A dimerization-activated proximity labeling system for direct characterization of cadherin cis interactions

E-cadherins (Ecads) are essential transmembrane cell-cell adhesion proteins that mediate epithelial tissue formation and maintenance. Robust adhesion requires cis dimerization between neighboring Ecads on the cell surface and previous structural and biophysical studies have proposed conflicting models on the role of specific and nonspecific interactions in mediating cis dimerization. However, since these studies were carried out with isolated Ecad extracellular regions in cell-free systems, it is unknown if specific and nonspecific cis binding modes also occur with transmembrane Ecads in live cells, and if specific and nonspecific cis dimers recruit different sets of cytoplasmic proteins to Ecad junctions. To directly address these knowledge gaps, we developed a Dimerization Activated TuboID (DAT) proximity labeling system that reports on different modes of Ecad cis binding and their corresponding proteomes. Using DAT, fluorescence measurements, diffusion-reaction simulations, and adhesion assays, we demonstrate that Ecads in live cells form cis dimers via both specific and non-specific interactions and that cis dimerization does not require either prior trans contacts or an intact actin cytoskeleton. However, we show that the loss of specific cis interactions results in increased junctional instability and Ecad mobility, which leads to dysregulated peripheral protein interactions without affecting recruitment of core junctional proteins. Our study provides key mechanistic insights on cadherin cis interactions and also presents a toolkit that can be used to study a broad range of protein cis dimers in live cells.