Desmosomal Adhesion In Vivo

Abstract

Desmosomes are intercellular junctions that provide strong adhesion or hyper-adhesion in tissues. Here, we discuss the molecular and structural basis of this with particular reference to the desmosomal cadherins (DCs), their isoforms and evolution. We also assess the role of DCs as regulators of epithelial differentiation. New data on the role of desmosomes in development and human disease, especially wound healing and pemphigus, are briefly discussed, and the importance of regulation of the adhesiveness of desmosomes in tissue dynamics is considered.

Keywords::

• desmosome
• cell–cell adhesion
• desmocollin
• desmoglein
• desmosomal cadherin
• development
• wound healing
• pemphigus

INTRODUCTION

Animal body tissues are amazingly tough. This is evident in human epidermis, which ranges from 0.07 mm to 0.12 mm in thickness over the general body surface, increasing to 0.8 mm on the palms and 1.4 mm on the soles. The epidermis is subjected to continuous rubbing and pressure. The living layers of the epidermis are composed of cells, keratinocytes (˜20 μm in diameter) surrounded by plasma membranes (˜70-nm thick). How can such a tissue resist physical stress? There are three contributory structures: (1) the underlying dermis, composed of resilient proteins, collagen, elastin and fibrillin, to which the epidermis is anchored by hemidesmosomes; (2) the cytoskeletal proteins, mainly keratins, which give the cells internal strength; and (3) the adhesive links, which both cement the cells together and provide surface attachments for the keratin filaments, creating a tissue-wide scaffolding support.

The principal adhesive links are intercellular junctions called desmosomes, and the purpose of this article is to discuss their structure and function in tissues. Desmosomes are abundant in stress-bearing tissues including cardiac muscle and stratified epithelia. They are less abundant in simple epithelia, meninges and follicular dendritic cells, but are absent in nervous tissue, skeletal and smooth muscle and endothelia, although they are present in at least some endothelia of fish (Fawcett, 1961).

Tissues are dynamic structures, and there is therefore a conflict between toughness, which is vital for survival of the organism, and the necessity for malleability in development, turnover and damage repair. On the one hand, it seems crucial for the cells to be locked together to provide strength and resilience; on the other, relative cellular motility seems desirable for modelling, replacement and remodelling. The desmosomal lock, which binds cells together, must be capable of unlocking.

Since this article deals only with specific aspects, we refer the reader to other recent reviews for broader coverage of the topic. These include desmosome assembly and dynamics (Nekrasova & Green, 2013), mechanistic basis of heart diseases involving desmosomes (Al-Jassar et al., 2013) pemphigus (Kitajima, 2013; Waschke, 2008), desmosomes and cancer (Chidgey & Dawson, 2007), as well as more general reviews (Dubash & Green, 2011; Kowalczyk & Green, 2013; Thomason et al., 2010).

DESMOSOMAL ADHESION

Adhesion molecules and some evolutionary considerations

Adhesion in desmosomes is mediated by the desmosomal cadherins (DCs), desmocollin (Dsc) and desmoglein (Dsg). In mammals, there are multiple DC isoforms; for example, in humans, there are three Dscs (1–3) and four Dsgs (1–4) (Figure 1). Dsc2 and Dsg2 are ubiquitous in desmosome-bearing tissues, although there is now some disagreement as to whether Dsg2 occurs in the basal layer of interfollicular epidermis or is confined to hair follicles (Hartlieb et al., 2013). The other isoforms are primarily expressed in stratified epithelia; Dsc1, Dsg1 and Dsg4 predominate in the upper layers, and Dsc3 and Dsg3 in the lower layers. In the zebrafish, however, which also has stratified epithelia, there is only one Dsc (zDsc) and two closely related zDsgs (zDsgα and zDsgβ) (Goonesinghe et al., 2012). Curiously, zDsc is the orthologue of mammalian Dsc1 and the both zDsgs are orthologues of mammalian Dsg2.

Figure 1. Diagram showing the distributions of the major desmosomal components and their isoforms in human epidermis. In the cornified layer, desmosomal adhesion is mediated by corneodesmosin, which is not discussed in this review.

Dscs and Dsgs show ˜30% amino acid identity to each other and to Type1 cadherins, which they resemble more closely in their extracellular (EC) domains. Cytoplasmically, Dscs show alternative splicing of a stop codon-containing mini exon that, when spliced out, produces a longer “a” form, containing binding sites for other plaque components, mainly plakoglobin (PG), plakophilin (PKP) and desmoplakin (DP), and, when spliced in, a shorter “b” form that supports PKP but not PG and DP binding (Figures 2 and 4). The “a” form supports desmosomal plaque formation, but the “b” form does not (Troyanovsky et al., 1993), leaving the function of the “b” form unresolved. Mice lacking Dsc1 have adhesion defects in the upper epidermis, but mice lacking the “a” and “b” cytoplasmic domains of Dsc1 do not show such defects, suggesting that the EC and transmembrane domains are sufficient to maintain adhesion (Cheng et al., 2004; Chidgey et al., 2001). Human Dsc genes contain 17 exons, exon 16 encoding the mini exon, but the zebrafish gene contains only 16 exons, lacking the mini exon and therefore a Dsc “b” form (Goonesinghe et al., 2012). Yet, zebrafish form functional desmosomes. ENSEMBL data suggest that Xenopus, the chicken and the zebra finch, also lack a mini exon and thus a “b” form, whereas a mini exon and two spliced forms are present in the duck-billed platypus (D. Garrod; unpublished). Thus, the “b” form may be strictly mammalian, making it even more intriguing to speculate about its function.

Figure 2. Stick diagrams of the DCs, Dsc2 and Dsg2, showing features referred to in text. EC, extracellular; EA, extracellular anchor; TM, transmembrane; ICS, intracellular cadherin segment; IPL, intracellular proline-rich linker; RUD, repeat unit domain; TD, terminal domain.

Figure 3. Graph showing reciprocal exponential distributions of Dsc1 and Dsc3 in bovine nasal epidermis as determined by quantitative analysis of immuno-gold labelling (North et al., 1996).

Figure 4. Desmosome structure. Electron micrograph of two desmosomes from human chronic wound epidermis showing the midline between the plasma membranes, the outer dense plaques and the inner dense plaques with attached keratin filaments (bar = 100 nm). Diagram showing the quadratic array of DCs the intercellular region. This had a periodicity of ˜70Å. Diagram (not to scale) showing transverse lamellar arrangement of desmosome. The locations of the molecules are based on immuno-gold labelling and electron tomography (See text).

The cytoplasmic domains of Dsgs consist of a series of subdomains, an intracellular anchor, an intracellular cadherin segment, an intracellular proline-rich linker, a repeat unit domain (RUD) and a terminal domain (TD) (Figure 2). This unique organisation is present in zebrafish and therefore appears fundamental to desmosome structure and function; true desmosomes do not appear to be present in invertebrates. Apparently normal desmosomes can be formed by cells lacking Dsc and therefore having Dsg as the only DC (Koeser et al., 2003). This suggests that the cytoplasmic domain of Dsg, like Dsc “a”, is sufficient to support desmosome plaque formation.

Possible functions of the DC isoforms

It is not clear why mammals require different isoforms of DCs. Studies of human skin diseases and gene knock-out in mice indicate that the different isoforms have functions that, broadly speaking, correlate with their expression patterns. Thus, in the blistering disease pemphigus foliaceus autoantibodies that target Dsg1 cause loss of cell–cell adhesion (acantholysis) in the granular layer (Mahoney et al., 1999). Bacterial exfoliative toxins, serine proteases, show exquisite specificity for Dsg1 and cause blistering only in the superficial epidermis (Hanakawa et al., 2004). Autosomal dominant DSG1 mutations cause striate palmoplantar keratoderma, the formation of hyperkeratotic plaques on the fingers, palms and soles (Rickman et al., 1999), and homozygous mutations in DSG1 cause severe dermatitis, multiple allergies and metabolic wasting syndrome (SAM), which involves acantholysis in upper epidermis (Samuelov et al., 2013). Knockout of Dsc1 from mice causes acantholysis in the granular layer and epidermal fragility (Chidgey et al., 2001).

By contrast, autoantibodies against Dsg3 cause acantholysis between the basal and first suprabasal layers of epidermis and oral mucosa in the blistering disease pemphigus vulgaris (PV) (autoantibodies to Dsg1are generally also required to produce epidermal blistering) (Mahoney et al., 1999; Shirakata et al., 1998). Knockout of Dsg3 from mice also causes suprabasal acantholysis in oral mucosa and epidermis (Koch et al., 1997). Global knockout of Dsc3 from mice causes death at E2.5, before the first desmosomes form, but conditional knockout from the epidermis gives rise to PV-like suprabasal acantholysis (Chen et al., 2008; Den et al., 2006). Pathogenic antibodies to Dsc3 have also been demonstrated in PV (Mao et al., 2010), and it is likely that such antibodies also cause pemphigus vegetans (Saruta et al., 2013).

The cleavage planes due to loss of cell adhesion are quite specifically localised, much more so than expression of the various targeted isoforms of DCs. In fact, the territories in which the different isoforms are expressed overlap extensively and in at least one case, the bovine nasal epidermis, Dsc3 and Dsc1 show exquisite exponential reciprocally graded expression (Figure 3) (North et al., 1996). The reason for this is unknown. Does it mean that the adhesive properties of desmosomes change gradually with level in the epidermis? For example, is Dsc1 more strongly adhesive than Dsc3, giving stronger adhesion in the upper epidermis than in the lower regions, and if so, why should this be? Surely the epidermis needs to be strong throughout. In the human keratinocyte cell line HaCaT, which expresses Dsg2 and Dsg3, roughly Dsg3 appears equally more important for adhesion than Dsg2, except at high shear rates when Dsg2 is also important (Hartlieb et al., 2013). This may suggest differential adhesive properties for the different isoforms, thus supporting a previous suggestion based on differential sorting of mammary epithelial cells (Runswick et al., 2001). More work is needed on the differential adhesive properties of DC isoforms.

Reciprocally graded protein distributions have a widely recognised role in developmental pattern formation (Hironaka & Morishita, 2012; Kicheva et al., 2012; St Johnston & Nusslein-Volhard, 1992). Is it possible that the reciprocally graded distributions of DCs play a signalling role in regulating differentiation of stratified epithelia (North et al., 1996)? A series of studies in which the DCs Dsg2, Dsg3 and Dsc3 were mis-expressed in mouse epidermis under the involucrin, keratin 1 or keratin 14 promoters showed perturbation of differentiation and proliferation, thus tending to support a role for these proteins in regulating differentiation (Allen et al., 1996; Brennan et al., 2007; Elias et al., 2001; Hardman et al., 2005; Merritt et al., 2002). However, mis-expression of Dsc1a under the K14 promoter produced no phenotype (Henkler et al., 2001). Inv-Dsg2 keratinocytes showed enhanced survival and anchorage-independent growth dependent on epidermal growth factor receptor (EGFR) activation and NF-κB activity and activation of multiple cell survival pathways, in the mice which were more susceptible to tumour promotion (Brennan et al., 2007). K14-Dsc3 keratinocytes showed enhanced beta-catenin signalling (Hardman et al., 2005).

A powerful case has been made from culture experiments for a role of Dsg1 in regulating epidermal differentiation. Thus, Dsg1 promotes differentiation by suppressing EGFR/extracellular signaling-regulating kinases (ERK) signalling, an effect mediated though the cytoplasmic domain of the DC (Getsios et al., 2009). Further, Dsg1 interacts with Erbin, another differentiation-related protein, to disrupt Ras-SHOC2 complexes, thus diminishing ERK signalling and driving differentiation (Harmon et al., 2013). In turn, Dsg1 promotion of differentiation is regulated by the GEF breakpoint cluster region (Bcr) which regulates Rho/MAL control of Dgs1 expression (Dubash et al., 2013). In this context, it is interesting that striate palmoplantar keratoderma patients deficient in Dsg1 show increased Ras-SHOC2 activity and decreased SHOC2-Erbin co-localisation (Harmon et al., 2013).

Thus, there is considerable evidence that DCs can participate in the regulation of differentiation signals. Do they regulate epidermal differentiation in vivo? Knockout of mouse Dsg3 or Dsc3 produced adhesive defects in interfollicular epidermis, but no defects in epidermal differentiation (Chen et al., 2008; Koch et al., 1997). Knockout of Dsc1 also produced adhesion defects and slight differentiation defects, probably secondary consequences of trauma caused by the adhesion defects (Chidgey et al., 2001). There has not yet been a knockout of Dsg1 but failure of Dsg1 membrane expression in SAM syndrome results in psoriasiform dermatitis with alternating para- and orthokeratosis and hypo- and hypergranulosis (Samuelov et al., 2013). On the basis of knockout evidence so far available, it would seem then that the DCs are not major regulators of epidermal differentiation in mice. On the other hand, the association of mutations in human DSG1 and DSC2 with palmoplantar keratoderma (PPK), and the latter with woolly hair, suggests that they contribute to fine regulation of differentiation (Rickman et al., 1999; Simpson et al., 2009).

Little is known about how DC gene expression is regulated. Dsg1, Dsc3 and Dsc2 genes possess epithelial-specific promoters containing binding sites for transcription factors, the AP-1, AP-2 and Sp1 families, that regulate keratinocyte-specific gene expression (Adams et al., 1998; Marsden et al., 1997; Silos et al., 1996). Smith et al. (2004) showed that Dsc1 and Dsc3 are differentially activated by epidermally expressed members of the CCAAT/enhancer-binding protein (C/EBP) family; both are activated by C/EBPδ, but Dsc1 is specifically activated by C/EBPα and Dsc3 by C/EBPβ (Smith et al., 2004). This may contribute to the differential expression of these genes in epidermis. Furthermore, Dsc2 and Dsc3 expression are differentially regulated by PG; PG activates both genes but suppresses Dsc3 expression in the presence of Lef-1 (Tokonzaba et al., 2013). Additionally, c-rel a component of the ectodysplasin-A (EDA)/ectodysplasin-A receptor (EDAR)/NF-κB signalling cascade activates Dsc2 expression in conjunction with PG/Lef-1. This is seen as a mechanism for differential regulation of Dsc2/Dsc3 particularly in hair follicle development.

The mechanism of adhesion

Adhesive binding by the DCs is directly mediated by their EC domains, which resemble those of Type1 cadherins. As shown by sequence analysis, homology modelling and determination of the NMR structure of hDsg2, the EC domains consist of five subdomains (EC1–5) of which at least the first four have a Type1 cadherin-like β-barrel structure. Furthermore, the inter-domain regions possess low-affinity calcium-binding sites. These are occupied at physiological EC calcium concentrations of > 1 mM and are functional because they maintain the molecules in an extended, adhesion-competent configuration (Koch et al., 1997; Nagar et al., 1996; Pokutta et al., 1994). When the sites are unoccupied, the EC domains adopt a flexible, non-adhesive conformation.

Because of this, cadherin and DC adhesion is conventionally referred to as “calcium-dependent.” Calcium dependence provides a useful, if artificial, tool for the cell biologist, enabling manipulation of cell adhesion and junction formation in culture (Hennings & Holbrook, 1983). Cells generally adhere to surfaces and proliferate when the calcium concentration of the medium ≤ 0.1 mM but neither desmosomes nor adherens junctions form. Restoring a physiological calcium concentration then promotes adhesion and initiates rapid junction formation. Similarly, chelation of EC calcium can be used to dissociate cells. However, calcium dependence is unlikely to provide an adhesion-regulatory mechanism in vivo because EC calcium is maintained relatively constant, always above that necessary for cadherin-mediated adhesion. The normal range of calcium concentration in human serum is 2.12–2.62 mM, and remedial treatment is given if it falls below about 1.9 mM.

Trans interaction (i.e. cell–cell) by classical cadherins involves strand-swapping by the EC1 domains of where the side chains of conserved tryptophan residues (Trp2) are inserted into hydrophobic pockets of apposed molecules. The Trp residue and hydrophobic pocket are conserved in DCs, and we have shown that mutations affecting either abolish adhesive binding (Nie et al., 2011). Thus, similar strand-swapping is likely to be involved in desmosomal adhesion.

Type 1 cadherins participate in homophilic adhesion, which appears to be energetically favoured by the strand-swapping mechanism (Chen et al., 2005). Adhesion between DCs has long been regarded as both homo- and heterophilic, but the evidence for this was based on studies of the interactions of complete or incomplete recombinant EC domains by biophysical methods or of incomplete molecules in cells that do not form desmosomes (Chitaev & Troyanovsky, 1997; Syed et al., 2002; Waschke et al., 2007). It seems that all desmosomes in vivo contain at least one Dsc and one Dsg, and their ratio appears to be critical for adhesion (Getsios et al., 2004). Furthermore, where different isoforms of the DCs are co-expressed in tissues, they are mixed in the same desmosomes (North et al., 1996). Thus, multiple types of homo- and heterophilic binding are potentially possible. For example, in HaCaT cells, which express similar amounts of Dsc2, Dsc3, Dsg2 and Dsg3, all co-localised in desmosomes, nonspecific binding would allow 10 possible interactions. Could desmosomes really be that disorganised?

To determine the nature of DC binding in desmosome-forming cells, we treated HaCaT cells with a membrane-impermeable, homobifunctional cross-linker (Nie et al., 2011). Since the desmosomes were in their calcium-dependent phase, we could show, by chelating calcium, that the desmosomes were extracellularly cross-linked. The cells were then solubilised, and immunoprecipitation was carried out with antibodies to each of the DCs. Blotting of the immunoprecipitates showed that each was recognised only by the antibody as used for IP. In other words, each DC bound exclusively to itself. Further experiments showed that the binding was in trans. We concluded that adhesive binding by the DCs was homophilic and isoform-specific; of the 10 possible combinations only four were found, Dsc2-Dsc2, Dsc3-Dsc3, Dsg2-Dsg2 and Dsg3-Dsg3. We do not understand the significance of this adhesive specificity but speculate that it is fundamental to the structural organisation and strength of desmosomal adhesion.

DESMOSOME STRUCTURE

Desmosomes were first identified as densities between adhering cells by Bizzozero (1864) but no further understanding was made until the advent of electron microscopy. Desmosomes have an extremely regular structure (Figure 4). In the transverse or “z” direction, desmosomes have a symmetrical, layered structure (Odland, 1958). The space between the plasma membranes of the two cells contributing to the desmosome is sometimes called the desmosomal core or desmoglea. Halfway between the membranes lies a density, the midline, where adhesive binding occurs because the N-termini of the DCs are located there (Al-Amoudi et al., 2007; Shimizu et al., 2005). Lanthanum infiltration and electron tomography of vitreous sections have demonstrated that the DC EC domains are arranged in the en face or “x-y” plane in a quadratic array with a repeat of ˜70 Å, which is very close to that found in the C-cadherin crystal structure (Al-Amoudi et al., 2007; Boggon et al., 2002; Garrod et al., 2005; Rayns et al., 1969). The inter-membrane distance is ˜35 nm, also very close to the 38.5 nm determined from the C-cadherin crystal structure (Al-Amoudi et al., 2007).

The desmosomal plaques also have a layered structure, resolved into an outer dense plaque(ODP), with its inner face about 20 nm from the membrane, and an inner dense plaque (IDP), which joins the IFs about 50 nm from the membrane (Al-Amoudi et al., 2011; North et al., 1999). Immuno-gold labelling showed the ODP as a region of multiple protein–protein interactions (North et al., 1999). The key observations were as follows: (i) PKP lies close to the plasma membrane; (ii) PG and the N-terminus of DP are further from the membrane and overlap with the C-terminus of Dsc “a”, as well as with the entire cytoplasmic domain of Dsg3, which lies in the ODP; (iii) the C-terminus of Dsc “b” is closer to the membrane and spatially separated from PG and DP; (iv) the C-terminus of DP lies in the IDP, roughly 40 nm from the membrane. This is consistent with the predicted length of the shorter sliced form, DPII, and suggests that DPI is coiled or folded. Broadly speaking, the locations of these molecules are consistent with their interactions determined in vitro. We have since determined that the RUD domain of Dsg1/2 (which have larger cytoplasmic domains than Dsg3) is located in the ODP near its inner face and the TD internal to the ODP but not extending to the IDP (Scothern and Garrod; unpublished).

Electron tomography of the ODP produced a molecular map with a resolution of 3.2 nm (Al-Amoudi et al., 2011). This showed a 2D interconnected quasiperiodic lattice with a similar spatial organisation to the EC side. On the basis of the above-mentioned immuno-gold labelling, the transverse organisation was resolved into an outer 4 nm-thick PKP layer and an inner, denser, 8 nm-thick PG layer, which also contains the N-termini of DP molecules.

HYPER-ADHESION

We have coined the term hyper-adhesion to distinguish the strong adhesion mediated by mature desmosomes from what we believe to be the weaker adhesion mediated by Type1 cadherins at adherens junctions (Garrod et al., 2005; Kimura et al., 2007). Hyper-adhesion is defined experimentally by the complete resistance of desmosomes to disruption by chelation of EC calcium with EGTA (Garrod, 2013). This property is characteristic of most, possibly all, desmosomes in adult mammalian tissues (Garrod et al., 2005; Kimura et al., 2007; Thomason et al., 2012; Wallis et al., 2000). Newly formed desmosomes in culture are calcium dependent. At this time chelation of EC calcium causes desmosomes to split into halves through the plane of adhesion. However, when epithelial cells are maintained in confluent culture, desmosomes acquire hyper-adhesion without changing their molecular composition (Kimura et al., 2007). HaCaT cells with hyper-adhesive desmosomes were found to be more strongly adhesive than those with calcium-dependent desmosomes (Kimura et al., 2007).

The molecular basis of hyper-adhesion is not understood. However, studies of epidermal wound healing and mouse embryonic development suggest that it is somehow linked to the presence of a midline; hyper- adhesive desmosomes appear to have a prominent midline whereas calcium dependent desmosomes do not (Garrod et al., 2005; Kimura et al., 2012; Thomason et al., 2012) and see above and below). This implies that the arrangement of the DC EC domains in the intercellular space can be dynamically regulated, in turn regulating the adhesive strength of the desmosomes. It seems probable that transmembrane signals emanating from the desmosomal plaque are involved in this regulation. Thus, desmosomal adhesiveness is regulated by protein kinase C alpha (PKCα), activation-promoting calcium dependence and inhibition-promoting hyper-adhesion (Garrod et al., 2005; Kimura et al., 2007; Thomason et al., 2012; Wallis et al., 2000). Multiple signals may be involved since inhibition of tyrosine phosphatases (i.e. promotion of tyrosine phosphorylation) also promotes hyper-adhesion (Garrod et al., 2008).We suggest that phosphorylation of one or more plaque components causes rearrangement within the plaque and transmits a signal to the EC domains. Desmosomes lacking PKP1 or PG do not seem to acquire hyper-adhesion (South et al., 2003) (McHarg, Mueller and Garrod: unpublished) so these plaque components may be important in its regulation. On the other hand, desmosomes lacking DP or carrying DP mutations that prevent IF attachment seem to acquire a midline (Jonkman et al., 2005; Sumigray & Lechler, 2012). Phosphorylation does, however, regulate DP-keratin binding and keratins have been shown to regulate PKCα activity and DP phosphorylation (Hobbs & Green, 2012; Kroger et al., 2013). Enhanced association of keratin with DP through blocking DP phosphorylation provides another mechanism for strengthening desmosomal adhesion (Hobbs & Green, 2012).

The importance of hyper-adhesion in vivo: desmosomes in wound healing

Hyper-adhesion, which locks epidermal cells together, seems incompatible with cell migration and therefore with wound re-epithelialisation. There is also some evidence for desmosome down-regulation in wound healing (Allen & Potten, 1975; Croft & Tarin, 1970; Garrod et al., 2005). A series of studies have now shown that desmosomal adhesion changes from hyper-adhesive to calcium dependent in wound edge epithelium.

First, wounding a hyper-adhesive monolayer of MDCK cells caused desmosomes at the wound edge to become rapidly calcium dependent and this change was propagated to cells hundreds of micrometres from the edge (Wallis et al., 2000). Furthermore, the change appeared to be signalled by PKCα. Second, on wounding mouse epidermis, desmosomes in the wound-edge epithelium lost hyper-adhesiveness, becoming calcium dependent (Garrod et al., 2005). In doing so, they lost the midline and the intercellular space narrowed by 10% (Garrod et al., 2005). Transition to calcium dependence was accompanied by translocation of PKCα from cytosol to desmosomal plaques suggesting its activation. Third, Thomason et al. (2012) provided direct evidence of the role of PKCα in wound re-epithelialisation. Thus, PKCα −/− mice showed delayed re-epithelialisation, whereas bitransgenic mice over-expressing constitutively active PKCα showed accelerated re-epithelialisation. Furthermore, these effects were associated with delayed loss of hyper-adhesion in PKCα−/− mice and accelerated loss of hyper-adhesion in CA-PKCα mice. Moreover, in acute human epidermal wounds, PKCα localised to desmosomes at the wound edge. This was associated with switch of desmosomes to calcium dependence. However, in chronic wounds, desmosomes failed to switch from the hyper-adhesive state and PKCα remained cytoplasmic. These results suggest that manipulation of PKC signalling could provide a novel therapeutic approach for chronic wounds.

The normal presence and distribution of desmosomal structural proteins is important to desmosomes functions. Beaudry et al. (2010) used mice lacking the desmosomal structural component Perp in order to demonstrate the contribution of desmosomal adhesion to wound healing (Beaudry et al., 2010). Perp conditional knockout mice showed delayed wound healing relative to controls. Furthermore, loss of Perp did not impair keratinocyte proliferation and actually enhanced keratinocyte migration in vitro. Thus, Perp's role in cell adhesion is essential for wound closure.

The importance of hyper-adhesion in vivo: PV

In PV, a potentially fatal blistering disease of skin and mucous membranes, autoantibodies to Dsg1 and Dsg3 cause acantholysis (Amagai et al., 1991; Ding et al., 1999). Suggested alternative, but not necessarily mutually exclusive mechanisms, for acantholysis include direct disruption of desmosomal adhesion due to steric hindrance by the autoantibodies and activation of outside–in signalling by autoantibody binding (Muller et al., 2008; Waschke, 2008).

Ultrastructural analysis of human PV appears to show that direct disruption of desmosomal adhesion is not the primary event (Diercks et al., 2009). Rather, there is extensive loss of cell–cell adhesion in inter-desmosomal regions and possible intracellular cleavage behind the desmosomal plaque that might indicate a weakening of the cytoskeleton, perhaps through a signalling mechanism involving plakoglobin (Diercks et al., 2009; Muller et al., 2008). By contrast, abundant split desmosomes with inserted keratin filaments were found in a mouse model of pemphigus (Shimizu et al., 2004).

Work in which keratinocytes were treated PV autoantibodies in culture showed that desmosomes were down-regulated and Dsg 3 was depleted and internalised (Calkins et al., 2006; Cirillo et al., 2008; Cirillo et al., 2006; Delva et al., 2008; Yamamoto et al., 2007). However, all of this work was presumably carried out with keratinocytes possessing calcium-dependent rather than hyper-adhesive desmosomes. A study in which keratinocytes with calcium dependent desmosomes and those with hyper-adhesive desmosomes were compared showed that hyper-adhesion inhibited PV autoantibody-induced acantholysis and internalisation of adhesion molecules including Dsg 3 and E-cadherin (Cirillo et al., 2010). Furthermore, over-expression of PKP1 in keratinocytes caused the desmosomes to become hyper-adhesive and protected them from dissociation by PV antibodies (Tucker et al., 2013). This result is intriguing in relation to previous observations that loss of PKP1 destabilises desmosomes (South et al., 2003). Moreover, PKP1 (Figure 1) is predominantly expressed in the upper layers of epidermis where acantholysis does not occur in PV. However, Kimura et al. found that keratinocytes became hyper-adhesive in confluent culture without any change in the level of PKP1 expression (Kimura et al., 2007).

While the primary effect of PV autoantibodies does not appear to be direct disruption of existing desmosomal adhesion, they could inhibit de novo desmosome assembly, which must be a continuous process in stratified epithelia. During the progress of disease, this would be expected to result in gradual down-regulation of desmosomes and loss of cell–cell adhesion. It would therefore be interesting to know whether the above-mentioned results obtained with calcium-dependent keratinocytes in culture have any counterpart in vivo. It could also be the case that the primary loss of inter-desmosomal adhesion found in PV might resemble epidermal wounding and thus cause activation of PKC and consequent weakening of desmosomal adhesion as we have described (Garrod et al., 2005). In this case, inhibition of PKC could provide a novel therapy for pemphigus (Cirillo et al., 2010; Kitajima, 2013).

DESMOSOMES IN DEVELOPMENT

Desmosomes are essential for embryonic development. Deletion of the genes for PG, PKP2 or DP in mice results in embryonic death principally because of failure of intercellular adhesion (Bierkamp et al., 1996; Gallicano et al., 1998; Grossmann et al., 2004; Ruiz et al., 1996). On the other hand, the early embryonic lethal effects caused by deletion of Dgs2 or Dsc3 are more probably due to signalling defects. Deletion of Dsg2 causes death at around the time of implantation because of a defect in embryonic stem cell proliferation, while that of Dsc3 causes death prior to E2.5, which precedes first desmosome assembly in the trophectoderm (Den et al., 2006; Eshkind et al., 2002). Of further developmental significance may be the demonstration that desmosomal adhesion regulates cell positioning during mammary gland morphogenesis (Runswick et al., 2001).

Since desmosomes appear early in mouse tissue development and since developing tissues must remain malleable to participate in morphogenetic movements, we hypothesised that initial weak adhesion would be followed by acquisition of hyper-adhesion (Kimura et al., 2012). We showed that epidermal desmosomes were calcium dependent until embryonic day 12 (E12) and became hyper-adhesive by E14. Similarly, blastocyst trophectodermal desmosomes were calcium-dependent on E3 but became hyper-adhesive by E4.5. In both, development of hyper-adhesion was accompanied by the appearance of a midline supporting previous evidence that hyper-adhesiveness depends on the organised arrangement of DCs. By contrast, adherens junctions remained calcium dependent throughout development, but tight junctions became calcium independent as desmosomes mature. Using protein kinase C (PKC) activation and PKCα−/− mice, we provide evidence suggesting that conventional PKC isoforms are involved in developmental progression to hyper-adhesiveness. Furthermore, regulation of desmosomal adhesion by PKC may be important in trophoblast migration during implantation. It appears that tissue stabilisation is one of several roles played by desmosomes in animal development.

Because desmosomes are essential in mammalian development and they appear at the mid gastrula stage of Fundulus development, it seemed likely that they may also be important in fish early development. We therefore examined the role of DCs in zebrafish early development (Goonesinghe et al., 2012). Both zfDsc and zfDsgα were present as maternal and zygotic transcripts, whereas zfDsgβ was first expressed from 8 hours post-fertilisation (hpf). All three transcripts were present throughout subsequent stages. Morpholino knockdown of both zfDsc and zfDsgα expression produced similar defects in epiboly, axis elongation and somite formation, associated with abnormal desmosomes or reduced desmosome numbers. These results demonstrate an important role for DCs and desmosomes in the early morphogenesis of the zebrafish embryo and provide a basis for more detailed analysis of this role. Knockdown of PG in zebrafish gave rise to heart defects at 48 hpf (Martin et al., 2009).

CONCLUSION

The year 2014 is the 150th anniversary of the first brief description of these intercellular connections with the light microscope (Bizzozero, 1864), and we still know rather little about their role in tissues. They are quite difficult to study in vivo because they are extremely insoluble and can only really be resolved with the electron microscope. We still need to understand many things. In particular, how are desmosomal dynamics regulated in vivo and how does that relate to the dynamics of tissues during normal homeostasis, during development and during disease? What precisely is the role of desmosomes in cellular signalling and the regulation of differentiation? Why do mammals have multiple isoforms of DCs and what impact do they have on the two previous questions?

Declaration of interest: The authors report no declaration of interest. The authors alone are responsible for the content and writing of the paper.