Protocol to study starvation-induced autophagy in developing rat neurons.
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ABSTRACT:
SUBMITTER: Wojnacki J
PROVIDER: S-EPMC8358452 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Autophagy measurement has been challenging due to the transient nature of autophagy vesicles, in which degradation of cargo occurs. Here, we present a protocol to monitor starvation-induced autophagy using a live high-throughput microscopy system in a fast and automated manner without the need for sample preparation. We provide a detailed protocol describing the generation of turboGFP-LC3B expressing mouse embryonic fibroblasts (MEFs), the measurement of autophagy over time and the analysis of data. For complete details on the use and execution of this protocol, please refer to Nowosad et al. (2020, 2021).
| S-EPMC8605097 | biostudies-literature
Project description:BackgroundAutophagy is a vesicular trafficking process responsible for the degradation of long-lived, misfolded or abnormal proteins, as well as damaged or surplus organelles. Abnormalities of the autophagic activity may result in the accumulation of protein aggregates, organelle dysfunction, and autophagy disorders were associated with various diseases. Hence, mechanisms of autophagy regulation are under exploration.MethodsOver-expression of hsa-miR-376a1 (shortly MIR376A) was performed to evaluate its effects on autophagy. Autophagy-related targets of the miRNA were predicted using Microcosm Targets and MIRanda bioinformatics tools and experimentally validated. Endogenous miRNA was blocked using antagomirs and the effects on target expression and autophagy were analyzed. Luciferase tests were performed to confirm that 3' UTR sequences in target genes were functional. Differential expression of MIR376A and the related MIR376B was compared using TaqMan quantitative PCR.ResultsHere, we demonstrated that, a microRNA (miRNA) from the DLK1/GTL2 gene cluster, MIR376A, played an important role in autophagy regulation. We showed that, amino acid and serum starvation-induced autophagy was blocked by MIR376A overexpression in MCF-7 and Huh7 cells. MIR376A shared the same seed sequence and had overlapping targets with MIR376B, and similarly blocked the expression of key autophagy proteins ATG4C and BECN1 (Beclin 1). Indeed, 3' UTR sequences in the mRNA of these autophagy proteins were responsive to MIR376A in luciferase assays. Antagomir tests showed that, endogenous MIR376A was participating to the control of ATG4C and BECN1 transcript and protein levels. Moreover, blockage of endogenous MIR376A accelerated starvation-induced autophagic activity. Interestingly, MIR376A and MIR376B levels were increased with different kinetics in response to starvation stress and tissue-specific level differences were also observed, pointing out to an overlapping but miRNA-specific biological role.ConclusionsOur findings underline the importance of miRNAs encoded by the DLK1/GTL2 gene cluster in stress-response control mechanisms, and introduce MIR376A as a new regulator of autophagy.
| S-EPMC3864973 | biostudies-literature
Project description:Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a de novo double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome facilitates the degradation and recycling of the cytoplasmic cargo. In yeast, the upstream signals that regulate the induction of starvation-induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyperphosphorylation of Atg13. However, in mammals, the ortholog complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian autophagy is unknown. Here we report the identification and characterization of novel nutrient-regulated phosphorylation sites on ATG13: Ser-224 and Ser-258. mTOR directly phosphorylates ATG13 on Ser-258 while Ser-224 is modulated by the AMPK pathway. In ATG13 knock-out cells reconstituted with an unphosphorylatable mutant of ATG13, ULK1 kinase activity is more potent, and amino acid starvation induced more rapid ATG13 and ULK1 translocation. These events culminated in a more rapid starvation-induced autophagy response. Therefore, ATG13 phosphorylation plays a crucial role in autophagy regulation.
| S-EPMC4786734 | biostudies-literature
Project description:Autophagy is an important degradation pathway, which is induced after starvation, where it buffers nutrient deprivation by recycling macromolecules in organisms from yeast to man. While the classical pathway mediating this response is via mTOR inhibition, there are likely to be additional pathways that support the process. Here, we identify Annexin A2 as an autophagy modulator that regulates autophagosome formation by enabling appropriate ATG9A trafficking from endosomes to autophagosomes via actin. This process is dependent on the Annexin A2 effectors ARP2 and Spire1. Annexin A2 expression increases after starvation in cells in an mTOR-independent fashion. This is mediated via Jun N-terminal kinase activation of c-Jun, which, in turn, enhances the trans-activation of the Annexin A2 promoter. Annexin A2 knockdown abrogates starvation-induced autophagy, while its overexpression induces autophagy. Hence, c-Jun-mediated transcriptional responses support starvation-induced autophagy by regulating Annexin A2 expression levels.
| S-EPMC4560779 | biostudies-literature
Project description:Starvation induces autophagy to preserve cellular homeostasis in virtually all eukaryotic organisms. However, the mechanisms by which starvation induces autophagy are not completely understood. In mammalian cells, the antiapoptotic protein, Bcl-2, binds to Beclin 1 during nonstarvation conditions and inhibits its autophagy function. Here we show that starvation induces phosphorylation of cellular Bcl-2 at residues T69, S70, and S87 of the nonstructured loop; Bcl-2 dissociation from Beclin 1; and autophagy activation. In contrast, viral Bcl-2, which lacks the phosphorylation site-containing nonstructured loop, fails to dissociate from Beclin 1 during starvation. Furthermore, the stress-activated signaling molecule, c-Jun N-terminal protein kinase 1 (JNK1), but not JNK2, mediates starvation-induced Bcl-2 phosphorylation, Bcl-2 dissociation from Beclin 1, and autophagy activation. Together, our findings demonstrate that JNK1-mediated multisite phosphorylation of Bcl-2 stimulates starvation-induced autophagy by disrupting the Bcl-2/Beclin 1 complex. These findings define a mechanism that cells use to regulate autophagic activity in response to nutrient status.
| S-EPMC2478643 | biostudies-literature
Project description:How energy deprivation induces macroautophagy/autophagy is not fully understood. Here, we show that Atg11, a receptor protein for cargo recognition in selective autophagy, is required for the initiation of glucose starvation-induced autophagy. Upon glucose starvation, Atg11 facilitates the interaction between Snf1 and Atg1, thus is required for Snf1-dependent Atg1 activation. Phagophore assembly site (PAS) formation requires Atg11 via its control of the association of Atg17 with Atg29-Atg31. The binding of Atg11 with Atg9 is crucial for recruiting Atg9 vesicles to the PAS and, thus, glucose starvation-induced autophagy. We propose Atg11 as a key initiation factor controlling multiple key steps in energy deprivation-induced autophagy. Abbreviations: AMPK: AMP-activated protein kinase; Ams1: α-mannosidase; Ape1: aminopeptidase I; Cvt: cytoplasm-to-vacuole targeting; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; MBP: myelin basic protein; MMS: methanesulfonate; PAS: phagophore assembly site; PNBM: p-nitrobenzyl mesylate; SD-G: glucose starvation medium; SD-N: nitrogen starvation medium; ULK1, unc-51 like autophagy activating kinase 1; WT: wild type.
| S-EPMC7751503 | biostudies-literature
Project description:Autophagy is a catabolic process conserved among eukaryotes. Under nutrient starvation, a portion of the cytoplasm is non-selectively sequestered into autophagosomes. Consequently, ribosomes are delivered to the vacuole/lysosome for destruction, but the precise mechanism of autophagic RNA degradation and its physiological implications for cellular metabolism remain unknown. We characterized autophagy-dependent RNA catabolism using a combination of metabolome and molecular biological analyses in yeast. RNA delivered to the vacuole was processed by Rny1, a T2-type ribonuclease, generating 3'-NMPs that were immediately converted to nucleosides by the vacuolar non-specific phosphatase Pho8. In the cytoplasm, these nucleosides were broken down by the nucleosidases Pnp1 and Urh1. Most of the resultant bases were not re-assimilated, but excreted from the cell. Bulk non-selective autophagy causes drastic perturbation of metabolism, which must be minimized to maintain intracellular homeostasis.
| S-EPMC4337068 | biostudies-literature
Project description:Polymorphic variants of the FTO (fat mass and obesity) gene associate with body mass index in humans, but the underlying molecular mechanisms have not been firmly determined. FTO is linked to energy homeostasis via amino acid sensing and is thought to activate the mammalian target of rapamycin complex 1, a negative regulator of autophagy. FTO localises both to the nucleus and the cytoplasm, and in this study we identify a functional nuclear localisation signal (NLS) in the N-terminus of FTO, as well as nuclear localization information in its very C-terminus. Inhibition of FTO nuclear transport has no effect on autophagy and in contrast to a previously proposed role of FTO in autophagy, we find no difference in starvation-induced autophagy in control cells compared to a panel of cell types depleted of FTO. Future studies that further characterise the cellular functions of FTO will be important to understand why variants in FTO are associated with body weight.
| S-EPMC5347998 | biostudies-literature
Project description:Hexokinase-II (HK-II) catalyzes the first step of glycolysis and also functions as a protective molecule; however, its role in protective autophagy has not been determined. Results showed that inhibition of HK-II diminished, while overexpression of HK-II potentiated, autophagy induced by glucose deprivation in cardiomyocyte and noncardiomyocyte cells. Immunoprecipitation studies revealed that HK-II binds to and inhibits the autophagy suppressor, mTOR complex 1 (TORC1), and that this binding was increased by glucose deprivation. The TOS motif, a scaffold sequence responsible for binding TORC1 substrates, is present in HK-II, and mutating it blocked its ability to bind to TORC1 and regulate protective autophagy. The transition from glycolysis to autophagy appears to be regulated by a decrease in glucose-6 phosphate. We suggest that HK-II binds TORC1 as a decoy substrate and provides a previously unrecognized mechanism for switching cells from a metabolic economy, based on plentiful energy, to one of conservation, under starvation.
| S-EPMC3943874 | biostudies-literature
Project description:AimsInsulin-like growth factor 1 (IGF-1) is known to exert cardioprotective actions. However, it remains unknown if autophagy, a major adaptive response to nutritional stress, contributes to IGF-1-mediated cardioprotection.Methods and resultsWe subjected cultured neonatal rat cardiomyocytes, as well as live mice, to nutritional stress and assessed cell death and autophagic rates. Nutritional stress induced by serum/glucose deprivation strongly induced autophagy and cell death, and both responses were inhibited by IGF-1. The Akt/mammalian target of rapamycin (mTOR) pathway mediated the effects of IGF-1 upon autophagy. Importantly, starvation also decreased intracellular ATP levels and oxygen consumption leading to AMP-activated protein kinase (AMPK) activation; IGF-1 increased mitochondrial Ca(2+) uptake and mitochondrial respiration in nutrient-starved cells. IGF-1 also rescued ATP levels, reduced AMPK phosphorylation and increased p70(S6K) phosphorylation, which indicates that in addition to Akt/mTOR, IGF-1 inhibits autophagy by the AMPK/mTOR axis. In mice harbouring a liver-specific igf1 deletion, which dramatically reduces IGF-1 plasma levels, AMPK activity and autophagy were increased, and significant heart weight loss was observed in comparison with wild-type starved animals, revealing the importance of IGF-1 in maintaining cardiac adaptability to nutritional insults in vivo.ConclusionOur data support the cardioprotective actions of IGF-1, which, by rescuing the mitochondrial metabolism and the energetic state of cells, reduces cell death and controls the potentially harmful autophagic response to nutritional challenges. IGF-1, therefore, may prove beneficial to mitigate damage induced by excessive nutrient-related stress, including ischaemic disease in multiple tissues.
| S-EPMC3286200 | biostudies-literature