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.

Celsr1 adhesive interactions mediate the asymmetric organization of planar polarity complexes.

To orchestrate collective polarization across tissues, planar cell polarity (PCP) proteins localize asymmetrically to cell junctions, a conserved feature of PCP that requires the atypical cadherin Celsr1. We report that mouse Celsr1 engages in both trans- and cis-interactions, and organizes into dense and highly stable punctate assemblies. We provide evidence suggesting that PCP-mutant variant of Celsr1, Celsr1Crsh, selectively impairs lateral cis-interactions. Although Celsr1Crsh mediates cell adhesion in trans, it displays increased mobility, diminishes junctional enrichment, and fails to engage in homophilic adhesion with the wild-type protein, phenotypes that can be rescued by ectopic cis-dimerization. Using biochemical and super-resolution microscopy approaches, we show that although Celsr1Crsh physically interacts with PCP proteins Frizzled6 and Vangl2, it fails to organize these proteins into asymmetric junctional complexes. Our results suggest mammalian Celsr1 functions not only as a trans-adhesive homodimeric bridge, but also as an organizer of intercellular Frizzled6 and Vangl2 asymmetry through lateral, cis-interactions.

The desmosome is a mesoscale lipid raft-like membrane domain.

Desmogleins (Dsgs) are cadherin family adhesion molecules essential for epidermal integrity. Previous studies have shown that desmogleins associate with lipid rafts, but the significance of this association was not clear. Here, we report that the desmoglein transmembrane domain (TMD) is the primary determinant of raft association. Further, we identify a novel mutation in the DSG1 TMD (G562R) that causes severe dermatitis, multiple allergies, and metabolic wasting syndrome. Molecular modeling predicts that this G-to-R mutation shortens the DSG1 TMD, and experiments directly demonstrate that this mutation compromises both lipid raft association and desmosome incorporation. Finally, cryo-electron tomography indicates that the lipid bilayer within the desmosome is ∼10% thicker than adjacent regions of the plasma membrane. These findings suggest that differences in bilayer thickness influence the organization of adhesion molecules within the epithelial plasma membrane, with cadherin TMDs recruited to the desmosome via the establishment of a specialized mesoscale lipid raft-like membrane domain.

E-cadherin binds to desmoglein to facilitate desmosome assembly.

Desmosomes are adhesive junctions composed of two desmosomal cadherins: desmocollin (Dsc) and desmoglein (Dsg). Previous studies demonstrate that E-cadherin (Ecad), an adhesive protein that interacts in both trans (between opposing cells) and cis (on the same cell surface) conformations, facilitates desmosome assembly via an unknown mechanism. Here we use structure-function analysis to resolve the mechanistic roles of Ecad in desmosome formation. Using AFM force measurements, we demonstrate that Ecad interacts with isoform 2 of Dsg via a conserved Leu-175 on the Ecad cis binding interface. Super-resolution imaging reveals that Ecad is enriched in nascent desmosomes, supporting a role for Ecad in early desmosome assembly. Finally, confocal imaging demonstrates that desmosome assembly is initiated at sites of Ecad mediated adhesion, and that Ecad-L175 is required for efficient Dsg2 and desmoplakin recruitment to intercellular contacts. We propose that Ecad trans interactions at nascent cell-cell contacts initiate the recruitment of Dsg through direct cis interactions with Ecad which facilitates desmosome assembly.

Molecular organization of the desmosome as revealed by direct stochastic optical reconstruction microscopy.

Desmosomes are macromolecular junctions responsible for providing strong cell-cell adhesion. Because of their size and molecular complexity, the precise ultrastructural organization of desmosomes is challenging to study. Here, we used direct stochastic optical reconstruction microscopy (dSTORM) to resolve individual plaque pairs for inner and outer dense plaque proteins. Analysis methods based on desmosomal mirror symmetry were developed to measure plaque-to-plaque distances and create an integrated map. We quantified the organization of desmoglein 3, plakoglobin and desmoplakin (N-terminal, rod and C-terminal domains) in primary human keratinocytes. Longer desmosome lengths correlated with increasing plaque-to-plaque distance, suggesting that desmoplakin is arranged with its long axis at an angle within the plaque. We next examined whether plaque organization changed in different adhesive states. Plaque-to-plaque distance for the desmoplakin rod and C-terminal domains decreased in PKP-1-mediated hyperadhesive desmosomes, suggesting that protein reorganization correlates with function. Finally, in human epidermis we found a difference in plaque-to-plaque distance for the desmoplakin C-terminal domain, but not the desmoplakin rod domain or plakoglobin, between basal and suprabasal cells. Our data reveal the molecular organization of desmosomes in cultured keratinocytes and skin as defined by dSTORM.

Super-Resolution Microscopy Reveals Altered Desmosomal Protein Organization in Tissue from Patients with Pemphigus Vulgaris.

Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease in which autoantibodies (IgG) are directed against the desmosomal cadherin desmoglein 3. To better understand how PV IgG alters desmosome morphology and function in vivo, biopsies from patients with PV were analyzed by structured illumination microscopy, a form of superresolution fluorescence microscopy. In patient tissue, desmosomal proteins were aberrantly clustered and patient IgG colocalized with markers for lipid rafts and endosomes. Additionally, steady-state levels of desmoglein 3 were decreased and desmosomes were reduced in size in patient tissue. Desmosomes at blister sites were occasionally split, with PV IgG decorating the extracellular faces of split desmosomes. Desmosome splitting was recapitulated in vitro by exposing cultured keratinocytes both to PV IgG and to mechanical stress, demonstrating that splitting at the blister interface in patient tissue is due to compromised desmosomal adhesive function. These findings indicate that desmoglein 3 clustering and endocytosis are associated with reduced desmosome size and adhesion defects in tissue of patients with PV. Further, this study reveals that superresolution optical imaging is a powerful approach for studying epidermal adhesion structures in normal and diseased skin.

Desmosomes in acquired disease.

Desmosomes are cell-cell junctions that mediate adhesion and couple the intermediate filament cytoskeleton to sites of cell-cell contact. This architectural arrangement integrates adhesion and cytoskeletal elements of adjacent cells. The importance of this robust adhesion system is evident in numerous human diseases, both inherited and acquired, which occur when desmosome function is compromised. This review focuses on autoimmune and infectious diseases that impair desmosome function. In addition, we discuss emerging evidence that desmosomal genes are often misregulated in cancer. The emphasis of our discussion is placed on the way in which human diseases can inform our understanding of basic desmosome biology and in turn, the means by which fundamental advances in the cell biology of desmosomes might lead to new treatments for acquired diseases of the desmosome.

Desmosome assembly and disassembly are membrane raft-dependent.

Strong intercellular adhesion is critical for tissues that experience mechanical stress, such as the skin and heart. Desmosomes provide adhesive strength to tissues by anchoring desmosomal cadherins of neighboring cells to the intermediate filament cytoskeleton. Alterations in assembly and disassembly compromise desmosome function and may contribute to human diseases, such as the autoimmune skin blistering disease pemphigus vulgaris (PV). We previously demonstrated that PV auto-antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3) cause loss of adhesion by triggering membrane raft-mediated Dsg3 endocytosis. We hypothesized that raft membrane microdomains play a broader role in desmosome homeostasis by regulating the dynamics of desmosome assembly and disassembly. In human keratinocytes, Dsg3 is raft associated as determined by biochemical and super resolution immunofluorescence microscopy methods. Cholesterol depletion, which disrupts rafts, prevented desmosome assembly and adhesion, thus functionally linking rafts to desmosome formation. Interestingly, Dsg3 did not associate with rafts in cells lacking desmosomal proteins. Additionally, PV IgG-induced desmosome disassembly occurred by redistribution of Dsg3 into raft-containing endocytic membrane domains, resulting in cholesterol-dependent loss of adhesion. These findings demonstrate that membrane rafts are required for desmosome assembly and disassembly dynamics, suggesting therapeutic potential for raft targeting agents in desmosomal diseases such as PV.

Plakophilin-1 protects keratinocytes from pemphigus vulgaris IgG by forming calcium-independent desmosomes.

Plakophilin-1 (PKP-1) is an armadillo family protein critical for desmosomal adhesion and epidermal integrity. In the autoimmune skin-blistering disease pemphigus vulgaris (PV), autoantibodies (IgG) target the desmosomal cadherin desmoglein 3 (Dsg3) and compromise keratinocyte cell-cell adhesion. Here, we report that enhanced expression of PKP-1 protects keratinocytes from PV IgG-induced loss of cell-cell adhesion. PKP-1 prevents loss of Dsg3 and other desmosomal proteins from cell-cell borders and prevents alterations in desmosome ultrastructure in keratinocytes treated with PV IgG. Using a series of Dsg3 chimeras and deletion constructs, we find that PKP-1 clusters Dsg3 with the desmosomal plaque protein desmoplakin in a manner dependent on the plakoglobin-binding domain of the Dsg3 tail. Furthermore, PKP-1 expression transforms desmosome adhesion from a calcium-dependent to a calcium-independent and hyperadhesive state. These results demonstrate that manipulating the expression of a single desmosomal plaque protein can block the pathogenic effects of PV IgG on keratinocyte adhesion.

Signaling dependent and independent mechanisms in pemphigus vulgaris blister formation.

Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease caused by autoantibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3). Significant advances in our understanding of pemphigus pathomechanisms have been derived from the generation of pathogenic monoclonal Dsg3 antibodies. However, conflicting models for pemphigus pathogenicity have arisen from studies using either polyclonal PV patient IgG or monoclonal Dsg3 antibodies. In the present study, the pathogenic mechanisms of polyclonal PV IgG and monoclonal Dsg3 antibodies were directly compared. Polyclonal PV IgG cause extensive clustering and endocytosis of keratinocyte cell surface Dsg3, whereas pathogenic mouse monoclonal antibodies compromise cell-cell adhesion strength without causing these alterations in Dsg3 trafficking. Furthermore, tyrosine kinase or p38 MAPK inhibition prevents loss of keratinocyte adhesion in response to polyclonal PV IgG. In contrast, disruption of adhesion by pathogenic monoclonal antibodies is not prevented by these inhibitors either in vitro or in human skin explants. Our results reveal that the pathogenic activity of polyclonal PV IgG can be attributed to p38 MAPK-dependent clustering and endocytosis of Dsg3, whereas pathogenic monoclonal Dsg3 antibodies can function independently of this pathway. These findings have important implications for understanding pemphigus pathophysiology, and for the design of pemphigus model systems and therapeutic interventions.

Mutation of the conserved polyadenosine RNA binding protein, ZC3H14/dNab2, impairs neural function in Drosophila and humans.

Here we report a human intellectual disability disease locus on chromosome 14q31.3 corresponding to mutation of the ZC3H14 gene that encodes a conserved polyadenosine RNA binding protein. We identify ZC3H14 mRNA transcripts in the human central nervous system, and we find that rodent ZC3H14 protein is expressed in hippocampal neurons and colocalizes with poly(A) RNA in neuronal cell bodies. A Drosophila melanogaster model of this disease created by mutation of the gene encoding the ZC3H14 ortholog dNab2, which also binds polyadenosine RNA, reveals that dNab2 is essential for development and required in neurons for normal locomotion and flight. Biochemical and genetic data indicate that dNab2 restricts bulk poly(A) tail length in vivo, suggesting that this function may underlie its role in development and disease. These studies reveal a conserved requirement for ZC3H14/dNab2 in the metazoan nervous system and identify a poly(A) RNA binding protein associated with a human brain disorder.