المؤلفون: Per Ottar Seglen, Nikolai Engedal, Paula Szalai, Morten Luhr

المصدر: Methods in Molecular Biology ISBN: 9781493988723

مصطلحات موضوعية: 0303 health sciences, 030303 biophysics, Autophagy, Leupeptin, Bafilomycin, Vacuole, Cell biology, 03 medical and health sciences, Cytosol, chemistry.chemical_compound, medicine.anatomical_structure, chemistry, Cytoplasm, Lysosome, Organelle, medicine, 030304 developmental biology

الوصف: 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.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::3b1ddca2232d7623f6c4379764b8139fTest
https://doi.org/10.1007/978-1-4939-8873-0_20Test

المؤلفون: Nikolai Engedal, Per Ottar Seglen

المصدر: Autophagy. 12:439-441

مصطلحات موضوعية: 0301 basic medicine, Cytoplasm, Thapsigargin, Cycloheximide, Biology, 03 medical and health sciences, chemistry.chemical_compound, Cell Line, Tumor, Lysosome, Lactate dehydrogenase, LNCaP, Autophagy, medicine, Views and Commentaries, Animals, Humans, Molecular Biology, Adenine, Cell Biology, Rats, Cell biology, 030104 developmental biology, medicine.anatomical_structure, chemistry, Hepatocyte, embryonic structures, Hepatocytes, biological phenomena, cell phenomena, and immunity, Microtubule-Associated Proteins, Flux (metabolism)

الوصف: To investigate the role of LC3 in bulk autophagy we compared its autophagic-lysosomal processing (using an improved quantitative immunoblotting method) with autophagic-lysosomal bulk cargo flux (measured by our established LDH [lactate dehydrogenase] sequestration assay) in amino acid-starved rat hepatocytes treated with cycloheximide to prevent new LC3 influx. Block-release experiments with the reversible autophagy inhibitors 3-methyladenine (3MA) and thapsigargin (TG) showed that while only 3MA suppressed phagophoric LC3 attachment (lipidation), both inhibitors prevented phagophore closure (cargo sequestration). Upon release from closure blockade, some autophagic-lysosomal LC3 flux was resumed even in the presence of 3MA, i.e., without an accompanying bulk cargo flux. Conversely, whereas the autophagic-lysosomal flux of LC3 halted within ∼100 min of cycloheximide treatment, the bulk cargo flux continued at a high rate. siRNA-mediated knockdown of LC3 family proteins in LNCaP prostate carcinoma cells confirmed that autophagy of cytoplasmic bulk cargo was completely LC3 independent also in these cells, and in the absence of cycloheximide. However, a strong requirement for GABARAP family proteins was evident. Since bulk autophagy of cytoplasm (macroautophagy) and autophagic-lysosomal LC3 processing may apparently be mutually independent, LC3 would seem to be unsuitable as a general indicator of autophagy.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::4eb5a1531968862e99ce9daa402b6e1cTest
https://doi.org/10.1080/15548627.2015.1076606Test

المؤلفون: Nikolai Engedal, L. Gerner, Frank Sætre, Paula Szalai, Per Ottar Seglen, Morten Luhr

مصطلحات موضوعية: 0301 basic medicine, Autophagosome, Cell type, Endosome, Autophagy, Vacuole, Biology, Cell biology, Cell membrane, Cellular material, 03 medical and health sciences, Cytosol, 030104 developmental biology, medicine.anatomical_structure, medicine

الوصف: Autophagy (self-eating) is a common term for various processes by which cellular components are transferred to lysosomes for degradation. In macroautophagy, intracellular membrane structures termed "phagophores" expand to encapsulate autophagic cargo into sealed, double-membrane vacuoles termed "autophagosomes," which subsequently may fuse with endosomes to form intermediary vacuoles called "amphisomes," and finally with lysosomes to have their contents degraded and recycled. Autophagy is frequently analyzed by monitoring phagophore- and autophagosome-associated markers such as LC3. Although useful, it is becoming increasingly clear that very few, if any, of these marker proteins are entirely specific to the autophagic process. Moreover, phagophore/autophagosome markers cannot be used to measure autophagic activity since they are part of the autophagic machinery, or "cart," rather than autophagic cargo. Thus, there is a great need for functional assays in autophagy research. Here, we describe a method that quantitatively measures the nonselective autophagic sequestration of endogenous cytosolic cargo. The method is based on a crude separation of sedimentable cellular material from cytosol and a subsequent measurement of the fraction of a cytosolic enzyme activity transferred to the sedimentable fraction by autophagic sequestration. The original assay was first developed in 1990, but during the last few years we have systematically downscaled and simplified the method into the time- and cost-efficient procedure presented here, which can be performed with standard laboratory equipment and is suitable for any cell type.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::ff4d6a3344a7b5dbf96662a30d271461Test
https://doi.org/10.1016/bs.mie.2016.09.064Test

المؤلفون: Hiroyuki Miyoshi, Ken Cadwell, Thaddeus S. Stappenbeck, Per Ottar Seglen, Tatsuya Saitoh, Khushbu Patel, Richard D. Head, Wandy L. Beatty, Jun-Lin Guan, Nicole P. Malvin, Herbert W. Virgin, Mary C. Dinauer, Shizuo Akira

المصدر: The EMBO Journal. 32:3130-3144

مصطلحات موضوعية: Goblet cell, NADPH oxidase, General Immunology and Microbiology, biology, Endosome, General Neuroscience, Autophagy, Mucin granule, Vacuole, Mucus, General Biochemistry, Genetics and Molecular Biology, Cell biology, medicine.anatomical_structure, Biochemistry, medicine, biology.protein, Secretion, Molecular Biology

الوصف: Delivery of granule contents to epithelial surfaces by secretory cells is a critical physiologic process. In the intestine, goblet cells secrete mucus that is required for homeostasis. Autophagy proteins are required for secretion in some cases, though the mechanism and cell biological basis for this requirement remain unknown. We found that in colonic goblet cells, proteins involved in initiation and elongation of autophagosomes were required for efficient mucus secretion. The autophagy protein LC3 localized to intracellular multi-vesicular vacuoles that were consistent with a fusion of autophagosomes and endosomes. Using cultured intestinal epithelial cells, we found that NADPH oxidases localized to and enhanced the formation of these LC3-positive vacuoles. Both autophagy proteins and endosome formation were required for maximal production of reactive oxygen species (ROS) derived from NADPH oxidases. Importantly, generation of ROS was critical to control mucin granule accumulation in colonic goblet cells. Thus, autophagy proteins can control secretory function through ROS, which is in part generated by LC3-positive vacuole-associated NADPH oxidases. These findings provide a novel mechanism by which autophagy proteins can control secretion.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::52e2c1451c778482dd29f5c2be68d211Test
https://doi.org/10.1038/emboj.2013.233Test

المؤلفون: Harald Thidemann Johansen, Frank Sætre, Linda Korseberg Hagen, Per Ottar Seglen, Anders Øverbye

المصدر: Autophagy. 7:1011-1027

مصطلحات موضوعية: Male, Time Factors, Cell Survival, Leupeptins, Proteolysis, Molecular Sequence Data, Vacuole, Legumain, Models, Biological, Mass Spectrometry, chemistry.chemical_compound, Ammonia, Sequence Analysis, Protein, Lysosome, Lactate dehydrogenase, Autophagy, medicine, Animals, Humans, Amino Acid Sequence, Asparagine, Rats, Wistar, Molecular Biology, L-Lactate Dehydrogenase, medicine.diagnostic_test, biology, Leupeptin, Cell Biology, Peptide Fragments, Basic Research Paper, Rats, Cysteine Endopeptidases, Cytosol, medicine.anatomical_structure, Betaine-Homocysteine S-Methyltransferase, chemistry, Biochemistry, Hepatocytes, biology.protein, Lysosomes

الوصف: To investigate the stepwise autophagic-lysosomal processing of hepatocellular proteins, the abundant cytosolic enzyme, betaine:homocysteine methyltransferase (BHMT) was used as a probe. Full-length (45 kDa) endogenous BHMT was found to be cleaved in an autophagy-dependent (3-methyladenine-sensitive) manner in isolated rat hepatocytes to generate a novel N-terminal 10-kDa fragment (p10) identified and characterized by mass spectrometry. The cleavage site was consistent with cleavage by the asparaginyl proteinase, legumain and indeed a specific inhibitor of this enzyme (AJN-230) was able to completely suppress p10 formation in intact cells, causing instead accumulation of a 42-kDa intermediate. To prevent further degradation of p10 or p42 by the cysteine proteinases present in autophagic vacuoles, the proteinase inhibitor leupeptin had to be present. Asparagine, an inhibitor of amphisome-lysosome fusion, did not detectably impede either p42 or p10 formation, indicating that BHMT processing primarily takes place in amphisomes rather than in lysosomes. Lactate dehydrogenase (LDH) was similarly degraded primarily in amphisomes by leupeptin-sensitive proteolysis, but some additional leupeptin-resistant LDH degradation in lysosomes was also indicated. The autophagic sequestration of BHMT appeared to be nonselective, as the accumulation of p10 (in the presence of leupeptin) or of its precursors (in the additional presence of AJN-230) proceeded at approximately the same rate as the model autophagic cargo, LDH. The complete lack of a cytosolic background makes p10 suitable for use in a "fragment assay" of autophagic activity in whole cells. Incubation of hepatocytes with ammonium chloride, which neutralizes amphisomes as well as lysosomes, caused rapid, irreversible inhibition of legumain activity and stopped all p10 formation. The availability of several methods for selective targeting of legumain in intact cells may facilitate functional studies of this enigmatic enzyme, and perhaps suggest novel ways to reduce its contribution to cancer cell metastasis or autoimmune disease.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::84547638f57adc593bb2bffc1fb554c2Test
https://doi.org/10.4161/auto.7.9.16436Test

المؤلفون: Egil S. Erichsen, Trond Berg, Takashi Ueno, Camilla Raiborg, Per E. Stromhaug, Monica Fengsrud, Per Ottar Seglen

المصدر: Biochemical Journal. 352:773-781

مصطلحات موضوعية: Autophagosome, Endosome, Endoplasmic reticulum, Autophagy, Cell Biology, Biology, Mitochondrion, Biochemistry, Cell biology, Cytosol, medicine.anatomical_structure, Lysosome, medicine, Molecular Biology, Peptide sequence

الوصف: In a search for autophagosome-associated proteins, two-dimensional gel separations of proteins from purified autophagosomes, postnuclear supernatant, cytosol, lysosomes, mitochondria, endosomes and a cytomembrane fraction (mostly endoplasmic reticulum) were compared. Three proteins, with monomeric molecular masses of 43, 35 and 31kDa, were enriched in total or sedimentable fractions of autophagosomes relative to the corresponding fractions of postnuclear supernatant, suggesting an association with the autophagosomal delimiting membrane. These proteins were also present on lysosomal membranes, but they were absent from mitochondria, and detected only in small amounts in the cytomembrane fraction and in endosomes, indicating that they were not associated with organelles sequestered by autophagy. However, all three proteins were present in the cytosol, suggesting that they were cytosolic proteins binding peripherally to the delimiting membrane of autophagosomes, probably to its innermost surface as indicated by their resistance to treatment of intact autophagosomes with proteinase or protein-stripping agents. Amino acid sequencing identified these proteins as an isoform of argininosuccinate synthase, an N-truncated variant of glyceraldehyde-3-phosphate dehydrogenase, and a sequence variant of short-chain 2-enoyl-CoA hydratase.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::1ae3aeca1674f70f6adebf61eda6fe9bTest
https://doi.org/10.1042/bj3520773Test

المؤلفون: Hamid R. Samari, Per Ottar Seglen

المصدر: Journal of Biological Chemistry. 273:23758-23763

مصطلحات موضوعية: biology, Autophagy, AMPK, Cell Biology, Adenosine kinase, Riboside, Biochemistry, Adenosine, chemistry.chemical_compound, Adenosine deaminase, AMP-activated protein kinase, chemistry, biology.protein, medicine, Protein kinase A, Molecular Biology, medicine.drug

الوصف: To examine the role of AMP-activated protein kinase (AMPK; EC 2.7.1.109) in the regulation of autophagy, rat hepatocytes were incubated with the AMPK proactivators, adenosine, 5-amino-4-imidazole carboxamide riboside (AICAR), orN 6-mercaptopurine riboside. Autophagic activity was inhibited by all three nucleosides, AICAR andN 6-mercaptopurine riboside being more potent (IC50 = 0.3 mm) than adenosine (IC50 = 1 mm). 2′-Deoxycoformycin, an adenosine deaminase (EC 3.5.4.4) inhibitor, increased the potency of adenosine 5-fold, suggesting that the effectiveness of adenosine as an autophagy inhibitor was curtailed by its intracellular deamination. 5-Iodotubercidin, an adenosine kinase (EC 2.7.1.20) inhibitor, abolished the effects of all three nucleosides, indicating that they needed to be phosphorylated to inhibit autophagy. A 5-iodotubercidin-suppressible phosphorylation of AICAR to 5-aminoimidazole-4-carboxamide riboside monophosphate was confirmed by chromatographic analysis. AICAR, up to 0.4 mm, had no significant effect on intracellular ATP concentrations. Because activated AMPK phosphorylates and inactivates 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (EC 1.1.1.88), the rate-limiting enzyme in cholesterol synthesis, the strong inhibition of hepatocytic cholesterol synthesis by all three nucleosides confirmed their ability to activate AMPK under the conditions used. Lovastatin and simvastatin, inhibitors of HMG-CoA reductase, strongly suppressed cholesterol synthesis while having no effect on autophagic activity, suggesting that AMPK inhibits autophagy independently of its effects on HMG-CoA reductase and cholesterol metabolism.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::9b71eb6b6a62831a024c3d6798bd0c26Test
https://doi.org/10.1074/jbc.273.37.23758Test

المؤلفون: Per Ottar Seglen

المصدر: Cell Biology and Toxicology. 13:301-315

مصطلحات موضوعية: Liver cytology, Cell growth, Health, Toxicology and Mutagenesis, Autophagy, Cell Biology, Protein degradation, Biology, Toxicology, medicine.disease_cause, Cell biology, medicine.anatomical_structure, Hepatocyte, Immunology, Cancer cell, medicine, Stem cell, Carcinogenesis

الوصف: Hepatocytes have the ability to go through specialized cell cycles, which, during normal developmental liver growth, result in the formation of binuclear and polyploid cells. In the adult rat liver, the majority of the hepatocytes (about 70%) are tetraploid, 15-20% are octoploid, and only 10-15% are diploid (about 50% in humans). One-third of the hepatocytes in either rats or humans are binuclear (with two diploid or two tetraploid nuclei). Among cultured rat hepatocytes stimulated with growth factors (EGF and insulin), one-half of the mitoses are of the binucleating type (suggesting a "quantal" mechanism), causing one-third of the postmitotic cells to become binuclear. In contrast, regenerative liver growth, induced by partial hepatectomy, is predominantly nonbinucleating. During rat liver carcinogenesis, the early populations of phenotypically altered cells (foci) are predominantly diploid, as are the later neoplastic nodules and carcinomas, which can be shown to have a regeneration-like, largely nonbinucleating growth pattern. A negative correlation between growth capacity and ploidy can be demonstrated in cultured hepatocytes, regenerating livers, neoplastic nodules, and hepatocellular carcinomas, suggesting that suppression of binucleation and polyploidization may carry a growth advantage, in addition to helping to maintain a large population of diploid, potential stem cells. Since a diploid genome is less protected against mutagenic change than a polyploid genome, diploid tumor cells may, furthermore, be more prone than polyploid cells to undergo mutation-based progression toward increasing malignancy. The ability of liver tumor promoters like 2-acetylaminofluorene, cyproterone acetate, alpha-hexachlorocyclohexane and methylclofenapate to induce nonbinucleating hepatocyte growth may, therefore, cooperate with the selective growth stimulation of cancer cells and cancer cell precursors to promote liver carcinogenesis. Autophagy, a mechanism for the bulk degradation of cytoplasm, contributes to intracellular protein turnover and serves to restrict cellular growth. Rat liver carcinogenesis is accompanied by a progressive reduction of autophagic capacity, preneoplastic livers having 50% and hepatocellular carcinoma cells only 20% as much autophagy as normal hepatocytes. The ascites hepatoma cell line AH-130 has virtually no autophagy during logarithmic growth, but some autophagy is turned on when the cells become growth-arrested at high cell density. Ascitic fluid from AH-130 cells is able to completely inhibit autophagy in normal hepatocytes, suggesting that the cancer cells may improve their growth ability through an autocrine, autophagy-suppressive mechanism. Hepatocytes from preneoplastic livers similarly maintain a low autophagic activity under restrictive culture conditions, thereby surviving much better than normal hepatocytes, which switch on their autophagy. In the presence of an autophagy inhibitor (3-methyladenine), normal and preneoplastic hepatocytes survive equally well, testifying to the importance of autophagy as a determinant of cell survival and growth.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::c8dab4e06e897492e058dc826a5cd77dTest
https://doi.org/10.1023/a:1007487425047Test

المؤلفون: Trond Olav Berg, Per Ottar Seglen, Per E. Stromhaug, Trond Berg

المصدر: European Journal of Biochemistry. 221:595-602

مصطلحات موضوعية: Male, Leupeptins, Centrifugation, Vacuole, Cell Fractionation, Vinblastine, Biochemistry, Cathepsin C, chemistry.chemical_compound, Methionine, Phagosomes, Lysosome, Autophagy, Centrifugation, Density Gradient, medicine, Animals, Rats, Wistar, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases, Phagosome, L-Lactate Dehydrogenase, biology, Leupeptin, Acid phosphatase, Dipeptides, Rats, Cytosol, medicine.anatomical_structure, Liver, chemistry, biology.protein, Cell fractionation, Lysosomes

الوصف: In density-gradient analyses of autophagic vacuoles from isolated rat hepatocytes, autophagosomes could be recognized by the presence of an autophagically sequestered cytosolic enzyme, lactate dehydrogenase (LDH). Lysosomes were identified by marker enzymes such as acid phosphatase, or by degradation products from 125I-tyramine-cellobiose-asialoorosomucoid (125I-TC-AOM) loaded into the lysosomes by an intravenous injection in vivo 18 h prior to cell isolation. Autophagosomes and lysosomes showed similar, largely overlapping, density distributions both in hypertonic sucrose gradients and in isotonic Nycodenz gradients. As a step towards the purification of autophagosomes, we investigated the possibility of using lysosomal enzyme substrates to achieve selective destruction of lysosomes by swelling. Hepatocytes were first incubated for 2 h at 37 degrees C with vinblastine (50 microM) to obtain an accumulation of autophagosomes (to 3-5-times above the control level). The cells were then electrodisrupted and the disruptates incubated with a variety of substrates for lysosomal enzymes. Among these, glycyl-phenylalanine-2-naphthylamide (GPN), a cathepsin-C substrate, and methionine-O-methylester (MetOMe), an esterase substrate, turned out to induce extensive rupture of lysosomes, as measured by a strongly reduced sedimentability of acid phosphatase and a nearly complete loss of 125I-TC-AOM sedimentability in substrate-treated preparations from control or vinblastine-treated cells. The lysosomes of cells treated with leupeptin or asparagine were largely resistant to the action of GPN, probably as a result of interference with cathepsin-C activity or lysosomal function in general. Autophagosomes were partially destroyed by MetOMe, as indicated by a reduction in sedimentable LDH, but GPN had no effect on either autophagosomes or mitochondria. The ability of GPN to selectively destroy lysosomes without affecting the autophagosomes of vinblastine-treated cells should make GPN treatment a useful aid in the purification of rat liver autophagosomes.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::2fc1e421dad7ff5a6e07b706e6cff75bTest
https://doi.org/10.1111/j.1432-1033.1994.tb18771.xTest

المؤلفون: Camilla Raiborg, Monica Fengsrud, Egil S. Erichsen, Trond Berg, Per Ottar Seglen

المصدر: European journal of cell biology. 79(12)

مصطلحات موضوعية: Autophagosome, Histology, Endosome, Biotin, Vacuole, Biology, Pathology and Forensic Medicine, Lysosome, Phagosomes, Organelle, medicine, Autophagy, Animals, Freeze Fracturing, Cryoelectron Microscopy, Membrane Proteins, Cell Biology, General Medicine, Intracellular Membranes, Transmembrane protein, Cell biology, Rats, Membrane, medicine.anatomical_structure, Membrane protein, Vacuoles

الوصف: The delimiting membranes of isolated autophagosomes from rat liver had extremely few transmembrane proteins, as indicated by the paucity of intramembrane particles in freeze-fracture images (about 20 particles/microm2, whereas isolated lysosomes had about 2000 particles/microm2). The autophagosomes also appeared to lack peripheral surface membrane proteins, since attempts to surface-biotinylate intact autophagosomes only yielded biotinylation of proteins from contaminating damaged mitochondria. All the membrane layers of multilamellar autophagosomes were equally particle-poor; the same was true of the autophagosome-forming, sequestering membrane complexes (phagophores). Isolated amphisomes (vacuoles formed by fusion between autophagosomes and endosomes) had more intramembrane particles than the autophagosomes (about 90 particles/microm2), and freeze-fracture images of these organelles frequently showed particle-rich endosomes fusing with particle-poor or particle-free autophagosomes. The appearence of multiple particle clusters suggested that a single autophagic vacuole could undergo multiple fusions with endosomes. Only the outermost membrane of bi- or multilamellar autophagic vacuoles appeared to engage in such fusions.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::9ca1d291f557fe97bf580b3bb6fa8bfaTest
https://pubmed.ncbi.nlm.nih.gov/11152279Test