Stable and Unstable Cadherin Dimers: Mechanisms of Formation and Roles in Cell Adhesion

Abstract

Numerous attempts to elucidate the strength of cadherin dimerization that mediates intercellular adhesion have produced controversial and inconclusive results. To clarify this issue, we compared E-cadherin dimerization on the surface of living cells with how the same process unfolds on agarose beads. In both cases, dimerization was monitored by the same site-specific cross-linking assay, greatly simplifying data interpretation. We showed that on the agarose surface under physiological conditions, E-cadherin produced a weak dimer that immediately dissociated after the depletion of calcium ions. However, either at pH 5 or in the presence of cadmium ions, E-cadherin produced a strong dimer that was unable to dissociate upon calcium depletion. Both types of dimers were W156-dependent. Remarkably, only the strong dimer was found on the surface of living cells. We also showed that the intracellular cadherin region, the clustering of which through catenins had been proposed as stabilizer of weak intercadherin interactions, was not needed, in fact, for cadherin junction assembly. Taken together, our data present convincing evidence that cadherin adhesion is based on high-affinity cadherin-cadherin interactions.

Spontaneous assembly and active disassembly balance adherens junction homeostasis

“…This imbalance between cadherin recruitment and its release in the ATP-depleted cells led to the junctional entrapment of nearly the entire cadherin pool present on the cell surface. This work together with our previous biochemical data (13,15), compellingly show that the cadherin extracellular region is able to produce relatively stable junctional structures.…”

“…Another possibility is that cadherin release from the junctions is driven by clathrin-mediated endocytosis. This possibility is suggested by recent FRAP experiments (16), a pharmacological study of junction disintegration in low calcium (20), and our previous work with the catenin-uncoupled Ec-Δ748-Myc mutant (13). In that work we showed that the Ec-Δ748-Myc cadherin is mostly cytosolic and cannot form junctions in control cells.…”

“…The very low affinity of cadherin adhesive interactions also suggests that other cellular proteins facilitate efficient cadherin clustering (3). However, recent data (12)(13)(14) are also consistent with an alternative point of view, which holds that cadherin clustering can proceed via a diffusion-trap mechanism. By this mechanism, cadherin molecules first form a small number of adhesion bonds that maintain the resulting nascent intercellular contact long enough for new cadherin molecules to be diffused into this contact.…”

“…To confirm one of these possibilities, we blocked active cellular processes in Ec-Dendra-expressing cells using the combination of the oxidative phosphorylation and glycolysis inhibitors (NaN 3 or antimycin and 2-deoxyglucose, correspondingly). Such treatments were shown to rapidly deplete cells for ATP (13). Junctional cadherin turnover would be ATP independent if it is based on the weak affinity of cadherin-cadherin interactions.…”

Resolving cadherin interactions and binding cooperativity at the single-molecule level

“…Although the cadherin cis interaction site was initially localized to the EC1 domains (10,24), it was later proposed that EC1 and EC2 domains of neighboring cadherin molecules are involved in these interactions (11,26). However, other studies using protein cross-linking and coimmunoprecipitation demonstrated that cis cadherin dimers were formed only in cells grown artificially at low calcium concentrations, and that cis and trans interactions shared the same adhesive interface (27)(28)(29). Although some electron tomography experiments have shown trans interactions involving both cadherin monomers and cis dimers (13), other cryoelectron tomographs suggest that cis dimerization is mandatory for trans adhesion (12).…”