A systems and molecular analysis of G protein-mediated signalling
Croft, Wayne D. (2012) A systems and molecular analysis of G protein-mediated signalling. PhD thesis, University of Warwick.
WRAP_THESIS_Croft_2012.pdf - Submitted Version
Download (169Mb) | Preview
Official URL: http://webcat.warwick.ac.uk/record=b2581632~S1
The ability of cells to respond correctly to signals from their microenvironment is an essential prerequisite of life. Many external signals are detected through G protein-coupled receptor (GPCR) signalling pathways, which control all aspects of eukaryotic physiology. Ligand-bound GPCRs initiate signalling by promoting exchange of GDP for GTP on the Gα subunit of heterotrimeric G proteins, thereby facilitating activation of downstream effectors. Signalling is terminated by the hydrolysis of GTP to GDP through intrinsic GTPase activity of the Gα subunit, in a reaction catalysed by the regulator of G protein signalling (RGS) proteins. Due to the problem of complexity in higher eukaryotic GPCR signalling, the matingresponse in Schizosaccharomyces pombe has been used to study GPCR signalling in isolation. In vivo data from quantitative assays of reporter strains and live-cell uorescence microscopy informs the development of an ordinary differential equation model of the signalling pathway, first described by Smith et al., 2009. The rate of nucleotide exchange on the Gα (Gpa1) is a key molecular mechanism controlling duration and amplitude of signalling response. The in uence of this is investigated through characterisation of Gpa1 nucleotide exchange mutants and perturbation of reaction rate parameters in the computational model. Further, this thesis also presents data relating to the temporal and spatial regulation of Rgs1 (the sole RGS protein for Gpa1). Using an inter-disciplinary approach, evidence is provided to suggest that an interaction between Rgs1 and the C-terminal tail of the GPCR (Mam2) tethers Rgs1 to the plasma membrane to facilitate its function. Finally, quantification of signalling at the single cell level is described. Time-lapse livecell imaging of uorescent reporter cells is optimised and single cell signalling response quantified using image analysis software. Single cell quantification provides greater insight into temporal dynamics, cell-to-cell variability, and highlights the existence of mechanisms for cellular decision-making.
|Item Type:||Thesis or Dissertation (PhD)|
|Subjects:||Q Science > QP Physiology|
|Library of Congress Subject Headings (LCSH):||G proteins, Cellular signal transduction, Schizosaccharomyces pombe -- Physiology|
|Institution:||University of Warwick|
|Theses Department:||Warwick Systems Biology Centre|
|Supervisor(s)/Advisor:||Ladds, Graham Robert ; Rand, David A.|
|Extent:||xviii, 283 leaves : ill., charts|
Actions (login required)