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1991 Fiscal Year Final Research Report Summary

A New Rapid Freezing Apparatus for Electron Microscopy

Research Project

Project/Area Number 02558026
Research Category

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

Allocation TypeSingle-year Grants
Research Field 分子遺伝学・分子生理学
Research InstitutionNational Institute for Physiological Sciences

Principal Investigator

TSUKITA Shoichiro  National Institute for Physiological Sciences, Professor, 生理学研究所, 教授 (50155347)

Co-Investigator(Kenkyū-buntansha) TSUKITA Sachiko  National Institute for Physiological Sciences, Assistant Professor, 生理学研究所, 助手 (00188517)
Project Period (FY) 1990 – 1991
Keywordscaged compounds / rapid freezing / electron microscopy / actin / myosin / liquid helium / etching / replica / liquid helium
Research Abstract

The interaction between myosin subfragment 1 (S1) and actin filaments after the photolysis of P^3-1-(2-nitropheny1)ethy1 ester of ATP (caged ATP) was analyzed with a newly-developed freezing system using liquid helium. Actin and S1 (100muM each) formed a rope-like double helix characteristic of rigor in the presence of 5 mM caged ATP at room temperature. At 15 ms after photolysis, the rope-like double helix was partially disintegrated. The number of S1 attached to actin filaments gradually decreased up to 35 ms after photolysis, and no more changes were detected from 35 to 200 ms. After depletion of ATP. the rope-like double helix was reformed. Taking recent analyses of actomyosin kinetics into consideration, we concluded that most S1 observed on actin filaments at 25-200 ms are so called "weakly-bound S1" (S1.ATP or S1.ADP.Pi) and that the weakly-bound S1 under a rapid association-dissociation equilibrium with actin filaments can be captured by electron microscopy by means of our newly-developed freezing system.
This enabled us to directly compare the conformation of weakly- and strongly-bound S1. Within the resolution of deep-etch replica technique, there were no significant conformational differences between weakly- and strongly-bound S1, and neither types of S1 showed any positive cooperativity in their binding to actin filaments. Close comparison revealed that the weakly- and strongly-bound S1 have different angles of attachment. As compared to strongly-bound S1, weakly-bound S1 showed broad distribution of attachment angle and a decreased tilt from the perpendicular to the filaments. These results discussed with special reference to the molecular mechanism of acto-myosin interaction in the presence of ATP.

  • Research Products

    (17 results)

All Other

All Publications (17 results)

  • [Publications] Sato,N.: "Radixin,a barbed end-capping actin-modulating protein,is concentrated at the cleavage furrow during cytokinesis." Journal of Cell Biology. 113. 321-330 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Moto,E.: "Double-rowed organization of inner dynein arms in chlamydomonas flgaella revealed by tilt-series thin-section electron microscopy." Journal of Cell Science. 99. 57-66 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tsukita,S.: "Specific proto-oncogenic tyrosine kinases of src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylatio is elevated." Journal of Cell Biology. 113. 867-879 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Yonemura,S.: "Mass isolation of cleavage furrows from dividing sea urchin eggs." Journal of Cell Science. 100. 73-84 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Nagafuchi,A.: "The 102kd cadherin-associated protein: Similarity to vinculin and posttranscriptional regulation of expression." Cell. 65. 1-20 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Funayama,N.: "Radixin is a novel member of the band 4.1 family." Journal of Cell Biology. 115. 1039-1048 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tsukita. Sh., Tukita, Sa. & Nagafuchi. A.: "The undercoat of adherens junctions: A Key specialized structure in organogenesis and carcinogenesis." Cell Struct. Funct.15. 7-12 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Nishikawa. S., Tsukita, Sa. & Tsukita. Sh.: "Localization of adherens junction proteins along the possible sliding interface between secretory ameloblasts of the rat incisor." Cell Struct. Funct.15. 245-250 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Funatsu, T., Higuchi, H. & Ishiwata. S.: "Elastic filaments in skeletal muscle revealed by selective removal of thin filaments with plasma gelsolin." J. Cell Biol.110. 53-62 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Sato, N., Yonemura, S., Obinata. T., Tsukita. Sa. & Tsukita. Sh.: "Radixin. a barbedendcapping actin-modulating protein. is concentrated at the cleavage furrow during cytokinesis" J. Cell Biol.113. 321-330 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Muto, E., Kamiya, R. & Tsukita. Sh.: "Double-rowed organization of inner dynein arms in Chlamybomonas flagella revealed by tilt-series thin-section electron microscopy." J. Cell Sei.99. 57-66 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tsukita, Sa., Oishi. K., Akiyama. T., Yamanashi. Y., Yamamoto. T. & Tsukita. Sh.: "Specific proto-oncogenic tyrosine kinases of src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylation is elevated." J. Cell Biol.113. 867-879 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yonemura, S., Mabuchi. I. & Tsukita. Sh.: "Mass isolation of cleavage furrows from dividing sea urchin eggs." J. Cell Sei.100. 73-84 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Nagafuchi, A., Takeichi. M. & Tsukita. Sh.: "The 102kd cadherin-associated protein: Similarity to vinculin and posttranscriptional regulation of expression." Cell 65. 1-20 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Funayama, N., Nagafuchi, A., Sato. N., Tsukita, Sh.: "Radixin is a novel member of the band 4.1 family." J. Cell Biol.115. 1039-1048 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Itoh, M., Yonemura, S., Nagafuchi. A., Tsukita. Sa. & Tsukita. Sh.: "A 2200kD undercoat-constitutive protein: Its specific localization at cadherin-based cell-to-cell adhesion sites." J. Cell Biol.115. 1449-1462 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tsukita, Sh.: "Signal transduction through the undercoat of adherens junctions which is tightly associated with actin filaments. Yamakawa. T. ed." RINSHOKEN 15 Years of Research. Tokyo. 19-29 (1991)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1994-03-18  

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