Ubiquitylation is a versatile post-translational modification that participates in regulation of protein stability, via proteasomal and lysosomal degradative pathways, regulation of membrane protein internalisation and other trafficking events, and regulating the biological activity of some proteins independent of degradation. The diverse functions of ubiquitylation as a post- translational protein modification allow speculation that regulation of protein ubiquitylation status may be of crucial importance during the dynamic process of development. A screen of known, and suspected, ubiquitin pathway enzymes was designed to test this hypothesis. Whole mount in situ hybridisation was conducted on early post implantation mouse embryos to determine expression patterns of the ubiquitin pathway enzyme targets. This screen was not pursued in depth due to difficulties in resolving doubts regarding the sensitivity of the method and the validity of weak ubiquitous staining patterns. The FAM deubiquitylating enzyme is a known developmentally regulated ubiquitin pathway enzyme, and although believed to antagonise the conjugation of ubiquitin to specific substrates its cell biology remains poorly characterised. In different cellular contexts FAM has been reported to localise to points of cell-cell contact or to endosomes, and circumstantial evidence suggests a role in regulating trafficking of a cell-cell adhesion complex (Murray et al., 2004; Taya et al., 1999; Taya et al., 1998). It was sought to further investigate the role of FAM in cell-cell adhesion in the well characterized polarized epithelial cell line, MDCKII, by creating clonal MDCKII cell lines that overexpress FAM. These cell lines were to be analysed for alterations in cell-cell adhesive properties and the biochemistry of proposed FAM substrates, which include the cell adhesion molecules β-catenin, E-cadherin and AF-6. MDCKII cell lines expressing exogenous V5-tagged murine FAM were successfully isolated, but failed to show changes in cell-cell adhesive properties. Generation of an antibody that reliably recognised both the canine and murine FAM protein demonstrated that the FAM-V5 expressing cell lines did not have increased total FAM protein. Other approaches taken to facilitate the study of FAM include attempts to express GFP-FAM fusion proteins and to generate an inducible FAM overexpressing cell line. Further alternative approaches are discussed. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2005.
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