Destabilization of AJs and TCJs in Pk2-overexpressing ectoderm. (A and B) Representative images of YFP-C-cadherin in the control (A) or Pk2-overexpressing (B) ectoderm. Snapshots from Video 8 (durations of 14 min 30 s at 30-s time intervals). Arrowheads indicate C-cadherin puncta in the cytoplasm. Imaging was initiated at stage 11.5. (C) Representative kymographs of YFP-C-cadherin at BJs from Video 8. Single z-plane images. (D) Representative kymographs of GFP-C-cadherin FRAP experiments. Arrowheads indicate the timing of photobleaching. Brackets show the sites of photobleaching. The recovery of GFP-C-cadherin fluorescence signals was quantified in areas outlined by the white-dotted line. In Pk2 #1 junctions, the recovery of GFP-C-cadherin did not reach the prebleach level. In Pk2 #2 junctions, the recovery of GFP-C-cadherin exceeded the prebleach level. (E) Representative curve fitting of GFP-C-cadherin FRAP in the control (blue) and Pk2 OE (red) ectoderms. The light blue and red regions represent the S.D. values. GraphPad was used for curve fitting. (F and G) Percent GFP-C-cadherin recovery (F) and the halftime of recovery (G) in the control and Pk2 OE ectoderms. The percent recovery was based on calculated plateau values. AJs with unstable plateau values were excluded. 28 AJs (control) and 28 AJs (+Flag-Pk2) from more than five embryos were used. The Mann‒Whitney U test was used to compare mean ranks. (H) Representative images of GFP-C-cadherin at TCJs in control and Pk2 OE cells. The white circles indicate TCJs. (I) Quantification of YFP-C-cadherin variability among TCJs. The integrated fluorescence intensity of YFP-C-cadherin at TCJs was quantified in still images. 186 TCJs (control) and 107 TCJs (+Flag-Pk2) from three embryos were used. (J) Representative snapshot images of GFP-C-cadherin fluctuations at single TCJs from time-lapse imaging in the control and Pk2 OE ectoderms. (K) Quantification of YFP-C-cadherin fluctuations at individual TCJs. Changes in YFP-C-cad intensity at individual TCJs were quantified after time-lapse imaging. 10 TCJs (control) and 10 TCJs (+Flag-Pk2) per embryo were used. Three embryos per group were used. The Kolmogorov‒Smirnov test was used to compare distributions in I and K. (L–M′) Representative images of GFP-tricellulin in stage 11 control (L and L′) and Flag-Pk2 OE (M and M′) ectoderms. The square areas in L and M are enlarged in L′ and M′. The arrowheads indicate ectopic enrichment of GFP-tricellulin at BJs. The arrows indicate tricellulin accumulation at TCJs. (N) Line plots of GFP-tricellulin fluorescence intensity at three junctions. The pairs of orange, blue, and green arrows indicate the TCJs. The arrowheads indicate mislocalization of tricellulin at the BJs. (O) Quantification of GFP-tricellulin accumulation at TCJs in the control and Pk2 OE ectoderms. The fluorescence intensity of GFP-tricellulin at individual TCJs was normalized to the average fluorescence intensity of GFP-tricellulin at BJs from the same images. (P and Q) Variations in GFP-tricellulin intensity at the TCJs (P) and BJs (Q) are shown as S.Ds. 83 TCJs (control) and 87 TCJs (Pk2 KD) from three embryos per group were used. The Mann‒Whitney U test was used to compare mean ranks (O‒Q). ***P < 0.001, **P < 0.01, *P < 0.05. Scale bars: 20 μm (A, B, L, and M), 5 μm (C and D), 10 μm (H), and 2 μm (J, L′, and M′).