title: A new probe to measure autophagic flux in vitro and in vivo.
abstract: Macroautophagy is a catabolic process that delivers cytoplasmic components via the autophagosome to lysosomes for degradation. Measuring autophagic activity is critical to dissect molecular mechanisms and functions of autophagy but remains challenging due to the lack of a definitive method. We have recently developed a new fluorescent probe, GFP-LC3-RFP-LC3ΔG, to assess autophagic flux. Upon intracellular expression, the probe is cleaved by ATG4 family proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. The former is degraded by autophagy while the latter persists as an internal control in the cytosol. Autophagic flux can thus be quantified by obtaining the ratio of GFP:RFP signals. Using this method, we have identified several autophagy-modulating drugs by screening an approved drug library. We have also demonstrated that induced and basal autophagic flux can be monitored in zebrafish and mice.
authors.affiliation: a Department of Biochemistry and Molecular Biology , Graduate School and Faculty of Medicine, the University of Tokyo , Tokyo , Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Morishita
authors.affiliation: a Department of Biochemistry and Molecular Biology , Graduate School and Faculty of Medicine, the University of Tokyo , Tokyo , Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kaizuka
authors.affiliation: a Department of Biochemistry and Molecular Biology , Graduate School and Faculty of Medicine, the University of Tokyo , Tokyo , Japan.
authors.forename: Yutaro
authors.initials: Y
authors.lastname: Hama
authors.affiliation: a Department of Biochemistry and Molecular Biology , Graduate School and Faculty of Medicine, the University of Tokyo , Tokyo , Japan.
authors.forename: Noboru
authors.initials: N
authors.lastname: Mizushima
mesh_terms: D000818:Animals; D001343:Autophagy; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D015335:Molecular Probes / Q000737:chemistry*; D015027:Zebrafish
chemical_list: D008869:Microtubule-Associated Proteins; D015335:Molecular Probes; D049452:Green Fluorescent Proteins
keywords: LC3; autophagic flux assay; autophagy; mice; zebrafish