Grant: Discovery Grants Program - Individual

Autophagy is an evolutionary conserved cellular process in which cells degrade their own components to generate nutrients. It is induced by nutrient starvation that is sensed by mTORC1, a master regulator of cellular metabolism and homeostasis. Autophagy starts with the assembly and recruitment of the core autophagy machinery to Endoplasmic Reticulum (ER) subdomains, known as omegasomes, from which phagophores are formed. Phagophores engulf cargos and then mature into autophagosomes that can fuse with lysosomes in order to degrade and recycle the cargos. Ion channels are membrane proteins that selectively conduct ions across cell membranes. They are found not only in the plasma membrane but also in organelles that are associated with autophagy, such as the ER, phagophores, and lysosomes. The long-term objective of my research program is to understand how these ion channels regulate lysosome physiology and autophagy. TRPMLs are a group of Ca2+ release channels primarily localized at endosomes and lysosomes. Their activities are tightly controlled by luminal pH of the compartments and compartment-specific phosphoinositides (PIPs) that act as key determinants of the identity of endolysosomes and spatiotemporal cues to regulate autophagy. In addition to endosomes, TRPML3 is also highly expressed in lysosomes where it is inactivated by lysosomal low pH. On the other hand, TRPML3 is activated by PI3P, a PIP enriched at the ER omegasomes that have higher luminal pH. Interestingly, our recent results suggest that mTORC1 inhibition upon starvation stimulates TRPML3 by increasing PI3P, and this further inhibits mTORC1 to facilitate autophagy induction. However, it remains unclear how TRPML3 is involved in mTORC1 suppression and autophagy induction. The short-term objective of my research program is to study the activation mechanism of TRPML3 upon starvation and the molecular mechanisms underlying TRPML3's regulation of mTORC1 and autophagy induction. Because 1) lysosomes and ER omegasomes form physical contacts, 2) starvation activates the Class III PI3K VPS34 to produce PI3P at the ER omegasomes, 3) Ca2+ acts as a cofactor of the Class II PI3K PI3KC2ß that resides in lysosomes, and 4) starvation stimulates lysosomal PI3KC2ß to produce PI3,4P2 at lysosomes where PI3,4P2 suppresses mTORC1, we hypothesize that upon starvation lysosomal TRPML3 is recruited to less acidic ER omegasomes where TRPML3 is activated by PI3P to release ER Ca2+; TRPML3-mediated ER Ca2+ release activates lysosomal PI3KC2ß to generate PI3,4P2 which further inhibits mTORC1. Eventually, mTORC1 is greatly reduced to promote autophagy induction. These studies will significantly facilitate our understanding of the molecular mechanisms underlying autophagy induction, ER Ca2+ signaling, and lysosome-ER crosstalk. More importantly, individuals who acquire expertise with a diversity of techniques and skills in my lab will advance the research of organellar biology in Canada.