) adhesions from 10 to 11 cells, pooled from two independent experiments; *** indicates P < 0.001 (t test). (F) Representative images of NIH3T3 cells expressing a tensin3 IDR construct lacking the H1 motif (GFP-tensin3-IDR∆H1) or a tensin3 construct lacking both the H1 motif and the C-terminal SH2-PTB domains (GFP-tensin3-∆H1∆Cterm). Note that neither of these constructs shows localization to vinculin-positive cell-matrix adhesion sites. (G) Amino acid sequence alignment of tensin1, tensin2 and tensin3 (using Clustal Omega) shows that there is some homology between the tensin3 H1 region and a corresponding stretch of amino acids in tensin1. No homology is observed in tensin2. Scale bars in B, C, D, and F indicate 10 µm.$

A helical motif in the tensin3 IDR mediates the association with talin and vinculin. (A) Secondary structure prediction of the tensin3 IDR reveals two stretches of amino acids predicted to form a helix, termed “H1” and “H2.” (B) Co-expression of mCh-vinFL-cBAK together with a tensin3-IDR construct lacking either H1 (GFP-tensin3-IDR∆H1) or H2 (GFP-tensin3-IDR∆H2) in NIH3T3 cells shows that the H1 motif is responsible for the interaction between the tensin3 IDR and vinculin. (C) Immunostaining of NIH3T3 cells expressing mCh-tensin3-∆H1-cBAK shows that the H1 motif is responsible for the recruitment of endogenous vinculin and talin to mitochondria. (D) Tensin3 lacking the H1 motif (GFP-tensin3-FL∆H1) expressed in NIH3T3 cells localization to cell-matrix adhesions. Line profiles show that this construct has a higher cytoplasmic fraction compared to GFP-tensin3-FL. (E) FRAP experiments in NIH3T3 cells show the H1 motif regulates the turnover of tensin3 within cell-matrix adhesion sites; mobile fraction 36.49% ± 1.62 and 56.51% ± 1.34. Error bars are SEM, n = 74 (GFP-tensin3-FL) or 99 (GFP-tensin3-FL∆H1) adhesions from 10 to 11 cells, pooled from two independent experiments; *** indicates P < 0.001 (t test). (F) Representative images of NIH3T3 cells expressing a tensin3 IDR construct lacking the H1 motif (GFP-tensin3-IDR∆H1) or a tensin3 construct lacking both the H1 motif and the C-terminal SH2-PTB domains (GFP-tensin3-∆H1∆Cterm). Note that neither of these constructs shows localization to vinculin-positive cell-matrix adhesion sites. (G) Amino acid sequence alignment of tensin1, tensin2 and tensin3 (using Clustal Omega) shows that there is some homology between the tensin3 H1 region and a corresponding stretch of amino acids in tensin1. No homology is observed in tensin2. Scale bars in B, C, D, and F indicate 10 µm.