Detection of pemphigus vulgaris and pemphigus foliaceus antigens by immunoblot analysis using different antigen sources.

In an immunoblot analysis with human epidermal extract as a source of antigens, all (28/28) pemphigus vulgaris (Pv) sera showed a specific reactivity with a 130-kD protein. Several, but not all, Pv sera reacted with similar antigens in both a bovine muzzle desmosome preparation and extract of cultured human squamous carcinoma cells. On the other hand, some pemphigus foliaceus (Pf) sera exhibited reactivity with a 150-kD protein, which is most likely desmoglein I, in both the human epidermal extract and the bovine desmosome preparation, but no Pf serum reacted with this antigen in the squamous carcinoma cell extract. Furthermore, 4/16 Pv sera also reacted with a 150-kD protein in the desmosome preparation, which seemed to be the same as Pf antigen. These results show a relationship between antigens of both Pf and Pv and desmosomes, as well as heterogeneities of both Pv and Pf antigens in terms of antigenic molecules or epitopes. Furthermore, this study presents the possibility that immunoblot analysis can be routinely used for differentiation of Pv and Pf antibodies.

Early expression of desmosomal components during kidney tubule morphogenesis in human and murine embryos.

Developing kidneys of human and murine fetuses have been stained with monoclonal antibodies to desmosomal proteins 1 and 2 (desmoplakins) (dp 1&2), desmosomal glycoprotein 1 (desmoglein) and a polyclonal antiserum to desmosomal glycoproteins 2 and 3 (desmocollins). All three antibodies stain the mesenchymal condensates that represent the first stage in kidney tubule development, indicating that desmosomal antigens are expressed very early in tubule morphogenesis. Desmosomal antigens are continuously expressed throughout the developing tubule being concentrated at the apical and basal regions of the lateral membranes of cells. Staining is also present in both visceral and parietal membranes of the developing Bowman's capsule. In the mature tubule, desmosomal staining becomes restricted to a discontinuous apico-lateral ring around the cells. Staining is completely lost from the visceral membrane of the mature Bowman's capsule (the podocytes) but persists in the parietal membrane. At the condensate stage, staining for dp1&2 is much more intense than staining for simple epithelial keratin. Electron microscopy showed the presence of small (ca 0.1 microns) punctate junctions in the developing tubule. These may be immature desmosomes. No fully mature desmosomes such as are present in mature kidney were found. The results suggest that desmosomal proteins and glycoproteins are involved in the early development of adhesive contacts between cells of the kidney tubule. The changing pattern of antigen expression, the loss of desmosomal staining from the podocytes and the immaturity of junctions suggest that desmosomal adhesion is labile during tubule morphogenesis, perhaps in order to facilitate changes of cell-cell contact.

Immunoblot analyses of Brazilian pemphigus foliaceus antigen using different antigen sources.

We investigated the Brazilian pemphigus foliaceus (BPf) antigen applying the immunoblotting method to two different antigen sources using 27 patients' sera. Twelve BPf sera reacted specifically with a 150 kD protein in extract of dispase separated human epidermis, while 18 sera yielded a similar protein band in bovine muzzle desmosomal preparation. The diversity of staining intensities between the two samples suggested the heterogeneity of BPf antigens in terms of epitopes. Japanese sporadic pemphigus foliaceus (Pf) sera showed similar results but Japanese pemphigus vulgaris (Pv) sera recognized different antigens of 130 kD or 135 kD, suggesting that BPf is similar to Japanese Pf but is distinct from Pv in respect to the antigenic substance. Furthermore, the present study showed that immunoblot analysis using different antigen sources should be a valuable tool to determine clinical types of pemphigus.

Immunohistochemical localization of desmosomal and cytoskeletal proteins in the epidermis of healthy individuals and patients with Hailey-Hailey's disease.

We have studied the immunohistochemical localization of desmosomal and cytoskeletal proteins in the epidermis of healthy individuals and patients with Hailey-Hailey's disease. It was found that the localization of desmosomal proteins (desmoplakin, desmoglein) and keratin filaments in the involved epidermis of patients with Hailey-Hailey's disease was almost same as that in the normal epidermis of healthy individuals. In contrast, the localization of actin filaments in the involved epidermis of patients with Hailey-Hailey's disease differed from that in the normal epidermis of healthy individuals.

Demonstration of an adhering-junction molecule (plakoglobin) in the autoantigens of pemphigus foliaceus and pemphigus vulgaris.

Pemphigus foliaceus and pemphigus vulgaris are skin diseases in which antibodies against the cell surface of keratinocytes destroy the adhesion between epidermal cells, producing blisters. Patients with pemphigus foliaceus have antibodies to a complex of three polypeptides of 260, 160, and 85 kd (the foliaceus complex), whereas patients with pemphigus vulgaris have antibodies to a complex of 210-kd, 130-kd, and 85-kd polypeptides (the vulgaris complex). The 160-kd polypeptide of the foliaceus complex has been identified as desmoglein, a desmosomal glycoprotein. We suspected that the 85-kd component in both these antigenic complexes might be plakoglobin, another molecule in the adhering junctions of cells. To characterize these antigenic complexes, we used the serum of five patients with pemphigus foliaceus, that of four patients with pemphigus vulgaris, and monoclonal antiplakoglobin antibodies. We found that monoclonal antibodies to plakoglobin immunoprecipitated the 85-kd polypeptide from the dissociated foliaceus and vulgaris complexes and precipitated both complexes from epidermal extracts. Serum from patients with pemphigus foliaceus or pemphigus vulgaris (but not from four normal controls) bound desmoglein and the 130-kd polypeptide, respectively, showing that these peptides (and not plakoglobin) are the antigenic binding sites in these disorders. We conclude that plakoglobin, a protein of the adhering junctions of epidermal cells, is the 85-kd molecule in the antigenic complexes found in both pemphigus foliaceus and pemphigus vulgaris, although it is not the binding site in either disorder.

Desmoplakin I and desmoplakin II. Purification and characterization.

Desmoplakins I and II (DP1 and DP2), major cytoskeletal structural proteins concentrated in desmosomes, have been purified in milligram quantities from keratomed pig tongue epithelium. DP1 and DP2 extracted from purified desmosomes in 4 M urea were chromatographed on DEAE-cellulose and remained soluble after removal of urea during subsequent chromatography. The two proteins differed by only about 15% in molecular weight (Mr = 285,000 for DP1 and 225,000 for DP2 on sodium dodecyl sulfate-polyacrylamide gels) were found to have similar Svedberg constants, 6.7 S (DP1) and 6.4 S (DP2); nevertheless, separation was readily achieved by gel filtration, since DP1 has a Stokes radius (Rs) of 164 nm, but DP2 has a Rs = 90 nm. Calculated molecular mass was 462,000 daltons for DP1 and 242,000 daltons for DP2, suggesting that DP1 may be a dimer in solution and DP2 a monomer. Cross-linking by disuccinimidyl suberate of 125I-labeled DP1 or DP2 at nanomolar concentrations confirmed that DP1 is a dimer by doubling of its apparent Mr on sodium dodecyl sulfate gels and indicated that DP2, which failed to become cross-linked, is a monomer. DP1 in the presence of 8 M urea could not be cross-linked, indicating that urea dissociated the dimers. Calculated frictional ratios (f/f0 = 3 for DP1 and 2 for DP2) indicate that both proteins are highly asymmetric. Rotary shadowing of DP1 demonstrated flexible dumbbell-like extended shapes with a maximal length of about 180 nm with a central rod and coiled or folded end domains. DP2 showed variable extended shapes of maximal length of 78-93 nm. The increased length and Rs of desmoplakin I is probably accounted for by formation of tail-to-tail dimers. Two-dimensional peptide maps and amino acid analysis showed very similar profiles for the two proteins. Purified keratin filaments failed to bind DP1 or DP2, and prekeratins polymerized in vitro and sedimented failed to remove desmoplakins, suggesting that desmoplakins do not bind keratins directly. These studies provide a basis for functional and detailed structural studies with purified native desmosomal proteins.

Changes to desmosomal antigens and lectin-binding sites during differentiation in normal human epidermis: a quantitative ultrastructural study.

During epidermal differentiation, desmosomes undergo a series of changes in their abundance, structure and properties, which has previously been defined by conventional electron microscopy and the use of antibodies to desmosomal proteins at the light-microscope level. Such changes in a major adhesive organelle would be expected to have a significant role in the maintenance of epidermal organization, and therefore require more detailed characterization. In the present study, modifications to certain desmosomal components in normal human epidermis have been located and quantified by immunogold electron microscopy. Antibodies to desmosomal protein dp3 and glycoprotein dg1 were used to label the cytoplasmic regions of the junctions and lectins concanavalin A (ConA) and wheat germ agglutinin (WGA) to probe the extracellular glycosylated material. Binding was measured at histologically defined levels and expressed as gold particles per microns of desmosome length (linear particle density: LPD). In addition, desmosome frequency, expressed as the percentage of the cell membrane length occupied by desmosomes, was measured. Highly significant changes in desmosome frequency, diameter and LPD were observed between epidermal strata and, in basal and upper horny cells, between different regions of the same cell surface. These parameters rose to a maximum in the spinous or granular layers: their subsequent decrease continued without interruption across the interface between the living and terminally differentiated horny layers. Remaining reactivity with antibodies, but not lectins, was almost completely abolished immediately before the final disintegration of the desmosome structure in the lower horny layer. In contrast, numerous large, highly immunoreactive desmosomes were retained up to the outer surface in the grossly thickened horny layer found in callus. Though the overall pattern of a rise followed by a fall was similar for all parameters measured, differences were observed between probes. Thus, the extent of the rise in available antigen between the lateral and apical surfaces of the basal cell was greater for dg1 than for dp3; the subsequent decrease in dp3 antigens in upper epidermal layers was more rapid than that for dg1, and changes to both antigens preceded those to lectin-binding sites. These results show that differences in desmosome frequency and in the size and antibody-binding characteristics of individual junctions underlie the heterogeneous distribution of desmosomal components within epidermis that is found by light-microscope immunocytochemistry. They further suggest that the disintegration of desmosomes within normal horny layer, which is an essential preliminary to desquamation, is the culmination of a sequence of events that begins in the upper living tissue and initially involves cytoplasmic components.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of desmoplakin in normal cultured human keratinocytes and in basal cell carcinoma cells.

In cultured human keratinocytes (NHEK) maintained in medium containing low levels of Ca2+ (0.04 mM) desmoplakin is a component of certain electron-dense bodies in the cytoplasm. These bodies are associated with bundles of intermediate filaments. Upon elevation of the level of Ca2+ in the culture medium to 1.2 mM, desmoplakin first appears at sites of cell-cell contact in association with bundles of intermediate filaments. Subsequently, desmoplakin becomes incorporated into desmosomes in a manner comparable to that seen in mouse keratinocytes (Jones and Goldman: Journal of Cell Biology 101:506-517, 1985). NHEK cells maintained for 24 hr at Ca2+ concentrations between 0.04 mM and 0.18 mM were processed for immunofluorescence, immunoelectron, and conventional electron microscopical analysis. In NHEK cells grown at Ca2+ concentrations of 0.11 mM, desmoplakin appears to be localized in electron-dense bodies associated with intermediate filaments at sites of cell-cell contact in the absence of formed desmosomes. At a Ca2+ concentration of 0.13 mM desmoplakin is arrayed like beads on a "string" of intermediate filaments at areas of cell-cell association. At 0.15 mM, desmosome formation occurs, and desmoplakin is associated with the desmosomal plaque. In basal cell carcinoma cells desmoplakin is not restricted to desmosomes but also occurs in certain electron-dense bodies morphologically similar to those seen in NHEK maintained in low levels of Ca2+ and during early stages of desmosome assembly. We discuss the possibility of "cycling" of desmoplakin through these bodies in proliferative cells.

Immunochemical analysis of the distribution of the desmosomal protein desmoglein I in different layers of plantar epidermis.

An antiserum raised against the bovine desmosomal protein desmoglein I (DGI), Mr approximately 160 kDa, was used in an immunochemical analysis of human plantar epidermis. Different layers of the tissue were prepared by means of horizontal freeze sectioning. Loosely attached surface layers were obtained by means of scraping of the skin surface with a scalpel. Tissue extracts were analysed by means of sodium dodecylsulphate polyacrylamide gel electrophoresis followed by immunoblotting. Significant amounts of a component with Mr approximately 160 kDa, reactive with the DG I-antiserum, were found in all layers except the loosely attached surface layers. In these layers the antiserum detected a component with Mr approximately 80 kDa, not found in other layers. This component may be a degradation product of DG I. Since DG I belongs to the group of transmembrane desmosomal proteins that is believed to constitute the link between the intracellular parts of desmosomes of opposing cells, it is concluded that desmosomes may play an important role in plantar stratum corneum cell cohesion, and that degradation of desmosomes may be an important step in desquamation in plantar epidermis.

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 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.

Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. II. Morphological analysis.

Biochemical analysis of the kinetics of assembly of two cytoplasmic plaque proteins of the desmosome, desmoplakins I (250,000 Mr) and II (215,000 Mr), in Madin-Darby canine kidney (MDCK) epithelial cells, demonstrated that these proteins exist in a soluble and insoluble pool, as defined by their extract ability in a Triton X-100 high salt buffer (CSK buffer). Upon cell-cell contact, there is a rapid increase in the capacity of the insoluble pool at the expense of the soluble pool; subsequently, the insoluble pool is stabilized, while proteins remaining in the soluble pool continue to be degraded rapidly (Pasdar, M., and W. J. Nelson. 1988. J. Cell Biol. 106:677-685). In this paper, we have sought to determine the spatial distribution of the soluble and insoluble pools of desmoplakins I and II, and their organization in the absence and presence of cell-cell contact by using differential extraction procedures and indirect immunofluorescence microscopy. In the absence of cell-cell contact, two morphologically and spatially distinct patterns of staining of desmoplakins I and II were observed: a pattern of discrete spots in the cytoplasm and perinuclear region, which is insoluble in CSK buffer; and a pattern of diffuse perinuclear staining, which is soluble in CSK buffer, but which is preserved when cells are fixed in 100% methanol at -20 degrees C. Upon cell-cell contact, in the absence or presence of protein synthesis, the punctate staining pattern of desmoplakins I and II is cleared rapidly and efficiently from the cytoplasm to the plasma membrane in areas of cell-cell contact (less than 180 min). The distribution of the diffuse perinuclear staining pattern remains relatively unchanged and becomes the principal form of desmoplakins I and II in the cytoplasm 180 min after induction of cell-cell contact. Thereafter, the relative intensity of staining of the diffuse pattern gradually diminishes and is completely absent 2-3 d after induction of cell-cell contact. Significantly, double immunofluorescence shows that during desmosome assembly on the plasma membrane both staining patterns coincide with a subpopulation of cytokeratin intermediate filaments. Taken together with the preceding biochemical analysis, we suggest that the assembly of desmoplakins I and II in MDCK epithelial cells is regulated at three discrete stages during the formation of desmosomes.

Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. I. Biochemical analysis.

The functional interaction of cells in the formation of tissues requires the establishment and maintenance of cell-cell contact by the junctional complex. However, little is known biochemically about the mechanism(s) that regulates junctional complex assembly. To address this problem, we have initiated a study of the regulation of assembly of one component of the junctional complex, the desmosome, during induction of cell-cell contact in cultures of Madin-Darby canine kidney epithelial cells. Here we have analyzed two major protein components of the desmosomal plaque, desmoplakins I (Mr of 250,000) and II (Mr of 215,000). Analysis of protein levels of desmoplakins I and II by immunoprecipitation with an antiserum that reacts specifically with an epitope common to both proteins revealed that desmoplakins I and II are synthesized and accumulate at steady state in a ratio of 3-4:1 (in the absence or presence of cell-cell contact). The kinetics of desmoplakins I and II stabilization and assembly were analyzed after partitioning of newly synthesized proteins into a soluble and insoluble protein fraction by extraction of whole cells in a Triton X-100 high salt buffer. In the absence of cell-cell contact, both the soluble and insoluble pools of desmoplakins I and II are unstable and are degraded rapidly (t1/2 approximately 8 h). Upon induction of cell-cell contact, the capacity of the insoluble pool increases approximately three-fold as a proportion of the soluble pool of newly synthesized desmoplakins I and II is titrated into the insoluble pool. The insoluble pool becomes relatively stable (t1/2 greater than 72 h), whereas proteins remaining in the soluble pool (approximately 25-40% of the total) are degraded rapidly (t1/2 approximately 8 h). Furthermore, we show that desmoplakins I and II can be recruited from this unstable soluble pool of protein to the stable insoluble pool upon induction of cell-cell contact 4 h after synthesis; significantly, the stabilization of this population of newly synthesized desmoplakins I and II is blocked by the addition of cycloheximide at the time of cell-cell contact, indicating that the coordinate synthesis of another protein(s) is required for protein stabilization.

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.

Characterization of a 125K glycoprotein associated with bovine epithelial desmosomes.

An analysis of the concanavalinA binding polypeptide components of bovine tongue epithelial desmosomes reveals that in addition to the known desmosomal glycoproteins of 100/115K (the 'desmocollins'), 140K and 160/165K ('desmoglein 1') there is an uncharacterized glycoprotein of 125K (K = Mr x 10(-3). This latter polypeptide is immunologically distinct from known desmosomal glycoproteins, as determined by Western immunoblotting, but is recognized by an antibody preparation directed against the epithelial cell adhesion molecule E-cadherin. Moreover, the cadherin antibodies recognize a polypeptide present in bovine muzzle desmosomes that co-migrates with the 125K glycoprotein component of bovine tongue epithelial desmosomes. Upon treatment of bovine tongue desmosomes with a solution containing 9.5 M-urea, the 125K polypeptide becomes enriched in a urea-insoluble, membrane-enriched pelletable desmosomal fraction. Cadherin antibodies and antibodies directed against the 100/115K and 160/165K desmosomal glycoproteins generate similar immunofluorescence staining patterns in cryostat sections of bovine tongue epithelium. However, immunoelectron microscopic analysis of bovine tongue epithelium reveals that cadherin antibodies recognize components located both along the intercellular region of the desmosome and along nondesmosomal cell surfaces whereas antibodies directed against the 100/115K and the 160/165K desmosomal glycoproteins bind specifically to desmosomes. These results suggest that a cadherin-like glycoprotein component may play a role in the adhesive properties of the desmosomes of stratified squamous epithelia.

Evidence for heterogeneity in the 160/165 x 10(3) Mr glycoprotein components of desmosomes.

We have prepared both monoclonal and polyclonal antibody preparations directed against the 160/165 x 10(3) Mr glycoproteins (desmogleins) of bovine tongue epithelial desmosomes. The polyclonal antibody preparation recognizes desmosomes in a number of mouse tissues, e.g. mouse skin, heart, bladder and trachea, as determined by immunofluorescence microscopy. Furthermore, the polyclonal antibodies recognize polypeptide(s), present in the high salt, Triton-insoluble residues ('cytoskeleton preparations') of mouse skin, heart, bladder and trachea, which comigrate with the 160/165 x 10(3) Mr glycoproteins of bovine tongue epithelial desmosomes as determined by 'Western' immunoblotting. Conversely, the monoclonal 160/165 x 10(3) Mr antibody preparation recognizes desmosomes of stratified squamous epithelial tissues but not desmosomes in other tissue types. Moreover, whereas the monoclonal antibodies recognize 160/165 x 10(3) Mr polypeptides in mouse skin cell cytoskeletons they show no immunoreactivity with the cytoskeleton preparations of mouse bladder, trachea and heart following immunoblotting. These results suggest therefore that although there are conserved epitopes of the 160/165 x 10(3) Mr glycoproteins there are also epitopes of these molecules which vary from tissue to tissue. Double label immunofluorescence observations of cryostat sections of mouse skin using the monoclonal antibodies and antibodies directed against desmoplakin, a plaque component of desmosomes, reveal that the monoclonal antibodies do not recognize certain desmosomes in basal cells which are recognized by desmoplakin antibodies. Indeed, double label observations of cryostat sections of mouse skin using the monoclonal antibodies and human autoantibodies which react with hemidesmosomal components suggest that the monoclonal antibodies stain desmosomes located along the apical surfaces of basal cells but fail to recognize desmosomes along the lateral surfaces of these same cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical characterization of the soluble form of the junctional plaque protein, plakoglobin, from different cell types.

A polypeptide of identical molecular mass (Mr 83,000) and charge to desmosomal plakoglobin from bovine snout epidermis was identified in soluble and pelletable fractions from diverse tissues and cells of different mammalian species, including cells and tissues devoid of desmosomes (e.g. endothelial, retinal, lenticular cells, fibroblasts). The protein, however, was not detected in erythrocytes and platelets and in myeloma cells, nor in smooth muscle tissue. In all cells examined, the plakoglobin soluble upon cell lysis in buffers of near-physiological pH and ionic strength (21-31% of the total plakoglobin in the different cell types) was found to exist in a distinct molecular form. On sucrose gradient centrifugation it appeared at about 7 S and gel filtration chromatography revealed a Stokes radius of about 5.0 nm, from which an Mr of about 170,000 was estimated. By using isoelectric focusing under non-denaturing conditions, soluble approximately equal to 7-S plakoglobin had an isoelectric point at about pH 5.3. The plaque-bound and the soluble form of plakoglobin were indistinguishable by electrical charge and molecular mass, regardless of the source, indicating molecular identity. Cross-linking of soluble proteins with cupric 1,10-phenanthroline resulted in the formaton of a cross-linked product of plakoglobin with similar physical properties as the native approximately equal to 7-S particle, which is compatible with the interpretation that the soluble plakoglobin particle is a dimer. While a major proportion of the plakoglobin in the desmosomal plaque was resistant to various extraction procedures, plakoglobin present in the plaques of non-desmosome-containing cells and tissues was readily extractable under low and high salt conditions. This indicates that differences exist in the binding of plakoglobin to desmosomal plaques and the plaques of non-demosomal junctions.

Immunocytochemical analysis of Ewing's tumors. Patterns of expression of intermediate filaments and desmosomal proteins indicate cell type heterogeneity and pluripotential differentiation.

Examples of classical Ewing's tumors ("Ewing's sarcomas") of both skeletal and extraskeletal locations were analyzed for the expression of intermediate filament (IF) and cell junction proteins, with the use of immunofluorescence and immunoelectron microscopy as well as gel electrophoresis. In all 11 tumors examined vimentin filaments were abundant. A type of plaque-bearing small cell junction, which is common in these tumors but difficult to classify by morphologic criteria, was identified by antibodies to desmoplakins as true desmosomes. These were found in all cases, although in a very variable proportion of cells. Some of these junctions were associated with vimentin IFs. In addition, 9 of the cases examined showed scattered or clustered cells expressing the simple-epithelium type cytokeratins 8 and 18. Moreover, 3 cases displayed dispersed or clustered cells producing neurofilaments. The value of these observations, notably the cell type heterogeneity, for the diagnosis of tumors of this group is discussed. The results further indicate that Ewing's tumors are derived from a primitive, pluripotential cell that may differentiate, in variable proportions, into cells with mesenchymal, epithelial, and, more rarely, even neural features, suggesting that this tumor should be regarded as a blastoma, rather than as a true sarcoma.

Desmosomal plaque-associated vimentin filaments in human ovarian granulosa cell tumors of various histologic patterns.

Proteins of intermediate-sized filaments and desmosomal plaques (desmoplakins) of four human ovarian granulosa cell tumors were studied by immunofluorescence and immunoelectron microscopy and by two-dimensional gel electrophoresis of microdissected tissue samples. All tumor cells, irrespective of their specific histologic patterns, contained both vimentin and desmoplakins. Cytokeratin-positive structures were absent or very scant in most tumor regions, but more common in trabecular, insular, macro- and microfollicular structures. Biochemical analysis revealed the presence of Cytokeratin Polypeptides 8 and 18. Desmin filaments, neurofilaments, and glial filaments were not detected. Immunoelectron microscopy showed vimentin filaments attached to desmoplakin-positive plaques of desmosomes. These results indicate that granulosa cell tumors contain true desmosomes, which are associated primarily with vimentin filaments. This phenomenon has so far only been described in meningiomas and in blastema cells of nephroblastomas. Our observations suggest that in most neoplastic granulosa cells one epithelial feature, ie, cytokeratin expression, is greatly reduced, whereas desmosomes are still formed in appreciable frequencies. This unusual constellation of cytoskeletal elements in granulosa cell tumors may be useful in the differential diagnosis from other ovarian neoplasms, especially undifferentiated carcinomas. The importance of the use of antibodies specific for exclusively desmosomal proteins in classifying morphologically ill-defined junctional structures (eg, "rudimentary junctions," "primitive junctions," "desmosome-like junctions") is emphasized.