title: Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3.
abstract: During the process of autophagy, autophagosomes undergo a maturation process consisting of multiple fusions with endosomes and lysosomes, which provide an acidic environment and digestive function to the interior of the autophagosome. Here we found that a fusion protein of monomeric red-fluorescence protein and LC3, the most widely used marker for autophagosomes, exhibits a quite different localization pattern from that of GFP-LC3. GFP-LC3 loses fluorescence due to lysosomal acidic and degradative conditions but mRFP-LC3 does not, indicating that the latter can label the autophagic compartments both before and after fusion with lysosomes. Taking advantage of this property, we devised a novel method for dissecting the maturation process of autophagosomes. mRFP-GFP tandem fluorescent-tagged LC3 (tfLC3) showed a GFP and mRFP signal before the fusion with lysosomes, and exhibited only the mRFP signal subsequently. Using this method, we provided evidence that overexpression of a dominant negative form of Rab7 prevented the fusion of autophagosomes with lysosomes, suggesting that Rab7 is involved in this step. This method will be of general utility for analysis of the autophagosome maturation process.
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Kimura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Noda
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Yoshimori
mesh_terms: D000818:Animals; D001343:Autophagy / Q000502:physiology*; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000502:physiology; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000502:physiology*; D008954:Models, Biological; D010588:Phagosomes / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D000097573:Red Fluorescent Protein
chemical_list: D017931:DNA Primers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C477012:MAP1LC3A protein, human; C480860:Map1lc3b protein, mouse