Defining domain-specific orientational order in the desmosomal cadherins.

Desmosomes are large, macromolecular protein assemblies that mechanically couple the intermediate filament cytoskeleton to sites of cadherin-mediated cell adhesion, thereby providing structural integrity to tissues that routinely experience large forces. Proper desmosomal adhesion is necessary for the normal development and maintenance of vertebrate tissues, such as epithelia and cardiac muscle, while dysfunction can lead to severe disease of the heart and skin. Therefore, it is important to understand the relationship between desmosomal adhesion and the architecture of the molecules that form the adhesive interface, the desmosomal cadherins (DCs). However, desmosomes are embedded in two plasma membranes and are linked to the cytoskeletal networks of two cells, imposing extreme difficulty on traditional structural studies of DC architecture, which have yielded conflicting results. Consequently, the relationship between DC architecture and adhesive function remains unclear. To overcome these challenges, we utilized excitation-resolved fluorescence polarization microscopy to quantify the orientational order of the extracellular and intracellular domains of three DC isoforms: desmoglein 2, desmocollin 2, and desmoglein 3. We found that DC ectodomains were significantly more ordered than their cytoplasmic counterparts, indicating a drastic difference in DC architecture between opposing sides of the plasma membrane. This difference was conserved among all DCs tested, suggesting that it may be an important feature of desmosomal architecture. Moreover, our findings suggest that the organization of DC ectodomains is predominantly the result of extracellular adhesive interactions. We employed azimuthal orientation mapping to show that DC ectodomains are arranged with rotational symmetry about the membrane normal. Finally, we performed a series of mathematical simulations to test the feasibility of a recently proposed antiparallel arrangement of DC ectodomains, finding that it is supported by our experimental data. Importantly, the strategies employed here have the potential to elucidate molecular mechanisms for diseases that result from defective desmosome architecture.

Inhibition of retinoid X receptor improved the morphology, localization of desmosomal proteins and paracellular permeability in three-dimensional cultures of mouse keratinocytes.

Retinoic acid (RA) plays an important role in epithelial homeostasis and influences the morphology, proliferation, differentiation and permeability of epithelial cells. Mouse keratinocytes, K38, reconstituted non-keratinized stratified epithelium in three-dimensional (3D) cultures with serum, which contains retinol (a source of RA), but the morphology was different from in vivo epithelium. The formed epithelium was thick, with loosened cell-cell contacts. Here, we investigated whether the inhibition of RA receptor (RAR)/retinoid X receptor (RXR)-mediated signaling by an RXR antagonist, HX 531, improved K38 3D cultures in terms of morphology and intercellular junctions. The epithelium formed by 0.5 µM HX531 was thin, and the intercellular space was narrowed because of the restoration of the layer-specific distribution of desmoglein (DSG)-1, DSG3 and plakoglobin (PG). Moreover, the levels of desmosomal proteins and tight junction proteins, including DSG1, DSG2, DSG3, PG, claudin (CLDN)-1 and CLDN4 increased, but the adherens junction protein, E-cadherin, did not show any change. Furthermore, CLDN1 was recruited to occludin-positive cell-cell contacts in the superficial cells and transepithelial electrical resistance was increased. Therefore, K38 3D cultures treated with 0.5 µM HX531 provides a useful in vitro model to study intercellular junctions in the non-keratinized epithelium.

Differential Pathomechanisms of Desmoglein 1 Transmembrane Domain Mutations in Skin Disease.

Dominant and recessive mutations in the desmosomal cadherin, desmoglein (DSG) 1, cause the skin diseases palmoplantar keratoderma (PPK) and severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome, respectively. In this study, we compare two dominant missense mutations in the DSG1 transmembrane domain (TMD), G557R and G562R, causing PPK (DSG1PPK-TMD) and SAM syndrome (DSG1SAM-TMD), respectively, to determine the differing pathomechanisms of these mutants. Expressing the DSG1TMD mutants in a DSG-null background, we use cellular and biochemical assays to reveal the differences in the mechanistic behavior of each mutant. Super-resolution microscopy and functional assays showed a failure by both mutants to assemble desmosomes due to reduced membrane trafficking and lipid raft targeting. DSG1SAM-TMD maintained normal expression levels and turnover relative to wildtype DSG1, but DSG1PPK-TMD lacked stability, leading to increased turnover through lysosomal and proteasomal pathways and reduced expression levels. These results differentiate the underlying pathomechanisms of these disorders, suggesting that DSG1SAM-TMD acts dominant negatively, whereas DSG1PPK-TMD is a loss-of-function mutation causing the milder PPK disease phenotype. These mutants portray the importance of the DSG TMD in desmosome function and suggest that a greater understanding of the desmosomal cadherin TMDs will further our understanding of the role that desmosomes play in epidermal pathophysiology.

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) (Dsc2ERΔIEC). 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.

Enteroids Generated from Patients with Severe Inflammation in Crohn's Disease Maintain Alterations of Junctional Proteins.

BACKGROUND: The mechanisms underlying loss of intestinal epithelial barrier [IEB] function in Crohn's disease [CD] are poorly understood. We tested whether human enteroids generated from isolated intestinal crypts of CD patients serve as an appropriate in vitro model to analyse changes of IEB proteins observed in patients' specimens. METHODS: Gut samples from CD patients and healthy individuals who underwent surgery were collected. Enteroids were generated from intestinal crypts and analyses of junctional proteins in comparison to full wall samples were performed. RESULTS: Histopathology confirmed the presence of CD and the extent of inflammation in intestinal full wall sections. As revealed by immunostaining and Western blot analysis, profound changes in expression patterns of tight junction, adherens junction and desmosomal proteins were observed in full wall specimens when CD was present. Unexpectedly, when enteroids were generated from specimens of CD patients with severe inflammation, alterations of most tight junction proteins and the majority of changes in desmosomal proteins but not E-cadherin were maintained under culture conditions. Importantly, these changes were maintained without any additional stimulation of cytokines. Interestingly, qRT-PCR demonstrated that mRNA levels of junctional proteins were not different when enteroids from CD patients were compared to enteroids from healthy controls. CONCLUSIONS: These data indicate that enteroids generated from patients with severe inflammation in CD maintain some characteristics of intestinal barrier protein changes on a post-transcriptional level. The enteroid in vitro model represents an appropriate tool to gain further cellular and molecular insights into the pathogenesis of barrier dysfunction in CD.

Development and regeneration dynamics of the Medaka notochord.

The notochord is an embryonic tissue that acts as a hydrostatic skeleton until ossification begins in vertebrates. It is composed of outer sheath cells and inner vacuolated cells, which are generated from a common pool of disc-shaped precursors. Notochord extension during early embryogenesis is driven by the growth of vacuolated cells, reflecting in turn the expansion of their inner vacuole. Here we use desmogon, a novel desmosomal cadherin, to follow notochord development and regeneration in medaka (Oryzias latipes). We trace desmogon â€‹+ disc-shaped precursors at the single cell level to demonstrate that they operate as unipotent progenitors, giving rise to either sheath or vacuolated cells. We reveal that once specified, vacuolated cells grow asynchronously and drive notochord expansion bi-directionally. Additionally, we uncover distinct regenerative responses in the notochord, which depend on the nature of the injury sustained. By generating a desmogon CRISPR mutant we demonstrate that this cadherin is essential for proper vacuolated cell shape and therefore correct notochord and spine morphology. Our work expands the repertoire of model systems to study dynamic aspects of the notochord in vivo, and provides new insights in its development and regeneration properties.

Integrative prognostic subtype discovery in high-grade serous ovarian cancer.

OBJECTIVE: We sought to identify novel molecular subtypes of high-grade serous ovarian cancer (HGSC) by the integration of gene expression and proteomics data and to find the underlying biological characteristics of ovarian cancer to improve the clinical outcome. METHODS: The iCluster method was utilized to analysis 131 common HGSC samples between TCGA and Clinical Proteomic Tumor Analysis Consortium databases. Kaplan-Meier survival curves were used to estimate the overall survival of patients, and the differences in survival curves were assessed using the log-rank test. RESULTS: Two novel ovarian cancer subtypes with different overall survival (P = .00114) and different platinum status (P = .0061) were identified. Eighteen messenger RNAs and 38 proteins were selected as differential molecules between subtypes. Pathway analysis demonstrated arrhythmogenic right ventricular cardiomyopathy pathway played a critical role in the discrimination of these two subtypes and desmosomal cadherin DSG2, DSP, JUP, and PKP2 in this pathway were overexpression in subtype I compared with subtype II. CONCLUSION: Our study extended the underlying prognosis-related biological characteristics of high-grade serous ovarian cancer. Enrichment of desmosomal cadherin increased the risk for HGSC prognosis among platinum-sensitive patients, the results guided the revision of the treatment options for platinum-sensitive ovarian cancer patients to improve outcomes.

Depletion of the cellular cholesterol content reduces the dynamics of desmosomal cadherins and interferes with desmosomal strength.

Desmosomal cadherins, desmocollins, and desmogleins are cholesterol-dependent entities responsible for the stable adhesion of desmosomes in epithelial cells. Here, we investigated the influence of cellular cholesterol depletion on the dynamic properties of the desmosomal cadherin desmocollin, particularly the lateral mobility and distribution of desmocollin 2 (Dsc2-YFP) in the plasma membrane, and how these properties influence the adhesion strength of desmosomes. Depletion of cellular cholesterol decreased the lateral mobility of Dsc2-YFP and caused dispersion of Dsc2-YFP in the plasma membrane of epithelial MDCK cells. As a consequence of the altered Dsc2-YFP dynamics, the adhesive strength of desmosomes was weakened. Moreover, our study is the first to show and quantify the co-association of desmosomes with cholesterol/sphingomyelin-enriched membrane domains at the ultrastructural level. Taken together, our data emphasize a critical role for the cellular cholesterol content in regulating the lateral mobility and distribution of Dsc2 and show that cholesterol depletion reduces the strength of desmosomal adhesions.

Characteristics of multicellular tumor spheroids formed by pancreatic cells expressing different adhesion molecules.

AIMS: Multicellular tumor spheroids (MCTS) produced by different methods vary in forms, sizes, and properties. The aim of this work was to characterize MCTS formed by six pancreatic cell lines on a non-adherent surface. MATERIALS AND METHODS: Human pancreatic cells were grown in 2D and 3D conditions and compared for the expression of E- and desmosomal cadherins (PCR, confocal microscopy), growth, cell cycling, apoptosis (flow cytometry), and a response to antitumor drugs doxorubicin and gemcitabine (MTT-assay). KEY FINDINGS: Three types of MCTS were identified: BxPC-3, T3M4 formed small number of large and dense spheroids representing type I MCTS; COLO-357 and AsPC-1 generated type II multiple and loose MCTS of different sizes while MiaPaCa-2 and PANC-1 represented type III cultures which grew almost as floating monolayer films. Formation of type I MCTS depended on the simultaneous expression of DSG3 and several DSC proteins; II MCTS expressed solely DSG2-DSC2 but not DSG3, while type III cells either did not express E-cadherin or a pair of DSG and DSC proteins. Cells in type I MCTS but not in types II and III ones quickly became quiescent which correlated with a decrease in the proliferation, increased apoptosis, and a higher resistance to antitumor drugs doxorubicin and gemcitabine. SIGNIFICANCE: Taken collectively, pancreatic cells significantly vary in the expression of desmosomal cadherins, resulting in the formation of MCTS with different characteristics. The sensitivity of MCTS to various drugs depends on the type of cells and the method of spheroid preparation used.

Clinical and molecular implications of structural changes to desmosomes and corneodesmosomes.

Desmosomes provide the main intercellular adhesive properties between epidermal keratinocytes. Their distribution becomes uneven in severe dermatitis, multiple allergies and metabolic wasting syndrome due to desmoglein 1 deficiency and the loss of intercellular adhesion or acantholysis. When keratinocytes differentiate from granular cells into cornified cells, desmosomes are transformed into corneodesmosomes and can provide stronger intercellular adhesion. Degradation of corneodesmosomes is a tightly regulated process involving a number of proteases and their inhibitors. Peripheral corneodesmosomes are protected from proteolytic degradation by the tight junction-related structures around them, and this construction provides the basis for the normal basket weave-like structure of the stratum corneum. In Netherton syndrome, which is caused by an absence of the protease inhibitor lymphoepithelial Kazal-type-related inhibitor, premature degradation of corneodesmosomes occurs due to the overactivation of proteases involved in corneodesmosome degradation. Inflammatory peeling skin disease is caused by the absence of corneodesmosin, a unique component of corneodesmosomes. In this disease, corneodesmosomes are structurally abnormal, and their adhesiveness is compromised, which leads to intercellular splitting between the stratum corneum and stratum granulosum. The better we understand desmosome and corneodesmosome ultrastructure in normal and diseased skin, the clearer the physiological and pathological mechanisms of epidermal integrity become.

ER-to-Golgi blockade of nascent desmosomal cadherins in SERCA2-inhibited keratinocytes: Implications for Darier's disease.

Darier's disease (DD) is an autosomal dominantly inherited skin disorder caused by mutations in sarco/endoplasmic reticulum Ca2+ -ATPase 2 (SERCA2), a Ca2+ pump that transports Ca2+ from the cytosol to the endoplasmic reticulum (ER). Loss of desmosomes and keratinocyte cohesion is a characteristic feature of DD. Desmosomal cadherins (DC) are Ca2+ -dependent transmembrane adhesion proteins of desmosomes, which are mislocalized in the lesional but not perilesional skin of DD. We show here that inhibition of SERCA2 by 2 distinct inhibitors results in accumulation of DC precursors in keratinocytes, indicating ER-to-Golgi transport of nascent DC is blocked. Partial loss of SERCA2 by siRNA has no such effect, implicating that haploinsufficiency is not sufficient to affect nascent DC maturation. However, a synergistic effect is revealed between SERCA2 siRNA and an ineffective dose of SERCA2 inhibitor, and between an agonist of the ER Ca2+ release channel and SERCA2 inhibitor. These results suggest that reduction of ER Ca2+ below a critical level causes ER retention of nascent DC. Moreover, colocalization of DC with ER calnexin is detected in SERCA2-inhibited keratinocytes and DD epidermis. Collectively, our data demonstrate that loss of SERCA2 impairs ER-to-Golgi transport of nascent DC, which may contribute to DD pathogenesis.

Desmosomal cadherins and signaling: lessons from autoimmune disease.

Autoantibodies from patients suffering from the autoimmune blistering skin disease pemphigus can be applied as tools to study desmosomal adhesion. These autoantibodies targeting the desmosomal cadherins desmoglein (Dsg) 1 and Dsg3 cause disruption of desmosomes and loss of intercellular cohesion. Although pemphigus autoantibodies were initially proposed to sterically hinder desmosomes, many groups have shown that they activate signaling pathways which cause disruption of desmosomes and loss of intercellular cohesion by uncoupling the desmosomal plaque from the intermediate filament cytoskeleton and/or by interfering with desmosome turnover. These studies demonstrate that desmogleins serve as receptor molecules to transmit outside-in signaling and demonstrate that desmosomal cadherins have functions in addition to their adhesive properties. Two central molecules regulating cytoskeletal anchorage and desmosome turnover are p38MAPK and PKC. As cytoskeletal uncoupling in turn enhances Dsg3 depletion from desmosomes, both mechanisms reinforce one another in a vicious cycle that compromise the integrity and number of desmosomes.

Desmosomal adhesion in vivo.

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.

Desmosomal cadherins are decreased in explanted arrhythmogenic right ventricular dysplasia/cardiomyopathy patient hearts.

AIMS: Arrhythmogenic right ventricular Dysplasia/cardiomyopathy (ARVD/C) is an autosomal dominant inherited cardiomyopathy associated with ventricular arrhythmia, heart failure and sudden death. Genetic studies have demonstrated the central role of desmosomal proteins in this disease, where 50% of patients harbor a mutation in a desmosmal gene. However, clinical diagnosis of the disease remains difficult and molecular mechanisms appears heterogeneous and poorly understood. The aim of this study was to characterize the expression profile of desmosomal proteins in explanted ARVD/C heart samples, in order to identify common features of the disease. METHODS AND RESULTS: We examined plakophilin-2, desmoglein-2, desmocollin-2, plakoglobin and ß-catenin protein expression levels from seven independent ARVD/C heart samples compared to two ischemic, five dilated cardiomyopathy and one healthy heart sample as controls. Ventricular and septum sections were examined by immunoblot analysis of total heart protein extracts and by immunostaining. Immunoblots indicated significant decreases in desmoglein-2 and desmocollin-2, independent of any known underlying mutations, whereas immune-histochemical analysis showed normal localization of all desmosomal proteins. Quantitative RT-PCR revealed normal DSG2 and DSC2 mRNA transcript levels, suggesting increased protein turn-over rather than transcriptional down regulation. CONCLUSION: Reduced cardiac desmoglein-2 and desmocollin-2 levels appear to be specifically associated with ARVD/C, independent of underlying mutations. These findings highlight a key role of desmosomal cadherins in the pathophysiology of ARVD/C. Whether these reductions could be considered as specific markers for ARVD/C requires replication analysis.

Structure, function, and regulation of desmosomes.

Desmosomes are adhesive intercellular junctions that mechanically integrate adjacent cells by coupling adhesive interactions mediated by desmosomal cadherins to the intermediate filament cytoskeletal network. Desmosomal cadherins are connected to intermediate filaments by densely clustered cytoplasmic plaque proteins comprising members of the armadillo gene family, including plakoglobin and plakophilins, and members of the plakin family of cytolinkers, such as desmoplakin. The importance of desmosomes in tissue integrity is highlighted by human diseases caused by mutations in desmosomal genes, autoantibody attack of desmosomal cadherins, and bacterial toxins that selectively target desmosomal cadherins. In addition to reviewing the well-known roles of desmosomal proteins in tissue integrity, this chapter also highlights the growing appreciation for how desmosomal proteins are integrated with cell signaling pathways to contribute to vertebrate tissue organization and differentiation.

Interplay between Rab35 and Arf6 controls cargo recycling to coordinate cell adhesion and migration.

Cells inversely adjust the plasma membrane levels of integrins and cadherins during cell migration and cell-cell adhesion but the regulatory mechanisms that coordinate these trafficking events remain unknown. Here, we demonstrate that the small GTPase Rab35 maintains cadherins at the cell surface to promote cell-cell adhesion. Simultaneously, Rab35 supresses the activity of the GTPase Arf6 to downregulate an Arf6-dependent recycling pathway for ß1-integrin and EGF receptors, resulting in inhibition of cell migration and attenuation of signaling downstream of these receptors. Importantly, the phenotypes of decreased cell adhesion and increased cell migration observed following Rab35 knock down are consistent with the epithelial-mesenchymal transition, a feature of invasive cancer cells, and we show that Rab35 expression is suppressed in a subset of cancers characterized by Arf6 hyperactivity. Our data thus identify a key molecular mechanism that efficiently coordinates the inverse intracellular sorting and cell surface levels of cadherin and integrin receptors for cell migration and differentiation.

p120-Catenin: a novel regulator of innate immunity and inflammation.

p120-Catenin is the prototypic member of a subfamily of armadillo repeat domain proteins. Like its structural homologues, ß- and γ-catenin, p120-catenin is an essential component of adherens junctions in endothelial cells and other polarized adherent cells. p120-Catenin binds directly to the cytoplasmic domain of cadherin and contributes to the regulation of cell-cell junctional integrity. Studies have demonstrated that p120-catenin plays important roles in cell-cell adhesion, embryonic development, cell proliferation and polarity, tumor cell migration, and cancer progression. However, recent insights have generated an entirely new perspective, suggesting that p120-catenin is implicated in the anti-inflammatory responses in the absence and presence of infection. This review summarizes the present knowledge and recent progress toward elucidating the novel role of p120-catenin in the regulation of innate immunity and inflammation.

Cell-cell connectivity: desmosomes and disease.

Cell-cell connectivity is an absolute requirement for the correct functioning of cells, tissues and entire organisms. At the level of the individual cell, direct cell-cell adherence and communication is mediated by the intercellular junction complexes: desmosomes, adherens, tight and gap junctions. A broad spectrum of inherited, infectious and auto-immune diseases can affect the proper function of intercellular junctions and result in either diseases affecting specific individual tissues or widespread syndromic conditions. A particularly diverse group of diseases result from direct or indirect disruption of desmosomes--a consequence of their importance in tissue integrity, their extensive distribution, complex structure, and the wide variety of functions their components accomplish. As a consequence, disruption of desmosomal assembly, structure or integrity disrupts not only their intercellular adhesive function but also their functions in cell communication and regulation, leading to such diverse pathologies as cardiomyopathy, epidermal and mucosal blistering, palmoplantar keratoderma, woolly hair, keratosis, epidermolysis bullosa, ectodermal dysplasia and alopecia. Here, as well as describing the importance of the other intercellular junctions, we focus primarily on the desmosome, its structure and its role in disease. We will examine the various pathologies that result from impairment of desmosome function and thereby demonstrate the importance of desmosomes to tissues and to the organism as a whole.

The three-dimensional molecular structure of the desmosomal plaque.

The cytoplasmic surface of intercellular junctions is a complex network of molecular interactions that link the extracellular region of the desmosomal cadherins with the cytoskeletal intermediate filaments. Although 3D structures of the major plaque components are known, the overall architecture remains unknown. We used cryoelectron tomography of vitreous sections from human epidermis to record 3D images of desmosomes in vivo and in situ at molecular resolution. Our results show that the architecture of the cytoplasmic surface of the desmosome is a 2D interconnected quasiperiodic lattice, with a similar spatial organization to the extracellular side. Subtomogram averaging of the plaque region reveals two distinct layers of the desmosomal plaque: a low-density layer closer to the membrane and a high-density layer further away from the membrane. When combined with a heuristic, allowing simultaneous constrained fitting of the high-resolution structures of the major plaque proteins (desmoplakin, plakophilin, and plakoglobin), it reveals their mutual molecular interactions and explains their stoichiometry. The arrangement suggests that alternate plakoglobin-desmoplakin complexes create a template on which desmosomal cadherins cluster before they stabilize extracellularly by binding at their N-terminal tips. Plakophilins are added as a molecular reinforcement to fill the gap between the formed plaque complexes and the plasma membrane.