Afadin KD dramatically alters the shape of ZO KD cells. (A–C) ZO KD versus ZO/afadin KD (A vs. B). (B) At many BCJs, the myosin array was unaltered (yellow arrows), but rosette-like cell arrangements pointing toward sets of hyperconstricted junctions (C, arrows and arrowheads) and areas of reduced or disrupted apical Ecad staining (asterisks and green arrows) were observed in other areas. (B and C) Maximum-intensity projections (5-µm apical) covering apical half of the tissue. (D) Major/minor axis aspect ratios. Note shift from isotropic to elongated in ZO/afadin KD. (E) Scatter plot of axis ratios. Both mean and coefficient of variance (CV) were significantly higher in ZO/afadin KD. (F) Mean velocity calculated based on velocity field (v) via PIV analysis of phase-contrast videos. (G) Spatial correlation of velocity fields. (H) Heat maps of divergence fields (∇.v) superimposed to cell velocity fields showing coherent patterns of extending (positive, red) and contracting (negative, blue) regions. The correlation curves of the divergence field below the heat maps represent the distance over which divergence patterns extend or contract. (I) Histogram of the mean magnitude of divergence. In F, G, and I, error bars are SDs of 300 cells (n = 3).