المؤلفون: Andreas Brech, T. Berg, Rune Kjeken, Trond Berg, Seyed Ali Mousavi, Per Ottar Seglen

المصدر: Biochimica et Biophysica Acta (BBA) - Biomembranes. 1510:243-257

مصطلحات موضوعية: Male, Endosome, Endocytic cycle, Biophysics, Asialoglycoproteins, Endosomes, Vacuole, In Vitro Techniques, Biology, Endocytosis, Biochemistry, Iodine Radioisotopes, Organelle, Autophagy, Animals, Enzyme Inhibitors, Rats, Wistar, Receptor, Horseradish Peroxidase, L-Lactate Dehydrogenase, Pinocytosis, Proton Pump Inhibitors, Serum Albumin, Bovine, Orosomucoid, Cell Biology, Anti-Bacterial Agents, Rats, Cell biology, Microscopy, Electron, Liver, Asialoglycoprotein receptor, Macrolides, Lysosomes

الوصف: Bafilomycin A(1) (BAF) and concanamycin A (ConcA) are selective inhibitors of the H(+)-ATPases of the vacuolar system. We have examined the effects of these inhibitors on different steps in endocytic pathways in rat hepatocytes, using [(125)I]tyramine-cellobiose-labeled asialoorosomucoid ([(125)I]TC-AOM) and [(125)I]tyramine-cellobiose-labeled bovine serum albumin ([(125)I]TC-BSA) as probes for respectively receptor-mediated endocytosis and pinocytosis (here defined as fluid phase endocytosis). The effects of BAF and ConcA were in principle identical, although ConcA was more effective than BAF. The main findings were as follows. (1) BAF/ConcA reduced the rate of uptake of both [(125)I]TC-AOM and [(125)I]TC-BSA. The reduced uptake of [(125)I]TC-AOM was partly due to a redistribution of the asialoglycoprotein receptors (ASGPR) such that the number of surface receptors was reduced approximately 40% without a change in the total number of receptors. (2) BAF/ConcA at the same time increased retroendocytosis (recycling) of both probes. The increased recycling of the ligand ([(125)I]TC-AOM) is partly a consequence of the enhanced pH in endosomes, which prevents dissociation of ligand. (3) It was furthermore found that the ligand remained bound to the receptor in presence of BAF/ConcA and that the total amount of ligand molecules internalized in BAF/ConcA-treated cells was only slightly in excess of the total number of receptors. These data indicate that reduced pH in endosomes is the prime cause of receptor inactivation and release of ligand in early endosomes. (4) Subcellular fractionation experiments showed that [(125)I]TC-AOM remained in early endosomes, well separated from lysosomes in sucrose gradients. The fluid phase marker, [(125)I]TC-BSA, on the other hand, seemed to reach a later endosome in the BAF/ConcA-treated cells. This organelle coincided with lysosomes in the gradient, but hypotonic medium was found to selectively release a lysosomal enzyme (beta-acetylglucosaminidase), indicating that even [(125)I]TC-BSA remained in a prelysosomal compartment in the BAF/ConcA-treated cells. (5) Electron microscopy using horseradish peroxidase (HRP) as a fluid phase marker verified that BAF/ConcA inhibited transfer of material from late endosomes ('multivesicular bodies'). (6) BAF/ConcA led to accumulation of lactate dehydrogenase (LDH) in autophagic vacuoles, but although the drugs partly inhibited fusion between autophagosomes and lysosomes a number of autolysosomes was formed in the presence of BAF/ConcA. This observation explains the reduced buoyant density of lysosomes (revealed in sucrose density gradients). In conclusion, BAF/ConcA inhibit transfer of endocytosed material from late endosomes to lysosomes, but do not at the same time prevent fusion between autophagosomes and lysosomes.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::8c411aca5e36cc61fa9f59ce83982df5Test
https://doi.org/10.1016/s0005-2736Test(00)00354-0

المؤلفون: Norbert Roos, Monica Fengsrud, Trond Berg, Willisa Liou, Per Ottar Seglen, Jan W. Slot

المصدر: Experimental Cell Research. 221:504-519

مصطلحات موضوعية: Male, Leupeptins, Endosome, Endocytic cycle, Gold Colloid, Vacuole, Biology, Vinblastine, Endocytosis, Microtubules, chemistry.chemical_compound, Antigens, CD, Lysosomal-Associated Membrane Protein 1, Organelle, Autophagy, Animals, Protease Inhibitors, Rats, Wistar, Carbonic Anhydrases, Membrane Glycoproteins, L-Lactate Dehydrogenase, Superoxide Dismutase, Leupeptin, Lysosome-Associated Membrane Glycoproteins, Serum Albumin, Bovine, Cell Biology, Rats, Cell biology, Cytosol, Liver, Biochemistry, chemistry, Vacuoles, Cattle, Asparagine, Lysosomes, Biomarkers

الوصف: The interactions between the autophagic and the endocytic degradation pathways were investigated by means of immunogold labeling of autophagic vacuoles (AVs) in ultrathin frozen sections from isolated rat hepatocytes. AVs were identified by their autophagocytosed contents of the degradation-resistant cytosolic enzyme CuZn-superoxide dismutase (SOD). Another cytosolic enzyme, carbonic anhydrase (CAIII), was rapidly degraded in the lysosomes, making the vacuolar CAIII/SOD ratio useful as a rough indicator of the progress of autophagic-lysosomal degradation. Lysosomes could be recognized by the presence of the lysosomal membrane glycoprotein lgp120, which was absent from hepatocytic endosomes. Endocytic inputs into the AVs were detected by the presence of gold-conjugated bovine serum albumin (BSA-gold), taken up by fluid-phase endocytosis. All vacuoles recognized morphologically as AVs were SOD-positive, as were essentially all of the lysosomes (96%). The majority (72%) of the lysosomes also labeled positively for BSA within 2 h of endocytosis. The data are thus compatible with the notion that all lysosomes can engage in both autophagic and endocytic degradation. Lgp120 appeared to distinguish well between lysosomes and nonlysosomal AVs: the lgp120-negative AVs (nonlysosomes) had a CAIII/SOD ratio identical to that of the cytosol, indicating that no degradation had occurred. In the lgp120-positive AVs (lysosomes), the ratio was only 43% of the cytosolic value, consistent with substantial CAIII degradation. Among the nonlysosomal AVs (about one-third of all AVs), one-half were BSA-positive, suggesting that early AVs (autophagosomes) and suggesting that early AVs (autophagosomes) and intermediary AVs (amphisomes) that had fused with endosomes were equally abundant. These morphological data thus support previous biochemical evidence for a prelysosomal meeting of the autophagic and endocytic pathways. The microtubule inhibitor vinblastine inhibited the autophagic influx to the lysosomes, causing an accumulation of autophagosomes and a reduction in average lysosomal size. Vinblastine also inhibited the endocytic flux, thereby precluding the formation of amphisomes and of BSA-positive lysosomes. High concentrations (20 mM) of asparagine induced swelling of amphisomes and of BSA-positive lysosomes, probably reflecting an acidotropic effect of ammonia generated by asparagine deamination. Asparagine also caused an accumulation of autophagosomes, amphisomes, and BSA-negative lysosomes, presumably as a result of impaired fusion with the swollen BSA-positive lysosomes. The two agents thus appear to perturb the autophagic-endocytic-lysosomal vacuole dynamics by different mechanisms, making them useful in the further study of these complex organelle interactions.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::364dd30d236af8468ac2f1136e7ba2a2Test
https://doi.org/10.1006/excr.1995.1402Test

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

المصدر: The Biochemical journal. 283

مصطلحات موضوعية: Male, Sucrose, Glycoside Hydrolases, Endocytic cycle, Vacuole, Biology, Endocytosis, Tritium, Vinblastine, Biochemistry, chemistry.chemical_compound, Raffinose, Lysosome, medicine, Autophagy, Animals, Asparagine, Carbon Radioisotopes, Molecular Biology, Cells, Cultured, beta-Fructofuranosidase, Rats, Inbred Strains, Cell Biology, Cell biology, Rats, Kinetics, Invertase, medicine.anatomical_structure, chemistry, Liver, Lysosomes, Research Article

الوصف: In isolated rat hepatocytes electroloaded with [14C]sucrose, autophaged sugar accumulated in lysosomes under control conditions, and in prelysosomal autophagic vacuoles (amphisomes) in the presence of asparagine, an inhibitor of autophagic-lysosomal fusion. Endocytic uptake of the sucrose-cleaving enzyme invertase resulted in rapid and complete degradation of autophaged sucrose in both amphisomes and lysosomes. Pre-accumulated sucrose was degraded equally well in both compartments, regardless of amphisomal-lysosomal flux inhibition by asparagine, suggesting that endocytic entry into the autophagic pathway can take place both at the lysosomal and at the amphisomal level. The completeness of sucrose degradation by endocytosed invertase furthermore indicates that all lysosomes involved in autophagy can also engage in endocytosis. Endocytosed invertase reached the amphisomes even when autophagy was blocked by 3-methyladenine, and autophaged sucrose reached this compartment even when endocytic influx was blocked by vinblastine, suggesting that amphisomes may exhibit some degree of permanence independently of either pathway.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::593cdfa4a315c24d76d168ff441cd148Test
https://pubmed.ncbi.nlm.nih.gov/1575680Test

المؤلفون: Paul B. Gordon, Per Ottar Seglen

المصدر: Biochemical and biophysical research communications. 151(1)

مصطلحات موضوعية: Male, Endocytic cycle, Biophysics, Lactose, Vacuole, Biology, Vinblastine, Biochemistry, chemistry.chemical_compound, Phagocytosis, Autophagy, Animals, Asparagine, Molecular Biology, Cells, Cultured, chemistry.chemical_classification, Rats, Inbred Strains, Cell Biology, Compartment (chemistry), beta-Galactosidase, Endocytosis, Cell biology, Rats, Cytosol, Enzyme, chemistry, Liver, Vacuoles, Lysosomes

الوصف: [14C]Lactose, electroinjected into the cytosol of isolated rat hepatocytes, was sequestered by autophagy, transferred to lysosomes and eventually hydrolysed. Asparagine prevented the fusion between prelysosomal autophagic vacuoles and lysosomes, and caused lactose to accumulate in the former. However, if the hepatocytes were simultaneously allowed to endocytose added β-galactosidase, no lactose accumulation occurred. These results suggest that autophagically sequestered lactose and endocytosed β-galactosidase were delivered to the same prelysosomal vacuole, where the lactose was hydrolysed by the enzyme. The name amphisome is suggested for this new functional compartment, common to the autophagic and endocytic pathways.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::a5669d65cf3dfb38c3cff5f8e20b7dedTest
https://pubmed.ncbi.nlm.nih.gov/3126737Test