Cadherin cis and trans interactions are mutually cooperative

Cadherin transmembrane proteins are responsible for intercellular adhesion in all biological tissues and modulate tissue morphogenesis, cell motility, force transduction, and macromolecular transport. The protein-mediated adhesions consist of adhesive trans interactions and lateral cis interactions. Although theory suggests cooperativity between cis and trans bonds, direct experimental evidence of such cooperativity has not been demonstrated. Here, the use of superresolution microscopy, in conjunction with intermolecular single-molecule Förster resonance energy transfer, demonstrated the mutual cooperativity of cis and trans interactions. Results further demonstrate the consequent assembly of large intermembrane junctions, using a biomimetic lipid bilayer cell adhesion model. Notably, the presence of cis interactions resulted in a nearly 30-fold increase in trans-binding lifetimes between epithelial-cadherin extracellular domains. In turn, the presence of trans interactions increased the lifetime of cis bonds. Importantly, comparison of trans-binding lifetimes of small and large cadherin clusters suggests that this cooperativity is primarily due to allostery. The direct quantitative demonstration of strong mutual cooperativity between cis and trans interactions at intermembrane adhesions provides insights into the long-standing controversy of how weak cis and trans interactions act in concert to create strong macroscopic cell adhesions.

Cadherin adhesion proteins are essential for the hierarchical organization of all multicellular organisms, and their dysfunction is associated with several pathologies (1–8). For example, deficiencies in cadherin-mediated adhesion are correlated with the onset and metastasis of multiple cancers and tissue diseases (6–8). Cadherin-mediated adhesion involves the formation of adherens junctions (9, 10), which entail interactions between cadherin extracellular domains in cis and trans configurations, where cis interactions occur between proteins on the same cell membrane, and trans interactions occur between proteins on opposing membranes (11–26). Theory suggests that these cis and trans bonds form cooperatively (11–26). For example, cis interactions are believed to enhance molecular ordering (15) and may increase intercellular adhesion through cluster avidity (16, 27, 28), with potential applications related to angiogenesis and therefore cancer therapies (29–31). Early studies suggested that cis interactions enhanced the cadherin adhesive function (16). However, observations of lateral cis interactions between cadherin extracellular domains have been elusive because of their low affinity and the challenges of studying membrane-bound proteins (32–34). Observations of cis interactions in crystal structures and the disruption of cadherin organization within junctions by putative cis mutants suggested that they operate in tandem with trans interactions (15, 35). One hypothesis was that initial trans binding enhances the cis-binding affinity, leading to lateral clustering, junction nucleation, and growth (36). Such trans → cis cooperativity was predicted theoretically but not verified experimentally (37).Recent single-molecule (SM) studies successfully demonstrated that cis interactions induced clustering between cadherin extracellular domains on a supported lipid bilayer (SLB) (38, 39). The latter result suggested that conformational constraints associated with membrane immobilization increased the cis-binding affinity sufficiently to induce clustering, even in the absence of trans interactions. This observation raised the possibility of the reciprocal cooperativity (i.e., cis → trans), in which initial cis binding may enhance adhesion. Although the probability (but not the strength) of trans binding was found to increase for cadherin dimer constructs, relative to the monomer (34), the connection to cis interactions, if any, remains unclear. Demonstrating cis/trans cooperativity would require demonstrating that the presence of cis interactions alters the strength of trans bonds quantitatively and vice versa.In this study, we systematically identified and quantified cis/trans cooperativity, using dynamic SM Förster resonance energy transfer (FRET). These measurements determine whether cis interactions increase trans-binding lifetimes and, conversely, whether trans interactions increase cis-binding lifetimes. They further elucidated the putative role of cis/trans cooperativity in the formation and growth of cadherin junctions between opposing membranes. We find that cis and trans interactions are strongly and mutually cooperative. Most importantly, results show that cis interactions dramatically increase trans-binding lifetimes by more than an order of magnitude, and these cooperative interactions are shown to facilitate the assembly of large junctions. A detailed analysis of trans-binding kinetics as a function of cluster size provide insight into the molecular mechanism of the elevated trans lifetimes. The results presented suggest that specific cis interactions allosterically activate trans-binding interactions.