Investigation into the role of plakoglobin in Xenopus development

Desmosomes are rivet-like intercellular junctions which link intermediate filament networks of adjacent cells, dissipating mechanical stress over the tissue as a whole. They are composed of a number of different proteins and glycoproteins including the transmembrane adhesive molecules (the desmosomal cadherins) and the plaque-localised armadillo family protein, plakoglobin (PG). Following preliminary experiments showing a desmosomal cadherin was expressed during early Xenopus development, before fully formed desmosomes had been observed, it was decided to investigate the potential morphogenetic role of desmosomal cadherins during early development. Initially it was decided to characterise the expression pattern of desmosomal components through early Xenopus development using a panel of antibodies, which were screened for cross-reactivity by Western blotting. Sections were prepared using a variety of techniques, and conditions optimised for immunostaining. These optimal conditions, however, did not maintain tissue integrity at early stages of development, and the proposed time course study was abandoned. In parallel an attempt was made to clone a Xenopus desmosomal cadherin using a variety of methods, for use in complementary in situ analysis and intervention studies, unfortunately without success, and so after two years this project was abandoned. Following published experiments which illustrated the role of β-catenin (a molecule closely related to the desmosomal protein PG) in the Wnt signalling pathway, it was decided to further elucidate the potential signalling role of PG in early pattern formation. GST-fusion proteins of the unique N- and C-terminal domains of Xenopus PG and of the N-terminal two fifths of human PG were made, and antibodies generated against them. These were used to study PG through early Xenopus development by Western blotting, and revealed an interesting non-uniform expression pattern. When Fab fragments were made and microinjected into embryos, they caused non-specific toxic effects that were not significantly more severe than those generated by Fab fragments from control antisera. No double axis embryos were generated above background levels, which would have indicated a specific activation of the Wnt signalling pathway, although these were produced when positive control anti-β-catenin Fab fragments were injected. Little is known about the function of the terminal regions of PG, or indeed whether any other molecules bind to them. PG becomes tyrosine phosphorylated after certain stimuli and it has been postulated that the phosphorylation site lies in one of the terminal regions The fusion proteins were therefore used for in vitro binding and kinase studies in an attempt to address these questions. Of the molecules tested, only α-catenin was found to associate with the N-terminal domain of PG, in a phosphorylation-independent manner, and the site of tyrosine phosphorylation could not be identified