PUBLICATION

An Autophagic Flux Probe that Releases an Internal Control

Authors
Kaizuka, T., Morishita, H., Hama, Y., Tsukamoto, S., Matsui, T., Toyota, Y., Kodama, A., Ishihara, T., Mizushima, T., Mizushima, N.
ID
ZDB-PUB-161108-6
Date
2016
Source
Molecular Cell   64(4): 835-849 (Journal)
Registered Authors
Mizushima, Noboru, Morishita, Hideaki
Keywords
none
MeSH Terms
  • Lysosomes/drug effects*
  • Lysosomes/metabolism
  • Microtubule-Associated Proteins/genetics
  • Microtubule-Associated Proteins/metabolism
  • Genes, Reporter
  • Cysteine Endopeptidases/genetics
  • Cysteine Endopeptidases/metabolism
  • Mice
  • Gene Expression Regulation
  • High-Throughput Screening Assays*
  • Animals
  • Zebrafish
  • Phagosomes/drug effects*
  • Phagosomes/metabolism
  • HeLa Cells
  • Small Molecule Libraries/pharmacology*
  • Embryo, Nonmammalian
  • Molecular Probes/genetics*
  • Molecular Probes/metabolism
  • Nuclear Proteins/genetics
  • Nuclear Proteins/metabolism
  • DNA-Binding Proteins/genetics
  • DNA-Binding Proteins/metabolism
  • Spectrometry, Fluorescence
  • Humans
  • Autophagy/drug effects*
  • Autophagy/genetics
  • Green Fluorescent Proteins/genetics
  • Green Fluorescent Proteins/metabolism
(all 29)
PubMed
27818143 Full text @ Mol. Cell
Abstract
Macroautophagy is an intracellular degradation system that utilizes the autophagosome to deliver cytoplasmic components to the lysosome. Measuring autophagic activity is critically important but remains complicated and challenging. Here, we have developed GFP-LC3-RFP-LC3ΔG, a fluorescent probe to evaluate autophagic flux. This probe is cleaved by endogenous ATG4 proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. GFP-LC3 is degraded by autophagy, while RFP-LC3ΔG remains in the cytosol, serving as an internal control. Thus, autophagic flux can be estimated by calculating the GFP/RFP signal ratio. Using this probe, we re-evaluated previously reported autophagy-modulating compounds, performed a high-throughput screen of an approved drug library, and identified autophagy modulators. Furthermore, we succeeded in measuring both induced and basal autophagic flux in embryos and tissues of zebrafish and mice. The GFP-LC3-RFP-LC3ΔG probe is a simple and quantitative method to evaluate autophagic flux in cultured cells and whole organisms.
Genes / Markers
Figures
Figure Gallery (3 images)
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Expression
Phenotype
Fish Conditions Stage Phenotype Figure
jt2Tgchemical treatment by environment: torin 1Prim-5
jt2Tgchemical treatment by environment: torin 1Prim-5
rb1cc1jt1/jt1standard conditionsPrim-5
rb1cc1jt1/jt1standard conditionsPrim-5
rb1cc1jt1/jt1standard conditionsPrim-5
rb1cc1jt1/jt1standard conditionsPrim-5
1 - 6 of 6
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Mutations / Transgenics
Allele Construct Type Affected Genomic Region
jt1
    		Small Deletion
    jt2TgTransgenic Insertion
      1 - 2 of 2
      Show
      Human Disease / Model
      No data available
      Sequence Targeting Reagents
      Target Reagent Reagent Type
      rb1cc1CRISPR1-rb1cc1CRISPR
      1 - 1 of 1
      Show
      Fish
      Fish
      jt2Tg
      rb1cc1jt1/jt1
      1 - 2 of 2
      Show
      Antibodies
      No data available
      Orthology
      No data available
      Engineered Foreign Genes
      Marker Marker Type Name
      GFPEFGGFP
      mRFPEFGmRFP
      1 - 2 of 2
      Show
      Mapping
      No data available
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      Citation
      Kaizuka, T., Morishita, H., Hama, Y., Tsukamoto, S., Matsui, T., Toyota, Y., Kodama, A., Ishihara, T., Mizushima, T., Mizushima, N. (2016) An Autophagic Flux Probe that Releases an Internal Control. Molecular Cell. 64(4):835-849.