Talin is a retention signal for tensin3 that controls mechanosensitive tensin3 condensation and the client protein recruitment. (A) Representative images of NIH3T3 cells expressing GFP-TNS3 at a lower (0 μm) and a higher (0.6 μm) focal plane, with dashed boxes zoomed to the right. Note that TNS3 spheres are observed in proximity to the adhesion plane and in the cytoplasm. (B) Representative images of NIH3T3 cells expressing GFP-TNS3 (green) with immunostaining of endosomal marker EEA1, lysosomal marker LAMP1, or active β1 integrins (magenta). (C) Images of an NIH3T3 cell forming GFP-TNS3 condensates, labeled with fluorescently conjugated WGA (shown in blue). (D) Correlation between the relative protein level of mCh-TNS3-WT or mCh-TNS3-L702E and the cellular coverage (%) of TNS3 condensates in NIH3T3 cells, represented by linear regressions with 95% confidence intervals. n = 181 cells (WT) and 168 cells (L702E); nonparametric Spearman’s correlation r = 0.8773 (WT) and 0.9013 (L702E). (E) Representative background-subtracted images of a HFF cell expressing GFP-LIMD1 with staining for endogenous tensin3. The dashed box is zoomed in on the right, with a yellow arrow above two tensin3 condensates. (F) Line profile for the yellow arrow in E. (G) Background-subtracted images of HFF cells plated overnight on FN-coated 5 or 50 kPa PAA hydrogels. Endogenous tensin3, green; paxillin, magenta. (H) Quantification of the mean condensate number in G. n = 40 (5 kPa) and 41 (50 kPa) cells. (I) Representative images of NIH3T3 cells expressing GFP-TNS3-WT in green with exogenously co-expressed proteins in magenta. The black box indicates recruitment to the TNS3 condensates, and the gray box indicates no recruitment. (J and K) Images of NIH3T3 cells forming the TNS3 condensates in green with immunofluorescence staining for stress granule protein G3BP1 and TDP43 (J), and for tyrosine-phosphorylated proteins (K) with an antibody that probes phosphotyrosine (clone 4G10) in magenta. (L) Images of NIH3T3 cells forming the TNS3-L702E condensates in green with exogenously co-expressed proteins in magenta. Note that the summary table for I–L is shown in Fig. 8 C. **** indicates P < 0.0001 (D: ANCOVA; H: Mann–Whitney test). Scale bars are 10 µm (A–C, G, and I–L) or 5 µm (E). ANCOVA, analysis of covariance.
Figure S4.

Talin is a retention signal for tensin3 that controls mechanosensitive tensin3 condensation and the client protein recruitment. (A) Representative images of NIH3T3 cells expressing GFP-TNS3 at a lower (0 μm) and a higher (0.6 μm) focal plane, with dashed boxes zoomed to the right. Note that TNS3 spheres are observed in proximity to the adhesion plane and in the cytoplasm. (B) Representative images of NIH3T3 cells expressing GFP-TNS3 (green) with immunostaining of endosomal marker EEA1, lysosomal marker LAMP1, or active β1 integrins (magenta). (C) Images of an NIH3T3 cell forming GFP-TNS3 condensates, labeled with fluorescently conjugated WGA (shown in blue). (D) Correlation between the relative protein level of mCh-TNS3-WT or mCh-TNS3-L702E and the cellular coverage (%) of TNS3 condensates in NIH3T3 cells, represented by linear regressions with 95% confidence intervals. n = 181 cells (WT) and 168 cells (L702E); nonparametric Spearman’s correlation r = 0.8773 (WT) and 0.9013 (L702E). (E) Representative background-subtracted images of a HFF cell expressing GFP-LIMD1 with staining for endogenous tensin3. The dashed box is zoomed in on the right, with a yellow arrow above two tensin3 condensates. (F) Line profile for the yellow arrow in E. (G) Background-subtracted images of HFF cells plated overnight on FN-coated 5 or 50 kPa PAA hydrogels. Endogenous tensin3, green; paxillin, magenta. (H) Quantification of the mean condensate number in G. n = 40 (5 kPa) and 41 (50 kPa) cells. (I) Representative images of NIH3T3 cells expressing GFP-TNS3-WT in green with exogenously co-expressed proteins in magenta. The black box indicates recruitment to the TNS3 condensates, and the gray box indicates no recruitment. (J and K) Images of NIH3T3 cells forming the TNS3 condensates in green with immunofluorescence staining for stress granule protein G3BP1 and TDP43 (J), and for tyrosine-phosphorylated proteins (K) with an antibody that probes phosphotyrosine (clone 4G10) in magenta. (L) Images of NIH3T3 cells forming the TNS3-L702E condensates in green with exogenously co-expressed proteins in magenta. Note that the summary table for I–L is shown in Fig. 8 C. **** indicates P < 0.0001 (D: ANCOVA; H: Mann–Whitney test). Scale bars are 10 µm (A–C, G, and I–L) or 5 µm (E). ANCOVA, analysis of covariance.

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