Generation of atomic model of more intact doublet microtubule structure

• Project/Area Number: 20K15733
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 43020:Structural biochemistry-related
• Research Institution: Nara Institute of Science and Technology
• Principal Investigator: Ichikawa Muneyoshi 奈良先端科学技術大学院大学, 先端科学技術研究科, 助教 (80844662)
• Project Period (FY): 2020-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2021: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000); Fiscal Year 2020: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000)
• Keywords: ダイニン / 繊毛 / クライオ電子顕微鏡法 / モータータンパク質 / クライオ電顕 / X線結晶構造解析 / 微小管 / クライオ電子顕微鏡

Outline of Research at the Start

繊毛の基本構造であるダブレット微小管は、そのチューブリン格子の内側及び外側に多くのタンパク質が結合している。近年、繊毛のダブレット微小管のチューブリン格子構造とその内側に結合したタンパク質の近原子分解能の立体構造が得られたが、その原子モデルは未だ得られていない。また、ダブレット微小管の外側に結合しているタンパク質の構造は低分解能でしか得られていない。そこで、本研究では、外側のタンパク質複合体を保持した、より生体内の状態に近いダブレット微小管の構造を、クライオ電子顕微鏡法や、X線結晶構造解析などの手法を用いて原子分解能で得ることで、その構築・機能についての知見を得る。

Outline of Final Research Achievements

Cilia are tiny hair-like structures that drive cell motility. There are nine doublet microtubules in cilia, and axonemal dynein, a motor protein, is periodically bound to these doublet microtubules. Among axonemal dynein, outer-arm dynein is the main motor that produces ciliary motion. Outer arm dynein is a complex composed of many subunits, but the details of the structure of outer arm dynein bound to doublet microtubules were to be eluciated. In this study, subunit structure of the outer-arm dynein complex bound to the doublet microtubule was obtained by cryo-electron microscopy. Furthermore, by comparing the structure of the outer arm dynein complex with that of the inactive outer arm dynein complex before its incorporation into the cilia, the activation mechanisms of outer arm dynein was revealed.

Academic Significance and Societal Importance of the Research Achievements

繊毛は、ヒトの体内のほぼ全ての細胞に存在しており、繊毛タンパク質の異常は、内蔵逆位や不妊などヒトにおける繊毛病の症状を引き起こすことも知られている。そのため、繊毛の運動機構の理解は重要な課題である。本研究において繊毛内の外腕ダイニンの立体構造及び活性化機構を解明できたことは、これらの繊毛病の病態の理解に重要な基盤となる。また、本研究において明らかになった外腕ダイニンの高分解能の構造は、今後、外腕ダイニンを改変し、新たなナノデバイスを開発することにも応用できるだろう。

Research Products

• [Int'l Joint Research] McGill大学(カナダ)
• [Int'l Joint Research] McGill大学(カナダ)
• [Journal Article] Remodeling and activation mechanisms of outer arm dyneins revealed by cryo‐EM (2021)
  • Author(s): Kubo Shintaroh、Yang Shun Kai、Black Corbin S、Dai Daniel、Valente-Paterno Melissa、Gaertig Jacek、Ichikawa Muneyoshi、Bui Khanh Huy
  • Journal Title: EMBO reports Volume: 22 Issue: 9
  • DOI: 10.15252/embr.202152911
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Crystal structure of human PACRG in complex with MEIG1 reveals roles in axoneme formation and tubulin binding (2021)
  • Author(s): Nimra Khan, Dylan Pelletier, Thomas S. McAlear, Nathalie Croteau, Simon Veyron, Andrew N. Bayne, Corbin Black, Muneyoshi Ichikawa, Ahmad Abdelzaher Zaki Khalifa, Sami Chaaban, Igor Kurinov, Gary Brouhard, Susanne Bechstedt, Khanh Huy Bui, Jean-Francois Trempe
  • Journal Title: Structure Volume: 29 Issue: 6 Pages: 1-15
  • DOI: 10.1016/j.str.2021.01.001
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] The Complex of Outer-arm Dynein Light Chain-1 and the Microtubule-binding Domain of the Heavy Chain Shows How Axonemal Dynein Tunes Ciliary Beating (2021)
  • Author(s): Toshiki Yagi, Akiyuki Toda, Muneyoshi Ichikawa, Genji Kurisu
  • Journal Title: Seibutsu Butsuri Volume: 61 Issue: 1 Pages: 020-022
  • DOI: 10.2142/biophys.61.020
  • NAID: 130007976664
  • ISSN: 0582-4052, 1347-4219
  • Peer Reviewed / Open Access
• [Journal Article] Identification and mapping of central pair proteins by proteomic analysis (2020)
  • Author(s): Daniel Dai, Muneyoshi Ichikawa, Katya Peri, Reid Rebinsky, Khanh Huy Bui
  • Journal Title: Biophysics and Physicobiology Volume: 17 Issue: 0 Pages: 71-85
  • DOI: 10.2142/biophysico.bsj-2019048
  • NAID: 130007879483
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] クライオ電顕で明らかになった外腕ダイニンの構造変化と活性化機構 (2021)
  • Author(s): Kubo Shintaroh、Yang Shun Kai、Black Corbin S、Dai Daniel、Valente-Paterno Melissa、Gaertig Jacek、Ichikawa Muneyoshi、Bui Khanh Huy
  • Organizer: 第59会生物物理学会年会
• [Presentation] Remodeling and activation mechanisms of the outer arm dynein complex. (2021)
  • Author(s): Kubo Shintaroh、Yang Shun Kai、Black Corbin S、Dai Daniel、Valente-Paterno Melissa、Gaertig Jacek、Ichikawa Muneyoshi、Bui Khanh Huy
  • Organizer: Dynein 2021
  • Int'l Joint Research
• [Presentation] How Rib43a and Acetylation of K40 control the rigidity of Microtubules (2021)
  • Author(s): Shintaroh Kubo, Ahmad Khalifa, Muneyoshi Ichikawa, Daniel Dai, Corbin Black, Katya Peri, Shun Kai Yang, Jaiver Vargas, Khanh-Huy Bui
  • Organizer: 65th Biophysical Society Annual Meeting
  • Int'l Joint Research
• [Presentation] Remodeling and activation mechanisms of outer arm dynein revealed by cryo-EM (2021)
  • Author(s): Muneyoshi Ichikawa
  • Organizer: 18th BSCB GenSoc UK Cilia Network e-symposium
  • Int'l Joint Research / Invited
• [Presentation] How Rib43a and Acetylation of K40 control the rigidity of Microtubules (2020)
  • Author(s): Shintaroh Kubo, Ahmad Khalifa, Muneyoshi Ichikawa, Daniel Dai, Corbin Black, Katya Peri, Shun Kai Yang, Jaiver Vargas, Khanh-Huy Bui
  • Organizer: EMBO WORKSHOP
  • Int'l Joint Research
• [Presentation] ダブレット微小管上での外腕ダイニンの高分解能構造 (2020)
  • Author(s): 市川宗厳
  • Organizer: 第10回分子モーター討論会
• [Presentation] Cryo-EM revealed unique and diverse binding schemes of the microtubule inner proteins at the inner junction region of cilia (2020)
  • Author(s): Muneyoshi Ichikawa, Ahmad Khalifa, Daniel Dai, Shintaroh Kubo, Corbin Black, Katya Peri, Thomas McAlear, Simon Veyron, Shun Kai Yang, Javier Vargas, Susanne Bechstedt, Jean-Francois Trempe, Khanh Huy Bui
  • Organizer: 第58回日本生物物理学会年会
• [Presentation] Characterization of properties of microtubule inner protein FAP85. (2020)
  • Author(s): Yoshitoki Shibao, Corbin Black, Muneyoshi Ichikawa, Junya Kirima, Kazuhiro Oiwa, Khanh Huy Bui, Tomoya Tsukazaki
  • Organizer: 第58回日本生物物理学会年会
• [Presentation] クライオ電顕によるダブレット微小管の近原子分解能構造 (2020)
  • Author(s): Muneyoshi Ichikawa, Ahmad Khalifa, Daniel Dai, Shintaroh Kubo, Corbin Black, Katya Peri, Thomas McAlear, Simon Veyron, Shun Kai Yang, Javier Vargas, Susanne Bechstedt, Jean-Francois Trempe, Khanh Huy Bui
  • Organizer: 日本顕微鏡学会 第76回学術講演会 日本－カナダ 二国間交流シンポジウム
  • Int'l Joint Research / Invited