Pkp4-promoted E-cadherin clusters are ineffective in adhesion. (a) Dispase-based cohesion assay of A431 cells expressing EcGFP (EcGFP) and their derivative lines lacking pkp4 (pkp4-KO) or p120 (p120-KO). Left: Representative images showing cell sheets detached from the culture dishes before and after application of mechanical stress (shaking). Right: Quantification (n = 4) of sheet areas (pixels) before shaking (left) and the number of sheet fragments generated after shaking (right). Data represent mean ± SD. (b) Maximum-intensity projections of all x–y optical slices of αCat/pkp2/3-KO cells expressing GFP-pkp4, stained for pkp4 (green) and F-actin (red). Bar, 20 μm. The boxed region is enlarged at right. Bar, 8 μm. Note the precise alignment of pkp4 clusters along actin-rich cell–cell protrusions. (c) Co-culture of αCat/pkp2/3-KO cells expressing GFPpkp4 with αCat-KO cells expressing αCatCH-Δ259. Cells were stained for GFP (green) and mCH (red). Bar, 20 μm. The boxed region is enlarged at right. Heterochromatic GFP/mCH clusters are evident at the interfaces between the two cell populations. (d) Dispase-based cohesion assay of parental αCat/pkp2/3-KO cells and the corresponding line expressing GFP-pkp4 (GFPpkp4-αCat/pkp2/3-KO). Left: Representative images showing detached cell sheets before and after mechanical stress. Note that cell sheets from both lines fail to contract. Right: Quantification (n = 4) of sheet areas (pixels) prior to shaking. The number of fragments after shaking was not quantified. Data represent mean ± SD. Statistical significance in a and d was assessed using a two-tailed Student’s t test: ns, not significant; *P < 0.05; ****P < 0.0001. αCat, α-catenin; mCH, mCherry tag.