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1.
J Biol Chem ; 294(20): 8197-8217, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-30926605

RESUMO

Endoplasmic reticulum (ER) stress is thought to activate autophagy via unfolded protein response (UPR)-mediated transcriptional up-regulation of autophagy machinery components and modulation of microtubule-associated protein 1 light chain 3 (LC3). The upstream UPR constituents pancreatic EIF2-α kinase (PERK) and inositol-requiring enzyme 1 (IRE1) have been reported to mediate these effects, suggesting that UPR may stimulate autophagy via PERK and IRE1. However, how the UPR and its components affect autophagic activity has not been thoroughly examined. By analyzing the flux of LC3 through the autophagic pathway, as well as the sequestration and degradation of autophagic cargo, we here conclusively show that the classical ER stressor tunicamycin (TM) enhances autophagic activity in mammalian cells. PERK and its downstream factor, activating transcription factor 4 (ATF4), were crucial for this induction, but surprisingly, IRE1 constitutively suppressed autophagic activity. TM-induced autophagy required autophagy-related 13 (ATG13), Unc-51-like autophagy-activating kinases 1/2 (ULK1/ULK2), and GABA type A receptor-associated proteins (GABARAPs), but interestingly, LC3 proteins appeared to be redundant. Strikingly, ATF4 was activated independently of PERK in both LNCaP and HeLa cells, and our further examination revealed that ATF4 and PERK regulated autophagy through separate mechanisms. Specifically, whereas ATF4 controlled transcription and was essential for autophagosome formation, PERK acted in a transcription-independent manner and was required at a post-sequestration step in the autophagic pathway. In conclusion, our results indicate that TM-induced UPR activates functional autophagy, and whereas IRE1 is a negative regulator, PERK and ATF4 are required at distinct steps in the autophagic pathway.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Morte Celular Autofágica/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/genética , Morte Celular Autofágica/genética , Autofagossomos/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Células PC-3 , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Resposta a Proteínas não Dobradas/genética , eIF-2 Quinase/genética
2.
Methods Mol Biol ; 1880: 307-313, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610706

RESUMO

The nonselective bulk sequestration of cytoplasm and its subsequent delivery to lysosomes for degradation was originally defined as autophagy or macroautophagy. However, both terms are now increasingly being applied in a generic sense to encompass the many recently described mechanisms for selective sequestration and degradation of individual cellular elements. We will therefore use the term bulk autophagy to denote the non-exclusive and largely nonselective process.Bulk autophagy can be measured directly by a cargo sequestration assay, using a cargo marker representative of total cytoplasm. The assay described here measures the sequestration and accumulation of the ubiquitous cytosolic protein lactate dehydrogenase (LDH) in the sedimentable autophagic vacuoles of cells incubated with an inhibitor of intravacuolar degradation such as bafilomycin or leupeptin. Electrodisruption of the plasma membrane followed by centrifugal sedimentation of the "cell corpses" (which retain their organelles in an intact state, bound to the cytoskeleton) is a convenient, efficient, and reproducible way to separate the small fraction of sequestered, sedimentable LDH from the major pool of cytosolic LDH.


Assuntos
Autofagossomos/metabolismo , Autofagia , L-Lactato Desidrogenase/metabolismo , Animais , Técnicas de Cultura de Células/métodos , Citosol/metabolismo , Eletroporação/métodos , Ensaios Enzimáticos/métodos , Humanos , L-Lactato Desidrogenase/análise , Leupeptinas/farmacologia , Lisossomos/metabolismo , Macrolídeos/farmacologia
3.
J Vis Exp ; (137)2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-30102280

RESUMO

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.


Assuntos
L-Lactato Desidrogenase/metabolismo , Animais , Autofagia , Bioensaio , Células Cultivadas
4.
Autophagy ; 14(8): 1435-1455, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29940786

RESUMO

Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for degradation. Autophagy is involved in the pathophysiology of numerous diseases and its modulation is beneficial for the outcome of numerous specific diseases. Several lysosomal inhibitors such as bafilomycin A1 (BafA1), protease inhibitors and chloroquine (CQ), have been used interchangeably to block autophagy in in vitro experiments assuming that they all primarily block lysosomal degradation. Among them, only CQ and its derivate hydroxychloroquine (HCQ) are FDA-approved drugs and are thus currently the principal compounds used in clinical trials aimed to treat tumors through autophagy inhibition. However, the precise mechanism of how CQ blocks autophagy remains to be firmly demonstrated. In this study, we focus on how CQ inhibits autophagy and directly compare its effects to those of BafA1. We show that CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle. Furthermore, CQ induces an autophagy-independent severe disorganization of the Golgi and endo-lysosomal systems, which might contribute to the fusion impairment. Strikingly, HCQ-treated mice also show a Golgi disorganization in kidney and intestinal tissues. Altogether, our data reveal that CQ and HCQ are not bona fide surrogates for other types of late stage lysosomal inhibitors for in vivo experiments. Moreover, the multiple cellular alterations caused by CQ and HCQ call for caution when interpreting results obtained by blocking autophagy with this drug.


Assuntos
Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Cloroquina/farmacologia , Lisossomos/metabolismo , Fusão de Membrana/efeitos dos fármacos , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/ultraestrutura , Linhagem Celular Tumoral , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/ultraestrutura , Receptores ErbB/metabolismo , Feminino , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Hidroxicloroquina/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/ultraestrutura , Macrolídeos/farmacologia , Camundongos Endogâmicos C57BL , Proteólise/efeitos dos fármacos , Proteína Sequestossoma-1/metabolismo
5.
Bio Protoc ; 8(9): e2836, 2018 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-34286043

RESUMO

Autophagy is a key player in the maintenance of cellular homeostasis in eukaryotes, and numerous diseases, including cancer and neurodegenerative disorders, are associated with alterations in autophagy. The interest for studying autophagy has grown intensely in the last two decades, and so has the arsenal of methods utilised to study this highly dynamic and complex process. Changes in the expression and/or localisation of autophagy-related proteins are frequently assessed by Western blot and various microscopy techniques. Such analyses may be indicative of alterations in autophagy-related processes and informative about the specific marker being investigated. However, since these proteins are part of the autophagic machinery, and not autophagic cargo, they cannot be used to draw conclusions regarding autophagic cargo flux. Here, we provide a protocol to quantitatively assess bulk autophagic flux by employing the long-lived protein degradation assay. Our procedure, which traces the degradation of 14C valine-labelled proteins, is simple and quick, allows for processing of a relatively large number of samples in parallel, and can in principle be used with any adherent cell line. Most importantly, it enables quantitative measurements of endogenous cargo flux through the autophagic pathway. As such, it is one of the gold standards for studying autophagic activity.

6.
Artigo em Inglês | MEDLINE | ID: mdl-28516064

RESUMO

Bacterial membrane vesicle (MV) production has been mainly studied in Gram-negative species. In this study, we show that Listeria monocytogenes, a Gram-positive pathogen that causes the food-borne illness listeriosis, produces MVs both in vitro and in vivo. We found that a major virulence factor, the pore-forming hemolysin listeriolysin O (LLO), is tightly associated with the MVs, where it resides in an oxidized, inactive state. Previous studies have shown that LLO may induce cell death and autophagy. To monitor possible effects of LLO and MVs on autophagy, we performed assays for LC3 lipidation and LDH sequestration as well as analysis by confocal microscopy of HEK293 cells expressing GFP-LC3. The results revealed that MVs alone did not affect autophagy whereas they effectively abrogated autophagy induced by pure LLO or by another pore-forming toxin from Vibrio cholerae, VCC. Moreover, Listeria monocytogenes MVs significantly decreased Torin1-stimulated macroautophagy. In addition, MVs protected against necrosis of HEK293 cells caused by the lytic action of LLO. We explored the mechanisms of LLO-induced autophagy and cell death and demonstrated that the protective effect of MVs involves an inhibition of LLO-induced pore formation resulting in inhibition of autophagy and the lytic action on eukaryotic cells. Further, we determined that these MVs help bacteria to survive inside eukaryotic cells (mouse embryonic fibroblasts). Taken together, these findings suggest that intracellular release of MVs from L. monocytogenes may represent a bacterial strategy to survive inside host cells, by its control of LLO activity and by avoidance of destruction from the autophagy system during infection.


Assuntos
Autofagia/efeitos dos fármacos , Toxinas Bacterianas/farmacologia , Morte Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proteínas de Choque Térmico/farmacologia , Proteínas Hemolisinas/farmacologia , Listeria monocytogenes/metabolismo , Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Membrana Celular/microbiologia , Citoplasma/metabolismo , Células HEK293 , Células HeLa , Humanos , Listeria monocytogenes/citologia , Listeria monocytogenes/patogenicidade , Listeriose/metabolismo , Macrófagos/microbiologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Naftiridinas/farmacologia , Células RAW 264.7 , Fatores de Virulência/metabolismo
7.
Exp Cell Res ; 333(1): 21-38, 2015 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-25684710

RESUMO

LC3, a mammalian homologue of yeast Atg8, is assumed to play an important part in bulk sequestration and degradation of cytoplasm (macroautophagy), and is widely used as an indicator of this process. To critically examine its role, we followed the autophagic flux of LC3 in rat hepatocytes during conditions of maximal macroautophagic activity (amino acid depletion), combined with analyses of macroautophagic cargo sequestration, measured as transfer of the cytosolic protein lactate dehydrogenase (LDH) to sedimentable organelles. To accurately determine LC3 turnover we developed a quantitative immunoblotting procedure that corrects for differential immunoreactivity of cytosolic and membrane-associated LC3 forms, and we included cycloheximide to block influx of newly synthesized LC3. As expected, LC3 was initially degraded by the autophagic-lysosomal pathway, but, surprisingly, autophagic LC3-flux ceased after ~2h. In contrast, macroautophagic cargo flux was well maintained, and density gradient analysis showed that sequestered LDH partly accumulated in LC3-free autophagic vacuoles. Hepatocytic macroautophagy could thus proceed independently of LC3. Silencing of either of the two mammalian Atg8 subfamilies in LNCaP prostate cancer cells exposed to macroautophagy-inducing conditions (starvation or the mTOR-inhibitor Torin1) confirmed that macroautophagic sequestration did not require the LC3 subfamily, but, intriguingly, we found the GABARAP subfamily to be essential.


Assuntos
Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Animais , Proteína 5 Relacionada à Autofagia , Células Cultivadas , Masculino , Camundongos , Proteínas/metabolismo , Ratos Wistar , Vacúolos/metabolismo
8.
Methods ; 75: 25-36, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25576638

RESUMO

Macroautophagy, the process responsible for bulk sequestration and lysosomal degradation of cytoplasm, is often monitored by means of the autophagy-related marker protein LC3. This protein is linked to the phagophoric membrane by lipidation during the final steps of phagophore assembly, and it remains associated with autophagic organelles until it is degraded in the lysosomes. The transfer of LC3 from cytosol to membranes and organelles can be measured by immunoblotting or immunofluorescence microscopy, but these assays provide no information about functional macroautophagic activity, i.e., whether the phagophores are actually engaged in the sequestration of cytoplasmic cargo and enclosing this cargo into sealed autophagosomes. Moreover, accumulating evidence suggest that macroautophagy can proceed independently of LC3. There is therefore a need for alternative methods, preferably effective cargo sequestration assays, which can monitor actual macroautophagic activity. Here, we provide an overview of various approaches that have been used over the last four decades to measure macroautophagic sequestration activity in mammalian cells. Particular emphasis is given to the so-called "LDH sequestration assay", which measures the transfer of the autophagic cargo marker enzyme LDH (lactate dehydrogenase) from the cytosol to autophagic vacuoles. The LDH sequestration assay was originally developed to measure macroautophagic activity in primary rat hepatocytes. Subsequently, it has found use in several other cell types, and in this article we demonstrate a further validation and simplification of the method, and show that it is applicable to several cell lines that are commonly used to study autophagy.


Assuntos
Autofagia/genética , Lisossomos/metabolismo , Biologia Molecular/métodos , Células Cultivadas , Citoplasma/metabolismo , Células HeLa , Humanos , Immunoblotting/métodos , L-Lactato Desidrogenase/imunologia , L-Lactato Desidrogenase/metabolismo , Lisossomos/genética , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Associadas aos Microtúbulos/metabolismo
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