Konishi Yoshinobu, Terai Kenta, Furuta Yasuhide, Kiyonari Hiroshi, Abe Takaya, Ueda Yoshihiro, Kinashi Tatsuo, Hamazaki Yoko, Takaori-Kondo Akifumi, Matsuda Michiyuki
Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan; Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan.
iScience. 2018 Dec 21;10:98-113. doi: 10.1016/j.isci.2018.11.025. Epub 2018 Nov 20.
Extracellular signal-regulated kinase (ERK) plays critical roles in T cell development in the thymus. Nevertheless, the dynamics of ERK activity and the role of ERK in regulating thymocyte motility remain largely unknown due to technical limitations. To visualize ERK activity in thymocytes, we here developed knockin reporter mice expressing a Förster/fluorescence resonance energy transfer (FRET)-based biosensor for ERK from the ROSA26 locus. Live imaging of thymocytes isolated from the reporter mice revealed that ERK regulates thymocyte motility in a subtype-specific manner. Negative correlation between ERK activity and motility was observed in CD4/CD8 double-positive thymocytes and CD8 single-positive thymocytes, but not in CD4 single-positive thymocytes. Interestingly, however, the temporal deviations of ERK activity from the average correlate with the motility of CD4 single-positive thymocytes. Thus, live-cell FRET imaging will open a window to understanding the dynamic nature and the diverse functions of ERK signaling in T cell biology.
细胞外信号调节激酶（ERK）在胸腺中T细胞发育过程中发挥着关键作用。然而，由于技术限制，ERK活性的动态变化以及ERK在调节胸腺细胞运动性方面的作用仍 largely未知。为了可视化胸腺细胞中的ERK活性，我们在此开发了敲入报告基因小鼠，该小鼠从ROSA26位点表达一种基于Förster/荧光共振能量转移（FRET）的ERK生物传感器。对从报告基因小鼠中分离出的胸腺细胞进行实时成像显示，ERK以亚型特异性方式调节胸腺细胞运动性。在CD4/CD8双阳性胸腺细胞和CD8单阳性胸腺细胞中观察到ERK活性与运动性之间呈负相关，但在CD4单阳性胸腺细胞中未观察到。然而，有趣的是，ERK活性与平均值的时间偏差与CD4单阳性胸腺细胞的运动性相关。因此，活细胞FRET成像将为理解ERK信号在T细胞生物学中的动态性质和多样功能打开一扇窗口。
