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Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.

Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, Tanida I, Ezaki J, Mizushima N, Ohsumi Y, Uchiyama Y, Kominami E, Tanaka K, Chiba T - J. Cell Biol. (2005)

Bottom Line: Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation.Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies.Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japan.

ABSTRACT
Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation. This mechanism is involved in the preservation of nutrients under starvation condition as well as the normal turnover of cytoplasmic component. Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies. Here, we generated conditional knockout mice of Atg7, an essential gene for autophagy in yeast. Atg7 was essential for ATG conjugation systems and autophagosome formation, amino acid supply in neonates, and starvation-induced bulk degradation of proteins and organelles in mice. Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates. Our results indicate the important role of autophagy in starvation response and the quality control of proteins and organelles in quiescent cells.

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Accumulation of ubiquitin-positive aggregates in Atg7-deficient liver. (A and B) Immunofluorescent detection of ubiquitin in the Atg7F/+:Mx1 (A) and Atg7F/F:Mx1 (B) liver. (C–F) Immunoelectron micrographs of ubiquitin in a representative mutant liver. The high-magnification view shows ubiquitin particles near the lipid dropletlike structure (D–F). Bars, 0.5 μm. (G) Immunoblot analysis of the liver. PNS fractions of the liver at 90 d after injection were immunoblotted with the indicated antibodies. Data shown are representative of three separate experiments.
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fig7: Accumulation of ubiquitin-positive aggregates in Atg7-deficient liver. (A and B) Immunofluorescent detection of ubiquitin in the Atg7F/+:Mx1 (A) and Atg7F/F:Mx1 (B) liver. (C–F) Immunoelectron micrographs of ubiquitin in a representative mutant liver. The high-magnification view shows ubiquitin particles near the lipid dropletlike structure (D–F). Bars, 0.5 μm. (G) Immunoblot analysis of the liver. PNS fractions of the liver at 90 d after injection were immunoblotted with the indicated antibodies. Data shown are representative of three separate experiments.

Mentions: Autophagy has been implicated in not only organelle turnover but also in elimination of protein aggregates (Kopito, 2000). Protein aggregates are often ubiquitinated. In the next step, we immunostained the liver with an ubiquitin antibody to examine the presence of such aggregates. Several ubiquitin-positive particles of various sizes were detected in the Atg7F/F:Mx1 but not in Atg7F/+:Mx1 hepatic cells at both 10 and 90 d after pIpC injection (Fig. 7, A and B; and Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200412022/DC1). The immunoblots of the liver lysates revealed the accumulation of high-molecular mass polyubiquitinated proteins in the mutant liver (Fig. 7 G and Fig. S5), suggesting that the ubiquitin particles are aggregates of polyubiquitinated proteins. To further determine the localization of ubiquitin-positive dots, analysis of immunoelectron micrographs was performed. Numerous particles of colloidal gold, indicative of ubiquitin, were detected on lipid dropletlike structures, membranous structures, and amorphous substances in the cytoplasm (Fig. 7, C–F). Such signals were not observed in the wild-type liver (unpublished data).


Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.

Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, Tanida I, Ezaki J, Mizushima N, Ohsumi Y, Uchiyama Y, Kominami E, Tanaka K, Chiba T - J. Cell Biol. (2005)

Accumulation of ubiquitin-positive aggregates in Atg7-deficient liver. (A and B) Immunofluorescent detection of ubiquitin in the Atg7F/+:Mx1 (A) and Atg7F/F:Mx1 (B) liver. (C–F) Immunoelectron micrographs of ubiquitin in a representative mutant liver. The high-magnification view shows ubiquitin particles near the lipid dropletlike structure (D–F). Bars, 0.5 μm. (G) Immunoblot analysis of the liver. PNS fractions of the liver at 90 d after injection were immunoblotted with the indicated antibodies. Data shown are representative of three separate experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171928&req=5

fig7: Accumulation of ubiquitin-positive aggregates in Atg7-deficient liver. (A and B) Immunofluorescent detection of ubiquitin in the Atg7F/+:Mx1 (A) and Atg7F/F:Mx1 (B) liver. (C–F) Immunoelectron micrographs of ubiquitin in a representative mutant liver. The high-magnification view shows ubiquitin particles near the lipid dropletlike structure (D–F). Bars, 0.5 μm. (G) Immunoblot analysis of the liver. PNS fractions of the liver at 90 d after injection were immunoblotted with the indicated antibodies. Data shown are representative of three separate experiments.
Mentions: Autophagy has been implicated in not only organelle turnover but also in elimination of protein aggregates (Kopito, 2000). Protein aggregates are often ubiquitinated. In the next step, we immunostained the liver with an ubiquitin antibody to examine the presence of such aggregates. Several ubiquitin-positive particles of various sizes were detected in the Atg7F/F:Mx1 but not in Atg7F/+:Mx1 hepatic cells at both 10 and 90 d after pIpC injection (Fig. 7, A and B; and Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200412022/DC1). The immunoblots of the liver lysates revealed the accumulation of high-molecular mass polyubiquitinated proteins in the mutant liver (Fig. 7 G and Fig. S5), suggesting that the ubiquitin particles are aggregates of polyubiquitinated proteins. To further determine the localization of ubiquitin-positive dots, analysis of immunoelectron micrographs was performed. Numerous particles of colloidal gold, indicative of ubiquitin, were detected on lipid dropletlike structures, membranous structures, and amorphous substances in the cytoplasm (Fig. 7, C–F). Such signals were not observed in the wild-type liver (unpublished data).

Bottom Line: Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation.Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies.Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japan.

ABSTRACT
Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation. This mechanism is involved in the preservation of nutrients under starvation condition as well as the normal turnover of cytoplasmic component. Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies. Here, we generated conditional knockout mice of Atg7, an essential gene for autophagy in yeast. Atg7 was essential for ATG conjugation systems and autophagosome formation, amino acid supply in neonates, and starvation-induced bulk degradation of proteins and organelles in mice. Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates. Our results indicate the important role of autophagy in starvation response and the quality control of proteins and organelles in quiescent cells.

Show MeSH
Related in: MedlinePlus