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theses

Over the last few years, extensive studies have linked the activity of the transcription factor EB (TFEB) to lysosomal function. These observations proposed an intriguing integration of cellular catabolism, sustained by lysosomes, with anabolic processes largely controlled by the mechanistic target of rapamycin complex 1 (mTORC1) signaling. Interestingly, lysosomal function directly affects mTORC1 activity and is regulated by TFEB, a transcription factor and direct substrate of mTORC1. Thus, the lysosomal-mTOR signaling complex represents a hub of cellular energy metabolism, and its dysregulation underlies the onset of several multifaceted disorders including neurodegeneration, cancer, and Type 2 diabetes. Recent evidence has suggested that under nutrient-deficient conditions TFEB controls mTORC1 reactivation and induction of anabolic processes upon nutrient replenishment by modulating the transcriptional levels of small GTPases to re-tether mTORC1 to lysosomal membranes. Although very significant, these studies did not address the subsequent intracellular mechanisms that lead to the stimulation of mTORC1 kinase activity and autophagy during starvation. Here, I demonstrated that TFEB promotes assembly of a mTORC1-containing nutrient sensing complex through the formation of endosomes carrying activated phosphoinositide-dependent kinase 1 (p-PDK1), its activated substrate, protein kinase B (p-AKT), and the mTORC1-tethering factor RagD. Therefore, TFEB-induced signaling endosomes en route to lysosomes are required to dissociate lysosomal-mTORC1 inhibitor, Tuberous Sclerosis complex (TSC), and re-tether mTORC1 to endolysosomal membranes. Chromatin immunoprecipitation assays identified 623 endocytic genes regulated by TFEB, of which 46 were core endocytic genes including Clathrin, Caveolin, early and the late endosomal markers, Rab5 and Rab7, respectively. Validation of expression of these endocytic proteins in blots and endocytosis assays using Dextran-Rhodamine and EGF-Rhodamine showed that TFEB stimulates endocytosis. Pharmacological and genetic inhibition of endocytosis or retrograde trafficking abolished mTORC1 activity without affecting total levels of pT308-Akt and despite the presence of active TFEB in the nucleus. These TFEB-induced endosomes are required to assemble the so-called lysosomal nutrient sensing complex (LYNUS), composed of vATPase, Ragulator and the small GTPases RagA/B and C/D. LYNUS tethers mTORC1 to lysosomal membranes. I show that during starvation, TFEB-triggered endocytosis reactivates mTORC1 and is required for autophagy. I found that TFEB-triggered endocytosis is required for delivery of mTORC1 activators, lysosome biogenesis, and autophagy flux. This study identifies TFEB-mediated endocytosis as a critical process driving lysosome biogenesis and mTORC1 activation and emphasizes the role of the endolysosomal system in molecular clearance.

ETD_8607

Ph.D.

Includes bibliographical references

Includes vita

by Israel Chukwudi Nnah

doi:10.7282/T3JS9TMD

ETD doctoral

The author owns the copyright to this work.