Desmosomes are cadherin-based intercellular junctions that primarily provide mechanical stability to tissues such as epithelia and cardiac muscle. Desmosomal cadherins, which are Ca2+-dependent adhesion molecules, are of central importance in mediating direct intercellular interaction. The close association of these proteins, with intracellular components of desmosomes ultimately linked to the cytoskeleton, is believed to play an important role in tissue morphogenesis during development and wound healing. Elucidation of the binding mechanism of adhesive interfaces between the extracellular domains of cadherins has been approached by structural, biophysical and biochemical methods. X-ray crystal structures of isolated extracellular domains of cadherins have provided compelling evidence of the mutual binding of the highly conserved N-terminal residue, Trp2, from opposing proteins. This binding interface was also implicated by biochemical and cell-adhesion assays and mutagenesis data to be the primary adhesive interface between cells. Recent results based on electron tomography of epidermal desmosomes were consistent with this view, showing cadherin molecules interacting at their N-terminal tips. An integrative structural approach involving X-ray crystallography, cryo-electron tomography and immuno-electron microscopy should give the complete picture of the architecture of this important junction; identifying its various proteins and showing their arrangements and binding interfaces under native conditions. Together with these ‘static’ approaches, live-cell imaging of cultured keratinocytes should provide important insights into the dynamic property of the assembly and disassembly of desmosomes.
- adherens junction
- electron tomography
- vitreous section
- X-ray crystallography
Intercellular adhesion junctions are crucial for multicellular systems because they provide mechanical stability and mediate signal transduction between cells. Among the variety of specialized intercellular junctions, desmosomes are considered to be the most dedicated to mechanical stability. They are found in tissues that are subjected to mechanical stress, such as cardiac muscle and skin. Recent data underline the importance of desmosomes, which not only maintain tissue integrity, but also play an important role in tissue morphogenesis (reviewed in ). The importance of desmosomes is emphasized by genetic and autoimmune diseases associated with defects in various desmosomal components. For example, disruption in expression or function of the desmosomal intercellular proteins results in skin-blistering diseases, such as the autoimmune pemphigus, in which the tissue integrity is severely compromised [2,3].
Desmosomes are symmetrical disc-shaped junctions comprising three distinct gene families: cadherins, armadillo proteins and plakins [4,5] (Figure 1). Extracellularly, desmosomes use physical associations between cadherin family members of Ca2+-dependent transmembrane proteins characterized by five tandem extracellular domains (EC1–EC5), which are homologous with the extracellular domains of classical cadherins. Unlike the adherens junction, another type of intercellular junction which involves homophilic extracellular interactions between one type of classical cadherin, desmosomes require interaction between two types of cadherins called desmocollin (Dsc) and desmoglein (Dsg). Whether this interaction is homophilic or heterophilic remains controversial [6–9].
Intracellularly, desmosomes are strictly distinct from adherens junctions, making up a set of proteins which constitute an electron-dense plaque that is ultimately connected to the cell cytoskeleton. This plaque consists of an ODP (outer dense plaque) and an IDP (inner dense plaque) situated closer to and further from the plasma membrane respectively . Many efforts to deconstruct these plaques to understand how the constituent proteins interact have been hampered by the highly insoluble nature of the desmosome. However, a combination of in vivo protein–protein interaction and reconstitution studies has provided important information about the interactions of proteins in the intracellular plaques of desmosomes (reviewed in ). Dsc and Dsg cytoplasmic domains interact with a member of the armadillo signalling proteins, namely plakoglobin (PG) and plakophilin (PP). PG associates via an intracellular domain which resembles the classical cadherin-binding site for β-catenin. PG is notable for being common to both desmosomes and adherens junctions . PG binds via its central armadillo repeat domain to the N-terminal of desmoplakin (DP), a major plakin protein, which ultimately links, through its C-terminus, to the cytoskeletal intermediate filaments. PP has much more complex associations with desmosomal components, including desmosomal cadherins, PG, the N-terminal of DP and also directly with intermediate filaments, thus contributing to a complex network of lateral interactions in the cytoplasmic plaques [12,13]. These cytoplasmic interactions have been shown to be essential for strong cellular adhesion through their role in molecular clustering and signal transduction [13–15].
Cadherin ectodomain interactions
Classical cadherins have been most extensively studied using structural, biophysical and mutagenesis methods to identify the molecular interfaces that mediate the interactions of their extracellular domains. Initial X-ray studies of the first domain (EC1) of N-cadherins (neural cadherins) provided the first compelling model for cadherin interactions . The EC1s of N-cadherins interact at their N-terminal tips forming a ‘linear zipper’ (Figure 2). In this zipper arrangement, the laterally associated cadherin ‘strand’ dimers engage similarly oriented dimers on the neighbouring cells. This important interaction involves the domain swap of the highly conserved Trp2. This zipper model was supported by EM (electron microscopy) data of recombinant E-cadherins (epithelial cadherins), in which Ca2+-dependent two-step association of cis and trans interactions at the N-terminal tips were observed respectively . Subsequent crystallographic studies of the first two domains (EC1–EC2) of E- and N-cadherins revealed contact interfaces different from the zipper model [18,19]. For example, the two-domain E-cadherin dimers are aligned in parallel orientation with the closest contacts seen in the Ca2+-binding region, leaving the N-terminals of EC1 far less intimately associated than is required for strand exchange to occur, as described in the crystal structure of Shapiro et al. . Also, the crystal structure of EC1–EC2 of E-cadherin  showed the molecules in an intertwisted X-shaped form with similar contact regions to the previous study  but different to the zipper model in that Trp2 was docked in the hydrophobic pocket of its own molecule. These two models [18,20], called ‘calcium-site’ models, are now considered as crystallization artefacts (discussed in ).
The first crystal structure of the full ectodomain from C-cadherin (representative of classical cadherin) from Xenopus showed that the molecules adopt a stable curved conformation , which is consistent with EM studies of isolated native and recombinant Ca2+-bound E-cadherin extracellular domains [17,23]. These molecules are engaged in the domain swap of Trp2 in the EC1, but this time mediating trans interactions (Figure 2) and not cis interactions as originally interpreted . There is now compelling evidence supported by mutagenesis data and cell-adhesion assays that such Trp2 trans interactions play a pivotal role in cadherin-mediated adhesion . In contrast with the EC1 domain swapping, direct force measurements between cadherin ectodomains have shown extensive domain interactions [24–26]. The cadherin ectodomains bearing C-terminal affinity tags were bound to a lipid monolayer that had been deposited on to a mica surface. Using a surface-force apparatus, both the magnitude and the distance dependence of the adhesive force were measured as the surfaces were brought together and separated. Assuming elongated straight cadherin molecules, these measurements revealed three distinct binding states corresponding to three bilayer separations. The maximum adhesive force develops when the bilayer separation corresponds to the full length of the ectodomain, suggesting that the cadherins from opposed bilayers are fully interdigitated. This implies alternate interactions of EC1 with the EC2–EC5 domains. However, the use of force measurements alone can not identify specific domain–domain interactions. Utilization of highly sensitive intermolecular-force microscopy provides an alternative explanation for the molecular interdigitation . This study revealed four distinct bound states of cadherin interactions, each identified by its unique bond strength, defined as the force required to break the interaction between paired cadherin molecules. On the basis of these findings, a cell-adhesion model involving multiple domain interactions was proposed , which, for the first time, supports the domain swapping observed in the strand dimer and also explains the molecular interdigitation proposed previously by the surface-force measurements. The characteristic of this model is that the naturally curved cadherin molecules have an increased bending capacity, so that the corresponding cadherin domains from opposed cell membranes overlap involving the Trp2 domain swapping of the EC1 domains.
EM and electron tomography of desmosomes
Desmosomes are readily identified in transmission electron micrographs by their lamellar structure characterized by a prominent dense midline between opposing cell surfaces and by electron-dense cytoplasmic plaques into which putative keratin intermediate filaments are seen to insert .
A previous study, using electron tomography of freeze-substituted desmosomes from neonatal mouse epidermis, revealed a disorganized three-dimensional array of cadherin molecules . Modelling of the crystal structure of the C-cadherin ectodomain  on to the tomographic maps revealed a stochastic interaction of the cadherins, which was described as three prominent arrangements that resemble the letters W, S and λ in which the molecules interact at their N-terminal tips. These molecular interactions imply high flexibility of EC1 domain dimers. Although a W shape implies the symmetrical exchange of the Trp2 residues, similar to the structure by Boggon et al. , an S shape implies the interaction between Trp2 of one molecule and the non-active site of the neighbouring molecule without inserting Trp2 into the hydrophobic pocket of the neighbouring molecule.
In contrast with freeze-substituted skin samples, vitrified samples showed, originally by two dimensional EM and later by electron tomography, the periodic nature of the desmosome [30,31]. In the two-dimensional electron micrographs, the extracellular domains of desmosomal cadherins were visualized as densely packed and periodically arranged, but protruding in a straight manner from the corresponding cell membranes (Figure 3), and hence the curved nature of cadherin ectodomains could not be resolved. The three-dimensional reconstruction of electron tomograms verified the quasi-periodic arrangement of the cadherins at ∼7 nm intervals along the midline with a curved shape resembling the X-ray structure of C-cadherin (Figure 3). The periodicity is in agreement with the molecular packing observed in the X-ray structure of the EC1 of N-cadherins reported by Shapiro et al. , and also with the periodicity seen in freeze-substitution images . Sub-tomogram averaging revealed also the periodic arrangement of the cadherin molecules as well as the predominant alternating trans and cis interactions (Figure 4) similar to those shown by Shapiro et al. . The resulting model explains the two-dimensional projection images observed previously with CEMOVIS (cryo-EM of vitreous sections) projection images at various orientations.
This highly organized structure of the desmosome, as is the natural state in vivo, has been shown to be Ca2+-independent, and is referred to as hyper-adhesive [33,34]. This state may seem, at first, counterintuitive, because Ca2+-independent cell adhesion arises from Ca2+-dependent molecules. However, this might be taken as strong evidence for a possible role of Ca2+-site domains in maximizing cell adhesion in the absence of Ca2+. Upon wounding in epidermis and confluent epithelial sheets, desmosomes adopt another state characterized by Ca2+-dependence and loss of the midline, suggesting a less-organized structure [33,34]. This state was shown to mimic the characteristic of adherens junctions which are Ca2+-dependent . Hence it was hypothesized that the extracellular region of adherens junctions are naturally disorganized . Electron micrographs based on freeze–fracture and deep etching of adherens junctions showed a disorganized array of oligomers forming globule-like structures in the midline of the extracellular space , which was thought to support this hypothesis . On the other hand, cryo-EM of artificial VE-cadherin (vascular endothelial cadherin)-based adherens junctions revealed an organized structure of cadherins, self-assembled into hexamers , which was confirmed by single-particle averaging of negatively stained recombinants of VE-cadherin . It is appealing to speculate that adherens junctions and desmosomes could adopt different arrangements of cadherins given the marked difference in the respective intermembrane distance. This is likely to be the case given the molecular flexibility of the EC1 domain dimers in classical and desmosomal cadherins. It is well known that there are substantial differences between the binding interfaces of both classical and desmosomal cadherins and type II cadherins as they are unable to bind one another . Most notably, classical and desmosomal cadherins have a single conserved tryptophan residue in the A-strands of their EC1 domains, whereas type II cadherins have two conserved tryptophan residues involved in the domain swap of the EC1 domain, resulting in a rigidified dimer interface of type II cadherins compared with classical and desmosomal cadherins . The high flexibility of EC1 domain dimers has, in fact, been reported within members of classical cadherins. In particular, analysis of various X-ray crystals of several types of classical cadherins (N-, E- and C-cadherins) has shown intermolecular angles of the EC1 domain dimers ranging between 54° and 88° [16,22].
Despite their overall structural stability ensuring strong adhesion between cells, desmosomes are highly dynamic structures, capable of rapid assembly and disassembly. This is necessary because individual keratinocytes need to migrate and differentiate during embryonic development or wound healing. Therefore considerable changes of cell–cell contact have to occur, involving breakdown and formation of intercellular junctions. If the junctions were not capable of a continuous turnover, the patterning of tissue would be lost. Therefore desmosomes are functionally flexible intercellular junctions that ensure strong adhesion and, at the same time, facilitate cell movements for proper morphogenesis and patterning during tissue development.
Adherens junctions are formed before desmosomes, which indicates a potential hierarchical relationship in junction formation . In addition to this temporal order, a spatial order of the different types of junctions was also observed at an early stage of embryonic development in mice, in which desmosomes characterized by the dense midline and the electron-dense plaques were localized to the basolateral cell–cell contacts . The same order was observed in cultured MDCK (Madin–Darby canine kidney) cells using specific inhibitory antibodies in combination with Ca2+-switch assays . Furthermore, classical cadherins have been shown to be essential for regulation and assembly of desmosomes in keratinocytes [44,45]. Conversely, defects in desmosomal proteins disrupt the function of adherens junctions. Dp-deficient epidermis inhibits desmosome formation and adhesion mediated by adherens junctions . Conclusively, adherens junctions initiate cell–cell contacts that are later stabilized by desmosomes.
Detailed analysis at early adhesion of keratinocytes revealed that desmosome assembly starts with zippering of actin-containing junctions at the tips of filopodia of adjacent cells, generating a double row of punctate adherens junctions, and then desmosomes stabilize adhesion by forming between the lateral filopodial surfaces . Therefore the role of the adherens junction is to bring the membranes into close proximity to allow for desmosomal cadherin clustering and adhesion, thereby forming the desmosome. Clustering was shown to be an important step of junction formation given the intrinsic weakness of interaction between single cadherin molecules [17,47–49]. Therefore one might postulate that the plasticity of desmosomes, and, in general, cadherin-mediated junctions, is gained from the intrinsic weakness of individual cadherin interactions. In fact, a recent study based on numerical calculations showed that this weak affinity of cadherin interactions arises from competition of the domain swapping of Trp2 between monomeric and dimeric species, because the same interface is formed by the swapped domain in both cases . Furthermore, it was shown that, besides this low affinity, it is necessary for the cadherin concentration at the initial cell–cell contact to be kept low enough so that the binding specificity of cadherins is preserved. Therefore strong adhesion arises only from a high concentration of cadherins expressed on cell surfaces which leads to numerous cadherin interactions. In the case of desmosomes, it is not clear whether the presence of two types of cadherins, Dsc and Dsg, is essential for maximal adhesion, as it is not clear whether these molecules interact at all [6–8]. Heterodimeric interaction between Dsc and Dsg was shown to be required in transfected cells . On the other hand, it was shown that peptides against both Dsc and Dsg were required to block adhesion in epithelial cells, suggesting that interactions between desmosomal cadherins are mainly homophilic . This is also supported by the analysis of cadherin specificity by Chen et al. , which shows that, at equilibrium conditions, homophilic dimerization is preferred to heterophilic dimerization. Therefore desmosomal cadherins are unlikely to form cis heterodimers and perhaps not trans heterodimers either.
Conclusions and future directions
Over the last 20 years, much work has been done to elucidate the protein–protein interactions underlying the desmosome adhesion. Although biochemical studies have significantly increased our understanding of how desmosomes are built up, we still do not know how these interactions are temporally and spatially co-ordinated in living cells in order to regulate assembly, disassembly and wound healing in tissues. Understanding the structural and functional aspects of these processes will most probably require hybrid approaches, such as correlative live-cell imaging and cryo-electron tomography of vitreous sections.
Detailed analysis of human diseases and knockout mice model systems has shown important roles for desmosomes in heart and skin integrity. Disruption in the function of desmosomal proteins often results in tissue fragility, with significant clinical consequences. Ultrastructural and tomographic data on the architectural consequences of these diseases will have an important impact not only on basic cell research, but also on therapeutic efforts to interfere with the pathogenesis of heart and skin diseases caused by defects in desmosomal components.
We thank Margot Scheffer for critically reading the manuscript. This work was supported by grants from the FP6 Marie Curie mobility network and EMBO (European Molecular Biology Organization) fellowships to A.A.-A. and from the FP6 3DEM (three-dimensional electron microscopy) network of excellence to A.S.F.
Structure and Function in Cell Adhesion: Biochemical Society Annual Symposium No. 75 held at The Palace Hotel, Manchester, U.K., 3–5 December 2007. Organized and Edited by David Garrod (Manchester, U.K.).
Abbreviations: C-cadherin, representative of classical cadherin; CEMOVIS, cryo-electron microscopy of vitreous sections; DP, desmoplakin; Dsc, desmocollin; Dsg, desmoglein; EC1(etc.), extracellular domain 1 (etc.); E-cadherin, epithelial cadherin; EM, electron microscopy; IDP, inner dense plaque; N-cadherin, neural cadherin; ODP, outer dense plaque; PG, plakoglobin; PP, plakophilin; VE-cadherin, vascular endothelial cadherin
- © The Authors Journal compilation © 2008 Biochemical Society