Cadherins play a key role in the dynamics of cell-cell contact formation and remodeling of junctions and tissues. Cadherincadherin interactions are gated by extracellular Ca 2+ , which serves to rigidify the cadherin extracellular domains and promote trans junctional interactions. Here we describe the direct visualization and quantification of spatiotemporal dynamics of N-cadherin interactions across intercellular junctions in living cells using a genetically encodable FRET reporter system. Direct measurements of transjunctional cadherin interactions revealed a sudden, but partial, loss of homophilic interactions (τ = 1.17 ± 0.06 s ), suggesting two types of native cadherin interactions-one that is rapidly modulated by changes in extracellular Ca 2+ and another with relatively stable adhesive activity that is Ca 2+ independent. The Ca 2+-sensitive dynamics of cadherin interactions were transmitted to the cell interior where β-catenin translocated to N-cadherin at the junction in both cells. These data indicate that cadherins can rapidly convey dynamic information about the extracellular environment to both cells that comprise a junction.cell adhesion | fluorescence resonance energy transfer | trans binding T he junctions between cells are populated with a variety of cell adhesion molecules that drive the recognition, assembly, and dynamics of cell-cell interactions. Parsing the distinct functions of different adhesion molecules has been challenging, in part due to a paucity of truly revealing in vivo assays. Among cell adhesion molecule families, the classic cadherins exhibit a unique dependence on extracellular Ca 2+ to rigidify the extracellular domains and enable trans junctional homophilic interactions. Three Ca 2+ ions bind with different affinities to each of the pockets between cadherin extracellular domains (1, 2). The affinity of the various Ca 2+ binding sites is in the micromolar to millimolar range (3-8), suggesting the possibility that cadherins can respond dynamically to changes in junctional Ca 2+ levels, and by virtue of physical interactions with cytoplasmic molecules, signal information about the junctional status to the cell interior.3D reconstructions of desmosomes by cryoelectron tomography reveal cadherins interact across intercellular interfaces (9, 10), and crystallographic data of several cadherin domains (4, 11, 12) implicate the first two extracellular repeats (ECs 1 and 2) as critical for homophilic interactions. Previous studies have indicated the importance of a highly conserved tryptophan residue (Trp2), present in the first EC domain, for cadherin-dependent adhesion (2, 12). This tryptophan inserts into the hydrophobic pocket of the partner N-cadherin (Ncad) molecule to form a strand-swapped dimeric structure (11, 12). Mutation of Trp2 to an alanine residue (W2A) prevents strand swapping (13) and results in a loss of adhesive function (2, 14, 15). Although extensive work has elucidated the structure (4, 9-14), biochemistry (2, 16) and single-molecule characterization (15, 17) of ...