Fine morphology and molecular basis of liquid-liquid phase separation caused by the p62 mutation

• Project/Area Number: 20H03415
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 48010:Anatomy-related
• Research Institution: Fukushima Medical University
• Principal Investigator: Waguri Satoshi 福島県立医科大学, 医学部, 教授 (30244908)
• Co-Investigator(Kenkyū-buntansha): 田村 直輝 福島県立医科大学, 医学部, 講師 (70745992)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥17,550,000 (Direct Cost: ¥13,500,000、Indirect Cost: ¥4,050,000); Fiscal Year 2022: ¥5,720,000 (Direct Cost: ¥4,400,000、Indirect Cost: ¥1,320,000); Fiscal Year 2021: ¥5,720,000 (Direct Cost: ¥4,400,000、Indirect Cost: ¥1,320,000); Fiscal Year 2020: ¥6,110,000 (Direct Cost: ¥4,700,000、Indirect Cost: ¥1,410,000)
• Keywords: p62液滴 / SQSTM1 / 液－液相分離 / ストレス顆粒 / 高浸透圧ストレス / 選択的オートファジー / Keap1 / 液滴 / CLEM / 免疫電顕 / 液-液相分離 / オートファジー / 高浸透圧 / p62 / 乳癌 / ゲル / 凝集 / 電子顕微鏡解析

Outline of Research at the Start

様々な分子がひしめく細胞内では、液―液相分離によりできる分子の集合体が重要な役割りを担う。しかし生体における相分離の同定は困難であり、化学固定した組織で認められる集合体が液体様の「液滴」か、それよりも固い「ゲル」か「凝集体」かを区別する術はない。そこで本研究では、相分離を引き起こすp62タンパク質を対象とし、その疾患関連遺伝子変異とp62集合体の微細構造を対比させることで、その形態学的検出法を開発する。これにより、凝集体構造を伴う病態の理解を深める。

Outline of Final Research Achievements

To investigate the physiological significance of p62 droplets, the influence of disease-related genetic mutations in p62 on droplet structure was investigated. However, as a similar study was reported by others, the research plan was changed and detailed analyses of p62-droplets in various experimental systems were carried out. We found that p62-droplets and stress granules induced under hyperosmotic stress showed distinct behavior and fine structures. We also found molecules that undergo autophagy degradation via p62-droplets. In addition, inhibition of LC3-dependent selective autophagy in hepatocytes resulted in the formation of p62-droplets with numerous small vesicles, where Keap1 was also localized. Together with other results, the p62-droplets serve as a platform for both autophagy and stress response signaling.

Academic Significance and Societal Importance of the Research Achievements

p62の遺伝子変異や封入体形成は、神経変性疾患、非アルコール性脂肪肝炎、肝細胞癌等で報告されている。これら封入体の形成は液－液相分離から始まると考えられるが、その病態生理学的な意義は不明であった。また、LC3に依存した選択的オートファジーの基質分解機序は明確ではなかった。今回、p62液滴の形成が選択的オートファジーの重要なプロセスを成すこと、その発動がストレス応答に直接関連していることを示した。本成果は、選択的オートファジーの新たな意義の解明につながると共に、p62液滴形成を制御することが上記疾患の病態の理解と、治療開発の糸口に繋がることを意味する。

Research Products

• [Journal Article] STING signalling is terminated through ESCRT-dependent microautophagy of vesicles originating from recycling endosomes. (2023)
  • Author(s): Kuchitsu Y, Mukai K, Uematsu R, Takaada Y, Shinojima A, Shindo R, Shoji T, Hamano S, Ogawa E, Sato R, Miyake K, Kato A, Kawaguchi Y, Nishitani-Isa M, Izawa K, Nishikomori R, Yasumi T, Suzuki T, Dohmae N, Uemura T, Barber GN, Arai H, Waguri S, Taguchi T.
  • Journal Title: Nature Cell Biology Volume: 25 Issue: 3 Pages: 453-466
  • DOI: 10.1038/s41556-023-01098-9
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3. (2022)
  • Author(s): Ishimura R, El-Gowily AH, Noshiro D, Komatsu-Hirota S, Ono Y, Shindo M, Hatta T, Abe M, Uemura T, Lee-Okada HC, Mohamed TM, Yokomizo T, Ueno T, Sakimura K, Natsume T, Sorimachi H, Inada T, Waguri S, Noda NN, Komatsu M.
  • Journal Title: Nature Communications Volume: 13 Issue: 1 Pages: 7857-7857
  • DOI: 10.1038/s41467-022-35501-0
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Lack of Hepatic Autophagy Promotes Severity of Liver Injury but Not Steatosis (2022)
  • Author(s): Ding WX, Ni HM, Waguri S, Komatsu M.
  • Journal Title: J Hepatol. Volume: 77(5) Issue: 5 Pages: 1458-1459
  • DOI: 10.1016/j.jhep.2022.05.015
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Loss of <i>Atg2b</i> and <i>Gskip</i> Impairs the Maintenance of the Hematopoietic Stem Cell Pool Size (2022)
  • Author(s): Sakai Shun-suke、Hasegawa Atsushi、Ishimura Ryosuke、Tamura Naoki、Kageyama Shun、Komatsu-Hirota Satoko、Abe Manabu、Ling Yiwei、Okuda Shujiro、Funayama Manabu、Kikkawa Mika、Miura Yoshiki、Sakimura Kenji、Narita Ichiei、Waguri Satoshi、Shimizu Ritsuko、Komatsu Masaaki
  • Journal Title: Molecular and Cellular Biology Volume: 42 Issue: 1 Pages: 1-17
  • DOI: 10.1128/mcb.00024-21
  • Peer Reviewed / Open Access
• [Journal Article] Clathrin adapters AP-1 and GGA2 support expression of epidermal growth factor receptor for cell growth. (2021)
  • Author(s): Takefumi Uemura, Takehiro Suzuki, Naoshi Dohmae, Satoshi Waguri
  • Journal Title: Oncogenesis Volume: 10 Issue: 11 Pages: 80-80
  • DOI: 10.1038/s41389-021-00367-2
  • Peer Reviewed / Open Access
• [Journal Article] Arf GTPase-activating proteins SMAP1 and AGFG2 regulate the size of Weibel-Palade bodies and exocytosis of von Willebrand factor (2021)
  • Author(s): Asano Watanabe, Hikari Hataida, Naoya Inoue, Kosuke Kamon, Keigo Baba, Kuniaki Sasaki, Rika Kimura, Honoka Sasaki, Yuka Eura, Wei-Fen Ni, Yuji Shibasaki, Satoshi Waguri, Koichi Kokame and Yoko Shiba
  • Journal Title: Biology open Volume: 10 Issue: 9
  • DOI: 10.1242/bio.058789
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] p62/SQSTM1-droplet serves as a platform for autophagosome formation and anti-oxidative stress response (2021)
  • Author(s): Kageyama S, Gudmundsson SR, Sou YS, Ichimura Y, Tamura N, Kazuno S, Ueno T, Miura Y, Noshiro D, Abe M, Mizushima T, Miura N, Okuda S, Motohashi H, Lee JA, Sakimura K, Ohe T, Noda NN, Waguri S, Eskelinen EL, Komatsu M.
  • Journal Title: Nature Communications Volume: 12 Issue: 1 Pages: 16-16
  • DOI: 10.1038/s41467-020-20185-1
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] ERdj8 governs the size of autophagosomes during the formation process (2020)
  • Author(s): Yamamoto Yo-hei、Kasai Ayano、Omori Hiroko、Takino Tomoe、Sugihara Munechika、Umemoto Tetsuo、Hamasaki Maho、Hatta Tomohisa、Natsume Tohru、Morimoto Richard I.、Arai Ritsuko、Waguri Satoshi、Sato Miyuki、Sato Ken、Bar-Nun Shoshana、Yoshimori Tamotsu、Noda Takeshi、Nagata Kazuhiro
  • Journal Title: Journal of Cell Biology Volume: 219 Issue: 8 Pages: 1-12
  • DOI: 10.1083/jcb.201903127
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Autophagy is involved in the sclerotic phase of systemic sclerosis (2020)
  • Author(s): Mori Tatsuhiko、Tamura Naoki、Waguri Satoshi、Yamamoto Toshiyuki
  • Journal Title: FUKUSHIMA JOURNAL OF MEDICAL SCIENCE Volume: 66 Issue: 1 Pages: 17-24
  • DOI: 10.5387/fms.2019-28
  • NAID: 130007834283
  • ISSN: 0016-2590, 2185-4610
  • Peer Reviewed / Open Access
• [Presentation] 高浸透圧ストレス下で形成される非膜性オルガネラの解析 (2023)
  • Author(s): 田村直輝、和栗聡
  • Organizer: 第128回日本解剖学会総会・全国学術総会
  • Invited
• [Presentation] ATG9Aは、オートファジー非依存的にクラミジア増殖に関与する (2023)
  • Author(s): 鈴木倫毅、和栗聡
  • Organizer: 第128回日本解剖学会総会・全国学術総会
• [Presentation] 高浸透圧ストレス下で形成される非膜性オルガネラの比較解析 (2022)
  • Author(s): 田村直輝、和栗聡
  • Organizer: 第74回日本細胞生物学会大会
• [Presentation] UBXD8flox/flox・CNP1-Creマウスの解析 (2022)
  • Author(s): 鈴木倫毅、和栗聡
  • Organizer: 日本顕微鏡学会第78回学術講演会
• [Presentation] Ultrastructural analysis on the process of isolation membrane formation during piecemeal mitophagy (2022)
  • Author(s): Ritsuko Arai, Shun-ichi Yamashita, Tatsuya Sugisaki, Wu Huajui, Tomotake Kanki, Satoshi Waguri
  • Organizer: 日本顕微鏡学会第78回学術講演会
  • Invited
• [Presentation] 高浸透圧ストレス下で形成される非膜性オルガネラの微細構造解析 (2022)
  • Author(s): 田村直輝、和栗聡
  • Organizer: 日本顕微鏡学会第78回学術講演会
• [Presentation] Characterization of p62-body induced under hyperosmotic stress in mammalian cells (2022)
  • Author(s): Naoki Tamura、Satoshi Waguri
  • Organizer: The 10th International Symposium on Autophagy
  • Int'l Joint Research
• [Presentation] Non-autophagic function of ATG9A is involved in the proliferation of Chlamydia trachomatis (2022)
  • Author(s): Michitaka Suzuki、Satoshi Waguri
  • Organizer: The 10th International Symposium on Autophagy
  • Int'l Joint Research
• [Presentation] 哺乳類細胞におけるSQSTM1/p62液滴の形成と分解 (2022)
  • Author(s): 和栗 聡、田村 直輝
  • Organizer: 第127回日本解剖学会総会・全国学術大会
  • Invited
• [Presentation] ピースミールマイトファジー隔離膜形成プロセスの微細形態学的解析 (2021)
  • Author(s): 荒井 律子、山下 俊一、杉崎 達也、Wu Huajui、神吉 智丈、和栗 聡
  • Organizer: 第44回日本分子生物学会年会
  • Invited
• [Presentation] Recycling endosome-localized clathrin adaptors AP-1 and GGA2 regulate cell surface expression of EGFR for cell growth (2021)
  • Author(s): 植村 武文、和栗 聡
  • Organizer: 第80回日本癌学会学術総会
• [Presentation] ピースミールマイトファジー隔離膜構築プロセスの電子顕微鏡解析 (2021)
  • Author(s): 荒井 律子、山下 俊一、杉崎 達也、Wu Huajui、神吉 智丈、和栗 聡
  • Organizer: 日本解剖学会第67回東北・北海道連合支部学術集会
• [Presentation] Ultrastructural analysis on the process of isolation membrane formation during piecemeal mitophagy (2021)
  • Author(s): Ritsuko Arai, Shun-ichi Yamashita, Tatsuya Sugisaki, Wu Huajui, Tomotake Kanki, Satoshi Waguri
  • Organizer: 126th Annual meeting of The Japanese Association of Anatomists
  • Invited
• [Presentation] 高浸透圧ストレス変動に応答したp62顆粒およびストレス顆粒の形成・消失機序 (2020)
  • Author(s): 田村直輝、和栗聡
  • Organizer: 第72回日本細胞生物学会大会
• [Presentation] 高浸透圧ストレス下における非膜性オルガネラの形成と分解 (2020)
  • Author(s): 田村直輝、和栗聡
  • Organizer: 第66回日本解剖学会 東北・北海道連合支部学術集会
• [Remarks] 福島県立医科大学 解剖・組織学講座 業績
  • URL: https://www.fmu.ac.jp/home/anatomy2/achieve_2022.html
• [Remarks] 福島県立医科大学 解剖・組織学講座
  • URL: https://www.fmu.ac.jp/home/anatomy2/