Continuous sensing of nutrients and growth factors by the mTORC1-TFEB axis
Abstract
mTORC1 senses nutrients and growth factors and phosphorylates downstream targets, including the transcription factor TFEB, to coordinate metabolic supply and demand. These functions position mTORC1 as a central controller of cellular homeostasis, but the behavior of this system in individual cells has not been well characterized. Here, we provide measurements necessary to refine quantitative models for mTORC1 as a metabolic controller. We developed a series of fluorescent protein-TFEB fusions and a multiplexed immunofluorescence approach to investigate how combinations of stimuli jointly regulate mTORC1 signaling at the single-cell level. Live imaging of individual MCF10A cells confirmed that mTORC1-TFEB signaling responds continuously to individual, sequential, or simultaneous treatment with amino acids and the growth factor insulin. Under physiologically relevant concentrations of amino acids, we observe correlated fluctuations in TFEB, AMPK, and AKT signaling that indicate continuous activity adjustments to nutrient availability. Using partial least squares regression modeling, we show that these continuous gradations are connected to protein synthesis rate via a distributed network of mTORC1 effectors, providing quantitative support for the qualitative model of mTORC1 as a homeostatic controller and clarifying its functional behavior within individual cells.
... Interestingly, following this early response, nuclear fluorescence gradually drops with a distinct 'overshoot', indicating a partial cytoplasmic relocalisation of TFEB. A similar observation was recently reported 27,28 , and it has been suggested to be caused by a rapid rheostatic response mediated by mTOR reactivation, which allows cells to quickly adapt to metabolic changes 27 . In the pulse experiment, upon refeeding, TFEB nuclear level quickly drops but then slightly increases generating a small but distinct 'undershoot', as shown in Fig. 1J. ...
Transcription Factor EB (TFEB) controls lysosomal biogenesis and autophagy in response to nutritional status and other stress factors. Although its regulation by nuclear translocation is known to involve a complex network of well-studied regulatory processes, the precise contribution of each of these mechanisms is unclear. Using microfluidics technology and real-time imaging coupled with mathematical modelling, we explored the dynamic regulation of TFEB under different conditions. We found that TFEB nuclear translocation upon nutrient deprivation happens in two phases: a fast one characterised by a transient boost in TFEB dephosphorylation dependent on transient calcium release mediated by mucolipin 1 (MCOLN1) followed by activation of the Calcineurin phosphatase, and a slower one driven by inhibition of mTORC1-dependent phosphorylation of TFEB. Upon refeeding, TFEB cytoplasmic relocalisation kinetics are determined by Exportin 1 (XPO1). Collectively, our results show how different mechanisms interact to regulate TFEB activation and the power of microfluidics and quantitative modelling to elucidate complex biological mechanisms.
... Cell-to-cell variation in cellular metabolism was also observed in vivo using intravital imaging of an AMPK biosensor [2]. Multiplexed analysis of AMPK, mTOR, and AKT biosensors within the same cells demonstrated concerted fluctuations in all of these pathways, revealing the dynamics by which these regulators help to maintain cellular homeostasis [50]. Further extending these analyses, a new AMPK biosensor design has enabled high dynamic range measurements of AMPK activity in subcellular locations [7]. ...
It is increasingly clear that cellular metabolic function varies not just between cells of different tissues, but also within tissues and cell types. In this essay, we envision how differences in central carbon metabolism arise from multiple sources, including the cell cycle, circadian rhythms, intrinsic metabolic cycles, and others. We also discuss and compare methods that enable such variation to be detected, including single-cell metabolomics and RNA-sequencing. We pay particular attention to biosensors for AMPK and central carbon metabolites, which when used in combination with metabolic perturbations, provide clear evidence of cellular variance in metabolic function.
... ; https://doi.org/10.1101/2024.05.06.592787 doi: bioRxiv preprint promotes lysosomal biogenesis (Lapierre et al. 2013;Settembre et al. 2011). In the presence amino acids, mTORC1 is activated and phosphorylates TFEB, preventing its nuclear localization (Martina et al. 2012;Roczniak-Ferguson et al. 2012;Sparta et al. 2023). It is possible that signaling through mTORC1 is diminished in flr-4 and flr-1 mutants due to impaired absorption of dipeptides (Hara et al. 1998). ...
Ion channels are necessary for proper water and nutrient absorption in the intestine, which supports cellular metabolism and organismal growth. While a role for Na+ co-transporters and pumps in intestinal nutrient absorption is well defined, how individual K+ uniporters function to maintain ion homeostasis is poorly understood. Using Caenorhabditis elegans , we show that a gain-of-function mutation in twk-26 , which encodes a two-pore domain K+ ion channel orthologous to human KCNK3, facilitates nutrient absorption and suppresses the metabolic and developmental defects displayed by impaired intestinal MAP Kinase (MAPK) signaling. Mutations in drl-1 and flr-4 , which encode two components of this MAPK pathway, cause severe growth defects, reduced lipid storage, and a dramatic increase in autophagic lysosomes, which mirror dietary restriction phenotypes. Additionally, these MAPK mutants display structural defects of the intestine and an impaired defecation motor program. We find that activation of TWK-26 reverses the dietary restriction-like state of the MAPK mutants by restoring intestinal nutrient absorption without correcting the intestinal bloating or defecation defects. This study provides unique insight into the mechanisms by which intestinal K+ ion channels support intestinal metabolic homeostasis.
Cellular function is controlled through intricate networks of signals, which lead to the myriad pathways governing cell fate. Fluorescent biosensors have enabled the study of these signaling pathways in living systems across temporal and spatial scales. Over the years there has been an explosion in the number of fluorescent biosensors, as they have become available for numerous targets, utilized across spectral space, and suited for various imaging techniques. To guide users through this extensive biosensor landscape, we discuss critical aspects of fluorescent proteins for consideration in biosensor development, smart tagging strategies, and the historical and recent biosensors of various types, grouped by target, and with a focus on the design and recent applications of these sensors in living systems.
Pleural mesothelioma (PM) is an aggressive malignancy with poor prognosis. Although histology and pathologic stage are important prognostic factors, better prognostic biomarkers are needed. The ribosomal protein S6 is a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway involved in protein synthesis and cell proliferation. In previous studies, low phosphorylated S6 (pS6) immunoreactivity was significantly correlated with longer progression-free survival (PFS) and overall survival (OS) in PM patients. We aimed to correlate pS6 expression to clinical data in a large multi-centre PM cohort as part of the European Thoracic Oncology Platform (ETOP) Mesoscape project. Tissue Micro Arrays (TMAs) of PM were constructed and expression of pS6 was evaluated by a semi-quantitatively aggregate H-score. Expression results were correlated to patient characteristics as well as OS/PFS. pS6 IHC results of 364 patients from 9 centres, diagnosed between 1999 and 2017 were available. The primary histology of included tumours was epithelioid (70.3%), followed by biphasic (24.2%) and sarcomatoid (5.5%). TMAs included both treatment-naïve and tumour tissue taken after induction chemotherapy. High pS6 expression (181 patients with H-score>1.41) was significantly associated with less complete resection. In the overall cohort, OS/PFS were not significantly different between pS6-low and pS6-high patients. In a subgroup analysis non-epithelioid (biphasic and sarcomatoid) patients with high pS6 expression showed a significantly shorter OS (p < 0.001, 10.7 versus 16.9 months) and PFS (p < 0.001, 6.2 versus 10.8 months). In subgroup analysis, in non-epithelioid PM patients high pS6 expression was associated with significantly shorter OS and PFS. These exploratory findings suggest a clinically relevant PI3K pathway activation in non-epithelioid PM which might lay the foundation for future targeted treatment strategies.
Activating mutations in RAS are present in ~ 30% of human tumors, and the resulting aberrations in ERK/MAPK signaling play a central role in oncogenesis. However, the form of these signaling changes is uncertain, with activating RAS mutants linked to both increased and decreased ERK activation in vivo. Rationally targeting the kinase activity of this pathway requires clarification of the quantitative effects of RAS mutations. Here, we use live-cell imaging in cells expressing only one RAS isoform to quantify ERK activity with a new level of accuracy. We find that despite large differences in their biochemical activity, mutant KRAS isoforms within cells have similar ranges of ERK output. We identify roles for pathway-level effects, including variation in feedback strength and feedforward modulation of phosphatase activity, that act to rescale pathway sensitivity, ultimately resisting changes in the dynamic range of ERK activity while preserving responsiveness to growth factor stimuli. Our results reconcile seemingly inconsistent reports within the literature and imply that the signaling changes induced by RAS mutations early in oncogenesis are subtle.
The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates1–3. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy4,5, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases. Owing to this mechanism, the phosphorylation of TFEB—unlike other substrates of mTORC1, such as S6K and 4E-BP1— is strictly dependent on the amino-acid-mediated activation of RagC and RagD GTPases, but is insensitive to RHEB activity induced by growth factors. This mechanism has a crucial role in Birt–Hogg–Dubé syndrome, a disorder that is caused by mutations in the RagC and RagD activator folliculin (FLCN) and is characterized by benign skin tumours, lung and kidney cysts and renal cell carcinoma6,7. We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and mTORC1 hyperactivity in a mouse model of Birt–Hogg–Dubé syndrome. Accordingly, depletion of TFEB in kidneys of these mice fully rescued the disease phenotype and associated lethality, and normalized mTORC1 activity. Our findings identify a mechanism that enables differential phosphorylation of mTORC1 substrates, the dysregulation of which leads to kidney cysts and cancer. Dysregulation of an mTORC1 substrate-specific mechanism leads to constitutive activation of TFEB, and promotes kidney cystogenesis and tumorigenesis in a mouse model of Birt–Hogg–Dubé syndrome.
All transcription factors are equal, but some are more equal than others. In the 25 yr since the gene encoding the microphthalmia-associated transcription factor (MITF) was first isolated, MITF has emerged as a key coordinator of many aspects of melanocyte and melanoma biology. Like all transcription factors, MITF binds to specific DNA sequences and up-regulates or down-regulates its target genes. What marks MITF as being remarkable among its peers is the sheer range of biological processes that it appears to coordinate. These include cell survival, differentiation, proliferation, invasion, senescence, metabolism, and DNA damage repair. In this article we present our current understanding of MITF's role and regulation in development and disease, as well as those of the MITF-related factors TFEB and TFE3, and highlight key areas where our knowledge of MITF regulation and function is limited.
During starvation the transcriptional activation of catabolic processes is induced by the nuclear translocation and consequent activation of transcription factor EB (TFEB), a master modulator of autophagy and lysosomal biogenesis. However, how TFEB is inactivated upon nutrient refeeding is currently unknown. Here we show that TFEB subcellular localization is dynamically controlled by its continuous shuttling between the cytosol and the nucleus, with the nuclear export representing a limiting step. TFEB nuclear export is mediated by CRM1 and is modulated by nutrient availability via mTOR-dependent hierarchical multisite phosphorylation of serines S142 and S138, which are localized in proximity of a nuclear export signal (NES). Our data on TFEB nucleo-cytoplasmic shuttling suggest an unpredicted role of mTOR in nuclear export.
Mutations in RAS or BRAF are associated with poor prognosis and resistance to epidermal growth factor receptor (EGFR)-targeted therapy in colorectal cancer (CRC). Despite their common ability to activate downstream genes such as MEK and ERK, the therapeutic benefit of MEK inhibitors for patients with RAS/BRAF mutant CRC is limited, highlighting the need for biomarkers to predict the efficacy of MEK inhibition. Previously, we reported that a change in phosphorylation of ribosomal protein S6 (pS6) after MEK inhibition was significantly associated with sensitivity to MEK inhibition in gastric cancer cells. Here, we investigated the value of the response in pS6 for predicting the efficacy of trametinib, a MEK inhibitor, in patients with RAS/BRAF mutant CRC using patient-derived CRC organoids. We found that a subset of CRC cell lines and organoids were sensitive to trametinib. The change in phosphorylated ERK, a downstream molecule of the RAS/RAF/MEK pathway, was not significantly associated with trametinib sensitivity. On the other hand, only those with sensitivity showed a reduction of pS6 levels in response to trametinib. The change in pS6 after trametinib treatment was detectable by Western blotting, immunohistochemistry or immunocytochemistry. We also demonstrated an impact of MEK inhibition on pS6 in vivo using a xenograft model. Our data suggest that, in combination with patient-derived organoids, immunostaining-based detection of pS6 could be useful for prediction of trametinib sensitivity. Reduced pS6 correlates with sensitivity to MEK inhibition in colorectal cancer organoids as well as cell lines. This study shows the potential applicability of MEK inhibitors for a subset of colorectal cancer patients with RAS/BRAF mutation by using the change in pS6 levels as a predictive diagnostic marker.
Cell-to-cell heterogeneity in metabolism plays an unknown role in physiology and pharmacology. To functionally characterize cellular variability in metabolism, we treated cells with inhibitors of oxidative phosphorylation (OXPHOS) and monitored their responses with live-cell reporters for ATP, ADP/ATP, or activity of the energy-sensing kinase AMPK. Across multiple OXPHOS inhibitors and cell types, we identified a subpopulation of cells resistant to activation of AMPK and reduction of ADP/ATP ratio. This resistant state persists transiently for at least several hours and can be inherited during cell divisions. OXPHOS inhibition suppresses the mTORC1 and ERK growth signaling pathways in sensitive cells, but not in resistant cells. Resistance is linked to a multi-factorial combination of increased glucose uptake, reduced protein biosynthesis, and G0/G1 cell-cycle status. Our results reveal dynamic fluctuations in cellular energetic balance and provide a basis for measuring and predicting the distribution of cellular responses to OXPHOS inhibition.
The mechanistic target of rapamycin (mTOR) signaling pathway coordinates environmental and intracellular cues to control eukaryotic cell growth. As a pivot point between anabolic and catabolic processes, mTOR complex 1 (mTORC1) signaling has established roles in regulating metabolism, translation and autophagy. Hyperactivity of the mTOR pathway is associated with numerous human diseases, including diabetes, cancer and epilepsy. Pharmacological inhibition of the mTOR pathway can extend lifespan in a variety of model organisms. Given its broad control of essential cellular processes and clear relevance to human health, there is extensive interest in elucidating how upstream inputs regulate mTORC1 activation. In this Cell Science at a Glance article and accompanying poster, we summarize our understanding of how extracellular and intracellular signals feed into the mTOR pathway, how the lysosome acts as an mTOR signaling hub, and how downstream signaling controls autophagy and lysosome biogenesis.
The protein kinase complex mechanistic target of rapamycin complex 1 (mTORC1) serves as a key conduit between growth signals and the metabolic processes underlying cell growth. The activation state of mTORC1 is controlled by intracellular nutrients and energy, as well as exogenous hormones and growth factors, thereby integrating local and systemic growth signals. Here we discuss the molecular logic of the mTORC1 signalling network and its importance in coupling growth signals to the control of cellular metabolism. After activation, mTORC1 promotes the conversion of available nutrients and energy into the major macromolecular species contributing to cellular mass, including proteins, nucleic acids and lipids, while suppressing the autophagic recycling of these macromolecules back into their nutrient constituents. Given that uncoupling of mTORC1 from its normal regulatory inputs contributes to many diseases—including cancer, genetic tumour syndromes, metabolic diseases, autoimmune diseases and neurological disorders—understanding the molecular logic of the mTORC1 network and how to modulate it may present therapeutic opportunities for treatment of a broad range of diseases and potentially even for the extension of lifespan.
Making multiplexed subcellular protein maps
Being able to visualize protein localizations within cells and tissues by means of immuno-fluorescence microscopy has been key to developments in cell biology and beyond. Gut et al. present a high-throughput method that achieves the detection of more than 40 different proteins in biological samples across multiple spatial scales. This allows the simultaneous quantification of their expression levels in thousands of single cells; captures their detailed subcellular distribution to various compartments, organelles, and cellular structures within each of these single cells; and places all this information within a multicellular context. Such a scale-crossing dataset empowers artificial intelligence–based computer vision algorithms to achieve a comprehensive profiling of intracellular protein maps to measure their responses to different multicellular, cellular, and pharmacological contexts, and to reveal new cellular states.
Science , this issue p. eaar7042