mCherry-GFP-LC3 Addgene, Dr. Autophagy and mTOR signaling, C. The mTORC1 Autopgagy a sifnaling of DEPTOR, Raptor, PRAS40, mLST8, mTOR, and phosphorylate downstream targets to regulate protein synthesis or mRNA translation, lipid synthesis, nucleotide synthesis, lysosomal biogenesis, and autophagy.

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Autophagy - Macroautophagy \u0026 Importance in Health

Autophagy and mTOR signaling -

Many components of the autophagic machinery and autophagy receptors such as p62 present in these organelles are themselves substrates of the process being subjected to lysosomal degradation However, Gαq is not a substrate of autophagy under nutrient stress conditions, since serum removal did not promote a significant decrease of Gαq protein levels in WT MEFs, or in cells deficient for Atg5, a well-established model of macroautophagy deficiency, contrary to what was observed for the known autophagy substrate p62 Supplementary Fig.

Overall, the unexpected location of Gαq in autophagic compartments and lysosomes and the fact that this protein is not degraded under nutrient stress conditions in these organelles suggested that the observed changes in autophagy upon Gαq modulation may result from a regulatory function of this protein on autophagy.

A representative blot of four independent experiments is shown. A representative blot of five independent experiments is shown.

For a and b , autophagic markers LC3-II and p62 and the activation levels of the mTORC1 and AMPK pathways were analyzed by assessing the phosphorylation status of the downstream target of mTORC1 S6 ribosomal protein see Supplementary Fig.

Tubulin and GAPDH for the p-AMPK and AMPK blots in panel a were used as loading controls. The main cellular nutrient sensor pathways converge in the AMPK and mTORC1 cascades to regulate metabolism and autophagy.

The expected attenuation of the mTORC1 cascade in WT cells upon low serum Fig. Of note, in serum-starved cells where downmodulation of mTORC1 pathway and upregulation of autophagy takes place Fig. a Experiment outline diagram. Tubulin, actin, or GAPDH were used as loading controls. Blots are representative of three panels b , d , and panel e lower blots or five panels c and e independent experiments.

See Supplementary Fig. In control cells without CNO stimulation , this pattern persists in the subsequent hours, as assessed by the decreased status of p-S6 Fig. A similar pattern of autophagy modulation was detected when testing the evolution of p62 levels Supplementary Fig.

Once again, we did not detect changes in the AMPK pathway. It is worth noting that the reactivation of the mTORC1 pathway triggered by CNO does not completely parallel the pattern of Akt activation Fig. These data were consistent with the occurrence of alternative routes of Gq-mediated mTORC1 stimulation.

CNO-mediated preservation of mTORC1 activation also attenuated the increase in LC3-II levels triggered by amino acid removal in control cells Fig. Consistent with this notion, overexpression of constitutively active mutants of Gαq Gq-RC and Gq-QL was able to maintain mTORC1 pathway activation even under low-serum conditions Supplementary Fig.

This mTORC1 reactivation is essential to inactivate ongoing autophagy and return to basal conditions. Two different refeeding paradigms were used. WT vs.

Tubulin was used as a loading control. Representative blots are shown. a , b Statistical significance was analyzed using two-sided unpaired t -test. Image on the right shows the quantification analysis of lysosome distribution.

Representative blots of three independent experiments are shown. Control vs. Immunofluorescence with LAMP1 to highlight the endolysosomal compartment revealed that WT MEFs displayed the expected switch of lysosomes from the cell periphery to a more perinuclear location upon exposure to amino acid-free medium 25 , whereas the re-exposure to amino acids restored their original location Fig.

These results are consistent with their inability to stimulate mTORC1 in response to nutrient recovery. Intriguingly, immunoprecipitation of either endogenous Gαq Fig. Importantly, immunoprecipitation of endogenous Raptor, a specific component of mTORC1, also revealed the presence in the same complex of Gαq, p62, and mTOR Fig.

The mTORC1 activation complex formation undergoes a dynamic modulation upon nutrient fluctuations 15 , Following amino acid deprivation, we found dissociation of p62 from mTOR, while Raptor and Gαq remained in the mTOR complex Fig.

In response to amino acid replenishment, p62 re-associated to the mTORC1 complex. Blots are representative of three a , c and four b independent experiments.

c WT vs. d Control vs. We searched for potential molecular links between Gαq and mTORC1 activation. A novel acidic region in Gαq responsible for its direct interaction with the PB1-containing protein Protein kinase C ζ PKCζ has been reported, and glutamic acid residues and proven to be critical for such interaction Remarkably, the stimulation of the mTORC1 pathway triggered by transient expression of Gαq WT in low-serum conditions was not mimicked upon overexpression of the Gq-EEAA mutant Fig.

Overall, these results suggested that the PB1-binding region of Gαq is involved in mTORC1 pathway modulation. A representative blot of three independent experiments is shown.

c — f CHO cells were transiently transfected with the indicated combinations of HA-p62, Gαq WT or the indicated Gαq mutants constitutively active Gq-QL or GqRC, active but PLC-β activation-defective GqQL-AA, defective in binding to PB1-domain-containing proteins Gq-EEAA and constitutively active GqRC-EEAA , or the PB1 domain of p62 GFP-Flag-PB1-p Gαq WT vs.

We, therefore, used transient overexpression of Gαq and HA-p62 constructs to characterize their association. Of note, p62 displays features of a bona fide Gαq effector reviewed in ref. Consistent with data in Fig.

Of note, Gαq mutants Gαq-YF, Gαq-TE, Gαq-WD unable to bind GRK2 co-immunoprecipitated with p62 to an even greater extent than wild-type Gαq Supplementary Fig. Consistent with a PB1-like association between Gαq and p62, the overexpression of the PB1 domain of p62 clearly inhibited the complex formation Fig.

a CHO cells were transfected with HA-p62 and Gαq constructs, starved with 0. c Basal activation status of the mTORC1 cascade and autophagy levels correlates with the ability of Gαq mutants to interact with p mTOR immunoprecipitates and total lysates were analyzed by western blot with specific antibodies to determine the components of the complex and overall cell expression levels, respectively.

The blots shown are representative of three independent experiments. e mTORC1 reactivation and autophagy modulation in response to amino acid recovery after starvation correlate with the ability of Gαq to interact with p Statistical significance was analyzed using two-way ANOVA with Bonferroni multiple comparison test.

Basal activation status of the mTORC1 cascade and autophagy levels correlated with the ability of Gαq mutants to interact with p Overall, these results support the notion that the interaction between Gαq and p62 in response to nutrients is key for the stimulation of the mTORC1 pathway and the control of autophagy.

Our data suggest that Gαq acts as a general and relevant modulator of mTORC1 signaling over autophagy in response to fluctuations in different types of nutrients. The metabolic modulation of autophagy is mainly orchestrated by the balance between the opposite inputs provided by the mTORC1 negative and AMPK positive pathways 12 , 31 , Moreover, constitutively active mutations in Gαq maintain mTORC1 activated in absence of serum.

Fifth, consistent with its role as an autophagy modulator, we demonstrate the presence of an endogenous Gαq pool in autophagic compartments and lysosomes in rat liver, where it co-localizes with mTOR, Raptor, and p62, and immunoprecipitation of endogenous mTOR or Raptor from WT MEFs in the presence of nutrients shows that Gαq is part of the mTORC1 multimolecular complex.

This signaling adaptor contains several functional regions and displays a variety of subcellular locations, including autophagosomes and lysosomes reviewed in refs.

p62 plays a central role as an autophagy receptor by forming PB1-linked homo-oligomers, recruiting ubiquitinated cargos, and inducing nucleation of the autophagosome membrane via its interaction with LC3 33 being itself a substrate of autophagy p62 is also involved in other cellular processes 33 , including the modulation of mTORC1 see below.

The mTORC1 signaling hub displays an intricate spatiotemporal regulation, involving both upstream signaling pathways and timely recruitment to lysosomes 12 , Major components of the mTORC1 complex include mTOR kinase, mLST8, DEPTOR, and the specific regulatory protein Raptor, which plays a relevant role in facilitating substrate recruitment and in controlling mTORC1 subcellular localization 12 , Importantly, the presence of amino acids, acting through different sensors, is key for full mTORC1 activation by facilitating its recruitment to the lysosomal membrane.

This is achieved via the Ras-related GTP-binding protein Rag family of small GTPases. Glucose-dependent mTORC1 stimulation is also partially regulated through Rag-GTPases by complex mechanisms reviewed in refs. Thus, Rags appear to integrate multiple nutrient inputs converging in mTORC1 modulation.

Importantly, in the presence of amino acids, p62 has been reported to interact with Raptor, thus helping mTORC1 complex translocation to the lysosome The presence of p62 in the mTORC1 multimolecular complex might also contribute to the full activation of kinase activity via TRAF6-mediated K63 ubiquitination of mTOR First, p62, mTOR, Raptor, and Gαq are present in the same multimolecular complex in endogenous conditions in the presence of nutrients, whereas cells lacking Gαq display a reduced presence of p62 in mTOR and Raptor immunoprecipitates along with decreased mTORC1 pathway activation.

Gαq and p62 appear to associate through a PB1-like interaction, involving the PB1 domain of p62 and the acidic PB1-binding region of Gαq, different from the specific interface involved in the association of Gαq with the canonical PLCβ or p63RhoGEF effectors This acidic Gαq region has been shown to interact with the PB1 domain of PKCζ 29 or with the cold-activated channel TRPM8 42 , consistent with this Gαq region being a functional module.

Such core Gαq scaffolding role would be consistent with its general role in autophagy control in response to different types of nutrient stress see scheme in Fig. In nutrient-sufficiency conditions, the presence of serum, amino acids, or glucose would foster the PB1-like interaction between Gαq and p62, leading to basal mTORC1 stimulation and preservation of low, homeostatic autophagy levels.

Upper part: In nutrient-sufficiency conditions, the presence of serum, amino acids, or glucose would foster the PB1-like interaction between Gαq and p62, leading to basal mTORC1 stimulation and preservation of homeostatic autophagy levels.

Lower part: Autophagy levels diagram. In response to starvation, there is a controlled and transient upregulation of autophagy, with a subsequent return to basal levels upon the partial recovery of nutrients. An increasing number of nutrients and metabolites are being identified as endogenous ligands for Gq-coupled GPCR 16 , 17 , On the other hand, relevant serum components as lysophosphatidic acid or sphingosinephosphate and factors as insulin or IGF-1 have been reported to directly stimulate Gαq signaling The presence of Gαq in lysosomal and autophagy-related organelles may be subsequent to receptor-mediated activation at the plasma membrane or represent a stable pool able to undergo nutrient-mediated stimulation at such locations.

Recent data indicate the presence of Gαq in endosomes and other organelles, such as mitochondria 49 , The agonist-dependent presence of GPCR and heterotrimeric G proteins in endosomes as a consequence of GPCR recycling or degradation mechanisms has also been reported Since endocytosis-derived organelles, can form elongation membranes to finally form autophagosomes in response to starvation 54 , endosomes might constitute a source for Gαq in autophagy-related organelles.

Alternatively, activation of Gαq by nutrient-sensitive GPCR could take place directly at locations different from the plasma membrane via molecules that can gain access to the intracellular milieu via specific transporters, as is the case for amino acids, glucose, and other nutrients.

In sum, our results postulate Gαq as a key hub in the autophagy machinery. All rats were housed under pathogen-free conditions and handled according to protocols approved by the Institutional Animal Care and Use Committee of Albert Einstein College of Medicine New York, USA.

Since for these experiments mice were not treated with tamoxifen, we refer to them as WT mice as they display endogenous Gαq levels. We did not use randomization in our animal studies.

We were not blinded to the group in our animal studies. Offermanns Max-Planck-Institute for Heart and Lung Research, Germany. Atg5 WT and Atg5 KO MEFs were provided by Dr.

Mizushima University of Tokyo, Tokyo, Japan and DREADD-Gq-HEK cells human female by Dr. Silvio Gutkind University of San Diego, California, USA CHO cells overexpressing the muscarinic M3 acetylcholine receptor CHO-M3 mice female were a kind gift from Dr.

Tobin University of Glasgow, UK. Cell lines were authenticated by short tandem repeat STR or DNA barcoding analysis and tested for mycoplasma contamination. Human umbilical vein endothelial cells HUVEC human male were grown in Medium Life Technologies, without NaHCO 3 supplemented with 2.

Explants were homogenized using metal beads in a Tissue Lyser Qiagen, Hilden, Germany 60 , and lysates used for immunoblot analysis of mTORC1 pathway modulation. For autophagy modulation by nutrient depletion assays, cells were maintained in 0. Nutrient recovery experiments in MEF cells were carried out by adding increasing percentages of bovine serum or a full amino acid mix MEM amino acids solution 50× Sigma-Aldrich.

For degradation assays, cells were treated with the lysosomal inhibitors mentioned and the proteasomal inhibitor MG 0. The Gαq and Gαq-RC cDNA were from our laboratories Dr. Aragay, CSIC, Barcelona, Spain. Lin Stony Brook University, New York, USA. The Gαq mutant proteins that lack the ability to interact with GRK2 Gαq-TE, WD, YF were a gift from Dr.

Kozasa The University of Tokyo, Japan. GRK2, GRK2-KR, GRK2-DA cDNAs were a gift from Dr. Benovic The Kimmel Cancer Center, USA. The GRK2-RH domain cDNA 61 was generated in our laboratory. The HA-p62 construct was provided by Dr.

Campbell University of Illinois at Chicago, USA. GFP-Flag-PB1-p62 was provided by Dr. Berk University of Rochester, NY, USA. mCherry-GFP-LC3 Addgene, Dr.

Mizushima, constructed by T. Kaizuka University of Tokyo, Japan. cDNAs for lentiviral construction pMD2.

G, pSD. Iggo University of St Andrews, Edinburgh, UK. Gq-wt and psd Gq-EEAA were created in our laboratory from psd GqQL Addgene, Dr. All primers' information has been included in Supplementary Table 1. Gq-EEAA DNA constructs were transfected together with psPAX2 and pMD2. All generated MEF cells lines, psd.

Cells were seeded in a six-well plate a day before transduction. Then, a 2× polybrene mixture was prepared by adding 0. Depending on the cell type these stable cell lines were used until passage 3—4.

Immunoprecipitation was performed with agarose-conjugated anti-HA antibodies Santa Cruz, F-7, scAC. Endogenous immunoprecipitation assays were performed by incubating 0.

Protein concentration was measured using the DC Protein Assay Reagents A, B, and S Bio-Rad. Immunoreactivity bands were quantified by laser densitometry with a Biorad GS scanner and using de Bio-Rad provided Image Lab 5. Vertical lines in western blots indicate juxtaposed lanes that come from the same gel but were nonadjacent.

Cells were mounted with Fluoromount-G-Dapi Southern Biotech. Sample images were acquired using a confocal laser microscope LSM Zeiss at ×63 magnification. Image J software NIH was used for general image analysis and manipulations. Imaging of the mCherry-GPF-LC3 reporter was carried out using high-content microscopy.

The total number of autophagic vacuoles AV, autophagosomes plus autolysosomes was estimated from the number of mCherry-positive puncta, whereas puncta positive for GFP and mCherry were scored as autophagosomes APG.

Autophagic flux was calculated as the number of mCherry only fluorescent puncta autolysosomes, AL per cell. Before quantification, the maximum intensity projection was obtained for each cell image, and the cell and nucleus boundaries were extracted by respectively manual and automatic segmentation.

The fluorescence intensity density per ring was then measured and normalized with respect to the total intensity density of the cell see Appendix Fig.

S4 for details. At least 30 cells were measured for each condition. The xCELLigence system RTCA SP instrument Roche Applied Science monitors changes in the cell index a measure of cell attachment to the plate which has been shown to effectively correlate to proliferation, adhesion, and viability changes In no case was cell death due to excessive confluence as confirmed by plate inspection with a microscope.

To quantitatively measure apoptosis, the PE Annexin V Apoptosis Detection kit I BD-Bioscience was utilized. Samples were analyzed by flow cytometry on a BD FacsCalibur flow cytometer BD-Bioscience. To determine the apoptotic stage of the different cell populations, 7-AAD- and Annexin V-positive cells were determined with the CellQuest Pro Version 4.

Cells treated with staurosporine 2. Samples were analyzed by flow cytometry on a BD FacsCalibur flow cytometer BD-Bioscience , and data were analyzed with the FlowJo software. After several washes, cells were scraped and collected in the same buffer, centrifuged and the cell pellets were processed for embedding in the Epoxy, TAAB resin TAAB Laboratories according to standard procedures Autophagic vacuoles were identified using established criteria Autophagic vacuoles vesicles of 0.

Total autophagy vacuoles were composed of the sum of autophagosomes and autolysosomes. The maturation of autophagy vacuoles was calculated as the percent of autolysosomes and autophagosomes out of the total number of autophagy vacuoles.

Autophagic vacuoles were isolated from rat liver using discontinuous metrizamide density gradient Briefly, after homogenization, livers were subjected to sequential differential centrifugations to separate a fraction enriched in autophagy-related compartments and mitochondrial.

This fraction was placed at the bottom of a discontinuous three layers gradient of metrizamide and upon centrifugation, mitochondria remained at the bottom whereas a fraction enriched in autophagosomes is recovered in the top band and in autolysosomes in the second band from the top.

A lysosomal enriched fraction is also recovered from the third band of the gradient from the top. Upon extensive washing with 0. com or by the software Application Software Version 3. Flow cytometry data were analyzed with the FlowJo Software V7. Image J software v1. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

List of Figures that have associated raw data are Figs. Other data that support the findings of this study are available from the corresponding authors upon reasonable request. No datasets were generated or analyzed during this study. All unique materials generated are readily available from the authors.

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The mechanistic sugnaling of Autophagy and mTOR signaling mTORmaster regulator of cellular metabolism, exists in two distinct Autophagy and mTOR signaling mTOR complex Auophagy and mTOR Autophgy 2 mTORC1 and signaliny. MTORC1 is a master switch for most energetically Fuel Management Software processes in Autophagy and mTOR signaling cell, driving cell growth and building cellular biomass in instances of nutrient sivnaling, and conversely, allowing autophagic recycling of cellular components upon nutrient limitation. The means by which the mTOR kinase blocks autophagy include direct inhibition of the early steps of the process, and the control of the lysosomal degradative capacity of the cell by inhibiting the transactivation of genes encoding structural, regulatory, and catalytic factors. Upon inhibition of mTOR, autophagic recycling of cellular components results in the reactivation of mTORC1; thus, autophagy lies both downstream and upstream of mTOR. The functional relationship between the mTOR pathway and autophagy involves complex regulatory loops that are significantly deciphered at the cellular level, but incompletely understood at the physiological level.

Autophagy and mTOR signaling -

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Would you like to visit your country specific website? YES NO. Save This Selection. All Rights Reserved. Pathway Description Legend Pathway Description: Macroautophagy, often referred to as autophagy, is a catabolic process that results in the autophagosomic-lysosomal degradation of bulk cytoplasmic contents, abnormal protein aggregates, and excess or damaged organelles.

Codogno P, Mehrpour M, Proikas-Cezanne T Canonical and non-canonical autophagy: variations on a common theme of self-eating? Cell Biol. Ding WX, Yin XM Mitophagy: mechanisms, pathophysiological roles, and analysis.

Feng D, Liu L, Zhu Y, Chen Q Molecular signaling toward mitophagy and its physiological significance. Cell Res. Papinski D, Kraft C Atg1 kinase organizes autophagosome formation by phosphorylating Atg9.

Autophagy 10 7 , — Schneider JL, Cuervo AM Autophagy and human disease: emerging themes. created September revised September Request Permission for Pathway View PDF.

Learn More With PhosphoSite Plus ®. Image on the right shows the quantification analysis of lysosome distribution. Representative blots of three independent experiments are shown.

Control vs. Immunofluorescence with LAMP1 to highlight the endolysosomal compartment revealed that WT MEFs displayed the expected switch of lysosomes from the cell periphery to a more perinuclear location upon exposure to amino acid-free medium 25 , whereas the re-exposure to amino acids restored their original location Fig.

These results are consistent with their inability to stimulate mTORC1 in response to nutrient recovery. Intriguingly, immunoprecipitation of either endogenous Gαq Fig. Importantly, immunoprecipitation of endogenous Raptor, a specific component of mTORC1, also revealed the presence in the same complex of Gαq, p62, and mTOR Fig.

The mTORC1 activation complex formation undergoes a dynamic modulation upon nutrient fluctuations 15 , Following amino acid deprivation, we found dissociation of p62 from mTOR, while Raptor and Gαq remained in the mTOR complex Fig.

In response to amino acid replenishment, p62 re-associated to the mTORC1 complex. Blots are representative of three a , c and four b independent experiments. c WT vs. d Control vs. We searched for potential molecular links between Gαq and mTORC1 activation. A novel acidic region in Gαq responsible for its direct interaction with the PB1-containing protein Protein kinase C ζ PKCζ has been reported, and glutamic acid residues and proven to be critical for such interaction Remarkably, the stimulation of the mTORC1 pathway triggered by transient expression of Gαq WT in low-serum conditions was not mimicked upon overexpression of the Gq-EEAA mutant Fig.

Overall, these results suggested that the PB1-binding region of Gαq is involved in mTORC1 pathway modulation. A representative blot of three independent experiments is shown. c — f CHO cells were transiently transfected with the indicated combinations of HA-p62, Gαq WT or the indicated Gαq mutants constitutively active Gq-QL or GqRC, active but PLC-β activation-defective GqQL-AA, defective in binding to PB1-domain-containing proteins Gq-EEAA and constitutively active GqRC-EEAA , or the PB1 domain of p62 GFP-Flag-PB1-p Gαq WT vs.

We, therefore, used transient overexpression of Gαq and HA-p62 constructs to characterize their association. Of note, p62 displays features of a bona fide Gαq effector reviewed in ref.

Consistent with data in Fig. Of note, Gαq mutants Gαq-YF, Gαq-TE, Gαq-WD unable to bind GRK2 co-immunoprecipitated with p62 to an even greater extent than wild-type Gαq Supplementary Fig.

Consistent with a PB1-like association between Gαq and p62, the overexpression of the PB1 domain of p62 clearly inhibited the complex formation Fig. a CHO cells were transfected with HA-p62 and Gαq constructs, starved with 0.

c Basal activation status of the mTORC1 cascade and autophagy levels correlates with the ability of Gαq mutants to interact with p mTOR immunoprecipitates and total lysates were analyzed by western blot with specific antibodies to determine the components of the complex and overall cell expression levels, respectively.

The blots shown are representative of three independent experiments. e mTORC1 reactivation and autophagy modulation in response to amino acid recovery after starvation correlate with the ability of Gαq to interact with p Statistical significance was analyzed using two-way ANOVA with Bonferroni multiple comparison test.

Basal activation status of the mTORC1 cascade and autophagy levels correlated with the ability of Gαq mutants to interact with p Overall, these results support the notion that the interaction between Gαq and p62 in response to nutrients is key for the stimulation of the mTORC1 pathway and the control of autophagy.

Our data suggest that Gαq acts as a general and relevant modulator of mTORC1 signaling over autophagy in response to fluctuations in different types of nutrients.

The metabolic modulation of autophagy is mainly orchestrated by the balance between the opposite inputs provided by the mTORC1 negative and AMPK positive pathways 12 , 31 , Moreover, constitutively active mutations in Gαq maintain mTORC1 activated in absence of serum.

Fifth, consistent with its role as an autophagy modulator, we demonstrate the presence of an endogenous Gαq pool in autophagic compartments and lysosomes in rat liver, where it co-localizes with mTOR, Raptor, and p62, and immunoprecipitation of endogenous mTOR or Raptor from WT MEFs in the presence of nutrients shows that Gαq is part of the mTORC1 multimolecular complex.

This signaling adaptor contains several functional regions and displays a variety of subcellular locations, including autophagosomes and lysosomes reviewed in refs. p62 plays a central role as an autophagy receptor by forming PB1-linked homo-oligomers, recruiting ubiquitinated cargos, and inducing nucleation of the autophagosome membrane via its interaction with LC3 33 being itself a substrate of autophagy p62 is also involved in other cellular processes 33 , including the modulation of mTORC1 see below.

The mTORC1 signaling hub displays an intricate spatiotemporal regulation, involving both upstream signaling pathways and timely recruitment to lysosomes 12 , Major components of the mTORC1 complex include mTOR kinase, mLST8, DEPTOR, and the specific regulatory protein Raptor, which plays a relevant role in facilitating substrate recruitment and in controlling mTORC1 subcellular localization 12 , Importantly, the presence of amino acids, acting through different sensors, is key for full mTORC1 activation by facilitating its recruitment to the lysosomal membrane.

This is achieved via the Ras-related GTP-binding protein Rag family of small GTPases. Glucose-dependent mTORC1 stimulation is also partially regulated through Rag-GTPases by complex mechanisms reviewed in refs.

Thus, Rags appear to integrate multiple nutrient inputs converging in mTORC1 modulation. Importantly, in the presence of amino acids, p62 has been reported to interact with Raptor, thus helping mTORC1 complex translocation to the lysosome The presence of p62 in the mTORC1 multimolecular complex might also contribute to the full activation of kinase activity via TRAF6-mediated K63 ubiquitination of mTOR First, p62, mTOR, Raptor, and Gαq are present in the same multimolecular complex in endogenous conditions in the presence of nutrients, whereas cells lacking Gαq display a reduced presence of p62 in mTOR and Raptor immunoprecipitates along with decreased mTORC1 pathway activation.

Gαq and p62 appear to associate through a PB1-like interaction, involving the PB1 domain of p62 and the acidic PB1-binding region of Gαq, different from the specific interface involved in the association of Gαq with the canonical PLCβ or p63RhoGEF effectors This acidic Gαq region has been shown to interact with the PB1 domain of PKCζ 29 or with the cold-activated channel TRPM8 42 , consistent with this Gαq region being a functional module.

Such core Gαq scaffolding role would be consistent with its general role in autophagy control in response to different types of nutrient stress see scheme in Fig. In nutrient-sufficiency conditions, the presence of serum, amino acids, or glucose would foster the PB1-like interaction between Gαq and p62, leading to basal mTORC1 stimulation and preservation of low, homeostatic autophagy levels.

Upper part: In nutrient-sufficiency conditions, the presence of serum, amino acids, or glucose would foster the PB1-like interaction between Gαq and p62, leading to basal mTORC1 stimulation and preservation of homeostatic autophagy levels.

Lower part: Autophagy levels diagram. In response to starvation, there is a controlled and transient upregulation of autophagy, with a subsequent return to basal levels upon the partial recovery of nutrients.

An increasing number of nutrients and metabolites are being identified as endogenous ligands for Gq-coupled GPCR 16 , 17 , On the other hand, relevant serum components as lysophosphatidic acid or sphingosinephosphate and factors as insulin or IGF-1 have been reported to directly stimulate Gαq signaling The presence of Gαq in lysosomal and autophagy-related organelles may be subsequent to receptor-mediated activation at the plasma membrane or represent a stable pool able to undergo nutrient-mediated stimulation at such locations.

Recent data indicate the presence of Gαq in endosomes and other organelles, such as mitochondria 49 , The agonist-dependent presence of GPCR and heterotrimeric G proteins in endosomes as a consequence of GPCR recycling or degradation mechanisms has also been reported Since endocytosis-derived organelles, can form elongation membranes to finally form autophagosomes in response to starvation 54 , endosomes might constitute a source for Gαq in autophagy-related organelles.

Alternatively, activation of Gαq by nutrient-sensitive GPCR could take place directly at locations different from the plasma membrane via molecules that can gain access to the intracellular milieu via specific transporters, as is the case for amino acids, glucose, and other nutrients.

In sum, our results postulate Gαq as a key hub in the autophagy machinery. All rats were housed under pathogen-free conditions and handled according to protocols approved by the Institutional Animal Care and Use Committee of Albert Einstein College of Medicine New York, USA.

Since for these experiments mice were not treated with tamoxifen, we refer to them as WT mice as they display endogenous Gαq levels. We did not use randomization in our animal studies. We were not blinded to the group in our animal studies.

Offermanns Max-Planck-Institute for Heart and Lung Research, Germany. Atg5 WT and Atg5 KO MEFs were provided by Dr. Mizushima University of Tokyo, Tokyo, Japan and DREADD-Gq-HEK cells human female by Dr.

Silvio Gutkind University of San Diego, California, USA CHO cells overexpressing the muscarinic M3 acetylcholine receptor CHO-M3 mice female were a kind gift from Dr.

Tobin University of Glasgow, UK. Cell lines were authenticated by short tandem repeat STR or DNA barcoding analysis and tested for mycoplasma contamination.

Human umbilical vein endothelial cells HUVEC human male were grown in Medium Life Technologies, without NaHCO 3 supplemented with 2.

Explants were homogenized using metal beads in a Tissue Lyser Qiagen, Hilden, Germany 60 , and lysates used for immunoblot analysis of mTORC1 pathway modulation. For autophagy modulation by nutrient depletion assays, cells were maintained in 0. Nutrient recovery experiments in MEF cells were carried out by adding increasing percentages of bovine serum or a full amino acid mix MEM amino acids solution 50× Sigma-Aldrich.

For degradation assays, cells were treated with the lysosomal inhibitors mentioned and the proteasomal inhibitor MG 0. The Gαq and Gαq-RC cDNA were from our laboratories Dr. Aragay, CSIC, Barcelona, Spain. Lin Stony Brook University, New York, USA. The Gαq mutant proteins that lack the ability to interact with GRK2 Gαq-TE, WD, YF were a gift from Dr.

Kozasa The University of Tokyo, Japan. GRK2, GRK2-KR, GRK2-DA cDNAs were a gift from Dr. Benovic The Kimmel Cancer Center, USA. The GRK2-RH domain cDNA 61 was generated in our laboratory. The HA-p62 construct was provided by Dr.

Campbell University of Illinois at Chicago, USA. GFP-Flag-PB1-p62 was provided by Dr. Berk University of Rochester, NY, USA. mCherry-GFP-LC3 Addgene, Dr. Mizushima, constructed by T. Kaizuka University of Tokyo, Japan. cDNAs for lentiviral construction pMD2. G, pSD. Iggo University of St Andrews, Edinburgh, UK.

Gq-wt and psd Gq-EEAA were created in our laboratory from psd GqQL Addgene, Dr. All primers' information has been included in Supplementary Table 1. Gq-EEAA DNA constructs were transfected together with psPAX2 and pMD2. All generated MEF cells lines, psd. Cells were seeded in a six-well plate a day before transduction.

Then, a 2× polybrene mixture was prepared by adding 0. Depending on the cell type these stable cell lines were used until passage 3—4. Immunoprecipitation was performed with agarose-conjugated anti-HA antibodies Santa Cruz, F-7, scAC.

Endogenous immunoprecipitation assays were performed by incubating 0. Protein concentration was measured using the DC Protein Assay Reagents A, B, and S Bio-Rad. Immunoreactivity bands were quantified by laser densitometry with a Biorad GS scanner and using de Bio-Rad provided Image Lab 5. Vertical lines in western blots indicate juxtaposed lanes that come from the same gel but were nonadjacent.

Cells were mounted with Fluoromount-G-Dapi Southern Biotech. Sample images were acquired using a confocal laser microscope LSM Zeiss at ×63 magnification. Image J software NIH was used for general image analysis and manipulations. Imaging of the mCherry-GPF-LC3 reporter was carried out using high-content microscopy.

The total number of autophagic vacuoles AV, autophagosomes plus autolysosomes was estimated from the number of mCherry-positive puncta, whereas puncta positive for GFP and mCherry were scored as autophagosomes APG.

Autophagic flux was calculated as the number of mCherry only fluorescent puncta autolysosomes, AL per cell. Before quantification, the maximum intensity projection was obtained for each cell image, and the cell and nucleus boundaries were extracted by respectively manual and automatic segmentation.

The fluorescence intensity density per ring was then measured and normalized with respect to the total intensity density of the cell see Appendix Fig. S4 for details. At least 30 cells were measured for each condition. The xCELLigence system RTCA SP instrument Roche Applied Science monitors changes in the cell index a measure of cell attachment to the plate which has been shown to effectively correlate to proliferation, adhesion, and viability changes In no case was cell death due to excessive confluence as confirmed by plate inspection with a microscope.

To quantitatively measure apoptosis, the PE Annexin V Apoptosis Detection kit I BD-Bioscience was utilized. Samples were analyzed by flow cytometry on a BD FacsCalibur flow cytometer BD-Bioscience. To determine the apoptotic stage of the different cell populations, 7-AAD- and Annexin V-positive cells were determined with the CellQuest Pro Version 4.

Cells treated with staurosporine 2. Samples were analyzed by flow cytometry on a BD FacsCalibur flow cytometer BD-Bioscience , and data were analyzed with the FlowJo software. After several washes, cells were scraped and collected in the same buffer, centrifuged and the cell pellets were processed for embedding in the Epoxy, TAAB resin TAAB Laboratories according to standard procedures Autophagic vacuoles were identified using established criteria Autophagic vacuoles vesicles of 0.

Total autophagy vacuoles were composed of the sum of autophagosomes and autolysosomes. The maturation of autophagy vacuoles was calculated as the percent of autolysosomes and autophagosomes out of the total number of autophagy vacuoles.

Autophagic vacuoles were isolated from rat liver using discontinuous metrizamide density gradient Briefly, after homogenization, livers were subjected to sequential differential centrifugations to separate a fraction enriched in autophagy-related compartments and mitochondrial.

This fraction was placed at the bottom of a discontinuous three layers gradient of metrizamide and upon centrifugation, mitochondria remained at the bottom whereas a fraction enriched in autophagosomes is recovered in the top band and in autolysosomes in the second band from the top.

A lysosomal enriched fraction is also recovered from the third band of the gradient from the top. Upon extensive washing with 0. com or by the software Application Software Version 3. Flow cytometry data were analyzed with the FlowJo Software V7.

Image J software v1. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

List of Figures that have associated raw data are Figs. Other data that support the findings of this study are available from the corresponding authors upon reasonable request. No datasets were generated or analyzed during this study.

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Thank you for visiting nature. You are using a browser version with signalin support for CSS. To nad the Autophagy and mTOR signaling experience, we recommend Sports dietary analysis use Autophagy and mTOR signaling more up to date browser or turn aand compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. The mTORC1 node plays a major role in autophagy modulation. We report a role of the ubiquitous Gαq subunit, a known transducer of plasma membrane G protein-coupled receptors signaling, as a core modulator of mTORC1 and autophagy. They are also unable to reactivate mTORC1 and thus inactivate ongoing autophagy upon nutrient recovery. Autophagy and mTOR signaling SLProud CGWyttenbach Signalibg. mTOR's role Autophaagy ageing: protein synthesis Autophayg autophagy?. Autophagy and mTOR signaling Albany NY. Copyright: signalign Hands et al. This is an Autophayy article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The molecular and cellular mechanisms that regulate ageing are currently under scrutiny because ageing is linked to many human diseases. The nutrient sensing TOR pathway is emerging as a key regulator of ageing.