Live cell imaging of regulated necrosis

2018 Fiscal Year Final Research Report

• Project/Area Number: 16K01378
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Biomedical engineering/Biomaterial science and engineering
• Research Institution: Toho University
• Principal Investigator: MURAI Shin 東邦大学, 医学部, 助教 (90287540)
• Co-Investigator(Kenkyū-buntansha): 中野 裕康 東邦大学, 医学部, 教授 (70276476)
• Research Collaborator: YAMAGUCHI Yoshifumi
• Project Period (FY): 2016-04-01 – 2019-03-31
• Keywords: MLKL / RIPK3 / Necroptosis / FRET

Outline of Final Research Achievements

Regulated necrosis is a regulated form of necrosis that depends on RIPK3 and MLKL. To monitor the induction of regulated necrosis, we have designed FRET biosensor (KLN) which inserted the linker sequence derived from MLKL between CFP and YFP. Since KLN expression suppressed the cell proliferation, the KLN linker sequence was modified. As the result, FRET biosensor without cytotoxicity was developed and termed as SMART. SMART can monitor regulated necrosis, but not apoptosis or necrosis in vitro, indicating that SMART is a specific sensor to regulated necrosis. FRET was not observed by the knockdown of RIPK3 expression or the inhibition of RIPK3 activity, suggesting that SMART can monitor RIPK3 activity which is required the induction of regulated necrosis. Since the unphosphorylated SMART mutant can also monitor RIPK3 activation by its FRET, it was concluded that FRET of SMART is required for the interaction of RIPK3 but not phosphorylation by RIPK3.

Free Research Field

細胞死　細胞生物学　生化学

Academic Significance and Societal Importance of the Research Achievements

疾患により損傷をうけた組織では、アポトーシスや計画的ネクローシスに加え、貪食されずに残った死細胞による二次的ネクローシス、ウイルス感染によるパイロトーシスや膜脂質の酸化によるフェロトーシスなど様々な細胞死が混在していることが予想される。病態の増悪を抑制するためには、それぞれの細胞死に特異的な阻害剤が有用である。本研究の成果により、計画的ネクローシスを他の細胞死と区別して検出することで、効果的な治療薬の選択が可能となる。さらに薬剤投与による計画的ネクローシスの抑制効果の解析が可能であるため、ドラッグ・スクリーニングによる新規治療薬の開発をはじめとする医療面への貢献も期待できる。