| Summary: | In this thesis I set out to characterise the interaction of 14-3-3 with lipid raft-like membrane domains. 14-3-3 associated with DRMs isolated from rate brain extract in a 14-3-3 isoform specific manner. Inhibition of 14-3-3 target protein interactions suggested that this association was reliant on binding of 14-3-3 to a membrane protein. The interaction of 14-3-3 with DRMs was further investigated in N2a and PC12 cells and also be detergent-free method. There are, however, some concerns over whether DRMs represent physiological membrane domains. Therefore I also investigated quantitatively the colocalisation of 14-3-3 with membrane domains in intact cells by confocal microscopy, using the lipid raft marker cholera toxin B subunit (CTXB). For comparison, other DRM-resident proteins, Thy-1, syntaxin-1a and SNAP-25, were also examined. SNAP-25 and Thy-1 showed a high degree of coincidence with CTXB in PC12 cells, 14-3-3 also colocalised with CTXB but to a much lower extent. As lipid rafts have been implicated in the control of regulated exocytosis the high coincidence of SNAP-25 and lack of coincidence of syntaxin-1 with CTXB is of interest. Cholesterol depletion, which affects the integrity of lipid raft-like domains, revealed some discrepancies between the association of 14-3-3., Thy-1, SNAP-25 and syntaxin-1a with DRMs and CTXB domains imaged in intact PC12 and N2a cells. CTXB clusters were partially disrupted in N2a cells and the coincidence of SNAP-25, but not 14-3-3, with CTXB was reduced. These findings indicate a role for lipid raft-like domains in controlling the spatial distribution of SNAP-25 on the plasma membrane. However, the lack of cholesterol dependent 14-3-3 localisation indicates that other membrane compartmentalisation mechanism may affect 14-3-3 distribution.
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