Defining the mechanisms of equal-sized cell division using advanced genetic and optogenetic technologies

• Project/Area Number: 17H05002
• Research Category: Grant-in-Aid for Young Scientists (A)
• Allocation Type: Single-year Grants
• Research Field: Cell biology
• Research Institution: Okinawa Institute of Science and Technology Graduate University (2020); Nagoya University (2017-2019)
• Principal Investigator: Kiyomitsu Tomomi 沖縄科学技術大学院大学, 細胞分裂動態ユニット, 准教授 (10503443)
• Project Period (FY): 2017-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥26,260,000 (Direct Cost: ¥20,200,000、Indirect Cost: ¥6,060,000); Fiscal Year 2020: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000); Fiscal Year 2019: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2018: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000); Fiscal Year 2017: ¥11,700,000 (Direct Cost: ¥9,000,000、Indirect Cost: ¥2,700,000)
• Keywords: 細胞分裂 / 紡錘体 / 光操作 / オーキシン誘導デグロン / NuMA / Ran-GTP / ダイニン / 等分裂 / dynein / 紡錘体配置 / 光遺伝学

Outline of Final Research Achievements

Mitotic spindle assembly and positioning are required for equal-sized division. In this study, we analyzed their mechanisms using CRISPR/Cas9, auxin-inducible degron (AID), and optogenetic technologies. Using optogenetic manipulation of NuMA, we identified the functional unit of the cortical force-generating machinery which controls spindle position in human cells. In addition, we improved our understanding of mitotic Ran-GTP functions for spindle assembly using AID. We published these studies in eLife and Current Biology, respectively.

Academic Significance and Societal Importance of the Research Achievements

多細胞生物の発生過程において、細胞は「紡錘体の細胞内配置」を巧みに制御することによって、細胞内外の極性因子の分配や娘細胞のサイズを決め、多様な組織を形成している。紡錘体の配置は、細胞表層に構成される紡錘体牽引装置によって制御されるが、本研究によって、その機能中枢は、Dynein-Dynactin-NuMAの集合体であることを明らかにした。また紡錘体の配置を光で操作することにも成功したため、今後、細胞分裂の対称性・非対称性制御の理解を進めることができる。またAID法を用いて、分裂期特異的に標的タンパクを分解する系の樹立にも成功した。本成果は、今後様々な紡錘体関連因子の機能の理解に貢献できる。

Research Products

• [Journal Article] Ran-GTP Is Non-essential to Activate NuMA for Mitotic Spindle-Pole Focusing but Dynamically Polarizes HURP Near Chromosomes (2021)
  • Author(s): Tsuchiya Kenta、Hayashi Hisato、Nishina Momoko、Okumura Masako、Sato Yoshikatsu、Kanemaki Masato T.、Goshima Gohta、Kiyomitsu Tomomi
  • Journal Title: Current Biology Volume: 31 Issue: 1 Pages: 115-127.e3
  • DOI: 10.1016/j.cub.2020.09.091
  • Peer Reviewed / Open Access
• [Journal Article] Importin-β targets HURP to kinetochore-fibers in coordination with Ran-GTP in human mitotic cells (2018)
  • Author(s): Tsuchiya K, Hayashi H, Nishina M, Okumura M, Kanemaki MT, Goshima G, Kiyomitsu T
  • Journal Title: bioRxiv Volume: 1 Pages: 1-31
  • DOI: 10.1101/473538
  • Open Access
• [Journal Article] Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble (2018)
  • Author(s): Okumura Masako、Natsume Toyoaki、Kanemaki Masato T、Kiyomitsu Tomomi
  • Journal Title: eLife Volume: 7 Pages: 1-24
  • DOI: 10.7554/elife.36559
  • Peer Reviewed / Open Access
• [Presentation] Mechanisms of Ran-based mitotic spindle assembly revealed by auxin-mediated rapid protein knock-down (2020)
  • Author(s): Tomomi Kiyomitsu
  • Organizer: The 43rd Annual Meeting of the Molecular Biology Society of Japan
  • Int'l Joint Research / Invited
• [Presentation] The development of mitosis-specific rapid protein-degradation assays in human cells (2019)
  • Author(s): Tomomi Kiyomitsu
  • Organizer: EMBO/EMBL symposium, Seeing is Believing
  • Int'l Joint Research
• [Presentation] 光操作と急速分解、二刀流でダイニンの分裂期機能に迫る (2019)
  • Author(s): 清光智美
  • Organizer: 分子モーター討論会
  • Invited
• [Presentation] Mechanisms of dynein-based force generation at the cell cortex and spindle poles during metaphase (2018)
  • Author(s): Kiyomitsu T
  • Organizer: The 41st Annual Meeting of the Molecular Biology Society of Japan
  • Int'l Joint Research / Invited
• [Presentation] Optogenetic reconstitution reveals that Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble. (2018)
  • Author(s): Kiyomitsu T
  • Organizer: EMBO microtubule meeting
  • Int'l Joint Research
• [Book] 実験医学-クローズアップ実験法 (2019)
  • Author(s): 清光智美
  • Publisher: 羊土社
• [Remarks] 生命の核心を探る細胞分裂研究
  • URL: https://www.oist.jp/ja/news-center/news/2021/2/3/35890
• [Remarks] 世界初! 光で細胞分裂装置の操作に成功~体作りに重要な細胞分裂の仕組みの理解促進に期待~
  • URL: http://www.nagoya-u.ac.jp/about-nu/public-relations/researchinfo/upload_images/20180709_sci_1.pdf