The Notch signalling pathway plays an essential role in cell-fate decisions and morphogenesis, and is frequently ectopically activated in human cancers. The signal is initiated through DSL ligand-dependent Notch proteolysis, which releases its intracellular domain. However, over-expression of the E3 ubiquitin ligase Deltex can bypass the requirement for Notch ligands and ectopically activate Notch by directing it into the endocytic pathway. It has been shown that Deltex induced endocytic-Notch signalling, but not DSL-induced signalling, requires both HOPS and AP-3 complex components that mediate trafficking to lysosomes and related organelles. I showed through a combination of the analysis of the dxsm mutant allele and by expression of mutant forms of Deltex in cell culture, that the C-terminal region of Deltex is important for Notch signalling, but is not required for Notch endocytosis. This suggests that the C-terminal region contains an interaction site that may direct endocytosed Notch to the correct endocytic compartment. A key question however was whether this endocytic pathway is utilised for full Notch signaling in normal development alongside the canonical activation mechanism. I therefore investigated the expression of Drosophila midline single-minded (sim), which is a Notch signal reporter gene, and embryonic neurogenesis which is repressed by Notch signalling. I found that deltex, HOPS and AP-3 mutants displayed gaps in sim expression and also a neurogenic phenotype similar to Notch loss-of-function, and consistent with a role for ligand-independent Notch activation in normal development. I found that the penetrance of these phenotypes increased when flies were cultured at higher temperature. These results suggest that Dx/HOPS/AP-3-dependent Notch activation provides a developmental robustness to the Notch signalling network. It was also found that AP-3 and HOPS components have an additional role in regulating cell survival, which is partially separable in time using a temperature shift assay. I also revealed a requirement of maternal Notch for cell survival in early embryonic development, which may be related to the HOPS-dependent function. Comparison of deltex, AP-3 and HOPS mutant phenotypes suggested that there may be functional redundancy of deltex with components that regulate Notch endocytosis, and of AP-3 with proteins that mediate subsequent trafficking to the late endosome/lysosome. Finally I characterised the molecular lesions of an allele of the HOPS component light and the AP-3 component ruby and identified lesions which were consistent with the loss-of-function of these genes.
|Date of Award
|1 Aug 2011
- The University of Manchester
|Martin Baron (Supervisor)
- Notch, Deltex, HOPS, AP-3, endocytosis, neurogenesis