An Autophagic Flux Probe that Releases an Internal Control.
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.
Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
Takeshi
T
Kaizuka
Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
Hideaki
H
Morishita
Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
Yutaro
Y
Hama
Laboratory Animal and Genome Sciences Section, National Institute of Quantum and Radiological Science and Technology, Anagawa, Inage-ku, Chiba 263-8555, Japan.
Satoshi
S
Tsukamoto
Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
Takahide
T
Matsui
Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan; School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan.
Yuichiro
Y
Toyota
Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
Akihiko
A
Kodama
Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki, Kanagawa 216-8512, Japan.
Tomoaki
T
Ishihara
LTT Bio-Pharma Co., LTD., Tokyo 105-0022, Japan.
Tohru
T
Mizushima
Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan. Electronic address: nmizu@m.u-tokyo.ac.jp.
Noboru
N
Mizushima
D000818:Animals; D001343:Autophagy / Q000187:drug effects* / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004625:Embryo, Nonmammalian; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D015335:Molecular Probes / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010588:Phagosomes / Q000187:drug effects* / Q000378:metabolism; D054852:Small Molecule Libraries / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D044767:Ubiquitin-Protein Ligases; D015027:Zebrafish; D000070584:Tripartite Motif Proteins
D003546:Cysteine Endopeptidases; D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins; D008869:Microtubule-Associated Proteins; D015335:Molecular Probes; D009687:Nuclear Proteins; D054852:Small Molecule Libraries; D044767:Ubiquitin-Protein Ligases; C480860:Map1lc3b protein, mouse; C489296:Trim27 protein, mouse; D000070584:Tripartite Motif Proteins; C000599119:Atg4a protein, mouse