| Popis: |
The serine/threonine kinase glycogen synthase kinase-3 (GSK-3) was initially identified as a key regulator of insulin-dependent glycogen synthesis. GSK-3 was subsequently shown to function in a wide range of cellular processes including differentiation, growth, motility and apoptosis. A number of mechanisms have been shown to allow differential regulation of GSK-3 these include modulation by upstream signals, control of substrate specificity and GSK-3 localisation. The aim of this project was to better characterise the role of GSK-3 in the social amoeba Dictyostelium discoideum and to investigate the possibility that GSK-3 activity is directed through interactions with specific GSK-3 binding proteins. In this study I generated both mammalian and Dictyostelium cell lines expressing GSK-3 homologues tagged with GFP. I demonstrated that the addition of this tag did not obviously alter cellular localization or the ability of GSK-3 to interact with known protein binding partners. I also confirmed that these fusion proteins retained kinase activity and that the levels of GSK-3 (3 over-expression used in this study were not detrimental to cell viability. I further characterized the role of GSK-3 in the social amoebae Dictyostelium, using both knockout and over-expression cell lines to demonstrate that this protein fulfils multiple roles during early development. Dark-field time-lapse microscopy techniques were employed to identify a role for GSK-3 in co-ordinating chemotaxis and cellular polarization. During the course of this project I also identified a requirement for GskA in cytokinesis when cells were grown in shaking culture. Finally, both the mammalian and Dictyostelium cell lines generated in this study were used for affinity chromatography experiments and a number of potential GSK-3 binding partners were identified. |
