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
-
- Autophagy/drug effects*
- Autophagy/genetics
- Cysteine Endopeptidases/genetics
- Cysteine Endopeptidases/metabolism
- Humans
- Nuclear Proteins/genetics
- Nuclear Proteins/metabolism
- Zebrafish
- Spectrometry, Fluorescence
- HeLa Cells
- Green Fluorescent Proteins/genetics
- Green Fluorescent Proteins/metabolism
- Animals
- Genes, Reporter
- Molecular Probes/genetics*
- Molecular Probes/metabolism
- Phagosomes/drug effects*
- Phagosomes/metabolism
- DNA-Binding Proteins/genetics
- DNA-Binding Proteins/metabolism
- Gene Expression Regulation
- Lysosomes/drug effects*
- Lysosomes/metabolism
- Embryo, Nonmammalian
- Microtubule-Associated Proteins/genetics
- Microtubule-Associated Proteins/metabolism
- Mice
- High-Throughput Screening Assays*
- Small Molecule Libraries/pharmacology*
- PubMed
- 27818143 Full text @ Mol. Cell
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.
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
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping