Autophagy, a process of lysosomal degradation essential for cellular homeostasis, is transcriptionally regulated by Transcription Factor EB (TFEB) [
1‐
3], which coordinates the expression of genes involved in lysosome biogenesis, autophagy and endocytosis [
1,
2,
4]. Under normal growth conditions TFEB is transiently recruited to the lysosomes through its interaction with active RAG GTPases at the lysosomal membrane [
5]. Active RAG GTPases also recruit the anabolic kinase complex mTOR, which phosphorylates TFEB at serine S211 to promote its dissociation from the lysosome and binding with 14-3-3 protein family members, which retain TFEB in the cytoplasm and inhibit its transcriptional activity [
5‐
7]. Upon amino acid starvation, RAG GTPases are inactivated [
8] resulting in the loss of lysosomal recruitment of TFEB and mTOR. Consequently, the cytoplasmic pool of TFEB becomes dephosphorylated, leading to the dissociation from 14-3-3 proteins and ultimately to nuclear accumulation of TFEB. Besides amino acid starvation, pharmacological inhibition of mTOR and lysosomal stresses result in TFEB dephosphorylation and nuclear accumulation [
7,
9]. In the nucleus, TFEB activates the transcription of the CLEAR network (Coordinated Lysosomal Expression and Regulation), which is composed of at least 471 direct targets, including a battery of lysosomal and autophagy genes [
1].
Abnormalities in autophagic processes can lead to neurodegenerative diseases and cancer [
10]. Moreover, recent studies have identified TFEB and other family members as key players for metabolic reprogramming in pancreatic cancer [
11,
12]. Thus, TFEB presents an attractive target for manipulating the cellular autophagic capacity in disease treatment. To date, studies on TFEB have primarily focused on the role of mTOR-mediated regulation of nuclear-cytoplasmic TFEB shuttling. Intriguingly, transcription of TFEB-controlled autophagosomal and lysosomal genes is increased in cells overexpressing TFEB [
1,
2,
6] and overall cellular TFEB protein levels are reduced following TFEB activation via long-term (15 hours) chloroquine-induced lysosomal stress [
7], suggesting TFEB concentration changes may contribute to the regulation of TFEB signaling. However, the relationship between mTOR activity states and temporal changes in TFEB subcellular localization and concentration has not been elucidated. To that end, we performed time course analysis over 15 hours of TFEB levels and localization by quantitative Western blotting and imaging cytometry. We activated mTOR by fresh addition of fully-supplemented medium (FM), or inhibited mTOR by Torin1 treatment [
13] or nutrient deprivation [
14]. We report that overall cellular TFEB levels transiently decrease in response to small increases in mTOR activation, and transiently increase in response to mTOR inhibition. Both Western blot and population-averaged imaging results displayed high variability, suggesting that heterogeneous TFEB responses within the cell population may cache important information on these complex dynamics. We therefore analyzed single-cell imaging cytometry data using spanning-tree progression analysis of density-normalized events (SPADE) agglomerative clustering [
15], as a basis for unbiased and quantitative detection of spatial and temporal dynamics of subpopulations. Using unsupervised clustering, we identified three TFEB phenotype subpopulations, with low, medium and high nuclear TFEB concentrations. We found that total cellular TFEB levels and subcellular localization are directly under control of a short-term rheostat controlled by mTOR. mTOR inhibition rapidly activates TFEB in a fraction of cells, for a limited duration, with distinct TFEB subpopulation re-inactivation dynamics in response to Torin1
vs. nutrient deprivation. Moreover, time course subpopulation analysis identified a correlation between TFEB protein levels and nuclear localization, and revealed differences between HeLa and MCF7 cells in the sensitivity of TFEB to mTOR regulation. Finally, subpopulation analysis revealed that in response to mTOR inhibition, maximal nuclear localization of TFEB is negatively regulated by the proteasome, independently of TFEB concentration.