Fig 2: Position of Dsg1 at the cell–cell interface regulates adherens junction contractility. a Immunofluorescence staining of vinculin and Flag (insets) in undifferentiated NHEKs retrovirally transduced with GFP, Dsg1-FL-Flag, or Dsg1-909-Flag. b Quantification of vinculin intensity at the cell borders of undifferentiated NHEKs expressing GFP, Dsg1-FL-Flag (FL), or Dsg1-909-Flag (909) (three independent experiments, at least 40 borders from each experiment were analyzed per condition; F = 40.497, ***p < 0.001 one-way Anova with Tukey test). c Structured illumination microscopy (SIM) images of vinculin and Flag in undifferentiated NHEKs expressing GFP, Dsg1-FL-Flag, or Dsg1-909-Flag (Flag staining, insets). d Quantification of the total area of vinculin puncta at the cell–cell borders from SIM images (three independent experiments; F = 16.081, ***p < 0.001 one-way Anova with Tukey test). e An E-cadherin FRET tension sensor (E-cad-TSmod) was co-expressed in undifferentiated NHEKs with mCherry, Dsg1-FL-Flag, or Dsg1-909-Flag. Immunofluorescence analysis for Flag, fluorescent images of the YFP channel demonstrating E-cad-TSmod expression and corresponding maps of the FRET index are shown. f FRET index for E-cad-TSmod was measured at cell–cell contacts in undifferentiated NHEKs expressing mCherry, Dsg1-FL-Flag (FL), or Dsg1-909-Flag (909) (five independent experiments; F = 19.358, ***p < 0.001 one-way Anova with Tukey test). g Western blot of undifferentiated NHEKs retrovirally transduced with E-cad-TSmod together with mCherry or Dsg1-FL-Flag and treated with control or Tctex-1 siRNA. GAPDH is a loading control. h Quantification of the FRET index for E-cad-TSmod at cell–cell junctions in undifferentiated NHEKs expressing mCherry or Dsg1-FL with or without silencing of Tctex-1 (four independent experiments; ns: non-significant; ***p < 0.001 unpaired two-tail t test). Scale bars = 10 µm. Error bars represent SEM

Fig 3: Tctex-1 dependent Dsg1 localization at the cell–cell interface promotes extrusion of MDCK cells from a polarized monolayer. a X–Z scanned images of MDCK polarized monolayers expressing GFP, Dsg1-FL-Flag, or Dsg1-909-Flag. Monolayers were stained for Flag (green), F-actin (red), and DAPI (marks nuclei). Scale bar = 20 µm. b Quantification of the amount of MDCK cells in a second layer on top of the monolayers expressing GFP, Dsg1-FL-Flag, or Dsg1-909-Flag (three independent experiments; F = 59.503, ***p < 0.001 one-way Anova with Tukey test). The levels of ectopic Dsg1 proteins expression and endogenous E-cadherin and Tctex-1 are shown on the corresponding western blot. GAPDH is loading control. c Quantification of the amount of MDCK cells remaining in a second layer on top of the monolayers expressing Dsg1-FL-Flag with or without Tctex-1 knockdown (three independent experiments; ***p < 0.001 unpaired two-tail t test). The level of ectopic Dsg1-FL expression and Tctex-1 knockdown is shown on the corresponding western blot. GAPDH is loading control. Error bars represent SEM

Fig 4: Dsg1–Tctex-1 interactions promote perijunctional actin polymerization. Quantification of the cell area of differentiated NHEKs treated with control (siNeg) or Tctex-1 siRNA (a) or undifferentiated NHEKs infected with GFP, Dsg1-FL-Flag, or Dsg1-909-Flag (b) shown in Whisker plots. Box boundaries indicate the range of cell areas measured, middle bars depict the mean of compiled data sets, whiskers represent the maximum and the minimum of the measured areas (collected from three independent experiments and areas of at least 125 cells were measured per condition, ***p < 0.001, unpaired two-tailed t test in a and one-way Anova with Tukey test in b, F = 68.519). c Z-volume reconstructions of the cell–cell borders of undifferentiated NHEKs retrovirally transduced with Dsg1-FL-Flag or Dsg1-909-Flag. NHEKs were stained for Flag (green) and E-cadherin (red) and imaged using structured illumination microscopy (SIM). Rectangles mark zoomed in areas. The lateral distance between E-cadherin and Dsg1 at cell contacts of NHEKs is quantified to the right (three independent experiments; ***p < 0.001, unpaired two-tailed t test). d GFP, Dsg1-FL-Flag, or Dsg1-909-Flag-infected NHEKs were stained for F-actin and Flag (green). e Line scan analyses of cortical F-actin intensities at the cell–cell interface in undifferentiated NHEKs expressing GFP or ectopic Dsg1-Flag constructs (three independent experiments, at least 50 borders were analyzed per condition in each experiment (F = 46.432, **p < 0.01, one-way Anova with Tukey test). f Representative SIM images of F-actin (gray/red) and Flag (green) at the cell–cell interface of undifferentiated NHEKs expressing GFP, Dsg1-FL-Flag, or Dsg1-909-Flag. g Barbed-end labeling (G-actin incorporation) at cell junctions in undifferentiated NHEKs expressing GFP, Dsg1-FL-Flag, or Dsg1-909-Flag (Flag staining shown in top insets, green). Rectangles mark zoomed junctional regions, which are shown to the right, barbed ends (red) and Flag (green). h Quantification of barbed-end labeling in undifferentiated NHEKs expressing GFP or ectopic Dsg1 constructs (at least 40 borders per condition were analyzed from each experiment, three independent experiments; ***p < 0.001, one-way Anova with Tukey test). Scale bars = 10 µm. Error bars represent SEM

Fig 5: Tctex-1 interacts with Dsg1 in keratinocytes. a Yeast 2-hybrid (Y2H) analysis of interactions between Sos-tagged Dsg1 and Myr-Tctex-1 protein. Growth on galactose (GAL) at 37 °C indicates an interaction. Incubation on glucose (GLU) at 25 °C represents a permissive growth condition, while incubation at 37 °C on GLU is a control for temperature reversion. IA: intracellular anchor domain, ICS: intracellular cadherin-like sequence domain, PL: intracellular proline-rich linker domain, RUD: repeat unit domain, TD: desmoglein terminal domain. b Immunofluorescence analysis of Tctex-1, Dsg1, and E-cadherin localization in differentiating NHEK cells. Scale bar = 5 µm. c Immunofluorescence analysis of Dsg1 and Tctex-1 in epidermis. Scale bar = 10 µm. d High-resolution analysis of Dsg1, Ecad, and Tctex-1 in the basal layer of the epidermis shows Tctex-1 localization in the cytoplasm and at the cell periphery in close proximity with Dsg1 and E-cad. Scale bar = 5 µm. e Proximity ligation assay (PLA) using primary antibodies directed against Dsg1 or E-cad and Tctex-1 performed in differentiated NHEKs with or without Dsg1 or Tctex-1 knockdown. Plakoglobin staining in green marks cell–cell borders. Scale bar = 20 µm. f PLA signal area for e was counted per field for each condition (>10 cells per field) and averages were determined. Data for one representative experiment out of three biological repeats, F = 153.581, ***p < 0.001, one way Anova with Tukey test. g Western blot showing level of Dsg1 and Tctex-1 knockdown. h PLA using primary antibodies directed against Dsg1 and Tctex-1, E-cadherin and Tctex-1, or Dsg2 and Tctex-1 was performed on undifferentiated NHEKs with or without Tctex-1 knockdown. Scale bar = 10 µm. i PLA signal area for h was counted per field for each condition (>10 cells per field) and averages were determined. Data for one representative experiment out of three biological repeats is shown in the graph (***p < 0.001, unpaired two-tailed t test). j The level of cadherin proteins and Tctex-1 knockdown is shown on the corresponding western blot. k Diagram of Dsg1 Flag-tagged constructs used in the study. l Western blot of ectopic Dsg1 Flag-tagged constructs immunoprecipitated from NHEK lysates and probed for Tctex-1. GFP was used as a control. m PLA using primary antibody directed against Flag and Tctex-1 was performed on undifferentiated NHEKs retrovirally transduced with GFP, Dsg1-FL-Flag, Dsg1-909-Flag, or Dsg1-ICS-Flag. Scale bar = 10 µm. n PLA signal area for m was counted per field for each condition (>10 cells per field) (three independent experiments; F = 47.792, ***p < 0.001, one-way Anova with Tukey test). PLA signal pseudocolored yellow. DAPI staining marks nuclei. Error bars represent standard error of the mean (SEM)