Lataster Levin, Huber Hanna Mereth, Böttcher Christina, Föller Stefanie, Takors Ralf, Radziwill Gerald
Faculty of Biology, Institute of Biology II, University of Freiburg, 79098 Freiburg, Germany.
Institute of Biochemical Engineering, University of Stuttgart, 70569 Stuttgart, Germany.
Biology (Basel). 2023 Aug 31;12(9):1194. doi: 10.3390/biology12091194.
The progression through the cell cycle phases is driven by cyclin-dependent kinases and cyclins as their regulatory subunits. As nuclear protein, the cell cycle inhibitor p21/CDKN1A arrests the cell cycle at the growth phase G1 by inhibiting the activity of cyclin-dependent kinases. The G1 phase correlates with increased cell size and cellular productivity. Here, we applied an optogenetic approach to control the subcellular localization of p21 and its nuclear functions. To generate light-controllable p21, appropriate fusions with the blue light switch cryptochrome 2/CIBN and the AsLOV-based light-inducible nuclear localization signal, LINuS, were used. Both systems, p21-CRY2/CIB1 and p21-LINuS, increased the amounts of cells arrested in the G1 phase correlating with the increased cell-specific productivity of the reporter-protein-secreted alkaline phosphatase. Varying the intervals of blue LED light exposure and the light dose enable the fine-tuning of the systems. Light-controllable p21 implemented in producer cell lines could be applied to steer the uncoupling of cell proliferation and cell cycle arrest at the G1 phase optimizing the production of biotherapeutic proteins.
细胞周期各阶段的进程由细胞周期蛋白依赖性激酶以及作为其调节亚基的细胞周期蛋白驱动。作为一种核蛋白，细胞周期抑制剂p21/CDKN1A通过抑制细胞周期蛋白依赖性激酶的活性，使细胞周期停滞在生长阶段G1期。G1期与细胞体积增大和细胞生产力提高相关。在此，我们应用光遗传学方法来控制p21的亚细胞定位及其核功能。为了生成光控p21，我们使用了与蓝光开关隐花色素2/CIBN以及基于AsLOV的光诱导核定位信号LINuS进行适当融合。p21-CRY2/CIB1和p21-LINuS这两个系统都增加了停滞在G1期的细胞数量，这与报告蛋白分泌型碱性磷酸酶的细胞特异性生产力提高相关。改变蓝光发光二极管光照的间隔时间和光剂量能够对系统进行微调。在生产细胞系中实现的光控p21可用于调控细胞增殖与G1期细胞周期停滞的解偶联，从而优化生物治疗蛋白的生产。
