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Single-molecule visualisation of kinetochore-microtubule interface using the in vitro reconstitution system

Research Project

Project/Area Number 16K14672
Research Category

Grant-in-Aid for Challenging Exploratory Research

Allocation TypeMulti-year Fund
Research Field Molecular biology
Research InstitutionHiroshima University

Principal Investigator

Toda Takashi  広島大学, 先端物質科学研究科, 特任教授 (50197894)

Co-Investigator(Kenkyū-buntansha) 湯川 格史  広島大学, 先端物質科学研究科, 特任助教 (50403605)
Research Collaborator Matsuo Yuzy  
Project Period (FY) 2016-04-01 – 2019-03-31
Project Status Completed (Fiscal Year 2018)
Budget Amount *help
¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000)
Fiscal Year 2018: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2017: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2016: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Keywords染色体 / 紡錘体微小管 / in vitro再構成 / 微小管ポリメラーゼ / 微小管結合タンパク質 / 全反射照明蛍光顕微鏡 / 動原体 / 分裂酵母 / 紡錘体(スピンドル)微小管 / 動原体構成タンパク質 / 微小管重合 / 分裂装置 / キネトコア / 微小管 / in vitro / TIRF / スピンドル / 染色体分配 / 再構築 / TIRF顕微鏡 / 1分子視覚化
Outline of Final Research Achievements

Equal partition of genetic material (chromosomes) lies at the heart of cell reproduction. The kinetochore is a large proteinaceous structure formed on the chromosome. The microtubule is a major component of division apparatus. For each pair of chromosomes to segregate equally, the microtubule captures and pulls the kinetochore towards each pole. The aim of this program was the in vitro reconstitution of the interface that recapitulates the kinetochore-microtubule interaction. Using fission yeast, we first showed that Ndc80 (a core kinetochore component) and Dis1 (a microtubule polymerase) genetically interact. The following in vitro study indicated that these two proteins do not interact directly; instead, the third component, the third protein Mal3/EB1 is necessary for Ndc80 to interact with Dis1. These result have suggested that three factors, namely Ndc80, Dis1 and Mal3, comprise the minimal requirement for the in vitro reconstruction of the kinetochore-microtubule interface.

Academic Significance and Societal Importance of the Research Achievements

染色体の娘細胞への均等分配は、生命の継承、生物の生存にとって最も重要なプロセスである。染色体不分離、非均等分配が起こると、生殖細胞では流産あるいはダウン症候群、常細胞では、細胞ガン化が惹起される。本研究により、染色体分配には3つのタンパク質が必要最小限であることが、明らかになった。この成果により、基礎生命科学分野におけて長年の謎であった染色体分配機構の全容解明への道程が示された。一方、医学、医療分野に対しては、3つのタンパク質をターゲットとする活性小分子の開発、創薬を可能とし、ヒト疾患の予防、治癒に寄与する。よって本研究成果は、学術的、社会的意義、両面から重要な知見を提供したと云える。

Report

(4 results)
  • 2018 Annual Research Report   Final Research Report ( PDF )
  • 2017 Research-status Report
  • 2016 Research-status Report
  • Research Products

    (45 results)

All 2018 2017 2016 Other

All Int'l Joint Research (6 results) Journal Article (10 results) (of which Int'l Joint Research: 7 results,  Peer Reviewed: 9 results,  Open Access: 10 results,  Acknowledgement Compliant: 2 results) Presentation (25 results) (of which Int'l Joint Research: 6 results,  Invited: 9 results) Remarks (4 results)

  • [Int'l Joint Research] CSIR/Central Drug Research Institute/Sector 10(インド)

    • Related Report
      2018 Annual Research Report
  • [Int'l Joint Research] The Francis Crick Institute(英国)

    • Related Report
      2017 Research-status Report
  • [Int'l Joint Research] University of California, San Diego(米国)

    • Related Report
      2017 Research-status Report
  • [Int'l Joint Research] Universitat Pompeu Fabra (UPF)(Spain)

    • Related Report
      2017 Research-status Report
  • [Int'l Joint Research] The Francis Crick Institute(英国)

    • Related Report
      2016 Research-status Report
  • [Int'l Joint Research] Centre for Genomic Regulation (CRG)(Spain)

    • Related Report
      2016 Research-status Report
  • [Journal Article] Fission yeast cells overproducing HSET/KIFC1 provides a useful tool for identification and evaluation of human kinesin-14 inhibitors2018

    • Author(s)
      Yukawa Masashi、Yamauchi Tomoaki、Kurisawa Naoaki、Ahmed Shakil、Kimura Ken-ichi、Toda Takashi
    • Journal Title

      Fungal Genetics and Biology

      Volume: 116 Pages: 33-41

    • DOI

      10.1016/j.fgb.2018.04.006

    • NAID

      120006800807

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Suppressor Analysis Uncovers That MAPs and Microtubule Dynamics Balance with the Cut7/Kinesin-5 Motor for Mitotic Spindle Assembly in Schizosaccharomyces pombe2018

    • Author(s)
      Yukawa Masashi、Yamada Yusuke、Toda Takashi
    • Journal Title

      G3 (Genes|Genomes|Genetics)

      Volume: 9 Issue: 1 Pages: 269-280

    • DOI

      10.1534/g3.118.200896

    • NAID

      120006784517

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Two spatially distinct kinesin-14 proteins, Pkl1 and Klp2, generate collaborative inward forces against kinesin-5 Cut7 in S. pombe.2018

    • Author(s)
      Masashi Yukawa, Yusuke Yamada, Tomoaki Yamauchi, and Takashi Toda
    • Journal Title

      Journal of Cell Science

      Volume: 131 Issue: 1 Pages: 1-11

    • DOI

      10.1242/jcs.210740

    • NAID

      120006784518

    • Related Report
      2017 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast.2017

    • Author(s)
      Masashi Yukawa, Tomoki Kawakami, Masaki Okazaki, Kazunori Kume, Ngang Heok Tang, and Takashi Toda
    • Journal Title

      Molecular Biology of the Cell

      Volume: 28 Issue: 25 Pages: 3647-3659

    • DOI

      10.1091/mbc.e17-08-0497

    • NAID

      120006784519

    • Related Report
      2017 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Generation and characterisation of temperature sensitive mutants of genes encoding the fission yeast spindle pole body2017

    • Author(s)
      Tang, N.H., Fong, C.S., Jourdain, I., Masuda, H., Yukawa, M., and Toda, T.
    • Journal Title

      PeerJ Preprints

      Volume: 5

    • DOI

      10.7287/peerj.preprints.3377v1

    • Related Report
      2017 Research-status Report
    • Open Access / Int'l Joint Research
  • [Journal Article] Purification and characterisation of the fission yeast Ndc80 complex2017

    • Author(s)
      Matsuo, Y., Maurer, S.P., Surrey, T., and Toda, T.
    • Journal Title

      Protein expression and purification

      Volume: 135 Pages: 61-69

    • DOI

      10.1016/j.pep.2017.05.002

    • NAID

      120006822634

    • Related Report
      2017 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Regulation of centriolar satellite integrity and its physiology2017

    • Author(s)
      Hori, A., and Toda, T.
    • Journal Title

      Cell Mol Life Sci

      Volume: 74 Issue: 2 Pages: 213-229

    • DOI

      10.1007/s00018-016-2315-x

    • Related Report
      2017 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] Identification of three signaling molecules required for calcineurin-dependent monopolar growth induced by the DNA replication checkpoint in fission yeast2017

    • Author(s)
      Kume K, Hashimoto T, Suzuki M, Mizunuma M, Toda T, Hirata D
    • Journal Title

      Biochem Biophys Res Commun.

      Volume: 491 Issue: 4 Pages: 883-889

    • DOI

      10.1016/j.bbrc.2017.07.129

    • Related Report
      2017 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] An unconventional interaction between Dis1/TOG and Mal3/EB1 in fission yeast promotes the fidelity of chromosome segregation2016

    • Author(s)
      Matsuo, Y., Maurer, S. P., Yukawa, M., Zakian, S., Singleton, M. R., Surrey, T. and Toda,
    • Journal Title

      J Cell Sci

      Volume: 129 Issue: 24 Pages: 4592-4606

    • DOI

      10.1242/jcs.197533

    • NAID

      120006811255

    • Related Report
      2016 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
  • [Journal Article] The hairpin region of Ndc80 is important for the kinetochore recruitment of Mph1/MPS1 in fission yeast.2016

    • Author(s)
      Chmielewska, A. E., Tang N-H. and Toda, T.
    • Journal Title

      Cell Cycle

      Volume: 15 Issue: 5 Pages: 740-747

    • DOI

      10.1080/15384101.2016.1148842

    • NAID

      120006822684

    • Related Report
      2016 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
  • [Presentation] Screening for anti-tumor compounds using HSET-overproduction fission yeast and isolated kolavenic acid analog from Solidago altissima2018

    • Author(s)
      Kurisawa N., Koshino H.,Yukawa M., Toda T., Kimura K
    • Organizer
      International Symposium on Innovative Agriculture and Fishery(ISIAF 2018)
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Exploring the molecular pathways leading to bipolar spindle formation.2018

    • Author(s)
      Toda T., Okazaki M., Yamauchi T., Yamada Y., Kawakami T., Teratani Y., Oishi M., Yukawa M.
    • Organizer
      Joint Annual Meeting of 51st JSDB and 70th JSCB (第70回細胞生物学会・第51回発生生物学会合同大会)
    • Related Report
      2018 Annual Research Report
  • [Presentation] Motor-dependent and independent pathways leading to bipolar spindle assembly2018

    • Author(s)
      Toda T.
    • Organizer
      10th HiHA International Workshop “Toward an understanding of chromosome dynamics, segregation and function”
    • Related Report
      2018 Annual Research Report
  • [Presentation] Towards achieving healthy ageing society-from basic biology to applied science2018

    • Author(s)
      Toda T.
    • Organizer
      MIRAI Seminars
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] ヒトキネシン-14(HSET)過剰発現分裂酵母株を用いて単離されたkolavenic acid analogの生物活性2018

    • Author(s)
      栗澤尚瑛、湯川格史、越野広雪、登田 隆、木村賢一
    • Organizer
      第91回日本生化学会大会
    • Related Report
      2018 Annual Research Report
  • [Presentation] 分裂酵母を用いたヒト14型キネシン阻害剤の探索2018

    • Author(s)
      登田 隆、Ahmed Shakil、栗澤尚瑛,木村賢一、湯川格史
    • Organizer
      第51回 酵母遺伝学フォーラム
    • Related Report
      2018 Annual Research Report
  • [Presentation] 分裂酵母Kinesin-5/Cut7と拮抗的に機能する微小管関連因子群の同定と解析2018

    • Author(s)
      山田侑亮、湯川格史、登田 隆
    • Organizer
      第51回 酵母遺伝学フォーラム
    • Related Report
      2018 Annual Research Report
  • [Presentation] 双極性紡錘体形成における力発生装置としての微小管ポリメラーゼ複合体Alp7-Alp14の新規機能2018

    • Author(s)
      河上友基, 湯川格史、登田 隆
    • Organizer
      第51回 酵母遺伝学フォーラム
    • Related Report
      2018 Annual Research Report
  • [Presentation] PCR法を用いた分裂酵母5型キネシンcut7変異体の包括的分離と解析2018

    • Author(s)
      寺谷康宏, 湯川格史、登田 隆
    • Organizer
      第51回 酵母遺伝学フォーラム
    • Related Report
      2018 Annual Research Report
  • [Presentation] M期紡錘体形成に必須な分裂酵母5型キネシンcut7変異を抑制するRNA結合タンパク質遺伝子nrp1の解析2018

    • Author(s)
      大石充輝, 湯川格史、登田 隆
    • Organizer
      第51回 酵母遺伝学フォーラム
    • Related Report
      2018 Annual Research Report
  • [Presentation] 分裂酵母6型キネシンモーターKlp9の機能ドメインと細胞周期制御2018

    • Author(s)
      湯川格史,登田 隆
    • Organizer
      第51回 酵母遺伝学フォーラム
    • Related Report
      2018 Annual Research Report
  • [Presentation] スピンドル微小管はいかに形成されるのか-基礎研究から応用まで2018

    • Author(s)
      登田 隆
    • Organizer
      第22回 酵母合同シンポジウム
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] 分裂酵母5型キネシンcut7変異を抑圧する多コピーサプレッサーの取得と解析2018

    • Author(s)
      武藤 慧、湯川格史、登田 隆
    • Organizer
      第36回イーストワークショップ
    • Related Report
      2018 Annual Research Report
  • [Presentation] 分裂酵母を利用した新規生理活性物質の探索と解析2018

    • Author(s)
      森山 亮、稲田彩花、湯川格史、登田 隆
    • Organizer
      第36回イーストワークショップ
    • Related Report
      2018 Annual Research Report
  • [Presentation] 染色体分配に必須な紡錘体微小管構造を規定するキネシン依存的及び非依存的経路の同定2018

    • Author(s)
      登田 隆, 山田侑亮, 河上友基, 寺谷康宏, 大石充輝, 湯川格史
    • Organizer
      第41回 日本分子生物学会年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] 分裂酵母を用いた抗がん剤シードとなる分子標的阻害物質の新規探索法2018

    • Author(s)
      湯川格史, 栗澤尚瑛,Ahmed Shakil,木村賢一,登田 隆
    • Organizer
      第41回 日本分子生物学会年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] 分裂酵母の新規RNA結合タンパク質Nrp1の機能解析2018

    • Author(s)
      湯川格史, 山田侑亮, 大石充輝, 登田 隆
    • Organizer
      先進ゲノム解析研究推進プラットフォーム拡大班会議
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] キネトコア-スピンドル微小管相互作用インターフェイスのin vitro再構築と可視化の試み2017

    • Author(s)
      登田 隆
    • Organizer
      第34回染色体ワークショップ
    • Place of Presentation
      ホテル、アカデミア、上総、千葉
    • Year and Date
      2017-01-11
    • Related Report
      2016 Research-status Report
    • Invited
  • [Presentation] Exploring the Mitotic Roles of Kinesin-8 in Fission Yeast2017

    • Author(s)
      Corinne Pinder and Takashi Toda
    • Organizer
      Pombe2017:9th International Fission Yeast Meeting
    • Related Report
      2017 Research-status Report
    • Int'l Joint Research / Invited
  • [Presentation] Assembly of mitotic bipolar spindle in the absence of kinesin-5 Cut72017

    • Author(s)
      Masaki Okazaki, Yusuke Yamada, Tomoaki Yamauchi, Tomoki Kawakami, ※Masashi Yukawa and Takashi Toda
    • Organizer
      Pombe2017:9th International Fission Yeast Meeting
    • Related Report
      2017 Research-status Report
    • Int'l Joint Research / Invited
  • [Presentation] Towards the in vitro Reconstitution of the Kinetochore-Microtubule Interface2017

    • Author(s)
      Yuzy Matsuo, Martin R Singleton, Silva Zakian, Sebastian P. Maurer, Thomas Surrey and Takashi Toda
    • Organizer
      Pombe2017:9th International Fission Yeast Meeting
    • Related Report
      2017 Research-status Report
    • Int'l Joint Research
  • [Presentation] 5型キネシンを必要としない新規M期紡錘体構造形成経路2017

    • Author(s)
      登田 隆, 岡崎雅紀, 山内智瑛, 山田侑亮, 河上友基, 湯川格史
    • Organizer
      第40回日本分子生物学会年会(2017年度生命科学系学会合同年次大会, ConBio2017)
    • Related Report
      2017 Research-status Report
  • [Presentation] Kinesin-5に依存しない新規紡錘体形成経路の探索2017

    • Author(s)
      登田 隆, 岡崎雅紀, 山内智瑛, 山田侑亮, 河上友基, 湯川格史
    • Organizer
      第69回日本細胞生物学会大会
    • Related Report
      2017 Research-status Report
    • Invited
  • [Presentation] 型キネシンに依存しない新規紡錘体形成経路の探索2017

    • Author(s)
      湯川格史、河上友基、岡崎雅紀、登田 隆
    • Organizer
      酵母遺伝学フォーラム第50回研究報告会
    • Related Report
      2017 Research-status Report
    • Invited
  • [Presentation] Exploring the molecular mechanism of mitotic spindle assembly and chromosome segregation2016

    • Author(s)
      登田 隆
    • Organizer
      14th International Conference on Yeast (ICY)
    • Place of Presentation
      Yume Butai, Awaji Island, Hyogo
    • Year and Date
      2016-09-11
    • Related Report
      2016 Research-status Report
    • Int'l Joint Research / Invited
  • [Remarks] Toda laboratory HP

    • URL

      http://mccb.hiroshima-u.ac.jp/

    • Related Report
      2018 Annual Research Report
  • [Remarks] HiHA HP

    • URL

      http://hiha.hiroshima-u.ac.jp/

    • Related Report
      2018 Annual Research Report
  • [Remarks]

    • URL

      https://www.crick.ac.uk/research/a-z-researchers/researchers-p-s/thomas-surrey/

    • Related Report
      2016 Research-status Report
  • [Remarks]

    • URL

      https://www.crick.ac.uk/research/a-z-researchers/researchers-p-s/martin-singleton/

    • Related Report
      2016 Research-status Report

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Published: 2016-04-21   Modified: 2022-02-22  

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