Controlling notch protein interaactions in stem cell differentiation. (360G-Wellcome-084070_Z_07_Z)

The proposed project will focus on understanding and manipulating protein:protein interactions controlling stem cell differentiation with a particular focus on Notch receptors and ligands. Notch signalling occurs via direct cell:cell contact, links the fate of cells to their neighbours and plays a controlling role throughout development. Many aspects of the pleiotropic actions of Notch are recapitulated during neurogenesis from embryonic stem cells and this will form the biological focus of these studies. The project will build on existing work where we have demonstrate that blocking antibodies can be generated either by binding ligand (dll4) or by targeting EGF domains 11-14 of Notch 1. We will generate blocking and potentially activating antibodies to all members of the receptor/ligand family. This will provide specificity in blocking/activating Notch signalling which is currently unavailable using ligand based activation systems. The mechanism by which ligand binding permits release of the intracellular signalling domain is unknown. The project will test the hypothesis that activation of notch involves a ligand dependant release of a protective intra-molecular interaction, by screening directly for interactions between isolated groups of EGF domains. A number of Notch interacting proteins have recently been identified, including novel activators, inhibitors and molecules contributing to sub-cellular localisation. In addition we and others have generated evidence of ectopic distribution of Notch in some cells. We will specifically investigate the potential association of notch with supra-molecular signalling complexes, with a view to identifying alternative routes to manipulate notch signalling for research and potentially therapeutic applications.