STR is responsible for the functional difference between Pk1 and Pk2 on AJs and actomyosin. (A–E) Representative images of Xenopus gastrula ectoderms mosaically expressing myrGFP only (A), with HA-Pk1 (B), with Flag-Pk2 (C), with HA-Pk1(2STR) (D), or with Flag-Pk2(1STR) (E). myrGFP and Pk mutant RNA were injected into one ventral-animal blastomere in stage four to eight embryos. myrRFP RNA was injected into the other ventral-animal blastomere to label neighboring cells. Pigmented wild-type embryos were used for image acquisition and quantification. Imaging was initiated at stage 11. (F) 2D plots of the cell shapes in A–E. The x axis represents the cell perimeter. The y axis represents the apical domain size. Individual dots represent cells expressing myrGFP only (blue), HA-Pk1 (green), Flag-Pk2 (red), HA-Pk1(2STR) (light blue), or Flag-Pk2(1STR) (purple). Mosaic cells were those that shared cell–cell boundaries with at least three myrRFP-injected cells. (G and H) Circularity (G) and solidity (H) of Pk mutant-expressing cells. The same cells in F were used for quantification. An ImageJ plugin, Particle Analyzer, was used for the calculations. Circularity = 4pi (area/perimeter^2). Solidity (convexity) = apical domain size/convex hull area. A maximum value of 1 represents a perfect circle. Cells with <100 μm2 apical domain area were excluded to increase the reliability of the assays. One-way ANOVA was used to compare means, with the Bonferroni correction for multiple pairwise comparisons. Three embryos per group were used. The total number of cells was as follows: 298 cells (myrGFP only); 116 cells (+Pk1); 165 cells (+Pk1(2STR)); 249 cells (+Pk2); 187 cells (+Pk2(1STR)). (I–M) Representative images of Xenopus gastrula ectoderms expressing myrGFP only (I), with Pk1 (J), with Pk1(2VBD) (K), with Pk2 (L), or with Pk2(1VBD) (M). Imaging was initiated at stage 11.5. Pigmented wild-type embryos were used for image acquisition and quantification. (N–P) Quantification of hexagonal cell packing according to the variation in AJ linearity (N), AJ length (O), and the number of neighboring cells (P). The dots indicate the average AJ linearity in individual embryos. The lines indicate the average AJ linearity of three embryos. Statistical analyses of N and O were based on the original numeric data. One-way ANOVA was used to compare means, with the Bonferroni correction for multiple pairwise comparisons in N. The Kolmogorov‒Smirnov test was used to compare distributions in O. The chi-square test was used in P. Three embryos per group were used. The total number of cells and AJs was as follows: 123 cells and 409 AJs (myrGFP only); 123 cells and 374 AJs (+Pk1); 100 cells and 366 AJs (+Pk1(1VBD)); 115 cells and 355 AJs (+Pk2); and 146 cells and 290 AJs (+Pk2(1VBD)). *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. Scale bars: 20 μm.
