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2015 年度 実績報告書

イメージングと時系列統計解析による細胞死制御機構の定量的な解析

研究課題

研究課題/領域番号 15J08196
研究機関京都大学

研究代表者

三浦 晴子  京都大学, 生命科学研究科, 特別研究員(DC1)

研究期間 (年度) 2015-04-24 – 2018-03-31
キーワードcell death / p38 / JNK / single cell analysis
研究実績の概要

The stress activated protein kinases (SAPKs), namely JNK and p38, are crucial determinants of cell fate, when cells are exposed to environmental stress signals. Although the SAPKs and their role in cell death have been extensively studied, it is still unclear when and how they cooperatively regulate cell death at the single cell level. To address this issue, we developed a multiplexed live-cell imaging system to simultaneously monitor JNK and p38 activities and caspase activation at the single cell level. Cell lines stably expressing all reporters were established. Simultaneous imaging revealed that exposure to various stresses resulted in different kinetics of JNK and p38 activation. Interestingly, p38 inhibition enhanced JNK activation in all tested stress conditions, suggesting that p38 negatively regulated JNK. This crosstalk inhibition from p38 to JNK was also observed in DNA damage induced cell death. Upon etoposide treatment, sustained p38 activity correlated with cell death, while pulsatile JNK activation was observed in dying cells. However, when p38 was inhibited, sustained JNK activation was induced by etoposide and correlated with cell death. These results indicate that crosstalk inhibition of JNK by p38 might play an important role for the balance between life and death. Consistently, co-treatment with p38 and JNK inhibitors reduced cell death. In contrast, knockout of p38a or JNK1/2 resulted in increased cell death, which might be due to long-term changes in gene expression downstream of p38 and JNK.

現在までの達成度 (区分)
現在までの達成度 (区分)

2: おおむね順調に進展している

理由

According to the initial plan, I succeeded in the preparation of reporters for stress and cell death related signaling molecules, establishment of cell lines which simultaneously express all reporters, linear unmixing, and multicolor imaging.
I constructed translocation based reporters for p38 and JNK kinases, a nuclear marker based on nuclear localization sequences, and FRET biosensors for caspase 3 and 8. These probes slightly differ from the initially planned probes, but proved very specific and useful for the study of stress signaling dynamics during cell death.

今後の研究の推進方策

(1) Further multicolor imaging will be performed, when cell death is induced by various stress conditions and anticancer drugs, and an automated imaging analysis tool should be developed.
(2) Perturbation studies will target the molecular mechanism of the cross inhibition of JNK by p38 and the differential effects of p38 and JNK revealed by inhibitor and knockout experiments.
(3) The in (1) obtained signaling dynamics will be studied by statistical analysis to extract parameters which are determinant for cell death. Furthermore, a mathematical model should be established to validate the imaging data.

  • 研究成果

    (4件)

すべて 2016

すべて 学会発表 (4件) (うち国際学会 1件)

  • [学会発表] Single cell analysis of stress signaling dynamics in the life-death decision2016

    • 著者名/発表者名
      Haruko Miura
    • 学会等名
      The 68th Annual Meeting of the Japan Society for Cell Biology
    • 発表場所
      Kyoto Terrsa, Kyoto, Japan
    • 年月日
      2016-06-15 – 2016-06-17
  • [学会発表] Single cell analysis of stress signaling dynamics in a life-death decision2016

    • 著者名/発表者名
      Haruko Miura
    • 学会等名
      FASEB Conference, Cell Signaling in Cancer: from Mechanisms to Therapy
    • 発表場所
      Base Village Conference Center, Snowmass Village, CO, USA
    • 年月日
      2016-06-05 – 2016-06-10
    • 国際学会
  • [学会発表] Single cell analysis of stress signaling dynamics in the life-death decision2016

    • 著者名/発表者名
      Haruko Miura
    • 学会等名
      生命動態の分子メカニズムと数理, 生命動態システム科学四拠点・CREST・PRESTO・QBiC 合同シンポジウム
    • 発表場所
      Sheraton Hotel Hiroshima, Hiroshima, Japan
    • 年月日
      2016-03-25 – 2016-03-26
  • [学会発表] Single cell dynamics of stress signaling in the life-death decision2016

    • 著者名/発表者名
      Haruko Miura
    • 学会等名
      The 14th International Student Seminar, Kyoto University
    • 発表場所
      Kyoto University Shiran-kaikan, Kyoto, Japan
    • 年月日
      2016-03-10 – 2016-03-11

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公開日: 2016-12-27  

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