The Experts below are selected from a list of 324 Experts worldwide ranked by ideXlab platform
Yuji Moriyasu - One of the best experts on this subject based on the ideXlab platform.
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Isolation of Autolysosomes from Tobacco BY-2 Cells.
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: Chihiro Takatsuka, Yuko Inoue-aono, Yuji MoriyasuAbstract:Autolysosomes are organelles that sequester and degrade a portion of the cytoplasm during autophagy. Although autophagosomes are short lived compared to other organelles such as mitochondria, plastids, and peroxisomes, many Autolysosomes accumulate in tobacco BY-2 cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor. We here describe our methodology for isolating Autolysosomes from BY-2 cells by conventional cell fractionation using a Percoll gradient. The Autolysosome fraction separates clearly from fractions containing mitochondria and peroxisomes. It contains acid phosphatase, vacuolar H+-ATPase, and protease activity. Electron micrographs show that the fraction contains partially degraded cytoplasm seen in Autolysosomes before isolation although an Autolysosome structure is only partially preserved.
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detecting autophagy in arabidopsis roots by membrane permeable cysteine protease inhibitor e 64d and endocytosis tracer fm4 64
Plant Signaling & Behavior, 2011Co-Authors: Yuumi Ohye, Yuko Inoue, Yuji MoriyasuAbstract:Autophagy is the process by which cells degrade their own components in lysosomes or vacuoles. Autophagy in tobacco BY-2 cells cultured in sucrose-free medium takes place in formed, Autolysosomes in the presence of a cysteine protease inhibitor. The Autolysosomes in BY-2 cells are located in the endocytotic pathway and thus can be stained with fluorescent endocytosis marker FM4-64. In the present study, in order to detect autophagy in the root cells of Arabidopsis, we incubated root tips from Arabidopsis seedlings in culture medium containing the membrane-permeable cysteine protease inhibitor E-64d and FM4-64, and examined whether Autolysosomes stained with FM4-64 are accumulated. The results suggest that autophagy accompanying the formation of Autolysosomes also occurs in Arabidopsis root cells. Such autophagy appeared to occur constitutively in the root cells in nutrient-sufficient culture medium. Even in atg5 mutants in which an autophagy-related gene is disrupted, accumulation of the structures stained with FM4-64, which likely correspond to Autolysosomes, was seen although at lower level than in wild type roots.
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Autophagy in Tobacco BY-2 Cells Cultured under Sucrose Starvation Conditions: Isolation of the Autolysosome and its Characterization
Plant & cell physiology, 2011Co-Authors: Chihiro Takatsuka, Yuko Inoue, Tomoya Higuchi, Stefan Hillmer, David G. Robinson, Yuji MoriyasuAbstract:Tobacco culture cells carry out a large-scale degradation of intracellular proteins in order to survive under sucrose starvation conditions. We have previously suggested that this bulk degradation of cellular proteins is performed by autophagy, where Autolysosomes formed de novo act as the major lytic compartments. The digestion process in Autolysosomes can be retarded by addition of the cysteine protease inhibitor E-64c to the culture medium, resulting in the accumulation of Autolysosomes. In the present study, we have investigated several properties of Autolysosomes in tobacco cells. Electron microscopy showed that the Autolysosomes contain osmiophilic particles, some of which resemble partially degraded mitochondria. It also revealed the presence of two kinds of Autolysosome precursor structures; one resembled the isolation membrane and the other the autophagosome of mammalian cells. Immunofluorescence microscopy showed that Autolysosomes contain acid phosphatase, in accordance with cytochemical enzyme analyses by light and electron microscopy in a previous study. Autolysosomes isolated by cell fractionation on Percoll gradients showed the localization of acid phosphatase, vacuolar H + -ATPase and cysteine protease. These results show that starvation-induced autophagy in tobacco cells follows a macroautophagic-type response similar to that described for other eukaryotes. However, our results indicate that, although the plant vacuole is often described as being equivalent to the lysosome of the animal cell, a new low pH lytic compartment—the Autolysosome—also contributes to proteolytic degradation when tobacco cells are subjected to sucrose deprivation.
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Detecting autophagy in Arabidopsis roots by membrane-permeable cysteine protease inhibitor E-64d and endocytosis tracer FM4–64
Plant Signaling & Behavior, 2011Co-Authors: Yuko Inoue, Yuji MoriyasuAbstract:Autophagy is the process by which cells degrade their own components in lysosomes or vacuoles. Autophagy in tobacco BY-2 cells cultured in sucrose-free medium takes place in formed, Autolysosomes in the presence of a cysteine protease inhibitor. The Autolysosomes in BY-2 cells are located in the endocytotic pathway and thus can be stained with fluorescent endocytosis marker FM4-64. In the present study, in order to detect autophagy in the root cells of Arabidopsis, we incubated root tips from Arabidopsis seedlings in culture medium containing the membrane-permeable cysteine protease inhibitor E-64d and FM4-64, and examined whether Autolysosomes stained with FM4-64 are accumulated. The results suggest that autophagy accompanying the formation of Autolysosomes also occurs in Arabidopsis root cells. Such autophagy appeared to occur constitutively in the root cells in nutrient-sufficient culture medium. Even in atg5 mutants in which an autophagy-related gene is disrupted, accumulation of the structures stained with FM4-64, which likely correspond to Autolysosomes, was seen although at lower level than in wild type roots.
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Contribution of the plasma membrane and central vacuole in the formation of Autolysosomes in cultured tobacco cells.
Plant & cell physiology, 2004Co-Authors: Kanako Yano, Sumiko Matsui, Tomohiro Tsuchiya, Masayoshi Maeshima, Natsumaro Kutsuna, Seiichiro Hasezawa, Yuji MoriyasuAbstract:Autolysosomes accumulate in tobacco cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor. We characterized these plant Autolysosomes using fluorescent dyes and green fluorescent protein (GFP). Observation using the endocytosis markers, FM4-64 and Lucifer Yellow CH, suggested that there is a membrane flow from the plasma membrane to Autolysosomes. Using these dyes as well as GFP-AtVam3p, sporamin-GFP and gamma-VM23-GFP fusion proteins as markers of the central vacuole, we found transport of components of the central vacuole to Autolysosomes. Thus endocytosis and the supply from the central vacuole may contribute to the formation of Autolysosomes.
Jonathan D. Smith - One of the best experts on this subject based on the ideXlab platform.
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Abstract 323: Macrophages From the Atherosclerosis-prone DBA/2 Mouse Strain Carry a Null Allele of the Gpnmb Gene That Reduces Lysosome Function
Arteriosclerosis Thrombosis and Vascular Biology, 2019Co-Authors: Peggy Robinet, Brian Ritchey, Shuhui Wang Lorkowski, Alexander M. Alzayed, Sophia Degeorgia, Eve Schodowski, Jonathan D. SmithAbstract:In a previous study, we identified Autolysosome formation as the limiting step for turnover of cholesterol ester in lipid droplets of macrophage foam cells from the athero-sensitive DBA/2 strain co...
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Abstract 536: Lysosome Insufficiency in Atherosclerosis Prone DBA/2 Mouse Macrophages Associated With Impaired Autolysosome Formation and Lipid Drop Clearance
Arteriosclerosis Thrombosis and Vascular Biology, 2015Co-Authors: Peggy Robinet, Jonathan D. SmithAbstract:In a previous study, we identified Autolysosome formation as the limiting step for turnover of cholesterol esters in lipid droplets of macrophage foam cells from the atherosclerosis sensitive DBA/2 strain compared to the atherosclerosis resistant AKR mouse strain. As autophagosome formation was similar in these two strains, we wanted to evaluate the role of lysosome biogenesis and function on Autolysosome formation in AKR and DBA/2 cells. The transcription factor TFEB is a key regulator for lysosome biogenesis and function that positively regulates the expression of lysosomal enzymes and structural proteins, and controls lysosomes number. For all our studies, we cultured AKR and DBA/2 macrophages with or without acetylated LDL (AcLDL) for 24h. First, we analyzed TFEB protein expression by western blot. Upon loading, TFEB was increased in AKR (48%, p
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abstract 536 lysosome insufficiency in atherosclerosis prone dba 2 mouse macrophages associated with impaired Autolysosome formation and lipid drop clearance
Arteriosclerosis Thrombosis and Vascular Biology, 2015Co-Authors: Peggy Robinet, Jonathan D. SmithAbstract:In a previous study, we identified Autolysosome formation as the limiting step for turnover of cholesterol esters in lipid droplets of macrophage foam cells from the atherosclerosis sensitive DBA/2 strain compared to the atherosclerosis resistant AKR mouse strain. As autophagosome formation was similar in these two strains, we wanted to evaluate the role of lysosome biogenesis and function on Autolysosome formation in AKR and DBA/2 cells. The transcription factor TFEB is a key regulator for lysosome biogenesis and function that positively regulates the expression of lysosomal enzymes and structural proteins, and controls lysosomes number. For all our studies, we cultured AKR and DBA/2 macrophages with or without acetylated LDL (AcLDL) for 24h. First, we analyzed TFEB protein expression by western blot. Upon loading, TFEB was increased in AKR (48%, p<0.01) but not DBA/2 cells leading to a 45% higher TFEB level in AKR vs. DBA/2 foam cells (p<0.05), suggesting that lysosome number and function may be impaire...
Tobias B. Huber - One of the best experts on this subject based on the ideXlab platform.
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The class III phosphatidylinositol 3-kinase PIK3C3/VPS34 regulates endocytosis and autophagosome-Autolysosome formation in podocytes
Autophagy, 2013Co-Authors: Wibke Bechtel, Martin Helmstädter, Jan Balica, Björn Hartleben, Christoph Schell, Tobias B. HuberAbstract:Phosphatidylinositol phosphates are key regulators of vesicle identity, formation and trafficking. In mammalian cells, the evolutionarily conserved class III PtdIns 3-kinase PIK3C3/VPS34 is part of a large multiprotein complex that catalyzes the localized phosphorylation of phosphatidylinositol to phosphatidylinositol-3-phosphate (PtdIns3P). We demonstrate that PIK3C3 has a key function in vesicular trafficking, endocytosis and autophagosome-Autolysosome formation in the highly specialized glomerular podocytes.
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the class iii phosphatidylinositol 3 kinase pik3c3 vps34 regulates endocytosis and autophagosome Autolysosome formation in podocytes
Autophagy, 2013Co-Authors: Wibke Bechtel, Martin Helmstädter, Jan Balica, Björn Hartleben, Christoph Schell, Tobias B. HuberAbstract:Phosphatidylinositol phosphates are key regulators of vesicle identity, formation and trafficking. In mammalian cells, the evolutionarily conserved class III PtdIns 3-kinase PIK3C3/VPS34 is part of a large multiprotein complex that catalyzes the localized phosphorylation of phosphatidylinositol to phosphatidylinositol-3-phosphate (PtdIns3P). We demonstrate that PIK3C3 has a key function in vesicular trafficking, endocytosis and autophagosome-Autolysosome formation in the highly specialized glomerular podocytes.
Ji Eun Lee - One of the best experts on this subject based on the ideXlab platform.
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TRIM31 promotes Atg5/Atg7-independent autophagy in intestinal cells
Nature Communications, 2016Co-Authors: Taeyun A. Lee, Insook Jang, Hyun Jin Choi, Areum Park, Sujin Kang, Seung Won Kim, Jae Hee Cheon, Jin Won Cho, Ji Eun LeeAbstract:Autophagy is responsible for the bulk degradation of cytosolic constituents and plays an essential role in the intestinal epithelium by controlling beneficial host-bacterial relationships. Atg5 and Atg7 are thought to be critical for autophagy. However, Atg5- or Atg7-deficient cells still form autophagosomes and Autolysosomes, and are capable of removing proteins or bacteria. Here, we report that human TRIM31 (tripartite motif), an intestine-specific protein localized in mitochondria, is essential for promoting lipopolysaccharide-induced Atg5/Atg7-independent autophagy. TRIM31 directly interacts with phosphatidylethanolamine in a palmitoylation-dependent manner, leading to induction of Autolysosome formation. Depletion of endogenous TRIM31 significantly increases the number of intestinal epithelial cells containing invasive bacteria. Crohn's disease patients display TRIM31 downregulation. Human cytomegalovirus-infected intestinal cells show a decrease in TRIM31 expression as well as a significant increase in bacterial load, reversible by the introduction of wild-type TRIM31. We provide insight into an alternative autophagy pathway that protects against intestinal pathogenic bacterial infection.
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trim31 promotes atg5 atg7 independent autophagy in intestinal cells
Nature Communications, 2016Co-Authors: Taeyun A. Lee, Insook Jang, Hyun Jin Choi, Areum Park, Sujin Kang, Seung Won Kim, Jae Hee Cheon, Jin Won Cho, Ji Eun LeeAbstract:Autophagy is responsible for the bulk degradation of cytosolic constituents and plays an essential role in the intestinal epithelium by controlling beneficial host-bacterial relationships. Atg5 and Atg7 are thought to be critical for autophagy. However, Atg5- or Atg7-deficient cells still form autophagosomes and Autolysosomes, and are capable of removing proteins or bacteria. Here, we report that human TRIM31 (tripartite motif), an intestine-specific protein localized in mitochondria, is essential for promoting lipopolysaccharide-induced Atg5/Atg7-independent autophagy. TRIM31 directly interacts with phosphatidylethanolamine in a palmitoylation-dependent manner, leading to induction of Autolysosome formation. Depletion of endogenous TRIM31 significantly increases the number of intestinal epithelial cells containing invasive bacteria. Crohn's disease patients display TRIM31 downregulation. Human cytomegalovirus-infected intestinal cells show a decrease in TRIM31 expression as well as a significant increase in bacterial load, reversible by the introduction of wild-type TRIM31. We provide insight into an alternative autophagy pathway that protects against intestinal pathogenic bacterial infection.
Peggy Robinet - One of the best experts on this subject based on the ideXlab platform.
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Abstract 323: Macrophages From the Atherosclerosis-prone DBA/2 Mouse Strain Carry a Null Allele of the Gpnmb Gene That Reduces Lysosome Function
Arteriosclerosis Thrombosis and Vascular Biology, 2019Co-Authors: Peggy Robinet, Brian Ritchey, Shuhui Wang Lorkowski, Alexander M. Alzayed, Sophia Degeorgia, Eve Schodowski, Jonathan D. SmithAbstract:In a previous study, we identified Autolysosome formation as the limiting step for turnover of cholesterol ester in lipid droplets of macrophage foam cells from the athero-sensitive DBA/2 strain co...
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Abstract 536: Lysosome Insufficiency in Atherosclerosis Prone DBA/2 Mouse Macrophages Associated With Impaired Autolysosome Formation and Lipid Drop Clearance
Arteriosclerosis Thrombosis and Vascular Biology, 2015Co-Authors: Peggy Robinet, Jonathan D. SmithAbstract:In a previous study, we identified Autolysosome formation as the limiting step for turnover of cholesterol esters in lipid droplets of macrophage foam cells from the atherosclerosis sensitive DBA/2 strain compared to the atherosclerosis resistant AKR mouse strain. As autophagosome formation was similar in these two strains, we wanted to evaluate the role of lysosome biogenesis and function on Autolysosome formation in AKR and DBA/2 cells. The transcription factor TFEB is a key regulator for lysosome biogenesis and function that positively regulates the expression of lysosomal enzymes and structural proteins, and controls lysosomes number. For all our studies, we cultured AKR and DBA/2 macrophages with or without acetylated LDL (AcLDL) for 24h. First, we analyzed TFEB protein expression by western blot. Upon loading, TFEB was increased in AKR (48%, p
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abstract 536 lysosome insufficiency in atherosclerosis prone dba 2 mouse macrophages associated with impaired Autolysosome formation and lipid drop clearance
Arteriosclerosis Thrombosis and Vascular Biology, 2015Co-Authors: Peggy Robinet, Jonathan D. SmithAbstract:In a previous study, we identified Autolysosome formation as the limiting step for turnover of cholesterol esters in lipid droplets of macrophage foam cells from the atherosclerosis sensitive DBA/2 strain compared to the atherosclerosis resistant AKR mouse strain. As autophagosome formation was similar in these two strains, we wanted to evaluate the role of lysosome biogenesis and function on Autolysosome formation in AKR and DBA/2 cells. The transcription factor TFEB is a key regulator for lysosome biogenesis and function that positively regulates the expression of lysosomal enzymes and structural proteins, and controls lysosomes number. For all our studies, we cultured AKR and DBA/2 macrophages with or without acetylated LDL (AcLDL) for 24h. First, we analyzed TFEB protein expression by western blot. Upon loading, TFEB was increased in AKR (48%, p<0.01) but not DBA/2 cells leading to a 45% higher TFEB level in AKR vs. DBA/2 foam cells (p<0.05), suggesting that lysosome number and function may be impaire...
