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: Frank Sætre, Per Ottar Seglen, Nikolai Engedal, Linda Korseberg Hagen

المصدر: FEBS Journal. 282:2202-2214

مصطلحات موضوعية: Male, Thapsigargin, Lipid-anchored protein, Vacuole, Biology, Biochemistry, Cathepsin B, chemistry.chemical_compound, Autophagy, Animals, Rats, Wistar, Molecular Biology, Cells, Cultured, Differential centrifugation, Adenine, Cell Biology, Cell biology, Cytosol, chemistry, Cytoplasm, Proteolysis, Hepatocytes, Lysosomes, Microtubule-Associated Proteins, Protein Processing, Post-Translational

الوصف: Autophagy is the process by which portions of cytoplasm are enclosed by membranous organelles, phagophores, which deliver the sequestered cytoplasm to degradative autophagic vacuoles. Genes and proteins involved in phagophore manufacture have been extensively studied, but little is known about how mature phagophores proceed through the subsequent steps of expansion, closure and fusion. Here we have addressed these issues by combining our unique autophagic cargo sequestration assay (using the cytosolic enzyme lactate dehydrogenase as a cargo marker) with quantitative measurements of the lipidation-dependent anchorage and turnover of the phagophore-associated protein LC3. In isolated rat hepatocytes, amino acid starved to induce maximal autophagic activity, the two unrelated reversible autophagy inhibitors 3-methyladenine (3MA) and thapsigargin (TG) both blocked cargo sequestration completely. However, whereas 3MA inhibited LC3 lipidation, TG did not, thus apparently acting at a post-lipidation step to prevent phagophore closure. Intriguingly, the resumption of cargo sequestration seen upon release from a reversible TG block was completely suppressed by 3MA, revealing that 3MA not only inhibits LC3 lipidation but also (like TG) blocks phagophore closure at a post-lipidation step. 3MA did not, however, prevent the resumption of lysosomal LC3 degradation, indicating that phagophores could fuse directly with degradative autophagic vacuoles without carrying cytosolic cargo. This fusion step was clearly blocked by TG. Furthermore, density gradient centrifugation revealed that a fraction of the LC3-marked phagophores retained by TG could be density-shifted by the acidotropic drug propylamine along with the lysosomal marker cathepsin B, suggesting physical association of some phagophores with lysosomes prior to cargo sequestration.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::f338d395fb4cc96eff5a86e2cfcc0d3bTest
https://doi.org/10.1111/febs.13268Test

المؤلفون: Morten Luhr, Ian G. Mills, Nikolai Engedal, Frank Sætre, Paula Szalai, Per Ottar Seglen

المصدر: Methods. 75:25-36

مصطلحات موضوعية: Cytoplasm, L-Lactate Dehydrogenase, Immunoblotting, Autophagy, Lipid-anchored protein, Vacuole, Biology, General Biochemistry, Genetics and Molecular Biology, Green fluorescent protein, Cell biology, Cytosol, Biochemistry, Organelle, Humans, Lysosomes, Microtubule-Associated Proteins, Molecular Biology, Cells, Cultured, PI3K/AKT/mTOR pathway, HeLa Cells

الوصف: 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.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::aef44310176571d80775a04c77fd407cTest
https://doi.org/10.1016/j.ymeth.2014.12.021Test

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: Daniel J. Klionsky, Per Ottar Seglen

المصدر: Autophagy. 6:1017-1031

مصطلحات موضوعية: Psychoanalysis, Autophagy, Perspective (graphical), Cell Biology, Protein degradation, Biology, Molecular Biology, Physiological responses, Phagophore assembly site

الوصف: Macroautophagy was first detected in the late 1950s and was pursued briefly in the 1960s, primarily in regard to physiological responses to glucagon versus insulin. Relatively few people studied this process throughout the subsequent two decades. One of those people, however, was Per Seglen. At the first Gordon Research Conference on “Autophagy in Stress, Development and Disease,” John Lemasters referred to Per as the “Norse god of autophagy” (hence, the title of this perspective). Lest some people in that audience or some readers of this article, think that title a tad presumptuous, I encourage them to read the following interview.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::d45d62db32bbaa6d6aa196dcde9117f9Test
https://doi.org/10.4161/auto.6.8.13092Test

المؤلفون: Monica F. Brinchmann, Per Ottar Seglen

المصدر: Autophagy. 6:542-547

مصطلحات موضوعية: Male, Autophagosome, Endosome, Cell Separation, Biology, Cell Fractionation, symbols.namesake, Phagosomes, Autophagy, Animals, Rats, Wistar, Molecular Biology, Cell Nucleus, Differential centrifugation, Endoplasmic reticulum, Vesicle, Cell Biology, Golgi apparatus, Rats, Cell biology, Hepatocytes, symbols, Cell fractionation, Lysosomes, Microtubule-Associated Proteins, Percoll

الوصف: To facilitate the purification of rat liver autophagosomes, isolated rat hepatocytes are first incubated for 2 h at 37°C with vinblastine, which induces autophagosome accumulation by blocking the fusion of these organelles with endosomes and lysosomes. The hepatocytes are then electrodisrupted and homogenized, and the various cellular organelles sequentially removed by subcellular fractionation. A brief incubation of the homogenate with the cathepsin C substrate, glycyl-phenylalanine-naphthylamide (GPN), causes rapid osmotic disruption of the lysosomes due to intralysosomal accumulation of GPN cleavage products. Nuclei are removed by differential centrifugation, and the postnuclear supernatant subsequently fractionated on a two-step Nycodenz density gradient. Autophagosomes are recovered in an intermediate density fraction, free from cytosol and mitochondria. The autophagosomes are finally separated from the membranes and vesicles of the endoplasmic reticulum, Golgi, endosomes, etc. by sieving through a density gradient of colloidal silica particles (Percoll). The final preparation contains about 95% autophagosomes and 5% amphisomes according to morphological and biochemical criteria.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::12d6dd3ef0a2cd8f6ab6758a539af510Test
https://doi.org/10.4161/auto.6.4.11272Test

المؤلفون: Monica Fengsrud, Marianne Lunde Sneve, Per Ottar Seglen, Anders Øverbye

المصدر: Autophagy. 1:157-162

مصطلحات موضوعية: Male, Proteomics, Autophagosome, Molecular Sequence Data, Dehydrogenase, In Vitro Techniques, Silver stain, 3-alpha-Hydroxysteroid Dehydrogenase (B-Specific), Phagosomes, Organelle, Autophagy, Animals, Electrophoresis, Gel, Two-Dimensional, Amino Acid Sequence, Rats, Wistar, Molecular Biology, Glyceraldehyde 3-phosphate dehydrogenase, biology, Intracellular Membranes, Cell Biology, Molecular biology, Rats, Isoenzymes, Cytosol, Biochemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Hepatocytes, biology.protein, Lysosomes, Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+), Immunostaining

الوصف: Immunoblotting of two-dimensional polyacrylamide gels (pI 3-10) revealed six cytosolic molecular forms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat hepatocytes. Two of the four full-length (approximately 37 kDa) forms exhibited some binding to sedimentable cellular elements (but not to mitochondria), whereas one full-length and two short (approximately 35 kDa) forms selectively bound to the membranes of autophagosomes and lysosomes. Tryptic fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed the identity of the major full-length forms as GAPDH, but attempts to identify the major short form consistently suggested that this spot represented a different enzyme, 3-alpha-hydroxysteroid dehydrogenase (3alphaHSD). Silver staining indicated that this 3alphaHSD form selectively bound to autophagosomal and lysosomal membranes. Immunoblotting of more focused 2D gels (pI 6-9) with an antibody raised against 3alphaHSD demonstrated immunostaining of four 3alphaHSD forms with masses of about 35 kDa. Autophagosomal membrane preparations were highly and selectively enriched with respect to all of these 3alphaHSD forms. One of them comigrated with the major short form of GAPDH, accounting for the paradoxical mass spectrometric identification of 3alphaHSD from this spot. Proteomic analysis by a combination of immunological and mass spectrometric identification methods was thus capable of resolving two comigrating dehydrogenases selectively associated with autophagic organelles.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::ca6a8da4dd2990056ffdde4c6ad79309Test
https://doi.org/10.4161/auto.1.3.2037Test