2016 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 | p38 / JNK / cell death / crosstalk / live cell imaging |
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| Outline of Annual Research Achievements |
The stress activated protein kinases p38 and JNK play important roles in the cellular response to diverse stress conditions. Although p38 and JNK have been studied for decades, it is still unknown how they regulate each other and how they regulate cell fate decisions at the single cell level.
To address these issues, we performed simultaneous imaging of p38 and JNK activities in single living cells using our previously developed multiplexed fluorescent imaging system. Interestingly, we found that p38 negatively regulated JNK in all tested stress conditions, which was mediated by both transcriptional and posttranslational mechanisms.
Furthermore, p38 and JNK activities seem to have both, pro-death and pro-survival roles, which probably depends on the timing and strength of the activities.
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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
I succeeded in multiplexed imaging of JNK, p38, and also caspase 8 activities in single living cells. We used this imaging system in human cancer cell lines stably expressing all reporters and analyzed their activities in response to inflammatory inputs, stresses, and chemotherapeutic drugs. By specific image analysis programs, we successfully extracted activity time courses from the raw image data of each each reporter. Perturbation experiments using inducible expression, small molecule inhibitors, and CRISPR Cas9 knock out revealed regulatory mechanisms between these pathways and implicated complex roles of JNK and p38 in cell death. To study the dual roles of JNK and p38, we performed gene expression studies. In summary, my research made good progress according to the initial plans.
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| Strategy for Future Research Activity |
In future, I want to address following points.
(1) Statistical analysis of the p38 and JNK activity time-courses will extract parameters which are determinant for cell death.
(2) A synthetic and specific activation system of p38 and JNK should be developed, e.g. based on optogenetics. This will help to clarify the role of p38 and JNK, in especially whether their activities are sufficient to induce cell death.
(3) I want to titrate p38 and JNK activation by drug titration or synthetic activation and analyze the amount of induced cell death, in order to find a putative threshold of p38 and JNK activities, which separates survival and cell death.
(4) Finally, I will summarize my results in two papers, which I will submit to peer-reviewed scientific journals.
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