Tensin3 LLPS is controlled by talin activity in response to rigidity sensing. (A) Confocal images of NIH3T3 cells expressing mCh-TNS3-WT or mCh-TNS3-L702E plated overnight on FN-coated 1.5 or 28 kPa PDMS dishes. (B) Quantification of the mean TNS3 condensate number in A. n = 83 (WT/1.5 kPa), 80 (WT/28 kPa), 83 (L702E/1.5 kPa), and 89 (L702E/28 kPa) cells. Note that condensates that are larger than 0.1 μm2 with a circularity between 0.7 and 1 were quantified. (C) Images of endogenous tensin3 or GFP-TNS3 (green) with endogenous paxillin (magenta) in HFF cells (top panel) and NIH3T3 cells (bottom panel) plated on FN-coated glass, respectively. The dashed boxes are zoomed to the right, with condensates indicated by yellow arrows. (D) Quantification of the circularity of overexpressed GFP-TNS3 condensates in NIH3T3 cells, endogenous tensin3 condensates in HFF cells, and tensin3 adhesions in HFF cells. The boxes represent the 25–75th percentiles with the median indicated; the whiskers indicate the range of values. n = 717 (overexpressed condensates, 17 cells), 192 (endogenous condensates, 29 cells), and 4,883 (tensin3 adhesions, 33 cells). (E) Representative images of HFF cells plated on FN-coated glass (left panel). The dashed boxes are zoomed to the right panel, with tensin3 condensates (green) indicated by white arrows and a line profile below for the yellow arrow line. The lipid membrane is labeled with WGA (blue); the active β1 integrin (magenta) is stained using 9EG7 antibody. Note that the tensin3 condensate is not colocalized with vesicle structures indicated by WGA and active β1 integrin. (F) Images (background-subtracted) of HFF cells plated overnight on FN-coated 1.5 or 28 kPa PDMS dishes. Endogenous tensin3 (green) and paxillin (magenta) were visualized by staining. The dashed boxes are zoomed in below, with condensates indicated by yellow arrows. (G) Quantification of the mean number of endogenous tensin3 condensates in F. n = 33 (1.5 kPa) and 40 (28 kPa) cells. (H) Mitochondrial pulldown experiments using HEK293T cells expressing GFP-TLN1-cBAK (WT or E1770A) and mCh-IDR. (I) Quantification of H pooled from three replicates. Data are normalized to WT. Error bars are the SEM. All data are collected from three independent experiments. * indicates P < 0.05, and **** indicates P <0.0001 (B and D: Kruskal–Wallis test with Dunn’s multiple comparisons; G: Mann–Whitney test; I: unpaired t test). Scale bar: 10 µm (A, C, and F) and 5 µm (E). Source data are available for this figure: SourceData F7.
Figure 7.

Tensin3 LLPS is controlled by talin activity in response to rigidity sensing. (A) Confocal images of NIH3T3 cells expressing mCh-TNS3-WT or mCh-TNS3-L702E plated overnight on FN-coated 1.5 or 28 kPa PDMS dishes. (B) Quantification of the mean TNS3 condensate number in A. n = 83 (WT/1.5 kPa), 80 (WT/28 kPa), 83 (L702E/1.5 kPa), and 89 (L702E/28 kPa) cells. Note that condensates that are larger than 0.1 μm2 with a circularity between 0.7 and 1 were quantified. (C) Images of endogenous tensin3 or GFP-TNS3 (green) with endogenous paxillin (magenta) in HFF cells (top panel) and NIH3T3 cells (bottom panel) plated on FN-coated glass, respectively. The dashed boxes are zoomed to the right, with condensates indicated by yellow arrows. (D) Quantification of the circularity of overexpressed GFP-TNS3 condensates in NIH3T3 cells, endogenous tensin3 condensates in HFF cells, and tensin3 adhesions in HFF cells. The boxes represent the 25–75th percentiles with the median indicated; the whiskers indicate the range of values. n = 717 (overexpressed condensates, 17 cells), 192 (endogenous condensates, 29 cells), and 4,883 (tensin3 adhesions, 33 cells). (E) Representative images of HFF cells plated on FN-coated glass (left panel). The dashed boxes are zoomed to the right panel, with tensin3 condensates (green) indicated by white arrows and a line profile below for the yellow arrow line. The lipid membrane is labeled with WGA (blue); the active β1 integrin (magenta) is stained using 9EG7 antibody. Note that the tensin3 condensate is not colocalized with vesicle structures indicated by WGA and active β1 integrin. (F) Images (background-subtracted) of HFF cells plated overnight on FN-coated 1.5 or 28 kPa PDMS dishes. Endogenous tensin3 (green) and paxillin (magenta) were visualized by staining. The dashed boxes are zoomed in below, with condensates indicated by yellow arrows. (G) Quantification of the mean number of endogenous tensin3 condensates in F. n = 33 (1.5 kPa) and 40 (28 kPa) cells. (H) Mitochondrial pulldown experiments using HEK293T cells expressing GFP-TLN1-cBAK (WT or E1770A) and mCh-IDR. (I) Quantification of H pooled from three replicates. Data are normalized to WT. Error bars are the SEM. All data are collected from three independent experiments. * indicates P < 0.05, and **** indicates P <0.0001 (B and D: Kruskal–Wallis test with Dunn’s multiple comparisons; G: Mann–Whitney test; I: unpaired t test). Scale bar: 10 µm (A, C, and F) and 5 µm (E). Source data are available for this figure: SourceData F7.

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