报告题目：Fluorescent andbioluminescent sensors for single cell analysis
报告人：Takeaki Ozawa
报告时间：11月25日（周二）下午3点
报告地点：401号楼一楼会议室
欧洲杯官网
放射医学及交叉学科研究院
2014年11月24日
Abstract:
Acurrent focus of biological research is to quantify and image cellularprocesses in living subjects. To analyze such cellular processes,genetically-encoded reporters have been extensively used. The most commonreporters are firefly luciferase, renilla luciferase, green fluorescent protein(GFP) and its variants with various spectral properties. Herein, novel designof split GFP and split luciferase will be described; the principle is based onreconstitution of the split-reporter fragments. I will focus on recent advancesin fluorescence and bioluminescence imaging using the reconstitutiontechnology.
Takeaki Ozawa
Department of Chemistry
Graduate School of Science
The University of Tokyo
Hongo, Bunkyo-ku, Tokyo113-0033, Japan
Phone: +81-3-5841-4351
Fax: +81-3-5802-2989
E-mail : ozawa@chem.s.u-tokyo.ac.jp
Takeaki Ozawa received his B.S. degree in 1993 andM.S. degree in 1995 from The University of Tokyo, Japan. He completed thedoctoral course at the University of Tokyo, Graduate Schoolof Science, and received his Ph.D in 1998. He served as research associate atThe University of Tokyo, and started an independent position of associateprofessor at the Institute for Molecular Science, Japan. He is in current position ofprofessor at Department of Chemistry, Graduate School of Science, TheUniversity of Tokyo. His research interest is “to develop analytical methodsfor visualizing and controlling functions of molecules in living subjects”.
Awards
The Chemical Society of Japan (CSJ)Awards for Young Chemists (2004).
Young Scientists Awardfrom Minister of MEXT (Ministry of Education, Culture, Science and Technology)(2005).
Japan Society for thePromotion of Science (JSPS) Prize (2010).
Publications(selected papers)
(1) Sustained Accurate Recording of IntracellularAcidification in Living Tissues with a Photo-controllable BioluminescentProtein. M. Hattori, S. Haga, H. Takakura, M.Ozaki and T. Ozawa, Proc. Nat.Acad. Sci. USA, 110, 9332-9337(2013).
(2) Rapid and high-sensitivity cell-based assays ofprotein–protein interactions using split click beetle luciferasecomplementation: An approach to the study of G protein-coupled receptors. N.Misawa, A.K.M. Kafi, M. Hattori, K. Miura, K. Masuda and T. Ozawa, Anal. Chem., 82, 2552-2560 (2010).
(3) High-Sensitivity Real-Time Imaging of DualProtein-Protein Interactions in Living Subjects Using Multicolor Luciferases.N. Hida, M. Awais, M. Takeuchi, N. Ueno, M. Tashiro, T. Singh, M. Hayashi, K.Ohmiya and T. Ozawa, PLoS ONE,4, e5868 (2009).
(4) Cyclic Luciferase for Real-Time Sensing ofCaspase-3 Activities in Living Mammals. A. Kanno, Y. Yamanaka, H. Hirano, Y.Umezawa and T. Ozawa, Angew. Chem.Int. Ed. 46, 7595-7599 (2007).
(5) Imaging Dynamics of Endogenous Mitochondrial RNAin Single Living Cells. T. Ozawa, Y. Natori, M. Sato and Y. Umezawa, Nature Methods, 4, 413-419 (2007).
(6) High-throughput Sensing and Non-invasive Imagingof Protein Nuclear Transport by Using Reconstitution of Split RenillaLuciferase. S. B. Kim*, T.Ozawa*, S. Watanabe and Y. Umezawa, Proc. Natl. Acad. Sci. U.S.A., 101,11542-11547 (2004). (*equal contribution to this work)
(7) A genetic Approach to Identifying MitochondrialProteins. T. Ozawa, Y. Sako, M.Sato, T. Kitamura, and Y. Umezawa, NatureBiotechnol., 21, 287-293 (2003).
(8) Fluorescent Indicators for ImagingProtein Phosphorylation in Single Living Cells. M. Sato, T. Ozawa, K. Inukai,T. Asano, and Y. Umezawa, NatureBiotechnol., 20, 287-294 (2002).
Publications(all)
Original Papers (Pee-Reviewed Publications)
(1) Multimodal and multiplexspectral imaging of rat cornea ex vivousing a white-light laser source. H.Segawa, Y. Kaji, P. Leproux, V. Couderc, T.Ozawa, T. Oshika, and H. Kano, J.Biophotonics, in press (2014).
(2) Structure-biasrelationships for fenoterol stereoisomers in six molecular and cellular assaysat the β2-adrenoceptor. M.T. Reinartz, S. K?lble, T. Littmann, T. Ozawa, S.Dove, V. Kaever, I.W. Wainer, R. Seifert, NaunynSchmiedebergs Arch Pharmacol., in press. (2014 Oct 24.)
(3) p62/SQSTM1 plays aprotective role in oxidative injury of steatotic liver in a mouse hepatectomymodel. S. Haga, T. Ozawa, Y. Yamada, N. Morita, I. Nagashima, H. Inoue,Y. Inaba, N. Noda, R. Abe, K. Umezawa, M. Ozaki, Antioxid. Redox Signal., in press.
(4) Electronically resonantthird-order sum frequency generation spectroscopy using a nanosecondwhite-light supercontinuum. H. Segawa, N. Fukutake, P. Leproux, V. Couderc, T.Ozawa and H. Kano, Opt. Express, 22, 10416-10429 (2014).
(5) Developing a potentiallyimmunologically-inert tetracycline-regulatable viral vector for gene therapy inthe peripheral nerve. S. Hoyng, S. Gnavi, F. Winter, R. Eggers, T. Ozawa,A. Zaldumbide, R. Hoeben, M. Malessy, and J. Verhaagen, Gene Therapy, in press.
(6) Long noncoding RNANEAT1-dependent SFPQ relocation between nuclear body paraspeckle and promotermediates IL8 expression in response to immune stimuli. K. Imamura, N. Imamachi,G. Akizuki, M. Kumakura, A. Kawaguchi, K. Nagata, A. Kato, Y. Kawaguchi, H.Sato, M. Yoneda, C. Kai, T. Yada, Y. Suzuki, T. Yamada, T. Ozawa, K.Kaneki, T. Inoue, M. Kobayashi, T. Kodama, Y. Wada, K. Sekimizu, N. Akimitsu, Mol. Cell, 53, 393-406 (2014).
(7) Assay methods forSUMO-SIM interactions in vivo and in vitro using a split-luciferase complementationsystem. M. Hirohama, A.R. Voet, T. Ozawa, H. Saitoh, Y. Nakao, K.Y.Zhang, A. Ito and M. Yoshida, Anal.Biochem., 448, 92-94 (2013).
(8) Bioluminescent Probes toAnalyze Ligand-induced Phosphatidylinositol 3,4,5-trisphosphate Production withSplit Luciferase Complementation. L.Z. Yang, Y. Nasu, M. Hattori, H. Yoshimura,A. Kanno, T.Ozawa, Anal.Chem., 85, 11352-11359 (2013).
(9) ROS stress resetscircadian clocks to coordinate pro-survival signals. T. Tamaru, M. Hattori, Y.Ninomiya, G. Kawamura, G. Varès, K. Honda, D. P. Mishra, B. Wang, I. Benjamin,P. Sassone-Corsi, T. Ozawa and K. Takamatsu, PLoS ONE, 8, e82006 (2013).
(10)Folding coupled with assembly in split green fluorescent proteins studiedby structure-based molecular simulations. M. Ito, T. Ozawa and S. Takada, J. Phys. Chem., 117,13212-13218 (2013).
(11)Long-time Accurate Recording of Intracellular Acidification in LivingTissues with a Photo-controllable Bioluminescent Protein. M. Hattori, S. Haga, H. Takakura, M. Ozaki and T. Ozawa, Proc. Nat.Acad. Sci. USA, 110, 9332-9337(2013).
(12)Spatiotemporal visualisation of proHB-EGF ectodomain shedding in livingcells. H. Inoue, T.Ozawa and S. Higashiyama, J. Biochem., 154, 67-76 (2013).
(13)Analysis of temporal patterns of GPCR–β-arrestin interactions using splitluciferase-fragment complementation. M. Hattori, M. Tanaka, H. Takakura, K,Aoki, K. Miura, T. Anzai and T. Ozawa, Mol. Biosystems, 9,957-964 (2013).
(14)Longitudinal Bioluminescence Imaging of the Dynamics of Doxorubicin InducedApoptosis. G. Niu, L. Zhu, D.N. Ho, F. Zhang, H. Gao, Q. Quan, N. Hida, T.Ozawa, G. Liu and X. Chen, Theranostics,3, 190-200 (2013).
(15)Rational Design and Development of Near-infrared-emitting FireflyLuciferins Available in vivo. R. Kojima, H. Takakura, T. Ozawa, Y. Tada,T. Nagano and Y. Urano, Angew. Chem. Int.Ed., 52,1175-1179 (2013).
(16)Rational Design and Development of Near-infrared-emitting FireflyLuciferins Available in vivo. R. Kojima, H. Takakura, T. Ozawa, Y. Tada, T. Nagano and Y. Urano, Angew. Chem. Int. Ed., in press.
(17)Development of 5'- and 7'-Substituted Luciferin Analogues as Acid-TolerantSubstrates of Firefly Luciferase. H. Takakura, R. Kojima, T. Ozawa, T. Nagano and Y. Urano, ChemBioChem, 13, 1424-1427 (2012).
(18)Measuring CREB activation using bioluminescent probes that detect KID-KIXinteraction in living cells. T. Ishimoto, H. Mano, T. Ozawa and H. Mori, BioconjugateChem., 23, 923-932 (2012).
(19)Fluorescent Probes for Imaging Endogenous β-actin mRNA in Living Cells Using FluorescentProtein-tagged Pumilio. H. Yoshimura, A. Inaguma, T. Yamada and T. Ozawa, ACS Chem.Biol., 7, 999-1005 (2012).
(20)Visualization and Quantitative Analysis of G Protein-Coupled Receptor?β-Arrestin Interaction in Single Cells and SpecificOrgans of Living Mice Using SplitLuciferase Complementation. H. Takakura, M. Hattori, M. Takeuchi and T. Ozawa, ACS Chem.Biol., 7, 901-910 (2012).
(21)In vivo monitoring of liver damage using caspase-3 probe. M. Ozaki, S. Hagaand T. Ozawa, Theranostics, 2, 207-214 (2012).
(22) Synchronization ofcircadian Per2 rhythms and HSF1-BMAL1:CLOCK interaction in mouse fibroblastsafter short-term heat shock pulse. T. Tamaru, M. Hattori, K. Honda, I.Benjamin, T. Ozawa and K. Takamatsu, PLoSONE, 6, e24521 (2011).
(23) Visualization ofnon-engineered single mRNAs in living cells using genetically encodedfluorescent probes. T. Yamada, H. Yoshimura, A. Inaguma and T. Ozawa, Anal. Chem., 83, 5708-5714 (2011).
(24) Real-time monitoring ofactin polymerization in living cells using split luciferase. T. Ishimoto, T.Ozawa and H. Mori, Bioconjugate Chem.,22, 1136-1144 (2011).
(25) Dual-ColorBioluminescence Analysis for Quantitatively Monitoring G-Protein-CoupledReceptor and b-Arrestin Interactions. A.K.M. Kafi, M. Hattori, N. Misawa and T.Ozawa, Pharmaceuticals, 4, 457-469 (2011).
(26) RatiometricBioluminescence Indicators for Monitoring Cyclic AMP in Live Cells Based onLuciferase-Fragment Complementation. M. Takeuchi, Y. Nagaoka, T. Yamada, H.Takakura and T. Ozawa, Anal. Chem.,82, 9306-9313 (2010).
(27) Luciferases for the Studyof Protein-Protein Interactions in Live Cells and Animals. A.K.M. Kafi, M.Hattori and T. Ozawa, Nano Life,1, 45-62 (2010).
(28) p66(Shc) has a PivotalFunction in Impaired Liver Regeneration in Aged Mice by a Redox-DependentMechanism. S. Haga, N. Morita, K. Irani, M.Fujiyoshi, T. Ogino, T. Ozawa and M. Ozaki, Lab Invest., 90,1718-1726 (2010).
(29) Creating BioluminescentIndicators to Visualize Biological Events in Living Cells and Animals. S. B.Kim and T. Ozawa, Supramol. Chem.,22, 439-448 (2010).
(30) Rapid andhigh-sensitivity cell-based assays of protein–protein interactions using splitclick beetle luciferase complementation: An approach to the study of Gprotein-coupled receptors. N. Misawa, A.K.M. Kafi, M. Hattori, K. Miura, K.Masuda and T. Ozawa, Anal. Chem.,82, 2552-2560 (2010).