A systematic approach for cataloging mTORC1 regulators

Abstract

mTORC1 is a major regulator of eukaryotic cell growth. As a cellular decision-maker, mTORC1 surveils internal levels of basic biomolecules such as amino acids, ATP, cholesterol, and external levels of growth factors. Under sufficient nutrient conditions, mTORC1 is active and drives anabolic processes while inhibiting catabolic ones. Dysregulation of mTORC1 activation has far-reaching consequences for human health, and understanding how upstream regulation occurs is of great interest.

Toward generating a complete list of mTORC1 regulators, we developed a strategy to identify them by CRISPR-Cas9 FACS-based screening. As a proof-of-principle screen, we first identified negative regulators of the mTORC1 pathway. Then, we adapted the technique to uncover positive regulators of mTORC1. These screening results revealed many novel genes as mTORC1 regulators as well as highlighted the importance of mitochondrial health for mTORC1 activation.

Following validation studies, we investigated the long-standing question of how mitochondrial stress impinges upon the mTORC1 signaling pathway. We observed short- and long-term responses to mitochondrial distress through time course experiments with the mitochondrial inhibitor, oligomycin. Furthermore, we attributed these responses to the kinases, AMPK and HRI, respectively.