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"Autophagic flux" in normal mouse tissues: focus on endogenous LC3A processing.

Identifieur interne : 001480 ( Main/Exploration ); précédent : 001479; suivant : 001481

"Autophagic flux" in normal mouse tissues: focus on endogenous LC3A processing.

Auteurs : Christos E. Zois [Grèce] ; Alexandra Giatromanolaki ; Efthimios Sivridis ; Marina Papaiakovou ; Heikki Kainulainen ; Michael I. Koukourakis

Source :

RBID : pubmed:21997374

Descripteurs français

English descriptors

Abstract

Autophagy is a major intracellular pathway for the degradation and recycling of long-lived proteins, mature ribosomes and even entire organelles. The best studied autophagic marker is the LC3B and it is believed that only the amount of the LC3B-II correlates with the amount of the autophagic membranes. Whether the LC3A processing, aside to LC3B, is a valuable endogenous 'autophagic flux' marker is far less clear. The specificity of rabbit polyclonal antibodies to the LC3A and the LC3B was tested against the commercial available human recombinant proteins LC3A and LC3B. In order to measure 'autophagic flux' in mouse liver, lung, kidney and heart we used: (1) a lysosomotropic reagent chloroquine, which inhibit the intra-lysosomal acidification or their fusion with autophagosome, (2) nutrient starvation as an autophagic stimulus and (3) ionizing radiation, which is known to destabilize lysosomes. According to the immunoblotting work the LC3A protein follows discrete patterns of LC3A-I and LC3A-II changes in liver, lung, kidney and heart tissues of mice, whereas the LC3B protein didn't follow the same pattern under stressor conditions. We conclude that the endogenous LC3A processing is a major marker of autophagy flux in mouse model. Fractionated samples (soluble vs. membrane fractions) should be used in immunoblotting to allow discrimination between the LC3-I soluble and the LC3-II membrane protein and kinetics. Further, when dealing with in vivo models it is necessary to check the specificity of the antibodies used against the LC3A and LC3B proteins as their expression and responsiveness is not overlapping.

DOI: 10.4161/auto.7.11.16664
PubMed: 21997374


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Le document en format XML

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<term>Apoptosis Regulatory Proteins (metabolism)</term>
<term>Autophagy (drug effects)</term>
<term>Autophagy (radiation effects)</term>
<term>Beclin-1</term>
<term>Chloroquine (pharmacology)</term>
<term>Gene Expression Regulation (drug effects)</term>
<term>Gene Expression Regulation (radiation effects)</term>
<term>Heat-Shock Proteins (metabolism)</term>
<term>Hepatocytes (cytology)</term>
<term>Hepatocytes (drug effects)</term>
<term>Hepatocytes (metabolism)</term>
<term>Hepatocytes (radiation effects)</term>
<term>Humans</term>
<term>Immunohistochemistry</term>
<term>Liver (cytology)</term>
<term>Liver (drug effects)</term>
<term>Liver (metabolism)</term>
<term>Male</term>
<term>Mice</term>
<term>Mice, Inbred BALB C</term>
<term>Microtubule-Associated Proteins (genetics)</term>
<term>Microtubule-Associated Proteins (immunology)</term>
<term>Microtubule-Associated Proteins (metabolism)</term>
<term>Organ Specificity (drug effects)</term>
<term>Organ Specificity (radiation effects)</term>
<term>Protein Processing, Post-Translational (drug effects)</term>
<term>Protein Processing, Post-Translational (radiation effects)</term>
<term>RNA, Messenger (genetics)</term>
<term>RNA, Messenger (metabolism)</term>
<term>Radiation, Ionizing</term>
<term>Sequestosome-1 Protein</term>
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<term>ARN messager (génétique)</term>
<term>ARN messager (métabolisme)</term>
<term>Animaux</term>
<term>Autophagie ()</term>
<term>Autophagie (effets des radiations)</term>
<term>Bécline-1</term>
<term>Chloroquine (pharmacologie)</term>
<term>Foie ()</term>
<term>Foie (cytologie)</term>
<term>Foie (métabolisme)</term>
<term>Humains</term>
<term>Hépatocytes ()</term>
<term>Hépatocytes (cytologie)</term>
<term>Hépatocytes (effets des radiations)</term>
<term>Hépatocytes (métabolisme)</term>
<term>Immunohistochimie</term>
<term>Maturation post-traductionnelle des protéines ()</term>
<term>Maturation post-traductionnelle des protéines (effets des radiations)</term>
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<term>Protéines adaptatrices de la transduction du signal (métabolisme)</term>
<term>Protéines associées aux microtubules (génétique)</term>
<term>Protéines associées aux microtubules (immunologie)</term>
<term>Protéines associées aux microtubules (métabolisme)</term>
<term>Protéines du choc thermique (métabolisme)</term>
<term>Protéines régulatrices de l'apoptose (métabolisme)</term>
<term>Rayonnement ionisant</term>
<term>Régulation de l'expression des gènes ()</term>
<term>Régulation de l'expression des gènes (effets des radiations)</term>
<term>Souris</term>
<term>Souris de lignée BALB C</term>
<term>Spécificité d'organe ()</term>
<term>Spécificité d'organe (effets des radiations)</term>
<term>Spécificité des anticorps (immunologie)</term>
<term>Séquestosome-1</term>
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<term>Microtubule-Associated Proteins</term>
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<term>Adaptor Proteins, Signal Transducing</term>
<term>Apoptosis Regulatory Proteins</term>
<term>Heat-Shock Proteins</term>
<term>Microtubule-Associated Proteins</term>
<term>RNA, Messenger</term>
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<term>Foie</term>
<term>Hépatocytes</term>
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<keywords scheme="MESH" qualifier="cytology" xml:lang="en">
<term>Hepatocytes</term>
<term>Liver</term>
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<term>Autophagy</term>
<term>Gene Expression Regulation</term>
<term>Hepatocytes</term>
<term>Liver</term>
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<term>Autophagie</term>
<term>Hépatocytes</term>
<term>Maturation post-traductionnelle des protéines</term>
<term>Régulation de l'expression des gènes</term>
<term>Spécificité d'organe</term>
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<term>ARN messager</term>
<term>Protéines associées aux microtubules</term>
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<term>Protéines associées aux microtubules</term>
<term>Spécificité des anticorps</term>
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<term>Antibody Specificity</term>
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<term>Liver</term>
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<term>Foie</term>
<term>Hépatocytes</term>
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<term>Protéines associées aux microtubules</term>
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<term>Protéines régulatrices de l'apoptose</term>
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<term>Gene Expression Regulation</term>
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<term>Organ Specificity</term>
<term>Protein Processing, Post-Translational</term>
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<term>Hépatocytes</term>
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<term>Régulation de l'expression des gènes</term>
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<front>
<div type="abstract" xml:lang="en">Autophagy is a major intracellular pathway for the degradation and recycling of long-lived proteins, mature ribosomes and even entire organelles. The best studied autophagic marker is the LC3B and it is believed that only the amount of the LC3B-II correlates with the amount of the autophagic membranes. Whether the LC3A processing, aside to LC3B, is a valuable endogenous 'autophagic flux' marker is far less clear. The specificity of rabbit polyclonal antibodies to the LC3A and the LC3B was tested against the commercial available human recombinant proteins LC3A and LC3B. In order to measure 'autophagic flux' in mouse liver, lung, kidney and heart we used: (1) a lysosomotropic reagent chloroquine, which inhibit the intra-lysosomal acidification or their fusion with autophagosome, (2) nutrient starvation as an autophagic stimulus and (3) ionizing radiation, which is known to destabilize lysosomes. According to the immunoblotting work the LC3A protein follows discrete patterns of LC3A-I and LC3A-II changes in liver, lung, kidney and heart tissues of mice, whereas the LC3B protein didn't follow the same pattern under stressor conditions. We conclude that the endogenous LC3A processing is a major marker of autophagy flux in mouse model. Fractionated samples (soluble vs. membrane fractions) should be used in immunoblotting to allow discrimination between the LC3-I soluble and the LC3-II membrane protein and kinetics. Further, when dealing with in vivo models it is necessary to check the specificity of the antibodies used against the LC3A and LC3B proteins as their expression and responsiveness is not overlapping.</div>
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<name sortKey="Sivridis, Efthimios" sort="Sivridis, Efthimios" uniqKey="Sivridis E" first="Efthimios" last="Sivridis">Efthimios Sivridis</name>
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