Extracellularly truncated desmoglein 1 compromises desmosomes in MDCK cells

The formation and stability of epithelial tissue involves cell adhesion and the connection of the intermediate filaments of contiguous cells, mediated by desmosomes. The cadherin family members Desmocollins (Dsc) and Desmogleins (Dsg) mediate desmosome extracellular adhesion. The main intracellular molecules identified linking Dscs and Dsgs with the intermediate filament network are Plakoglobin (PG), Plakophilins (PPs) and Desmoplakin (DP). Previous studies on desmosome-mediated adhesion have focused on the intracellular domains of Dsc and Dsg because of their capacity to interact with PG, PPs and DP. This study examines the role of the extracellular domain of Dsg1 upon desmosome stability in MDCK cells. Dsg1 was constructed containing an extracellular deletion (Dsg delta 1EC) and was expressed in MDCK cells. A high expressor Dsg delta 1EC/MDCK clone was obtained and analysed for its capacity to form desmosomes in cell monolayers and when growing under mechanical stress in three-dimensional collagen cultures. Phenotypic changes associated with the ectopic expression of Dsg1 delta EC in MDCK cells were: disturbance of the cytokeratin network, a change in the quality and number of desmosomes and impairment of the formation of cysts in suspension cultures. Interestingly, Dsg1 delta EC was not localized in desmosomes, but was still able to maintain its intracytoplasmic interaction with PG, suggesting that the disruptive effects were largely due to PG and/or PP sequestration.     

The binding of plakoglobin to desmosomal cadherins: patterns of binding sites and topogenic potential

Plakoglobin is the only protein that occurs in the cytoplasmic plaques of all known adhering junctions and has been shown to be crucially involved in the formation and maintenance of desmosomes anchoring intermediate-sized filaments (IFs) by its interaction with the desmosomal cadherins, desmoglein (Dsg), and desmocollin (Dsc). This topogenic importance of plakoglobin is now directly shown in living cells as well as in binding assays in vitro. We show that, in transfected human A-431 carcinoma cells, a chimeric protein combining the vesicle-forming transmembrane glycoprotein synaptophysin, with the complete human plakoglobin sequence, is sorted to small vesicles many of which associate with desmosomal plaques and their attached IFs. Immunoprecipitation experiments have further revealed that the chimeric plakoglobin-containing transmembrane molecules of these vesicles are tightly bound to Dsg and Dsc but not to endogenous plakoglobin, thus demonstrating that the binding of plakoglobin to desmosomal cadherins does not require its soluble state and is strong enough to attach large structures such as vesicles to desmosomes. To identify the binding domains and the mechanisms involved in the interaction of plakoglobin with desmosomal cadherins, we have developed direct binding assays in vitro in which plakoglobin or parts thereof, produced by recombinant DNA technology in E. coli, are exposed to molecules containing the "C- domains" of several cadherins. These assays have shown that plakoglobin associates most tightly with the C-domain of Dsg, to a lesser degree with that of Dsc and only weakly with the C-domain of E-cadherin. Three separate segments of plakoglobin containing various numbers of the so- called arm repeats exhibit distinct binding to the desmosomal cadherins comparable in strength to that of the entire molecule. The binding pattern of plakoglobin segments in vitro is compared with that in vivo. Paradoxically, in vitro some internal plakoglobin fragments bind even better to the C-domain of E-cadherin than the entire molecule, indicating that elements exist in native plakoglobin that interfere with the interaction of this protein with its various cadherin partners.     

Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins

In epidermal cells, keratin intermediate filaments connect with desmosomes to form extensive cadherin-mediated cytoskeletal architectures. Desmoplakin (DPI), a desmosomal component lacking a transmembrane domain, has been implicated in this interaction, although most studies have been conducted with cells that contain few or no desmosomes, and efforts to demonstrate direct interactions between desmoplakin and intermediate filaments have not been successful. In this report, we explore the biochemical nature of the connections between keratin filaments and desmosomes in epidermal keratinocytes. We show that the carboxy terminal "tail" of DPI associates directly with the amino terminal "head" of type II epidermal keratins, including K1, K2, K5, and K6. We have engineered and purified recombinant K5 head and DPI tail, and we demonstrate direct interaction in vitro by solution- binding assays and by ligand blot assays. This marked association is not seen with simple epithelial type II keratins, vimentin, or with type I keratins, providing a possible explanation for the greater stability of the epidermal keratin filament architecture over that of other cell types. We have identified an 18-amino acid residue stretch in the K5 head that is conserved only among type II epidermal keratins and that appears to play some role in DPI tail binding. This finding might have important implications for understanding a recent point mutation found within this binding site in a family with a blistering skin disorder.     

Coordinated expression of desmoglein 1 and desmocollin 1 regulates intercellular adhesion

Desmoglein 1 (Dsg1) is a component of desmosomes present in the upper epidermis and can be targeted by autoimmune antibodies or bacterial toxins, resulting in skin blistering diseases. These defects in tissue integrity are believed to result from compromised desmosomal adhesion; yet, previous attempts to directly test the adhesive roles of desmosomal cadherins using normally non-adherent L cells have yielded mixed results. Here, two complementary approaches were used to better resolve the molecular determinants for Dsg1-mediated adhesion: (1) a tetracycline-inducible system was used to modulate the levels of Dsg1 expressed in L cell lines containing desmocollin 1 (Dsc1) and plakoglobin (PG) and (2) a retroviral gene delivery system was used to introduce Dsg1 into normal human epidermal keratinocytes (NHEK). By increasing Dsg1 expression relative to Dsc1 and PG, we were able to demonstrate that the ratio of Dsg1:Dsc1 is a critical determinant of desmosomal adhesion in fibroblasts. The distribution of Dsg1 was organized at areas of cell-cell contact in the multicellular aggregates that formed in these suspension cultures. Similarly, the introduction of Dsg1 into NHEKs was capable of increasing the aggregation of single cell suspensions and further enhanced the adhesive strength of intact epithelial sheets. Endogenous Dsc1 levels were also increased in NHEKs containing Dsg1, providing further support for the coordination of these two desmosomal cadherins in regulating adhesive structures. These Dsg1-mediated effects on intercellular adhesion were directly related to the presence of an intact extracellular domain as ETA, a toxin that specifically cleaves this desmosomal cadherin, inhibited adhesion in both fibroblasts and keratinocytes. Collectively, these observations demonstrate that Dsg1 promotes the formation of intercellular adhesion complexes and suggest that the relative level of Dsg and Dsc expressed at the cell surface regulates this adhesive process.     

Interaction of the bullous pemphigoid antigen 1 (BP230) and desmoplakin with intermediate filaments is mediated by distinct sequences within their COOH terminus

The bullous pemphigoid antigen 1 (BP230) and desmoplakin (DP) are members of the plakin protein family of cytolinkers. Despite their homology, their COOH termini selectively bind distinct intermediate filaments (IFs). We studied sequences within their COOH termini required for their interaction with the epidermal keratins K5/K14, the simple epithelial keratins K8/K18, and type III IF vimentin by yeast three-hybrid, cell transfection, and overlay assays. The results indicate that BP230 interacts with K5/K14 but not with K8/K18 or vimentin via a region encompassing both the B and C subdomains and the COOH extremity, including a COOH-terminal eight-amino-acid stretch. In contrast, the C subdomain with the COOH-terminal extremity of DP interacts with K5/K14 and K8/K18, and its linker region is able to associate with K8/K18 and vimentin. Furthermore, the potential of DP to interact with IF proteins in yeast seems to be regulated by phosphorylation of Ser 2849 within its COOH terminus. Strikingly, BP230 and DP interacted with cytokeratins only when both type I and type II keratins were present. The head and tail domains of K5/K14 keratins were dispensable for their interaction with BP230 or DP. On the basis of our findings, we postulate that (1) the binding specificity of plakins for various IF proteins depends on their linker region between the highly homologous B and C subdomains and their COOH extremity and (2) the association of DP and BP230 with both epidermal and simple keratins is critically affected by the tertiary structure induced by heterodimerization and involves recognition sites located primarily in the rod domain of these keratins.     

Assessment of splice variant-specific functions of desmocollin 1 in the skin

Desmocollin 1 (Dsc1) is part of a desmosomal cell adhesion receptor formed in terminally differentiating keratinocytes of stratified epithelia. The dsc1 gene encodes two proteins (Dsc1a and Dsc1b) that differ only with respect to their COOH-terminal cytoplasmic amino acid sequences. On the basis of in vitro experiments, it is thought that the Dsc1a variant is essential for assembly of the desmosomal plaque, a structure that connects desmosomes to the intermediate filament cytoskeleton of epithelial cells. We have generated mice that synthesize a truncated Dsc1 receptor that lacks both the Dsc1a- and Dsc1b-specific COOH-terminal domains. This mutant transmembrane receptor, which does not bind the common desmosomal plaque proteins plakoglobin and plakophilin 1, is integrated into functional desmosomes. Interestingly, our mutant mice did not show the epidermal fragility previously observed in dsc1-null mice. This suggests that neither the Dsc1a- nor the Dsc1b-specific COOH-terminal cytoplasmic domain is required for establishing and maintaining desmosomal adhesion. However, a comparison of our mutants with dsc1-null mice suggests that the Dsc1 extracellular domain is necessary to maintain structural integrity of the skin.     

Regulation of intestinal epithelial intercellular adhesion and barrier function by desmosomal cadherin desmocollin-2

The role of desmosomal cadherin desmocollin-2 (Dsc2) in regulating barrier function in intestinal epithelial cells (IECs) is not well understood. Here, we report the consequences of silencing Dsc2 on IEC barrier function in vivo using mice with inducible intestinal–epithelial-specific Dsc2 knockdown (KD) (Dsc2^ERΔIEC). While the small intestinal gross architecture was maintained, loss of epithelial Dsc2 influenced desmosomal plaque structure, which was smaller in size and had increased intermembrane space between adjacent epithelial cells. Functional analysis revealed that loss of Dsc2 increased intestinal permeability in vivo, supporting a role for Dsc2 in the regulation of intestinal epithelial barrier function. These results were corroborated in model human IECs in which Dsc2 KD resulted in decreased cell–cell adhesion and impaired barrier function. It is noteworthy that Dsc2 KD cells exhibited delayed recruitment of desmoglein-2 (Dsg2) to the plasma membrane after calcium switch-induced intercellular junction reassembly, while E-cadherin accumulation was unaffected. Mechanistically, loss of Dsc2 increased desmoplakin (DP I/II) protein expression and promoted intermediate filament interaction with DP I/II and was associated with enhanced tension on desmosomes as measured by a Dsg2-tension sensor. In conclusion, we provide new insights on Dsc2 regulation of mechanical tension, adhesion, and barrier function in IECs.     

Defining desmosomal plakophilin-3 interactions

Plakophilin 3 (PKP3) is a recently described armadillo protein of the desmosomal plaque, which is synthesized in simple and stratified epithelia. We investigated the localization pattern of endogenous and exogenous PKP3 and fragments thereof. The desmosomal binding properties of PKP3 were determined using yeast two-hybrid, coimmunoprecipitation and colocalization experiments. To this end, novel mouse anti-PKP3 mAbs were generated. We found that PKP3 binds all three desmogleins, desmocollin (Dsc) 3a and -3b, and possibly also Dsc1a and -2a. As such, this is the first protein interaction ever observed with a Dsc-b isoform. Moreover, we determined that PKP3 interacts with plakoglobin, desmoplakin (DP) and the epithelial keratin 18. Evidence was found for the presence of at least two DP-PKP3 interaction sites. This finding might explain how lateral DP-PKP interactions are established in the upper layers of stratified epithelia, increasing the size of the desmosome and the number of anchoring points available for keratins. Together, these results show that PKP3, whose epithelial and epidermal desmosomal expression pattern and protein interaction repertoire are broader than those of PKP1 and -2, is a unique multiprotein binding element in the basic architecture of a vast majority of epithelial desmosomes.     

Identification of the plakoglobin-binding domain in desmoglein and its role in plaque assembly and intermediate filament anchorage

The carboxyterminal cytoplasmic portions (tails) of desmosomal cadherins of both the desmoglein (Dsg) and desmocollin type are integral components of the desmosomal plaque and are involved in desmosome assembly and the anchorage of intermediate-sized filaments. When additional Dsg tails were introduced by cDNA transfection into cultured human epithelial cells, in the form of chimeras with the aminoterminal membrane insertion domain of rat connexin32 (Co32), the resulting stably transfected cells showed a dominant-negative defect specific for desmosomal junctions: despite the continual presence of all desmosomal proteins, the endogenous desmosomes disappeared and the formation of Co32-Dsg chimeric gap junctions was inhibited. Using cell transfection in combination with immunoprecipitation techniques, we have examined a series of deletion mutants of the Dsg1 tail in Co32-Dsg chimeras. We show that upon removal of the last 262 amino acids the truncated Dsg tail still effects the binding of plakoglobin but not of detectable amounts of any catenin and induces the dominant-negative phenotype. However, further truncation or excision of the next 41 amino acids, which correspond to the highly conserved carboxyterminus of the C-domain in other cadherins, abolishes plakoglobin binding and allows desmosomes to reform. Therefore, we conclude that this short segment provides a plakoglobin-binding site and is important for plaque assembly and the specific anchorage of either actin filaments in adherens junctions or IFs in desmosomes.     

Dissection of protein linkage between keratins and pinin, a protein with dual location at desmosome-intermediate filament complex and in the nucleus

Pinin is a cell adhesion-associated and nuclear protein that has been shown to localize in the vicinity of intermediate filament (IF) convergence upon the cytoplasmic face of the desmosomal plaque as well as in the nucleus. The localization of pinin to the desmosomes has been correlated with the reinforcement of intercellular adhesion and increased IF organization. In this study, keratins 18, 8, and 19 were identified to interact with the amino end domain of pinin in a two-hybrid screening. Further truncation analyses indicated that the 2B domain of keratin contains the sequence responsible for interacting with pinin. The amino end of pinin (residues 1-98) is sufficient to bind to keratin. Point mutation analyses revealed two essential residues within the pinin fragment 1-98, leucine 8 and leucine 19, for the interaction with keratin. Finally, in vitro protein overlay binding assays confirmed the direct interaction of the amino end domain of pinin with keratins, while pinin mutant L8P GST fusion protein failed to bind to keratins in the overlay assay. Coupled with our previous morphological observations and transfection studies, these data suggest that pinin may play a role in epithelial cell adhesion and the IF complex through a direct interaction with the keratin filaments.     

Direct Ca2+-dependent Heterophilic Interaction between Desmosomal Cadherins, Desmoglein and Desmocollin, Contributes to Cell–Cell Adhesion

Human fibrosarcoma cells, HT-1080, feature extensive adherens junctions, lack mature desmosomes, and express a single known desmosomal protein, Desmoglein 2 (Dsg2). Transfection of these cells with bovine Desmocollin 1a (Dsc1a) caused dramatic changes in the subcellular distribution of endogenous Dsg2. Both cadherins clustered in the areas of the adherens junctions, whereas only a minor portion of Dsg2 was seen in these areas in the parental cells. Deletion mapping showed that intact extracellular cadherin-like repeats of Dsc1a (Arg¹-Thr¹⁷⁰) are required for the translocation of Dsg2. Deletion of the intracellular C-domain that mediates the interaction of Dsc1a with plakoglobin, or the CSI region that is involved in the binding to desmoplakin, had no effect. Coimmunoprecipitation experiments of cell lysates stably expressing Dsc1a with anti-Dsc or -Dsg antibodies demonstrate that the desmosomal cadherins, Dsg2 and Dsc1a, are involved in a direct Ca²⁺-dependent interaction. This conclusion was further supported by the results of solid phase binding experiments. These showed that the Dsc1a fragment containing cadherin-like repeats 1 and 2 binds directly to the extracellular portion of Dsg in a Ca²⁺-dependent manner. The contribution of the Dsg/ Dsc interaction to cell–cell adhesion was tested by coculturing HT-1080 cells expressing Dsc1a with HT-1080 cells lacking Dsc but expressing myc-tagged plakoglobin (MPg). In the latter cells, MPg and the endogenous Dsg form stable complexes. The observed specific coimmunoprecipitation of MPg by anti-Dsc antibodies in coculture indicates that an intercellular interaction between Dsc1 and Dsg is involved in cell–cell adhesion.     

The head domain of plakophilin-1 binds to desmoplakin and enhances its recruitment to desmosomes. Implications for cutaneous disease.

The contribution of desmosomes to epidermal integrity is evident in the inherited blistering disorder associated with the absence of a functional gene for plakophilin-1. To define the function of plakophilin-1 in desmosome assembly, interactions among the desmosomal cadherins, desmoplakin, and the armadillo family members plakoglobin and plakophilin-1 were examined. In transient expression assays, plakophilin-1 formed complexes with a desmoplakin amino-terminal domain and enhanced its recruitment to cell-cell borders; this recruitment was not dependent on the equimolar expression of desmosomal cadherins. In contrast to desmoplakin-plakoglobin interactions, the interaction between desmoplakin and plakophilin-1 was not mediated by the armadillo repeat domain of plakophilin-1 but by the non-armadillo head domain, as assessed by yeast two-hybrid and recruitment assays. We propose a model whereby plakoglobin serves as a linker between the cadherins and desmoplakin, whereas plakophilin-1 enhances lateral interactions between desmoplakin molecules. This model suggests that epidermal lesions in patients lacking plakophilin-1 are a consequence of the loss of integrity resulting from a decrease in binding sites for desmoplakin and intermediate filaments at desmosomes.     

Desmosomal cadherins utilize distinct kinesins for assembly into desmosomes

The desmosomal cadherins, desmogleins (Dsgs) and desmocollins (Dscs), comprise the adhesive core of intercellular junctions known as desmosomes. Although these adhesion molecules are known to be critical for tissue integrity, mechanisms that coordinate their trafficking into intercellular junctions to regulate their proper ratio and distribution are unknown. We demonstrate that Dsg2 and Dsc2 both exhibit microtubule-dependent transport in epithelial cells but use distinct motors to traffic to the plasma membrane. Functional interference with kinesin-1 blocked Dsg2 transport, resulting in the assembly of Dsg2-deficient junctions with minimal impact on distribution of Dsc2 or desmosomal plaque components. In contrast, inhibiting kinesin-2 prevented Dsc2 movement and decreased its plasma membrane accumulation without affecting Dsg2 trafficking. Either kinesin-1 or -2 deficiency weakened intercellular adhesion, despite the maintenance of adherens junctions and other desmosome components at the plasma membrane. Differential regulation of desmosomal cadherin transport could provide a mechanism to tailor adhesion strength during tissue morphogenesis and remodeling.     

Genomic organization of mouse desmocollin genes reveals evolutionary conservation.

Desmosomal cadherins are a family of calcium regulated proteins involved in the formation of desmosomes, a type of cell junction important in maintaining cell adhesion and tissue stability. The desmosomal plaque consists of members of the desmosomal cadherin, plakin and armadillo family of proteins. Desmosomal cadherins are transmembrane glycoproteins that interact with desmosomal cadherins of the adjacent cells via their extracellular repeat domains and are divided in two subfamilies, the desmogleins (Dsg) and the desmocollins (Dsc). On the cytoplasmic side, the cadherins connect to the intermediate filament (IF) network indirectly by interacting with plakin and armadillo proteins. Here, we report the elucidation of the genomic structure of two mouse desmocollin genes, Dsc2 and Dsc3. Interestingly, at the genomic level, desmocollins show a higher degree of similarity to the classical cadherins, such as E-cadherin, than to the desmogleins.     

Desmocollin 3-mediated Binding Is Crucial for Keratinocyte Cohesion and Is Impaired in Pemphigus

Desmocollin (Dsc) 1–3 and desmoglein (Dsg) 1–4, transmembrane proteins of the cadherin family, form the adhesive core of desmosomes. Here we provide evidence that Dsc3 homo- and heterophilic trans-interaction is crucial for epidermal integrity. Single molecule atomic force microscopy (AFM) revealed homophilic trans-interaction of Dsc3. Dsc3 displayed heterophilic interaction with Dsg1 but not with Dsg3. A monoclonal antibody targeted against the extracellular domain reduced homophilic and heterophilic binding as measured by AFM, caused intraepidermal blistering in a model of human skin, and a loss of intercellular adhesion in cultured keratinocytes. Because autoantibodies against Dsg1 are associated with skin blistering in pemphigus, we characterized the role of Dsc3 binding for pemphigus pathogenesis. In contrast to AFM experiments, laser tweezer trapping revealed that pemphigus autoantibodies reduced binding of Dsc3-coated beads to the keratinocyte cell surface. These data indicate that loss of heterophilic Dsc3/Dsg1 binding may contribute to pemphigus skin blistering.Desmogleins (Dsg)² and desmocollins (Dsc) are members of the Ca²⁺-dependent cadherin family of adhesion molecules that extend with their outer domains into the extracellular core of desmosomes. Desmosomal cadherins include four Dsg (Dsg1–4) and three Dsc3 isoforms (Dsc1–3) (1, 2). Desmosomal cadherins share a common domain organization with five N-terminally located extracellular subdomains (EC1–5). The membrane-distal EC1 domain is thought to contain the adhesive interface necessary for trans-interaction as could be concluded from structural analysis and blocking studies using peptides and antibodies (3–5). By establishing trans- and cis-interacting adhesive complexes, desmosomal cadherins participate in providing mechanical strength to stratified epithelia (6). In human epidermis Dsg1 and Dsc1 expression decreases from the outermost granular layer toward deeper layers, whereas Dsg3 and Dsc3 are primarily found in the basal layer and display an inverse expression gradient (7, 8). In contrast to classical cadherins present in adherens junctions that primarily undergo homophilic trans-interaction, desmosomal cadherins are generally believed to mediate both homo- and heterophilic binding (9). Recently, an important role of Dsc3 for integrity of murine epidermis was demonstrated in animals with conditional epidermal Dsc3 deficiency that suffered from severe intraepidermal blister formation (10) comparable with the phenotype of the autoimmune bullous skin disease pemphigus vulgaris (PV) (11). PV is associated with antibodies (Abs) against Dsg3, in part combined with Abs targeting Dsg1, whereas Dsg1 Abs alone are associated with pemphigus foliaceus (PF). However, PV and PF sera usually do not contain autoantibodies targeting Dsc3 (12). In view of the apparently important role of Dsc3 in epidermal adhesion, we addressed whether Dsg1 and Dsg3 might heterophilically interact with Dsc3 and whether Abs in pemphigus might interfere with such type of interaction.     

Desmoglein-2: a novel regulator of apoptosis in the intestinal epithelium

Intestinal epithelial intercellular junctions regulate barrier properties, and they have been linked to epithelial differentiation and programmed cell death (apoptosis). However, mechanisms regulating these processes are poorly defined. Desmosomes are critical elements of intercellular junctions; they are punctate structures made up of transmembrane desmosomal cadherins termed desmoglein-2 (Dsg2) and desmocollin-2 (Dsc2) that affiliate with the underlying intermediate filaments via linker proteins to provide mechanical strength to epithelia. In the present study, we generated an antibody, AH12.2, that recognizes Dsg2. We show that Dsg2 but not another desmosomal cadherin, Dsc2, is cleaved by cysteine proteases during the onset of intestinal epithelial cell (IEC) apoptosis. Small interfering RNA-mediated down-regulation of Dsg2 protected epithelial cells from apoptosis. Moreover, we report that a C-terminal fragment of Dsg2 regulates apoptosis and Dsg2 protein levels. Our studies highlight a novel mechanism by which Dsg2 regulates IEC apoptosis driven by cysteine proteases during physiological differentiation and inflammation.     

Membrane-impermeable cross-linking provides evidence for homophilic, isoform-specific binding of desmosomal cadherins in epithelial cells

Desmosomes and adherens junctions are cadherin-based protein complexes responsible for cell-cell adhesion of epithelial cells. Type 1 cadherins of adherens junctions show specific homophilic adhesion that plays a major role in developmental tissue segregation. The desmosomal cadherins, desmocollin and desmoglein, occur as several different isoforms with overlapping expression in some tissues where different isoforms are located in the same desmosomes. Although adhesive binding of desmosomal cadherins has been investigated in a variety of ways, their interaction in desmosome-forming epithelial cells has not been studied. Here, using extracellular homobifunctional cross-linking, we provide evidence for homophilic and isoform-specific binding between the Dsc2, Dsc3, Dsg2, and Dsg3 isoforms in HaCaT keratinocytes and show that it represents trans interaction. Furthermore, the cross-linked adducts are present in the detergent-insoluble fraction, and electron microscopy shows that extracellular cross-linking probably occurs in desmosomes. We found no evidence for either heterophilic or cis interaction, but neither can be completely excluded by our data. Mutation of amino acid residues Trp-2 and Ala-80 that are important for trans interaction in classical cadherin adhesive binding abolished Dsc2 binding, indicating that these residues are also involved in desmosomal adhesion. These interactions of desmosomal cadherins may be of key importance for their ordered arrangement within desmosomes that we believe is essential for desmosomal adhesive strength and the maintenance of tissue integrity.     

Desmoglein 2 can undergo Ca2+-dependent interactions with both desmosomal and classical cadherins including E-cadherin and N-cadherin

Desmoglein (Dsg) 2 is a ubiquitously expressed desmosomal cadherin. Particularly, it is present in all cell types forming desmosomes, including epithelial cells and cardiac myocytes and is upregulated in the autoimmune skin disease pemphigus. Thus, we here characterized the binding properties of Dsg2 in more detail using atomic force microscopy (AFM). Dsg2 exhibits homophilic interactions and also heterophilic interactions with the desmosomal cadherin desmocollin (Dsc) 2, and further with the classical cadherins E-cadherin (E-Cad) and N-cadherin (N-Cad), which may be relevant for cross talk between desmosomes and adherens junctions in epithelia and cardiac myocytes. We found that all homo- and heterophilic interactions were Ca²⁺-dependent. All binding forces observed are in the same force range, i.e., 30 to 40 pN, except for the Dsg2/E-Cad unbinding force, which with 45 pN is significantly higher. To further characterize the nature of the interactions, we used tryptophan, a critical amino acid required for trans-interaction, and a tandem peptide (TP) designed to cross-link Dsg isoforms. TP was sufficient to prevent the tryptophan-induced loss of Dsg2 interaction with the desmosomal cadherins Dsg2 and Dsc2; however, not with the classical cadherins E-Cad and N-Cad, indicating that the interaction modes of Dsg2 with desmosomal and classical cadherins differ. TP rescued the tryptophan-induced loss of Dsg2 binding on living enterocytes, suggesting that interaction with desmosomal cadherins may be more relevant. In summary, the data suggest that the ubiquitous desmosomal cadherin Dsg2 enables the cross talk with adherens junctions by interacting with multiple binding partners with implications for proper adhesive function in healthy and diseased states.     

Suprabasal desmoglein 3 expression in the epidermis of transgenic mice results in hyperproliferation and abnormal differentiation

The desmoglein 1 (Dsg1) and desmocollin 1 (Dsc1) isoforms of the desmosomal cadherins are expressed in the suprabasal layers of epidermis, whereas Dsg3 and Dsc3 are more strongly expressed basally. This differential expression may have a function in epidermal morphogenesis and/or may regulate the proliferation and differentiation of keratinocytes. To test this hypothesis, we changed the expression pattern by overexpressing human Dsg3 under the control of the keratin 1 (K1) promoter in the suprabasal epidermis of transgenic mice. From around 12 weeks of age, the mice exhibited flaking of the skin accompanied by epidermal pustules and thinning of the hair. Histological analysis of affected areas revealed acanthosis, hypergranulosis, hyperkeratosis, localized parakeratosis, and abnormal hair follicles. This phenotype has some features in common with human ichthyosiform diseases. Electron microscopy revealed a mild epidermal spongiosis. Suprabasally, desmosomes showed incorporation of the exogenous protein by immunogold labeling but were normal in structure. The epidermis was hyperproliferative, and differentiation was abnormal, demonstrated by expression of K14 in the suprabasal layer, restriction of K1, and strong induction of K6 and K16. The changes resembled those found in previous studies in which growth factors, cytokines, and integrins had been overexpressed in epidermis. Thus our data strongly support the view that Dsg3 contributes to the regulation of epidermal differentiation. Our results contrast markedly with those recently obtained by expressing Dsg3 in epidermis under the involucrin promoter. Possible reasons for this difference are considered in this paper.     

Desmoplakin Is Required Early in Development for Assembly of Desmosomes and Cytoskeletal Linkage

Desmosomes first assemble in the E3.5 mouse trophectoderm, concomitant with establishment of epithelial polarity and appearance of a blastocoel cavity. Throughout development, they increase in size and number and are especially abundant in epidermis and heart muscle. Desmosomes mediate cell–cell adhesion through desmosomal cadherins, which differ from classical cadherins in their attachments to intermediate filaments (IFs), rather than actin filaments. Of the proteins implicated in making this IF connection, only desmoplakin (DP) is both exclusive to and ubiquitous among desmosomes. To explore its function and importance to tissue integrity, we ablated the desmoplakin gene. Homozygous −/− mutant embryos proceeded through implantation, but did not survive beyond E6.5. Mutant embryos proceeded through implantation, but did not survive beyond E6.5. Surprisingly, analysis of these embryos revealed a critical role for desmoplakin not only in anchoring IFs to desmosomes, but also in desmosome assembly and/or stabilization. This finding not only unveiled a new function for desmoplakin, but also provided the first opportunity to explore desmosome function during embryogenesis. While a blastocoel cavity formed and epithelial cell polarity was at least partially established in the DP (−/−) embryos, the paucity of desmosomal cell–cell junctions severely affected the modeling of tissue architecture and shaping of the early embryo.