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• W2093962878 abstract "Amphisomes, the autophagic vacuoles (AVs) formed upon fusion between autophagosomes and endosomes, have so far only been characterized in indirect, functional terms. To enable a physical distinction between autophagosomes and amphisomes, the latter were selectively density-shifted in sucrose gradients following fusion with AOM-gold-loaded endosomes (endosomes made dense by asialoorosomucoid-conjugated gold particles, endocytosed by isolated rat hepatocytes prior to subcellular fractionation). Whereas amphisomes, by this criterion, accounted for only a minor fraction of the AVs in control hepatocytes, treatment of the cells with leupeptin (an inhibitor of lysosomal protein degradation) caused an accumulation of amphisomes to about one-half of the AV population. A quantitative electron microscopic study confirmed that leupeptin induced a severalfold increase in the number of hepatocytic amphisomes (recognized by their gold particle contents; otherwise, their ultrastructure was quite similar to autophagosomes). Leupeptin caused, furthermore, a selective retention of endocytosed AOM-gold in the amphisomes at the expense of the lysosomes, consistent with an inhibition of amphisome-lysosome fusion. The electron micrographs suggested that autophagosomes could undergo multiple independent fusions, with multivesicular (late) endosomes to form amphisomes and with small lysosomes to form large autolysosomes. A biochemical comparison between autophagosomes and amphisomes, purified by a novel procedure, showed that the amphisomes were enriched in early endosome markers (the asialoglycoprotein receptor and the early endosome-associated protein 1) as well as in a late endosome marker (the cation-independent mannose 6-phosphate receptor). Amphisomes would thus seem to be capable of receiving inputs both from early and late endosomes. Amphisomes, the autophagic vacuoles (AVs) formed upon fusion between autophagosomes and endosomes, have so far only been characterized in indirect, functional terms. To enable a physical distinction between autophagosomes and amphisomes, the latter were selectively density-shifted in sucrose gradients following fusion with AOM-gold-loaded endosomes (endosomes made dense by asialoorosomucoid-conjugated gold particles, endocytosed by isolated rat hepatocytes prior to subcellular fractionation). Whereas amphisomes, by this criterion, accounted for only a minor fraction of the AVs in control hepatocytes, treatment of the cells with leupeptin (an inhibitor of lysosomal protein degradation) caused an accumulation of amphisomes to about one-half of the AV population. A quantitative electron microscopic study confirmed that leupeptin induced a severalfold increase in the number of hepatocytic amphisomes (recognized by their gold particle contents; otherwise, their ultrastructure was quite similar to autophagosomes). Leupeptin caused, furthermore, a selective retention of endocytosed AOM-gold in the amphisomes at the expense of the lysosomes, consistent with an inhibition of amphisome-lysosome fusion. The electron micrographs suggested that autophagosomes could undergo multiple independent fusions, with multivesicular (late) endosomes to form amphisomes and with small lysosomes to form large autolysosomes. A biochemical comparison between autophagosomes and amphisomes, purified by a novel procedure, showed that the amphisomes were enriched in early endosome markers (the asialoglycoprotein receptor and the early endosome-associated protein 1) as well as in a late endosome marker (the cation-independent mannose 6-phosphate receptor). Amphisomes would thus seem to be capable of receiving inputs both from early and late endosomes. Autophagy is a mechanism by which cells sequester and degrade parts of their own cytoplasm, including organelles (for a review, see Ref. 1Seglen P.O. Bohley P. Experientia. 1992; 48: 158-172Crossref PubMed Scopus (370) Google Scholar). Autophagy is basically a nonselective, bulk process; a number of cytosolic enzymes with widely different half-lives have been shown to be autophagically sequestered at identical rates (2Kopitz J. Kisen G.Ø. Gordon P.B. Bohley P. Seglen P.O. J. Cell Biol. 1990; 111: 941-953Crossref PubMed Scopus (191) Google Scholar). However, a selective organelle autophagy can apparently take place during the regression of hypertrophied organelles like the peroxisomes (3Luiken J.J.F.P. Van den Berg M. Heikoop J.C. Meijer A.J. FEBS Lett. 1992; 304: 93-97Crossref PubMed Scopus (58) Google Scholar) and, possibly, the smooth endoplasmic reticulum (4Bolender R.P. Weibel E.R. J. Cell Biol. 1973; 56: 746-761Crossref PubMed Scopus (188) Google Scholar). In the first recognizable autophagic step, single or multiple membrane cisternae with a distinct osmiophilic morphology (5Reunanen H. Punnonen E.-L. Hirsimäki P. Histochemistry. 1985; 83: 513-517Crossref PubMed Scopus (49) Google Scholar, 6Pfeifer U. Glaumann H. Ballard F.J. Lysosomes: Their Role in Protein Breakdown. Academic Press, London1987: 3-59Google Scholar, 7Marzella L. Glaumann H. Glaumann H. Ballard F.J. Lysosomes: Their Role in Protein Breakdown. Academic Press, London1987: 319-367Google Scholar), called phagophores (8Seglen P.O. Glaumann H. Ballard F.J. Lysosomes: Their Role in Protein Breakdown. Academic Press, London1987: 369-414Google Scholar, 9Fengsrud M. Roos N. Berg T. Liou W. Slot J.W. Seglen P.O. Exp. Cell Res. 1995; 221: 504-519Crossref PubMed Scopus (116) Google Scholar), wrap up a region of cytoplasm into a closed vacuolar organelle, an autophagosome. The phagophores, which form the autophagosome wall, may ultimately be derived from the endoplasmic reticulum (10Arstila A.U. Trump B.F. Am. J. Pathol. 1968; 53: 687-733PubMed Google Scholar, 11Dunn Jr., W.A. J. Cell Biol. 1990; 110: 1923-1933Crossref PubMed Scopus (515) Google Scholar, 12Dunn Jr., W.A. J. Cell Biol. 1990; 110: 1935-1945Crossref PubMed Scopus (371) Google Scholar, 13Furuno K. Ishikawa T. Akasaki K. Lee S. Nishimura Y. Tsuji H. Himeno M. Kato K. Exp. Cell Res. 1990; 189: 261-268Crossref PubMed Scopus (45) Google Scholar), perhaps representing highly modified endoplasmic reticulum cisternae that have acquired unique biochemical and structural properties (5Reunanen H. Punnonen E.-L. Hirsimäki P. Histochemistry. 1985; 83: 513-517Crossref PubMed Scopus (49) Google Scholar, 13Furuno K. Ishikawa T. Akasaki K. Lee S. Nishimura Y. Tsuji H. Himeno M. Kato K. Exp. Cell Res. 1990; 189: 261-268Crossref PubMed Scopus (45) Google Scholar, 14Ericsson J.L.E. Exp. Cell Res. 1969; 56: 393-405Crossref PubMed Scopus (77) Google Scholar, 15Reunanen H. Hirsimäki P. Histochemistry. 1983; 79: 59-67Crossref PubMed Scopus (20) Google Scholar, 16Punnonen E.-L. Pihakaski K. Mattila K. Lounatmaa K. Hirsimäki P. Cell Tissue Res. 1989; 258: 269-276Crossref PubMed Scopus (35) Google Scholar). Biochemical studies have indicated that autophagosomes can fuse with endosomes to form prelysosomal autophagic/endocytic vacuoles called amphisomes (17Gordon P.B. Seglen P.O. Biochem. Biophys. Res. Commun. 1988; 151: 40-47Crossref PubMed Scopus (214) Google Scholar, 18Høyvik H. Gordon P.B. Berg T.O. Strømhaug P.E. Seglen P.O. J. Cell Biol. 1991; 113: 1305-1312Crossref PubMed Scopus (77) Google Scholar, 19Gordon P.B. Høyvik H. Seglen P.O. Biochem. J. 1992; 283: 361-369Crossref PubMed Scopus (70) Google Scholar). (Autophagosomes, amphisomes, and lysosomes engaged in autophagy are often referred to collectively as autophagic vacuoles (AVs) 1The abbreviations used are: AVautophagic vacuole3MA3-methyladenine (6-amino-3-methylpurine)ASGPRasialoglycoprotein receptorLDHlactate dehydrogenaseLgp120lysosomal glycoprotein 120 (a lysosomal membrane protein)MPRcation-independent mannose 6-phosphate receptorTBSTris-buffered salineTCtyramine-cellobioseAOMasialoorosomucoidGPNglycyl-l-phenylalanine 2-naphthylamide. ). The amphisomes are apparently acidic (20Strømhaug P.E. Seglen P.O. Biochem. J. 1993; 291: 115-121Crossref PubMed Scopus (46) Google Scholar), probably due to the activity of a proton pump contributed by the endosomal fusion partner. Morphologically, hepatocytic amphisomes can be recognized as “intermediate” autophagic vacuoles (AVi/d) (i.e.autophagosome-like vacuoles containing endocytic markers like endocytosed gold or albumin-gold; acidic, yet lacking lysosomal enzymes or lysosomal membrane proteins like the glycoprotein Lgp120 (9Fengsrud M. Roos N. Berg T. Liou W. Slot J.W. Seglen P.O. Exp. Cell Res. 1995; 221: 504-519Crossref PubMed Scopus (116) Google Scholar, 12Dunn Jr., W.A. J. Cell Biol. 1990; 110: 1935-1945Crossref PubMed Scopus (371) Google Scholar,21Liou W. Geuze H.J. Geelen M.J.H. Slot J.W. J. Cell Biol. 1997; 136: 61-70Crossref PubMed Scopus (224) Google Scholar)). Amphisome formation has also been demonstrated in nonhepatic cells and tissues (22Tooze J. Hollinshead M. Ludwig T. Howell K. Hoflack B. Kern H. J. Cell Biol. 1990; 111: 329-345Crossref PubMed Scopus (152) Google Scholar, 23Punnonen E.-L. Autio S. Kaija H. Reunanen H. Eur. J. Cell Biol. 1993; 61: 54-66PubMed Google Scholar). autophagic vacuole 3-methyladenine (6-amino-3-methylpurine) asialoglycoprotein receptor lactate dehydrogenase lysosomal glycoprotein 120 (a lysosomal membrane protein) cation-independent mannose 6-phosphate receptor Tris-buffered saline tyramine-cellobiose asialoorosomucoid glycyl-l-phenylalanine 2-naphthylamide. There is good evidence that some of the autophagosomes may fuse directly with lysosomes (6Pfeifer U. Glaumann H. Ballard F.J. Lysosomes: Their Role in Protein Breakdown. Academic Press, London1987: 3-59Google Scholar, 12Dunn Jr., W.A. J. Cell Biol. 1990; 110: 1935-1945Crossref PubMed Scopus (371) Google Scholar, 19Gordon P.B. Høyvik H. Seglen P.O. Biochem. J. 1992; 283: 361-369Crossref PubMed Scopus (70) Google Scholar, 21Liou W. Geuze H.J. Geelen M.J.H. Slot J.W. J. Cell Biol. 1997; 136: 61-70Crossref PubMed Scopus (224) Google Scholar, 24Lawrence B.P. Brown W.J. J. Cell Sci. 1992; 102: 515-526PubMed Google Scholar, 25Yokota S. Himeno M. Kato K. Eur. J. Cell Biol. 1995; 66: 15-24PubMed Google Scholar). It has been suggested that autophagic delivery to the lysosomes may occur exclusively by autophagosome-lysosome fusion, i.e. without amphisome formation (24Lawrence B.P. Brown W.J. J. Cell Sci. 1992; 102: 515-526PubMed Google Scholar, 25Yokota S. Himeno M. Kato K. Eur. J. Cell Biol. 1995; 66: 15-24PubMed Google Scholar), but a recent morphometric study concluded that hepatocytic endosomes were 5–6 times more likely to enter the amphisome pathway than to fuse directly with lysosomes (21Liou W. Geuze H.J. Geelen M.J.H. Slot J.W. J. Cell Biol. 1997; 136: 61-70Crossref PubMed Scopus (224) Google Scholar). Differences in experimental approach, organelle markers, and organelle definitions make it difficult to resolve discrepant conclusions regarding the relative roles of the amphisomal and the direct autophagosomal pathway to the lysosome. Active lysosomes can be distinguished from the double- or multiple-membrane-enclosed autophagosomes by having only a single delimiting membrane, but amphisomes have not yet been unequivocally characterized in morphological terms. We therefore initiated the present study, using colloidal gold particles conjugated to asialoorosomucoid (AOM) both as an ultrastructural marker and as a density perturbant. AOM-gold is taken up by receptor-mediated endocytosis in isolated rat hepatocytes, and by means of different gold particle sizes and different loading times, a differential staining of lysosomes and prelysosomal endocytic vacuoles (which would include the amphisomes) can be obtained (26Holen I. Gordon P.B. Strømhaug P.E. Berg T.O. Fengsrud M. Brech A. Roos N. Berg T. Seglen P.O. Biochem. J. 1995; 311: 317-326Crossref PubMed Scopus (19) Google Scholar, 27Holen I. Strømhaug P.E. Gordon P.B. Fengsrud M. Berg T.O. Seglen P.O. J. Biol. Chem. 1995; 270: 12823-12831Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In addition, the high density of the gold particles would be expected to change the behavior of gold-laden endocytic vacuoles in density gradients, as has been shown in other cell types (28Futter C.E. Hopkins C.R. J. Cell Sci. 1989; 94: 685-694PubMed Google Scholar). By this approach, we have been able to identify the amphisomes both as a structural entities in sucrose density gradients and as distinct vacuoles in electron micrographs. Tyramine-cellobiose (TC) was a gift from professor Helge Tolleshaug (Nycomed Pharma A/S, Oslo, Norway). Na125I was from Amersham Pharmacia, Biotech (Little Chalfont, UK). 125I-TC-AOM was synthesized as described previously (29Berg T. Kindberg G.M. Ford T. Blomhoff R. Exp. Cell Res. 1985; 161: 285-296Crossref PubMed Scopus (52) Google Scholar), mixed with 20 times as much unlabeled AOM, and added to cell suspensions at a final concentration of 200 nm,i.e. 10 μg/ml (specific activity, 20,000 dpm/μg of protein). Leupeptin was purchased from Protein Research Foundation (Osaka, Japan); 3-methyladenine was from Fluka A.G. (Buchs, Switzerland); Metrizamide, Nycodenz, and iodixanol were from Nycomed Pharma A/S; and Percoll was from Amersham Pharmacia Biotech (Uppsala, Sweden). Kits for automated analysis of lactate dehydrogenase (LDH) and acid phosphatase were obtained from Boehringer (Mannheim, Germany). Antibodies against superoxide dismutase, endosome-associated protein 1, lysosomal glycoprotein Lgp120, and cathepsin B were kind gifts from Dr. Ling-Yi Chang (Duke University Medical Center, Durham, NC), Dr. Harald Stenmark (The Norwegian Radium Hospital, Oslo, Norway); Dr. William Dunn (University of Florida, Gainesville, FL), and Dr. David Buttle (University of Sheffield Medical School, Sheffield, UK), respectively. Antibodies against the asialoglycoprotein receptor (ASGPR), and the cation-independent mannose 6-phosphate receptor (MPR) were from Dr. Paul Weigel (University of Oklahoma Health Sciences Center, Oklahoma City, OK) and Dr. William Brown (Cornell University, Ithaca, NY), respectively. Other chemicals and biochemicals, including gold chloride (HAuCl4), vinblastine, and collagenase, were purchased from Sigma. Hepatocytes were prepared from 18-h starved male Wistar rats (250–300 g) by two-step collagenase perfusion (30Seglen P.O. Methods Cell Biol. 1976; 13: 29-83Crossref PubMed Scopus (5225) Google Scholar). The cells were incubated as suspensions (0.4-ml aliquots in shaking centrifuge tubes, 50–75-mg wet mass/ml) at 37 °C in suspension buffer (30Seglen P.O. Methods Cell Biol. 1976; 13: 29-83Crossref PubMed Scopus (5225) Google Scholar) fortified with pyruvate (20 mm) and Mg2+ (2 mm). AOM-gold,i.e. AOM conjugated to colloidal gold particles (3- or 10-nm diameter), was prepared according to established procedures (31Slot J.W. Geuze H.J. Eur. J. Cell Biol. 1985; 38: 87-93PubMed Google Scholar, 32Roth J. Bendayan M. Orci L. J. Histochem. Cytochem. 1978; 26: 1074-1081Crossref PubMed Scopus (677) Google Scholar). Colloidal gold was made by mixing, at 60 °C, 160 ml of 0.0125% gold chloride and 40 ml of tannic acid (0.125 or 0.0045%, for preparation of 3- and 10-nm gold particles, respectively) buffered with 0.2% trinatrium citrate dihydrate, keeping the mixture at 60 °C until a stable wine-red color had developed (instantly with 3-nm gold; 1 h with 10-nm gold). The volume was then reduced to one-half by boiling/evaporation (yielding, for 10-nm particles, a 520-nm absorbance of approximately 1.6, corresponding to 1.3·1013 gold particles/ml), and AOM or 125I-TC-AOM was thoroughly mixed into the suspension (10 min at room temperature) at a final concentration of 10 μg/ml. Bovine serum albumin (0.01%) was added as a stabilizer, and the AOM-coated gold particles were sedimented (45 min at 47,000 × g for 10-nm particles; 2 h at 47,000 × g for 3-nm particles) with a 95% recovery of gold and a 70% recovery of AOM, corresponding to about 3 molecules of AOM/gold particle. The pellet was resuspended with 0.9% NaCl to 1 ml and a 520-nm absorbance of 150–200 (AOM, 600–800 μg/ml, or 14–18 μm) and dialyzed against 0.9% NaCl overnight. Uptake of 125I-TC-AOM or 10-nm 125I-TC-AOM-gold was measured at “concentrations” of up to 200–400 nmAOM (520-nm absorbance of 2–4), referring to either free or gold-bound AOM. After incubation, cells were washed three times at 0 °C in 10% sucrose, and radioactivity in the cell pellets was measured by γ-counting. Intracellular AOM or AOM-gold “concentrations” were calculated on the basis of the measured wet mass of cell pellets, assuming the intracellular fluid volume to be 50% of the wet mass (33Gordon P.B. Høyvik H. Seglen P.O. Biochem. J. 1987; 243: 655-660Crossref PubMed Scopus (17) Google Scholar). For density shift experiments, high concentrations of AOM-gold were used (520-nm absorbance of about 20, i.e. about 2 μm AOM). After incubation, the hepatocytes in each tube were washed twice in 10% (w/v) sucrose and finally suspended in 0.5 ml of 10% sucrose. Four samples were pooled and subjected to electrodisruption by a single high voltage pulse (34Gordon P.B. Seglen P.O. Exp. Cell Res. 1982; 142: 1-14Crossref PubMed Scopus (76) Google Scholar). The resulting preparation is referred to as the disruptate. The disruptate was diluted with an equal volume of buffered sucrose (0.25 m sucrose, 10 mm HEPES, 1 mm EDTA, pH 7.3) and homogenized by 10 strokes in a Dounce homogenizer with a tightly fitting pestle. The homogenate was centrifuged for 2 min at 4,000 rpm (2,000 ×g) in a Sorvall SS-34 rotor and washed once (resuspended in 4 ml of buffered sucrose and recentrifuged), and the combined postnuclear fractions were layered on top of a 25-ml metrizamide density cushion (8% (w/v) metrizamide, 1 mmdithiothreitol, 1 mm EDTA, 50 mm potassium phosphate, pH 7.5; adjusted to 300 mosm with sucrose) and centrifuged at 50,000 × g for 1 h. The supernatant was removed by aspiration; the pellet (large granule fraction) was resuspended in 2 ml of buffered sucrose and homogenized (5 strokes); and 1.5 ml was layered on the top of a linear sucrose gradient. The sucrose gradients were prepared by mixing 63 and 15% sucrose solutions (5.5 ml of each) using a Biocomp Gradient Master (Nycomed Pharma A/S). The gradients were centrifuged at 285,000 ×g in a Beckman SW40 rotor at 4 °C for 105 min. The gradients were then divided into 20 0.67-ml fractions by upward displacement with Maxidens fluid (Nycomed Pharma A/S). The densities of the fractions were calculated from the refractive indices. Homogenizations and centrifugations were carried out at 0–4 °C. We have recently developed methods for the purification of hepatocytic autophagosomes and lysosomes, described in detail elsewhere. 2P. E. Strømhaug, T. O. Berg, M. Fengsrud, and P. O. Seglen, submitted for publication. In the present study, certain modifications were introduced in order to enrich the autophagosome preparation with respect to amphisomes. Hepatocyte homogenates were prepared in buffered sucrose as described above. For purification of autophagosomes (with only 5% amphisome contamination), homogenates from vinblastine-treated cells (hepatocytes incubated for 3 h at 37 °C with 50 μm vinblastine) were incubated with 0.5 mm glycyl-l-phenylalanine 2-naphthylamide (GPN) and 0.33% Me2SO (GPN solvent) for 6 min at 37 °C to disrupt the lysosomes (35Berg T.O. Strømhaug P.E. Berg T. Seglen P.O. Eur. J. Biochem. 1994; 221: 595-602Crossref PubMed Scopus (28) Google Scholar). For preparation of an approximately 50:50 mixture of amphisomes and autophagosomes, homogenates were prepared from leupeptin-treated cells (3 h at 37 °C with 0.3 mm leupeptin), and the GPN step was omitted (the enlarged lysosomes disappeared anyway, mostly in the nuclear fraction). For preparation of lysosomes, homogenates were prepared from cells in which autophagy had been blocked by incubation for 3 h at 37 °C with 10 mm 3-methyladenine (3MA); moreover, the GPN step was omitted. In either case, the homogenate was cooled to 4 °C, and further purification was performed at this temperature. The nuclei were sedimented by centrifugation for 2 min at 4,000 rpm (2,000 ×g) and washed once, and 14 ml of the combined postnuclear supernatants were placed on top of a discontinuous (two-step), isotonic Nycodenz gradient, containing 9.5% Nycodenz (in buffered sucrose diluted to isotonicity) in the 17-ml upper layer and 22.5% Nycodenz in the 7-ml bottom layer. After centrifugation for 1 h at 28,000 rpm (141,000 × g) in a Beckman SW28 rotor to remove mitochondria and peroxisomes, the vacuole-enriched interface band between the Nycodenz layers (5 ml) was diluted with an equal volume of buffered sucrose and layered on top of a Percoll two-step gradient (21 ml of 33% Percoll in buffered sucrose in the upper layer; 7 ml of 22.5% Nycodenz in buffered sucrose in the lower layer) and centrifuged for 30 min at 20,000 rpm (72,000 × g) to remove the endoplasmic reticulum. The autophagic vacuoles were recovered as a 5-ml fraction from the lower part of the Percoll gradient (near the Nycodenz interface) and diluted with 3.5 ml of isotonic 60% iodixanol, overlaid with 1.5 ml of 30% iodixanol and 2.5 ml of buffered sucrose, and centrifuged for 30 min in a Beckman SW40 rotor at 20,000 rpm (71,000 × g) to sediment the Percoll particles. The purified vacuoles (autophagosomes, autophagosomes plus amphisomes, or lysosomes, depending on the pretreatment of the cells or the homogenate) floated to the iodixanol/sucrose interface, where they could be recovered for morphological or biochemical analysis. Acid-soluble and acid-insoluble 125I radioactivity was measured by γ-counting of gradient fractions after precipitation with 10% trichloroacetic acid (27Holen I. Strømhaug P.E. Gordon P.B. Fengsrud M. Berg T.O. Seglen P.O. J. Biol. Chem. 1995; 270: 12823-12831Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Acid phosphatase (β-glycerophosphatase) was determined according to Ames (36Ames B.N. Methods Enzymol. 1966; 8: 115-118Crossref Scopus (3031) Google Scholar), and LDH was determined according to Bergmeyer (37Bergmeyer H.U. Berndt E. Methoden der enzymatischen Analyse. Verlag Chemie, Weinheim1974Google Scholar), using a Technicon RA-1000 autoanalyzer for both assays. In some particularly important experiments, the gradient resolution of LDH was improved by subtracting, from each fraction, the background of nonautophagocytosed LDH, measured in separate gradients as enzyme activity resistant to treatment of the cells with 10 mm of the autophagy inhibitor 3MA (19Gordon P.B. Høyvik H. Seglen P.O. Biochem. J. 1992; 283: 361-369Crossref PubMed Scopus (70) Google Scholar). Autophagic activity was measured in intact cells as the sequestration of cytosolic LDH into the sedimentable cell fraction (“cell corpses”) following electrodisruption and expressed as a percentage of the total cell-associated LDH (2Kopitz J. Kisen G.Ø. Gordon P.B. Bohley P. Seglen P.O. J. Cell Biol. 1990; 111: 941-953Crossref PubMed Scopus (191) Google Scholar). Equivalent amounts of protein from the various organelle fractions (whole cell homogenates, lysosomes, autophagosomes, amphisomes + autophagosomes) or from a “residual fraction” prepared as autophagosomes, but from cells incubated with 3MA and homogenates treated with GPN (and applied as an autophagosome-equivalent volume rather than as an equivalent amount of protein), were subjected to one-dimensional SDS-polyacrylamide gel electrophoresis, using the mini-Protean equipment from Bio-Rad. The residual fraction was essentially devoid of autophagosomes, amphisomes, or lysosomes, thus serving to define the background of contamination by endoplasmic reticulum, endosomes, etc. in the organelle fractions. The separated proteins were transferred onto Nitropure nitrocellulose supports (Micron Separations Inc., West Borough, MA) using a Bio-Rad Semidry Transblot apparatus. The blots were blocked overnight in 5% nonfat dried milk in Tris-buffered saline (TBS; 25 mm Tris, 0.8% NaCl, pH 7.4) and then incubated at room temperature for 2 h with primary antibody and for 30 min with horseradish peroxidase-conjugated secondary antibody (both in TBS with 2% dried milk). After each step, the blots were washed three times in TBS with 0.1% Tween 20. The antibodies were detected by chemiluminescence, using the SuperSignal peroxidase substrate (Pierce) and Kodak X-Omat LS films (Eastman Kodak Co.). The films were digitalized with a model 300A laser densitometer from Molecular Dynamics Inc. (Sunnyvale, CA). One ml of 3-nm AOM-gold suspension (absorbance value of 1.8 at 520 nm after 100-fold dilution) was injected intravenously into rats 24 h before cell isolation to prelabel hepatocytic lysosomes. To label vacuoles of the endocytic-lysosomal pathway in isolated hepatocytes, the cells were incubated for 3 h at 37 °C with a low concentration of 10-nm AOM-gold (final absorbance of 2.0 at 520 nm, i.e.110 of the concentration used for density shifting). The cells were washed once in washing buffer (30Seglen P.O. Methods Cell Biol. 1976; 13: 29-83Crossref PubMed Scopus (5225) Google Scholar), fixed in 2% glutaraldehyde, 0.1 m cacodylate buffer overnight at 4 °C, and postfixed for 60 min in 1% OsO4 reduced with 1.5% potassium ferrocyanide, followed by en bloc staining with 1.5% uranylacetate. After serial dehydration in ethanol and propylene oxide, specimens were embedded in Epon and then sectioned and poststained with 0.2% lead citrate. Sections were examined in a Phillips CM10 electron microscope at 60 kV. For quantitation of organelle numbers and gold contents, three blocks from each treatment group within each experiment were sectioned, and 2–4 cell profiles (with a nucleus) were examined on each grid. The number of endocytic and autophagic vacuoles per cell profile was counted in the microscope at × 13,500 magnification. For measurements of organelle diameters and volume fractions, 10 random micrographs for each treatment group were taken from four separate experiments (i.e. a total of 40 micrographs/treatment) and magnified to × 34,500, and the relative cytoplasmic volume fraction occupied by each type of organelle was determined morphometrically using a double lattice. Organelle profile diameters were generally recorded as the average of the shortest and longest diameter, except for small endocytic tubules and vesicles, where the smallest diameter was used. AOM-coated 10-nm gold particles (AOM-gold), used in the present experiments both as an ultrastructural vacuole marker and as a perturbant of vacuole density, were taken up into isolated hepatocytes by a saturable process, like free AOM (Fig.1 A). The uptake of radiolabeled AOM-gold was competed out by unlabeled AOM with the same efficiency as was radiolabeled, free AOM (Fig. 1 B), indicating that AOM-gold at the concentrations used (200–400 nm AOM) was taken up virtually exclusively by receptor-mediated endocytosis. The higher uptake efficiency of gold-bound AOM relative to free AOM in Fig. 1 A probably relates to the fact that each gold particle carries about three molecules of AOM, which may be internalized by a single receptor engagement, as opposed to only one molecule of free AOM per receptor engagement. Isolated rat hepatocytes were incubated at 37 °C under conditions of maximal autophagy (amino acid-free medium), with 125I-TC-AOM (200,000 dpm; 10 μg of protein/ml) included as a marker of endocytic-lysosomal vacuoles (26Holen I. Gordon P.B. Strømhaug P.E. Berg T.O. Fengsrud M. Brech A. Roos N. Berg T. Seglen P.O. Biochem. J. 1995; 311: 317-326Crossref PubMed Scopus (19) Google Scholar,27Holen I. Strømhaug P.E. Gordon P.B. Fengsrud M. Berg T.O. Seglen P.O. J. Biol. Chem. 1995; 270: 12823-12831Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). To shift the density of these vacuoles, colloidal 10-nm AOM-gold was added, replacing unlabeled AOM so as to leave the uptake of125I-TC-AOM unaltered. After 3 h of endocytosis in the absence of AOM-gold, virtually all of the 125I-TC-AOM banded in a sucrose density gradient as a single peak at 1.19 g/ml, mostly in acid-soluble (i.e. degraded) form (Fig.2 A), at the same position as the lysosomal marker enzyme, acid phosphatase (Fig. 2 B). When 10-nm AOM-gold was endocytosed along with the125I-TC-AOM, nearly all of the acid-soluble and acid-insoluble radioactivity, as well as most of the acid phosphatase, sedimented to the bottom of the gradient tube (>1.20 g/ml), indicating that the lysosomes had become extensively density-shifted by fusion with AOM-gold-loaded endosomes. Prelysosomal autophagic vacuoles (autophagosomes and amphisomes) can be recognized in density gradients by their contents of 3MA-sensitive,i.e. autophagocytosed, LDH (35Berg T.O. Strømhaug P.E. Berg T. Seglen P.O. Eur. J. Biochem. 1994; 221: 595-602Crossref PubMed Scopus (28) Google Scholar), which becomes degraded once these vacuoles fuse with lysosomes (2Kopitz J. Kisen G.Ø. Gordon P.B. Bohley P. Seglen P.O. J. Cell Biol. 1990; 111: 941-953Crossref PubMed Scopus (191) Google Scholar). As seen in Fig. 2 C, the peak of 3MA-sensitive LDH at 1.14 g/ml was only slightly shifted toward higher densities by endocytosed AOM-gold. This would suggest that in control cells, autophagocytosed LDH resides largely in autophagosomes rather than in amphisomes. Since so little prelysosomal LDH could be detected in control cells, an attempt was made to induce prelysosomal autophagic vacuole accumulation by incubating the cells with various inhibitors of late stages in the autophagic-lysosomal pathway (18Høyvik H. Gordon P.B. Berg T.O. Strømhaug P.E. Seglen P.O. J. Cell Biol. 1991; 113: 1305-1312Crossref PubMed Scopus (77) Google Scholar, 35Berg T.O. Strømhaug P.E. Berg T. Seglen P.O. Eur. J. Biochem. 1994; 221: 595-602Crossref PubMed Scopus (28) Google Scholar,38Seglen P.O. Exp. Cell Res. 1977; 107: 207-217Crossre" @default.
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