Desmosomes are the most prominent intercellular junctions in the epidermis where they provide an attachment site for the keratin intermediate filament (IF) cytoskeleton. During the current funding period we demonstrated that the obligate desmosomal component desmoplakin (DP) is required for linking IF to the desmosome and thus integrates the IF cytoskeleton into a supracellular network that joins all cells within the epidermis. The importance of DP in the epidermis was underscored by the discovery of an inherited form of palmoplantar keratoderma caused by haploinsufficiency of DP. Nevertheless, a number of questions remain which are key to understanding the etiology of human diseases targeting adhesive junctions. In the next funding period we propose to: 1) Determine the basis for tissue- and differentiation-specific interactions between IF and the DP C-terminus by a) carrying out yeast two hybrid analysis of associations between DP and simple epithelial versus epidermal keratins, by b) performing high resolution structural analysis of the DP C-terminus, and by c) analyzing the role of DP phosphorylation in desmosome assembly in living cells and tissues; 2) Determine how the DP N-terminus collaborates with cell type- and differentiation-specific armadillo proteins in desmosome assembly by defining the repertoire of DP binding partners in the arm family in cells and in vitro; 3) Address the importance of the IF-desmosome connection in keratinocyte behavior and mechanical integrity by using laser tracking and mechanical stretch assays coupled with gene microarray analysis to study cells inducibly expressing a dominant negative DP mutant that severs the DP-IF connection; 4) Address whether DP drives desmosome assembly in a classic cadherin dependent fashion by a) using fluorescently-tagged junction markers to establish the temporal and spatial events linking desmosome and adherens junction assembly in living cells, b) determining whether adherens junction proteins form a complex with DP, and c) examining whether desmosomal cadherins bind directly to downstream regions of the DP N-terminus, contributing to later stages of desmosome assembly. These experiments will provide new insight into how desmosomes are assembled and regulated, and will establish novel approaches towards defining the role of the DP-IF connection in keratinocyte integrity, intracellular signaling and behavior.
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