Establishment of single-cell readouts for the study of TORC1 signaling dynamics in budding yeast

The Target of Rapamycin Complex 1 (TORC1) is a key regulator of cell growth processes, including ribosome biogenesis, protein synthesis, and metabolism. Most current methods for measuring TORC1 activity in living organisms rely on immunoblotting to track phosphorylation of TORC1 substrates, which are limited in capturing rapid changes and single-cell dynamics in living organisms. Single-cell time-lapse microscopy offers a promising alternative but lacks suitable TORC1-specific live-cell imaging readouts in budding yeast. In this thesis, we aimed to develop and characterize new readouts for studying yeast TORC1 signaling dynamics using single-cell microscopy. We focused on the endogenous Sfp1 protein and mammalian TORC1 substrates as potential readouts. Our findings revealed that Sfp1 is regulated by both TORC1 and PKA pathways, which can independently control its subcellular localization via the control of a Nuclear Export Signal (NES) and zinc finger domains. Furthermore, detailed single-cell analysis throughout the cell cycle showed PKA as the main regulator of Sfp1 nuclear localization during the G1 phase, while neither TORC1 nor PKA influenced the G2/M phase peak, suggesting a cell cycle-dependent regulation. Additionally, we explored mammalian TORC1 substrates as potential readouts in yeast. Notably, 4E-BP1 phosphorylation was highly sensitive to TORC1 activity changes, indicating its potential as a translocation-based reporter for single-cell microscopy. However, other substrates like TORCAR and TFEB were inconsistent. This work provides insights into Sfp1 regulatory mechanisms and highlights 4E-BP1 as a promising readout for studying yeast TORC1 signaling dynamics, contributing to the broader understanding of cell growth and cycle progression.