INVERTEBRATE CONNECTIN, AN ELASTIC PROTEIN IN THE GIANT SARCOMERE OF INVERTEBRATE STRIATED MUSCLE

• Project/Area Number: 13440248
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 動物生理・代謝
• Research Institution: CHIBA UNIVERSITY
• Principal Investigator: KIMURA Sumiko CHIBA UNIVERSITY, DEPARTMENT OF BIOLOGY, FACULTY OF SCIENCE, ASSOCIATE PROFESSOR, 理学部, 助教授 (50093232)
• Co-Investigator(Kenkyū-buntansha): MARUYAMA Koscak NATIONAL CENTER FOR UNIVERSITY ENTRANCE EXAMINATIONS, PRESIDENT, センター長 (60012267)
• Project Period (FY): 2001 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥15,000,000 (Direct Cost: ¥15,000,000); Fiscal Year 2004: ¥1,100,000 (Direct Cost: ¥1,100,000); Fiscal Year 2003: ¥1,800,000 (Direct Cost: ¥1,800,000); Fiscal Year 2002: ¥1,800,000 (Direct Cost: ¥1,800,000); Fiscal Year 2001: ¥10,300,000 (Direct Cost: ¥10,300,000)
• Keywords: I-Connectin / Giant sarcomere / Elastic protein / PEVK / obliquely striated muscle / Titin / striated muscle / invertebrate / 1分子計測 / ザリガニ

Research Abstract

The giant filamentous protein, connectin (3000-3700 kDa) contributes to organize a contractile sarcomere by extending from the Z line over the thick filament up to the M line at the center of the sarcomere in vertebrate striated muscle. A molecule of connectin is very flexible and approximately 1 μm long in resting sarcomeres, and can be passively stretched resulting in tension generation.
In crayfish claw closer muscle, the giant sarcomeres are 8.3 μm long at rest, 3.5 times longer than vertebrate striated muscle sarcomeres and they are extensible up to 13 μm upon stretch. In order to clarify what kind of primary structure makes such a remarkable extension possible, we sequenced the cDNA encoding the Invertebrate-connectin (I-connectin) in crayfish claw closer muscle. The entire sequence of crayfish I-connectin was predicted from cDNA sequences of 53,424 bp (17,352 residues ; 1960 kDa). Crayfish I-connectin contains a novel SEK repeats, and also two PEVK domains and one kettin region. Kettin is a small isoform of I-connectin. Immunoblot tests using antibody to the SEK repeats, revealed the presence of I-connectin also in long sarcomeres of insect leg muscle and barnacle ventral muscle. The force measurements of the single recombinant SEK polypeptide were performed by intemolecular force microscopy(IFM), a refined AFM system. The force versus extension curves were well fit to the wormlike chain(WLC) model and the obtained persistence length of 0.37 nm indicates that the SEK region is a random coil for full length. Immunofluorescence microscopy demonstrated that the two repeats, the long spacer and the two PEVK domains contribute to sarcomere extension. These regions may allow a span of a 3.5 μm distance as a new class of composite spring.

Research Products

• [Journal Article] Partial sequence of 1200K connectin-like protein in obliquely striated muscle of a polychaete(Annelida) (2005)
  • Author(s): Izawa, N., Fukuzawa, A., Maruyama, K., Kimura, S.
  • Journal Title: J.Muscle Res.Cell Motil. (in press)
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Partial sequence of 1200K connectin-like protein in obliquely striated muscle of a polychaete (Annelida) : Evidence for structural diversity from vertebrate and invertebrate connectins. (2005)
  • Author(s): Izawa, N., Fukuzawa, A., Maruyama, K., Kimura, S.
  • Journal Title: J.Muscle Res.Cell Motil. (in press)
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Partial sequence of 1200K connectin-like protein in obliquely striated muscle of a polychaete (Annelida). (2005)
  • Author(s): Izawa, N., Fukuzawa, A., Kimura, S.
  • Journal Title: J.Muscle Res.Cell Motil. (in press)
• [Journal Article] Identification of high molecular weight proteins in squid muscle by Western blotting analysis and postmortem rheological changes. (2004)
  • Author(s): Kasamatsu, C., Kimura, S., Kagawa, M., Hatae, K.
  • Journal Title: Biosci.Biotechnol.Biochem. 68 Pages: 1119-1124
  • NAID: 10013144890
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Identification of high molecular weight proteins in squid muscle by western blotting analysis and postmortem rheological changes. (2004)
  • Author(s): Kasamatsu, C., Kimura, S., Kagawa, M., Hatae, K.
  • Journal Title: Biosci.Biotechnol.Biochem. 68 Pages: 1119-1124
  • NAID: 10013144890
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Single molecule measurement of elasticity of invertebrate connectin. (2004)
  • Author(s): Ikeda, K., Hiroshima, M., Fukuzawa, Tokunaga, M., Kimura, S.
  • Journal Title: Zool.Sci. 21 Pages: 1281-1281
• [Journal Article] Localization of Projectin in Locust Flight Muscle. (2003)
  • Author(s): Shimamura, J., Maruyama, K., Kimura, S.
  • Journal Title: Comp.Biochem.Physiol. 136 Pages: 419-423
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] The entire cDNA sequences of projectin isoforms of crayfish claw closer and flexor muscles and their localization. (2003)
  • Author(s): Oshino, T., Shimamura, J., Fukuzawa, A., Maruyama, K., Kimura, S.
  • Journal Title: J.Muscle Res.Cell Motil. 24 Pages: 431-438
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Localization of projectin in locust flight muscle. (2003)
  • Author(s): Shimamura, J., Maruyama, K., Kimura, S.
  • Journal Title: Comp.Biochem.Physiol. 136 Pages: 419-423
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] The entire cDNA sequences of projectin isoforms of crayfish claw closer and flexor muscles and their localization. (2003)
  • Author(s): Oshino, T., Shimamura, J., Fukuzawa, A, Maruyama, K., Kimura, S.
  • Journal Title: J.Muscle Res.Cell Motil. 24 Pages: 431-438
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Single Molecule Measurement of Elasticity of Serine-, Glutamate- and Lysine-Rich Repeats of Invertebrate Connectin Reveals That Its Elasticity. (2002)
  • Author(s): Fukuzawa, A., Hiroshima, M., Maruyama, K., Yonezawa, N., Tokunaga, M., Kimura, S.
  • Journal Title: J.Muscle Res.Cell Motil. 23 Pages: 449-453
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Glutamate-and Lysine-rich repeats of invertebrate connectin reveals that is elasticity is caused entropically by random coil structure. (2002)
  • Author(s): Fukuzawa, A., Hiroshima, M., Maruyama, K., Yonezawa, N., Tokunaga, M., Kimura, S.
  • Journal Title: J.Muscle Res.Cell.Motil. 23 Pages: 449-453
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Invertebrate connectin spans as much as 3.5μm in the giant sarcomeres of crayfish claw muscle. (2001)
  • Author(s): Fukuzawa, A., Shimamura, J., Takemori, S., Kanzawa, N., Yamaguchi, M., Sun, P., Maruyama, K., Kimura, S.
  • Journal Title: EMBO J. 20 Pages: 4826-4835
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Invertebrate connectin spans as much as 3.5 μm in the giant sarcomeres of crayfish claw muscle. (2001)
  • Author(s): Fukuzawa, A., Shimamura, J., Takemori, S., Kanzawa, N., Yamaguchi, M., Sun, P., Maruyama, K., Kimura, S.
  • Journal Title: EMBO J. 20 Pages: 4826-4835
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Shimamura, J.: "Localization of Projectin in Locust Flight Muscle."Comp.Biochem.Physiol.. 136. 419-423 (2003)
• [Publications] Oshino, T.: "The entire cDNA sequences of projectin isoforms of crayfish claw closer and flexor muscles and their localization."J.Muscle Res.Cell Motil.. 24. 431-438 (2003)
• [Publications] Noguchi, H.: "The Primary Structure of Chicken Breast Muscle Connectin (Titin)."Zool.Sci.. 20. 1542 (2003)
• [Publications] Kimura, S.: "Connectin (Titin)-like Giant Protein in Invertebrate Smooth Muscle (Molluscan ABRM)."Zool.Sci.. 20. 1542 (2003)
• [Publications] Fukuzawa, A.: "Single Molecule Measurement of Elasticity of Serine-, Glutamate-and Lysine-Rich Repeats of Invertebrate Connectin Reveals That Its Elasticity Is Caused Entropically by Random Coil Structure"J.Mus.Res.Cell Motil.. 23(in press). (2002)
• [Publications] Oshino, T.: "The entire cDNA sequences of projectin isoforms of crayfish claw closer and flexor muscles and their localization"J.Mus.Res.Cell Motil.. 24(in press). (2003)
• [Publications] Fukuzawa, A.: "Invertebrate connectin spans as much as 3.5 μm in the giant sarcomeres of crayfish claw muscle"EMBO Journal. 20. 4826-4835 (2001)
• [Publications] Shimamura, J.: "Localization of projectin in several arthropod striated muscles"Zoological Science. 18. 38 (2001)