X dimer mutations delay cadherin clustering in the calcium switch assay. (A) Time-lapse images (the first and the last frames from Videos 3 and 4) acquired at 20-s intervals (total duration 4 min) of cells expressing EcDendra-Δ748-KL (Δ748) and its X dimer K14E mutant (K14E) during the calcium switch assay in ATP depletion media (numbers indicate min after a switch; 0, immediately before the switch). Note that calcium induced the formation of numerous junctional clusters in cells expressing the parental, but not the X dimer, mutant. (B) Clustering kinetics of EcDendra-Δ748-KL (Δ748) and its X dimer mutants K14E (K14E) and Q101A/N143A (Q101/N143) after the addition of calcium (n = 10). The error bars indicate SD. (C) A-431 cells expressing the parental EcDendra-Δ748-KL (Δ748) mutant, X dimer mutant K14E (K14E), or X/strand-swap dimer–incompetent W2A/K14E mutant (W2A/K14E) were incubated in low calcium overnight. Then, to block morphological changes, they were depleted of ATP by a 10 min-long incubation in low-calcium/ATP-depletion media (0) and subsequently treated with high-calcium/ATP-depletion media for 10 min (10). Subcellular localization of the mutant and endogenous cadherin was determined by double staining as in Fig. 1. Note that the X dimer–incompetent K14E mutant inhibits the formation of adherens junctions. The inactivation of the strand-swap interface of this mutant by W2A mutation blocks this effect. Bars, 40 µM.