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. [Display omitted] •A novel autophagic flux probe, GFP-LC3-RFP-LC3ΔG, was developed•This method can be applicable for various types of laboratory equipment•Autophagy-modulating drugs were identified by screening an approved drug library•Induced and basal autophagic flux was monitored in zebrafish and mice Macroautophagy is a major intracellular degradation system. Kaizuka, Morishita et al. developed a novel autophagic flux probe. Using this probe, autophagy-modulating drugs were identified by screening an approved drug library, and autophagic flux was measured in zebrafish and mice.