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 LC3DG, a fluorescent probe to evaluate autophagic flux. This probe is cleaved by endogenous ATG4 proteases into equimolar amounts of GFP LC3 and RFP LC3DG. GFP LC3 is degraded by autophagy, while RFP LC3DG 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 LC3DG probe is a simple and quantitative method to evaluate autophagic flux in cultured cells and whole organisms.
Molecular cell