title: A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery.
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.
authors.affiliation: Life Function and Dynamics, ERATO, JST, Bunkyo-ku, Tokyo 113-8519, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Katayama
authors.forename: Takako
authors.initials: T
authors.lastname: Kogure
authors.forename: Noboru
authors.initials: N
authors.lastname: Mizushima
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Yoshimori
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
mesh_terms: D000818:Animals; D037421:Anthozoa / Q000737:chemistry / Q000235:genetics; D001343:Autophagy; D000071187:Autophagy-Related Protein 5; D002460:Cell Line; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D008869:Microtubule-Associated Proteins / Q000235:genetics; D008928:Mitochondria / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics
chemical_list: C495773:Atg5 protein, mouse; D000071187:Autophagy-Related Protein 5; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins