Journal
CHEMISTRY & BIOLOGY
Volume 18, Issue 8, Pages 1042-1052Publisher
CELL PRESS
DOI: 10.1016/j.chembiol.2011.05.013
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Funding
- MEXT of Japan
- KAKENHI [22700381]
- RIKEN
- Human Frontier Science Program
- Grants-in-Aid for Scientific Research [22700381, 23247034] Funding Source: KAKEN
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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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