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Cross-scale in-cell cryo-electron tomography and its application to epithelium

Planned Research

Project AreaNew cross-scale biology
Project/Area Number 21H05248
Research Category

Grant-in-Aid for Transformative Research Areas (A)

Allocation TypeSingle-year Grants
Review Section Transformative Research Areas, Section (III)
Research InstitutionThe University of Tokyo

Principal Investigator

吉川 雅英  東京大学, 大学院医学系研究科(医学部), 教授 (80272425)

Project Period (FY) 2021-09-10 – 2026-03-31
Project Status Granted (Fiscal Year 2025)
Budget Amount *help
¥163,540,000 (Direct Cost: ¥125,800,000、Indirect Cost: ¥37,740,000)
Fiscal Year 2025: ¥28,470,000 (Direct Cost: ¥21,900,000、Indirect Cost: ¥6,570,000)
Fiscal Year 2024: ¥28,990,000 (Direct Cost: ¥22,300,000、Indirect Cost: ¥6,690,000)
Fiscal Year 2023: ¥28,340,000 (Direct Cost: ¥21,800,000、Indirect Cost: ¥6,540,000)
Fiscal Year 2022: ¥29,120,000 (Direct Cost: ¥22,400,000、Indirect Cost: ¥6,720,000)
Fiscal Year 2021: ¥48,620,000 (Direct Cost: ¥37,400,000、Indirect Cost: ¥11,220,000)
Keywordsクライオ電子線トモグラフィー / Cryo-FIB-SEM / 形状標識 / CLEM / FIB-SEM / 上皮 / 繊毛 / 標識
Outline of Research at the Start

本領域の推進する「クロススケール細胞計測センター」の中で、我々のグループはクライオ電子線トモグラフィー(Cryo-ET)を、他の班の技術と統合することにより、細胞内のメゾ複雑体を含むクロススケール計測を可能とする技術開発を行う。これによって、メゾ複雑体の初期状態→遷移状態→最終状態のスナップショットを高解像度で可視化して、細胞内におけるタンパク質分子の分布・構造・状態を解明できると考える。

Outline of Annual Research Achievements

本研究では、クライオ電子線トモグラフィーを用い上皮細胞の形態形成のメカニズムを分子レベルから解明する。上皮細胞は、外界と臓器の「インターフェー ス」に当たり、臓器の機能に応じて様々な形態を取る。本提案では、これまで我々が研究してきた繊毛に加え、小腸の微絨毛や耳の不動毛など、これまで全く未開拓であった脊椎動物の上皮組織を対象とする。この目的のために、クライオ電子顕微鏡用の新しい細胞内標識「形状ラベル」を開発・応用することで、上皮の 形態形成のメカニズムを解明する。
本年度は、繊毛の構造解析、特にゼブラフィッシュを用いてカルシウムによる繊毛の制御機構、クラミドモナスを用いて中心対微小管の構造を解明した結果を論文として投稿した。前者はeLifeにて出版され、また、後者はrevision中である。ゼブラフィッシュを用いたカルシウムによる影響については、mutantを用いて生理学的な解析を行い、続報として投稿した。この論文も、現在revision中である。また、公募班の小田グループと共同で、膀胱上皮の構造解析と繊毛に於けるチューブリンC末端修飾に関する論文を出版した。計画班の稲葉グループとは、ゴルジ体で働く亜鉛トランスポーターの構造解析の論文を出版した。
さらに上皮に関する構造解析を行うためにFocused-Ion-Beamを用いたラメラの作成技術をハイスループット化したので、それらを用いて2024年度は上皮の構造をクライオ電子線トモグラフィーを用いて高解像度で解析していく予定である。

Current Status of Research Progress
Current Status of Research Progress

1: Research has progressed more than it was originally planned.

Reason

概要・実績にあるように、2023年度中に7報の論文を出版できたこと、さらに現在reivision中の論文も数多くある事から、計画以上に進展していると判断した。

Strategy for Future Research Activity

新たに開発しつつある形状ラベルや、ハイスループット化したFIB-SEMを用いて新たな上皮構造の解明を進める。また、2024年度に加わった公募班とも積極的に共同研究を進め、論文として投稿する。

Report

(3 results)
  • 2023 Annual Research Report
  • 2022 Annual Research Report
  • 2021 Annual Research Report
  • Research Products

    (58 results)

All 2024 2023 2022 2021 Other

All Int'l Joint Research (1 results) Journal Article (10 results) (of which Peer Reviewed: 8 results,  Open Access: 8 results) Presentation (46 results) (of which Int'l Joint Research: 21 results,  Invited: 11 results) Remarks (1 results)

  • [Int'l Joint Research] Purdue University(米国)

    • Related Report
      2023 Annual Research Report
  • [Journal Article] The role of the C-terminal tail region as a plug to regulate XKR8 lipid scramblase2024

    • Author(s)
      Sakuragi Takaharu、Kanai Ryuta、Otani Mayumi、Kikkawa Masahide、Toyoshima Chikashi、Nagata Shigekazu
    • Journal Title

      Journal of Biological Chemistry

      Volume: 300 Issue: 3 Pages: 105755-105755

    • DOI

      10.1016/j.jbc.2024.105755

    • Related Report
      2023 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Calaxin stabilizes the docking of outer arm dyneins onto ciliary doublet microtubule in vertebrates2023

    • Author(s)
      Yamaguchi Hiroshi、Morikawa Motohiro、Kikkawa Masahide
    • Journal Title

      eLife

      Volume: 12

    • DOI

      10.7554/elife.84860

    • Related Report
      2023 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] ATP‐Responsive Nanoparticles Covered with Biomolecular Machine “Chaperonin GroEL”2023

    • Author(s)
      Shen Hao K.、Morishita Kiyoshi、Hashim P. K.、Okuro Kou、Kashiwagi Daiki、Kimura Ayumi、Yanagisawa Haruaki、Kikkawa Masahide、Niwa Tatsuya、Taguchi Hideki、Aida Takuzo
    • Journal Title

      Angewandte Chemie International Edition

      Volume: 62 Issue: 31

    • DOI

      10.1002/anie.202304894

    • Related Report
      2023 Annual Research Report
    • Peer Reviewed
  • [Journal Article] α- and β-tubulin C-terminal tails with distinct modifications are crucial for ciliary motility and assembly2023

    • Author(s)
      Kubo Tomohiro、Tani Yuma、Yanagisawa Haru-Aki、Kikkawa Masahide、Oda Toshiyuki
    • Journal Title

      Journal of Cell Science

      Volume: 136 Issue: 16

    • DOI

      10.1242/jcs.261070

    • Related Report
      2023 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Cryo-EM structures of human zinc transporter ZnT7 reveal the mechanism of Zn2+ uptake into the Golgi apparatus2023

    • Author(s)
      Bui Han Ba、Watanabe Satoshi、Nomura Norimichi、Liu Kehong、Uemura Tomoko、Inoue Michio、Tsutsumi Akihisa、Fujita Hiroyuki、Kinoshita Kengo、Kato Yukinari、Iwata So、Kikkawa Masahide、Inaba Kenji
    • Journal Title

      Nature Communications

      Volume: 14 Issue: 1 Pages: 4770-4770

    • DOI

      10.1038/s41467-023-40521-5

    • Related Report
      2023 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] A quest to unravel the role of the stalk and microtubule‐binding domain of axonemal dyneins2023

    • Author(s)
      Kikkawa Masahide
    • Journal Title

      FEBS Letters

      Volume: 597 Issue: 17 Pages: 2147-2148

    • DOI

      10.1002/1873-3468.14707

    • Related Report
      2023 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Cryo-EM elucidates the uroplakin complex structure within liquid-crystalline lipids in the porcine urothelial membrane2023

    • Author(s)
      Yanagisawa Haruaki、Kita Yoshihiro、Oda Toshiyuki、Kikkawa Masahide
    • Journal Title

      Communications Biology

      Volume: 6 Issue: 1 Pages: 1018-1018

    • DOI

      10.1038/s42003-023-05393-x

    • Related Report
      2023 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Calaxin stabilizes the docking of outer arm dyneins onto ciliary doublet microtubule in vertebrates.2022

    • Author(s)
      Yamaguchi, H., Morikawa, M., Kikkawa, M.
    • Journal Title

      BioRxiv

      Volume: NA

    • DOI

      10.1101/2022.11.10.516068

    • Related Report
      2022 Annual Research Report
    • Open Access
  • [Journal Article] Identifying proteins in the cell by tagging techniques for cryo-electron microscopy2022

    • Author(s)
      Masahide Kikkawa, Haruaki Yanagisawa
    • Journal Title

      Microscopy (Oxf)

      Volume: 71 Issue: Supplement_1 Pages: i60-i65

    • DOI

      10.1093/jmicro/dfab059

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Parallel cryo electron tomography on in situ lamellae2022

    • Author(s)
      Fabian Eisenstein, Haruaki Yanagisawa, Hiroka Kashihara, Masahide Kikkawa, Sachiko Tsukita, adostin Danev
    • Journal Title

      bioRxiv

      Volume: 0 Pages: 0-0

    • DOI

      10.1101/2022.04.07.487557

    • Related Report
      2021 Annual Research Report
  • [Presentation] 第8回日本骨免疫学会 冬期学術集会2024

    • Author(s)
      吉川 雅英
    • Organizer
      クライオ電子顕微鏡によるクロススケール観察
    • Related Report
      2023 Annual Research Report
    • Invited
  • [Presentation] JST主催 第2回タンパク質シンポジウム プログラム2024

    • Author(s)
      吉川 雅英
    • Organizer
      クライオ電子顕微鏡による上皮構造のクロススケール観察
    • Related Report
      2023 Annual Research Report
  • [Presentation] 第129回日本解剖学会総会・全国学術集会2024

    • Author(s)
      牧野 司
    • Organizer
      精子形成におけるマンシェット輸送をになう新たなキネシンモーターの分子機構
    • Related Report
      2023 Annual Research Report
  • [Presentation] 日本顕微鏡学会第79回学術講演会2023

    • Author(s)
      吉川 雅英
    • Organizer
      クライオ電子顕微鏡による細胞骨格・モーター分子への応用
    • Related Report
      2023 Annual Research Report
    • Invited
  • [Presentation] 日本顕微鏡学会第79回学術講演会2023

    • Author(s)
      齊藤 知恵子
    • Organizer
      広域三次元光ー電子相関電子顕微鏡法で紐解く細胞内分解ダイナミクス
    • Related Report
      2023 Annual Research Report
  • [Presentation] 日本顕微鏡学会第79回学術講演会2023

    • Author(s)
      楊 景霏
    • Organizer
      The introduction and structural analysis with cryo-EM of M13 bacteriophage
    • Related Report
      2023 Annual Research Report
  • [Presentation] 日本顕微鏡学会第79回学術講演会2023

    • Author(s)
      佐々木 諒平
    • Organizer
      クライオ電子顕微鏡による微小管-ダイナミン複合体構造解析
    • Related Report
      2023 Annual Research Report
  • [Presentation] 第49回内藤コンファレンス2023

    • Author(s)
      吉川 雅英
    • Organizer
      Combination of cryo-EM and genetics for studying microtubule-binding proteins
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 第49回内藤コンファレンス2023

    • Author(s)
      谷 侑磨
    • Organizer
      FAP47, HYDIN, and CPC1 in the central pair apparatus of Chlamydomonas flagella
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 第49回内藤コンファレンス2023

    • Author(s)
      安 建成
    • Organizer
      GAS2 mediates F-actin and microtubule crosslinking
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 第49回内藤コンファレンス2023

    • Author(s)
      牧野 司
    • Organizer
      Analysis of a Novel Kinesin Motor Involved in Sperm specific Manchette-Transport
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 第49回内藤コンファレンス2023

    • Author(s)
      山口 博史
    • Organizer
      Cryo-electron tomography revealed that Calaxin stabilizes the docking of outer arm dyneins onto ciliary doublet microtubule in vertebrates
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 第23回日本蛋白質科学会年会2023

    • Author(s)
      佐々木 諒平
    • Organizer
      Prevention of particle adsorption at the air/water interface using DNA support structures for cryo-EM.
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 学術変革領域会議2023

    • Author(s)
      吉川 雅英
    • Organizer
      Combination of genetics and cryo-electron tomography to study various epitheliums
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 学術変革領域会議2023

    • Author(s)
      牧野 司
    • Organizer
      KIF6 Gene Deletion Causes Sperm Flagellar Immobility: Insights from KIF6 KO Mice
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 学術変革領域会議2023

    • Author(s)
      山口 博史
    • Organizer
      Cryo-electron tomography for structural/functional analysis of vertebrate ciliary axoneme
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 学術変革領域会議2023

    • Author(s)
      谷 侑磨
    • Organizer
      FAP47, HYDIN, and CPC1 in the central pair apparatus of Chlamydomonas flagella
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research
  • [Presentation] IMC20(The 20th International Microscopy Congress)2023

    • Author(s)
      吉川 雅英
    • Organizer
      Combination of genetics and cryo-electron tomography to study the docking of outer arm dyneins onto ciliary doublet microtubule in vertebrates
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Complete structure of M13 phage by cryo-electron microscopy.2023

    • Author(s)
      Masahide Kikkawa
    • Organizer
      第7回日本骨免疫学会 冬期学術集会
    • Related Report
      2022 Annual Research Report
    • Invited
  • [Presentation] Bioconjugation of Au25 nanocluster to trastuzumab.2023

    • Author(s)
      Katarzyna Malawska
    • Organizer
      The 103rd CSJ Annual Meeting (2023)
    • Related Report
      2022 Annual Research Report
  • [Presentation] Combination of cryo-electron tomography and genetics to study cilia/flagella2023

    • Author(s)
      Masahide Kikkawa
    • Organizer
      Cold Spring Harbor Conference Asia: Cilia and Centrosome
    • Related Report
      2022 Annual Research Report
    • Invited
  • [Presentation] Cryo-electron tomography revealed that Calaxin stabilizes the docking of outer arm dyneins onto ciliary doublet microtubule in vertebrates2023

    • Author(s)
      Hiroshi Yamaguchi
    • Organizer
      Cold Spring Harbor Conference Asia: Cilia and Centrosome
    • Related Report
      2022 Annual Research Report
    • Int'l Joint Research
  • [Presentation] FAP47, HYDIN, and CPC1 in the central pair apparatus of Chlamydomonas2023

    • Author(s)
      Yuma Tani
    • Organizer
      Cold Spring Harbor Conference Asia: Cilia and Centrosome
    • Related Report
      2022 Annual Research Report
    • Int'l Joint Research
  • [Presentation] GAS2 mediates F-actin and microtubule crosslinking2023

    • Author(s)
      Jiancheng An
    • Organizer
      Cold Spring Harbor Conference Asia: Cilia and Centrosome
    • Related Report
      2022 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 精子形成におけるマンシェット輸送をになう新たなキネシンモーターの解析2023

    • Author(s)
      牧野司
    • Organizer
      第128回 日本解剖学会総会・全国学術総会
    • Related Report
      2022 Annual Research Report
  • [Presentation] クライオ電子顕微鏡を用いた繊毛軸糸ダイニンコンポーネントCalaxinの機能解析2022

    • Author(s)
      山口 博史
    • Organizer
      第74回日本細胞生物学会大会
    • Related Report
      2022 Annual Research Report
  • [Presentation] Cryo-electron tomography revealed that Calaxin stabilizes the docking of outer arm dyneins onto ciliary doublet microtubule in vertebrate (クライオ電子顕微鏡を用いた繊毛軸糸ダイニンコンポーネント Calaxin の機能解析)2022

    • Author(s)
      Hiroshi Yamaguchi
    • Organizer
      第60回日本生物物理学会年会
    • Related Report
      2022 Annual Research Report
  • [Presentation] FAP47, HYDIN, and CPC1 in the central pair apparatus of Chlamydomonas2022

    • Author(s)
      Yuma Tani
    • Organizer
      第60回日本生物物理学会年会
    • Related Report
      2022 Annual Research Report
  • [Presentation] Near-atomic cryo-EM structure of GAR domain bound to the microtuble2022

    • Author(s)
      Jiancheng An
    • Organizer
      第60回日本生物物理学会年会
    • Related Report
      2022 Annual Research Report
  • [Presentation] FAP47, HYDIN, and CPC1 in the central pair apparatus of Chlamydomonas2022

    • Author(s)
      Yuma Tani
    • Organizer
      日本顕微鏡学会若手研究部会2022年度シンポジウム
    • Related Report
      2022 Annual Research Report
  • [Presentation] クライオ電子線トモグラフィーとcryo-FIB-SEMによる上皮構造の観察2022

    • Author(s)
      吉川雅英
    • Organizer
      統合生命科学研究科シンポジウム 分子原子レベルの統合生命科学研究
    • Related Report
      2022 Annual Research Report
    • Invited
  • [Presentation] クライオ電子顕微鏡によるクロススケール観察2022

    • Author(s)
      吉川雅英
    • Organizer
      リサーチトレンド2022 第4回クライオ電子顕微鏡ワークショップ
    • Related Report
      2022 Annual Research Report
    • Invited
  • [Presentation] The introduction and structural analysis with cryo-EM of M13 Bacteriophage2022

    • Author(s)
      Jingfei Yang
    • Organizer
      日本顕微鏡学会若手研究部会2022年度シンポジウム
    • Related Report
      2022 Annual Research Report
  • [Presentation] Prevention of particle denaturation on the air/water interface with the aid of DNA support structure for cryo-electron microscope.2022

    • Author(s)
      Ryohei Sasaki
    • Organizer
      日本顕微鏡学会若手研究部会2022年度シンポジウム
    • Related Report
      2022 Annual Research Report
  • [Presentation] cryo-ET を用いた繊毛軸糸ダイニンコンポーネント Calaxin の機能解析2022

    • Author(s)
      山口 博史
    • Organizer
      第12回繊毛研究会
    • Related Report
      2022 Annual Research Report
  • [Presentation] FAP47, HYDIN, and CPC1 in the central pair apparatus of Chlamydomonas2022

    • Author(s)
      Yuma Tani
    • Organizer
      第12回繊毛研究会
    • Related Report
      2022 Annual Research Report
  • [Presentation] FAP47, HYDIN, and CPC1 in the central pair apparatus of Chlamydomonas2022

    • Author(s)
      Yuma Tani
    • Organizer
      Cell Bio 2022 An ASCB | EMBO Meeting
    • Related Report
      2022 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Ultrastructural Analysis of Isolation Membrane by Cryo-Electron Tomography and Cryo-Focused Ion Beam2022

    • Author(s)
      Yoshiyuki Fukuda
    • Organizer
      日本顕微鏡学会第78回学術講演会
    • Related Report
      2022 Annual Research Report
  • [Presentation] Morphological analysis of the isolation membrane by cryo-electron tomography and cryo-FIB2022

    • Author(s)
      Yoshiyuki Fukuda
    • Organizer
      The 10th International Symposium on Autophagy
    • Related Report
      2022 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Ultrastructural Analysis of Isolation Membrane by Cryo-Electron Tomography and Cryo-Focused Ion Beam2022

    • Author(s)
      Yoshiyuki Fukuda
    • Organizer
      日本顕微鏡学会若手研究部会2022年度シンポジウム
    • Related Report
      2022 Annual Research Report
  • [Presentation] Morphological analysis of the isolation membrane by cryo-electron tomography and cryo-focused ion beam2022

    • Author(s)
      Yoshiyuki Fukuda
    • Organizer
      The 4th East-Asia Microscopy Conference
    • Related Report
      2022 Annual Research Report
    • Int'l Joint Research
  • [Presentation] クライオ電顕トモグラフィーによる脊椎動物の軸糸ダイニンリンカー構造の解析2021

    • Author(s)
      Hiroshi Yamaguchi
    • Organizer
      顕微鏡学会
    • Related Report
      2021 Annual Research Report
  • [Presentation] Combination of cryo-EM and genetics to study epithelial cells2021

    • Author(s)
      Masahide Kikkawa
    • Organizer
      Korean Society for Molecular and Cellular Biology 2021
    • Related Report
      2021 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Combination of cryo-EM ang genetics to study cilia/flagella2021

    • Author(s)
      Masahide Kikkawa
    • Organizer
      Computer simulations and Cryo-ET/EM of complex biomolecular systems
    • Related Report
      2021 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] クライオ電子顕微鏡によるクロススケール観察2021

    • Author(s)
      吉川雅英
    • Organizer
      第6回 日本骨免疫学会 ウィンタースクール
    • Related Report
      2021 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Cryo electron tomography of cellular specimens2021

    • Author(s)
      Yoshiyuki Fukuda
    • Organizer
      KSBMB International Conference 2021
    • Related Report
      2021 Annual Research Report
    • Int'l Joint Research / Invited
  • [Remarks] 東京大学・大学院・医学系研究科・生体構造学分野 ホームページ

    • URL

      https://structure.m.u-tokyo.ac.jp

    • Related Report
      2023 Annual Research Report

URL: 

Published: 2021-10-22   Modified: 2025-06-20  

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