An Autophagic Flux Probe that Releases an Internal Control

Mol Cell. 2016 Nov 17;64(4):835-849. doi: 10.1016/j.molcel.2016.09.037. Epub 2016 Nov 3.

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.

MeSH terms

  • Animals
  • Autophagy / drug effects*
  • Autophagy / genetics
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Embryo, Nonmammalian
  • Gene Expression Regulation
  • Genes, Reporter
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • High-Throughput Screening Assays*
  • Humans
  • Lysosomes / drug effects*
  • Lysosomes / metabolism
  • Mice
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism
  • Molecular Probes / genetics*
  • Molecular Probes / metabolism
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Phagosomes / drug effects*
  • Phagosomes / metabolism
  • Small Molecule Libraries / pharmacology*
  • Spectrometry, Fluorescence
  • Ubiquitin-Protein Ligases
  • Zebrafish

Substances

  • DNA-Binding Proteins
  • MAP1LC3 protein, mouse
  • Microtubule-Associated Proteins
  • Molecular Probes
  • Nuclear Proteins
  • Small Molecule Libraries
  • Green Fluorescent Proteins
  • Trim27 protein, mouse
  • Ubiquitin-Protein Ligases
  • Atg4a protein, mouse
  • Cysteine Endopeptidases