Molecular map of the desmosomal plaque

Recent biochemical and molecular approaches have begun to establish the pro tein interactions that lead to desmosome assembly, To determine whether the se associations occur in native desmosomes we have performed ultrastructura l localisation of specific domains of the major desmosomal components and h ave used the results to construct a molecular map of the desmosomal plaque. Antibodies directed against the amino- and carboxy-terminal domains of des moplakin, plakoglobin and plakophilin 1, and against the carboxy-terminal d omains of desmoglein 3, desmocollin 2a and desmocollin 2b, were used for im munogold labelling of ultrathin cryosections of bovine nasal epidermis, For each antibody, the mean distance of the gold particles, and thus the detec ted epitope, from the cytoplasmic surface of the plasma membrane was determ ined quantitatively, Results showed that: (i) plakophilin, although previou sly shown to bind intermediate filaments in vitro, is localised extremely c lose to the plasma membrane, rather than in the region where intermediate f ilaments 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 a cross the entire outer plaque, beyond both desmocollins; (iv) the amino ter minus of desmoplakin lies within the outer dense plaque and the carboxy ter minus some 40 nm distant in the zone of intermediate filament attachment, T his is consistent with a parallel arrangement of desmoplakin in dimers or h igher order aggregates and with the predicted length of desmoplakin II, ind icating that desmoplakin I may be folded or coiled. Thus several predictions from previous work were borne out by this study, b ut in other cases our observations yielded unexpected results. These result s have significant implications relating to molecular interactions in desmo somes and emphasise the importance of applying multiple and complementary a pproaches to biological investigations.