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Desmosomes are cell-cell adhesion complexes that are present in tissues that resist mechanical stress. They are mainly composed of two adhesive proteins, which are members of the cadherin superfamily of cell adhesion proteins, desmocollin (Dsc) and desmoglein (Dsg). However, the role of these proteins in desmosomal adhesion is unclear. Here, we use the single molecule force spectroscopy with an Atomic Force Microscope (AFM-FS) to characterize the interactions of type-2 isoforms of desmocollin (Dsc2) and desmoglein (Dsg2). We show that Dsc2 forms Ca2+ dependent homophilic bonds by swapping a conserved Tryptophan (Trp) residue between opposing binding partners; mutating this Trp inhibits Ca2+ dependent homophilic binding. In contrast, Dsg2 forms Ca2+ independent heterophilic bonds with Dsc2 via a mechanism that does not involve Trp strand-swapping.Previous studies suggest that desmosome formation requires the presence of classical cadherins at the site of desmosome assembly. This suggests a cross-talk between desmosomal and classical cadherins at cell-adhesion contacts. We therefore used AFM-FS to test if Dsc2 and Dsg2 interact with E-cadherin, a classical cadherin present in the epithelium. Our data shows that while Dsc2 does not bind to E-cadherin in the presence of Ca2+, Dsg2 forms Ca2+ independent complexes with E-cadherin. Using cadherin mutants we show that the interactions between Dsg2 and E-cadherin occur via a previously uncharacterized binding interface that does not involve either Trp strand-swapping or X-dimer formation (two well established classical cadherin binding mechanisms).

Adherens junctions (AJ) are mechano-sensitive protein complexes essential for many multicellular processes such as tissue morphogenesis and homeostasis. Genetic studies identified the AJ cell-cell adhesion complex of E-cadherin, beta-catenin, and the actin-binding protein alpha-catenin as a minimal ternary complex required for interactions with the actin cytoskeleton. However, attempts to reconstitute in vitro a direct linkage between the cadherin-catenin complex and actin filaments in bulk pelleting assays have been unsuccessful. Contemporary experiments in living cells indirectly showed that force, which was not reconstituted in vitro, is important for normal cell-cell adhesion. As a result, we developed a novel optical trapping assay to determine if and how the cadherin-catenin complex binds to actin filaments under mechanical load (see figure). We find that multiple cadherin-catenin complexes can together form a robust, load-resistant connection to actin that can withstand >10 pN of force for several seconds. Individual cadherin-catenin complexes also bind actin, but the interaction is relatively weak and transient. To our knowledge, these are the first data showing direct, mechanically robust binding between the cadherin-catenin complex and actin filaments. Our results provide essential support to models proposing that the AJ is a mechano-sensitive complex.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The cadherin family of Calcium-dependent cell adhesion proteins are critical for the morphogenesis and functional organization of tissues in multicellular organisms, but the molecular interactions that are at the core of cadherin binding are poorly understood. The generally accepted model is that cadherins adhere in three stages. First, the functional unit of cadherin adhesion is a cis-dimer formed by the binding of the extracellular regions of two 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, and third lateral clustering of cadherins cooperatively strengthens intercellular adhesion. Direct molecular proof 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 outermost 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. We also resolved the role of Tryptophan-2 a key amino acid in cadherin trans-binding. These results resolve conflicting data on trans- and cis-cadherin binding states, and provide quantitative evidence for cooperativity in trans-cadherin adhesion.

Adherens junctions (AJs) comprise protein complexes essential for integrating cell-cell adhesion and actin cytoskeleton functions, and their importance has been shown in many loss-of-function studies. Disruption of acto-myosin tension in living cells impairs formation of AJs, but attempts to reconstitute in bulk assays a direct linkage between a minimal AJ complex (E-cadherin/β-catenin/α-catenin) and actin filaments in vitro have been unsuccessful. Here we present a new optical trap-based single-molecule assay that tests whether this linkage requires tension (Fig. 1A). using a reconstituted E-cadherin/β-catenin/α-catenin complex (∼100 nM), an actin filament held between 2 optical traps, and moving the complex along the actin filament by oscillating the microscope stage, we observe binding events that likely involve multiple complexes and displacements of over 100 nm with loading rates of ∼45 pN/s that can sustain over 15 pN of force. The stepwise release mechanism may reflect sequential detachment of individual E-cadherin/catenin complexes (Fig. 1B). These data indicate that several E-cadherin/catenin complexes can form a robust, load-resistant connection to an actin filament under tension. In ongoing work, we are studying how mechanical load may regulate the affinity between the E-cadherin/catenin complexes and actin filaments.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Cadherins are homophilic adhesion molecules mediating Ca++ dependent cell-cell adhesion. It is not very clear how exactly cadherins interact and promote cell adhesion at the molecular level. We have previously characterized cadherin interactions with single molecule FRET and single molecule AFM and found cadherins interact mostly through outermost EC1 domains (Zhang & Sivasankar et al 2009). We then developed super-resolution localization microscopy that achieved sub-nanometer precision and accuracy and applied it to measurements of the end-to-end distances of E-cadherin dimers (Pertsinidis & Zhang et al 2010). The majority of the wild type cadherin dimers exhibits an extended EC5-EC5 distance of 32nm matching a strand-swapped trans-dimer model. A smaller population of the wild type dimers shows shorter distances of around 25nm suggesting an alternative binding configuration. We extended this work further by characterizing mutations in E-cadherin that prevent formation of the the strand-swapping interface. Interestingly, we observed that W2A dimers exist in a more compact conformation, similar to the minor population in the wild type dimers. The compact conformation is consistent with a so-called X-dimer interface discovered in recent crystal structures (Ciatto et al 2010, Harrison et al 2010). These new data refine our previous induced-fit dimerization pathway (Sivasankar & Zhang et al 2009) and are consistent with the X-dimer being the initial encounter complex. Furthermore, these results provide evidence that, at near-physiological Ca++ concentrations, E-cadherins can utilize two distinct binding interfaces to facilitate cell adhesion.

Desmocollin and Desmoglein are essential Ca2+ dependent cell adhesion proteins that mediate the integrity and functional organization of tissues in multi-cellular organisms; however, the molecular interactions that mediate their binding are not understood. It is currently believed that desmosomal cadherin cis-dimers are required to mediate adhesion, evidence for the role of cis-dimers is lacking. Furthermore, it is unclear if desmosomal cadherins interact via homophilic or heterophilic binding and the molecular mechanism by which desmosomal cadherins enhance their bond-strength. To resolve these questions we used Dynamic Force Spectroscopy with an Atomic Force Microscope to measure the homophilic and heterophilic binding of desmosomal cadherin monomers and cis-dimers at the single molecule level. Our measurements showed that desmosomal cadherin monomers alone mediate Ca2+ dependent adhesion, cis-dimers are not essential for adhesion. Furthermore, while desmocollin and desmoglein participate in Ca2+ dependent heterophilic binding, we could not measure Ca2+ dependent homophilic interactions between these cadherins. Dynamic Force Spectroscopy revealed that desmosomal cadherin interactions occur via a double barrier interaction potential; at low loading rates the outer barrier serves as the dominant impedance to unbinding while at higher loading rates the inner barrier serves as the primary kinetic barrier. These experiments resolve the molecular details of desmosomal cadherin binding and suggest a mechanism by which these essential cell adhesion molecules enhance bond strength and lifetime in the presence of force.