Characterisation of the PI3P-dependent signalling network responsible for nutrient sensing and autophagy

One of the most important requirements for healthy cellular growth is the ability to sense and respond to extracellular nutrients. When nutrient supply is plentiful, cells need only to fine tune their anabolic and catabolic rates so that their energy requirements match the available resources. When nutrients become limiting or are completely unavailable, a much stronger course of action is required to allow the cells to survive. During such times of nutrient withdrawal, the cells respond by initiating autophagy, a degradative pathway that allows the breakdown of intracellular proteins into amino acids that can be used subsequently for new protein synthesis or energy generation. An important question in autophagy concerns the mechanisms by which cells sense their extracellular nutrient content. Recent work from many groups has indicated that a small lipid molecule termed phosphatidylinositol 3 phosphate (PI3P) is an important signal for nutrient sensing. Our own work also indicates that autophagy is induced partially as a result of the formation of PI3P in specialised membrane compartments called omegasomes. Therefore, the mechanisms and signals that generate PI3P early during autophagy are likely to provide important information on the control of autophagy by nutrient sensing. The aim of this grant is to identify all human genes that are involved in this pathway. We plan to systematically silence all known human genes and then look at the effect that this will have in formation of PI3P during autophagy. By identifying all such genes we will be able to construct a wiring diagram of the cellular pathways that are implicated in nutrient sensing and respond during nutrient limitation by the induction of autophagy.

During nutrient withdrawal, cells respond by initiating autophagy, a degradative pathway that allows the breakdown of intracellular proteins into amino acids that can be used subsequently for new protein synthesis or ATP generation. In addition to this role in nutrient sensing, autophagy is important for elimination of unneeded organelles, for the defence against pathogens, for the pathogenesis of various diseases and for healthy ageing. Our recent work has suggested that at least some autophagosomes (double membrane vesicles mediating autophagy) are formed in novel membrane specializations termed omegasomes which are connected to the endoplasmic reticulum and are enriched in a phosphatidylinositol 3-phosphate (PI3P). This is consistent with earlier work showing that formation of PI3P is essential for the induction of autophagy. However, the signalling pathways leading from nutrient sensing to PI3P formation and autophagy induction are still unknown. The aim of this grant is to identify the main gene products whose activity is important for the PI3P-dependent induction of autophagy. We will use an siRNA screen of all known human genes, to identify those whose down-regulation (a) leads to omegasome formation in normal nutrient conditions or (b) inhibits amino acid starvation-induced formation of omegasomes. Based on our preliminary data we expect the following classes of molecules to be identified: (1) Those involved in amino acid sensing [AA sensor(s)] (2) Those involved in signalling to calcium following amino acid sensing (3) Those involved in signalling from calcium to Vps34 (4) Those involved in AA-dependent mTOR activation (5) Those involved in omegasome formation, expansion and collapse (5) Those involved in termination of PI3P signal (6) Those involved in feedback inhibition of autophagosome biogenesis. The ultimate aim of this grant is to construct a signalling diagram leading from nutrient sensing to PI3P formation and autophagy induction.