1993 Fiscal Year Final Research Report Summary
Time-lapse Electron Microscopy with Caged Compounds
| Project/Area Number |
04558034
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
分子遺伝学・分子生理学
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| Research Institution | National Institute for Physiological Sciences |
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Principal Investigator |
TSUKITA Shoichiro National Institute for Physiol.Sci. Prof., 生理学研究所, 教授 (50155347)
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| Co-Investigator(Kenkyū-buntansha) |
YONEMURA Shigenobu National Institute for Physiol.Sci. Assistant Prof., 生理学研究所, 助手 (60192811)
TSUKITA Sachiko National Institute for Physiol.Sci. Assistant Prof., 生理学研究所, 助手 (00188517)
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| Project Period (FY) |
1992 – 1993
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| Keywords | muscle / caged compounds / rapid freezing / liquid helium / platelet / electron microscopy / IP3 / temporal resolution |
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| Research Abstract |
The interaction between myosin subfragment 1 (S1) and actin filaments after the photolysis of P^3-1-(2-nitrophenyl)ethyl 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 analyzes 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 significatn 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 are discussed with special reference to the molecular mechanism of acto-myosin interaction in the presence of ATP.
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Research Products
(13 results)
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[Publications] Yonemura, S., Nagafuchi, A., Sato, N., and Tsukita, S.: "Concentration of an integral membrane protein, CD43 (leukosialin, sialophorin), in the cleavage furrow through the interaction of its cytoplasmic domain with actin-based cytoskeleton." J.Cell Biol.120. 437-449 (1993)
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「研究成果報告書概要(欧文)」より
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[Publications] Itoh, M., Nagafuchi, A., Yonemura, S., Yasuda-Kitani, T., Tsukita, S., and Tsukita, S.: "The 220kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells : cDNA cloning and immunoelectron microscopy." J.Cell Biol.121. 491-502 (1993)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Oda, H., Uemura, T., Shiomi, K., Nigafuchi, A., Tsukita, S., and Takeichi, M.: "Identification of a Drosophila homologue of alpha-catenin and its association with the armadillo protein." J.Cell Biol.121. 1133-1140 (1993)
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「研究成果報告書概要(欧文)」より
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[Publications] Furuse, M., Hirase, T., Itoh, M., Nagafuchi, A., Yonemura, S., Tsukita, S., and Tsukita, S.: "Occludin : A novel integral membrane protein localizing at tight junctions." J.Cell Biol.123. 1777-1788 (1993)
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「研究成果報告書概要(欧文)」より
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[Publications] Takeuchi, K., Sato, N., Kasahara, H., Yonemura, S., Nagafuchi, A., Tsukita, S., and Tsukita, s.: "Purturbation of cell adhesion and microvilli formation by antisence oligonucleotides to ERM family members." J.Cell Biol.(in press.).
Description
「研究成果報告書概要(欧文)」より
