Faculty Bibliography

We have established, by means of a monoclonal antibody and a cDNA clone, that a desmosomal polypeptide of Mr 83,000 also occurs at the plaques of other types of adhering junctions, including the vinculin-actin-associated intercellular junctions, e.g., the zonula adhaerens of epithelial cells and the endothelial, lens, and Sertoli cell junctions. This is the first component found in common among otherwise biochemically distinct plaque domains. Despite its concentration at these intercellular junctions, it is absent from the respective cell-substratum contact sites. In addition, it appears in a globular soluble 7S form in the cytoplasm. We discuss the significance of this protein, for which the name plakoglobin is proposed, in terms of its interaction with such biochemically diverse membrane domains and their different types of associated cytoskeletal 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

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

The seven major desmosomal polypeptides from isolated bovine muzzle desmosomes ranging from Mr 75 000 to 250 000 were separated by gel electrophoresis, isolated and characterized with respect to their amino acid composition and sugar content. The two largest polypeptides (bands 1 and 2), i.e. desmoplakins I and II, are similar in their amino acid composition, confirming our previous immunological and biochemical data, and display a relatively high glycine content. In contrast, the other two cytoplasmic components also believed to be associated with the desmosomal plaque, i.e. polypeptides of bands 5 (Mr 83 000) and 6 (Mr 75 000), differ significantly in their amino acid composition from the desmoplakins and from each other. All four candidate polypeptides for plaque association, i.e. bands 1, 2, 5, and 6, show no significant glycosylation. The glycoproteins 4a and 4b (Mr 115 000 and 130 000) are similar in their amino acid composition, peptide analysis and immunological reactivity. Both are relatively rich in mannose and galactose but also contain sialic acid. Our determinations also indicate that the two polypeptides differ significantly in their N-acetylglucosamine and mannose content. Most, if not all, of the sugar residues are associated with a water-soluble fragment of Mr 15 500 obtained after limited digestion with V8 protease. The glycopolypeptides obtained in band 3 (Mr 164 000-175 000) are distinct from the glycopolypeptides 4a and 4b in amino acid composition, sugar content, isoelectric pH values, certain antigenic determinants and in their pattern of cleavage products obtained by treatment with proteases or cyanogen bromide. The results identify polypeptides of bands 3, 4a and 4b as glycosylated with characteristic sugar compositions. It is suggested that the major glycoproteins (bands 3, 4a, 4b) of the desmosome are integral membrane components arranged in a special way conferring resistance to detergent treatment. The possible roles of these glycoproteins in cell recognition and in adhesive functions of the desmosome are discussed

Specific antibodies against the components of desmosomes, the adhesive junctions of epithelial cells, have been used to determine which components are located on the cell surface. Three criteria have been used: fluorescent antibody staining, immuno-gold labelling and electron microscopy, and quantitative measurements of antibody binding using [125I]protein A. When these techniques were applied to living Madin-Darby bovine kidney (MDBK) cells, antibodies against only two desmosomal components, glycoproteins of approximately 115 X 10(3) Mr and 100 X 10(3) Mr, bound to the cell surface. Antibodies against all other components, the 230 and 205 X 10(3) Mr proteins (desmoplakins), the 150 X 10(3) Mr glycoprotein and the 82 and 86 X 10(3) Mr proteins reacted in fluorescent antibody staining only after cells had been fixed and made permeable. MDBK cells were cultured in the presence of univalent fragments (Fab') of anti-desmosomal antibodies for periods from 24 h to 72 h. After these times cells were fixed, made permeable, and stained with anti-desmoplakin antibody to assay for desmosome formation. Fab' derived from anti-100 X 10(3) Mr protein specifically inhibited desmosome formation, whereas Fab's from anti-desmoplakin, anti-150 X 10(3) Mr and anti-82 and 86 X 10(3) Mr proteins were without effect. We conclude that the 100 X 10(3) Mr and the immunologically related 115 X 10(3) Mr components are located on the cell surface and are directly involved in cell-cell adhesion. We have named them desmocollins to denote that they are involved in the adhesive function of desmosomes. The modulation of desmocollin distribution during monolayer formation and establishment of epithelial polarity has also been studied. Fluorescent and immuno-gold labelling using Fab' or IgG at 4 degrees C revealed that desmocollins were initially evenly dispersed over the cell surface. Staining with IgG at 37 degrees C caused the desmocollins to 'patch' but not to 'cap'. With the establishment of confluency, desmocollins were gradually removed from the upper surfaces of the cells (or masked and rendered inaccessible to antibody) being confined to the lateral and probably basal regions of the cells. Treatment of confluent monolayers with 3 mM-EGTA rendered the desmocollins stainable, probably by causing their release from lateral constraint. Desmocollin staining at the cell surface was not appreciably reduced during 5 h of EGTA treatment, suggesting that desmocollins, unlike desmosomal plaques, may not be internalized after junction breakdown

In previous work we used immunofluorescent staining with specific antibodies to study the distribution of five desmosomal antigens in the epithelia of different vertebrate animals. We showed that all five antigens were present in all epithelia studied in human, bovine, rat, guinea pig, chick and frog (Rana pipiens) tissues. It was concluded that desmosomes are highly conserved structures. This paper extends those studies: by including three other species, a lizard (Lacerta viridis), the axolotl (Ambystoma mexicanum) and the trout (Salmo trutta), and by looking at several tissues in more detail. The principal results are as follows. The epidermis of all species down to the frog stain with equal intensity for all desmosomal antigens. In the epidermis of axolotl and trout, staining for desmosomal plaque constituents is present, but staining for the desmosomal glycoproteins is greatly reduced or absent. Within mammalian species as well as chick, lizard and frog, staining for the 115 X 10(3) and 100 X 10(3) molecular weight desmosomal glycoproteins is less intense in non-epidermal tissues than in the epidermis, while staining for desmosomal plaque constituents and for the 150 X 10(3) molecular weight glycoprotein is undiminished. It is possible, therefore, that slight differences exist between certain glycoproteins of epidermis and non-epidermal epithelia. The hearts of lower vertebrates (lizard, frog, axolotl and trout) stain only for individual desmosomal plaque antigens. The pillar cells of trout gill stain, adjacent to their collagenous columns, for one desmosomal plaque antigen. There is a fibrous cytoplasmic mat in this position but no desmosomes. Thus one of the desmosomal antigens may have a function outside the desmosome

Many workers regard cell adhesion as a highly specific phenomenon, believing that different molecular mechanisms are involved in the adhesion of cells of different tissues and different species. We believe that the evidence from cell behaviour is against this view and that cells share common adhesion mechanisms (for reviews see refs 1, 2); however, molecular evidence is lacking. As an approach to providing such evidence we have begun to study desmosomes, the cell-surface organelles responsible for strong intercellular adhesion in epithelia. We have raised antisera against each of five high-molecular weight (MW) desmosomal components. Having determined the specificity of our antisera by immunoblotting, we show here that each gives a staining pattern corresponding to the distribution of desmosomes in a range of tissues from different vertebrate species, demonstrating that desmosomal components are widely shared and highly conserved

Antibody to tubulin in man has been studied using a specific radioimmunoassay, affinity chromatography radioimmunoassay but markedly increased levels were noted in patients with infectious mononucleosis where the antibody was predominantly IgM in type. This finding was confirmed on fluorescence microscopy. Affinity chromatography purified antibody produced characteristic microtubular staining of fixed 3T3 cells, but in addition, produced weak staining of cryostat sections of rat tissue, similar in distribution to that of smooth muscle antibody. Our studies indicate that the IgM smooth muscle antibody found in infectious mononucleosis by IF techniques is at least in part due to an antitubulin antibody