Influence of Ca2+-chelation on the individual binding interfaces of N-cadherin.
(A) Disruption of the trans interaction of N-cadherin by BAPTA. Upon disruption of this interaction the fluorophores move further apart, reducing FRET. Examples for ratiometric FRET measurements of the WT (B), W2A (C), R14E (D) and V81D/V174D (E) before and after Ca2+-chelation are shown. After baseline recording, the Ca2+-chelator BAPTA (20 mM final concentration) was added. The fluorescence of the FRET acceptor Venus (yellow) and the donor Cerulean (cyan) as well as the ratio of the two (blue) are shown. (F) The quantification of the effect of BAPTA on all mutants is shown. While the mutants W2A and V81D/V174D showed a significantly higher decrease of the FRET signal due to Ca2+-chelation than the WT, no Ca2+-sensitivity was observed for the X-dimer mutant R14E. This confirms the hypothesis that the X-dimer is a Ca2+-dependent intermediate step in the formation of the strand-swapped dimer. n(WT) = 8, n(W2A) = 8, n(R14E) = 9, n(V81D/V174D) = 7 junctions. Statistics were conducted using either paired or unpaired t-test.