Regulation of desmosome assembly in MDCK epithelial cells: coordination of membrane core and cytoplasmic plaque domain assembly at the plasma membrane

Desmosomes are major components of the intercellular junctional complex in epithelia. They consist of at least eight different cytoplasmic and integral membrane proteins that are organized into two biochemically and structurally distinct domains: the cytoplasmic plaque and membrane core. We showed previously that in MDCK epithelial cells major components of the cytoplasmic plaque (desmoplakin I and II; DPI/II) and membrane core domains (desmoglein I; DGI) initially enter a pool of proteins that is soluble in buffers containing Triton X-100, and then titrate into an insoluble pool before their arrival at the plasma membrane (Pasdar, M., and W. J. Nelson. 1988. J. Cell Biol. 106:677- 685; Pasdar. M., and W. J. Nelson. 1989. J. Cell Biol. 109:163-177). We have now examined whether either the soluble or insoluble pool of these proteins represents an intracellular site for assembly and interactions between the domains before their assembly into desmosomes at the plasma membrane. Interactions between the Triton X-100-soluble pools of DPI/II and DGI were analyzed by sedimentation of extracted proteins in sucrose gradients. Results show distinct differences in the sedimentation profiles of these proteins, suggesting that they are not associated in the Triton X-100-soluble pool of proteins; this was also supported by the observation that DGI and DPI/II could not be coimmunoprecipitated in a complex with each other from sucrose gradient fractions. Immunofluorescence analysis of the insoluble pools of DPI/II and DGI, in cells in which desmosome assembly had been synchronized, showed distinct differences in the spatial distributions of these proteins. Furthermore, DPI/II and DGI were found to be associated with different elements of cytoskeleton; DPI/II were located along cytokeratin intermediate filaments, whereas DGI appeared to be associated with microtubules. The regulatory role of cytoskeletal elements in the intracellular organization and assembly of the cytoplasmic plaque and membrane core domains, and their integration into desmosomes on the plasma membrane is discussed.