Continuous sensing of nutrients and growth factors by the mTORC1-TFEB axis

Abstract mTORC1 senses nutrient and growth factor status and phosphorylates downstream targets, including the transcription factor TFEB, to coordinate metabolic supply and demand. The molecular mechanisms of mTORC1 activation are thought to enforce a strict requirement for simultaneous amino acid and growth factor stimuli, but this model has not been evaluated with quantitative or single-cell methods. Here, we develop a series of fluorescent protein-TFEB fusions and investigate how combinations of stimuli jointly regulate signaling from mTORC1 to TFEB at the single-cell level. Live-cell imaging of individual cells revealed that mTORC1-TFEB signaling responds with graded changes to individual amino acid and growth factor inputs, rather than behaving as a logical “AND” gate. We find that mTORC1 inputs can be sequentially sensed, with responses that vary between mTORC1 substrates and are amplified by input from other kinases, including GSK3β. In physiologically relevant concentrations of amino acids, we observe fluctuations in mTORC1-TFEB signaling that indicate continuous responsiveness to nutrient availability. Our results clarify how the molecular regulation of mTORC1 enables homeostatic processes at the cellular level and provide a more precise understanding of its behavior as an integrator of multiple inputs.