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Role of Cytoskeletal System and its Regulation by Ca^<2+> in Exocytosis

Research Project

Project/Area Number 63480124
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field General medical chemistry
Research InstitutionOsaka University

Principal Investigator

SOBUE Kenji  Osaka University Medical School, Department of Neurochemistry and Neuropharmacology, Professor, 医学部, 教授 (20112047)

Co-Investigator(Kenkyū-buntansha) TANAKA Junya  Osaka University Medical School, Department of Neurochemistry and Neuropharmacol, 医学部, 助手 (70217040)
INUI Makoto  Osaka University Medical School, Department of Neurochemistry and Neuropharmacol, 医学部, 助手 (70223237)
神田 啓子  大阪大学, 医学部, 助手
Project Period (FY) 1988 – 1990
Project Status Completed (Fiscal Year 1990)
Budget Amount *help
¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 1990: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1989: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1988: ¥4,200,000 (Direct Cost: ¥4,200,000)
KeywordsExocytosis / Synaptic vesicles / Chromaffin granules / Synapsis 1 / Calpactin I / 副腎髄質 / 神経シナプス / エクソサイトーシス / Ca^<2+>結合蛋白質 / カルモデュリン / 細胞骨格 / Ca^<2+>制御
Research Abstract

According to recent studies concerning the phenomena involved in transmitter release at nerve terminal, it has been shown that depolarization-dependent Ca^<2+> influx into the nerve terminal through Ca^<2+> channels triggers exocytosis of synaptic vesicles. Similar processes could occur on the occasion of exocytosis induced by Ca^<2+> influx into secretory cells such as adrenal chromaffin cells. However, the processes between Ca^<2+> influx and vesicle fusion to the plasma membranes are totally unknown. Of many cytoskeleton-and membrane-associated proteins, synapsis 1 was shown to be involved in the release of the transmitter at nerve terminals by a recent physiological study. Calpactin I, which exists in the adrenal medulla, is a Ca^<2+>-dependent phospholipid-binding protein, and aggregates phospholipid-containing liposomes in Ca^<2+>-dependent manner, suggesting that it may play a role in the process of exocytosis. Therefore, to elucidate the molecular mechanism of exocytosis, we ex … More amined the localization and function of synapsin 1 and calpactin I in presynaptic terminal and adrenal chromaffin cells, respectively.
We examined the molecular structure of synapsin 1 and its relationship with actin filaments, microtubules, and synaptic vesicles in presynaptic terminals using the low angle rotary shadowing technique, Quick-Freeze Deep Etch electron microscopy (QF-DE), immunoelectron microscopy. The synapsin 1, -47nm long, was composed of a head (-14nm diam) and a tail (-33nm long), having a tadpole-like appearance. The high resolution provided by QF-DE revealed that a single synapsin 1 cross-linked actin filaments and linked actin filaments with synaptic vesicles forming -30nm short strands. Synapsis 1 also connected a microtubule to synaptic vesicles, forming -30nm strands. These data suggest that synapsis 1 could be a main element of short linkage between actin filaments and synaptic vesicles, and between microtubules and syanptic vesicles, and between synaptic vesicles. Because phosphoryaltion of synapsis 1 by Ca^<2+>/calmodulin-dependent kinase detaches synapsis 1 from vesicles, it could release synaptic vesicles, and thus increase mobility of synaptic vesicles to the presynaptic membrane upon depolarization-dependent Ca^<2+> flux into the presynaptic terminal.
Calpactin I from adrenal medulla was found to be a globular molecule with a diameter of -11nm on mica. When liposomes were aggregated by calpactin, QF-DE revealed a fine thin strand of 6.5 nm long cross-linking opposing membrane in addition to the globules on liposomes. In cultured chromaffin cells, similar cross-linking short strands (6-10 nm) were found between chromaffin vesicles and the plasma membrane after stimulation with acetylcholine. Plasma membranes also revealed numerous globular structure 10 nm in diameter on their cytoplasmic surface. Immunoelectron microscopy showed that calpactin I was closely associated with the inner face of the plasma membrane and was especially conspicuous between plasma membrane and adjacent chromaffin vesicles. These data strongly suggest that clapactin I changes its conformation to cross-link vesicles and the plasma membrane after stimulation of cultured chromaffin cells and that it may play an important role in the binding of chromaffin vesicles to the plasma membrane during exocytosis. Less

Report

(4 results)
  • 1990 Annual Research Report   Final Research Report Summary
  • 1989 Annual Research Report
  • 1988 Annual Research Report
  • Research Products

    (56 results)

All Other

All Publications (56 results)

  • [Publications] Takao Nakata: "Conformational change and localization of calpactin I complex involved in exocytosis as revealed by quickーfreeze,deepーetch,electron microscopy and immunocytochemistry." Journal of Cell Biology. 110. 13-25 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Kenji Sobue: "Involvement of the membrane cytoskeletal proteins and the src gene product in growth cone abhesion and movement." Neuroscience Research. Supple.13. S80-91 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Akihiro Harada: "Developmental changes of synapsin I subcellular localization in rat cerebellar neurons." Cell Structure and Function. 15. 329-342 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Ken'ichiro Hayashi: "Stuctural and functional relationships between hーand lーcaldesmons." Journal of Biological Chemistry. 266. 355-361 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Toshihiko Tanaka: "Ca^<2+>_ーDependent of the spectrin/actin interaction by calmodulin and protein 4.1." Journal of Biological Chemistry. 266. 1134-1140 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Kenji Sobue: "Caldesmon: A novelーregulatory protein of smooth muscle and nonmuscle actinーmyosin interaction." Journal of Biological Chemistry. 266. (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Tadaharu Tsumoto: "Immunohistochemical localization of a membrane associated 4.1 protein in the rat visual cortex during postnatal development." Journal of Comparative Neurology. 271. 30-43 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Tadaharu Tsumoto: "A role of NMDA receptors and membrane associated Ca^<2+>/colmodulinbinding proteins in synaptic plasticity in the developing visualcortex." Biomedical Research. Supple.2. 43-52 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Nobutaka Hirokawa: "The cytoskeletal architecture of the presynaptic teminal and molecular structure of synapsin1." Journal of Cell Biology. 108. 111-126 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Hiroshi Yorifuji: "Localization of 4.1 related proteins in cerebellar neurons." European Journal of Cell Biology. 48. 104-115 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Takao Senda: "Ultrastructuctural and immunocytochemical studies on the cytoskeleton in the anterior pituitary of rats,with special regard to the relationship between actin filaments and secretory grands." Cell and Tissue Research. 258. 25-30 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] 祖父江 憲治: "シリ-ズ分子生物学の進歩II 分子神経生物学" 丸善, 396 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] 祖父江 憲治: "新生化学実験講座II 神経生化学" 東京化学同人, 542 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Sobue, K., Fujio, Y. and Kanda, K.: "A tumor promoter induces a reorganization of actin filaments and calspectin (fodrin or nonerythroid spectrin) in 3T3 cells." Proc. Natl. Acad. Sci. USA. 85. 482-486 (1988)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Minowa, O., Yazawa, M., Sobue, K., Itoh, K. and Yagi, K.: "Calmodulin fragments can not activate target enzymes." J. Biochem.103. 531-536 (1988)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Sobue, K., Kanda, K., Tanaka, T. and Ueki, N.: "Caldesmon : A common actin-linked regulatory protein in the smooth muscle and non-muscle contractile system." J. Cell. Biochem.37. 317-325 (1988)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Tsumoto, T., Sato, H. and Sobue, K.: "Immunohistochemical localization of a membrane-associated, 4.1-like protein in the rat visual cortex during postnatal development." J. Comp. Neurol.271. 30-43 (1988)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Sobue, K. and Kanda, K.: "Localization of pp60^<c-src> in growth cone of PC12 cell." Biochem. Biophys. Res. Commun.157. 1383-1389 (1988)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Tsumoto, T., Kimura, F., Hagihara, K., Sato, H. and Sobue, K.: "A role of NMDA receptors and membrane-associated, Ca^<2+>/calmodulin-binding proteins in synaptic plasticity in the developing visual cortex." Biomed. Res.9. 43-52 (1988)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Hirokawa, N., Sobue, K., Kanda, K., Harada, A. and Yorifuji, H.: "The cytoskeletal architecture of the presynaptic terminal and molecular structure of synapsis 1." J. Cell Biol.108. 111-126 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Sobue, K., Kanda, K., Miyamoto, I., Iida, K., Yahara, I., Hirai, R. and Hiragun, A.: "Comparison of the regional distribution of calspectin (nonerythroid spectrin or fodrin), alpha-actinin, vinculin nonerythroid protein 4.1, and calpactin in normal and avian sarcoma virus- or rous sarcoma virus-induced transformed cells." Exp. Cell Res.181. 256-262 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yorifuji, H., Kanda, K., Sobue, K. and Hirokawa, N.: "Localization of 4.1 related proteins in cerebellar neurons." Eur. J. Cell Biol.48. 104-115 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Hayashi, K., Yamada, S., Kanda, K., Kimizuka, F., Kato, I. and Sobue, 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)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Hoshimaru, M., Fujio, Y., Sobue, K., Sugimoto, T. and Nakanishi, S.: "Immunochemical evidence that myosin I heavy chain-like protein is identical to the 110-kilodalton brush-border protein." J. Biochem.106. 455-459 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Sobue, K. and Kanda, K.: "alpha-Actinins, calspectin (brain spectrin or fodrin) and actin participate in adhesion and movement of growth cones." Neuron. 3. 311-319 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Senda, T., Fujita, H., Ban, T., Zhong, C., Ishimura, K., Kanda, K. and Sobue, K.: "Ultrastructural and immunocytochemical studies on the cytoskeleton in the anterior pituitary of rats, with special regard to the relationship between actin filaments and secretory granules." Cell Tissue Res.258. 25-30 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Hayashi, K., Kanda, K., Kimizuka, F., Kato, I. and Sobue, K.: "Primary structure and functional expression of h-caldesmon complementary DNA." Biochem. Biophys. Res. Commun.164. 503-511 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Tanaka, T., Ohta, H., Kanda, K., Tanaka, T., Hidaka, H. and Sobue, K.: "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)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Nakata, T., Sobue, K. and Hirokawa, N.: "Conformational change and localization of calpactin I complex involved in exocytosis as revealed by quick-freeze, deep-etch, electron microscopy and immunocytochemistry." J. Cell Biol.110. 13-25 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Sobue, K.: "Involvement of the membrane cytoskeletal proteins and the src gene producing growth cone adhesion and movement." Neurosci. Res.Suppl. 13. S80-S91 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Harada, A., Sobue, K. and Hirokawa, N.: "Developmental changes of synapsis I subcellular localization in rat cerebellar neurons." Cell Struc. Func.15. 329-342 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Hayashi, K., Fujio, Y., Kato, I. and Sobue, K.: "Structural and functional relationships between h-and l-caldesmons." J. Biol. Chem.266. 355-361 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Tanaka, T., Kadowaki, K., Lazarides, E. and Sobue, K.: "Ca^<2+>-Dependent of the spectrin/actin interaction by calmodulin and protein 4.1." J. Biol. Chem.266. 1134-1140 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Sobue, K. and James R.: "Sellers Caldesmon : A novel-regulatory protein of smooth muscle and non-muscle actin-myosin interaction." J. Biol. Chem.(1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Takao Nakata: "Conformational change and localization of calpactin I complex involved in exocytosis as revealed by quickーfreeze,deepーetch,electron microscopy and immunocytochemistry." Journal of Cell Biology. 110. 13-25 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Kenji Sobue: "Involvement of the membrane cytoskeletal proteins and the src gene product in growth cone adhesion and movement." Neuroscience Research. Supple.13. S80-91 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Akihiro Harada: "Developmental changes of synapsin I subcellular localization in rat cerebellar neurons." Cell Structure and Function. 15. 329-342 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Ken'ichiro Hayashi: "Structural and functional relationships betweenhー and lーcaldesmons." Journal of Biological Chemistry. 266. 355-361 (1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] Toshihiko Tanaka: "Ca^<2+>ーDependent of the spectrin/actin interaction by calmodulin and protein4.1." Journal of Biological Chemistry. 266. 1134-1140 (1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] Kenji Sobue: "Caldesmon:A novelーregulatory protein of smooth muscle and nonmuscle actinーmyosin interaction" Journal of Biological Chemistry. 266. (1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] 祖父江 憲治: "シリ-ズ分子生物学の進歩II 分子神経生物学" 丸善, 396 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] 祖父江 憲治: "新生化学実験講座II 神経生化学" 東京化学同人, 542 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Takao Nakata: "Conformational Change in Localization of Calpactin I Complex Involved in Exocytosis as Revealed by Quick-Freeze,Deep-Etch Electron Microscopy and Immunocytochemistry" J.Cell.Biol.110. 13-25 (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] Kenji Sobue: "α-Actinins,Calspectin(Brain Spectrin or Fodrin),and Actin Participate in Adhesion and Movement of Growth Cones" Neuron. 3. 311-319 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Kenichiro Hayashi: "Primary Structure and Functional Expression of h-Caldesinon Complementary DNA" Biochem.Biophys.Res.Commun.164. 503-511 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Kenichiro Hayashi: "35kDa Fragment of h-Caldesinon Conserves Two Consensus Sequences of the Tropomyosin-Binding Domain in Troponin T." Biochem.Biophys.Res.Commun.161. 38-45 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Nobutaka Hirokawa: "The Cytoskeletal Architecture of the Presynaptic Terminal and Molecular Structure of Synapsin I" J.Cell.Biol.108. 111-126 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Toshihiko Tanaka: "Phosphorylation of high-Mr Caldesinon by Protein Kinase C Modulates the Regulatory Function of this Protein on the Interaction Between Actin and Myosin." Eur.J.Biochem.(1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] 祖父江憲治: "細胞骨格の機能" 共立出版, 342 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] 祖父江憲治: "シリ-ズ分子生物学の進歩11 分子神経生物学" 丸善株式会社, 396 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Sobue,K.: Proc.Natl.Acad.Sci.USA. 85. 482-486 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Tsumoto,T.: J.Comp.Neurol.271. 30-43 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Sobue,K.: Biochem.Biophys.Res.Commun.157. 1383-1389 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Sobue,K.: Exp.Cell Res.

    • Related Report
      1988 Annual Research Report
  • [Publications] Hirokawa,N.: J.Cell.Biol.108. 111-126 (1989)

    • Related Report
      1988 Annual Research Report
  • [Publications] Yorifuji,H.: Eur.J.Cell.Biol.

    • Related Report
      1988 Annual Research Report

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Published: 1988-04-01   Modified: 2016-04-21  

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