Functional analyses of actin filaments using dominant negative

• Project/Area Number: 18370067
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biophysics
• Research Institution: National Institute of Advanced Industrial Science and Technology
• Principal Investigator: UYEDA Taro National Institute of Advanced Industrial Science and Technology, セルエンジニアリング研究部門, 部門付き (90356551)
• Co-Investigator(Kenkyū-buntansha): 片山 栄作 東京大学, 医科学研究所, 教授 (50111505)
• Research Collaborator: 野口 太郎 筑波大学, 大学院・博士課程
• Project Period (FY): 2006 – 2008
• Project Status: Completed (Fiscal Year 2008)
• Budget Amount: ¥14,180,000 (Direct Cost: ¥11,300,000、Indirect Cost: ¥2,880,000); Fiscal Year 2008: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000); Fiscal Year 2007: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000); Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
• Keywords: アクチン / ミオシン / 協同的構造変化 / 優性変異 / 優性阻害 / 組換えタンパク質 / 細胞性粘菌 / 酵母 / 一分子力学測定 / カルシウム制御 / グリシンスキャニング / 発現系 / 構造変化 / グリシン / ショウジョウバエ / 生物物理 / 分子モーター / 蛋白質

Research Abstract

アクチンミオシン系の力発生において、アクチンフィラメントは線路として機能する一方、その協同的構造変化も何らかの重要な機能を果たすと考えられている。一般に協同的構造変化の機能の解明には、優性変異の解析が有力な研究手段となる。そこでAn and Mogami (1996) が同定した10 個の優性変異アクチンについて、われわれ独自のアクチン発現系を用いて6変異の精製に成功し機能解析を行った。その結果、4変異は重合に、2変異はCa2+依存的な協同的スイッチ機構に異常があることを発見した。さらにグリシンスキャニング法(後述)により、新たに5個の優性変異アクチンを同定した。

Research Products

• [Journal Article] Novel mode of cooperative binding between myosin and Mg2+-actin filaments in the presence of lowconcentrations of ATP (2009)
  • Author(s): Tokuraku, K., R. Kurogi, R. Toya, and T. Q. P. Uyeda
  • Journal Title: J. Mol. Biol 386 Pages: 149-162
• [Journal Article] Novel mode of cooperative binding between myosin and Mg^<2+>-actin filaments in the presence of low concentrations of ATP (2009)
  • Author(s): Kiyotaka Tokuraku, Rika Kurogi, Ryo Toya, Taro Q. P. Uyeda
  • Journal Title: Journal of Molecular Biology 386 Pages: 149-162
  • Peer Reviewed
• [Journal Article] A novel system for expressing toxic actin mutants in Dictyostelium and purification and characterization of a dominant lethal yeast actin mutant (2007)
  • Author(s): Noguchi T. Q. P., N. Kanzaki, H. Ueno, K. Hirose, and T. Q. P. Uyeda
  • Journal Title: J. Biol. Chem 282 Pages: 27721-27727
• [Journal Article] A novel system fbr expressing toxic actin mutants in Dictyostelium and purification and characterization of a dominant lethal veast acthl mutant. (2007)
  • Author(s): Noguchi T.Q.P., N Kanzakd, H Ueno, K.Hirose, and T.Q.P. Uyeda
  • Journal Title: The Journal of Biological chemistry 282 Pages: 27721-27727
  • Peer Reviewed
• [Presentation] Electoron Microscopy on The Comprehensive Structural Change during Actomyosin Sliding (2009)
  • Author(s): Eisaku Katayama, Yoshitaka Kimori, Norio Baba, Taro Q. P. Uyeda
  • Organizer: Biophysical Society 53rd Annual Meeting
  • Place of Presentation: Boston, USA
  • Year and Date: 2009-03-01
• [Presentation] アクチンG146の変異がミオシンとの相互作用に与える影響 (2009)
  • Author(s): 野口太郎, 小森智貴, 岩根敦子, 柳田敏雄, 上田太郎
  • Organizer: 生体運動研究合同班会議
  • Place of Presentation: 東京
  • Year and Date: 2009-01-10
• [Presentation] ショウジョウバエ飛翔能を優性に阻害する変異アクチンの機能解析 (2008)
  • Author(s): 野口太郎, 上野祐則, 広瀬恵子, 上田太郎
  • Organizer: 第31回日本分子生物学会第81回日本生化学会
  • Place of Presentation: 神戸
  • Year and Date: 2008-12-12
• [Presentation] Mutation at Gly146 of actin causes class-specific motility defect of myosin (2008)
  • Author(s): 野口太郎, 小森智貴, 上野裕則, 柳田敏雄, 広瀬恵子, 上田太郎
  • Organizer: 生物物理学会第46回年会
  • Place of Presentation: 福岡
  • Year and Date: 2008-12-03
• [Presentation] Characterization of dominant lethal D11K and D11Qmutant actins (2008)
  • Author(s): 中嶋潤, 野口太郎, 上田太郎
  • Organizer: 生物物理学会第46回年会
  • Place of Presentation: 福岡
  • Year and Date: 2008-12-03
• [Presentation] Characterization of dominant lethal D11K and D11Q mutant actins (2008)
  • Author(s): 中嶋潤, 野口太郎, 上田太郎
  • Organizer: 日本生物物理学会第46回年会
  • Place of Presentation: 福岡
  • Year and Date: 2008-12-03
• [Presentation] Mutation at Gly146 of actin causes class-specific motility defect of myosin (2008)
  • Author(s): 野口太郎, 小森智貴, 上野祐則, 柳田敏雄, 広瀬恵子, 上田太郎
  • Organizer: 日本生物物理学会第46回年会
  • Place of Presentation: 福岡
  • Year and Date: 2008-12-03
• [Presentation] The roles of actin filament and its state during actomyosin sliding (2008)
  • Author(s): 片山栄作
  • Organizer: 日本生物物理学会第46回年会
  • Place of Presentation: 福岡
  • Year and Date: 2008-12-03
• [Presentation] Evaluation of structural fluctuation within actin filament by fractal image analysis combined with a morphological filtering method (2008)
  • Author(s): 木森義隆, 児玉孝雄, 片山栄作
  • Organizer: 日本生物物理学会第46回年会
  • Place of Presentation: 福岡
  • Year and Date: 2008-12-03
• [Presentation] A novel expression system for toxic mutant actins, and characterization of mutant actins defective in motility (2008)
  • Author(s): 上田太郎
  • Organizer: 2nd International Symposium on Bio-nanosystems
  • Place of Presentation: 東京
  • Year and Date: 2008-10-31
• [Presentation] A novel expression system for dominant negative mutant actin in Dictyostelium (2008)
  • Author(s): 野口太郎, 神崎紀子, 上野祐則, 広瀬恵子, 上田太郎
  • Organizer: International Dietyostelium Conference 2008
  • Place of Presentation: つくば
  • Year and Date: 2008-09-17
• [Presentation] Revised behavior of myosin crossbridges during in vitro actin sliding (2008)
  • Author(s): E. Katayama, T. Kimori, M. Yamada, T. Eguchi, T. Sasaki, S. Maruta, N. Baba, and T. Uyeda
  • Organizer: Biophysical Society 51st Annual Meeting
  • Place of Presentation: Baltimore, USA
  • Year and Date: 2008-03-08
• [Presentation] Purification and characterization of a dominant lethal yeast actin, using a novel expression system for toxic actin mutants (2007)
  • Author(s): 上田太郎, 神崎紀子
  • Organizer: 日本生物物理学会第45回年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-21
• [Presentation] Glycine scanning mutational analysis of Dictyostelium actin (2007)
  • Author(s): 野口太郎, 上田太郎
  • Organizer: 日本生物物理学会第45回年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-21
• [Presentation] Further characterization of the configuration of actin-attached myosin-crossbridges during in vitro sliding (2007)
  • Author(s): 片山栄作, 木森義隆, 上田太郎
  • Organizer: 日本生物物理学会第45回年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-21
• [Presentation] Interaction of myosin with the filaments of acto-S1 chimera protein (2007)
  • Author(s): 和沢鉄一, シディック, 宮崎崇, 片山栄作, 上田太郎, 鈴木誠
  • Organizer: Interaction of myosin with the filaments of acto-S1 chimera protein
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-21
• [Presentation] Glycine scaming mutational analysis of Dictyostelium actin. (2007)
  • Author(s): 野口 太郎、上田 太郎
  • Organizer: 日本生物物理学会第45回年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-21
• [Presentation] Purification amd characterization of a dominant lethal yeast actin,usilganovel expression svstem fbr toxic actin mutarlts. (2007)
  • Author(s): 上田 太郎、神崎 紀子
  • Organizer: 日本生物物理学会第45回年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-21
• [Presentation] Ihteraction of myosill with the filaments of acto-S1 chimera protein. (2007)
  • Author(s): 和沢 鉄一、シディック、宮崎 崇、片山 栄作、上田 太郎、鈴木 誠
  • Organizer: 日本生物物理学会第45回年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-21
• [Presentation] A novel expression system for dominant negative mutant actins (2006)
  • Author(s): T. Noguchi, H, Ueno, K. Hirose, and T. Uyeda
  • Organizer: 5th East Asian Biophysics Symposium and 44th Annual Meeting of the Biophysical Society of Japan
  • Place of Presentation: Okinawa, Japan
  • Year and Date: 2006-11-16
• [Remarks]
  • URL: http://staff.aist.go.jp/t-uyeda/HP
• [Patent(Industrial Property Rights)] 毒性の強い組換えタンパク質の効率的発現法 (2006)
  • Inventor(s): 上田太郎, 野口太郎, 神崎紀子
  • Industrial Property Number: 2006-223042
  • Filing Date: 2006-08-18
• [Patent(Industrial Property Rights)] タンパク質の発現方法 (2006)
  • Inventor(s): 上田太郎, 野口太郎
  • Industrial Property Number: 2006-268914
  • Filing Date: 2006-09-29