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Title: Localization of the cytoplasmic sub-domains of the desmosomal cadherin desmoglein 2
Author: Scothern, Anthea
ISNI:       0000 0001 3557 1936
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2008
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Desmosomes are intercellular junctions found in epithelia and some other tissues. Their primary function is strong cell-cell adhesion. They also link the keratin intermediate filament (IF) cytoskeleton between cells and have roles in cell signalling, tissue morphogenesis and wound repair. Desmosomes are disc-like plaques arranged symmetrically on either side of the plasma membranes of adjacent cells. Each plaque consists of an outer dense plaque (ODP), an electron lucent zone and an inner dense plaque (IDP), the site of intermediate filament attachment. The plaques are joined to each other by the transmembrane desmosomal cadherins desmoglein and desmocollin which are adhesion molecules. These molecules have unique features in their cytoplasmic domains that may be important for desmosomal structure and function. Previous studies have predicted that Dsg extends from the extracellular core domain through the ODP and IDP and possibly bind to the IFs. However, immunogold localization ofthe C-terminus of the Dsg isoform with the shortest cytoplasmic domain, Dsg3, shows that this is not the case and that Dsg3 is entirely folded within the ODP. Dsg 2 has the largest cytoplasmic domain and is the most widespread isoform ofDsg. Its cytoplasmic domain (Dsg2Cyt) consists of483 amino acids divided into 5 sub-domains: the intracellular anchor, intracellular catenin site, internal proline-rich linker, repeat domain (RD) and terminal domain (TD). The aim ofthis project was to localize the RD and the TD within the desmosomal plaque by immunogold-electron microscopy. Recombinant N-terminally His-tagged Dsg2Cyt and GST-tagged Dsg2TD were expressed in E.coli, purified and used to raise polyclonal antibodies in rabbits. Characterization of these antibodies and further characterization of a mouse monoclonal antibody, 33-3D by immunoblotting showed that anti-Dsg2TD reacts exclusively with the TD and 33-3D reacts exclusively with the RD of Dsg2. Quantitative analysis of immunogold labelling of ultrathin cryosections of epidermis indicates that the repeat domain of Dsg2 is localized at the inner face of the outer dense plaque and the terminal domain is localized at the outer face of the inner dense plaque. This work contributes further to the elucidation of the structure of the desmosomal plaque by showing that the largest isoform ofDsg does extend beyond the ODP but it does not extend to the site ofIF attachment at the inner face ofthe IDP as suggested by other investigators.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available