Direct quantification of autophagic flux by a single molecule-based probe

Macroautophagy/autophagy remains a rapidly advancing research topic, and there continues to be a need for constantly evolving methodology to investigate this pathway at each individual step. Accordingly, new assays to measure autophagic flux in a robust and reliable manner are essential to understand the mechanism and physiological roles of autophagy. Kaizuka et al. recently reported a new fluorescence probe, GFP-LC3-RFP-LC3ΔG to directly demonstrate autophagic flux without being combined with lysosomal inhibitors (see the Kaizuka et al. punctum in this issue of the journal). When expressed in cells, the probe is cleaved into a degradable/quenchable part, GFP-LC3, and stable/cytosolic part, RFP-LC3ΔG. The latter serves as an internal control and a decrease of the GFP:RFP ratio indicates the occurrence of autophagy. Since the key index of this probe is the degradation of GFP-LC3, it can be used to measure the cumulative effect of basal autophagy. The assay is applicable to high-throughput drug discovery as well as in vivo analysis.