細胞内シグナルフローの可視化解析

• Project/Area Number: 12002003
• Research Category: Grant-in-Aid for Specially Promoted Research
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: The University of Tokyo
• Principal Investigator: 飯野 正光 東京大学, 大学院・医学系研究科, 教授 (50133939)
• Co-Investigator(Kenkyū-buntansha): 山澤 徳志子 (高橋 徳志子 / 高橋 徳志子(山澤 徳志子)) 東京大学, 大学院・医学系研究科, 助手 (00282616) ; 廣瀬 謙造 東京大学, 大学院・医学系研究科, 助教授 (00292730) ; 柿澤 昌 東京大学, 大学院・医学系研究科, 助手 (40291059) ; 竹島 浩 久留米大学, 分子生命科学研究所, 教授 (70212024)
• Project Period (FY): 2000 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥544,140,000 (Direct Cost: ¥439,800,000、Indirect Cost: ¥104,340,000); Fiscal Year 2004: ¥88,400,000 (Direct Cost: ¥68,000,000、Indirect Cost: ¥20,400,000); Fiscal Year 2003: ¥88,400,000 (Direct Cost: ¥68,000,000、Indirect Cost: ¥20,400,000); Fiscal Year 2002: ¥103,740,000 (Direct Cost: ¥79,800,000、Indirect Cost: ¥23,940,000); Fiscal Year 2001: ¥171,600,000 (Direct Cost: ¥132,000,000、Indirect Cost: ¥39,600,000); Fiscal Year 2000: ¥92,000,000 (Direct Cost: ¥92,000,000)
• Keywords: カルシウム / 一酸化窒素 / リン酸化 / ミオシン / イノシトール三リン酸 / イメージング / プルキンエ細胞 / 血管 / リシ酸化 / NFAT

Research Abstract

本研究では、重要なシグナル分子についてそれらを可視化するプローブの開発を推し進め、それを用いてシグナル分子の局在と機能の時間変化を生きた細胞でシグナル・カスケードをリアルタイムに可視化し、細胞機能の調節機構の解析を進める。これによって、シグナル分子が細胞機能を制御する機構を、全く新しい観点から明らかにすることを研究の目的としている。今年度の研究により以下の成果を得た。1)中枢シナプスにおけるIP_3シグナル機構の可視化解析:二光子励起顕微鏡法による細胞内IP_3濃度イメージングにより、シナプスにおいて、イオンチャネル型グルタミン酸受容体と代謝型グルタミン酸受容体が協同して下流のシグナル(IP_3産生)を誘導することを明らかにした。これによりシナプス後膜に2種のグルタミン酸受容体が共存する生理的意義を明らかにした。2)ミオシン軽鎖リン酸化インジケーターの開発:ミオシン軽鎖のリン酸化状態を経時的に測定するインジケーターを開発した。このインジケーターは、血管平滑筋の異常収縮などに関与すると考えられるRhoキナーゼ系作用薬の評価にも応用できる。3)中枢シナプスにおける一酸化窒素(NO)シグナルの可視化解析:神経終末から放出されるNOをシナプスにおいて初めて可視化した。これにより、従来の予想とは異なり、NOシグナルは高いシナプス特異性を有することを明らかにした。また、小脳の平行線維・プルキンエ細胞シナプスにおいて、NOシグナルが神経活動の頻度に二相性に依存していることを発見し、これに依存したシナプス可塑性を発見した(投稿中)。4)細胞周期と細胞死:アポトーシス関連分子BADと14-3-3分子の結合解離を生きた細胞内で経時的に観測することにより、増殖因子の除去と細胞周期の一致がアポトーシスを強く誘導することを明確にした(投稿中)。

Research Products

• [Journal Article] Real-time imaging of myosin II regulatory light chain phosphorylation using a new protein biosensor. (2005)
  • Author(s): Yamada, A.
  • Journal Title: Biochem.J. 385 Pages: 589-594
• [Journal Article] System-level identification of transcriptional circuits underlying mammalian circadian clocks. (2005)
  • Author(s): Ueda, H.R.
  • Journal Title: Nat.Genet. 37 Pages: 187-192
• [Journal Article] Molecular-timetable methods for detection of body time and rhythm disorders from single-time-point genome-wide expression profiles. (2004)
  • Author(s): Ueda, H.R.
  • Journal Title: Proc.Nat.Acad.Sci.U.S.A. 101 Pages: 11227-11232
• [Journal Article] Modification of intracellular Ca^<2+> dynamics by laser inactivation of inositol 1,4,5-trisphosphate receptor using membrane-permeant probes. (2004)
  • Author(s): Yogo, T.
  • Journal Title: Chem.Biol. 11 Pages: 1053-1058
• [Journal Article] Cross talk between metabotropic and ionotropic glutamate receptor-mediated signaling in parallel fiber-induced inositol 1,4,5-trisphosphateproduction in cerebellar Purkinje cells. (2004)
  • Author(s): Okubo, Y.
  • Journal Title: J.Neurosci. 24 Pages: 9513-9520
• [Publications] Inoue, T.: "Spatiotemporal laser inactivation of inositol 1,4,5-trisphosphate receptors using synthetic small-molecule probes."Chem.Biol.. 10. 503-509 (2003)
• [Publications] Namiki, S.: "Intracellular delivery of glutathione S-transferase into mammalian cells."Biochem.Biophys.Res.Commun.. 305. 592-597 (2003)
• [Publications] Tu, H.: "Functional and biochemical analysis of the type 1 inositol (1,4,5)-trisphosphate receptor calcium sensor."Biophys.J.. 85. 290-299 (2003)
• [Publications] Tomida, T.: "NFAT functions as a working memory of Ca^<2+> signals in decoding Ca^<2+> oscillation."EMBO J.. 22. 3825-3832 (2003)
• [Publications] Uchino, M.: "Isoform-specific phosphorylation of metabotropic glutamate receptor 5 by protein kinase C (PKC) blocks Ca^<2+> oscillation and oscillatory translocation of Ca^<2+>-dependent PKC."J.Biol.Chem.. 279. 2254-2261 (2004)
• [Publications] Shirane, D.: "Enzymatic production of RNAi libraries from cDNAs."Nature Genet.. 36. 190-196 (2004)
• [Publications] Ueda, H.R.: "Universality and flexibility in gene expression from bacteria to human."Proc.Nat.Acad.Sci.U.S.A.. (in press). (2004)
• [Publications] Tu, H.: "Functional characterization of the type 1 Inositol 1,4,5-trisphosphate receptor coupling domain SII({+/-}) splice variants and the opisthotonos mutant form"Biophys. J.. 82. 1995-2004 (2002)
• [Publications] Ueda, H.R.: "Genome-wide transcriptional orchestration of circadian rhythms in drosophila"J. Biol. Chem.. 277. 14048-14052 (2002)
• [Publications] Ueda, H.R.: "A transcription factor response element for gene expression during circadian night"Nature. 418. 534-539 (2002)
• [Publications] Nosyreva, E.: "The high-affinity calcium-calmodulin-binding site does not play a role in the modulation of type 1 inositol 1,4,5-trisphosphate receptor function by calcium and calmodulin"Biochem. J.. 365. 659-667 (2002)
• [Publications] Ueda, H.R.: "Intercellular coupling mechanism for synchronized and noise-resistant circadian oscillators"J. Theor. Biol.. 216. 501-512 (2002)
• [Publications] Nakanishi, W.: "Hydrophobic modifications at 1-phosphate of inositol 1,4,5-Trisphosphate analogues enhance receptor binding"Bioorg. Med. Chem. Lett.. 12. 911-913 (2002)
• [Publications] Noda, M: "Switch to anaerobic glucose metabolism with NADH accumulation in the b-cell model of mitochondrial diabetes : Characteristics of bHC9 cells deficient in mitochondrial DNA transcription"J. Biol. Chem.. 277. 41817-41826 (2002)
• [Publications] Kakizawa, S.: "Effects of insulin-like growth factor I on climbing fibre synapse elimination during cerebellar development"Eur. J. Neurosci.. 17. 545-554 (2003)
• [Publications] Miyakawa, T.: "Ca^<2+>-sensor region of IP_3 receptor controls intracellular Ca^<2+> signalling"EMBO J.. 20. 1674-1680 (2001)
• [Publications] Fujiwara, A.: "Reduced IP_3 sensitivity of IP_3 receptor in Purkinje neurons"NeuroReport. 12. 2647-2651 (2001)
• [Publications] Okubo, Y.: "Visualization of IP_3 dynamics reveals a novel AMPA receptor-triggered IP_3 production pathway mediated by voltage-dependent Ca^<2+> influx in Purkinje cells"Neuron. 32. 113-122 (2001)
• [Publications] Namikis, S.: "Mapping of heme-binding domains in soluble guanylyl cyclase β1 subunit"Biochem. Biophys. Res. Commun.. 288. 798-804 (2001)
• [Publications] Matsuzaki, M.: "Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons"Nature Neurosci. 4. 1086-1092 (2001)
• [Publications] Eto, K.: "Phosphatidylinositol 3-kinase suppresses glucose-stimulated insulin secretion by affecting post-cytosolic [Ca^<2+>] elevation signals"Diabetes. 51. 87-97 (2001)
• [Publications] Yamazawa, T.: "Simultaneous imaging of Ca^<2+> signals in interstitial cells of Cajal and longitudinal smooth muscle cells during rhythmic activity in mouse ileum"J. Physiol.. 538. 823-835 (2002)
• [Publications] Takahashi, N.: "Two-photon excitation imaging of pancreatic islets with various fluorescent probes"Diabetes. 51. S25-S28 (2002)
• [Publications] Oyamada, H.: "Novel mutations in C-terminal channel region of the ryanodine receptor in malignant hyperthermia patients"Jpn. J. Pharmacol.. 88. 159-166 (2002)
• [Publications] Miyakawa,T.: "Ca^<2+>-sensor region of IP_3 receptor controls intracellular Ca^<2+> signalling."EMBO J.. (in press). (2001)
• [Publications] Hashimoto,A.: "Negative control of store-operated Ca^<2+> influx by B cyll receptor crosslinking."J.Immunol.. 166. 1003-1008 (2001)
• [Publications] Takeshima,H.: "Junctophilins : A Novel Family of Junctional Membrane Complex Proteins."Molecular Cell. 6. 11-22 (2000)
• [Publications] Xu,X.: "Molecular cloning of cDNA encoding a Drosophila ryanodine receptor and functional studies of the carboxyl-terminal calcium release channel."Biophys.J.. 78. 1270-1281 (2000)
• [Publications] Kouzu,Y.: "Mutant mice lacking ryanodine receptor type 3 exhibit deficits of contextual fear conditioning and activation of calcium/calmodulin-dependent protein kinase II in hippocampus."Mol.Brain Res.. 76. 142-150 (2000)
• [Publications] Conklin,M.: "Comparison of Ca^<2+> sparks produced independently by two ryanodine receptor isoforms (type-1 of type-3)."Biophys.J.. 78. 1777-1785 (2000)
• [Publications] Nishi,M.: "Characterization of human junctophilin subtype genes."Biochem.Biophys.Res.Commun.. 273. 920-927 (2000)
• [Publications] Houtani,T.: "Distribution of nociceptin/orphanin FQ precursor protein and receptor in brain and spinal cord : A study using in situ hybridization and X-gal histochemistry in nociceptin receptor-deficient mice."J.Comp.Neurol.. 424. 489-508 (2000)
• [Publications] Wada,Y.: "A region of the sulfonylurea receptor critical for a modulation of ATP-sensitive K^+ channels by G-protein βγ-subunits."EMBO J.. 19. 4915-4925 (2000)
• [Publications] Hayek,S.A.: "Characterization of a calcium regulation domain of the skeletal muscle ryanodine receptor."Biochem.J.. 351. 57-65 (2000)
• [Publications] Ueda,H.: "Enhanced spinal nociceptin receptor expression develops morphine tolerance and dependence."J.Neurosci.. 20. 7640-7647 (2000)
• [Publications] Nagaraj,R.Y.: "Increased susceptibility to fatigue of slow- and fast-twitch muscles from mice lacking the MG29 gene."Physiol.Genomics. 4. 43-49 (2000)