Figure S2.
Vinculin is enriched at cadherin-based adhesions of mitotic cells in MDCK monolayers and intestinal organoids, dependent on E-cadherin force transmission. (A) Immunostaining of MDCK cells expressing GFP-vinculin for endogenous E-cadherin and paxillin, together with DAPI. While GFP-vinculin is able to localize to both adherens junctions (E-cadherin) and focal adhesions (paxillin), the observed mitotic enrichment is specific to adherens junctions. (B) WB of lysates from parental and vinculin knockout (KO) MDCK cells probed for vinculin and α-tubulin. (C) Representative image (z-projection) of MDCK vinculin KO cells expressing GFP-vinculin (Video 4), and quantification of the ratio of junctional versus cytosolic GFP-vinculin in interphase and following entry in mitosis (n = 26). ****, P < 0.0001; Wilcoxon matched-pairs signed-rank test. (D) Representative image (z-projection) of MDCK cells expressing GFP (Video 5), and quantification of the ratio of junctional versus cytosolic GFP in interphase and following entry in mitosis (n = 18). Data were pooled from three independent experiments. n.s., not significant (P = 0.09); Wilcoxon matched-pairs signed-rank test. (E) Representative image (z-projection) of MDCK cells expressing GFP–α-catenin (Video 1), and quantification of the ratio of junctional versus cytosolic GFP–α-catenin in interphase and following entry in mitosis (n = 30). Data were pooled from three independent experiments. n.s., not significant (P = 0.07); Wilcoxon matched-pairs signed-rank test. (F) Representative example of immunostaining for endogenous vinculin and E-cadherin in murine small intestinal organoids, together with DAPI. The green arrowhead indicates the cell–cell junction of a mitotic cell; gray arrowheads indicate cell–cell junctions of neighboring cells in interphase. Note that while vinculin accumulates at the lateral junction of mitotic cells, it is present at the apical junction of both mitotic and interphase cells, as well as in integrin adhesions in interphase cells. Scale bars, 10 µm. (G) Quantification of the ratio of junctional versus cytosolic vinculin in mitotic and interphase cells in murine small intestinal organoids (n = 19). Gray bars show the mean ratio with SD. Data were pooled from two independent experiments. ****, P < 0.0001; paired t test. (H) Quantification of the ratio of the intensity of junctional vinculin relative to the intensity of a junctional marker (α-catenin or β-catenin) in mitotic and interphase cells in murine small intestinal organoids (n = 19). Gray bars show the mean ratio with SD. Data were pooled from two independent experiments. ***, P = 0.0002; paired t test. (I) Immunostaining of MDCK cells, with and without induction of E-cadherinT151 expression, for endogenous E-cadherin and β-catenin, together with DAPI. E-cadherinT151 contains a truncated extracellular domain and can therefore not transmit forces between cells, but maintains an intact cytosolic tail that binds catenin proteins and the actin cytoskeleton (Troxell et al., 2000). The expression of E-cadherinT151 leads to the down-regulation of endogenous cadherins, but cells retain their monolayer due to the presence of other cell–cell adhesions (Troxell et al., 2000). Note that the antibody for E-cadherin recognizes the extracellular domain of E-cadherin, and therefore does not visualize E-cadherinT151. (J) Representative image (z-projection) of immunostaining of MDCK cells induced for E-cadherinT151 expression or uninduced (E-cadherinWT) for endogenous vinculin, together with DAPI. All scale bars, 10 µm. E-cad, E-cadherin; IB, immunoblot; Pax, paxillin; Vin, vinculin.

Vinculin is enriched at cadherin-based adhesions of mitotic cells in MDCK monolayers and intestinal organoids, dependent on E-cadherin force transmission. (A) Immunostaining of MDCK cells expressing GFP-vinculin for endogenous E-cadherin and paxillin, together with DAPI. While GFP-vinculin is able to localize to both adherens junctions (E-cadherin) and focal adhesions (paxillin), the observed mitotic enrichment is specific to adherens junctions. (B) WB of lysates from parental and vinculin knockout (KO) MDCK cells probed for vinculin and α-tubulin. (C) Representative image (z-projection) of MDCK vinculin KO cells expressing GFP-vinculin (Video 4), and quantification of the ratio of junctional versus cytosolic GFP-vinculin in interphase and following entry in mitosis (n = 26). ****, P < 0.0001; Wilcoxon matched-pairs signed-rank test. (D) Representative image (z-projection) of MDCK cells expressing GFP (Video 5), and quantification of the ratio of junctional versus cytosolic GFP in interphase and following entry in mitosis (n = 18). Data were pooled from three independent experiments. n.s., not significant (P = 0.09); Wilcoxon matched-pairs signed-rank test. (E) Representative image (z-projection) of MDCK cells expressing GFP–α-catenin (Video 1), and quantification of the ratio of junctional versus cytosolic GFP–α-catenin in interphase and following entry in mitosis (n = 30). Data were pooled from three independent experiments. n.s., not significant (P = 0.07); Wilcoxon matched-pairs signed-rank test. (F) Representative example of immunostaining for endogenous vinculin and E-cadherin in murine small intestinal organoids, together with DAPI. The green arrowhead indicates the cell–cell junction of a mitotic cell; gray arrowheads indicate cell–cell junctions of neighboring cells in interphase. Note that while vinculin accumulates at the lateral junction of mitotic cells, it is present at the apical junction of both mitotic and interphase cells, as well as in integrin adhesions in interphase cells. Scale bars, 10 µm. (G) Quantification of the ratio of junctional versus cytosolic vinculin in mitotic and interphase cells in murine small intestinal organoids (n = 19). Gray bars show the mean ratio with SD. Data were pooled from two independent experiments. ****, P < 0.0001; paired t test. (H) Quantification of the ratio of the intensity of junctional vinculin relative to the intensity of a junctional marker (α-catenin or β-catenin) in mitotic and interphase cells in murine small intestinal organoids (n = 19). Gray bars show the mean ratio with SD. Data were pooled from two independent experiments. ***, P = 0.0002; paired t test. (I) Immunostaining of MDCK cells, with and without induction of E-cadherinT151 expression, for endogenous E-cadherin and β-catenin, together with DAPI. E-cadherinT151 contains a truncated extracellular domain and can therefore not transmit forces between cells, but maintains an intact cytosolic tail that binds catenin proteins and the actin cytoskeleton (Troxell et al., 2000). The expression of E-cadherinT151 leads to the down-regulation of endogenous cadherins, but cells retain their monolayer due to the presence of other cell–cell adhesions (Troxell et al., 2000). Note that the antibody for E-cadherin recognizes the extracellular domain of E-cadherin, and therefore does not visualize E-cadherinT151. (J) Representative image (z-projection) of immunostaining of MDCK cells induced for E-cadherinT151 expression or uninduced (E-cadherinWT) for endogenous vinculin, together with DAPI. All scale bars, 10 µm. E-cad, E-cadherin; IB, immunoblot; Pax, paxillin; Vin, vinculin.

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