AID-mediated acute protein degradation system in human mitotic cells
| Project/Area Number |
16K14721
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Multi-year Fund |
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| Research Field |
Cell biology
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| Research Institution | Nagoya University |
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Principal Investigator |
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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Project Status |
Completed (Fiscal Year 2018)
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| Budget Amount *help |
¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
Fiscal Year 2017: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2016: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
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| Keywords | オーキシン誘導デグロン法 / 分裂期特異的分解 / Ran-GTP濃度勾配 / RCC1 / NuMA / HURP / オーキシン誘導デグロン / Ran-GTP / AID / ダイニン / Ran / ゲノム編集 / 細胞生物 |
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| Outline of Final Research Achievements |
Compared to DNA- or RNA-based depletion methods, acute degradation of proteins is more useful to understand protein functions in rapid biological processes such as mitosis. In this study, we sought to degrade target proteins within 30 min by combining auxin-mediated degradation (AID) technology and CRISPR/Cas-mediated genome editing in human cells. Importantly, we succeeded in establishing 3 AID cell lines for RCC1 (Ran GEF), RanGAP1, and importin-beta. Furthermore, we revealed that NuMA is not substantially affected by Ran-based network, whereas HURP is dynamically polarized and maintained on k-fibers near chromosomes by chromosome-derived Ran-GTP gradient (Tsuchiya et al., submitted). In addition, we demonstrated that clustering activity of NuMA is dispensable for spindle-pole focusing, but indispensable for spindle positioning/orientation via astral microtubule capture/pulling using AID-based replacement (Okumura et al., eLife 2018).
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| Academic Significance and Societal Importance of the Research Achievements |
これまで任意の標的タンパク質を分裂期に特異的に30分以内で急速に分解できる実験系は存在しなかった。この実験系の汎用性は極めて高く、核内外輸送因子Ran以外にも、分子モーターダイニンを含む様々な多機能タンパク質に応用でき、これらの鍵分子の特定のステージの機能を明らかにすることが期待できる。またRanによる制御システムは、ヒト卵母細胞において紡錘体形成に極めて重要な働きを示すが、体細胞ではいかに機能するか十分に検討されていなかった。本研究により、ヒト体細胞では、Ranの紡錘体形成機能は卵母細胞ほど優勢ではなく、並行する別経路がNuMA等の紡錘体形成因子を活性化する可能性も示唆された。
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Report
(4 results)
Research Products
(3 results)
