Architecture and dynamics of a desmosome-endoplasmic reticulum complex.

The endoplasmic reticulum (ER) forms a dynamic network that contacts other cellular membranes to regulate stress responses, calcium signalling and lipid transfer. Here, using high-resolution volume electron microscopy, we find that the ER forms a previously unknown association with keratin intermediate filaments and desmosomal cell-cell junctions. Peripheral ER assembles into mirror image-like arrangements at desmosomes and exhibits nanometre proximity to keratin filaments and the desmosome cytoplasmic plaque. ER tubules exhibit stable associations with desmosomes, and perturbation of desmosomes or keratin filaments alters ER organization, mobility and expression of ER stress transcripts. These findings indicate that desmosomes and the keratin cytoskeleton regulate the distribution, function and dynamics of the ER network. Overall, this study reveals a previously unknown subcellular architecture defined by the structural integration of ER tubules with an epithelial intercellular junction.

Desmosomes undergo dynamic architectural changes during assembly and maturation.

Desmosomes are macromolecular cell-cell junctions critical for maintaining adhesion and resisting mechanical stress in epithelial tissue. Desmosome assembly and the relationship between maturity and molecular architecture are not well understood. To address this, we employed a calcium switch assay to synchronize assembly followed by quantification of desmosome nanoscale organization using direct Stochastic Optical Reconstruction Microscopy (dSTORM). We found that the organization of the desmoplakin rod/C-terminal junction changed over the course of maturation, as indicated by a decrease in the plaque-to-plaque distance, while the plaque length increased. In contrast, the desmoplakin N-terminal domain and plakoglobin organization (plaque-to-plaque distance) were constant throughout maturation. This structural rearrangement of desmoplakin was concurrent with desmosome maturation measured by E-cadherin exclusion and increased adhesive strength. Using two-color dSTORM, we showed that while the number of individual E-cadherin containing junctions went down with the increasing time in high Ca2+, they maintained a wider desmoplakin rod/C-terminal plaque-to-plaque distance. This indicates that the maturation state of individual desmosomes can be identified by their architectural organization. We confirmed these architectural changes in another model of desmosome assembly, cell migration. Desmosomes in migrating cells, closest to the scratch where they are assembling, were shorter, E-cadherin enriched, and had wider desmoplakin rod/C-terminal plaque-to-plaque distances compared to desmosomes away from the wound edge. Key results were demonstrated in three cell lines representing simple, transitional, and stratified epithelia. Together, these data suggest that there is a set of architectural programs for desmosome maturation, and we hypothesize that desmoplakin architecture may be a contributing mechanism to regulating adhesive strength.

The Actin-Binding Protein α-Adducin Modulates Desmosomal Turnover and Plasticity.

Intercellular adhesion is essential for tissue integrity and homeostasis. Desmosomes are abundant in the epidermis and the myocardium-tissues, which are under constantly changing mechanical stresses. Yet, it is largely unclear whether desmosomal adhesion can be rapidly adapted to changing demands, and the mechanisms underlying desmosome turnover are only partially understood. In this study we show that the loss of the actin-binding protein α-adducin resulted in reduced desmosome numbers and prevented the ability of cultured keratinocytes or murine epidermis to withstand mechanical stress. This effect was not primarily caused by decreased levels or impaired adhesive properties of desmosomal molecules but rather by altered desmosome turnover. Mechanistically, reduced cortical actin density in α-adducin knockout keratinocytes resulted in increased mobility of the desmosomal adhesion molecule desmoglein 3 and impaired interactions with E-cadherin, a crucial step in desmosome formation. Accordingly, the loss of α-adducin prevented increased membrane localization of desmoglein 3 in response to cyclic stretch or shear stress. Our data demonstrate the plasticity of desmosomal molecules in response to mechanical stimuli and unravel a mechanism of how the actin cytoskeleton indirectly shapes intercellular adhesion by restricting the membrane mobility of desmosomal molecules.

Desmosome architecture derived from molecular dynamics simulations and cryo-electron tomography.

Desmosomes are cell-cell junctions that link tissue cells experiencing intense mechanical stress. Although the structure of the desmosomal cadherins is known, the desmosome architecture-which is essential for mediating numerous functions-remains elusive. Here, we recorded cryo-electron tomograms (cryo-ET) in which individual cadherins can be discerned; they appear variable in shape, spacing, and tilt with respect to the membrane. The resulting sub-tomogram average reaches a resolution of ∼26 Å, limited by the inherent flexibility of desmosomes. To address this challenge typical of dynamic biological assemblies, we combine sub-tomogram averaging with atomistic molecular dynamics (MD) simulations. We generate models of possible cadherin arrangements and perform an in silico screening according to biophysical and structural properties extracted from MD simulation trajectories. We find a truss-like arrangement of cadherins that resembles the characteristic footprint seen in the electron micrograph. The resulting model of the desmosomal architecture explains their unique biophysical properties and strength.

The desmosome as a model for lipid raft driven membrane domain organization.

Desmosomes are cadherin-based adhesion structures that mechanically couple the intermediate filament cytoskeleton of adjacent cells to confer mechanical stress resistance to tissues. We have recently described desmosomes as mesoscale lipid raft membrane domains that depend on raft dynamics for assembly, function, and disassembly. Lipid raft microdomains are regions of the plasma membrane enriched in sphingolipids and cholesterol. These domains participate in membrane domain heterogeneity, signaling and membrane trafficking. Cellular structures known to be dependent on raft dynamics include the post-synaptic density in neurons, the immunological synapse, and intercellular junctions, including desmosomes. In this review, we discuss the current state of the desmosome field and put forward new hypotheses for the role of lipid rafts in desmosome adhesion, signaling and epidermal homeostasis. Furthermore, we propose that differential lipid raft affinity of intercellular junction proteins is a central driving force in the organization of the epithelial apical junctional complex.

Cell-cell junctions as sensors and transducers of mechanical forces.

Epithelial and endothelial monolayers are multicellular sheets that form barriers between the 'outside' and 'inside' of tissues. Cell-cell junctions, made by adherens junctions, tight junctions and desmosomes, hold together these monolayers. They form intercellular contacts by binding their receptor counterparts on neighboring cells and anchoring these structures intracellularly to the cytoskeleton. During tissue development, maintenance and pathogenesis, monolayers encounter a range of mechanical forces from the cells themselves and from external systemic forces, such as blood pressure or tissue stiffness. The molecular landscape of cell-cell junctions is diverse, containing transmembrane proteins that form intercellular bonds and a variety of cytoplasmic proteins that remodel the junctional connection to the cytoskeleton. Many junction-associated proteins participate in mechanotransduction cascades to confer mechanical cues into cellular responses that allow monolayers to maintain their structural integrity. We will discuss force-dependent junctional molecular events and their role in cell-cell contact organization and remodeling.

Molecular organization, regulation and function of tricellular junctions.

Tricellular junctions are specialized cell-cell junctions formed at sites where three epithelial or endothelial cells make contact at their apical side. By holding three cells together, tricellular junctions contribute to the maintenance of epithelial barrier function and mechanical integrity. In addition, recent studies have uncovered new functions of tricellular junctions at both cellular and physiological levels. In this review, we describe the architecture and molecular components of tricellular junctions and discuss how tricellular junctions participate in various biological processes.

Distinct molecular signature of phospholamban p.Arg14del arrhythmogenic cardiomyopathy.

Phospholamban (PLN) p.Arg14del cardiomyopathy is characterized by a distinct arrhythmogenic biventricular phenotype that can be predominantly left ventricular, right ventricular, or both. Our aim was to further elucidate distinct features of this cardiomyopathy with respect to the distribution of desmosomal proteins observed by immunofluorescence (IF) in comparison to desmosomal arrhythmogenic cardiomyopathy and co-existent genetic variants. We studied eight explanted heart specimens from PLN p.Arg14del mutation carriers. Macro- and microscopic examination revealed biventricular presence of fibrofatty replacement and interstitial fibrosis. Five out of 8 (63%) patients met consensus criteria for both arrhythmogenic right ventricular cardiomyopathy (ARVC) and dilated cardiomyopathy (DCM). In four cases, targeted next-generation sequencing revealed one additional pathogenic variant and six variants of unknown significance. IF showed diminished junction plakoglobin signal intensity at the intercalated disks in 4 (67%) out of 6 cases fulfilling ARVC criteria but normal intensity in both cases fulfilling only DCM criteria. Notably, the four cases with diminished junction plakoglobin were also those where an additional gene variant was detected. IF for two proteins recently investigated in desmosomal arrhythmogenic cardiomyopathy (ACM), synapse-associated protein 97 and glycogen synthase kinase-3 beta, showed a distinct distributional pattern in comparison to desmosomal ACM. In 7 (88%) out of 8 cases we observed both a strong synapse-associated protein 97 signal at the sarcomeres and no glycogen synthase kinase-3 beta translocation to the intercalated discs. Phospholamban p.Arg14del cardiomyopathy is characterized by a distinct molecular signature compared to desmosomal ACM, specifically a different desmosomal protein distribution. This study substantiates the idea that additional genetic variants play a role in the phenotypical heterogeneity.

Intercalated discs: cellular adhesion and signaling in heart health and diseases.

Intercalated discs (ICDs) are highly orchestrated structures that connect neighboring cardiomyocytes in the heart. Three major complexes are distinguished in ICD: desmosome, adherens junction (AJ), and gap junction (GJ). Desmosomes are major cell adhesion junctions that anchor cell membrane to the intermediate filament network; AJs connect the actin cytoskeleton of adjacent cells; and gap junctions metabolically and electrically connect the cytoplasm of adjacent cardiomyocytes. All these complexes work as a single unit, the so-called area composita, interdependently rather than individually. Mutation or altered expression of ICD proteins results in various cardiac diseases, such as ARVC (arrhythmogenic right ventricular cardiomyopathy), dilated cardiomyopathy, and hypotrophy cardiomyopathy, eventually leading to heart failure. In this article, we first review the recent findings on the structural organization of ICD and their functions and then focus on the recent advances in molecular pathogenesis of the ICD-related heart diseases, which include two major areas: i) the ICD gene mutations in cardiac diseases, and ii) the involvement of ICD proteins in signal transduction pathways leading to myocardium remodeling and eventual heart failure. These major ICD-related signaling pathways include Wnt/ß-catenin pathway, p38 MAPK cascade, Rho-dependent serum response factor (SRF) signaling, calcineurin/NFAT signaling, Hippo kinase cascade, etc., which are differentially regulated in pathological conditions.

Mechanical loading of desmosomes depends on the magnitude and orientation of external stress.

Desmosomes are intercellular adhesion complexes that connect the intermediate filament cytoskeletons of neighboring cells, and are essential for the mechanical integrity of mammalian tissues. Mutations in desmosomal proteins cause severe human pathologies including epithelial blistering and heart muscle dysfunction. However, direct evidence for their load-bearing nature is lacking. Here we develop Förster resonance energy transfer (FRET)-based tension sensors to measure the forces experienced by desmoplakin, an obligate desmosomal protein that links the desmosomal plaque to intermediate filaments. Our experiments reveal that desmoplakin does not experience significant tension under most conditions, but instead becomes mechanically loaded when cells are exposed to external mechanical stresses. Stress-induced loading of desmoplakin is transient and sensitive to the magnitude and orientation of the applied tissue deformation, consistent with a stress absorbing function for desmosomes that is distinct from previously analyzed cell adhesion complexes.

Disturbed Desmoglein-2 in the intercalated disc of pediatric patients with dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) leads to disturbed contraction and force transduction, and is associated with substantial mortality in all age groups. Involvement of a disrupted composition of the intercalated disc (ID) has been reported. However, in children, little is established about such subcellular changes during disease, because of the pathological mix-up with the ongoing cardiac maturation. This leaves maladaptive remodeling often undetected. We aimed at illustrating subcellular alterations in children diagnosed with DCM compared to age-matched controls, focusing on ID proteins known to be crucially stable under healthy conditions and destabilized during cardiac injury in adults. Left ventricular or septal pediatric specimens were collected from 7 individuals diagnosed with DCM (age: 23 weeks in utero to 8 weeks postnatal) and age-matched controls that died of non-cardiovascular cause. We determined the amount of fibrosis and localization of ID proteins by immunohistochemistry. In pediatric DCM, most ID proteins follow similar spatiotemporal changes in localization as in controls. However, although no mutations were found, the signal of the desmosomal protein Desmoglein-2 was reduced in all pediatric DCM specimens, but not in controls or adult DCM patients. Endocardial and transmural fibrosis was increased in all pediatric DCM patients compared to age-matched controls. Composition of the ID in pediatric DCM patients is similar to controls, except for the localization of Desmoglein-2 and presence of severe fibrosis. This suggests that the architecture of desmosomes is already disturbed in the early stages of DCM. These findings contribute to the understanding of pediatric DCM.

Inhibition of p38MAPK signalling prevents epidermal blistering and alterations of desmosome structure induced by pemphigus autoantibodies in human epidermis.

BACKGROUND: Pemphigus vulgaris (PV) is a skin blistering disease caused by autoantibodies targeting the desmosomal adhesion proteins desmoglein (Dsg) 3 and 1. The mechanisms underlying pemphigus skin blistering are not fully elucidated but p38 mitogen-activated protein kinase (p38MAPK) activation is one of the signalling events necessary for full loss of cell cohesion. However, it is unclear whether ultrastructural hallmarks of desmosome morphology as observed in patients' lesions are mediated by p38MAPK signalling. OBJECTIVES: In this study, we tested the relevance of p38MAPK for blister formation and the ultrastructural changes induced by PV autoantibodies in human skin. METHODS: Human skin samples were injected with IgG fractions of one patient suffering from mucocutaneous PV (mcPV-IgG), one from mucosal-dominant PV (mdPV-IgG) or AK23, a pathogenic monoclonal Dsg3 antibody derived from a pemphigus mouse model. Samples were processed for histological and electron microscopy analyses. RESULTS: mcPV-IgG and AK23 but not mdPV-IgG reduced desmosome size, caused interdesmosomal widening and formation of split desmosomes, and altered keratin filament insertion. In contrast, full epidermal blister formation and lower desmosome number were evident in tissue samples exposed to mcPV-IgG only. Pharmacological inhibition of p38MAPK blunted the reduction of desmosome number and size, ameliorated interdesmosomal widening and loss of keratin insertion and prevented mcPV-IgG-induced blister formation. CONCLUSIONS: Our data demonstrate that blistering can be prevented by inhibition of p38MAPK in the human epidermis. Moreover, typical morphological alterations induced by mcPV-IgG such as interdesmosomal widening and the reduction of desmosome size at least in part require p38MAPK signalling.

CSPP-L Associates with the Desmosome of Polarized Epithelial Cells and Is Required for Normal Spheroid Formation.

Deleterious mutations of the Centrosome/Spindle Pole associated Protein 1 gene, CSPP1, are causative for Joubert-syndrome and Joubert-related developmental disorders. These disorders are defined by a characteristic mal-development of the brain, but frequently involve renal and hepatic cyst formation. CSPP-L, the large protein isoform of CSPP1 localizes to microtubule ends of the mitotic mid-spindle and the ciliary axoneme, and is required for ciliogenesis. We here report the microtubule independent but Desmoplakin dependent localization of CSPP-L to Desmosomes in apical-basal polarized epithelial cells. Importantly, siRNA conferred depletion of CSPP-L or Desmoplakin promoted multi-lumen spheroid formation in 3D-cultures of non-ciliated human colon carcinoma Caco-2 cells. Multi-lumen spheroids of CSPP1 siRNA transfectants showed disrupted apical cell junction localization of the cytoskeleton organizing RhoGEF ECT2. Our results hence identify a novel, non-ciliary role for CSPP-L in epithelial morphogenesis.

Cadherin flexibility provides a key difference between desmosomes and adherens junctions.

Desmosomes and adherens junctions are intercellular adhesive structures essential for the development and integrity of vertebrate tissue, including the epidermis and heart. Their cell adhesion molecules are cadherins: type 1 cadherins in adherens junctions and desmosomal cadherins in desmosomes. A fundamental difference is that desmosomes have a highly ordered structure in their extracellular region and exhibit calcium-independent hyperadhesion, whereas adherens junctions appear to lack such ordered arrays, and their adhesion is always calcium-dependent. We present here the structure of the entire ectodomain of desmosomal cadherin desmoglein 2 (Dsg2), using a combination of small-angle X-ray scattering, electron microscopy, and solution-based biophysical techniques. This structure reveals that the ectodomain of Dsg2 is flexible even in the calcium-bound state and, on average, is shorter than the type 1 cadherin crystal structures. The Dsg2 structure has an excellent fit with the electron tomography reconstructions of human desmosomes. This fit suggests an arrangement in which desmosomal cadherins form trans interactions but are too far apart to interact in cis, in agreement with previously reported observations. Cadherin flexibility may be key to explaining the plasticity of desmosomes that maintain tissue integrity in their hyperadhesive form, but can adopt a weaker, calcium-dependent adhesion during wound healing and early development.

Remodeling of keratin-coupled cell adhesion complexes.

Epithelial cells constitute the main barrier between the inside and outside of organs, acting as gatekeepers of their structure and integrity. Hemidesmosomes and desmosomes are respectively cell-matrix and cell-cell adhesions coupled to the intermediate filament cytoskeleton. These adhesions ensure mechanical integrity of the epithelial barrier. Although desmosomes and hemidesmosomes are essential in maintaining strong cell-cell and cell-matrix adhesions, there is an emerging view that they should be remodeled in order to maintain epithelial homeostasis. Here we review the adhesion properties of desmosomes and hemidesmosomes, as well as the mechanisms driving their remodeling. We also discuss recent data suggesting that keratin-coupled adhesion complexes can sense the biomechanical cellular environment and participate in the cellular response to such external cues.

Hyper-adhesion: a unique property of desmosomes.

Hyper-adhesion is a unique, strongly adhesive form of desmosomal adhesion that functions to maintain tissue integrity. In this short review, we define hyper-adhesion, summarise the evidence for it in culture and in vivo, discuss its role in development, wound healing, and skin disease, and speculate about its molecular and cellular basis.

Insights into desmosome biology from inherited human skin disease and cardiocutaneous syndromes.

The importance of desmosomes in tissue homeostasis is highlighted by natural and engineered mutations in desmosomal genes, which compromise the skin or heart and in some instances both. Desmosomal gene mutations account for 45-50% of cases of arrhythmogenic right ventricular cardiomyopathy, and are mutated in an array of other disorders such as striate palmoplantar keratoderma, hypotrichosis with or without skin vesicles and lethal acantholytic epidermolysis bullosa. Recently, we reported loss-of-function mutations in the human ADAM17 gene, encoding for the 'sheddase' ADAM17, a transmembrane protein which cleaves extracellular domains of substrate proteins including TNF-α, growth factors and desmoglein (DSG) 2. Patients present with cardiomyopathy and an inflammatory skin and bowel syndrome with defective DSG processing. In contrast, the dominantly inherited tylosis with oesophageal cancer appears to result from gain-of-function in ADAM17 due to increased processing via iRHOM2. This review discusses the heterogeneity of mutations in desmosomes and their regulatory proteins.

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.

Structural and functional diversity of desmosomes.

Desmosomes anchor intermediate filaments at sites of cell contact established by the interaction of cadherins extending from opposing cells. The incorporation of cadherins, catenin adaptors, and cytoskeletal elements resembles the closely related adherens junction. However, the recruitment of intermediate filaments distinguishes desmosomes and imparts a unique function. By linking the load-bearing intermediate filaments of neighboring cells, desmosomes create mechanically contiguous cell sheets and, in so doing, confer structural integrity to the tissues they populate. This trait and a well-established role in human disease have long captured the attention of cell biologists, as evidenced by a publication record dating back to the mid-1860s. Likewise, emerging data implicating the desmosome in signaling events pertinent to organismal development, carcinogenesis, and genetic disorders will secure a prominent role for desmosomes in future biological and biomedical investigations.