Fig 1: DAMGO and morphine differentially assemble protein-interaction networks to control MOR spatiotemporal signaling. A, DAMGO activation of the MOR leads to rapid recruitment of GRK2 and ß-arrestins and facilitates a redistribution of the MOR at the plasma membrane (66). The DAMGO-stimulated receptor is in close proximity to IQGAP1, a large scaffolding protein that is required for the activation of Rac1 and nuclear ERK. IQGAP1 has been previously shown to bind both ERK and Rac1 (38). The DAMGO-stimulated MOR also associates with the adaptor protein, CRKL, which is essential for the activation of both Rac1 and nuclear ERK. The activation of Rac1 and nuclear ERK (10 min) occurs prior to receptor internalization (30–60 min (8)). B, in contrast, morphine activation of the MOR stimulates a Gai/o–Gß?–PKCa pathway, which increases the proximity of MOR to desmosomal proteins. Desmosomes are formed by two transmembrane proteins, DSC and DSG, which in turn interact with the intracellular proteins plakoglobin/JUP, plakophilin (PKP) and desmoplakin (DSP) (15). The formation of stabilized desmosomal-like plasma membrane domains likely contributes to the inability of the morphine-stimulated MOR to redistribute and therefore controls the sustained increase in cytosolic ERK activity. Knockdown of DSC1 (green) or JUP (cyan) facilitated a transient increase in nuclear ERK in response to morphine, indicative of a redistribution of the receptor at the plasma membrane (8).
Fig 2: 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 3: Dsg1 recruits cortactin–Arp2/3 complexes to junctions to promote cortical actin polymerization. a Western blot of GFP immunoprecipitations from undifferentiated NHEKs expressing GFP, Dsg1-GFP or Dsg2-GFP probed for GFP, cortactin, Pg (positive control for Dsg1/Dsg2 interactions), and E-cadherin (negative control). b–e GFP, Dsg1-FL-Flag, or Dsg1-909-Flag-infected undifferentiated NHEKs were stained for cortactin and Flag (staining for Flag shown in top insets) (b) or Arp3 and Flag (d). Line scan analyses of cortactin intensity (c) or Arp3 intensity (e) at the cell–cell interfaces in undifferentiated NHEKs expressing GFP, Dsg1-FL-Flag, or Dsg1-909-Flag (at least 50 borders per condition were analyzed in each experiment, three independent experiments, ***p < 0.001, one-way Anova with Tukey test; F = 13.703 for c and F = 69.212 for e). f Barbed-end labeling at cell junctions of undifferentiated NHEKs expressing ectopic Dsg1-FL-Flag or Dsg1-909-Flag treated with DMSO or CK666. Flag staining is shown for each condition (insets, green). Rectangles mark zoomed in junctional regions, which are to the right of each panel, barbed ends (red) and Flag (green). g Quantification of barbed-end labeling in undifferentiated NHEKs expressing Dsg1-FL-Flag (FL) or Dsg1-909-Flag (909) treated with DMSO or CK666 (at least 60 borders were measured per condition from each experiment, three independent experiments; ***p < 0.001, unpaired two-tailed t test). Scale bars = 10 µm. Error bars represent SEM
Fig 4: 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 5: Diagram summarizing the findings of the present study. The sequence of events is required for initial steps of stratification. Newly synthesized Dsg1 is required for the Tctex-1–dynein complex to be localized at the cell–cell interface in the insoluble membrane compartment (1) where Dsg1 recruits cortactin–Arp2/3 actin nucleation complexes to promote actin polymerization/reorganization at the cell junctional area (2). Actin reorganization at the desmosomal junctions reduces tension at adherens junctions (AJ) (3) to promote basal cell delamination during the initial steps of stratification (4)
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