1991 Fiscal Year Annual Research Report

細胞骨格間架橋蛋白質の微量検出法とその制御解析法の開発

Research Project

Project/Area Number	01870106
Research Institution	Osaka University
Principal Investigator	祖父江憲治大阪大学, 医学部, 教授 (20112047)
Co-Investigator(Kenkyū-buntansha)	田中潤也大阪大学, 医学部, 助手 (70217040) 乾誠大阪大学, 医学部, 助手 (70223237)
Keywords	細胞骨格蛋白質 / 生化学的解析 / 超微細形態学的解析 / シナプシンI / カルパクチンI / アクチン / エクソサイト-シス
Research Abstract	本研究は、細胞骨格関連蛋白質による種々の細胞機能の制御機構の分子機作を生化学的手法と超微細形態学的手法を併用した新たな解析法で明かにすることを目指してきた。本年度は、カルパクチンIに注目し、分泌顆粒が細胞膜に結合する過程を副腎髄質細胞を対象に検討した。カルパクチンIは、アネキシンファミリ-に属する蛋白質で36KDaの重鎖と10KDaの軽鎖からなり、Ca^<2+>依存性にリン脂質と結合する。また、カルパクチンIは、リポゾ-ムや副腎髄質クロマフィン顆粒をCa^<2+>依存性に凝集させることが知られている。そこでカルパクチンIの分子形態及びエクソサイト-シスにおける副腎髄質細胞内の局在変化を電子顕微鏡を用いて詳細に検討した。精製されたカルパクチンIは、直径約10nmの球状分子として存在する。カルパクチンIによりリポゾ-ムをCa^<2+>存在下に凝集させると、球状構造とともに近接した2つのリポゾ-ム間に長さ約7nmの細いヒモ状の架橋構造が認められた。副腎髄質細胞では、細胞膜内側面に多数の球状構造が認められ、アセチルコリンによる分泌刺激を加えるとクロマフィン顆粒と細胞膜との間に細いヒモ状の架橋構造が観察された。さらに免疫電顕法でもカルパクチンIが細胞膜内側面に局在することが確認され、特に、細胞膜と近接するクロマフィン間に強い免疫反応性が認められた。以上の結果から、カルパクチンIは、Ca^<2+>・リン脂質依存性に構造変化を起こし細胞膜とクロマフィン顆粒間との架橋を行なっていると考えられる。このようにカルパクチンIがエクソサイト-シスにおける細胞膜と分泌顆粒との結合さらには膜の融合の過程に必須の役割を果たしている可能性が示された。

Research Products

(19 results)

All Other

All Publications (19 results)

[Publications] Hirokawa,N.: "The cytoskeletal architecture of the presynaptic terminal and molecular structure of synapsin 1." J.Cell Biol.108. 111-126 (1989)
[Publications] Sobue,K.: "Comparison of the regional distribution of calspectin(nonerythroid spectrin or fodrin),α-actinin,vinculin nonerythroid protein 4.1,and calpactin in normal and avian sarcoma virus-or rous sarcoma virusinduced transformed cells." Exp.Cell Res. 181. 256-262 (1989)
[Publications] Yorifuji,H.: "Localization of 4.1 related proteins in cerebellar neurons." Eur.J.Cell Biol.48. 104-115 (1989)
[Publications] Hayashi,K.: "35kDa fragment of h-caldesmon conserves two consensus sequences of the tropomyosin-binding domain in troponin T." Biochem.Biophys.Res.Commun.161. 38-45 (1989)
[Publications] Hoshimaru,M.: "Immunochemical evidence that myosin I heavy chain-like protein is identical to the 110-kilodalton brush-border protein." J.Biochem.106. 455-459 (1989)
[Publications] Sobue,K.: "α-Actinins,calspectin (brain spectrin or fodrin)and actin participate in adhesion and movement of growth cones." Neuron. 3. 311-319 (1989)
[Publications] Senda,T.: "Ultrastructural and immunocytochemical studies on the cytoskeleton in the anterior of rats,with special regard to the relationship between actin filaments and secretory granules." Cell Tissue Res.258. 25-30 (1989)
[Publications] Hayashi,K.: "Primary structure and functional expression of h-caldesmon complementary DNA." Biochem.Biophys.Res.Commun.164. 503-511 (1989)
[Publications] Tanaka,T.: "Phosphorylation of high-Mr caldesmons by protein kinase C modulates the regulatory function of this protein on the interaction between actin and myosin." Eur.J.Biochem.188. 495-500 (1990)
[Publications] Nakata,T.: "Conformational change and localization of calpactin I complexinvolved in exocytosis as revealed by quick-freeze,deep-etch,electron microscopy and immunocytochemistry." J.Cell Biol.110. 13-25 (1990)
[Publications] Sobue,K.: "Involvement of the membrane cyoriskeletal proteins and the src gene product in growth cone adhesion and movement." Neurosci.Res.Suppl.13. S80-S91 (1990)
[Publications] Harada,A.: "Developmental changes of synapsin I subcellular localization in rat cerebellar neurons." Cell Struc.Func.15. 329-342 (1990)
[Publications] Hayashi,K.: "Structural and functional relationships between h-and l-caldesmons." J.Biol.Chem.266. 355-361 (1991)
[Publications] Tanaka,T.: "Ca^<2+>-dependent of the spectrin/actin interaction by calmodulin and protein 4.1." J.Biol.Chem.266. 1134-1140 (1991)
[Publications] Sobue,K.: "A novel-regulatory protein of smooth muscle and non-muscle actinmyosin interaction." J.Biol.Chem.266. 12115-12118 (1991)
[Publications] Kitagawa,K.: "The synapsin I brain distribution in ischemia." Neuroscience. 46. 287-299 (1992)
[Publications] 祖父江憲治: "分子神経生物学,シリ-ズ分子生物学の進歩11 「神経系の形態形成-細胞骨格-」" 丸善, 396 (1989)
[Publications] Sobue,K.: "Calcium Protein Signaling Significances of two different Mr caldesmons." Plenum Publishing Corporation, 354 (1989)
[Publications] 祖父江憲治: "新生化学実験法講座11,神経生化学「神経系のCa^<2+>結合タンパク質」" 東京化学同人, 542 (1990)

1991 Fiscal Year Annual Research Report

細胞骨格間架橋蛋白質の微量検出法とその制御解析法の開発

Principal Investigator

祖父江 憲治 大阪大学, 医学部, 教授 (20112047)

Research Products

[Publications] Hirokawa,N.: "The cytoskeletal architecture of the presynaptic terminal and molecular structure of synapsin 1." J.Cell Biol.108. 111-126 (1989)

[Publications] Sobue,K.: "Comparison of the regional distribution of calspectin(nonerythroid spectrin or fodrin),α-actinin,vinculin nonerythroid protein 4.1,and calpactin in normal and avian sarcoma virus-or rous sarcoma virusinduced transformed cells." Exp.Cell Res. 181. 256-262 (1989)

[Publications] Yorifuji,H.: "Localization of 4.1 related proteins in cerebellar neurons." Eur.J.Cell Biol.48. 104-115 (1989)

[Publications] Hayashi,K.: "35kDa fragment of h-caldesmon conserves two consensus sequences of the tropomyosin-binding domain in troponin T." Biochem.Biophys.Res.Commun.161. 38-45 (1989)

[Publications] Hoshimaru,M.: "Immunochemical evidence that myosin I heavy chain-like protein is identical to the 110-kilodalton brush-border protein." J.Biochem.106. 455-459 (1989)

[Publications] Sobue,K.: "α-Actinins,calspectin (brain spectrin or fodrin)and actin participate in adhesion and movement of growth cones." Neuron. 3. 311-319 (1989)

[Publications] Senda,T.: "Ultrastructural and immunocytochemical studies on the cytoskeleton in the anterior of rats,with special regard to the relationship between actin filaments and secretory granules." Cell Tissue Res.258. 25-30 (1989)

[Publications] Hayashi,K.: "Primary structure and functional expression of h-caldesmon complementary DNA." Biochem.Biophys.Res.Commun.164. 503-511 (1989)

[Publications] Tanaka,T.: "Phosphorylation of high-Mr caldesmons by protein kinase C modulates the regulatory function of this protein on the interaction between actin and myosin." Eur.J.Biochem.188. 495-500 (1990)

[Publications] Nakata,T.: "Conformational change and localization of calpactin I complexinvolved in exocytosis as revealed by quick-freeze,deep-etch,electron microscopy and immunocytochemistry." J.Cell Biol.110. 13-25 (1990)

[Publications] Sobue,K.: "Involvement of the membrane cyoriskeletal proteins and the src gene product in growth cone adhesion and movement." Neurosci.Res.Suppl.13. S80-S91 (1990)

[Publications] Harada,A.: "Developmental changes of synapsin I subcellular localization in rat cerebellar neurons." Cell Struc.Func.15. 329-342 (1990)

[Publications] Hayashi,K.: "Structural and functional relationships between h-and l-caldesmons." J.Biol.Chem.266. 355-361 (1991)

[Publications] Tanaka,T.: "Ca^<2+>-dependent of the spectrin/actin interaction by calmodulin and protein 4.1." J.Biol.Chem.266. 1134-1140 (1991)

[Publications] Sobue,K.: "A novel-regulatory protein of smooth muscle and non-muscle actinmyosin interaction." J.Biol.Chem.266. 12115-12118 (1991)

[Publications] Kitagawa,K.: "The synapsin I brain distribution in ischemia." Neuroscience. 46. 287-299 (1992)

[Publications] 祖父江 憲治: "分子神経生物学,シリ-ズ分子生物学の進歩11 「神経系の形態形成-細胞骨格-」" 丸善, 396 (1989)

[Publications] Sobue,K.: "Calcium Protein Signaling Significances of two different Mr caldesmons." Plenum Publishing Corporation, 354 (1989)

[Publications] 祖父江 憲治: "新生化学実験法講座11,神経生化学 「神経系のCa^<2+>結合タンパク質」" 東京化学同人, 542 (1990)

祖父江憲治大阪大学, 医学部, 教授 (20112047)

[Publications] 祖父江憲治: "分子神経生物学,シリ-ズ分子生物学の進歩11 「神経系の形態形成-細胞骨格-」" 丸善, 396 (1989)

[Publications] 祖父江憲治: "新生化学実験法講座11,神経生化学「神経系のCa^<2+>結合タンパク質」" 東京化学同人, 542 (1990)