An Autophagic Flux Probe that Releases an Internal Control

Takeshi Kaizuka, Hideaki Morishita, Yutaro Hama, Satoshi Tsukamoto, Takahide Matsui, Yuichiro Toyota, Akihiko Kodama, Tomoaki Ishihara, Tohru Mizushima, Noboru Mizushima

Research output: Contribution to journalArticle

128 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)835-849
Number of pages15
JournalMolecular Cell
Volume64
Issue number4
DOIs
Publication statusPublished - 2016 Nov 17
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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    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. https://doi.org/10.1016/j.molcel.2016.09.037