Ultrastructural and biochemical identification of con A receptors in the desmosome

Correlated ultrastructural and biochemical methods were used to identify and localize Concanavalin A (Con A) receptors in the desmosomes of bovine epidermis. Specific carbohydrate residues were labeled with ferritin-Con A in thin sections of tissue embedded in a hydrophilic resin. Quantitative mapping of ferritin distribution in labeled desmosomes revealed that Con A receptors are localized in the intercellular zone and concentrated along the desmosomal midline or central dense stratum. Labeling was almost entirely absent when sections were treated with ferritin-Con A in the presence of 0.1 M α-methyl mannoside, a hapten-inhibitor of Con A. “Whole” desmosomes and desmosomal intercellular regions (desmosomal “cores”) were purified from bovine muzzle epidermis. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals a limited number of major desmosomal protein constituents. Certain of these are glycoproteins and are greatly enriched in the core fraction. Almost all the desmosomal glycoproteins are intensely labeled when electrophoretic gels of whole desmosome or core fractions are exposed to fluorescent Concanavalin A.     

Desmosomal Cadherins and Signaling: Lessons from Autoimmune Disease

Abstract

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.     

Isolation and symmetrical splitting of desmosomal structures in 9 M urea

A new way of isolating desmosomal structures from various epithelia is described which takes advantage of the unusual resistance of the desmosomal plaque and parts of the desmosomal membrane domain to denaturing agents such as 9 M urea and 5 M guanidinium hydrochloride (Gdn-HCl). The fractions obtained have been examined by electron microscopy and by gel electrophoresis. When cytoskeletal fractions from epithelial cells, notably those from multistratified epithelia such as bovine epidermis or tongue mucosa, are treated with urea or Gdn-HCl most of the cytoskeletal protein, including cytokeratin material, is removed. The desmosomal structures, however, are retained with well preserved plaque organization and desmoglea components and can be harvested by centrifugation. This simple and rapid procedure for the enrichment of desmosomal structures and proteins also express internal desmosomal domains as the result of "splitting" of the desmosome along the midline structure. These split desmosomal halves reveal regular arrays of desmogleal particles of 8 to 15 nm diameter projecting from the membrane surface. Gel electrophoresis of the polypeptides present in these residual structures has shown prominent amounts of desmoplakins I and II as well as components 3 and 5 whereas glycoproteins 4a and 4b are significantly reduced in relation to untreated or citric acid-treated fractions. Using immunoelectron microscopy on desmosomes split in urea we have also demonstrated the specific localization of desmoplakin on the cytoplasmic side. The observations suggest that the architectural components of the desmosome are among the cell structures most resistant to protein-denaturing treatments. The value of this procedure for preparations of desmosomal proteins and for the production of antibodies specifically reacting with internal domains of junctions, i.e., tools that may interfere with cell-to-cell coupling, is discussed.     

Down-Regulation of Desmosomes in Cultured Cells: The Roles of PKC,Microtubules and Lysosomal/Proteasomal Degradation

Desmosomes are intercellular adhesive junctions of major importance for tissue integrity. To allow cell motility and migration they are down-regulated in epidermal wound healing. Electron microscopy indicates that whole desmosomes are internalised by cells in tissues, but the mechanism of down-regulation is unclear. In this paper we provide an overview of the internalisation of half-desmosomes by cultured cells induced by calcium chelation. Our results show that: (i) half desmosome internalisation is dependent on conventional PKC isoforms; (ii) microtubules transport internalised half desmosomes to the region of the centrosome by a kinesin-dependent mechanism; (iii) desmosomal proteins remain colocalised after internalisation and are not recycled to the cell surface; (iv) internalised desmosomes are degraded by the combined action of lysosomes and proteasomes. We also confirm that half desmosome internalisation is dependent upon the actin cytoskeleton. These results suggest that half desmosomes are not disassembled and recycled during or after internalisation but instead are transported to the centrosomal region where they are degraded. These findings may have significance for the down-regulation of desmosomes in wounds.     

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.     

Pervanadate stabilizes desmosomes

Desmosomes are intercellular junctions responsible for strong cell-cell adhesion in epithelia and cardiac muscle. Numerous studies have shown that the other major type of epithelial cell adhesion, the adherens junction, is destabilized by src-induced tyrosine phosphorylation of two of its principal components, E-cadherin and β-catenin. Here we show that treatment of epithelial cells with the potent tyrosine phosphatase inhibitor sodium pervanadate causes tyrosine phosphorylation of the major desmosomal components desmoglein 2 and plakoglobin in both the non-ionic detergent soluble and insoluble cell fractions and, surprisingly, stabilizes desmosomal adhesion, inducing the hyper-adhesive form normally found in tissues and confluent cell sheets. Taken together with the few other studies on desmosomes these results suggest that the effects of tyrosine phosphorylation on desmosomal adhesion are complex.Key words: desmosome, cell-cell adhesion, intercellular junction, tyrosine phosphorylation, pervanadate, desmoglein, plakoglobin     

Intermediate filaments and the initiation of desmosome assembly

The desmosome junction is an important component in the cohesion of epithelial cells, especially epidermal keratinocytes. To gain insight into the structure and function of desmosomes, their morphogenesis has been studied in a primary mouse epidermal (PME) cell culture system. When these cells are grown in approximately 0.1 mM Ca2+, they contain no desmosomes. They are induced to form desmosomes when the Ca2+ level in the culture medium is raised to approximately 1.2 mM Ca2+. PME cells in medium containing low levels of Ca2+, and then processed for indirect immunofluorescence using antibodies directed against desmoplakins (desmosomal plaque proteins), display a pattern of discrete fluorescent spots concentrated mainly in the perinuclear region. Double label immunofluorescence using keratin and desmoplakin antibodies reveals that the desmoplakin-containing spots and the cytoplasmic network of tonofibrils (bundles of intermediate filaments [IFB]) are in the same juxtanuclear region. Within 1 h after the switch to higher levels of Ca2+, the spots move toward the cell surface, primarily to areas of cell-cell contact and not to free cell surfaces. This reorganization occurs at the same time that tonofibrils also move toward cell surfaces in contact with neighboring cells. Once the desmoplakin spots have reached the cell surface, they appear to aggregate to form desmosomes. These immunofluorescence observations have been confirmed by immunogold ultrastructural localization. Preliminary biochemical and immunological studies indicate that desmoplakin appears in whole cell protein extracts and in Triton high salt insoluble residues (i.e., cytoskeletal preparations consisting primarily of IFB) prepared from PME cells maintained in medium containing both low and normal Ca2+ levels. These findings show that certain desmosome components are preformed in the cytoplasm of PME cells. These components undergo a dramatic reorganization, which parallels the changes in IFB redistribution, upon induction of desmosome formation. The reorganization depends upon both the extracellular Ca2+ level and the establishment of cell-to-cell contacts. Furthermore, the data suggests that desmosomes do not act as organizing centers for the elaboration of IFB. Indeed, we postulate that the movement of IFB and preformed desmosomal components to the cell surface is an important initiating event in desmosome morphogenesis.     

Pemphigus vulgaris antigen, a desmoglein type of cadherin, is localized within keratinocyte desmosomes

Pemphigus vulgaris antigen (PVA) is a member of the desmoglein subfamily of cadherin cell adhesion molecules. Because autoantibodies in this disease cause blisters due to loss of epidermal cell adhesion, and because desmoglein is found in the desmosome cell adhesion junction, we wanted to determine if PVA is also found in desmosomes. By immunofluorescence, PV IgG bound, in a dotted pattern, to the cell surface of cultured human keratinocytes induced to differentiate with calcium, suggesting junctional staining. However, by preembedding, immunogold electron microscopic studies only slight labeling could be detected in desmosomes, presumably because of difficulty in gold penetration of intact desmosomes. We therefore treated the keratinocytes with 0.01% trypsin in 1 mM calcium, conditions known to preserve cadherin antigenicity but that caused slight separation of desmosomes, before immunogold staining. In this case there was extensive labeling of the extracellular part of desmosomes but not of the interdesmosomal cell membrane which was stained with anti-beta 2- microglobulin antibodies. To confirm the specificity of this binding we showed that antibodies raised in rabbits against the extracellular portions of PVA also bound desmosomes in these cultures. In intact mouse epidermis we could also show slight, but specific, immunogold desmosomal labeling with PV IgG. Furthermore, neonatal mice injected with PV IgG affinity purified on PVA showed desmosomal separation with the IgG localized to desmosomal cores. These results indicate that PVA is organized and concentrated within the desmosome where it presumably functions to maintain the integrity of stratifying epithelia.     

A role for plakophilin-1 in the initiation of desmosome assembly

Plakophilins (pkp-1, -2, and -3) comprise a family of armadillo-repeat containing proteins that are found in the desmosomal plaque and in the nucleus. Plakophilin-1 is most highly expressed in the suprabasal layers of the epidermis and loss of plakophilin-1 expression results in skin fragility-ectodermal dysplasia syndrome, which is characterized by a reduction in the number and size of desmosomes in the epithelia of affected individuals. To investigate the role of plakophilin-1 during desmosome formation, we fused plakophilin-1 to the hormone-binding domain of the estrogen receptor to create a fusion protein (plakophilin-1/ER) that can be activated in cell culture by the addition of 4-hydroxytamoxifen. When plakophilin-1/ER was expressed in A431 cells it was incorporated into endogenous desmosomes and did not disrupt desmosome formation. A derivative of A431 cells (A431D) do not form desmosomes, even though they express all the components believed to be necessary for desmosome assembly. Expression and activation of plakophilin-1/ER in A431D cells resulted in punctate desmoplakin staining on the cell surface. Co-expression of a classical cadherin (N-cadherin) and plakophilin-1/ER in A431D cells resulted in punctate desmoplakin staining at cell-cell borders. These data suggest that plakophilin-1 can induce assembly of desmosomal components in A431D cells in the absence of a classical cadherin; however a classical cadherin (N-cadherin) is required to direct assembly of desmosomes between adjacent cells. The activatable plakophilin-1/ER system provides a unique culture system to study the assembly of the desmosomal plaque in culture.     

Calcium-induced reorganization of desmosomal components in cultured human keratinocytes

We used antibodies raised against individual desmosomal components to study calcium-induced desmosome formation in human keratinocytes. When keratinocytes are forced to grow as a monolayer by reducing the level of calcium ions in the culture medium, there is little contact between adjacent cells. Raising the level of calcium ions rapidly induces desmosome formation, and stratification occurs within 24 h. We found that before addition of calcium the 115,000- and 100,000-mol-wt core glycoproteins were distributed over the entire cell surface, whereas the plaque proteins (205,000 and 230,000 mol wt), the 82,000- and 86,000-mol-wt proteins, and the 150,000-mol-wt glycoprotein were located throughout the cytoplasm. 15 min after increasing the calcium ion concentration, all of these molecules appeared at the cell margins. The intensity of peripheral staining increased over the next 2 h and during this time the distribution of keratin filaments changed from predominantly perinuclear to extend throughout the cytoplasm. Keratinocytes could be dissociated with EDTA for up to 2 h after exposure to calcium. After 3 h of exposure to calcium the cells were no longer susceptible to EDTA dissociation and staining for desmosomal plaque antigens persisted in regions of intercellular contact. Desmosomal staining in stratified cultures became greatly reduced within 24 h of lowering the calcium ion concentration again. We have preliminary evidence that stratification occurs by breakdown of desmosomes at lateral surfaces and reformation at surfaces of contact between basal and suprabasal cells, rather than by rearrangement of existing desmosomes. Involucrin-positive cells in the monolayer appeared to contain more 205,000- and 230,000-mol-wt proteins free in the cytoplasm than involucrin-negative cells.     

Maintenance of desmosomes in mouse hepatocytes after drug-induced rearrangement of cytokeratin filament material. Demonstration of independence of desmosomes and intermediate-sized filaments

The distribution of desmosomes and cytokeratin filaments (tonofilaments) in hepatocytes of normal mice and those intoxicated with griseofulvin was studied by immunofluorescence microscopy. Treatment with griseofulvin over prolonged periods of time resulted in the dissociation of cytokeratin filaments from the plasma membrane and the inclusions of cytokeratin material in typical cytoplasmic aggregates, i.e. "Mallory bodies". However, such hepatocytes still displayed typical desmosomal arrays, including rather regularly spaced desmosomes along the bile canaliculi. These observations show that, in this tissue, desmosomes are able to maintain their characteristic positions along the plasma membrane after disconnection of the intermediate filament cytoskeleton. This indicates that maintenance of desmosomal integrity and position is independent of desmosome anchorage to tonofilaments. The results are discussed in relation to current concepts of desmosome formation and turnover.     

Epidermal growth factor receptor inhibition promotes desmosome assembly and strengthens intercellular adhesion in squamous cell carcinoma cells

The epidermal growth factor receptor (EGFR) has been proposed as a key modulator of cadherin-containing intercellular junctions, particularly in tumors that overexpress this tyrosine kinase. Here the EGFR tyrosine kinase inhibitor PKI166 and EGFR blocking antibody C225, both of which are used clinically to treat head and neck cancers, were used to determine the effects of EGFR inhibition on intercellular junction assembly and adhesion in oral squamous cell carcinoma cells. EGFR inhibition resulted in a transition from a fibroblastic morphology to a more epithelial phenotype in cells grown in low calcium; under these conditions cadherin-mediated cell-cell adhesion is normally reduced, and desmosomes are absent. The accumulated levels of desmoglein 2 (Dsg2) and desmocollin 2 increased 1.7-2.0-fold, and both desmosomal cadherin and plaque components were recruited to cell-cell borders. This redistribution was paralleled by an increase in Dsg2 and desmoplakin in the Triton-insoluble cell fraction, suggesting that EGFR blockade promotes desmosome assembly. Importantly, E-cadherin expression and solubility were unchanged. Furthermore, PKI166 blocked tyrosine phosphorylation of Dsg2 and plakoglobin following epidermal growth factor stimulation, whereas no change in phosphorylation was detected for E-cadherin and beta-catenin. The increase in Dsg2 protein was in part due to the inhibition of matrix metalloproteinase-dependent proteolysis of this desmosomal cadherin. These morphological and biochemical changes were accompanied by an increase in intercellular adhesion based on functional assays at all calcium concentrations tested. Our results suggest that EGFR inhibition promotes desmosome assembly in oral squamous cell carcinoma cells, resulting in increased cell-cell adhesion.     

Involvement of Desmoplakin Phosphorylation in the Regulation of Desmosomes by Protein Kinase C,in HeLa Cells

In the present study, we have examined how modulation of protein kinase C (PKC) activity affected desmosome organization in HeLa cells. Immunofluorescence and electron microscopy showed that PKC activation upon short exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a reduction of intercellular contacts, splitting of desmosomes and dislocation of desmosomal components from the cell periphery towards the cytoplasm. As determined by immunoblot analysis of Triton X-100-soluble and -insoluble pools of proteins, these morphological changes were not correlated with modifications in the extractability of both desmoglein and plakoglobin, but involved almost complete solubilization of the desmosomal plaque protein, desmoplakin. Immunoprecipitation experiments and immunoblotting with anti-phosphoserine, anti-phosphothreonine and anti-phosphotyrosine antibodies revealed that desmoplakin was mainly phosphorylated on serine and tyrosine residues in both treated and untreated cells. While phosphotyrosine content was not affected by PKC activation, phosphorylation on serine residues was increased by about two-fold. This enhanced serine phosphorylation coincided with the increase in the protein solubility, suggesting that phosphorylation of desmoplakin may be a mechanism by which PKC mediates desmosome disassembly. Consistent with the loss of PKC activity, we also showed that down-modulation of the kinase (in response to prolonged TPA treatment) or its specific inhibition (by GF109203X) had opposite effects and increased desmosome formation. Taken together, these results clearly demonstrate an important role for PKC in the regulation of desmosomal junctions in HeLa cells, and identify serine phosphorylation of desmoplakin as a crucial event in this pathway.     

Involvement of desmoplakin phosphorylation in the regulation of desmosomes by protein kinase C, in HeLa cells.

In the present study, we have examined how modulation of protein kinase C (PKC) activity affected desmosome organization in HeLa cells. Immunofluorescence and electron microscopy showed that PKC activation upon short exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a reduction of intercellular contacts, splitting of desmosomes and dislocation of desmosomal components from the cell periphery towards the cytoplasm. As determined by immunoblot analysis of Triton X-100-soluble and -insoluble pools of proteins, these morphological changes were not correlated with modifications in the extractability of both desmoglein and plakoglobin, but involved almost complete solubilization of the desmosomal plaque protein, desmoplakin. Immunoprecipitation experiments and immunoblotting with anti-phosphoserine, antiphosphothreonine and anti-phosphotyrosine antibodies revealed that desmoplakin was mainly phosphorylated on serine and tyrosine residues in both treated and untreated cells. While phosphotyrosine content was not affected by PKC activation, phosphorylation on serine residues was increased by about two-fold. This enhanced serine phosphorylation coincided with the increase in the protein solubility, suggesting that phosphorylation of desmoplakin may be a mechanism by which PKC mediates desmosome disassembly. Consistent with the loss of PKC activity, we also showed that down-modulation of the kinase (in response to prolonged TPA treatment) or its specific inhibition (by GF 109203X) had opposite effects and increased desmosome formation. Taken together, these results clearly demonstrate an important role for PKC in the regulation ofdesmosomal junctions in HeLa cells, and identify serine phosphorylation of desmoplakin as a crucial event in this pathway.     

De novo formation of desmosomes in cultured cells upon transfection of genes encoding specific desmosomal components

Desmosomes are cell junctions and cytoskeleton-anchoring structures of epithelia, the myocardium, and dendritic reticulum cells of lymphatic follicles whose major components are known. Using cultured HT-1080 SL-1 fibrosarcoma-derived cells and transfection of cDNAs encoding specific desmosomal components, we have determined a minimum ensemble of proteins sufficient to introduce de novo structures, which, by morphology and functional competence, are indistinguishable from authentic desmosomes. In a more refined analysis, the influence of the desmosomal proteins desmoplakin (Dp), plakoglobin (Pg), and plakophilin 2 (Pp2) on the lateral clustering of the desmosomal transmembrane-glycoprotein desmoglein 2 (Dsg) was examined. We found that for efficient clustering of desmoglein 2 and desmosome structure formation, all three major plaque proteins-desmoplakin, plakoglobin, and plakophilin 2- were necessary. Furthermore, in this cell model, plakophilin 2 was capable of directing desmoplakin to adhaerens junctions (AJ), whereas plakoglobin was crucial for the segregation of desmosomal and AJ components. These results are discussed with respect to the variability in cell junction composition observed in various nonepithelial tissues.     

Loss of the p53/p63 regulated desmosomal protein Perp promotes tumorigenesis

Dysregulated cell-cell adhesion plays a critical role in epithelial cancer development. Studies of human and mouse cancers have indicated that loss of adhesion complexes known as adherens junctions contributes to tumor progression and metastasis. In contrast, little is known regarding the role of the related cell-cell adhesion junction, the desmosome, during cancer development. Studies analyzing expression of desmosome components during human cancer progression have yielded conflicting results, and therefore genetic studies using knockout mice to examine the functional consequence of desmosome inactivation for tumorigenesis are essential for elucidating the role of desmosomes in cancer development. Here, we investigate the consequences of desmosome loss for carcinogenesis by analyzing conditional knockout mice lacking Perp, a p53/p63 regulated gene that encodes an important component of desmosomes. Analysis of Perp-deficient mice in a UVB-induced squamous cell skin carcinoma model reveals that Perp ablation promotes both tumor initiation and progression. Tumor development is associated with inactivation of both of Perp's known functions, in apoptosis and cell-cell adhesion. Interestingly, Perp-deficient tumors exhibit widespread downregulation of desmosomal constituents while adherens junctions remain intact, suggesting that desmosome loss is a specific event important for tumorigenesis rather than a reflection of a general change in differentiation status. Similarly, human squamous cell carcinomas display loss of PERP expression with retention of adherens junctions components, indicating that this is a relevant stage of human cancer development. Using gene expression profiling, we show further that Perp loss induces a set of inflammation-related genes that could stimulate tumorigenesis. Together, these studies suggest that Perp-deficiency promotes cancer by enhancing cell survival, desmosome loss, and inflammation, and they highlight a fundamental role for Perp and desmosomes in tumor suppression. An understanding of the factors affecting cancer progression is important for ultimately improving the diagnosis, prognostication, and treatment of cancer.     

Fractionation of desmosomes and comparison of the polypeptide composition of desmosomes prepared from two bovine epithelial tissues

Desmosomes isolated from bovine tongue mucosa or muzzle epidermis appeared identical by ultrastructural analyses but had some differences in their polypeptide compositions as determined by SDS-PAGE. These preparations were extracted in 9 M urea, 10 mM Tris-HCl (pH 9), and 25 mM B-mercaptoethanol and then centrifuged at 240,000g for 30 min. The urea-soluble and insoluble fractions were analyzed by SDS-PAGE. The urea soluble fractions of both tongue and muzzle desmosomes were enriched in polypeptides of 240, 210, 81, and 75 kDa and also polypeptides (40 to 70 kDa) that were keratin-like, as determined by immunoblotting analyses with keratin antisera. The urea insoluble fraction of tongue desmosomes contained glycoproteins of 165, 160, 140, 110, and 100 kDa, while this fraction from muzzle contained glycoproteins of 165, 115, and 105 kDa. Ultrastructural examinations of insoluble pellets obtained from urea extracted tongue and muzzle desmosomes showed that most of the components at the cytoplasmic faces of the desmosomes were removed, while the membrane regions of the desmosomes resisted the treatment. The urea soluble proteins were dialyzed against 10 mM Tris-HCl (pH 7.6), and the resulting preparation was pelleted by centrifugation and examined by electron microscopy. Ultrastructural examination of this material revealed that it had assembled into a fibrillar meshwork, similar to the fibrillar region adjacent to the submembranous plaque of isolated desmosomes. Thus, treatment of isolated desmosomes with 9 M urea allowed the fractionation of membrane-associated desmosomal proteins from cytoplasmic desmosomal proteins. A comparison of these fractions from tongue and muzzle indicated that the polypeptide compositions of the desmosomes varied between tissues, especially with respect to the fractions enriched in either glycoproteins or keratin.     

ISOLATION OF EPIDERMAL DESMOSOMES

A method is reported for the isolation of desmosomes in a high yield and of a purity suitable for biochemical analysis. The procedure utilizes the selective solubilizing action of citric acid-sodium citrate (CASC) buffer, pH 2.6, on the non-cornified layers of cow nose epidermis, followed by discontinuous sucrose density gradient centrifugation. Electron microscopy with both thin sections of pellets and unfixed spread preparations reveals that after centrifugation, desmosomes are located mainly at the 55–60% sucrose interface. In the desmosome preparation thus obtained, the characteristic desmosome structure is well preserved, showing the midline, unit membranes, and dense plaques. Furthermore, removal of the epidermal filament bundles by the solubilizing action of CASC buffer has revealed a finely filamentous layer on the cytoplasmic surface of the plaques. The dimensions, location, and appearance of this layer correspond with those of the "connecting component" which has been previously suggested as being responsible for the attachment of epidermal filament bundles to the desmosome.     

Breaking the connection: displacement of the desmosomal plaque protein desmoplakin from cell-cell interfaces disrupts anchorage of intermediate filament bundles and alters intercellular junction assembly

The desmosomal plaque protein desmoplakin (DP), located at the juncture between the intermediate filament (IF) network and the cytoplasmic tails of the transmembrane desmosomal cadherins, has been proposed to link IF to the desmosomal plaque. Consistent with this hypothesis, previous studies of individual DP domains indicated that the DP COOH terminus associates with IF networks whereas NH2-terminal sequences govern the association of DP with the desmosomal plaque. Nevertheless, it had not yet been demonstrated that DP is required for attaching IF to the desmosome. To test this proposal directly, we generated A431 cell lines stably expressing DP NH2-terminal polypeptides, which were expected to compete with endogenous DP during desmosome assembly. As these polypeptides lacked the COOH-terminal IF-binding domain, this competition should result in the loss of IF anchorage if DP is required for linking IF to the desmosomal plaque. In such cells, a 70-kD DP NH2- terminal polypeptide (DP-NTP) colocalized at cell-cell interfaces with desmosomal proteins. As predicted, the distribution of endogenous DP was severely perturbed. At cell-cell borders where endogenous DP was undetectable by immunofluorescence, there was a striking absence of attached tonofibrils (IF bundles). Furthermore, DP-NTP assembled into ultrastructurally identifiable junctional structures lacking associated IF bundles. Surprisingly, immunofluorescence and immunogold electron microscopy indicated that adherens junction components were coassembled into these structures along with desmosomal components and DP-NTP. These results indicate that DP is required for anchoring IF networks to desmosomes and furthermore suggest that the DP-IF complex is important for governing the normal spatial segregation of adhesive junction components during their assembly into distinct structures.     

Desmoglein endocytosis and desmosome disassembly are coordinated responses to pemphigus autoantibodies

Desmosomes are adhesive intercellular junctions prominent in the skin and heart. Loss of desmosome function is associated with severe congenital and acquired disorders characterized by tissue fragility. Pemphigus vulgaris (PV) is an autoimmune disorder in which antibodies are directed against the desmosomal adhesion molecule Dsg3, resulting in severe mucosal erosions and epidermal blistering. To define the mechanisms by which Dsg3 autoantibodies disrupt keratinocyte adhesion, the fate of PV IgG and various desmosomal components was monitored in primary human keratinocytes exposed to PV patient IgG. PV IgG initially bound to keratinocyte cell surfaces and colocalized with desmosomal markers. Within 6 h after PV IgG binding to Dsg3, electron microscopy revealed that desmosomes were dramatically disrupted and keratinocyte adhesion was severely compromised. Immunofluorescence analysis indicated that PV IgG and Dsg3 were rapidly internalized from the cell surface in a complex with plakoglobin but not desmoplakin. Dsg3 internalization was associated with retraction of keratin filaments from cell-cell borders. Furthermore, the internalized PV IgG-Dsg3 complex colocalized with markers for both endosomes and lysosomes, suggesting that Dsg3 was targeted for degradation. Consistent with this possibility, biotinylation experiments demonstrated that soluble Dsg3 cell surface pools were rapidly depleted followed by loss of detergent-insoluble Dsg3. These findings demonstrate that Dsg3 endocytosis, keratin filament retraction, and the loss of keratinocyte cell-cell adhesion are coordinated responses to PV IgG.