Desmoplakins of Epithelial and Myocardial Desmosomes are Immunologically and Biochemically Related

Abstract. Guinea pig antibodies against desmoplakins from bovine muzzle epidermis showed specific reaction in several epithelial tissues with desmoplakin I (M_r 250,000) and desmoplakin II (M_r 215,000). By immunofluorescence microscopy, prominent punctate staining was observed in various lines of cultured epithelial cells, revealing desmosomal junctions at sites of established cell-to-cell contacts as well as hemidesmosomes and internalized desmosome-derived membrane domains. On frozen tissue sections punctate staining was observed along plasma membranes of epithelial cells, and electron microscopy using the immunoperoxidase technique revealed that the antibodies were specifically localized at the plaques associated with desmosomes and hemidesmosomes. Of a large number of non-epithelial cells examined positive staining was only observed on desmosome-like junctions of myocardial cells and Purkinje fiber cells. In both epithelial and myocardial tissues the antibodies showed a broad range of cross-reactivity between diverse vertebrate species such as man, cow, rodent, and chicken, indicating that desmoplakins contain determinants strongly conserved during evolution. When binding of these antibodies to cytoskeletal polypeptides separated by gel electrophoresis and blotted on nitrocellulose paper sheets was examined, specific reaction was noted with desmoplakin I and, to a variable degree, also desmoplakin II from various epithelial cells. Reaction was also observed with a myocardial polypeptide from bovine and human hearts which had a similar M_r value (250,000) and isoelectric pH range as desmoplakin I. We conclude that desmoplakins are the major proteins present in the desmosomal plaques of both epithelial and myocardial cells and that the desmoplakin polypeptides present in these two different cell types are very similar, if not identical.     

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.     

Specific attachment of desmin filaments to desmosomal plaques in cardiac myocytes

Intercellular junctions which are similar in ultrastructure and protein composition to typical desmosomes have so far only been found in epithelial cells and in heart tissue, specifically in the intercalated disks of cardiac myocytes and at cell boundaries between Purkinje fiber cells. In epithelial cells the cytoplasmic side of desmosomes, the 'desmosomal plaque', represents a specific attachment structure for the anchorage of intermediate filaments (IF) of the cytokeratin type. Cardiac myocytes do not contain cytokeratin filaments. In primary cultures of rat cardiac myocytes, we have examined by immunofluorescence and electron microscopy, using single and double label techniques, whether other types of IF are attached to the desmosomal plaques of the heart. Antibodies to desmoplakin, the major protein of the desmosomal plaque, have been used to label specifically the desmosomal plaques. It is shown that the desmoplakin-containing structures are often associated with IF stained by antibodies to desmin, i.e., the characteristic type of IF present in these cells. Like cytokeratin filaments in epithelial cells, desmin filaments attach laterally to the desmosomal plaque. They also remain attached to these plaques after endocytotic internalization of desmosomal domains by treatment of the cells with EGTA. These desmin filaments do not appear to attach to junctions of the fascia adherens type and to nexuses (gap junctions). These observations show that anchorage at desmosomal plaques is not restricted to IF of the cytokeratin type and that IF composed of either cytokeratin or desmin, specifically attach, in a lateral fashion, to desmoplakin-containing regions of the plasma membrane. We conclude that special domains exist in these two IF proteins that are involved in binding to the desmosomal plaque.     

A constitutive transmembrane glycoprotein of Mr 165,000 (desmoglein) in epidermal and non-epidermal desmosomes. II. Immunolocalization and microinjection studies

Using two monoclonal antibodies described in the preceding paper we determined by immunofluorescence microscopy the distribution of an integral membrane protein of the desmosomal domain, the major glycopolypeptide of Mr 165,000 (bovine muzzle epidermal desmosome band 3; desmoglein) in various normal tissues, tumors and cultured cell lines from several mammalian species. This protein was detected in dotted or streak-like arrays along cell boundary structures which were known to contain non-membrane-integrated desmosomal plaque proteins such as desmoplakins. This is true for epithelial, i.e. cytokeratin-expressing cell types, for the desmin-producing myocardiac and Purkinje fiber cells of the heart, and for certain vimentin-containing cells such as arachnoidal and meningiomal cells and dendritic follicular cells of lymph nodes. However, on the basis of both immunoblot and immunocytochemical reactions, the protein is absent from non-desmosomal adhering junctions, including those devoid of desmoplakin but containing another plaque protein, plakoglobin ("band 5 protein"). We have used these antibodies to localize their epitopes with respect to the cell membrane. By immunoelectron microscopy we found that both epitopes are located in the desmosomal plaques, and this was confirmed by microinjection of purified antibodies into living cultured cells which resulted in labelling of the plaques. From these findings, taken together with previous analyses and localizations of the carbohydrate moieties of this glycoprotein, we conclude that desmoglein is a transmembrane glycoprotein which projects into--and contributes to--the desmosomal plaque structure. This glycoprotein represents a general component of true desmosomes and it is coexpressed with obligatory desmosome-specific plaque proteins such as desmoplakin I. The potential value of this glycoprotein as a desmosomal and cell type marker in histology and tumor diagnosis is discussed.     

The complement of desmosomal plaque proteins in different cell types

Desmosomal plaque proteins have been identified in immunoblotting and immunolocalization experiments on a wide range of cell types from several species, using a panel of monoclonal murine antibodies to desmoplakins I and II and a guinea pig antiserum to desmosomal band 5 protein. Specifically, we have taken advantage of the fact that certain antibodies react with both desmoplakins I and II, whereas others react only with desmoplakin I, indicating that desmoplakin I contains unique regions not present on the closely related desmoplakin II. While some of these antibodies recognize epitopes conserved between chick and man, others display a narrow species specificity. The results show that proteins whose size, charge, and biochemical behavior are very similar to those of desmoplakin I and band 5 protein of cow snout epidermis are present in all desmosomes examined. These include examples of simple and pseudostratified epithelia and myocardial tissue, in addition to those of stratified epithelia. In contrast, in immunoblotting experiments, we have detected desmoplakin II only among cells of stratified and pseudostratified epithelial tissues. This suggests that the desmosomal plaque structure varies in its complement of polypeptides in a cell-type specific manner. We conclude that the obligatory desmosomal plaque proteins, desmoplakin I and band 5 protein, are expressed in a coordinate fashion but independently from other differentiation programs of expression such as those specific for either epithelial or cardiac cells.     

Different modes of internalization of proteins associated with adhaerens junctions and desmosomes: experimental separation of lateral contacts induces endocytosis of desmosomal plaque material.

The distribution and fate of two junctional complexes, zonula adhaerens and desmosomes, after dissociation of cell-cell contacts is described in MDBK cells. Junctions were split between adjacent cells by treatment with EGTA and proteins associated with the plaques of zonulae adhaerentes and desmosomes were localized by immunological methods. Splitting of these junctions is accompanied by the dislocation of desmosomal plaque protein from the cell periphery and its distribution in punctate arrays over the whole cytoplasm. By contrast, vinculin associated with zonulae adhaerentes is still seen at early times (0.5-1 h) in a conspicuous belt-like structure which, however, is displaced from the plasma membrane. Strong vinculin staining is maintained on leading edges of free cell surfaces. Electron microscopy of EGTA-treated cells exposed to colloidal gold particles reveals the disappearance of junctional structures from the cell periphery and the concomitant appearance of a distinct class of gold particle-containing vesicles which are coated by dense plaques. These vesicle plaques react with antibodies to desmosomal plaque proteins and are associated with filaments of the cytokeratin type. In the same cells, extended dense aggregates are seen which are most probably the membrane-detached vinculin-rich material from the zonula adhaerens . The experiments show that, upon release from their junction-mediated connections with adjacent cells, major proteins associated with the cytoplasmic side of the junctions remain, for several hours, clustered within plaques displaced from the cell surface. While plaque material of adhaerens junctions containing vinculin is recovered in large belt-like aggregates, desmosomal plaque protein remains attached to membrane structures and appears on distinct vesicles endocytotically formed from half-desmosomal equivalents.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Arm-Repeat Protein NPRAP (Neurojungin) Is a Constituent of the Plaques of the Outer Limiting Zone in the Retina, Defining a Novel Type of Adhering Junction

In the retina, special plaque-bearing adhering junctions are aligned to form a planar system (the "outer limiting zone," OLZ) of heterotypic connections between the photoreceptor cells and the surrounding glial cells ("Müller cells"), together with homotypic junctions. In the plaques of these junctions, which contain N-cadherin-and possibly also related cadherins-we have identified, by immunolocalization techniques, a recently discovered neural tissue-specific protein, neurojungin, a member of the plakoglobin/armadillo protein family. In these plaques we have also detected other adherens plaque proteins, such as alpha- and beta-catenin, protein p120, and vinculin, as well as proteins known as constituents of tight junction plaques, such as symplekin and protein ZO-1, and the desmosomal plaque protein plakophilin 2. This unusual combination of proteins and the demonstrated absence of plakoglobin define the OLZ junctions as a new and distinct category of adhering junction, which probably has special architectural functions.     

Targeted mutation of plakoglobin in mice reveals essential functions of desmosomes in the embryonic heart

Plakoglobin (gamma-catenin), a member of the armadillo family of proteins, is a constituent of the cytoplasmic plaque of desmosomes as well as of other adhering cell junctions, and is involved in anchorage of cytoskeletal filaments to specific cadherins. We have generated a null mutation of the plakoglobin gene in mice. Homozygous -/- mutant animals die between days 12-16 of embryogenesis due to defects in heart function. Often, heart ventricles burst and blood floods the pericard. This tissue instability correlates with the absence of desmosomes in heart, but not in epithelia organs. Instead, extended adherens junctions are formed in the heart, which contain desmosomal proteins, i.e., desmoplakin. Thus, plakoglobin is an essential component of myocardiac desmosomes and seems to play a crucial role in the sorting out of desmosomal and adherens junction components, and consequently in the architecture of intercalated discs and the stabilization of heart tissue.     

Significance of two desmosome plaque-associated polypeptides of molecular weights 75 000 and 83 000.

Isolated desmosomes from bovine epidermis contain two major polypeptides of mol. wts. 75 000 (D6) and 83 000 (D5) which, like the desmoplakins of mol. wt. greater than 200 000, are associated with the insoluble desmosomal plaque structure. We have characterized these two polypeptides and examined their significance by peptide map comparisons and translation of bovine epidermal mRNA in vitro. Polypeptide D5 is different from polypeptide D6 by its apparent mol. wt., its isoelectric pH (approximately 6.35, whereas D6 is a basic polypeptide isoelectric at pH approximately 8.5) and its peptide map. By all these criteria desmosomal polypeptides D5 and D6 are also different from cytokeratins, desmoplakins and the glycosylated desmosomal proteins. Both polypeptides are synthesized from different mRNAs separable by gel electrophoresis on agarose: mRNA coding for polypeptide D5 is approximately 3500 nucleotides long, that for D6 is significantly shorter (estimated to 3050 nucleotides), and both contain relatively large proportions of non-coding sequences. The translational products of these mRNAs co-migrate, on two-dimensional gel electrophoresis, with the specific polypeptides from bovine epidermis, indicating that they are genuine polypeptides and are not the result of considerable post-translational processing or modification of precursor molecules. The cell and tissue distribution of these two cytoskeletal proteins and possible functions are discussed.     

Isolation and characterization of desmosome-associated tonofilaments from rat intestinal brush border

Epithelial cells of the small intestine, like those of other internal organs, contain intermediate-sized filaments immunologically related to epidermal prekeratin which are especially concentrated in the cell apex. Brush-order fractions were isolated from rat small intestine, and apical tonofilaments attached to desmosomal plaques and terminal web residues were prepared therefrom by extraction in high salt (1.5 M KCl) buffer and Triton X-100. The structure of these filaments was indistinguishable from that of epidermal tonofilaments and, as with epidermal prekeratin, filaments could be reconstituted from solubilized, denatured intestinal tonofilament protein. On SDS polyacrylamide gel electrophoresis of proteins of the extracted desmosome-tonofilament fractions, a number of typical brush-border proteins were absent or reduced, and enrichment of three major polypeptides of Mr 55,000, 48,000, and 40,000 was noted. On two- dimensional gel electrophoresis, the three enriched major polypeptides usually appeared as pairs of isoelectric variants, and the two smaller components (Mr 48,000, and 40,000) were relatively acidic (isoelectric pH values of 5.40 and below), compared to the Mr 55,000 protein which focused at pH values higher than 6.4. The tonofilament proteins were shown to be immunologically related to epidermal prekeratin by immunoreplica and blotting techniques using antibodies to bovine epidermal prekeratins. Similar major polypeptides were found in desmosome-attached tonofilaments from small intestine of mouse and cow. However, comparisons with epidermal tissues of cow and rat showed that all major polypeptides of intestinal tonofilaments were different from the major prekeratin polypeptides of epidermal tonofilaments. The results present the first analysis of a defined fraction of tonofilaments from a nonepidermal cell. The data indicate that structurally identical tonofilaments can be formed, in different types of cells, by different polypeptides of the cytokeratin family of proteins and that tonofilaments of various epithelia display tissue- specific patterns of their protein subunits.     

Attachment of vimentin filaments to desmosomal plaques in human meningiomal cells and arachnoidal tissue

Desmosomal proteins are co-expressed with intermediate-sized filaments (IF) of the cytokeratin type in epithelial cells, and these IF are firmly attached to the desmosomal plaque. In meningiomal and certain arachnoidal cells, however, vimentin IF are attached to desmosomal plaques. Meningiomas obtained after surgery, arachnoid "membranes", and arachnoid granulations at autopsy, as well as meningiomal cells grown in short-term culture have been examined by single and double immunofluorescence and immunoelectron microscopy using antibodies to desmoplakins, vimentin, cytokeratins, glial filament protein, neurofilament protein, and procollagen. In addition, two-dimensional gel electrophoresis of the cytoskeletal proteins has been performed. Using all of these techniques, vimentin was the only IF protein that was detected in significant amounts. The junctions morphologically resembling desmosomes of epithelial cells have been identified as true desmosomes by antibodies specific for desmoplakins and they provided the membrane attachment sites for the vimentin IF. These findings show that anchorage of IF to the cell surface at desmosomal plaques is not restricted to cytokeratin IF as in epithelial cells and desmin IF as in cardiac myocytes, suggesting that binding to desmosomes and hemidesmosomes is a more common feature of IF organization. The co- expression of desmosomal proteins and IF of the vimentin type only defines a new class of cell ("desmofibrocyte") and may also provide an important histodiagnostic criterion.     

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.     

Identification of a basic protein of Mr 75,000 as an accessory desmosomal plaque protein in stratified and complex epithelia

Desmosomes are intercellular adhering junctions characterized by a special structure and certain obligatory constituent proteins such as the cytoplasmic protein, desmoglein. Desmosomal fractions from bovine muzzle epidermis contain, in addition, a major polypeptide of Mr approximately 75,000 ("band 6 protein") which differs from all other desmosomal proteins so far identified by its positive charge (isoelectric at pH approximately 8.5 in the denatured state) and its avidity to bind certain type I cytokeratins under stringent conditions. We purified this protein from bovine muzzle epidermis and raised antibodies to it. Using affinity-purified antibodies, we identified a protein of identical SDS-PAGE mobility and isoelectric pH in all epithelia of higher complexity, including representatives of stratified, complex (pseudostratified) and transitional epithelia as well as benign and malignant human tumors derived from such epithelia. Immunolocalization studies revealed the location of this protein along cell boundaries in stratified and complex epithelia, often resolved into punctate arrays. In some epithelia it seemed to be restricted to certain cell types and layers; in rat cornea, for example, it was only detected in upper strata. Electron microscopic immunolocalization showed that this protein is a component of the desmosomal plaque. However, it was not found in the desmosomes of all simple epithelia examined, in the tumors and cultured cells derived thereof, in myocardiac and Purkinje fiber cells, in arachnoideal cells and meningiomas, and in dendritic reticulum cells of lymphoid tissue, i.e., all cells containing typical desmosomes. The protein was also absent in all nondesmosomal adhering junctions. From these results we conclude that this basic protein is not an obligatory desmosomal plaque constituent but an accessory component specific to the desmosomes of certain kinds of epithelial cells with stratified tissue architecture. This suggests that the Mr 75,000 basic protein does not serve general desmosomal functions but rather cell type-specific ones and that the composition of the desmosomal plaque can be different in different cell types. The possible diagnostic value of this protein as a marker in cell typing is discussed.     

The plaque protein myozap identified as a novel major component of adhering junctions in endothelia of the blood and the lymph vascular systems

Recently the protein myozap, a 54-kD polypeptide which is not a member of any of the known cytoskeletal and junctional protein multigene families, has been identified as a constituent of the plaques of the composite junctions in the intercalated disks connecting the cardiomyocytes of mammalian hearts. Using a set of novel, highly sensitive and specific antibodies we now report that myozap is also a major constituent of the cytoplasmic plaques of the adherens junctions (AJs) connecting the endothelial cells of the mammalian blood and lymph vascular systems, including the desmoplakin-containing complexus adhaerentes of the virgultar cells of lymph node sinus. In light and electron microscopic immunolocalization experiments we show that myozap colocalizes with several proteins of desmosomal plaques as well as with AJ-specific transmembrane molecules, including VE-cadherin. In biochemical analyses, rigorous immunoprecipitation experiments have revealed N-cadherin, desmoplakin, desmoglein-2, plakophilin-2, plakoglobin and plectin as very stably bound complex partners. We conclude that myozap is a general component of cell-cell junctions not only in the myocardium but also in diverse endothelia of the blood and lymph vascular systems of adult mammals, suggesting that this protein not only serves a specific role in the heart but also a broader set of functions in the vessel systems. We also propose to use myozap as an endothelial cell type marker in diagnoses.     

Plakophilin-2: a cell-cell adhesion plaque molecule of selective and fundamental importance in cardiac functions and tumor cell growth

Within the characteristic ensemble of desmosomal plaque proteins, the armadillo protein plakophilin-2 (Pkp2) is known as a particularly important regulatory component in the cytoplasmic plaques of various other cell-cell junctions, such as the composite junctions (areae compositae) of the myocardiac intercalated disks and in the variously-sized and -shaped complex junctions of permanent cell culture lines derived therefrom. In addition, Pkp2 has been detected in certain protein complexes in the nucleoplasm of diverse kinds of cells. Using a novel set of highly sensitive and specific antibodies, both kinds of Pkp2, the junctional plaque-bound and the nuclear ones, can also be localized to the cytoplasmic plaques of diverse non-desmosomal cell-cell junction structures. These are not only the puncta adhaerentia and the fasciae adhaerentes connecting various types of highly proliferative non-epithelial cells growing in culture but also some very proliferative states of cardiac interstitial cells and cardiac myxomata, including tumors growing in situ as well as fetal stages of heart development and cultures of valvular interstitial cells. Possible functions and assembly mechanisms of such Pkp2-positive cell-cell junctions as well as medical consequences are discussed.     

The area composita of adhering junctions connecting heart muscle cells of vertebrates. V. The importance of plakophilin-2 demonstrated by small interference RNA-mediated knockdown in cultured rat cardiomyocytes

In the adult mammalian heart, the cardiomyocytes are connected by large polar arrays of closely spaced or even fused composite, plaque-bearing adhering junctions (areae compositae, ACs), in a region usually termed "intercalated disk" (ID). We have recently reported that during late embryogenesis and postnatally these polar assemblies of AC-junction structures are gradually formed as replacements of distinct embryonal junctions representing desmosomes and fasciae adhaerentes which then may amalgamate to the fused AC structures, in some regions occupying more than 90% of the total ID area. Previous gene knockout results as well as mutation analyses of specific human cardiomyopathies have suggested that among the various AC constituents, the desmosomal plaque protein, plakophilin-2, plays a particularly important role in the formation, architectural organization and stability of these junctions interconnecting mature cardiomyocytes. To examine this hypothesis, we have decided to study losses of--or molecular alterations in--such AC proteins with respect to their effects on myocardiac organization and functions. Here we report that plakophilin-2 is indeed of obvious importance for myocardial architecture and cell-cell coupling of rat cardiomyocytes growing in culture. We show that siRNA-mediated reduction of the cardiomyocyte content of plakophilin-2 but not of some other major plaque components such as desmoplakin results in progressive disintegration--and losses--of AC junction structures and that numerous variously sized vesicles appear, which are plaque protein-associated as demonstrable by immunofluorescence and immunoelectron microscopy. The importance of plakophilin-2 as a kind of "organizer" protein in the formation, stabilization and functions of the AC structure and the ID architecture is discussed in relation to other junction proteins and to causes of certain cardiomyopathies.     

Organization of desmosomal plaque proteins in cells growing at low calcium concentrations

Desmosomes are not formed in epithelial cell cultures growing in media with low (less than or equal to 0.1 mM) concentrations of Ca2+ (LCM) but appear rapidly upon shift to media of normal calcium concentrations (NCM). Previous authors using immunolocalization of desmoplakin, a marker protein for the desmosomal plaque, in LCM-grown cells have interpreted positively stained, dense, cytoplasmic aggregates on intermediate filaments (IF) bundles as preformed plaque units which upon NCM shift would move to the plasma membrane and contribute to desmosome formation. Studying various cell cultures, including primary mouse keratinocytes and human A-431 cells, we show that most, probably all, desmoplakin-positive aggregates in LCM-grown cells are associated with membranous structures, mostly vesicles, and also contain other desmosomal markers, including desmoglein, a transmembrane glycoprotein. We interpret such vesicles as residual desmosome-derived domains endocytosed upon cell dissociation. Only keratinocytes grown for long times (2-4 wk) in LCM are practically free from such vesicles. In addition, we demonstrate that certain cells such as A-431 cells, when passaged in LCM and in the absence of stable junctions, are able to continually assemble "half-desmosomes" on the plasma membrane which in turn can be endocytosed as plaque-bearing vesicles. We also show that in LCM the synthesis of several desmosomal proteins (desmoplakins I and II, plakoglobin, desmoglein, "band 6 protein") continues and that most of the plaque protein, desmoplakin, is diffusely spread over the cytoplasm, apparently in a soluble monodisperse form of approximately 9S. From our results we propose that the plaque proteins occur in small, discrete, diffusible entities in the cytoplasm, in concentrations that are relatively high in LCM and low in NCM, from which they assemble directly, i.e., without intermediate precursor aggregates on IFs in the cytoplasm, on certain plasma membrane domains in a Ca2+ dependent process.     

Identification of actin-, alpha-actinin-, and vinculin-containing plaques at the lateral membrane of epithelial cells

In this paper, a new type of spot desmosome-like junction (type II plaque) is described that is scattered along the entire lateral plasma membrane of rat and human intestinal epithelium. Ultrastructurally type II plaques differed from the classical type of epithelial spot desmosome ("macula adherens", further denoted as type I desmosome) by weak electron density of the membrane-associated plaque material, association of the plaques with microfilaments rather than intermediate filaments, and poorly visible material across the intercellular space. Thus, type II plaques resemble cross-sections of the zonula adherens. Immunofluorescence-microscopic studies were done using antibodies to a main protein associated with the plaques of type I desmosomes (desmoplakin I) and to the three major proteins located at the plaques of the zonula adherens (actin, alpha-actinin, and vinculin). Two types of plaques were visualized along the lateral surface of intestinal and prostatic epithelium: (a) the type I desmosomes, which were labeled with anti-desmoplakin but did not bind antibodies to actin, alpha-actinin, and vinculin, and (b) a further set of similarly sized plaques, which bound antibodies to actin, alpha-actinin, and vinculin but were not stained with anti-desmoplakin. Three-dimensional computer reconstruction of serial sections double-labeled with anti-desmoplakin and anti-alpha-actinin further confirmed that both types of plaques are spatially completely separated from each other along the lateral plasma membrane. The computer graphs further revealed that the actin-, alpha-actinin-, and vinculin-containing plaques have the tendency to form clusters, a feature also typical of type II plaques. It is suggested that the type II plaques represent spot desmosome-like intercellular junctions, which, like the zonula adherens, appear to be linked to the actin filament system. As the type II plaques cover a considerable part of the lateral cell surface, they might play a particular role in controlling cellular shape and intercellular 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.     

The area composita of adhering junctions connecting heart muscle cells of vertebrates. I. Molecular definition in intercalated disks of cardiomyocytes by immunoelectron microscopy of desmosomal proteins

Among sarcomeric muscles the cardiac muscle cells are unique by, inter alia, a systemic and extended cell-cell contact structure, the intercalated disk (ID), comprising frequent and closely spaced arrays of plaque-coated cell-cell adhering junctions (AJs). As some of these junctions may look somewhat like desmosomes and others like fasciae adhaerentes, the dogma has emerged in the literature that IDs contain - like epithelial cells - both kinds of AJs formed by - for the most - mutually exclusive molecular ensembles. This, however, is not the case. In comprehensive immunoelectron microscopic studies of mammalian (human, bovine, rat, mouse) and non-mammalian (chicken, amphibia, fishes) heart muscle tissues, we have localized major constituents of the desmosomal plaques of polar epithelia, desmoplakin, plakophilin-2 and plakoglobin, as well as the desmosomal cadherins, desmoglein Dsg2 and desmocollin Dsc2, in both kinds of ID AJs, independent of the specific morphological appearance. The desmosomal molecules are not restricted to the desmosome-like-looking junctions but can also be detected in junctions appearing similar to the zonula or fascia adhaerens structures. These AJs of cardiac ID are therefore subsumed under the collective term area composita. We discuss our results with respect to the importance of ID junction molecules for the formation, maintenance and function of the heart, particularly in relation to recent findings that deletions of - or mutations in - genes encoding such proteins can cause severe, sometimes lethal damages.