Molecular mechanism of AAA-type ATPase dynein

• Project/Area Number: 16370069
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biophysics
• Research Institution: The University of Tokyo
• Principal Investigator: TOYOSHIMA Yoko Y. The University of Tokyo, Department of Life Sciences, Associate Professor, 大学院・総合文化研究科, 助教授 (40158043)
• Co-Investigator(Kenkyū-buntansha): EDAMATSU Masaki The University of Tokyo, Department of Life Sciences, Research Associate, 大学院・総合文化研究科, 助手 (60251328)
• Project Period (FY): 2004 – 2005
• Project Status: Completed (Fiscal Year 2005)
• Budget Amount: ¥11,200,000 (Direct Cost: ¥11,200,000); Fiscal Year 2005: ¥4,100,000 (Direct Cost: ¥4,100,000); Fiscal Year 2004: ¥7,100,000 (Direct Cost: ¥7,100,000)
• Keywords: Dynein / Molecular motor / Microtubules / Stalk / AAA motif / ATPase activity / ATP binding site / Microtubule binding site / ATP結合部

Research Abstract

Motility of cytoplasmic dynein has been studied using in vitro microtubule-gliding assay system. Single molecules of dynein stalk head, as well as the stalk fragment composed of stalk head and the coiled-coil region, were observed to attach to the microtubule, move on the microtubule, and then detach from the microtubule. Analysis of the movement revealed to be Brownian motion, suggesting that the binding force of the stalk fragments is weak. Native dynein molecules purified from porcine brain were visualized by binding of the recombinant p150 fragment which contained dynein binding region and fused with gelsolin. In the presence of ATP, single molecules of native dynein did not show processive movement long enough to detect with a regular speed video camera (30fps). However, they behaved differently depending on the nucleotide conditions used. The native dynein molecules sit firmly on microtubules in the presence of AMP-PNP or ADP, and also in the absence of nucleotides. Meanwhile, in the presence of ADP-Vi, they moved around along microtubules very similar way to the movement of the stalk fragments. These results suggest that single molecules of dynein weakly bind to and keep contact with microtubules in ADP-Pi state, and the isolated stalk fragments mimic this state. The microtubule binding fragments of dynactin p150 also showed Brownian motion along microtubules, and the lifetime on microtubules were much longer than those of the stalk fragments and the native dynein molecules. These results show that dynactin p150 can contribute to increase the processivity of dynein movement without disturbing the motility in dynein-dynactin complex.

Research Products

• [Journal Article] Microtubule-binding properties of dynactin p150 expedient for dynein motility. (2006)
  • Author(s): Kobayashi, T., Shiroguchi, K., Edamatsu, M., Toyoshima Y.Y.
  • Journal Title: Biochem. Biophys. Res. Commun. 340 Pages: 23-28
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Overlapping hand-over-hand mecanism of single molecular motility of cytoplasmic dynein (2006)
  • Author(s): Toba, S., Watanabe, T.M., Yamaguchi-Okimoto, L., Toyoshima, Y.Y., Higuchi, H.
  • Journal Title: Peoc. Natl. Acad. Sci. USA 103 Pages: 5741-5745
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Microtubule-binding properties of dynactin p150 expedient for dynein motility (2006)
  • Author(s): Kobayashi, T., Shiroguchi, K., Edamatsu, M., Toyoshima Y.Y.
  • Journal Title: Biochem.Biophys.Res.Commun. 340 Pages: 23-28
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Overlapping hand-over-hand mechanism of single molecular motility of cytoplasmic dynein (2006)
  • Author(s): Toba, S., Watanabe, T.M., Yamaguchi-Okimoto, L., Toyoshima, Y.Y., Higuchi, H.
  • Journal Title: Proc.Natl Acad.Sci.USA 103 Pages: 5741-5745
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Recruitment of Katanin P60 by Phosphorylated NDEL1, a LTS1 interacting protein, is Essential for Mitotic Cell Division and Neuronal Migration (2005)
  • Author(s): Toyo-oka, K., Shinji Sasaki, S., Yano, Y., Mori, D., Kobayashi, T., Toyoshima, Y.Y., Tokuoka, S.M., Ishii, S., Shimizu, T., Muramatsu, M., Hiraiwa, N., Yoshiki, A., Wynshaw-Boris, A., Hirotsune, S.
  • Journal Title: Hum.Mol.Genet. 14 Pages: 3113-3128
• [Journal Article] Removal of tightly bound ADP induces distinct structural changes of the two tryptophan-containing regions of the ncd motor domain (2005)
  • Author(s): Morii, H., Shimizu, T., Mizuno, N., Edamatsu, M., Ogawa, K., Shimizu, Y., Toyoshima, Y.Y.
  • Journal Title: J.Biochem. 138 Pages: 95-104
  • NAID: 10017346315
• [Journal Article] Properties of the full-length heavy chains of Tetrahymena ciliary outer arm dynein separated by urea treatment (2004)
  • Author(s): Toba, S., Gibson, T.M., Shiroguchi, K., Toyoshima, Y.Y., Asai, D.J.
  • Journal Title: Cell Motil. Cytoskeleton 58 Pages: 30-38
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] A single-headed recombinant fragment of Dictyostelium cyoplasmic dynein can drive the robust sliding of microtubules (2004)
  • Author(s): Nishiura, N., Kon, T., Shiroguchi, K., Ohkura, R., Shima, T., Toyoshima, Y.Y., Sutoh, K.
  • Journal Title: J. Biol. Chem. 279 Pages: 22799-22802
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Dynein and kinesin share an overlapping microtubule-binding site (2004)
  • Author(s): Miuno, N., Toba, S., Edamatsu, M., Watai-Nishii, J., Hirokawa, N., Toyoshima, Y.Y., Kikkawa, M.
  • Journal Title: EMBO J. 23 Pages: 2459-2467
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Dissociation of double-headed cytoplasmic dynein into single-headed species and its motile properties (2004)
  • Author(s): Toba, S., Toyoshima, Y.Y.
  • Journal Title: Cell Motil. Cytoskeleton 58 Pages: 281-289
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Distinct functions of nucleotide-binding/hydrolysis sites in the four AAA modules of cytoplasmic dynein (2004)
  • Author(s): Kon, T., Nishiura, M., Ohkura, R., Toyoshima, Y.Y., Sutoh, K.
  • Journal Title: Biochemistry 43 Pages: 11266-11274
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Multiple ATP-hydrolyzing sites that potentiallty function in cytoplasmic dynein (2004)
  • Author(s): Takahashi, Y., Edamatsu, M., Toyoshima, Y.Y.
  • Journal Title: Proc. Natl Acad. Sci. USA 101 Pages: 12865-12869
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Properties of the full-length heavy chains of Tetrahymena ciliary outer arm dynein separated by urea treatment (2004)
  • Author(s): Toba, S., Gibson, T.M., Shiroguchi, K., Toyoshima, Y.Y., Asai, D.J.
  • Journal Title: Cell Motil.Cytoskeleton 58 Pages: 30-38
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] A single-headed recombinant fragment of Dictyostelium cytoplasmic dynein can drive the robust sliding of microtubules (2004)
  • Author(s): Nishiura, N., Kon, T., Shiroguchi, K., Ohkura, R., Shima, T., Toyoshima, Y.Y., Sutoh, K.
  • Journal Title: J.Biol. Chem. 279 Pages: 22799-22802
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Dynein and kinesin share an overlapping microtubule-binding site (2004)
  • Author(s): Mizuno, N., Toba, S., Edamatsu, M., Watai-Nishii, J., Hirokawa, N., Toyoshima, Y.Y., Kikkawa, M.
  • Journal Title: EMBO J. 23 Pages: 2459-2467
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Dissociation of double-headed cytoplasmic dynein into single-headed species and its motile properties (2004)
  • Author(s): Toba, S., Toyoshima, Y.Y.
  • Journal Title: Cell Motil.Cytoskeleton 58 Pages: 281-289
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Multiple ATP-hydrolyzing sites that potentiality function in cytoplasmic dynein. (2004)
  • Author(s): Takahashi, Y., Edamatsu, M., Toyoshima, Y.Y.
  • Journal Title: Proc.Natl Acad.Sci.USA 101 Pages: 12865-12869
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Properties of the full-length heavy chains of Tetrahymena ciliary outer arm dynein separated by urea treatment. (2004)
  • Author(s): Toba, S., Gibson, T.M., Shiroguchi, K., Toyoshima, Y.Y., Asai, D.J.
  • Journal Title: Cell Motil.Cytoskeleton 58 Pages: 30-38
• [Journal Article] A single-headed recombinant fragment of Dictyostelium cytoplasmic dynein can drive the robust sliding of microtubules. (2004)
  • Author(s): Nishiura, N., Kon, T., Shiroguchi, K., Ohkura, R., Shima, T., Toyoshima, Y.Y., Sutoh, K.
  • Journal Title: J.Biol.Chem. 279 Pages: 22799-22802
• [Journal Article] Dynein and kinesin share an overlapping microtubule-binding site. (2004)
  • Author(s): Mizuno, N., Toba, S., Edamatsu, M., Watai-Nishii, J., Hirokawa, N., Toyoshima, Y.Y., Kikkawa, M.
  • Journal Title: EMBO J. 23 Pages: 2459-2467
• [Journal Article] Dissociation of double-headed cytoplasmic dynein into single-headed species and its motile properties. (2004)
  • Author(s): Toba, S., Toyoshima, Y.Y.
  • Journal Title: Cell Motil.Cytoskeleton 58 Pages: 281-289
• [Journal Article] Distinct functions of nucleotide-binding/hydrolysis sites in the four AAA modules of cytoplasmic dynein. (2004)
  • Author(s): Kon, T., Nishiura, M., Ohkura, R., Toyoshima, Y.Y., Sutoh, K.
  • Journal Title: Biochemistry 43 Pages: 11266-11274
• [Journal Article] Multiple ATP-hydrolyzing sites that potentiallty function in cytoplasmic dynein. (2004)
  • Author(s): Takahashi, Y., Edamatsu, M., Toyoshima, Y.Y.
  • Journal Title: Proc.Natl.Acad.Sci.USA 101 Pages: 12865-12869