title: Visualization of mitochondria in living cells with a genetically encoded yellow fluorescent protein originating from a yellow-emitting luminous bacterium.
abstract: We have visualized redox and structural changes in the mitochondria of yeast Saccharomyces cerevisiae as a eukaryotic cell model using a genetically encoded yellow fluorescent protein (Y1-Yellow) and conventional fluorescence microscopy. Y1-Yellow originating from a yellow emitting luminous bacterium Aliivibrio sifiae Y1 was fused with a mitochondria-targeted sequence (mt-sequence). Y1-Yellow fluorescence arising only from the mitochondrial site and the color of yellow fluorescence could be easily differentiated from cellular autofluorescence and from that of conventional probes. Y1-Yellow expressing S. cerevisiae made the yellow fluorescence conspicuous at the mitochondrial site in response to reactive oxygen species (ROS) transiently derived in the wake of pretreatment with hydrogen peroxide. Based on our observation with Y1-Yellow fluorescence, we also showed that mitochondria rearrange to form a cluster structure surrounding chromosomal DNA via respiratory inhibition by cyanide, followed by the generation of ROS. In contrast, uptake of an uncoupler of oxidative phosphorylation is not responsible for mitochondrial rearrangement. These results indicate the utility of Y1-Yellow for visualization of mitochondrial vitality and morphology in living cells.
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, 1 Hashigami-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan. karatani@kit.ac.jp
authors.forename: Hajime
authors.initials: H
authors.lastname: Karatani
authors.forename: Yuki
authors.initials: Y
authors.lastname: Namikawa
authors.forename: Naomi
authors.initials: N
authors.lastname: Mori
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Nishikawa
authors.forename: Saki
authors.initials: S
authors.lastname: Imai
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Ihara
authors.forename: Aya
authors.initials: A
authors.lastname: Kinoshita
authors.forename: Kengo
authors.initials: K
authors.lastname: Kitadokoro
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Oyama
mesh_terms: D059706:Aliivibrio / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003486:Cyanides / Q000633:toxicity; D005947:Glucose / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007211:Indoles / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism; D059008:Time-Lapse Imaging; D014966:Xanthenes / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D003486:Cyanides; D007211:Indoles; D008164:Luminescent Proteins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D014966:Xanthenes; C121372:mitotracker orange; C054562:yellow fluorescent protein, Bacteria; C007293:DAPI; D006861:Hydrogen Peroxide; D005947:Glucose