Recent biochemical and molecular approaches have begun to establish the protein interactions that lead to desmosome assembly. To determine whether these associations occur in native desmosomes we have performed ultrastructural localisation of specific domains of the major desmosomal components and have used the results to construct a molecular map of the desmosomal plaque. Antibodies directed against the amino- and carboxy-terminal domains of desmoplakin, plakoglobin and plakophilin 1, and against the carboxy-terminal domains of desmoglein 3, desmocollin 2a and desmocollin 2b, were used for immunogold labelling of ultrathin cryosections of bovine nasal epidermis. For each antibody, the mean distance of the gold particles, and thus the detected epitope, from the cytoplasmic surface of the plasma membrane was determined quantitatively. Results showed that: (i) plakophilin, although previously shown to bind intermediate filaments in vitro, is localised extremely close to the plasma membrane, rather than in the region where intermediate filaments are seen to insert into the desmosomal plaque; (ii) while the ‘a’ form of desmocollin overlaps with plakoglobin and desmoplakin, the shorter ‘b’ form may be spatially separated from them; (iii) desmoglein 3 extends across the entire outer plaque, beyond both desmocollins; (iv) the amino terminus of desmoplakin lies within the outer dense plaque and the carboxy terminus some 40 nm distant in the zone of intermediate filament attachment. This is consistent with a parallel arrangement of desmoplakin in dimers or higher order aggregates and with the predicted length of desmoplakin II, indicating that desmoplakin I may be folded or coiled. Thus several predictions from previous work were borne out by this study, but in other cases our observations yielded unexpected results. These results have significant implications relating to molecular interactions in desmosomes and emphasise the importance of applying multiple and complementary approaches to biological investigations.

Desmocollins are cadherin-like glycoproteins involved in cell adhesion and plaque formation in desmosome junctions. Three distinct isoforms, the products of different genes, have been found in bovine tissues. We have reported previously that one of these, DSC3, is expressed only in basal and lower suprabasal layers of stratified epithelia. Using RT-PCR we have now obtained the complete cDNA coding sequence of mature bovine DSC3. It has alternatively spliced ‘a’ and ‘b’ forms found in other desmocollins but is unique in having a 43 instead of a 46 base pair exon. We have characterised a monoclonal antibody, 07–4G, which is specific for the Dsc3 protein, recognising an epitope in the extracellular domain. Immunofluorescent staining with 07–4G confirms that this isoform is found only in stratified epithelia, being strongly expressed in the basal cell layers of these tissues. The intensity of expression fades gradually in the suprabasal layers and disappears completely below the upper limit of desmosome expression. These results suggest that Dsc3 plays an important role in cell epithelial differentiation.