Structure, Function and Regulation of Desmosomes

I.A. Desmosome Structure and Morphology

Desmosomes are specialized and highly ordered membrane domains that mediate cell-cell contact and strong adhesion. Adhesive interactions at the desmosome are coupled to the intermediate filament cytoskeleton. By mediating both cell–cell adhesion and cytoskeletal linkages, desmosomes mechanically integrate cells within tissues and thereby function to resist mechanical stress [1-3]. This essential structural and mechanical function is highlighted by the prominent distribution of desmosomes in tissues that are routinely subjected to physical forces, such as the heart and skin, and the wide range of desmosomal diseases that result from disruption of desmosome function [4-6]. At the ultrastructural level, desmosomes appear as electron dense discs approximately 0.2–0.5 μm in diameter, which assemble into a mirror image arrangement at cell–cell interfaces [1, 7, 8] (Fig. 1). Large bundles of intermediate filaments extend from the nuclear surface and cell interior out toward the plasma membrane, where they attach to desmosomes by interweaving with the cytoplasmic plaque of the adhesive complex. The overall adhesive function of the desmosome is dependent upon the tethering of intermediate filaments to the desmosomal plaque, highlighting the integrated functions of adhesion and cytoskeletal elements. Thus, desmosomes are modular structures comprising adhesion molecules that bolt cells together, cytoskeletal cables that disperse forces, and linking molecules at the cytoplasmic plaque of the desmosome that carry mechanical load from the adhesion molecules to the intermediate filament cytoskeleton.

I.B. Desmosome Molecular Composition

Three major gene families encode desmosomal proteins. Desmosomal cadherins, comprising two subtypes called desmogleins and desmocollins, are a subfamily of the cadherin superfamily that mediate calcium-dependent cell–cell adhesion [1, 3]. In humans, four genes encode desmogleins (Dsg1-4) and three genes encode desmocollins (Dsc1-3) [2] (Fig. 2). These proteins include five extracellular cadherin repeats, each of which form Ig-like globular domains with calcium binding sites in between each pair of consecutive repeats [9]. The cytoplasmic domains of desmogleins and desmocollins both contain an intracellular anchor (IA) and a cadherin-like sequence (ICS), which is conserved in classical cadherins. The desmogleins have additional unique sequences with unknown functions, including a proline rich linker region (IPL), a repeat unit domain (RUD), and a desmoglein terminal domain (DTD) [10] (Fig. 2). Each of the three desmocollin RNAs can be alternatively spliced to yield an “a” and a “b” isoform. In the “b” splice variant, the region encoding the ICS domain is truncated and terminates with an additional 11 amino acids in Dsc1 and 2, and eight residues in Dsc3, not found in the “a” form [3]. The desmosomal cadherin genes are expressed in a tissue- and differentiation-specific manner [1, 3] (Table I), and emerging evidence suggests important roles for these expression patterns in driving epithelial patterning and differentiation.

Structural analysis of cadherin ectodomain interactions has yielded important insights into how cadherins mediate adhesive interactions between cells. In the case of classical cadherins, the defining molecular interaction mediating adhesion is termed a strand swapped dimer. This trans dimer is formed when a conserved tryptophan residue (Trp-2) on a cadherin from one cell is inserted into the hydrophobic pocket of a partnering cadherin on an adjacent cell. This adhesive dimer requires formation of an X-dimer intermediate, which lowers the energy needed for tryptophan swapping [9, 11]. Atomic level structural information on desmosomal cadherin ectodomain interactions is currently unavailable. However, the desmosomal cadherins, both desmogleins and desmocollins, possess the conserved tryptophan in their EC1 domain [9]. Furthermore, cryoelectron tomography and modeling based on classical cadherin ectodomain structures is consistent with EC1 domain interactions between desmosomal cadherins [12-14]. Lastly, crosslinking experiments suggest that the conserved Trp-2 residue in desmocollin is required for homophilic interactions of this desmosomal cadherin [15]. Nonetheless, we do not fully understand how this large and complex subfamily of cadherins interacts to facilitate desmosome assembly and adhesion.

Armadillo (Arm) family proteins represent cadherin binding partners that play important roles in tissue integrity and cell signaling. Two types of armadillo proteins are present in desmosomes [16, 17] (Fig. 2). Plakoglobin is a founding member of the armadillo gene family, defined by the presence of a 42 amino-acid repeat motif found in the closely related Drosophila armadillo protein and its vertebrate orthologue, β-catenin [18]. Plakoglobin binds to the ICS domain of desmogleins and desmocollins and functions as a bridge between the cadherin tail and intermediate filament binding proteins such as desmoplakin [2] (see below). Desmosomes also contain plakophilins, members of the p120-catenin subfamily, which have 9 Arm repeats in contrast to the 12 repeats in plakoglobin and β-catenin [17, 19].Humans express three plakophilins (PKP1-3) that are expressed differentially in simple and stratified epithelia, cardiomyocytes, endothelia, and other cell types [16].Biochemical and cryoelectron microscopy tomography suggest that desmoplakin, through interactions with both plakoglobin and plakophilins, drives clustering and lateral interactions between desmosomal cadherins [20-23].In addition to their important structural roles, both plakoglobin and plakophilins exhibit diverse junction-independent functions in the cytoplasm and nucleus [19, 24, 25].

The third major gene family encoding desmosomal proteins is the plakin family [26]. Among its members, desmoplakin is an obligate desmosomal protein that couples intermediate filaments to the desmosomal plaque [27, 28] (Fig. 2). The desmoplakin amino-terminus [26], binds directly to plakoglobin and the plakophilins [2, 20, 29, 30]. An extended alpha-helical coiled-coil rod links the plakin domain to a carboxyl terminal globular domain, which binds directly to intermediate filaments [31-33]. Humans have a single desmoplakin gene, which is alternatively spliced to yield desmoplakin I and desmoplakin II, with the desmoplakin II variant lacking about two-thirds of the rod domain [34]. The desmoplakin-I-specific sequences might facilitate association with microtubule binding proteins (see below and [35]), while the desmoplakin II variant appears to be particularly important in epidermal adhesion [36].

I.C. Desmosome-Associated Proteins

A variety of minor and/or differentiation-specific proteins are associated with desmosomes [37]. Corneodesmosin is a glycoprotein expressed primarily in the upper layers of the epidermis and hair follicles, where it contributes to keratinocyte cohesion and is a protease target during epidermal desquamation [38]. Another recently discovered desmosome protein with restricted expression is Perp (p53 apoptosis effector related to PMP-22) [39]. As a target of p53/p63 transcriptional regulation, Perp is expressed predominantly in stratifying epithelia where it plays an essential role in intercellular adhesion [39].Other reported desmosome-associated proteins include the calmodulin binding protein keratocalmin,the adaptor protein and RhoA regulator kazrin, a keratin-binding protein pinin, and several members of the plakin family [26, 37, 40, 41]. How these proteins contribute to desmosome function and the stoichiometry of their associations with the major desmosomal proteins is not well understood.

II.A. Desmosomal Cadherin Trafficking

Desmosomes are are constructed from distinct, cadherin- and plaque-associated complexes that form in the cytoplasm and are delivered to regions of cell-cell contact, where final assembly occurs. The calcium sensitive nature of desmosomal adhesion has served as tool to manipulate desmosome formation. When cells are cultured in low calcium, desmosomal proteins are recruited to the cell surface but are rapidly retrieved and degraded due to the inability of the desmosomal cadherins to mediate adhesion in the absence of calcium [2]. Upon addition of calcium, junction assembly is triggered and junctional proteins are stabilized. Early biochemical studies revealed that the desmosomal cadherins and plakoglobin co-fractionate, while desmoplakin is a part of distinct multiprotein complexes that separately traffic to cell–cell contacts [42]. This early work has been advanced by the use of fluorescently tagged desmosomal components and time-lapse imaging techniques, allowing direct visualization and analysis of desmosome molecule dynamics, including movement triggered by cells coming into contact with each other in response to calcium or at the edge of a scrape wound [43, 44].

Recent studies implicate the microtubule network and kinesin motor proteins in transporting desmogleins and desmocollins to sites of cell adhesion [45]. Interestingly, desmocollin delivery to the membrane requires kinesin-2, whereas desmoglein transport is dependent upon kinesin-1. Selective inactivation of either kinesin weakens cell–cell adhesion strength, with knockdown of both motors having additive effects. This dynamic arrangement points to a potential mechanism for regulating desmosome structure and adhesive strength through the independent control of individual cadherin subunits. PKP2 silencing interferes with long range microtubule-dependent trafficking of Dsc2 but not Dsg2, indicating that armadillo proteins might functionally couple desmosomal cadherins to the motor complex in some cases.

Advances have also been made in understanding how desmosomes are dismantled. Disruption of adhesion by calcium chelating agents was used in early studies to inactivate desmosomal cadherins, resulting in their rapid retrieval from the plasma membrane by the engulfment of entire junctions [1]. Similar mechanisms might be involved in the down-regulation of desmosomal adhesion upon epidermal injury [46]. More recent studies of desmosomal cadherin endocytosis have utilized antibody mediated disruption of adhesion, using IgG from patients afflicted with pemphigus vulgaris, a skin disease involving the generation of IgG against desmogleins (Dsg3 and Dsg1, see below). These auto-antibodies cluster desmogleins on the cell surface, leading to lipid raft mediated endocytosis and loss of cell surface desmogleins [47, 48]. Consistent with this pathway for desmoglein endocytosis, several recent studies have reported that desmosomal proteins co-fractionate with membrane raft microdomains [49, 50] (Stahley and Kowalczyk, unpublished). Thus, lipid raft carriers appear to play a role in regulating desmosomal cadherin trafficking, perhaps during both assembly and disassembly of desmosomes.

While sequences in the desmoglein cytoplasmic domain that regulate its association with lipid rafts or the endocytic machinery are not well understood, recent evidence suggests that intermolecular tail-tail interactions requiring the desmoglein unique region dampen Dsg2 internatlization.A mutation in this region associated with arrhythmogenic cardiomyopathy abrogates Dsg2 tail interactions and significantly enhances Dsg2 internalization [51].Components of the trafficking machinery could be targets in inherited diseases that cannot be explained by mutations in known desmosomal genes.

II.B. Desmoplakin and Plakophilins

Desmosomal plaque proteins in cultured keratinocytes exhibit a multi-phasic assembly process involving the formation and dynamic recruitment of non-membrane bound particles to sites of junction assembly. The initial wave of desmosome assembly is characterized by the localized accumulation of PKP2 at sites of cell-cell contact, followed closely by its binding partner desmoplakin [44, 52]. Desmoplakin accumulation at cell borders is followed by the de novo formation and translocation of desmoplakin/PKP2-containing cytoplasmic particles to the cell surface, a process requiring PKP2. The results suggest a model whereby de novo cell-cell contacts send signals to cytoplasmic plaque precursors, which are then translocated to regions of cell adhesion in an actin dependent manner [52].

To create a functional desmosome, desmoplakin needs to tightly couple desmosomal cadherins to the intermediate filament cytoskeleton. On the other hand, desmoplakin association with intermediate filaments needs to be highly dynamic in order for the protein to be transported efficiently to the cell surface during desmosome assembly. These competing requirements appear to be resolved by the ability of the plakophilins to scaffold serine/threonine kinases that modify desmoplakin association with the intermediate filament network. Supporting this idea, PKP2 associates with PKCα in a complex with desmoplakin, and PKP2 silencing disrupts this complex [53]. PKCα inhibition, PKP2 silencing, or mutation of S2849 downstream of the intermediate filament binding site in desmoplakin all result in retention of desmoplakin on intermediate filament networks and impair desmoplakin accumulation at sites of cell-cell contact. Desmoplakin translocation also requires actin and is regulated by RhoA through a PKP2-dependent mechanism [52]. Thus, plakophilins play critical roles in desmoplakin trafficking to sites of desmosome assembly through the regulation of both desmoplakin carboxyl-terminal domain phosphorylation and actin dynamics. We do not fully understand the relative contributions of the three plakophilin genes, which appear to drive desmosome assembly with different efficacies.

The regulation of desmoplakin dynamics might contribute to compromised epidermal adhesion observed in human Darier’s disease. This disease is caused by mutations in the ATPase that pumps calcium ions from the cytosol into the ER and is thus critical for maintaining calcium homeostasis [54]. Recent evidence suggests that silencing the Darier’s calcium pump interferes with desmoplakin transport to desmosomes as a result ofdefects in PKCα signaling and consequent retention of desmoplakin along keratin filaments [55] The accompanying defects in cell-cell adhesion could be reversed by activating PKC [55]. These studies highlight the importance of defining fundamental mechanisms of desmosome modulation in order to gain insights into disease pathomechanisms and to reveal new therapeutic targets.

II.C. Desmosome Maturation and Regulation of Adhesion

During the first several days after their formation, desmosomes continue to depend on extracellular calcium for their maintenance at the plasma membrane. However, over a period of a week or so they undergo a maturation process during which they become calcium-independent and achieve what has been coined a “hyperadhesive” state by Garrod and colleagues [56]. Upon wounding, desmosomes lose their hyperadhesive state in response to recruitment and activation of PKCα to the wound edge [57]. While the mechanism underlying the acquisition of hyperadhesion is unclear, one model is that desmosomal cadherin ectodomains undergo a conformational change that contributes to this state [56]. It has also been reported that keratinocyte cell sheets expressing a desmoplakin mutant with enhanced keratin-binding properties acquire strong adhesion over time and resist reduced calcium and activation of PKC [58]. Similarly, PKP1 over-expression (Tucker and Kowalczyk, unpublished) or loss of function mutations [59] alter desmosome adhesive strength and dependency on calcium. Together, these observations underscore the importance of desmosomal plaque protein composition in modulating desmosome adhesion.

Desmosomes in the epidermal stratum corneum are highly cross-linked and stabilized, creating a unique challenge for homeostatic regulation of desmosome turnover in the framework of a regenerating epithelium. In this context, desmosome turnover is thought to involve a carefully orchestrated process of degradation regulated by serine proteases and protease inhibitors expressed in the skin.Desmogleins are substrates for members of the kallikrein protease family, eight of which are expressed in the epidermis [60], as well as ADAM (A Disintegrin And Metalloprotease) family proteases [61]. Upsetting the balance of desmoglein synthesis and degradation and consequent alterations in desmosome function can result in human diseases such as Netherton syndrome, discussed in the section below.

III.A. Epidermal Autoimmune Disorders

The first studies to recognize a direct role for desmosomes in disease came from dermatologists studying pemphigus, a family of autoimmune disorders associated with epidermal fragility and blistering [4]. The hallmark of pemphigus is the loss of adhesion between keratinocytes (acantholysis) due to the generation of antibodies against desmogleins. In pemphigus foliaceus, autoantibodies (IgG) targeting Dsg1 are generated, resulting in superficial blisters in the granular layer of the epidermis. In pemphigus vulgaris, IgG targeting both Dsg3 and Dsg1 are produced, resulting in deep epidermal blisters and oral erosions [2, 62, 63]. The localization of blistering generally correlates with the expression patterns of the desmoglein antigens (Table I), providing important evidence that desmosomes are essential for maintaining strong cell-cell adhesion in stratifying epithelia. Pemphigus disease activity can be transferred by introducing IgG from the patient into mice or cell culture [64].This property has provided investigators with a tool to investigate disease pathomechanisms and basic cellular mechanisms of desmosome regulation. One proposed mechanism for antibody-mediated loss of adhesion is steric hindrance, which would be predicted to occur if Abs occupy sites on the cadherin extracellular domain that are involved in cell-cell adhesion [65, 66]. Evidence from atomic force microscopy and other approaches lend credence to this mechanism of pathogenicity [67]. However, other studies have shown that signal transduction pathways, particularly the p38 MAPK pathway, are activated downstream of antibody binding [68], and inhibitors of p38 MAPK prevent loss of adhesion [69, 70]. Furthermore, pemphigus antibodies cause desmoglein endocytosis, thereby depleting cell surface levels of the cadherin and compromising the adhesive potential of the cell surface [47, 48, 71, 72]. Desmoglein internalization and antibody-mediated loss of adhesion can be prevented by p38 MAPK and tyrosine kinase inhibitors [48, 73, 74]. If pemphigus antibodies cause loss of adhesion by steric hindrance alone, it is difficult to understand how inhibition of intracellular signaling pathways prevents loss of adhesion strength. These and other data suggest that these antibodies cause a range of effects on desmogleins and their associated regulatory pathways [70, 75-77]. Additional study of the pathomechansims of pemphigus should yield new therapeutic targets and insights into how keratinocytes regulate desmoglein cell surface levels and adhesion.

III.B. Desmosomes as Targets for Pathogens and Proteolysis

Whereas epidermal desmogleins are targets for autoimmune antibodies in skin disease, Dsg2 was recently identified as a receptor for a subclass of adenoviruses (serotypes 3, 7,11, and 14) that cause respiratory and urinary tract infections [78]. Adenoviral particle binding to Dsg2 triggered phenotypic changes in the target epithelial cells similar to those induced during epithelial-mesenchymal transitions. These data are interesting with respect to viral pathogenicity, and also provide evidence that desmosomal cadherins modulate the epithelial phenotype.

Desmogleins are also targets for both bacterially produced and endogenous proteases.A toxin produced by the staphylococcus bacteria that causes bullous impetigo, exfoliative toxin A (ETA), is a serine protease that cleaves Dsg1 after residue 381 between ECs 3 and 4 (Table I).This cleavage removes sequences required for cell-cell adhesion in the superficial epidermis, resulting in focal lesions that histologically resemble pemphigus foliaceus [79]. The specificity of the protease is quite remarkable; no other protein is known to be cleaved by ETA or the other closely related proteases produced by staphylococcal bacteria [80]. Dramatic evidence for this specificity is provided by staphylococcal scalded skin syndrome (SSSS), which is observed in infants and immune-compromised patients. In SSSS, the bacterial infection becomes systemic with extensive epidermal involvement. No other organ systems are affected by the protease, and the disorder can be successfully treated with antibiotic regimens to eliminate the bacterial infection. The discovery that Dsg1 is the target of ETA (Table I), provides unique verification for the role of Dsg1 in pemphigus foliaceus. Indeed, injection of either pemphigus foliaceus IgG or ETA into mouse epidermis produces an identical phenotype [62]. These observations, along with the genetic disorders discussed below, firmly establish the role of desmosomal cadherins in epidermal function and integrity.

Netherton syndrome is an autosomal recessive disease caused by mutations in the serine protease inhibitor LEKT1 (Lympho-epithelial Kazal-type related inhibitor) [81]. Loss of this protease inhibitor results in excessive tryptic and chymotryptic enzyme activity attributed to members of the kallikrein protease family.Reduced proteolysis of Dsg1 was proposed to be a central contributor to the aberrant desquamation and keratinization in this disorder [82]. Kallikrein-5 dependent proteolysis of Dsg1 [83] and ADAM-dependent proteolysis of Dsg2 have also been suggested to promote their turnover in oral squamous cell carcinoma cells [84]. Further, retention of Dsg2 was observed in the epidermis of patients with a recessive loss of function mutation in ADAM17 resulting in neonatal-onset inflammatory skin and bowel disease [85]. Finally, Dsg2 cleavage via cysteine proteinases was also reported to contribute to stimulus-induced apoptosis in intestinal epithelial cells [86]. Together, these observations suggest that aberrant proteolysis of desmogleins contributes to human disease pathogenesis.

III.C. Desmosomes as Targets of Inherited Disease of Skin and Heart

Mutations associated with desmosomal genes are frequently manifested in heart and skin, both of which are exposed to high mechanical stress [87] (Table I). Mutations in Dsg1, Dsg4, and PKP1, which are found in desmosomes of the differentiated layers of the epidermis, cause epidermal disorders without cardiac involvement. In contrast, mutations in genes such as Dsg2 and PKP2 lead to cardiac defects without epidermal disease. Other desmosomal components with more widespread expression patterns, such as desmoplakin, plakoglobin, and Dsc2, affect both heart and skin [5, 6]. Interestingly, the effects of desmosome gene mutations do not appear to be clinically manifest in internal organs, suggesting that desmosome function is most critical in stratifying epithelia and myocardium.

Common to most of the disorders affecting the epidermis is a loss of tissue integrity, likely resulting from a reduction in desmosome numbers and impaired intermediate filament attachment to the plasma membrane [5, 87]. Similarly, structural changes in cardiac intercellular junctions, the intercalated discs [88, 89], are a major contributing factor in the development of arrhythmogenic cardiomyopathy, which can result in sudden death in individuals with this “disease of the desmosome” [89, 90]. Advanced stages of the disease are accompanied by the loss of cardiac myocytes and replacement with fibrofatty tissue. How this transition occurs and the extent to which these changes in heart structure lead to arrhythmia is poorly understood. It now seems that loss of mechanical integrity alone cannot completely explain the arrhythmias that can occur in these individuals. Indeed, the mammalian intercalated disc is now known to remodel postnatally into hybrid junctions in which desmosome components associate not only with other mechanical junction components but also with gap junction components (e.g. Cx43) and the sodium channel protein Nav1.5 [88, 90]. The most commonly mutated gene associated with arrhythmogenic cardiomyopathy is the one encoding PKP2. Loss of this protein leads to changes in electrical coupling and excitability associated with redistribution of these channels from their normal sites at cardiomyocyte cell-cell junctions [91, 92]. It has also been reported that the redistribution of desmosomal armadillo proteins might interfere with nuclear transcription pathways driven by β-catenin, contributing to the fibrofatty replacement during disease pathogenesis [93]. This finding also prompted the suggestion that ectodermal defects such as woolly hair and changes in nails seen in cutaneous manifestations of desmosome disease involve reprogramming of cell fate through alterations in Wnt signaling [93, 94]. This idea is controversial, however, as alterations in β-catenin activity associated with desmosome gene mutations are variable [95].The finding that PKP2 deficiency in epithelial cells results in global activation of RhoA and PKCα [52, 53] suggests that desmosome impairment may affect multiple signaling pathways contributing toskin and heart disease progression. A role for desmosome molecules in signaling has also been implicated in normal tissue differentiation, as discussed below.