Analysis of the trans interaction of N-cadherin in living cells by acceptor bleach FRET experiments.

(A) Scheme depicting the insertion of Venus (FRET acceptor) and Cerulean (FRET donor) and the interactions of cadherin molecules across a cellular junction. If the two opposing molecules interact with each other in trans, the donor and acceptor are within the FRET distance, resulting in an increased acceptor emission and a donor quenching (upper panel). Upon bleaching of the FRET acceptor, the FRET donor is dequenched, leading to an increase in its fluorescence (lower panel). (B) Example for an acceptor bleach experiment with COS7-cells expressing either N-cadherin-Venus or –Cerulean. The upper two images show the Venus-channel before and after bleaching of the Venus signal in the junction (boxed region). The Cerulean channel is depicted in the lower two images. Bleaching of the Venus fluorescence leads to a dequenching of the FRET donor Cerulean, which can be observed in the lower two images. An enlargement of the junction (boxed region) is shown next to the images. (C) Quantitative comparison of the acceptor bleach experiments for N-cadherin-WT and its mutants. The bars represent the mean ± SEM. The mean of the W2A (n = 32) is significantly increased compared to the WT (n = 35, p < 0.0001, Mann Whitney, ***), while the mean of the mutant R14E was significantly lower (n = 29, p=0.0001, two-tailed unpaired t-test , ***). The cis mutant V81D/V174D (n = 33) does not show a significant difference (p=0.1158, two-tailed unpaired t-test). Scale bar = 20 µm.