RRC ID 47625
Author Kaizuka T, Morishita H, Hama Y, Tsukamoto S, Matsui T, Toyota Y, Kodama A, Ishihara T, Mizushima T, Mizushima N.
Title An Autophagic Flux Probe that Releases an Internal Control.
Journal 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.
Volume 64(4)
Pages 835-849
Published 2016-11-17
DOI 10.1016/j.molcel.2016.09.037
PII S1097-2765(16)30589-5
PMID 27818143
MeSH Animals Autophagy / drug effects* Autophagy / genetics Cysteine Endopeptidases / genetics Cysteine Endopeptidases / metabolism DNA-Binding Proteins / genetics DNA-Binding Proteins / metabolism Embryo, Nonmammalian Gene Expression Regulation Genes, Reporter Green Fluorescent Proteins / genetics Green Fluorescent Proteins / metabolism HeLa Cells High-Throughput Screening Assays* Humans Lysosomes / drug effects* Lysosomes / metabolism Mice Microtubule-Associated Proteins / genetics Microtubule-Associated Proteins / metabolism Molecular Probes / genetics* Molecular Probes / metabolism Nuclear Proteins / genetics Nuclear Proteins / metabolism Phagosomes / drug effects* Phagosomes / metabolism Small Molecule Libraries / pharmacology* Spectrometry, Fluorescence Ubiquitin-Protein Ligases Zebrafish
IF 14.548
Times Cited 127
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY CELL BIOLOGY
Resource
DNA material pMRX-IP-GFP-LC3-RFP-LC3 delta G (RDB14600) pMRX-IP-GFP-LC3-RFP (RDB14601).