A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery

We sought to develop a sensitive and quantitative technique capable of monitoring the entire flux of Autophagy involving fusion of lysosomal membranes. We observed the accumulation inside lysosomal compartments of Keima, a coral-derived acid-stable Fluorescent protein that emits different-colored signals at acidic and neutral pHs. The cumulative fluorescent readout can be used to quantify Autophagy at a single time point. Remarkably, the technique led us to characterize an Autophagy pathway in Atg5-deficient cells, in which conventional LC3-based autophagosome probes are ineffective. Due to the large Stokes shift of Keima, this Autophagy probe can be visualized in conjunction with other green-emitting fluorophores. We examined Mitophagy as a selective autophagic process; time-lapse imaging of mitochondria-targeted Keima and GFP-Parkin allowed us to observe simultaneously Parkin recruitment to and autophagic degradation of mitochondria after membrane depolarization.