Analysis of nuclear pore translocation mechanism : single molecule analysis and biochemical approach
| Project/Area Number |
15370090
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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 |
Cell biology
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| Research Institution | RIKEN |
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Principal Investigator |
IMAMOTO Naoko RIKEN, Cellular Dynamics Laboratory, Chief Scientist, 今本細胞核機能研究室, 主任研究員 (20202145)
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| Co-Investigator(Kenkyū-buntansha) |
KOSE Shingo RIKEN, Cellular Dynamics Laboratory, Research Scientist, 今本細胞核機能研究室, 研究員 (90333278)
KOIKE Makiko RIKEN, Cellular Dynamics Laboratory, Contract Technical Scientist, 今本細胞核機能研究室, 協力研究員 (30391949)
谷口 直子 大阪大学, 大学院・生命機能研究科・神経可塑性生理学研究室, 特任教員(COE) (90360586)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥15,400,000 (Direct Cost: ¥15,400,000)
Fiscal Year 2004: ¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 2003: ¥8,700,000 (Direct Cost: ¥8,700,000)
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| Keywords | nuclear transport / single molecule imaging / nuclear pore comlex / importin β / semi-intact cells / 70kDa heat-shock cognate protein / Importinβ / 低分子量GTPase Ran / β-カテニン / 核局在化シグナル / 核膜孔複合体構成因子 / importin β / 輸送担体 |
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| Research Abstract |
Using novel microscopy based on total internal reflection fluorescent microscopy (TIRF), we were able to clearly visualize single fluorescent molecules of green fluorescent protein (GFP)-tagged importin β, a carrier protein, and GFP-tagged cargo protein during transport on the nuclear envelope in both permeabilized cells and in living cells. Image analysis of single nuclear pores showed that two point resolution of 70 nm was achieved. Kinetic parameters of the interactions between translocating molecules and NPCs were obtained through quantitative analysis. Two types of binding were found ; weaker binding sites, which gathers up to 〜100 molecules/NPC, concentrating substrates locally ; and stronger binding sites, where up to 〜8 molecules/NPC are bound. Retention times by single molecule analysis and translocation rates by single nuclear analysis showed a significant correlation with a coefficient of 〜8 molecules/NPC, which exhibits the stoichiometry of transport. We termed weaker binding sites as a "multiplex sites", which is likely to consist from FG-repeat containing nucleoporins. Based on correlation coefficient, we proposed presence of 8 "transit site" in nuclear pore complex. Whether this transit site is equivalent to strong binding sites observed remains to be elucidated in the further study. Whether the findings account for different pathways, which show a wide range of transport rates, also remains to be elucidated. In the parallel experiments, we have purified, and identified 70kDa heat-shock cognate protein (hsc70) as a molecule that facilitates recycling of importin β. The effect of hsc70 was observed, not only in the case of the nuclear export of importin β but also for that of another import receptors, transportin and importin α. These results suggest that hsc70 broadly modulates nucleocytoplasmic transport systems by regulating nuclear export of receptor proteins (Kose et al., J Cell Biol. 2005).
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Report
(3 results)
Research Products
(45 results)
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[Journal Article] Dissociation of heterochromatin protein 1 from lamin B receptor induced by human polyomavirus agnoprotein : role nuclear egress of viral particles2005
Author(s)
Yuki Okada, Tadaki Suzuki, Yuji Sunden, Yasuko Orba, Shingo Kose, Noko Imamoto, Hidehiro Takahashi, Shinya Tanaka, William W.Hall, Kazuo Nagashima, Hirofumi Sawa
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Journal Title
EMBO Rep. 6
Pages: 452-457
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] The structure of importin-β bound to SREBP-2 : Nuclear import of a transcription factor2003
Author(s)
Soo Jae Lee, Toshihiro Sekimoto, Eiki Yamashita, Emi Nagoshi, Atsushi Nakagawa, Naoko Imamoto, Masato Yoshimura, Hiroaki Sakai, Khoon Tee Chong, Tomitake Ysukihara, Yoshihiro Yoneda
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Journal Title
Science 302
Pages: 1571-1575
Description
「研究成果報告書概要(欧文)」より
Related Report
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