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.
D000818:Animals; D002417:Cattle; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000648:ultrastructure; D051180:Desmoplakins; D003896:Desmosomes / Q000276:immunology / Q000648:ultrastructure*; D004591:Electrophoresis, Polyacrylamide Gel; D004817:Epidermis / Q000032:analysis / Q000648:ultrastructure; D006023:Glycoproteins / Q000032:analysis; D007120:Immunochemistry; D007365:Intercellular Junctions / Q000648:ultrastructure; D008565:Membrane Proteins / Q000032:analysis; D009092:Mucous Membrane / Q000032:analysis / Q000648:ultrastructure
D003598:Cytoskeletal Proteins; D051180:Desmoplakins; D006023:Glycoproteins; D008565:Membrane Proteins