Endoplasmic Reticulum Membrane and Contact Site Dynamics in Autophagy Regulation and Stress Response.
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ABSTRACT:
SUBMITTER: Morel E
PROVIDER: S-EPMC7272771 | biostudies-literature | 2020
REPOSITORIES: biostudies-literature
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Project description:Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated ?-super-tether (?-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in ?-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in ?-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of ?-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.
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Project description:Duck enteritis virus (DEV) can infect ducks, geese, and many other poultry species and leads to acute, septic and highly fatal infectious disease. Autophagy is an evolutionarily ancient pathway that plays an important role in many viral infections. We previously reported that DEV infection induces autophagy for its own benefit, but how this occurs remains unclear. In this study, endoplasmic reticulum (ER) stress was triggered by DEV infection, as demonstrated by the increased expression of the ER stress marker glucose-regulated protein 78 (GRP78) and the dilated morphology of the ER. Pathways associated with the unfolded protein response (UPR), including the PKR-like ER protein kinase (PERK) and inositol-requiring enzyme 1 (IRE1) pathways, but not the activating transcription factor 6 (ATF6) pathway, were activated in DEV-infected duck embryo fibroblast (DEF) cells. In addition, the knockdown of both PERK and IRE1 by small interfering RNAs (siRNAs) reduced the level of LC3-II and viral yields, which suggested that the PERK-eukaryotic initiation factor 2α (eIF2α) and IRE1-x-box protein1 (XBP1) pathways may contribute to DEV-induced autophagy. Collectively, these data offer new insight into the mechanisms of DEV -induced autophagy through activation of the ER stress-related UPR pathway.
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Project description:Synthesis of phospholipids, sterols and sphingolipids is thought to occur at contact sites between the endoplasmic reticulum (ER) and other organelles because many lipid-synthesizing enzymes are enriched in these contacts. In only a few cases have the enzymes been localized to contacts in vivo and in no instances have the contacts been demonstrated to be required for enzyme function. Here, we show that plasma membrane (PM)--ER contact sites in yeast are required for phosphatidylcholine synthesis and regulate the activity of the phosphatidylethanolamine N-methyltransferase enzyme, Opi3. Opi3 activity requires Osh3, which localizes to PM-ER contacts where it might facilitate in trans catalysis by Opi3. Thus, membrane contact sites provide a structural mechanism to regulate lipid synthesis.
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Project description:Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased PM integrity under multiple abiotic stresses, such as freezing, high salt, osmotic stress, and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild-type while the levels of most glycerolipid species remain unchanged. In addition, the SYT1-green fluorescent protein fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.
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Project description:The syncytiotrophoblast (STB) is a multinuclear layer forming the outer surface of the fetal part of the placenta deriving from villous cytotrophoblastic cell (vCTB) fusion and differentiation. This syncytialization process is characterized by morphological and biochemical alterations of the trophoblast, which probably require removal of pre-existing structures and proteins to maintain cell homeostasis and survival. Interestingly, autophagy, which allows degradation and recycling of cellular components, was shown to be activated in syncytiotrophoblast. Here we examined the involvement of endoplasmic reticulum stress (ERS) response in autophagy activation during vCTB syncytialization. We first demonstrated the activation of ERS response and autophagy during the time course of trophoblastic cell fusion and differentiation. Alteration of autophagy activation in vCTB by chemical treatments or Beclin-1 expression modulation leads to a decrease in trophoblastic syncytialization. Furthermore, ERS response inhibition by chemical treatment or siRNA strategy leads to a default in syncytialization, associated with alteration of autophagy markers and cell survival. From these data, we suggest that ERS response, by fine regulation of autophagy activation, may serve as an adaptive mechanism to promote cell survival during trophoblastic syncytialization.
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Project description:Accumulation of misfolded proinsulin in the ?-cell leads to dysfunction induced by endoplasmic reticulum (ER) stress, with diabetes as a consequence. Autophagy helps cellular adaptation to stress via clearance of misfolded proteins and damaged organelles. We studied the effects of proinsulin misfolding on autophagy and the impact of stimulating autophagy on diabetes progression in Akita mice, which carry a mutation in proinsulin, leading to its severe misfolding. Treatment of female diabetic Akita mice with rapamycin improved diabetes, increased pancreatic insulin content, and prevented ?-cell apoptosis. In vitro, autophagic flux was increased in Akita ?-cells. Treatment with rapamycin further stimulated autophagy, evidenced by increased autophagosome formation and enhancement of autophagosome-lysosome fusion. This was associated with attenuation of cellular stress and apoptosis. The mammalian target of rapamycin (mTOR) kinase inhibitor Torin1 mimicked the rapamycin effects on autophagy and stress, indicating that the beneficial effects of rapamycin are indeed mediated via inhibition of mTOR. Finally, inhibition of autophagy exacerbated stress and abolished the anti-ER stress effects of rapamycin. In conclusion, rapamycin reduces ER stress induced by accumulation of misfolded proinsulin, thereby improving diabetes and preventing ?-cell apoptosis. The beneficial effects of rapamycin in this context strictly depend on autophagy; therefore, stimulating autophagy may become a therapeutic approach for diabetes.
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Project description:Metabolic disorders such as obesity and nonalcoholic fatty liver disease (NAFLD) are emerging disorders that affect the global population. One facet of the disorders is attributed to the disturbance of membrane lipid homeostasis. Perturbation of endoplasmic reticulum (ER) homeostasis through changes in membrane phospholipid composition results in activation of the unfolded protein response (UPR) and causes dramatic translational and transcriptional changes in cell. To restore cellular homeostasis, the three highly conserved UPR transducers ATF6, IRE1, and PERK mediate cellular processes upon ER stress. The roles of the UPR in proteostatic stress caused by the accumulation of misfolded protein is well understood but lipid perturbation-induced UPR remains elusive. We found that genetically attenuated PC synthesis in C. elegans causes lipid droplets accumulation if not for the intervention of the UPR program. Transcriptional profiling of lipid perturbation-induced ER stress animals shows a unique subset of genes modulated in an UPR-dependent manner that are unaffected by proteostatic stress. Among these, we identified IRE1-modulated autophagy genes that trigger liberation of free fatty acids from excess lipid droplets suggesting a stress release mechanism by which free fatty acids are rechannelling to restore lipid homeostasis. Considering the important role of lipid homeostasis and how its impairment contributes to the pathologies in metabolic diseases, our data uncovers the indispensable role of a fully functional UPR program in regulating lipid homeostais in the face of chronic ER stress.
2018-10-31 | GSE99763 | GEO
Project description:Roots are the frontier of plant body to perceive underground environmental change. Endoplasmic reticulum (ER) stress response represents circumvention of cellular stress caused by various environmental changes; however, a limited number of studies are available on the ER stress responses in roots. Here, we report the tunicamycin (TM) -induced ER stress response in Arabidopsis roots by monitoring expression patterns of immunoglobulin-binding protein 3 (BiP3), a representative marker for the response. Roots promptly responded to the TM-induced ER stress through the induction of similar sets of ER stress-responsive genes. However, not all cells responded uniformly to the TM-induced ER stress in roots, as BiP3 was highly expressed in root tips, an outer layer in elongation zone, and an inner layer in mature zone of roots. We suggest that ER stress response in roots has tissue specificity.
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Project description:Autophagy is an evolutionally conserved process for the bulk degradation of cytoplasmic proteins and organelles. Recent observations indicate that autophagy is induced in response to cellular insults that result in the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER). However, the signaling mechanisms that activate autophagy under these conditions are not understood. Here, we report that ER stress-induced autophagy requires the activation of protein kinase C (PKC), a member of the novel-type PKC family. Induction of ER stress by treatment with either thapsigargin or tunicamycin activated autophagy in immortalized hepatocytes as monitored by the conversion LC3-I to LC3-II, clustering of LC3 into dot-like cytoplasmic structures, and electron microscopic detection of autophagosomes. Pharmacological inhibition of PKC or small interfering RNA-mediated knockdown of PKC prevented the autophagic response to ER stress. Treatment with ER stressors induced PKC phosphorylation within the activation loop and localization of phospho-PKC to LC3-containing dot structures in the cytoplasm. However, signaling through the known unfolded protein response sensors was not required for PKC activation. PKC activation and stress-induced autophagy were blocked by chelation of intracellular Ca(2+) with BAPTA-AM. PKC was not activated or required for autophagy in response to amino acid starvation. These observations indicate that Ca(2+)-dependent PKC activation is specifically required for autophagy in response to ER stress but not in response to amino acid starvation.
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Project description:Background Glioblastoma (GBM; grade IV glioma) is characterized by a very short overall survival time and extremely low 5-year survival rates. We intend to promote experimental and clinical research on rationale and scientifically driven drug repurposing. This may represent a safe and often inexpensive way to propose novel pharmacological approaches to GBM. Our precedent work describes the role of chlorpromazine (CPZ) in hindering malignant features of GBM. Here, we investigate in greater detail the molecular mechanisms at the basis of the effect of CPZ on GBM cells. Methods We employed proteomics platforms, i.e., activity-based protein profiling plus mass spectrometry, to identify potential cellular targets of the drug. Then, by means of established molecular and cellular biology techniques, we assessed the effects of this drug on GBM cell metabolic and survival pathways. Results The experimental output indicated as putative targets of CPZ several of factors implicated in endoplasmic reticulum (ER) stress, with consequent unfolded protein response (UPR). Such a perturbation culminated in a noticeable reactive oxygen species generation and intense autophagic response that resulted in cytotoxic and abortive effects for six GBM cell lines, three of which growing as neurospheres, while it appeared cytoprotective for the RPE-1 human non-cancer neuro-ectodermal cell line. Conclusions This discrepancy could be central in explaining the lethal effects of the drug on GBM cells and the relatively scarce cytotoxicity toward normal tissues attributed to this compound. The data presented here offer support to the multicenter phase II clinical trial we have undertaken, which consists of the addition of CPZ to first-line treatment of GBM patients carrying a hypo- or un-methylated MGMT gene, i.e. those characterized by intrinsic resistance to temozolomide. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-021-02144-w.
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