title: Genetically Encoded Fluorescent Probe for Imaging Apoptosis in Vivo with Spontaneous GFP Complementation.
abstract: Apoptosis plays a pivotal role in development and tissue homeostasis in multicellular organisms. Dysfunction of apoptosis is involved in many fatal diseases such as cancer. Visualization of apoptosis in living animals is necessary to understand the mechanism of apoptosis-related diseases. Here, we describe a genetically encoded fluorescent probe for imaging apoptosis in living multicellular organisms, based on spontaneous complementation of two fragments of a green fluorescent protein (GFP) variant (GFP OPT). The probe is designed for detection of mitochondria-mediated apoptosis during which a mitochondrial protein of Smac is released into cytosol. The Smac is connected with a carboxy-terminal fragment of GFP OPT (GFP11), whereas the remainder of GFP OPT (GFP(1-10)) is located in the cytosol. Under an apoptotic condition, the Smac is released from mitochondria into cytosol, allowing complementation of the GFP-OPT fragments and the emission of fluorescence. Live-cell imaging demonstrates that the probe enables detection of apoptosis in living cells with a high signal-to-background ratio. We applied the probe to living zebrafish, in which apoptotic cells were visualized with fluorescence. The technique provides a useful tool for the study of apoptosis in living animals, facilitating elucidation of the mechanisms of apoptosis-related diseases.
authors.affiliation: Department of Chemistry, School of Science, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Nasu
authors.affiliation: Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University , 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Asaoka
authors.affiliation: Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University , 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
authors.forename: Misako
authors.initials: M
authors.lastname: Namae
authors.affiliation: Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University , 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nishina
authors.affiliation: Department of Chemistry, School of Science, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Yoshimura
authors.affiliation: Department of Chemistry, School of Science, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Takeaki
authors.initials: T
authors.lastname: Ozawa
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D002470:Cell Survival; D005456:Fluorescent Dyes / Q000378:metabolism*; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D015027:Zebrafish
chemical_list: D005456:Fluorescent Dyes; D024101:Mitochondrial Proteins; D049452:Green Fluorescent Proteins