Mapping Notch endocytic signalling pathways via quantitative proteomics in D. melanogaster

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Extracellular signalling allows cells to communicate with one another in the context of tissues and influence the fate of their neighbouring cells, transducing signals from the external environment to the nucleus to affect the expression of target genes. Such processes are critically active during development and cell differentiation. One of the key players in this process is the ubiquitous signalling receptor Notch, which underpins many physiological processes in the cell. Proteolytic cleavage of the protein at the cell membrane leads to release of its intracellular domain which can find its way to the nucleus via several signalling routes, often involving endosomes. These latter routes can occur in a Clathrin-dependent and independent manner, and lead to activation/suppression of different sets of genes. Exactly how the various Notch interacting partners influence it during these endosomal signalling pathways is, however, not known. Using Drosophila S2 cells as a model system, we have carried out two RNAi screens to determine a high-quality set of Notch interactors that influence Notch expression for the two endosomal signalling routes, and wish to integrate this data with wider bioinformatics data to better understand how the signalling networks are constituted. We will then carry out a comprehensive global quantitative proteomics experiment to determine how the two different signalling pathways affect the proteome, defining the downstream Notch targets that are differentially expressed. This will reinforce hypotheses generated by the bioinformatics network analyses, which we will subsequently validate using a combination of targeted proteomics experiments using bespoke protein standards, and attendant cell biology and biochemistry. We also aim to examine the knockdown effects on the most informative and potent Notch interactors in vivo. Our overall aim is therefore the characterise the different regulatory mechanisms promoted by the two Notch signalling routes, defining attendant changes in the target genes at the protein level, and placing this into a phenotypic context in the Drosophila. The project integrates quantitative proteomics methods with bioinformatics to delineate key cell signalling components at the protein level, and embodies a systems biology approach to deliver new understanding of the rules of life. Our collaboration is novel, bringing together molecular and cellular biology with cutting-edge analytical science, and consequently makes good use of the transformative technologies BBSRC espouses. By focusing on a ubiquitous signalling pathway in metazoan life, we seek to uncover fundamental principles that govern development and cell differentiation that are relevant to understanding health across the life course.