1996 年度実績報告書

細胞骨格の分子細胞生物学及び分子遺伝学的研究;細胞内物質輸送、情報伝達及び細胞の形作りの機構

研究課題

研究課題/領域番号	08CE2007
研究機関	東京大学
研究代表者	廣川信隆東京大学, 医学部, 教授 (20010085)
研究分担者	金井克光東京大学, 医学部, 助教授 (80214427) 岡田康志東京大学, 医学部, 助手 (50272430) 吉川雅英東京大学, 医学部, 助手 (80272425) 佐藤玲子東京大学, 医学部, 講師 (40230718) 中田隆夫東京大学, 医学部, 助教授 (50218004)
キーワード	微小管 / 細胞骨格 / 分子モーター / キネシンスーパーファミリーKIFs / 微小管関連蛋白(MAPs) / 分子細胞生物学 / 分子遺伝学 / 物質輸送
研究概要	1.新しいモーター分子群KIFsのさらなる探索とその分子細胞生物学的解析。微小管結合及びATP結合の共有配列をもちい、海馬・小脳・脊髄・腎臓・精巣・網膜等のcDNAライブラリーを使って、KIFIC,KIF6,7,8,9,10,11,12,13A,13B,14,15,16A,16B,17の15の新しいKIF遺伝子を発見しクローニングを行い、一次構造の決定と組織別の発現を解析した。 2.KIF3A/3Bヘテロダイマーに結合する関連蛋白KAP3(Kinesin superfamily associated protein 3)を同定しその遺伝子のクローニング、一次構造の決定を行いさらにKAP3を単独あるいはKIF3A/3Bと同時に発現させその構造を電子顕微鏡で観察、KAP3がKIF3A/3Bヘテロダイマーの尾部に結合し球状の蛋白でありおそらく膜小器官とKIF3A/3Bの結合を制御しているであろうことを示した。 3.C末型KIF,KIFC2の分子細胞生物学的解析。 KIFC2のcDNAクローニング及び一次構造を決定した(792a.a.)。KIFC2はホモダイマーを形成し、神経系特に神経細胞に特異に発現され、神経細胞の細胞体と樹状突起に多く局在し、軸索にはほとんどなく、Multivesicular body様膜小器官を輸送するモーター分子であることが分った。 4.細胞骨格蛋白の遅い輸送の分子機構を明らかにするためcaged fluorescein tubulinを培養神経細胞内へ微量注入しその後紫外線による蛍光活性化を行った。一方すぐれた抗蛍光抗体を作り、それを用いた電子顕微鏡レベルの免疫細胞化学で蛍光活性化されたチュブリン分子の動態を明らかにした。又ニューロフィラメントM蛋白遺伝子にmycの標識をつけ、生きたマウスの後根知覚神経細胞にアデノウィルスを用いて導入し、その運命を免疫細胞化学的に解析しチュブリン、ニューロフィラメント蛋白ともポリマーとしてではなく小さなオリゴマーとして微小菅に沿って輸送されることを明らかにした。 5.KIF1A,KIF3A,KIF5,Brain dyneinのgene targetingを行った。現在その分子細胞生物学的解析を行っている。KIF1A-/-マウスは生後24時間以内にほとんど死亡し、重度の運動障害と知覚障害を示した。神経系を解析するとシナプス密度及びシナプス内でのシナプス小胞密度の著名な減少がみとめられ、又中枢神経系にfocal neuronal cell deathがみとめられKIF1Aがシナプス小胞の前駆体を輸送し神経細胞の機能発現と生存に必須の役割を果たしていることが示された。 6.キネシン頭部と微小菅の複合体の微細構造をクライオ電子顕微鏡で解析し、ATP水解の各ステップの構造変化をAMP・PNP,ADP・ADPi存在下で解析し3次元の再構成を現在行っている。

研究成果

(27件)

すべてその他

すべて文献書誌 (27件)

[文献書誌] Takemura,R.et al.: "mRNA expression of KIF1A,KIF1B,KIF2,KIF3A,KIF3B,KIF4,KIF5and cytoplasmicdynein during axonal regeneration." Journal of Neuroscience. 16. 31-35 (1996)
[文献書誌] Hirokawa,et al.: "Selective stabilization of tau in axons and MAP2C in cell bodies and dendrites contributes to polarized localization of cytoskeletal proteins in mature neurons." Journal of Cell Biology. 132. 667-679 (1996)
[文献書誌] Hirokawa,N.: "Organelle transport along microtubules-the role of KIFs (Kinesin superfamily proteins)." Trends in Cell Biology. 6. 135-141 (1996)
[文献書誌] Beech,P.L.et al.: "Localization of kinesin superfamily proteins to the connecting cilium of fish photoreceptors." Journal of Cell Science. 109. 889-897 (1996)
[文献書誌] Funakoshi,T.et al.: "Active transport of photoactivated tubulin molecules in growing axons revealed by a new electron microscopic analysis." Journal of Cell Biology. 133. 1347-1353 (1996)
[文献書誌] Terada,S.et al.: "Visualization of slow axonal transport in vivo." Science. 273. 784-788 (1996)
[文献書誌] Yamazaki,H.et al.: "Cloning and characterization of KAP3 : a novel kinesin superfamily associated protein of KIF3A/3B." Proc.Natl.Acad.Sci.USA. 93. 8443-8448 (1996)
[文献書誌] Grayer Wolf,S.et al.: "Interpreting a medium-resolution model of tubulin : comparison of Zinc-sheet and microtubule structure." Journal of Molecular Biology. 262. 485-501 (1996)
[文献書誌] Sato-Harada,R.et al.: "Microtubule-associated proteins regulate microtubule function as the track for intracellular membrane organelle transports." Cell Structure and Function. 21. 283-295 (1996)
[文献書誌] Hirokawa,N.: "The molecular mechanisms of organelle transport along microtubules : the identification and characterization of KIFs." Cell Structure and Function. 21. 357-367 (1996)
[文献書誌] Saito,N.et al.: "KIFC2,a novel C-terminal motor domain type kinesin superfamily,is a neuron specific motor for the transport of multivesicular body-like organelles in dendrites." Neuron. (in presss.). (1997)
[文献書誌] Kutsuwada,T.et al.: "Impairment of suckling response,trigeminal neuronal pattern formation and hippocampal LTD in NMDA receptor e2 subunit mutant mice." Neuron. 16. 333-344 (1996)
[文献書誌] Hori,Y.et al.: "Long-lasting synaptic facilitation induced by secretion in superficial dorsal horn neurones of rat spinal cord." Journal of Physiology. 492. 867-876 (1996)
[文献書誌] Silver,R.A.et al.: "Non-NMDA glutamate receptor occupancy and open probability at a rat cerebellar synapse with single and multiple release sites." Journal of Physiology. 494. 231-250 (1996)
[文献書誌] Takahashi,T.et al.: "Functional correlation of NMDA receptor e subunis expression with the properties of single0channel and synaptic current in the developing cerebellum." Journal of Neuroscience. 16. 4376-4382 (1996)
[文献書誌] Yokoi,M.et al.: "Impariment of hippocampal mossy fiber LTD in mice lacking mGLUR2." Science. 273. 645-647 (1996)
[文献書誌] Kobayashi,K.et al.: "Presynaptic long-term depression at the hippocampal mossy fiber-CA3 synapse." Science. 273. 648-650 (1996)
[文献書誌] Takahashi,T.et al.: "Presynaptic calcium current moduration by a metabotropic glutamate receptor." Science. 274. 594-597 (1996)
[文献書誌] Morimoto,T.et al.: "Transient ischemia depletes free ubiquitin in the gerbil hippocampal CA1neurons." Am.J.Pathol.148. 249-257 (1996)
[文献書誌] Fukumoto,H.et al.: "Amyloid β protein (Aβ) deposition in normal aging has the same characteristics as that in Alzheimer's disease : Predominance of Aβ 42(43) and association of Aβ40 with cored plaques." Am.J.Pathol.148. 259-266 (1996)
[文献書誌] Mann,D.N.A.et al.: "MLC : predominant deposition of Aβ 42 (43) in plaques in cases of Alzheimer's disease and hereditary cerebral hemorrhage associated with mutants in the APP gene." Am.J.Pathol.148. 259-266 (1996)
[文献書誌] Iwatsubo,T.et al.: "Purification and characterization of Lewy bodies from the brains of patients with diffuse Lewy body disease." Am.J.Pathol.148. 1517-1529 (1996)
[文献書誌] Gu,Y.et al.: "τ is widely expressed in rat tissues." J.Neurochem.67. 1235-1244 (1996)
[文献書誌] Hasegawa,M.et al.: "Characterization of mAb AP422, anovel phosphorylation-dependent monoclonalantibody against tau protein." F EBS Lett.384. 25-30 (1996)
[文献書誌] Funato,H.et al.: "Proliferating cell nuclear antigen (PCNA) expresed in leptomenings." J.Histochem.Cytochem.44. 1261-1265 (1996)
[文献書誌] Kimura,T.et al.: "Sequential changes of tau-site-specific phosphorylation during development of paired helical filaments." Dementia. 7. 177-181 (1996)
[文献書誌] Kanemaru,K.et al.: "Comparable amyloid β-protein (Aβ) 42 (43) and Aβ 40 deposition in the aged monkey brain." Neurosci.Lett.214. 196-198 (1996)

1996 年度 実績報告書

細胞骨格の分子細胞生物学及び分子遺伝学的研究;細胞内物質輸送、情報伝達及び細胞の形作りの機構

研究代表者

廣川 信隆 東京大学, 医学部, 教授 (20010085)

研究成果

[文献書誌] Takemura,R.et al.: "mRNA expression of KIF1A,KIF1B,KIF2,KIF3A,KIF3B,KIF4,KIF5and cytoplasmicdynein during axonal regeneration." Journal of Neuroscience. 16. 31-35 (1996)

[文献書誌] Hirokawa,et al.: "Selective stabilization of tau in axons and MAP2C in cell bodies and dendrites contributes to polarized localization of cytoskeletal proteins in mature neurons." Journal of Cell Biology. 132. 667-679 (1996)

[文献書誌] Hirokawa,N.: "Organelle transport along microtubules-the role of KIFs (Kinesin superfamily proteins)." Trends in Cell Biology. 6. 135-141 (1996)

[文献書誌] Beech,P.L.et al.: "Localization of kinesin superfamily proteins to the connecting cilium of fish photoreceptors." Journal of Cell Science. 109. 889-897 (1996)

[文献書誌] Funakoshi,T.et al.: "Active transport of photoactivated tubulin molecules in growing axons revealed by a new electron microscopic analysis." Journal of Cell Biology. 133. 1347-1353 (1996)

[文献書誌] Terada,S.et al.: "Visualization of slow axonal transport in vivo." Science. 273. 784-788 (1996)

[文献書誌] Yamazaki,H.et al.: "Cloning and characterization of KAP3 : a novel kinesin superfamily associated protein of KIF3A/3B." Proc.Natl.Acad.Sci.USA. 93. 8443-8448 (1996)

[文献書誌] Grayer Wolf,S.et al.: "Interpreting a medium-resolution model of tubulin : comparison of Zinc-sheet and microtubule structure." Journal of Molecular Biology. 262. 485-501 (1996)

[文献書誌] Sato-Harada,R.et al.: "Microtubule-associated proteins regulate microtubule function as the track for intracellular membrane organelle transports." Cell Structure and Function. 21. 283-295 (1996)

[文献書誌] Hirokawa,N.: "The molecular mechanisms of organelle transport along microtubules : the identification and characterization of KIFs." Cell Structure and Function. 21. 357-367 (1996)

[文献書誌] Saito,N.et al.: "KIFC2,a novel C-terminal motor domain type kinesin superfamily,is a neuron specific motor for the transport of multivesicular body-like organelles in dendrites." Neuron. (in presss.). (1997)

[文献書誌] Kutsuwada,T.et al.: "Impairment of suckling response,trigeminal neuronal pattern formation and hippocampal LTD in NMDA receptor e2 subunit mutant mice." Neuron. 16. 333-344 (1996)

[文献書誌] Hori,Y.et al.: "Long-lasting synaptic facilitation induced by secretion in superficial dorsal horn neurones of rat spinal cord." Journal of Physiology. 492. 867-876 (1996)

[文献書誌] Silver,R.A.et al.: "Non-NMDA glutamate receptor occupancy and open probability at a rat cerebellar synapse with single and multiple release sites." Journal of Physiology. 494. 231-250 (1996)

[文献書誌] Takahashi,T.et al.: "Functional correlation of NMDA receptor e subunis expression with the properties of single0channel and synaptic current in the developing cerebellum." Journal of Neuroscience. 16. 4376-4382 (1996)

[文献書誌] Yokoi,M.et al.: "Impariment of hippocampal mossy fiber LTD in mice lacking mGLUR2." Science. 273. 645-647 (1996)

[文献書誌] Kobayashi,K.et al.: "Presynaptic long-term depression at the hippocampal mossy fiber-CA3 synapse." Science. 273. 648-650 (1996)

[文献書誌] Takahashi,T.et al.: "Presynaptic calcium current moduration by a metabotropic glutamate receptor." Science. 274. 594-597 (1996)

[文献書誌] Morimoto,T.et al.: "Transient ischemia depletes free ubiquitin in the gerbil hippocampal CA1neurons." Am.J.Pathol.148. 249-257 (1996)

[文献書誌] Fukumoto,H.et al.: "Amyloid β protein (Aβ) deposition in normal aging has the same characteristics as that in Alzheimer's disease : Predominance of Aβ 42(43) and association of Aβ40 with cored plaques." Am.J.Pathol.148. 259-266 (1996)

[文献書誌] Mann,D.N.A.et al.: "MLC : predominant deposition of Aβ 42 (43) in plaques in cases of Alzheimer's disease and hereditary cerebral hemorrhage associated with mutants in the APP gene." Am.J.Pathol.148. 259-266 (1996)

[文献書誌] Iwatsubo,T.et al.: "Purification and characterization of Lewy bodies from the brains of patients with diffuse Lewy body disease." Am.J.Pathol.148. 1517-1529 (1996)

[文献書誌] Gu,Y.et al.: "τ is widely expressed in rat tissues." J.Neurochem.67. 1235-1244 (1996)

[文献書誌] Hasegawa,M.et al.: "Characterization of mAb AP422, anovel phosphorylation-dependent monoclonalantibody against tau protein." F EBS Lett.384. 25-30 (1996)

[文献書誌] Funato,H.et al.: "Proliferating cell nuclear antigen (PCNA) expresed in leptomenings." J.Histochem.Cytochem.44. 1261-1265 (1996)

[文献書誌] Kimura,T.et al.: "Sequential changes of tau-site-specific phosphorylation during development of paired helical filaments." Dementia. 7. 177-181 (1996)

[文献書誌] Kanemaru,K.et al.: "Comparable amyloid β-protein (Aβ) 42 (43) and Aβ 40 deposition in the aged monkey brain." Neurosci.Lett.214. 196-198 (1996)

1996 年度実績報告書

廣川信隆東京大学, 医学部, 教授 (20010085)