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(BBSRC DTP) Dissecting distinct Notch signalosome compartments and their cellular responses


Project Description

Notch is an important cell signalling protein with numerous pivotal roles both in normal development and in the maintenance of adult stem cells, and hence healthy homoeostasis over the life course. Notch can be activated by several independent mechanisms (Shimizu et al. 2014, Cell 157:1160-74), and its misregulation is also frequently associated with disease, such as cancer. It is therefore important to understand the different mechanisms by which Notch is normally activated, since this is a prerequisite to selective targeting of a specific activation mechanism that may become misregulated in the cell.

Notch is a single span transmembrane protein which acts as a receptor in a highly conserved signalling pathway. The ligand for Notch is also present at the cell membrane, where at cell-cell contacts Notch and its ligands co-cluster to form an extended interaction interface. This acts as a signalosome environment in which proteolytic activation occurs, followed by release of the Notch intracellular domain into the cytoplasm and then subsequently to the nucleus where it can modify gene expression. Interestingly Notch can also be activated in a ligand-independent manner following endocytosis of full-length receptor through one of two distinct endocytic pathways, associated with clathrin-mediated or lipid raft mediated endocytic routes. In both cases Notch is also localised to a platform-like signalosome within the endosomal membrane, and the Notch intracellular domain is proteolytically released in this case by a mechanism that does not require ligand binding. Genetic, RNAi and microscopy studies have shown that each signalosome environment is unique and furthermore the Notch intracellular domain is differently modified by Tyrosine phosphorylation and ubiquitination. This has likely consequences on the downstream functions of Notch and may affect the repertoire of target genes induced. We are able to use the expression of specific regulators to separately focus Notch localisation into each of the activating compartments allowing the nature of the signalosome composition and the downstream cellular responses to be compared. This project will use cutting-edge quantitative proteomics, transcriptomics and bioinformatics methodologies, combined with manipulation of Notch intracellular trafficking and localisation to identify the molecular requirements of Notch activation by different routes and compare the consequent cellular responses. Studies will be extended to in vivo, physiological analyses of component functions by exploiting the Drosophila model organism.

Experienced will be gained in cell culture, mass spectrometry and multiple ’omics methods combined with bioinformatics, as well as manipulations using cell biology and in vivo using the Drosophila genetic model system

Funding Notes

This project is to be funded under the BBSRC Doctoral Training Programme. If you are interested in this project, please make direct contact with the Principal Supervisor to arrange to discuss the project further as soon as possible. You MUST also submit an online application form - full details on how to apply can be found on the BBSRC DTP website View Website

Applications are invited from UK/EU nationals only. Applicants must have obtained, or be about to obtain, at least an upper second class honours degree (or equivalent) in a relevant subject.

References

Shimizu H, Wilkin MB, Woodcock SA, Bonfini A, Hung Y, Mazaleyrat S, Baron M. (2017). The Drosophila ZO-1 protein Polychaetoid suppresses Deltex-regulated Notch activity to modulate germline stem cell niche formation. Open Biol. 7 pii: 160322.

Francavilla C, Papetti M, Rigbolt KT, Pedersen AK, Sigurdsson JO, Cazzamali G, Karemore G, Blagoev B, Olsen JV. Multilayered proteomics reveals molecular switches dictating ligand-dependent EGFR trafficking. Nat Struct Mol Biol. 2016 Jun;23(6):608-18.

Lawless C, Holman SW, Brownridge P, Lanthaler K, Harman VM, Watkins R, Hammond DE, Miller RL, Sims PF, Grant CM, Eyers CE, Beynon RJ, Hubbard SJ. (2016) Direct and Absolute Quantification of over 1800 Yeast Proteins via Selected Reaction Monitoring. Mol Cell Proteomics. 15:1309-22.

Bonfini A, Wilkin MB, Baron M. (2015) Reversible regulation of stem cell niche size associated with dietary control of Notch signalling. BMC Dev Biol. 31;15:8.

Shimizu H, Woodcock SA, Wilkin MB, Trubenová B, Monk NA, Baron M. (2014) Compensatory flux changes within an endocytic trafficking network maintain thermal robustness of Notch signaling. Cell 157:1160-74.

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