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, 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: 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: 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: Trond Berg, Per Ottar Seglen, Henrietta Blankson, Monica Fengsrud, Ingunn Holen, Per E. Stromhaug

المصدر: Intracellular Protein Catabolism ISBN: 9781461380030

مصطلحات موضوعية: Cytosol, Membrane, medicine.anatomical_structure, Chemistry, Hepatocyte, Organelle, Autophagy, medicine, Microsome, Peroxisome, Cathepsin C, Cell biology

الوصف: As a first step towards isolation of autophagic sequestering membranes (phagophores), we have purified autophagosomes from rat hepatocytes. Lysosomes were selectively destroyed by osmotic rupture, achieved by incubation of hepatocyte homogenates with the cathepsin C substrate glycyl-phenylalanyl-naphthylamide (GPN). Mitochondria and peroxisomes were removed by Nycodenz gradient centrifugation, and cytosol, microsomes and other organelles by rate sedimentation through metrizamide cushions. The purified autophagosomes were bordered by dual or multiple concentric membranes, suggesting that autophagic sequestration might be performed either by single autophagic cisternae or by cisternal stacks.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::2ed1426407d80af78eaeaf976720a95cTest
https://doi.org/10.1007/978-1-4613-0335-0_12Test

المؤلفون: Per Ottar Seglen, Helge Tolleshaug

المصدر: European Journal of Biochemistry. 153:223-229

مصطلحات موضوعية: Male, Sucrose, Density gradient, Acid Phosphatase, Digitonin, Mitochondria, Liver, In Vitro Techniques, Mitochondrion, Biochemistry, chemistry.chemical_compound, Lysosome, Organelle, Autophagy, Centrifugation, Density Gradient, medicine, Animals, biology, Adenine, Metrizamide, Temperature, Acid phosphatase, Rats, Inbred Strains, Rats, Cytosol, medicine.anatomical_structure, chemistry, Isopycnic, Chromatography, Gel, biology.protein, Lysosomes, Subcellular Fractions

الوصف: [14C]Sucrose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, was sequestered by sedimentable subcellular particles during incubation of the cells at 37 degrees C. The sedimentation characteristics of particle-associated [14C]sucrose were different from the lysosomal marker enzyme acid phosphatase, suggesting an involvement of organelles of greater size than the average lysosome. Isopycnic banding in isotonic metrizamide/sucrose density gradients resolved two major peaks of radioactivity: a light peak (1.08-1.10 g/ml) coinciding with lysosomal marker enzymes, and a dense peak (1.15 g/ml), coinciding with a mitochondrial marker enzyme. The dense peak was preferentially associated with large-size particles having the sedimentation properties of mitochondria, and it was resistant to the detergent digitonin at a concentration which extracted all of the radioactivity in the light peak. Similarly the autophagy inhibitor 3-methyladenine prevented accumulation of [14C]sucrose in the light peak, while the radioactivity in the dense peak was unaffected. We therefore tentatively conclude that the light peak represents autophagic sequestration of [14C]sucrose into lysosomes (and probably autophagosomes) while the dense peak represents a mitochondrial uptake unrelated to autophagy.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::dc3d7372c99e63010b184e801dd8ae7cTest
https://doi.org/10.1111/j.1432-1033.1985.tb09290.xTest

المؤلفون: Per Ottar Seglen, H. Hoyvik, P. B. Gordon

المصدر: Experimental cell research. 166(1)

مصطلحات موضوعية: Male, Sucrose, Density gradient, Lactose, Vacuole, Biology, Vinblastine, chemistry.chemical_compound, Phagocytosis, Lysosome, Phagosomes, Organelle, medicine, Autophagy, Animals, Phagosome, Adenine, Hydrolysis, Chloroquine, Rats, Inbred Strains, Cell Biology, Rats, Cytosol, medicine.anatomical_structure, Biochemistry, chemistry, Liver, Lysosomes

الوصف: [14C]Lactose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, is sequestered autophagically in the same way as the established sequestration probe, [14C]sucrose. However, unlike the inert sucrose molecule, lactose is rapidly hydrolysed in the lysosomes, and can therefore be used to probe the last step of the autophagic pathway (i.e. fusion with the lysosome). During autophagy lactose is present only at a low, steady-state level in pre-lysosomal vacuoles (probably autophagosomes), serving as a useful marker for these organelles. If autophagosome-lysosome fusion is blocked with vinblastine (Kovacs et al., Exp cell res 137 (1982) 191), [14C]lactose will accumulate continuously as a function of the sequestration rate, and reach a high level in the pre-lysosomal vacuoles. Density gradient analysis, using chloroquine (CLQ) to alter lysosomal density, suggests that these organelles have a broad density distribution (1.08-1.13 g/ml), thus differing significantly from the distribution of lysosomes.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::c46f164170981e9a73cc29b88622bbe1Test
https://pubmed.ncbi.nlm.nih.gov/3743649Test