Adherens junction assembly of WT-N-cadherin and its mutants.

(A) Expression pattern of WT-N-cadherin and its mutants in L cells. Confocal images of control L cells or L cell lines stably expressing the N-cadherin-Venus WT and its mutant forms. Membrane fluorescence can be seen for all mutants demonstrating plasma membrane localization and junction formation for all mutants. Top panel: Venus channel, bottom: Overlay of DIC and Venus channel. (B) Example images of junction assembly. Images were taken for all mutants at time 0, 1 and 2 h after imaging commenced. Established junctions can be seen for WT and R14E at 1 h and for all mutants at 2 h (arrowheads). (C) Quantification of junction assembly. The number of junctions formed in a field of view (FOV) was counted over time and plotted. The graph shows that the rate of junction formation for WT is fastest followed by R14E. The number of junctions for all mutants was compared to WT at 60 and 120 min. R14E was not significantly different while W2A and V81D/V174D had significantly lower number of junctions at 60 min and 120 min (n = 3 for all mutants, two-tailed unpaired t-test, P < 0.05). (D) Quantification of junction lifetime. The junction lifetime for junctions present in a FOV was calculated between 60 and 120 min. On average WT junctions were present for 82% of time, R14E = 75%, W2A and V81D/V174D had significantly lower average junction lifetimes of 39% and 6%, respectively, thus confirming that mutating the cis interface leads to highly volatile junctions (Mann-Whitney U test, P < 0.0001). Error bars indicate SD. n(WT) = 15, n(W2A) = 17, n(R14E) = 12, n(V81D/V174D) = 15 junctions. Scale bar (A) and (B) = 10 µm.