2015 Fiscal Year Annual Research Report
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
| Project/Area Number |
15J08196
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| Research Institution | Kyoto University |
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Principal Investigator |
三浦 晴子 京都大学, 生命科学研究科, 特別研究員(DC1)
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| Project Period (FY) |
2015-04-24 – 2018-03-31
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| Keywords | cell death / p38 / JNK / single cell analysis |
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| Outline of Annual Research Achievements |
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.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
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.
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| Strategy for Future Research Activity |
(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.
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