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Chem Biol. 2011 Aug 26;18(8):1042-52. doi: 10.1016/j.chembiol.2011.05.013.

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

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Life Function and Dynamics, ERATO, JST, Bunkyo-ku, Tokyo 113-8519, Japan.


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.

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