Correction: ESCRT-I fuels lysosomal degradation to restrict TFEB/TFE3 signaling via the Rag-mTORC1 pathway.
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ABSTRACT:
SUBMITTER: Wrobel M
PROVIDER: S-EPMC9175455 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Within the endolysosomal pathway in mammalian cells, ESCRT complexes facilitate degradation of proteins residing in endosomal membranes. Here, we show that mammalian ESCRT-I restricts the size of lysosomes and promotes degradation of proteins from lysosomal membranes, including MCOLN1, a Ca2+ channel protein. The altered lysosome morphology upon ESCRT-I depletion coincided with elevated expression of genes annotated to biogenesis of lysosomes due to prolonged activation of TFEB/TFE3 transcription factors. Lack of ESCRT-I also induced transcription of cholesterol biosynthesis genes, in response to inefficient delivery of cholesterol from endolysosomal compartments. Among factors that could possibly activate TFEB/TFE3 signaling upon ESCRT-I deficiency, we excluded lysosomal cholesterol accumulation and Ca2+-mediated dephosphorylation of TFEB/TFE3. However, we discovered that this activation occurs due to the inhibition of Rag GTPase-dependent mTORC1 pathway that specifically reduced phosphorylation of TFEB at S112. Constitutive activation of the Rag GTPase complex in cells lacking ESCRT-I restored S112 phosphorylation and prevented TFEB/TFE3 activation. Our results indicate that ESCRT-I deficiency evokes a homeostatic response to counteract lysosomal nutrient starvation, that is, improper supply of nutrients derived from lysosomal degradation.
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Project description:Within the endolysosomal pathway in mammalian cells, ESCRT complexes facilitate degradation of membrane proteins from limiting membranes of late endosomes. Recent studies revealed that yeast ESCRT proteins also sort for degradation, ubiquitinated proteins from vacuolar membrane. However, whether mammalian ESCRTs perform similar function at lysosomes remained unknown. Here, we studied the involvement of mammalian ESCRT-I in maintaining lysosomal membrane homeostasis and its implication in lysosome-related signaling. We show that ESCRT-I restricts the size of lysosomes and promotes degradation of lysosomal Ca2+ channel, MCOLN1 protein. ESCRT-I depletion induced transcriptional response related to MiT-TFE signaling and cholesterol biosynthesis, pointing to lysosomal dysfunction. The lack of ESCRT-I promoted abnormal cholesterol accumulation on lysosomes and activated TFEB/TFE3 transcription factors in Ca2+-MCOLN1-dependent, but lipid-independent manner. Hence, our study provides evidence that ESCRT-I maintains lysosomal homeostasis and elucidates MiT-TFE regulatory mechanism activated in response to ESCRT-I deprivation
2022-03-28 | GSE178665 | GEO
Project description:Myelin allows for fast and efficient axonal conduction, but much remains to be determined about the mechanisms that regulate myelin formation. To investigate the genetic basis of myelination, we carried out a genetic screen using zebrafish. Here, we show that the lysosomal G protein RagA is essential for CNS myelination. In rraga-/- mutant oligodendrocytes, target genes of the lysosomal transcription factor Tfeb are upregulated, consistent with previous evidence that RagA represses Tfeb activity. Loss of Tfeb function is sufficient to restore myelination in RagA mutants, indicating that hyperactive Tfeb represses myelination. Conversely, tfeb-/- single mutants exhibit ectopic myelin, further indicating that Tfeb represses myelination during development. In a mouse model of de- and remyelination, TFEB expression is increased in oligodendrocytes, but the protein is localized to the cytoplasm, and hence inactive, especially during remyelination. These results define essential regulators of myelination and may advance approaches to therapeutic remyelination.
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Project description:Cell growth is controlled by a lysosomal signalling complex containing Rag small GTPases and mammalian target of rapamycin complex 1 (mTORC1) kinase. Here, we carried out a microscopy-based genome-wide human short interfering RNA screen and discovered a lysosome-localized G protein-coupled receptor (GPCR)-like protein, GPR137B, that interacts with Rag GTPases, increases Rag localization and activity, and thereby regulates mTORC1 translocation and activity. High GPR137B expression can recruit and activate mTORC1 in the absence of amino acids. Furthermore, GPR137B also regulates the dissociation of activated Rag from lysosomes, suggesting that GPR137B controls a cycle of Rag activation and dissociation from lysosomes. GPR137B-knockout cells exhibited defective autophagy and an expanded lysosome compartment, similar to Rag-knockout cells. Like zebrafish RagA mutants, GPR137B-mutant zebrafish had upregulated TFEB target gene expression and an expanded lysosome compartment in microglia. Thus, GPR137B is a GPCR-like lysosomal regulatory protein that controls dynamic Rag and mTORC1 localization and activity as well as lysosome morphology.
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Project description:This SuperSeries is composed of the SubSeries listed below.
2023-04-30 | GSE230611 | GEO
Project description:Signaling pathways mediated by the mechanistic target of rapamycin (mTOR) play key roles in aging and age-related diseases. As a downstream protein of mTOR, transcription factor EB (TFEB) controls lysosome biogenesis and cellular trafficking, processes that are essential for the functions of phagocytic cells like the retinal pigment epithelium (RPE). In the current study, we show that a naturally occurring polyphenolic compound, quercetin, promoted TFEB nuclear translocation and enhanced its transcriptional activity in cultured RPE cells. Activated TFEB facilitated degradation of phagocytosed photoreceptor outer segments. Quercetin is a direct inhibitor of mTOR but did not influence the activity of Akt at the tested concentration range. Our data suggest that the dietary compound quercetin can have beneficial roles in neuronal tissues by improving the functions of the TFEB-lysosome axis and enhancing the capacities of cellular degradation and self-renewal.
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Project description:GGGGCC repeat expansion in C9orf72 is the most common genetic cause in both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), two neurodegenerative disorders in association with aging. Bidirectional repeat expansions in the noncoding region of C9orf72 have been shown to produce dipeptide repeat (DPR) proteins through repeat-associated non-ATG (RAN) translation and to reduce the expression level of the C9orf72 gene product, C9orf72 protein. Mechanisms underlying C9orf72-linked neurodegeneration include expanded RNA repeat gain of function, DPR toxicity, and C9orf72 protein loss of function. In the current study, we focus on the cellular function of C9orf72 protein. We report that C9orf72 can regulate lysosomal biogenesis and autophagy at the transcriptional level. We show that loss of C9orf72 leads to striking accumulation of lysosomes, autophagosomes, and autolysosomes in cells, which is associated with suppressed mTORC1 activity and enhanced nuclear translocation of MiT/TFE family members MITF, TFE3, and TFEB, three master regulators of lysosomal biogenesis and autophagy. We demonstrate that the DENN domain of C9orf72 specifically binds to inactive Rag GTPases, but not active Rag GTPases, thereby affecting the function of Rag/raptor/mTOR complex and mTORC1 activity. Furthermore, active Rag GTPases, but not inactive Rag GTPases or raptor rescued the impaired activity and lysosomal localization of mTORC1 in C9orf72-deficient cells. Taken together, the present study highlights a key role of C9orf72 in lysosomal and autophagosomal regulation, and demonstrates that Rag GTPases and mTORC1 are involved in C9orf72-mediated autophagy.
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Project description:Transcription factor EB (TFEB) is a master transcriptional regulator playing a key role in lysosomal biogenesis, autophagy and lysosomal exocytosis. TFEB activity is inhibited following its phosphorylation by mammalian target of rapamycin complex 1 (mTORC1) on the surface of the lysosome. Phosphorylated TFEB is bound by 14-3-3 proteins, resulting in its cytoplasmic retention in an inactive state. It was suggested that the calcium-dependent phosphatase calcineurin is responsible for dephosphorylation and subsequent activation of TFEB under conditions of lysosomal stress. We have recently demonstrated that TFEB is activated following exposure of cancer cells to lysosomotropic anticancer drugs, resulting in lysosome-mediated cancer drug resistance via increased lysosomal biogenesis, lysosomal drug sequestration, and drug extrusion through lysosomal exocytosis. Herein, we studied the molecular mechanism underlying lysosomotropic-drug-induced activation of TFEB. We demonstrate that accumulation of lysosomotropic drugs results in membrane fluidization of lysosome-like liposomes, which is strictly dependent on the acidity of the liposomal lumen. Lysosomal accumulation of lysosomotropic drugs and the consequent fluidization of the lysosomal membrane, facilitated the dissociation of mTOR from the lysosomal membrane and inhibited the kinase activity of mTORC1, which is necessary and sufficient for the rapid translocation of TFEB to the nucleus. We further show that while lysosomotropic drug sequestration induces Ca2+ release into the cytoplasm, facilitating calcineurin activation, chelation of cytosolic Ca2+, or direct inhibition of calcineurin activity, do not interfere with drug-induced nuclear translocation of TFEB. We thus suggest that lysosomotropic drug-induced activation of TFEB is mediated by mTORC1 inhibition due to lysosomal membrane fluidization and not by calcineurin activation. We further postulate that apart from calcineurin, other constitutively active phosphatase(s) partake in TFEB dephosphorylation and consequent activation. Moreover, a rapid export of TFEB from the nucleus to the cytosol occurs upon relief of mTORC1 inhibition, suggesting that dephosphorylated TFEB constantly travels between the nucleus and the cytosol, acting as a rapidly responding sensor of mTORC1 activity.
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Project description:A key step in nutrient sensing is activation of the master growth regulator, mTORC1 kinase, on the lysosomal membrane. Nutrients enable mTORC1 scaffolding by a complex composed of the Rag GTPases (Rags) and Ragulator, but the underlying mechanism of mTORC1 capture is poorly understood. Combining dynamic imaging in cells and reconstituted systems, we uncover an affinity switch that controls mTORC1 lifetime and activation at the lysosome. Nutrients destabilize the Rag-Ragulator interface, causing cycling of the Rags between lysosome-bound Ragulator and the cytoplasm, and rendering mTORC1 capture contingent on simultaneous engagement of two Rag-binding interfaces. Rag GTPase domains trigger cycling by coordinately weakening binding of the C-terminal domains to Ragulator in a nucleotide-controlled manner. Cancer-specific Rag mutants override release from Ragulator and enhance mTORC1 recruitment and signalling output. Cycling in the active state sets the Rags apart from most signalling GTPases, and provides a mechanism to attenuate mTORC1 signalling.
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Project description:Nearly 1.7 billion people are infected with Mycobacterium tuberculosis. Its ability to survive intracellularly is thought to be central to its success as a pathogen, but how it does this is poorly understood. Using a Drosophila model of infection, we identify three host cell activities, Rab7, CG8743, and the ESCRT machinery, that modulate the mycobacterial phagosome. In the absence of these factors the cell no longer restricts growth of the non-pathogen Mycobacterium smegmatis. Hence, we identify factors that represent unique vulnerabilities of the host cell, because manipulation of any one of them alone is sufficient to allow a nonpathogenic mycobacterial species to proliferate. Furthermore, we demonstrate that, in mammalian cells, the ESCRT machinery plays a conserved role in restricting bacterial growth.
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