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

Abstract

The cadherin family of Ca 2؉ -dependent cell adhesion proteins are critical for the morphogenesis and functional organization of tissues in multicellular organisms, but the molecular interactions between cadherins that are at the core of cell-cell adhesion are a matter of considerable debate. A widely-accepted model is that cadherins adhere in 3 stages. First, the functional unit of cadherin adhesion is a cis dimer formed by the binding of the extracellular regions of 2 cadherins on the same cell surface. Second, formation of low-affinity trans interactions between cadherin cis dimers on opposing cell surfaces initiates cell-cell adhesion. Third, lateral clustering of cadherins cooperatively strengthens intercellular adhesion. Evidence of these cadherin binding states during adhesion is, however, contradictory, and evidence for cooperativity is lacking. We used single-molecule structural (fluorescence resonance energy transfer) and functional (atomic force microscopy) assays to demonstrate directly that cadherin monomers interact via their N-terminal EC1 domain to form trans adhesive complexes. We could not detect the formation of cadherin cis dimers, but found that increasing the density of cadherin monomers cooperatively increased the probability of trans adhesive binding.atomic force microscope ͉ cell adhesion ͉ cis dimer ͉ fluorescence resonance energy transfer ͉ trans binding C adherins are Ca 2ϩ -dependent cell-cell adhesion proteins that play fundamental roles in the functional organization of cells and in maintaining the structural integrity of solid tissues (1, 2). Cadherin adhesion is modified during normal embryonic development, and disruption of adhesion is common in metastatic cancers (2-4). Despite detailed studies of cadherinmediated adhesion in multicellular organisms, a molecular understanding of the adhesive states of cadherin is less clear (5). Structural studies have shown that cadherin-cadherin contacts are mediated by the extracellular domain comprised of tandem repeats of 5 cadherin (EC) domains. A widely accepted model as summarized in textbooks (6) and recent review articles (1, 7-9) is that the functional unit of cadherin adhesion is a cis dimer formed by binding of the extracellular domains of 2 cadherins on the same cell surface. Cell-cell adhesion is initiated by the formation of trans adhesive complexes between the EC1 domains of cadherin cis dimers on opposing cell surfaces (1, 6-15). Strong cadherin adhesion may also require trans binding along additional EC domains (16)(17)(18)(19). Trans-cadherin binding is a lowaffinity interaction, but cell-cell adhesion is believed to be enhanced cooperatively by the lateral clustering of cadherins (20,21).Evidence for these trans-and cis-cadherin binding states, however, is controversial, and evidence for cooperativity in promoting adhesion is lacking. Models of cadherin cis dimerization are based on indirect evidence from the packing interactions in cadherin crystal structures (11,15,22,23), electron tomographs of desmosomes (12, 13), elect...

Resolving the molecular mechanism of cadherin catch bond formation

“…4b); Poisson statistics predicts that under these conditions, 497% of measured events occur because of the rupture of single X-dimers. Single-molecule fluorescence microscopy experiments have previously shown that under similar experimental conditions, a majority of the measured unbinding events occur because of rupture of single trans-dimers 29 . Approximately 1,000-2,000 single-molecule measurements were carried out at five different clamping forces.…”

“…Biotinylated cadherin mutants were immobilized on glass coverslips and AFM cantilevers (Olympus, Model: TR400PSA) using methods described previously 29 . Briefly, the cantilevers and coverslips were cleaned and functionalized with amine groups using a 2% v/v solution of 3-aminopropyltriethoxysilane (Sigma) dissolved in acetone.…”

“…The cadherins were site specifically biotinylated at their C terminus to a 15 amino-acid AviTag sequence, and immobilized on an AFM tip and substrate using a Polyethylene glycol (PEG) tether (Fig. 4a) 9,12,29 . The tip and substrate were brought into contact to allow opposing W2A cadherins to form X-dimers.…”

Adherens Junction: Molecular Architecture and Regulation

“…idea that a trans binding between cadherin monomers via their EC1 domains initiates their interactions (Zhang et al 2009). The calcium-sensitive sequences are highly conserved among the family members, whose sequences are the hallmarks for this family, and all classic cadherins show a similar calcium dependence.…”

Regulation of cell motile behavior by crosstalk between cadherin- and integrin-mediated adhesions

“…Cell-cell adhesion is mediated principally by cadherins, which form Ca 2+ -dependent trans-interactions between opposed extracellular domains on adjacent cells and bind a cytoplasmic complex of β-catenin and α-catenin (14). Adhesion of E-cadherin-expressing MDCK epithelial cells to surfaces functionalized with EcadFc mimics this interaction, because the bridging chemistry correctly orients the N terminus of the E-cadherin extracellular domain outward while still allowing rotation and short-range lateral clustering of the protein (13,15).To simultaneously mimic cell binding to ECM and other cells, we used unique, functionalized micropatterned surfaces comprised of alternating stripes of ECM (collagenIV) and adjustable amounts of EcadFc. Recruitment of cellular proteins to different stripes was monitored with GFP-tagged proteins that had been previously well characterized (8, 16).…”