CD2AP is required for the epithelial monolayer to withstand mechanical stress. (a) Projection of 70 deconvolved optical z slices of the entire 14-µm height of polarized MDCK cells showing a missing cell (white asterisks) within a cell monolayer transfected with CD2AP shRNA. (b) Phase-contrast microscopy showing a cell in mitosis (chromosome condensation, blue arrowheads) detaching from neighboring cells (red arrowheads) within a monolayer of cells transfected with CD2AP shRNA. (c) Time-lapse images of phase-contrast microscopy showing cells tearing away (red arrowheads) from each other within a monolayer of cells transfected with CD2AP shRNA. (d) BSA flux assays showing similar monolayer permeability between parental, CD2AP knockdown, and α-actinin-4 knockdown cells. (e) Occludin localization at the tight junction is normal in CD2AP knockdown cells. (f) Logistics of applying mechanical stress at intercellular junctions for the analysis of epithelial cohesion. (g) A pressure chamber apparatus that can generate hydraulic pressure across a cell monolayer to induce stress at intercellular junctions. (h) Pressure chamber apparatus is attached to a syringe for pumping fluid and a pressure gauge to monitor applied pressure. Transwell supports were held in position during pressure application by lids and screws at the top. (i) Schematics for the operation of the pressure chamber apparatus. Buffer is pumped through channels connecting the basal chambers to produce hydrostatic pressure. (j) Permeability assays showing increased tracer flux with increasing applied hydrostatic pressure to MDCK monolayers. (k) Permeability assays showing a low tolerance level and decreased resilience to mechanical stress in CD2AP knockdown cells. (l) Permeability assays showing greater increases in tracer permeability in CD2AP knockdown cells than in parental cells after mechanical stress. (m) E-cadherin localization at the cell–cell junction of MDCK cells showing intact monolayer after mechanical stress. (n) E-cadherin localization at the cell–cell junction of CD2AP knockdown cells showing holes (asterisks) in the monolayer after mechanical stress. All error bars indicate standard errors.