Imaging analysis of stress signaling and spatio-temporal dynamics of MAPKKK activity

(1)建立了自动化FRET分析系统，通过该系统可以同时实时成像来自1，000多个细胞的细胞内MAPK活性。(2)通过将MAPK活性FRET报告基因给予亚细胞组分，测定应激激活的MAPK活性的空间分布。我发现，核毒应激，紫外线，高渗应激和白细胞介素-1诱导p38 MAPK在细胞质中的激活。(3)在本研究之前，由于缺乏以良好的定量性确定个体细胞应答的工具，哺乳动物细胞中的个体细胞应答尚未得到很好的理解。使用自动FRET系统和MAPK活性指示剂，我发现在任何剂量下，单个细胞中的核糖毒性应激反应与细胞群体的平均反应相似。

(1) Automated FRET analysis system was established by which intracellular MAPK activity from more than 1,000 cells can be imaged simultaneously in real-time. (2) By administrating MAPK activity FRET reporter to subcellular components, spatial distribution of stress-activated MAPK activity was determined. I found that ribotoxic stress, UV, hyperosmotic stress, and interleukin-1 induces p38 MAPK activation in cytoplasm. (3) Before this study, individual cellular response in mammalian cells has been not well understood because of the lack of the tools to determine individual cellular response with a good quantitativity. Using automated FRET system and MAPK activity indicator, I identified that ribotoxic stress response in individual cell was similar to average response of cell population at any doses applied.