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Autophagy. 2017 Apr 3;13(4):757-758. doi: 10.1080/15548627.2016.1278094. Epub 2017 Jan 25.

A new probe to measure autophagic flux in vitro and in vivo.

Author information

1
a Department of Biochemistry and Molecular Biology , Graduate School and Faculty of Medicine, the University of Tokyo , Tokyo , Japan.

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.

KEYWORDS:

LC3; autophagic flux assay; autophagy; mice; zebrafish

PMID:
28121224
PMCID:
PMC5388228
DOI:
10.1080/15548627.2016.1278094
[Indexed for MEDLINE]
Free PMC Article

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