Reconstitution of Rab2 targeting to ER-Golgi intermediate compartment in semi-intact cells

• Project/Area Number: 17K07379
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Cell biology
• Research Institution: Tokyo Institute of Technology
• Principal Investigator: Kano Fumi 東京工業大学, 科学技術創成研究院, 准教授 (10361594)
• Project Period (FY): 2017-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000); Fiscal Year 2019: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2018: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2017: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: 再構成 / オルガネラ / タンパク質局在 / Rab2 / Rabタンパク質 / セミインタクト細胞 / ERGIC / ターゲティング / ゴルジ体

Outline of Final Research Achievements

Rab2, a member of small GTPase protein family, specifically targeted to ER-Golgi intermediate compartment (ERGIC). To reveal its regulatory mechanism, we reconstituted the targeting of GFP-tagged Rab2 recombinant protein to ERGIC in semi-intact HeLa cells, the plasma membrane of which was permeabilized with streptococcal pore forming toxin, streptolysin O. The targeting assay using the inhibitory antibodies revealed the involvement of two proteins, X and Y (the names of the proteins can not be open to the public since the paper relating to the results is now under submission), in the Rab2 targeting among the Rab2-interacting proteins. X was found interacting with Rab2 in cytoplasm and functioned for retaining the GTP-bound form of Rab2, while Y was bound to Rab2 near ERGIC membrane. These results suggested that Rab2 targeting to ERGIC was sequentially regulated by interacting with two distinct proteins.

Academic Significance and Societal Importance of the Research Achievements

セミインタクト細胞アッセイは細胞内構造の維持が必要不可欠なタンパク質の局在化研究に適した細胞側ツールであると言える。本研究で構築するアッセイは汎用性が高く、蛋白質の局在化と機能相関が重要である細胞生物学分野において学術的に意義がある。また、本申請研究で注目するRab2は、小胞輸送だけでなく、シグナル伝達、インスリン分泌・分解制御、オートファジーなどの細胞機能にも関与する。他にも自閉症スペクトラム障害や精神分裂病でRab2A点変異が報告されるなどRab2の生理機能の重要性はますます注目されており、その局在化機構解明はこれらの生命現象にも深く関わる重要なテーマである。

Research Products

• [Journal Article] Applications of cell resealing to reconstitute microRNA loading to extracellular vesicles (2021)
  • Author(s): Sonoda Yuki、Kano Fumi、Murata Masayuki
  • Journal Title: Scientific Reports Volume: 11 Issue: 1 Pages: 2900-2900
  • DOI: 10.1038/s41598-021-82452-5
  • Peer Reviewed / Open Access
• [Journal Article] リシール細胞技術を用いた“細胞編集” (2020)
  • Author(s): 村田昌之、加納ふみ
  • Journal Title: 医学のあゆみ Volume: 275 Pages: 144-148
• [Journal Article] Pyk2-dependent phosphorylation of LSR enhances localization of LSR and tricellulin at tricellular tight junctions (2019)
  • Author(s): Nakatsu, D., Kano, F., Shinozaki-Narikawa, N., Murata, M.
  • Journal Title: PLOS ONE Volume: 14 Issue: 10 Pages: e0223300-e0223300
  • DOI: 10.1371/journal.pone.0223300
  • Peer Reviewed / Open Access
• [Journal Article] The cell resealing technique for manipulating, visualizing, and elucidating molecular functions in living cells. (2019)
  • Author(s): Kunishige, R., Kano, F., Murata, M.
  • Journal Title: Biochem. Biophys. Acta (General Subjects) Volume: 印刷中 Issue: 2 Pages: 129329-129329
  • DOI: 10.1016/j.bbagen.2019.03.015
  • Peer Reviewed
• [Journal Article] Semi-intact cell system for reconstituting and analyzing cellular Golgi dynamics. (2019)
  • Author(s): Kano, F., Murata, M.
  • Journal Title: The Golgi Apparatus and Centriole Volume: 67 Pages: 233-250
  • DOI: 10.1007/978-3-030-23173-6_10
  • ISBN: 9783030231729, 9783030231736
  • Peer Reviewed
• [Journal Article] Phosphatidylinositol-3-phosphate-mediated actin domain formation linked to DNA synthesis upon insulin treatment in rat hepatoma-derived H4IIEC3 cells (2019)
  • Author(s): Kano, F., Murata, M.
  • Journal Title: Biochim. Biophys. Acta (BBA) - Mol. Cell Res. Volume: 1866 Issue: 5 Pages: 793-805
  • DOI: 10.1016/j.bbamcr.2019.02.005
  • Peer Reviewed
• [Journal Article] Changes in the asymmetric distribution of cholesterol in the plasma membrane influence streptolysin O pore formation (2019)
  • Author(s): Ogasawara, F., Kano, F., Murata, M., Kimura, Y., Kioka, N., Ueda, K
  • Journal Title: Sci. Rep. Volume: 9 Issue: 1 Pages: 4548-4548
  • DOI: 10.1038/s41598-019-39973-x
  • NAID: 120006644065
  • Peer Reviewed / Open Access
• [Journal Article] Cell Resealing Technique (2018)
  • Author(s): 村田昌之、加納ふみ
  • Journal Title: 生化学 Volume: 90 Issue: 4 Pages: 433-443
  • DOI: 10.14952/SEIKAGAKU.2018.900433
  • ISSN: 0037-1017
  • Year and Date: 2018-08-25
• [Journal Article] 特集 生体膜のバイオロジー Ⅴ.新技術 セミインタクト細胞リシール技術を用いた細胞機能解析 (2018)
  • Author(s): 國重 莉奈、加納 ふみ、村田 昌之
  • Journal Title: 生体の科学 Volume: 69 Issue: 3 Pages: 247-251
  • DOI: 10.11477/mf.2425200804
  • ISSN: 0370-9531, 1883-5503
  • Year and Date: 2018-06-15
• [Journal Article] Exosome-associated Shiga toxin 2 is released from cells and causes severe toxicity in mice (2018)
  • Author(s): Watanabe-Takahashi, M., Yamasaki, S., Murata, M., Kano, F., Motoyama, J., Yamate, J., Omi, J., Sato, W., Ukai, H., Shimasaki, K., Ikegawa, M., Tamura-Nakano, M., Yanoshita, R., Nishino, Y., Miyazawa, A., Natori, Y., Toyama-Sorimachi, N., Nishikawa, N.
  • Journal Title: Sci. Rep. Volume: 8 Issue: 1 Pages: 10776-10776
  • DOI: 10.1038/s41598-018-29128-9
  • Peer Reviewed / Open Access
• [Journal Article] LLO-mediated Cell Resealing System for Analyzing Intracellular Activity of Membrane-impermeable Biopharmaceuticals of Mid-sized Molecular Weight (2018)
  • Author(s): Murakami Masataka、Kano Fumi、Murata Masayuki
  • Journal Title: Scientific Reports Volume: 8 Issue: 1 Pages: 1946-1946
  • DOI: 10.1038/s41598-018-20482-2
  • Peer Reviewed / Open Access
• [Journal Article] The first successful observation of in-cell NMR signals of DNA and RNA in living human cells (2018)
  • Author(s): Yamaoki Yudai、Kiyoishi Ayaka、Miyake Masayuki、Kano Fumi、Murata Masayuki、Nagata Takashi、Katahira Masato
  • Journal Title: Phys Chem Chem Phys Volume: 20 Issue: 5 Pages: 2982-2985
  • DOI: 10.1039/c7cp05188c
  • NAID: 130007557810
  • Peer Reviewed / Open Access
• [Journal Article] セミインタクト細胞リシール技術を用いた糖尿病モデル細胞アレイとその解析法 (2018)
  • Author(s): 加納ふみ、野口誉之、村田昌之
  • Journal Title: 臓器チップの技術と開発動向（監修： 酒井康行,金森敏幸、シーエムシー出版） Volume: -
• [Journal Article] Establishment and phenotyping of disease model cells created by cell-resealing technique (2017)
  • Author(s): Kano Fumi、Noguchi Yoshiyuki、Murata Masayuki
  • Journal Title: Scientific Reports Volume: 7 Issue: 1 Pages: 15167-15167
  • DOI: 10.1038/s41598-017-15443-0
  • Peer Reviewed / Open Access
• [Journal Article] Pyruvate kinase M1 interacts with A-Raf and inhibits endoplasmic reticulum stress-induced apoptosis by activating MEK1/ERK pathway in mouse insulinoma cells (2017)
  • Author(s): Horiuchi Yuta、Nakatsu Daiki、Kano Fumi、Murata Masayuki
  • Journal Title: Cellular Signalling Volume: 38 Pages: 212-222
  • DOI: 10.1016/j.cellsig.2017.07.017
  • Peer Reviewed / Open Access
• [Journal Article] 病態モデル細胞の創成と新展開 －未来のナノメディシンに資する新しい細胞編集技術－」 (2017)
  • Author(s): 村田昌之、加納ふみ
  • Journal Title: キヤノン財団ライブラリー『ナノテクノロジーが拓く未来の医療』（片岡一則編著、丸善プラネット） Volume: - Pages: 228-249
• [Presentation] Cell-based analytical method for manipulating and evaluating protein network. (2020)
  • Author(s): Fumi Kano
  • Organizer: 第43回日本分子生物学会年会
  • Invited
• [Presentation] セミインタクト細胞リシール技術を用いた細胞機能解析・評価系の開発 (2019)
  • Author(s): 國重莉奈、園田雄輝、村上真隆、加納ふみ、村田昌之
  • Organizer: 日本ケミカルバイオロジー学会第１４回年会
• [Presentation] M型ピルビン酸産生酵素PKM1の減少は小胞体ストレス依存的な膵臓β細胞死を促進する (2019)
  • Author(s): 中津 大貴, 村田 昌之, 加納 ふみ
  • Organizer: 第19回日本蛋白質科学会年会 第71回日本細胞生物学会大会 合同年次大会
• [Presentation] 細胞内温度変動に関与する分子のスクリーニング法の開発 (2019)
  • Author(s): Takashi Mitsubori, Kohki Okabe, Fumi Kano, Takashi Funatsu
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] リシール細胞技術を用いた疾患モデル細胞の構築とそのフェノタイプ解析 (2018)
  • Author(s): 加納ふみ
  • Organizer: 電気化学会第85回大会
  • Invited