Shimada Mikio, Matsumoto Yoshihisa, Otsuka Kensuke
Zero-Carbon Energy Laboratory, Institute of Integrated Research, Institute of Science Tokyo, Tokyo, Japan.
Zero-Carbon Energy Laboratory, Institute of Integrated Research, Institute of Science Tokyo, Tokyo, Japan.
J Biol Chem. 2025 Jun 3;301(7):110328. doi: 10.1016/j.jbc.2025.110328.
Ionizing radiation induces DNA double-strand breaks, which compromise genomic stability and trigger programmed cell death. The cell's differentiation state modulates DNA damage response (DDR) mechanisms, including DNA repair pathways and cell cycle regulation. The accumulation of p53-binding protein 1 (53BP1) at DSB sites serves as a reliable biomarker for such damage. Previously, we developed a fluorescent live-cell imaging system, termed "Focicle," which monitors 53BP1 foci dynamics and cell cycle phases, utilizing fluorescent ubiquitination-based cell cycle indicators (hCdt1 and hGmnn) in mouse cells. In the current study, to investigate the relationship between differentiation state and DDR activity, we generated Focicle-integrated human induced pluripotent stem cells and further differentiated them into neural progenitors and mature neurons using an optimized Focicle cassette adapted for human cell lines. Using laser microirradiation, we observed differentiation-dependent alterations in 53BP1 foci accumulation dynamics and cell cycle progression. The newly established Focicle system represents a valuable tool for elucidating DDR activity during organ development.
电离辐射会导致DNA双链断裂，这会损害基因组稳定性并引发程序性细胞死亡。细胞的分化状态会调节DNA损伤反应（DDR）机制，包括DNA修复途径和细胞周期调控。p53结合蛋白1（53BP1）在双链断裂位点的积累是此类损伤的可靠生物标志物。此前，我们开发了一种名为“Focicle”的荧光活细胞成像系统，该系统利用基于荧光泛素化的细胞周期指示剂（hCdt1和hGmnn）在小鼠细胞中监测53BP1焦点动态和细胞周期阶段。在当前研究中，为了研究分化状态与DDR活性之间的关系，我们构建了整合Focicle的人诱导多能干细胞，并使用适用于人类细胞系的优化Focicle盒将其进一步分化为神经祖细胞和成熟神经元。通过激光微照射，我们观察到53BP1焦点积累动态和细胞周期进程中依赖分化的变化。新建立的Focicle系统是阐明器官发育过程中DDR活性的宝贵工具。
