RRC ID 50105
Author Katayama H, Kogure T, Mizushima N, Yoshimori T, Miyawaki A.
Title A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery.
Journal Chem Biol
Abstract 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.
Volume 18(8)
Pages 1042-52
Published 2011-8-26
DOI 10.1016/j.chembiol.2011.05.013
PII S1074-5521(11)00204-3
PMID 21867919
MeSH Animals Anthozoa / chemistry Anthozoa / genetics Autophagy* Autophagy-Related Protein 5 Cell Line Gene Deletion Green Fluorescent Proteins / analysis Green Fluorescent Proteins / genetics Luminescent Proteins / analysis* Luminescent Proteins / genetics Lysosomes / metabolism* Mice Microscopy, Confocal / methods* Microtubule-Associated Proteins / genetics Mitochondria / metabolism Mutagenesis, Site-Directed Recombinant Fusion Proteins / analysis Recombinant Fusion Proteins / genetics
IF 5.915
Times Cited 237
DNA material mKeima (h)/pCS2 (RDB15236) dKeima (h)/pCS2 (RDB15237) mt-mKeima (h)/pIND(SP1) (RDB15238) mt-mKeima/pcDNA3 (RDB15456).