A Sensitive and Quantitative Technique for Detecting Autophagic Events Based on Lysosomal Delivery

SummaryWe 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.

Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (191 K)Download as PowerPoint slideHighlights► We have developed a sensitive method for monitoring an entire flux of autophagy ► The method uses a coral fluorescent protein that accumulates inside the lysosomes ► The probe can detect selective autophagy, including mitophagy ► The probe can detect an autophagy pathway in Atg5-deficient cells