Exploiting Notch trafficking to probe mechanisms of endosomal sorting and compartmentation.

In eukaryotic cells the endo-lysosomal pathway is a vesicle mediated transport system that plays an important role in modulating the activity of membrane signalling receptors. It down regulates signalling by removing receptors from access to ligands at the cell surface, sequesters them from the cytoplasm by directing them into intraluminal vesicles of the endosomal lumen and then delivers them to the lysosome for degradation. However the endocytic pathway can also have a positive affect on signalling by bringing necessary components together on the endosomal perimeter membrane into an appropriate signal-competent environment or "signalosome". Hence endocytic flux and residency time of signalling components in different endosomal locations can shape the dynamics of signalling activity in different ways. Endosomes are comprised of different subdomains marked by a distinctive membrane and protein composition. However we still have only a limited understanding of how the sub-compartmentalisation of the endosomes, and the trafficking flux between their different domains, is linked to their different biological roles in signalling regulation, or how misregulation of endosomal architecture is linked to abberent signalling activity. The overarching aim of this proposal will be to take advantage of our new understanding of the endo-lysosomal regulation of Notch, a crucially important signalling receptor, as a model to address these important gaps in our knowledge.
Our recent work has revealed that Notch can be delivered to endosomes by two distinct routes to different as yet poorly understood, endosomal domains which are marked by enrichment or not for GPI-anchored proteins, which appear to be subcompartments of the larger organelle stuctures. Notch can be sorted in the endosomal domain between these compartments and also between the endosomal perimeter membrane and the intraluminal vesicles, making Notch regulation an ideal model with which to probe the functional compartments of the endolysosomal pathway. The challenge now is to better understand the compartmentalisation of the endolysosomal pathway into its functional regulatory units, understand how these units operate in the context of endosomal maturation and define how trafficking between different endosomal environments is linked to regulation of Notch signalling through the interplay of Notch regulatory and core endosomal sorting functions.
Drosophila is uniquely suited as a starting point for this research, being fast and inexpensive, and enabling us to capitalise on very efficient genetic strategies. Drosophila research has played a central role in establishing the foundations of modern biology, contributing to numerous important advances in cell biology, protein trafficking, cell signalling and development, and uncovering many of the conserved pathways that are fundamental to human health and disease. Importantly Drosophila provides a unique experimental platform to dissect the interaction between endosomal regulation and Notch signalling in vivo. This is important because there have been little detailed functional characterisation of endolysosomal compartmentation outside of cell culture studies. Numerous mutations are available that disrupt different components allowing the consequences on Notch to be discerned in the intact organism. The recent improvements in efficient gene editing techniques now mean that new mutations can be engineered into the Drosophila germline by design, to create site directed mutations and insert tags to facilitate bioimaging studies in vivo. This strategy is fast, cost-effective and likely to be highly translatable given that most aspects of fundamental biology are shared between humans and the fly.

Notch activation requires proteolytic release of its intracellular domain, which occurs on the endosomal limiting membrane. A key regulatory decision is whether Notch is transferred into the intra luminal vesicles (ILVs) and hence inactivated. How this decision is regulated is important because misregulation of Notch at this step can produce strong ectopic activation of Notch. Transfer of Notch off the limiting endosomal membrane requires the ESCRT 0,I,II and III complexes which recruit cargo and transfer it into the ILVs, which they also help to form. We have found that disruption of different ESCRT complexes results in Notch misctivation by distinct mechanisms that we have previously linked to pools of Notch in as yet uncharacterised GPI-protein positive or negative endosomal domains. Our data thus suggests unexpected links between ESCRT function and the subcompartmentalisation of Notch within the endosome. We propose that as Notch is trafficked through the endosomal sorting machinery then it becomes competent or refractory to activation by different mechanisms of activation. The latter defined by requirements for different degrees of endosomal maturation and dependence or not on the metalloprotease Kuzbanian/Adam10. Our challenge now is to understand endosomal subcompartmentation in a way that allows us to define the functional units of endolysososomal structure and understand how Notch is partitioned between these units. Our insights into the regulation of Notch provide us with an ideal model system with which to probe the functional compartmentation of the endolysosomal pathway. We will therefore use a combination of Drosophila genetics, biochemical and cell biological approaches along with EM and fluorescence imaging to uncover the hierarchy of interactions between the Notch, its endocytic trafficking regulators and the activities of the core endosomal sorting machinery in defining Notch endosomal compartmentalisation into different regulatory environments.

The endosomal trafficking system plays a central role in cellular physiology. Numerous medical and age related conditions have been linked to altered trafficking of proteins in the cell. Furthermore Notch signalling plays a crucial role in development and maintenance of the adult and its mis-regulation contributes substantially to various pathologies including aging related diseases such as neurodegenerative and most notably cancer. Notch is also a key player in regulation of the function and maintenance of stem cells in adult tissues and declines in Notch signalling have been linked to declining ability of repair of tissues in the elderly. Therefore our study will have a significant impact on society with relevance to possible developments of new treatments and in particular targeting those treatments to specific forms of its misactivation.

1:- Medical research
The endosomal trafficking system also plays a central role in cellular physiology exemplified by Notch regulation. Numerous medical and age related conditions have been linked to altered trafficking of proteins in the cell. To pursue routes of impact in the medical field I have established a collaboration with Dr. Keith Brennan of the Manchester breast centre who is already translating our findings from Drosophila into human cancer-research. A similar impact pathway yielded a patented IP relating to prognostic uses of expression of human Su(dx)-related protein in breast cancer patients. I have now established similar links to clinicians at the Christie Hospital and Patterson Institute who are interested in Notch activation in lung cancers. In addition, in collaboration with the Genetics department of St. Mary's hospital in Manchester, w)e have identified in flies, novel links between certain genes linked to neurodegenerative disorders and defective Notch endocytosis and activation. Our work on regulation of Notch may have an impact on understanding the mechanisms of these debilitating diseases. These collaborations will be managed through joint research meetings and an annual review which will assess how work arising from our programme can be best progressed towards clinically utility.

2: Public education
Key goals of this programme are:- to educate the public regarding how regulation of normal cell behaviour is vital to the body systems that normally function to keep us healthy. We further wish to educate the public as to how understanding these normal processes underpins our understanding and hope of medical intervention in pathological situations whereby normal means of control are corrupted. A further goal is to promote the importance of model organisms such as Drosophila as an alternative to animal models, as part of the NC3Rs replacement, refinement and reduction policy. I will regularly review teaching opportunities to communicate my scientific goals and outcomes to our undergraduate students who will go on to fulfil many diverse roles in society. I will utilise numerous opportunities to communicate with the public and schools using informative displays during annual outreach activities comprising of "meet a scientist" school visit days, Faculty of Life Science Open days, the Body Experience exhibition at the Manchester Museum, and the Manchester Science festival. I will further promote use in schools of simple practical experiments we have devised for teachers to demonstrate genetics and the utility of the fruit fly as a model organism. My recent election as parent governor to a local primary school will provide an excellent means to enhance contact with the teaching profession, understand how science teaching is performed in schools and to provide input and feedback on the design of the new science curriculum which emphasises ways of working scientifically.