Project Summary / Abstract Cells need to sense the nutrient availability to adjust their survival strategy. In eukaryotic cells, the mechanistic Target of Rapamycin Complex 1 (mTORC1) is a key regulator of cell growth and proliferation. Upon activation in a nutrient-rich environment, mTORC1 triggers anabolic reactions and inhibits catabolism. Amino acid signals are transmitted to mTORC1 through a series of protein complexes that ultimately converge on the Rag GTPases, a heterodimeric GTPase that recruits mTORC1 to the lysosomal surface. However, the molecular mechanisms of how these protein complexes dynamically transmit the amino acid signal are still elusive. In this proposal, we aim to develop biophysical tools to study the dynamics of the amino acid sensing process. Specifically, we aim to use single molecule FRET to study the conformational dynamics of the Rag GTPase heterodimer and its regulators (Project 1). Further, we plan to reconstitute an in vitro system to recapitulate Rag-dependent amino acid sensing on a membrane surface (Project 2), and use cryo-electron tomography (cryo-ET) to visualize the signaling complexes in their native environment (Project 3). The outcomes will yield novel insights into this important biological process and provide guidance to the development of therapeutic strategies.