2007 Fiscal Year Final Research Report Summary
Working Machinery of Dynein Molecules
| Project/Area Number |
18370059
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | The University of Tokyo |
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Principal Investigator |
TOYOSHIMA Yoko The University of Tokyo, Department of Life Sciences, Professor (40158043)
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| Project Period (FY) |
2006 – 2007
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| Keywords | dynein / microtubule / optical tweezers / single molecule measurement / unbinding force / protofilament / GFP / AMPPNP |
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| Research Abstract |
Dynein is a huge and complicated molecule and minus-end directed motor of microtubules. To elucidate its machinery as a motor protein, we measured the molecular behavior of single dynein molecules focusing of the interaction with microtubules. Because dynein motor domain is as large as about 15nm in diameter, which is twice of the size of tubulin heterodimer repeat, it is not clear whether dynein uses only a single protofilament or multiple protofilaments. To answer this question, we exploited the property of zinc-induced tubulin sheets that the motor binding sites are only exposed at either edge of the sheet. Previous study has shown that multiple dynein molecules on glass surface can glide zinc-sheets. However, it is possible that multiple dynein molecules interact with the sheets and the gliding doesn't necessarily mean that single dynein molecule can walk on a single protofilament processively. In this study, we investigated the single molecule motility assay on zinc-sheets by TIRF
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M. We tethered the zinc-sheets on the glass using antibody against tubulin or MAPs, so that the dynein binding site is exposed in the solution. To observe single dynein molecules, GFP-tagged yeast Cytoplasmic dynein was prepared. We successfully observed that the GFP-tagged dynein moves processively on zinc-sheet as well as on microtubules. However, the velocity on zinc-sheet was about half of that on microtubules. These observations suggest that two-headed dynein molecule is able to move on single protofilament processively, but it can move more effectively on the multiple protofilaments. We next measured the unbinding force of single-headed dynein by newly developed quick stretch type experiment. The results suggested that the unbinding force was high in no nucleotide or AMPPNP and low in ADP-Vi or ADP, and also polarity dependent; the binding was strong against the pulling force from the plus-end and weak against that from the minus-end. These results showed that rear head within the double-headed structure of dynein is detach faster than the front head, which enable the processive movement of dynein molecules. Less
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[Journal Article] NDEL1 phosphorylation by Aurora-A kinase is essential for centrosomal maturation, separation, and TACC3 recruitment2007
Author(s)
Mori D, Yano Y, Toyo-oka K, Yoshida N, Yamada M, Muramatsu M, Zhang D, Saya H, Toyoshima YY, Kinoshita K, Wynshaw-Boris A, Hirotsune S.
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Journal Title
Mol Cell Biol 27
Pages: 352-367
Description
「研究成果報告書概要(和文)」より
Peer Reviewed
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[Presentation] Fluctuation analysis of micorutubule sliding over kinesin thick filaments in vitro.2006
Author(s)
Hirata, D., Taba, T., Edamatsu, M., Toyoshima, Y.Y.、 Yamada, A., Imafuku, Y., & Tawada, K.
Organizer
Fifth East Asian Biophysics Symposium & 44th Annual Meeting of the Biophysical Society of Japan
Place of Presentation
Okinawa, Japan
Year and Date
20061112-16
Description
「研究成果報告書概要(和文)」より
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[Presentation] Fluctuation analysis of micorutubule sliding over kinesin thick filaments in vitro2006
Author(s)
Hirata, D., Taba, T., Edamatsu, M., Toyoshima, Y. Y., Yamada, A., Imafuku, Y., Tawada, K.
Organizer
Fifth East Asian Biophysics Symposium & 44th Annual Meeting of the Biophysical Society of Japan
Place of Presentation
Okinawa, Japan
Year and Date
20061112-16
Description
「研究成果報告書概要(欧文)」より
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