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Signaling to the actin cytoskeleton during chemotaxis and phagocytosis in Dictyostelium: The roles of Pten and the Dock180/Elmo proteins

Reference	BB/C006895/1
Principal Investigator / Supervisor	Professor Kees Weijer
Co-Investigators / Co-Supervisors
Institution	University of Dundee
Department	School of Life Sciences
Funding type	Research
Value (£)	301,705
Status	Completed
Type	Research Grant
Start date	01/03/2005
End date	31/12/2008
Duration	46 months

Abstract

Chemotaxis plays an important role during development, in wound healing, regeneration, and functioning of the immune system. When not properly controlled it caused/contributes to birth defects and disease. During chemotaxis cells have to measure and interpret chemical gradients and translocate up or down these gradients. Translocation up a gradient involves repeated cycles of extension of the front of the cell in the direction of the gradient of the attractant followed by pulling up of the rear end of the cell. Extension is driven by acting polymerisation, but also requires the simultaneous disassembly of the cortical layer of myosin thick filaments, to allow the extension to occur. We investigate signalling to the actin cytoskeleton in the genetically tractable organism Dictyostelium discoideum, which are chemotactically sensitive folate and cAMP and during phagocytosis of bacteria and yeasts. The transduction of the information provided by the often shallow folate and cAMP and during phagocytosis of bacteria and yeasts. The transduction of the information provided by the often shallow folate and cAMP gradients to localised actin polymerisation involves the sharply localised production of the phosphaltidylinositide(3,4,5)trisphosphate (PIP3). This involves the translocation of PI3 kinase which synthesises PIP3 from its precursor PI(4,5)P2, to the cells leading edge and the dissociation of the phosphatidyl 3 phosphatase Pten from the leading edge. PIP3 then acts as a binding site for PH domain containing proteins such as activators (Rac-gefs) of small GTPases of the Rac family, resulting in localised activation of Rac. Activated Rac then stimulates actin polymerisation. Deletion of Pten, results in an altered PIP3 signal and a reduced efficiency of chemotaxis. This can be rescued by reintroduction of Pten in the null mutant. However expression of a catalytically inactive Pten gene that has lost both its lipid and protein phosphatase activity also inhibits chemotaxisto folate and cAMP in wildtype and Pten null strains. It appears to act in a dominant negative manner. We will investigate whether this dominant negative effect is the result of altered PIP3 dynamics by investigation of PIP3 dynamics in these mutant strains. If Pip3 signalling is affected we will establish whether this through an action on Pten or on PI3 kinase activity. If PIP3 dynamics are not affected we will identify other potential interacting proteins using a variety of proteomic methods. We will investigate which domains mediate this effect and whether modification of Pten by phosphorylation plays a role in this process. We have shown that human Pten can rescue the developmental defect caused by knockout of the Dictyostelium Pten. This now opens up the possibility to characterise the importance of various functional domains of human Pten in the control of its localisation and its role in chemotaxis. Dictyostelium cells can still chemotax in absence of measurable PIP3 levels, which led us to investigate the function of the Dock/Elmo proteins, a novel class of bipartite Rac-Gefs. We have identified 8 possible Dock180 and 4 potential Elmo homologues. We have made knockout mutants in 6 Dock genes and shown that some play a role in the control of chemotaxis. We will now make knock out mutants of all these genes in various combinations to investigate their role in PIP3 independent signalling pathways to the actin cytoskeleton during chemotaxis and phagocytosis. We will characterise through which Racs they expert their function and start to investigate possible upstream signalling pathways. Together these studies will enhance our insight in the molecular mechanisms of the signalling pathways to the actin cytoskeleton during chemotaxis and phagocytosis.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Immunology, Microbiology, The 3 Rs (Replacement, Reduction and Refinement of animals in research)
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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