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3D-CLEM analysis of the membrane structure in autophagy

Research Project

Project/Area Number 17K15550
Research Category

Grant-in-Aid for Young Scientists (B)

Allocation TypeMulti-year Fund
Research Field General anatomy (including histology/embryology)
Research InstitutionJuntendo University

Principal Investigator

Kakuta Soichiro  順天堂大学, 医学(系)研究科(研究院), 准教授 (80551209)

Project Period (FY) 2017-04-01 – 2020-03-31
Project Status Completed (Fiscal Year 2019)
Budget Amount *help
¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2019: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2017: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Keywordsオートファジー / 電子顕微鏡 / 形態学 / 共焦点レーザー顕微鏡 / CLEM / 光-電子相関顕微鏡法 / 三次元電子顕微鏡解析 / メンブレントラフィック / Atg9 / 解剖学
Outline of Final Research Achievements

To better understand the morphology of autophagosome membrane formation, we performed a three-dimensional electron microscopy analysis. The primary objective of this research is to establish a method for selective observation of target structures and sites using light-electron correlative microscopy (CLEM). In addition to the labeling of the target of autophagy formation site, the mitochondria were fluorescently labeled. We developed the method for accurate positional correction by overlaying the fluorescence image with the electron microscope image using image analysis software. Normal cells showed autophagosomes and autolysosomes, and ATG7 KO cells showed more isolation membranes. Some isolation membrane-like structures were observed in ATG9A KO cells, where membrane formation is not supposed to occur.

Academic Significance and Societal Importance of the Research Achievements

オートファジーは様々な疾病への関与が盛んに報告されている細胞内機構である。顕微鏡を用いた形態学的な研究は初期から行われていたが、蛍光顕微鏡では微細形態までは捉えることが出来ず、一方で電子顕微鏡では立体的な情報を得ることが難しく、また形態だけでは目的の構造かどうか判断できないことも多い。本研究では蛍光顕微鏡像と「FIB/SEMによる3次元電子顕微鏡像」を組み合わせた解析を行うことでそれぞれの欠点を補うことが出来た。これによりオートファジー関連構造体の変化についての新しい知見が得られ、またこれを進めることによってオートファジー現象全体の理解へと繋がることが期待出来る。

Report

(4 results)
  • 2019 Annual Research Report   Final Research Report ( PDF )
  • 2018 Research-status Report
  • 2017 Research-status Report
  • Research Products

    (16 results)

All 2020 2019 2018 2017 Other

All Journal Article (9 results) (of which Int'l Joint Research: 3 results,  Peer Reviewed: 9 results,  Open Access: 6 results) Presentation (3 results) Remarks (4 results)

  • [Journal Article] Three-dimensional imaging of podocyte ultrastructure using FE-SEM and FIB-SEM tomography.2020

    • Author(s)
      Miyaki T, Kawasaki Y, Hosoyamada Y, Amari T, Kinoshita M, Matsuda H, Kakuta S, Sakai T, Ichimura K.
    • Journal Title

      Cell Tissue Res

      Volume: 379 Pages: 245-254

    • NAID

      120006883563

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Three-dimensional electron microscopy analysis reveals endopolygeny-like nuclear architecture segregation in Plasmodium oocyst development.2020

    • Author(s)
      Araki T, Kawai S, Kakuta S, Kobayashi H, Umeki Y, Saito-Nakano Y, Sasaki T, Nagamune K, Yasutomi Y, Nozaki T, Franke-Fayard B, Khan SM, Hisaeda H, Annoura T
    • Journal Title

      Parasitol Int.

      Volume: -

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Streptococcus pneumoniae triggers hierarchical autophagy through reprogramming of LAPosome-like vesicles via NDP52-delocalization2020

    • Author(s)
      Ogawa Michinaga、Takada Naoki、Shizukuishi Sayaka、Tomokiyo Mikado、Chang Bin、Yoshida Mitsutaka、Kakuta Soichiro、Tanida Isei、Ryo Akihide、Guan Jun-Lin、Takeyama Haruko、Ohnishi Makoto
    • Journal Title

      Communications Biology

      Volume: 3 Issue: 1

    • DOI

      10.1038/s42003-020-0753-3

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Autophagy Deficiency in Renal Proximal Tubular Cells Leads to an Increase in Cellular Injury and Apoptosis under Normal Fed Conditions2019

    • Author(s)
      Suzuki Chigure、Tanida Isei、Oliva Trejo Juan Alejandro、Kakuta Soichiro、Uchiyama Yasuo
    • Journal Title

      International Journal of Molecular Sciences

      Volume: 21 Issue: 1 Pages: 155-155

    • DOI

      10.3390/ijms21010155

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Cerebellar Neurodegeneration and Neuronal Circuit Remodeling in Golgi pH Regulator-Deficient Mice2019

    • Author(s)
      Sou Yu-shin、Kakuta Soichiro、Kamikubo Yuji、Niisato Kazue、Sakurai Takashi、Parajuli Laxmi Kumar、Tanida Isei、Saito Hiromitsu、Suzuki Noboru、Sakimura Kenji、Maeda Yusuke、Kinoshita Taroh、Uchiyama Yasuo、Koike Masato
    • Journal Title

      eNeuro

      Volume: 6 Issue: 3 Pages: 0427-18

    • DOI

      10.1523/eneuro.0427-18.2019

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Morphological processes of foot process effacement in puromycin aminonucleoside nephrosis revealed by FIB/SEM tomography.2019

    • Author(s)
      Ichimura K, Miyaki T, Kawasaki Y, Kinoshita M, Kakuta S, Sakai T.
    • Journal Title

      J Am Soc Nephrol

      Volume: 30(1) Issue: 1 Pages: 96-108

    • DOI

      10.1681/asn.2018020139

    • NAID

      120006883561

    • Related Report
      2018 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] NDP52 interacts with mitochondrial RNA poly(A) polymerase to promote mitophagy.2018

    • Author(s)
      Furuya N, Kakuta S, Sumiyoshi K, Ando M, Nonaka R, Suzuki A, Kazuno S, Saiki S, Hattori N.
    • Journal Title

      EMBO Reports

      Volume: 19 Issue: 12

    • DOI

      10.15252/embr.201846363

    • Related Report
      2018 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] Atg9a deficiency causes axon-specific lesions including neuronal circuit dysgenesis.2017

    • Author(s)
      Yamaguchi J, Suzuki C, Nanao T, Kakuta S, Ozawa K, Tanida I, Saitoh T, Sunabori T, Komatsu M, Tanaka K, Aoki S, Sakimura K, Uchiyama Y.
    • Journal Title

      Autophagy

      Volume: 17 Issue: 5 Pages: 1-14

    • DOI

      10.1080/15548627.2017.1314897

    • NAID

      120006881392

    • Related Report
      2018 Research-status Report 2017 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Small GTPase Rab1B is associated with ATG9A vesicles and regulates autophagosome formation.2017

    • Author(s)
      Kakuta S, Yamaguchi J, Suzuki C, Sasaki M, Kazuno S, Uchiyama Y.
    • Journal Title

      FASEB J

      Volume: 31(9) Issue: 9 Pages: 3757-3773

    • DOI

      10.1096/fj.201601052r

    • Related Report
      2017 Research-status Report
    • Peer Reviewed
  • [Presentation] FIB/SEMを用いた三次元光-電子相関顕微鏡解析によるオートファゴソーム関連構造体の解析2019

    • Author(s)
      角田宗一郎、オリバトレハンドロ、鈴木ちぐれ、内山安男
    • Organizer
      第124回日本解剖学会総会・全国学術集会
    • Related Report
      2018 Research-status Report
  • [Presentation] Three-dimensional correlative light and electron microscopy analysis of autophagy-related structures2018

    • Author(s)
      角田 宗一郎、鈴木 ちぐれ、オリバ アレハンドロ、内山 安男
    • Organizer
      第123回日本解剖学会全国学術集会(2018年3月、東京都武蔵野市)
    • Related Report
      2017 Research-status Report
  • [Presentation] 三次元光-電子相関顕微鏡法によるオートファゴソーム関連構造体の解析2017

    • Author(s)
      角田 宗一郎、鈴木 ちぐれ、オリバ アレハンドロ、内山 安男
    • Organizer
      2017年度 生命科学系学会合同年次大会
    • Related Report
      2017 Research-status Report
  • [Remarks] 順天堂大学 研究者情報データベース

    • URL

      https://www.juntendo.ac.jp/graduate/kenkyudb/search/researcher.php?MID=4852

    • Related Report
      2019 Annual Research Report
  • [Remarks] research map

    • URL

      https://researchmap.jp/skakuta/

    • Related Report
      2019 Annual Research Report
  • [Remarks] 順天堂大学・研究業績詳細

    • URL

      https://www.juntendo.ac.jp/graduate/kenkyudb/search/achievement.php?MID=4852

    • Related Report
      2018 Research-status Report
  • [Remarks]

    • URL

      https://www.juntendo.ac.jp/graduate/kenkyudb/search/achievement.php?MID=4852

    • Related Report
      2017 Research-status Report

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Published: 2017-04-28   Modified: 2021-02-19  

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