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

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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.