1999 Fiscal Year Annual Research Report

生物分子モーターの1分子計測と1分子操作

Research Project

Project/Area Number	09279104
Research Institution	Keio University
Principal Investigator	木下一彦慶應義塾大学, 理工学部, 教授 (30124366)
Co-Investigator(Kenkyū-buntansha)	上村慎治東京大学, 大学院・総合文化研究科, 助教授 (90177585) 大岩和弘郵政省通信総合研究所, 関西先端研究センター, 室長(研究職) 柳田敏雄大阪大学, 医学部, 教授 (30089883) 嶋本伸雄国立遺伝学研究所, 構造遺伝学研究センター, 教授 (20127658) 上田太郎工業技術院, 産業技術融合領域研究所, 主任研究官
Keywords	ミオシン / アクチン / ATP合成酵素 / ダイニン / trpリプレッサー / 光学顕微鏡 / 蛍光エネルギー移動 / レーザートラップ
Research Abstract	F_1-ATPaseが120度おきにステップする回転ステッパーモーターであることを、回転子サブユニットに結合させた蛍光色素1分子の偏光測定により確認した。昨年報告したアクチン線維を目印としての測定と比較することにより、ATP結合速度は負荷によらないことが分かった。また、小さなビーズを結合させて回転を観察した結果、無負荷回転速度は室温で毎秒100回転を越えること、その速度でもステップ動作がおき、ステップ自身には0.1ミリ秒程度しか時間がかからないこと、などがわかった。(木下) アクチン-ミオシン間に働く微小な相互作用力を非接触的に検出するために、一般のAFMの百倍以上の感度で力の分布を2次元的に画像化できるプローブ顕微鏡を開発した。純水中で基板上に吸着させたミオシンフィラメント静電力測定から、フィラメントは全体として負に帯電し、ベアゾーン付近の負電荷量が特に多いことがわかった。(柳田) クラミドモナスの内腕ダイニンの7つの亜種のうち、微小管を最も速く動かすことができる亜種cは8nmのステップ様変位を起こすステッピングモータであり、連続的な運動を起こすプロセッシブモータであった。また、亜種fは運動中に微小管に結合した状態を長くとる、すなわち従事率が高いモータであった。(大岩) 1分子蛍光エネルギー移動現象の実時間観測のための装置を開発した。レーザー光エバネッセント照明による蛍光像を適度なウェッジ角を持つプリズムを通すことによってスペクトルに分ける。10ミリ秒の時間分解能で画像として観測できる。(上村) 粘菌のG680V変異ミオシンは、いわゆる「プリストローク」状態が長時間持続すること示唆する結果を得た。またアクチンとミオシンの結合の協同性について、蛍光顕微鏡を用いた直接観察を進め、協同性には強弱二段階があることを明らかにした。(上田) 大腸菌trpリプレッサーは、DNA上をスライドすることにより特異的部位に対する親和性を1万-10万倍強めていることがわかった。スライディングがDNAの溝に沿ったラセン運動かどうかを調べるため、レーザートラップシステムを構築した。(嶋本)

Research Products

(29 results)

All Other

All Publications (29 results)

[Publications] K. Adachi, K. Kinosita, Jr. , T. Ando: "Single-fluorophore imaging with an unmodified epifluorescence microscope and conventional video camera"J. Microscop. 195. 125-132 (1999)
[Publications] H. Miyata, S. Nishiyama, et al.: "Protrusive growth from giant liposomes driven by actin polymerization"Proc. Natl. Acad. Sci. USA. 96. 2048-2053 (1999)
[Publications] Y. Arai, R. Yasuda, K. Akashi, et al.: "Tying a molecular knot with optical tweezers"Nature. 399. 446-448 (1999)
[Publications] H. Kato, T. Nishizaka, T. Iga, et al.: "Imaging of thermal activation of actomyosin motors"Proc. Natl. Acad. Sci. USA. 96. 9602-9606 (1999)
[Publications] H. Noji, K. Hasler, W. Junge, et al.: "Rotation of Escherichia coli Fl-ATPase"Biochem. Biophys. Res. Commun.. 260. 597-599 (1999)
[Publications] K. Kinosita, Jr.: "Real time imaging of rotating molecular machines"FASEB J.. 13 Suppl. 2. S201-S208 (1999)
[Publications] H. Miyata, G. Marriott, K. Akashi, et al.: "Cell deformation mechanism studied with actin-containing giant vesicle, a cell mimicking system"Giant Vesicles, Chapter. 24. 320-333 (2000)
[Publications] K. Akashi, H. Miyata, K. Kinosita, Jr.: "Observation of a variety of giant vesicles under an optical microscope"Giant Vesicles, Chapter. 5. 46-48 (2000)
[Publications] K. Kinosita, Jr. , R. Yasuda, H. Noji: "Fl-ATPase; a highly effiicient rotary ATP machine"Essays Biochem. 35(in press). (2000)
[Publications] K. Kinosita, Jr. , R. Yasuda, H. Noji, K.: "A rotary molecular motor that can work at near 100% efficiency"Philos. Trans. Biol. Sci.. (in press). (2000)
[Publications] Y. Ishii, Y. Kimura, H. Tanaka, et al.: "Imaging and nano-manipulation of single actomyosin motors at work"Proceedings of the Australian Physiological and Pharmacological Society. 30. 24-39 (1999)
[Publications] Y. Ishii, Y. Kimura, K. Kitamua, et al.: "Imaging and nano-manipulation of single actomyosin motors at work"Clinical and Experimental Pharmacology and Physiology. (in press). (2000)
[Publications] Y. Miyamoto, E. Muto, T. Mashimo, et al.: "Direct inhibition of microtubule-based kinesin motility by local anesthetics"Biophys. J.. 78(in press). (2000)
[Publications] L-H. Ye, H. Kishi, A. Nakamura, et al.: "Myosin light-chain kinase of smooth muscle stimulates myosin ATPase activity without phosphorylating myosin light chain"Proc. Natl. Acad. Sci. USA. 96. 6666-6671 (1999)
[Publications] H. Sakakibara, H. Kojima, Y. Sakai, et al: "Inner-arm dynein c of chlamydomonas flagella is a single-headed processive motor"Nature. 400. 586-590 (1999)
[Publications] A. Yamada, M. Yoshio, K. Oiwa, et al.: "Catchin, a novel protein in molluscan catch muscles, is produced by alterntive splicing from the myosin heavy chain gene"J. Mol. Biol.. 295. 169-178 (2000)
[Publications] S. Koyama, S. Kamimura: "Lowered sperm motility in subordinate social status of mice"Physiol. Behav.. 65. 665-669 (1999)
[Publications] T. Tani, S. Kamimura: "Dynein-ADP as a force generating intermediate revealed by a rapid reactivation of flagellar axoneme"Biophys. J.. 77. 1518-1527 (1999)
[Publications] M. L. W. Knetsch, T. Q. P. Uyeda, et al.: "Disturbed communication between actin- and nucleotide-binding sites in a myosin II with truncated 50/20-kDa junction"Journal of Biological Chemistry. 274. 20133-20138 (1999)
[Publications] S. Shu, R. J. Lee, J. M. LeBlanc-Straceki, et al.: "Role of myosin II tail sequences in its function and localization to the cleavage furrow"Journal of Cell Science. 112. 2195-2201 (1999)
[Publications] B. Patterson, T. Q. P. Uyeda: "構造遺伝学: 遺伝子内サプレッサー変異で構造と構造変化を解明する"細胞工学. 18. 1631-1640 (1999)
[Publications] Y. Fukui, T. Q. P. Uyeda, C. Kitayama, et al.: "Migration forces in dictyostelium measured by centrifugal DIC microscopy"Biological Bulletin. 197. 260-262 (1999)
[Publications] T. Q. P. Uyeda, S. Yumura: "Molecular biological approaches to study myosin functions in cytokinesis of Dictyostelium"Microscopy Research Technique. (in press). (2000)
[Publications] T. Q. P. Uyeda, C. Kitayama, S. Yumura: "Myosin II-independent cytokinesis in Dictyostelium its implications and mechanism"Cell Structure and Function. (in press). (2000)
[Publications] K. Mukherjee, H. Nagai, N. Shimamoto, et al.: "GroEL is involved in activation of Escherichia coli RNA polymerase devoid of omega subunit in vivo"Eur. J. Biochem.. 266. 228-235 (1999)
[Publications] N. Shimamoto: "One dimensional diffusion of proteins along DNA: its biological and chemical significance revealed by singlemolecule measurments"J. Biol. Chem.. 274. 15293-15296 (1999)
[Publications] R. Sen, H. Nagai, N. Shimamoto: "Polymerase-arrest at the lambdaPR promoter during transcription initiation"J. Biol. Chem.. 275(in press). (2000)
[Publications] 嶋本伸雄, 杵渕隆: "DNA上のタンパク質の滑り運動"DNA結合蛋白質の遠攻近交戦略科学. 69. 42-50 (1999)
[Publications] 嶋本伸雄: "分子生物学キーワード辞典田村秀明、饗場弘二編集"羊土社. (1999)

1999 Fiscal Year Annual Research Report

生物分子モーターの1分子計測と1分子操作

Principal Investigator

木下 一彦 慶應義塾大学, 理工学部, 教授 (30124366)

Research Products

[Publications] K. Adachi, K. Kinosita, Jr. , T. Ando: "Single-fluorophore imaging with an unmodified epifluorescence microscope and conventional video camera"J. Microscop. 195. 125-132 (1999)

[Publications] H. Miyata, S. Nishiyama, et al.: "Protrusive growth from giant liposomes driven by actin polymerization"Proc. Natl. Acad. Sci. USA. 96. 2048-2053 (1999)

[Publications] Y. Arai, R. Yasuda, K. Akashi, et al.: "Tying a molecular knot with optical tweezers"Nature. 399. 446-448 (1999)

[Publications] H. Kato, T. Nishizaka, T. Iga, et al.: "Imaging of thermal activation of actomyosin motors"Proc. Natl. Acad. Sci. USA. 96. 9602-9606 (1999)

[Publications] H. Noji, K. Hasler, W. Junge, et al.: "Rotation of Escherichia coli Fl-ATPase"Biochem. Biophys. Res. Commun.. 260. 597-599 (1999)

[Publications] K. Kinosita, Jr.: "Real time imaging of rotating molecular machines"FASEB J.. 13 Suppl. 2. S201-S208 (1999)

[Publications] H. Miyata, G. Marriott, K. Akashi, et al.: "Cell deformation mechanism studied with actin-containing giant vesicle, a cell mimicking system"Giant Vesicles, Chapter. 24. 320-333 (2000)

[Publications] K. Akashi, H. Miyata, K. Kinosita, Jr.: "Observation of a variety of giant vesicles under an optical microscope"Giant Vesicles, Chapter. 5. 46-48 (2000)

[Publications] K. Kinosita, Jr. , R. Yasuda, H. Noji: "Fl-ATPase; a highly effiicient rotary ATP machine"Essays Biochem. 35(in press). (2000)

[Publications] K. Kinosita, Jr. , R. Yasuda, H. Noji, K.: "A rotary molecular motor that can work at near 100% efficiency"Philos. Trans. Biol. Sci.. (in press). (2000)

[Publications] Y. Ishii, Y. Kimura, H. Tanaka, et al.: "Imaging and nano-manipulation of single actomyosin motors at work"Proceedings of the Australian Physiological and Pharmacological Society. 30. 24-39 (1999)

[Publications] Y. Ishii, Y. Kimura, K. Kitamua, et al.: "Imaging and nano-manipulation of single actomyosin motors at work"Clinical and Experimental Pharmacology and Physiology. (in press). (2000)

[Publications] Y. Miyamoto, E. Muto, T. Mashimo, et al.: "Direct inhibition of microtubule-based kinesin motility by local anesthetics"Biophys. J.. 78(in press). (2000)

[Publications] L-H. Ye, H. Kishi, A. Nakamura, et al.: "Myosin light-chain kinase of smooth muscle stimulates myosin ATPase activity without phosphorylating myosin light chain"Proc. Natl. Acad. Sci. USA. 96. 6666-6671 (1999)

[Publications] H. Sakakibara, H. Kojima, Y. Sakai, et al: "Inner-arm dynein c of chlamydomonas flagella is a single-headed processive motor"Nature. 400. 586-590 (1999)

[Publications] A. Yamada, M. Yoshio, K. Oiwa, et al.: "Catchin, a novel protein in molluscan catch muscles, is produced by alterntive splicing from the myosin heavy chain gene"J. Mol. Biol.. 295. 169-178 (2000)

[Publications] S. Koyama, S. Kamimura: "Lowered sperm motility in subordinate social status of mice"Physiol. Behav.. 65. 665-669 (1999)

[Publications] T. Tani, S. Kamimura: "Dynein-ADP as a force generating intermediate revealed by a rapid reactivation of flagellar axoneme"Biophys. J.. 77. 1518-1527 (1999)

[Publications] M. L. W. Knetsch, T. Q. P. Uyeda, et al.: "Disturbed communication between actin- and nucleotide-binding sites in a myosin II with truncated 50/20-kDa junction"Journal of Biological Chemistry. 274. 20133-20138 (1999)

[Publications] S. Shu, R. J. Lee, J. M. LeBlanc-Straceki, et al.: "Role of myosin II tail sequences in its function and localization to the cleavage furrow"Journal of Cell Science. 112. 2195-2201 (1999)

[Publications] B. Patterson, T. Q. P. Uyeda: "構造遺伝学: 遺伝子内サプレッサー変異で構造と構造変化を解明する"細胞工学. 18. 1631-1640 (1999)

[Publications] Y. Fukui, T. Q. P. Uyeda, C. Kitayama, et al.: "Migration forces in dictyostelium measured by centrifugal DIC microscopy"Biological Bulletin. 197. 260-262 (1999)

[Publications] T. Q. P. Uyeda, S. Yumura: "Molecular biological approaches to study myosin functions in cytokinesis of Dictyostelium"Microscopy Research Technique. (in press). (2000)

[Publications] T. Q. P. Uyeda, C. Kitayama, S. Yumura: "Myosin II-independent cytokinesis in Dictyostelium its implications and mechanism"Cell Structure and Function. (in press). (2000)

[Publications] K. Mukherjee, H. Nagai, N. Shimamoto, et al.: "GroEL is involved in activation of Escherichia coli RNA polymerase devoid of omega subunit in vivo"Eur. J. Biochem.. 266. 228-235 (1999)

[Publications] N. Shimamoto: "One dimensional diffusion of proteins along DNA: its biological and chemical significance revealed by singlemolecule measurments"J. Biol. Chem.. 274. 15293-15296 (1999)

[Publications] R. Sen, H. Nagai, N. Shimamoto: "Polymerase-arrest at the lambdaPR promoter during transcription initiation"J. Biol. Chem.. 275(in press). (2000)

[Publications] 嶋本 伸雄, 杵渕 隆: "DNA上のタンパク質の滑り運動"DNA結合蛋白質の遠攻近交戦略科学. 69. 42-50 (1999)

[Publications] 嶋本 伸雄: "分子生物学キーワード辞典 田村秀明、饗場弘二編集"羊土社. (1999)

木下一彦慶應義塾大学, 理工学部, 教授 (30124366)

[Publications] 嶋本伸雄, 杵渕隆: "DNA上のタンパク質の滑り運動"DNA結合蛋白質の遠攻近交戦略科学. 69. 42-50 (1999)

[Publications] 嶋本伸雄: "分子生物学キーワード辞典田村秀明、饗場弘二編集"羊土社. (1999)