Tensin3 undergoes LLPS in cells in a talin-regulated manner. (A) Schematic of human tensin3 with predicted disorder degree by IUPred (Dosztányi, 2018). Residues with a predicted disorder tendency higher than 0.5 are considered disordered. (B) Schematic of the tensin3 IDR deletion (ΔIDR, deletion of aa 351–1,152) construct used. Note that this construct is N-terminally labeled with mCh. (C) Representative images of NIH3T3 cells expressing mCh-TNS3-WT or mCh-TNS3-ΔIDR. The dashed boxes are zoomed in on the right. Note that deletion of the IDR abolished the formation of tensin3 condensates. (D i and ii) Image (i) of NIH3T3 cells co-expressing GFP-LIMD1 (green) and mCh-TNS3 (magenta). The dashed box is zoomed in on the right (ii), with a line profile below for the yellow arrow line. (E) Correlation between the relative protein level of mCh-TNS3 and the cellular coverage (%) of TNS3 condensates in individual NIH3T3 cells, represented by a linear regression with 95% confidence intervals. n = 181 cells; R2 = 0.6870; r = 0.8773 (nonparametric Spearman’s correlation). (F) Time-lapse images of a small GFP-TNS3 condensate (indicated by white arrows) derived from adhesion sites in NIH3T3 cells, related to Video 1. (G) Time-lapse images of GFP-TNS3 condensates (highlighted by dashed white circles) fusing in NIH3T3 cells, related to Video 2. (H) Images of NIH3T3 cells expressing mCh-TNS3-WT or mCh-TNS3-L702E. (I) Quantification of H pooled from triplicate experiments. Note that condensates that are larger than 0.1 μm2 with a circularity between 0.7 and 1 were quantified. Left panel: The percentage of transfected cells with TNS3 condensate formation in each area (610 × 499 μm). The boxes represent the 25–75th percentiles with the median indicated; the whiskers indicate the range of values; n = 56 (WT) and 41 (L702E) areas. Middle panel: The mean number of TNS3 condensates in cells. Error bars are the SEM. Right panel: The mean radius of TNS3 condensates in each cell. n = 68 (WT) and 100 (L702E) cells. (J) Time-lapse images of FRAP experiment in NIH3T3 cells expressing GFP-TNS3-WT or GFP-TNS3-L702E. (K) Quantification of recovery halftime (left and middle panels) and mobile fraction (right panel) in J pooled from triplicate experiments. n = 27 (WT, 15 cells) and 26 (L702E, 17 cells) adhesions. * indicates P < 0.05, and **** indicates P < 0.0001 (I right and left panel: Unpaired t test; I middle panel and K: Welch’s t test). Scale bar: 10 µm (C, D, and H); 5 μm (F and G); 3 μm (J).
Figure 6.

Tensin3 undergoes LLPS in cells in a talin-regulated manner. (A) Schematic of human tensin3 with predicted disorder degree by IUPred (Dosztányi, 2018). Residues with a predicted disorder tendency higher than 0.5 are considered disordered. (B) Schematic of the tensin3 IDR deletion (ΔIDR, deletion of aa 351–1,152) construct used. Note that this construct is N-terminally labeled with mCh. (C) Representative images of NIH3T3 cells expressing mCh-TNS3-WT or mCh-TNS3-ΔIDR. The dashed boxes are zoomed in on the right. Note that deletion of the IDR abolished the formation of tensin3 condensates. (D i and ii) Image (i) of NIH3T3 cells co-expressing GFP-LIMD1 (green) and mCh-TNS3 (magenta). The dashed box is zoomed in on the right (ii), with a line profile below for the yellow arrow line. (E) Correlation between the relative protein level of mCh-TNS3 and the cellular coverage (%) of TNS3 condensates in individual NIH3T3 cells, represented by a linear regression with 95% confidence intervals. n = 181 cells; R2 = 0.6870; r = 0.8773 (nonparametric Spearman’s correlation). (F) Time-lapse images of a small GFP-TNS3 condensate (indicated by white arrows) derived from adhesion sites in NIH3T3 cells, related to Video 1. (G) Time-lapse images of GFP-TNS3 condensates (highlighted by dashed white circles) fusing in NIH3T3 cells, related to Video 2. (H) Images of NIH3T3 cells expressing mCh-TNS3-WT or mCh-TNS3-L702E. (I) Quantification of H pooled from triplicate experiments. Note that condensates that are larger than 0.1 μm2 with a circularity between 0.7 and 1 were quantified. Left panel: The percentage of transfected cells with TNS3 condensate formation in each area (610 × 499 μm). The boxes represent the 25–75th percentiles with the median indicated; the whiskers indicate the range of values; n = 56 (WT) and 41 (L702E) areas. Middle panel: The mean number of TNS3 condensates in cells. Error bars are the SEM. Right panel: The mean radius of TNS3 condensates in each cell. n = 68 (WT) and 100 (L702E) cells. (J) Time-lapse images of FRAP experiment in NIH3T3 cells expressing GFP-TNS3-WT or GFP-TNS3-L702E. (K) Quantification of recovery halftime (left and middle panels) and mobile fraction (right panel) in J pooled from triplicate experiments. n = 27 (WT, 15 cells) and 26 (L702E, 17 cells) adhesions. * indicates P < 0.05, and **** indicates P < 0.0001 (I right and left panel: Unpaired t test; I middle panel and K: Welch’s t test). Scale bar: 10 µm (C, D, and H); 5 μm (F and G); 3 μm (J).

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