1999 Fiscal Year Annual Research Report

蛋白質の核-細胞質間輸送の可視化

Research Project

Project/Area Number	11242203
Research Category	Grant-in-Aid for Scientific Research on Priority Areas (B)
Research Institution	Osaka University
Principal Investigator	今本尚子大阪大学, 医学系研究科, 助手 (20202145)
Keywords	核蛋白質輸送 / importinα / importinβ / 核局在化シグナル / 低分子量GTPase Ran / 分子イメージング
Research Abstract	細胞内には多様な輸送経路が存在し、その輸送経路に乗って膨大な数の分子が核一細胞質間を流通する。多くの輸送経路に乗った分子が、核膜孔上を滞りなく流通する仕組みを知るには、分子が核膜孔を通過する基本概念を得る必要がある。分子が核膜孔を通過する動きをみることで、通過反応のステップ数や、通過の速度を知ることができれば、そうした基本概念に対して、有用な情報を得ることができる。一分子が一つの核膜孔を通過する過程を可視化するための準備を現在進めている。SV40T抗原のNLSを結合したCy3標識IgMや、GFPで標識した輸送担体importinβを作製し、セミインタクト細胞を用いたin vitro核蛋白質輸送再構成系において、基質や輸送担体が核内に集積する過程を対物レンズ型全反射照明法で観察したとき、どのくらいの感度で、基質と輸送担体が核膜孔複合休にターゲットし、ターゲットした核膜孔複合体をどの程度視野にとらえることができるかを調べた。IgM-NLS基質は2.5nM(Cy3で25nM)で、GFP-importinβは70pMで核膜孔にターゲットして核内に移入した。この条件下で、IgM-NLSは一つの核あたり、約700個の核膜孔複合体に結合することが確認された。対物レンズ型全反射照明法で、高感度で核膜孔複合体をとらえることができることが確認されたが、同時に、分子の核膜孔通過反応が非常に早いことが予想された。動いている1分子をとらえるためには、蛍光強度が強く、均一な基質を作製する必要がある。

Research Products
(12 results)

All Other

All Publications (12 results)

[Publications] Kose, S.: "β-Subunit of nuclear pore-targeting complex (importin-β) can be exported from the nucleus in a Ran-independent manner"J. Biol. Chem.. 274. 3946-3952 (1999)
[Publications] Hieda, M.: "A monoclonal antibody to the C-terminal acidic portion of Ran inhibits both the recycling of Ran and nuclear protein import in living cells"J. Cell Biol.. 144. 645-655 (1999)
[Publications] Yokoya, F.: "β-Catenin can be transported in to the nucleus in a Ran-unassisted manner"Mol. Biol. Cell. 10. 1119-1131 (1999)
[Publications] Nagoshi, E.: "Nuclear import of sterol regulatory element-binding protein-2, a basic helix-loop-helix-leucine zipper (bHLH-Zip)-containing transcription factor occurs through the direct interaction of importin β with HTH-Zip"Mol. Biol. Cell. 10. 2221-2233 (1999)
[Publications] Sahara, S.: "Acinus, a caspase-3-activated protein responsible for apoptotic chromatin condensation"Nature. 401. 168-173 (1999)
[Publications] Ohshima, T.: "CRM1 mediates nuclear export of nonstructural protein 2 from parvovirus minute virus of mine"Biochem. Biophys. Res. Commun.. 264. 144-150 (1999)
[Publications] Kose, S.: "Distinct energy requirement for nuclear import and export of importin β in living cells"FEBS Lett.. 463. 327-330 (1999)
[Publications] Haraguchi, T.: "Live fluorescence imaging reveals early recruitment of emerin, LBR HP1β and RanBP2 to reforming functional nuclear envelope"J. Cell Sci.. 113. 779-794 (2000)
[Publications] Matsubae, M.: "Characterization of the nuclear transport of a novel leucine-rich acidic nuclear protein-like protein"FEBS Lett.. (in press).
[Publications] Tachibana, T.: "Recycling of importin α from the nucleus is suppressed by loss of RCC1 fuction in living mammalian cells"Cell Struct Funct.. (in press).
[Publications] 小瀬真吾: "実験医学"羊土社. 7 (1999)
[Publications] 横屋史彦: "秀潤社"細胞工学. 7 (1999)

1999 Fiscal Year Annual Research Report

蛋白質の核-細胞質間輸送の可視化

Principal Investigator

今本 尚子 大阪大学, 医学系研究科, 助手 (20202145)

Research Products

[Publications] Kose, S.: "β-Subunit of nuclear pore-targeting complex (importin-β) can be exported from the nucleus in a Ran-independent manner"J. Biol. Chem.. 274. 3946-3952 (1999)

[Publications] Hieda, M.: "A monoclonal antibody to the C-terminal acidic portion of Ran inhibits both the recycling of Ran and nuclear protein import in living cells"J. Cell Biol.. 144. 645-655 (1999)

[Publications] Yokoya, F.: "β-Catenin can be transported in to the nucleus in a Ran-unassisted manner"Mol. Biol. Cell. 10. 1119-1131 (1999)

[Publications] Nagoshi, E.: "Nuclear import of sterol regulatory element-binding protein-2, a basic helix-loop-helix-leucine zipper (bHLH-Zip)-containing transcription factor occurs through the direct interaction of importin β with HTH-Zip"Mol. Biol. Cell. 10. 2221-2233 (1999)

[Publications] Sahara, S.: "Acinus, a caspase-3-activated protein responsible for apoptotic chromatin condensation"Nature. 401. 168-173 (1999)

[Publications] Ohshima, T.: "CRM1 mediates nuclear export of nonstructural protein 2 from parvovirus minute virus of mine"Biochem. Biophys. Res. Commun.. 264. 144-150 (1999)

[Publications] Kose, S.: "Distinct energy requirement for nuclear import and export of importin β in living cells"FEBS Lett.. 463. 327-330 (1999)

[Publications] Haraguchi, T.: "Live fluorescence imaging reveals early recruitment of emerin, LBR HP1β and RanBP2 to reforming functional nuclear envelope"J. Cell Sci.. 113. 779-794 (2000)

[Publications] Matsubae, M.: "Characterization of the nuclear transport of a novel leucine-rich acidic nuclear protein-like protein"FEBS Lett.. (in press).

[Publications] Tachibana, T.: "Recycling of importin α from the nucleus is suppressed by loss of RCC1 fuction in living mammalian cells"Cell Struct Funct.. (in press).

[Publications] 小瀬真吾: "実験医学"羊土社. 7 (1999)

[Publications] 横屋史彦: "秀潤社"細胞工学. 7 (1999)

今本尚子大阪大学, 医学系研究科, 助手 (20202145)