Quantitative Biology Research Group, Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan.
Division of Quantitative Biology, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan.
ACS Chem Biol. 2020 Nov 20;15(11):2896-2906. doi: 10.1021/acschembio.0c00477. Epub 2020 Nov 9.
Optogenetics is a powerful technique using photoresponsive proteins, and the light-inducible dimerization (LID) system, an optogenetic tool, allows to manipulate intracellular signaling pathways. One of the red/far-red responsive LID systems, phytochrome B (PhyB)-phytochrome interacting factor (PIF), has a unique property of controlling both association and dissociation by light on the second time scale, but PhyB requires a linear tetrapyrrole chromophore such as phycocyanobilin (PCB), and such chromophores are present only in higher plants and cyanobacteria. Here, we report that we further improved our previously developed PCB synthesis system (SynPCB) and successfully established a stable cell line containing a genetically encoded PhyB-PIF LID system. First, four genes responsible for PCB synthesis, namely, , , , and , were replaced with their counterparts derived from thermophilic cyanobacteria. Second, Fnr was truncated, followed by fusion with Fd to generate a chimeric protein, tFnr-Fd. Third, these genes were concatenated with P2A peptide cDNAs for polycistronic expression, resulting in an approximately 4-fold increase in PCB synthesis compared with the previous version. Finally, we incorporated the PhyB, PIF, and SynPCB system into drug inducible lentiviral and transposon vectors, which enabled us to induce PCB synthesis and the PhyB-PIF LID system by doxycycline treatment. These tools provide a new opportunity to advance our understanding of the causal relationship between intracellular signaling and cellular functions.
光遗传学是一种使用光响应蛋白和光诱导二聚化（LID）系统的强大技术，光遗传学工具允许操纵细胞内信号通路。红/远红响应 LID 系统之一的光敏色素 B（PhyB）-光敏色素相互作用因子（PIF）具有独特的性质，可以通过光在第二时间尺度上控制结合和解离，但 PhyB 需要线性四吡咯发色团，如藻蓝胆素（PCB），而这种发色团仅存在于高等植物和蓝细菌中。在这里，我们报告说，我们进一步改进了我们之前开发的 PCB 合成系统（SynPCB），并成功建立了一个含有遗传编码的 PhyB-PIF LID 系统的稳定细胞系。首先，将负责 PCB 合成的四个基因，即 、 、 、 和 ，替换为来自嗜热蓝细菌的对应基因。其次，Fnr 被截断，然后与 Fd 融合生成嵌合蛋白 tFnr-Fd。第三，这些基因与 P2A 肽 cDNA 串联进行多顺反子表达，与前一版本相比，PCB 合成增加了约 4 倍。最后，我们将 PhyB、PIF 和 SynPCB 系统整合到药物诱导的慢病毒和转座子载体中，这使我们能够通过强力霉素处理诱导 PCB 合成和 PhyB-PIF LID 系统。这些工具为深入了解细胞内信号与细胞功能之间的因果关系提供了新的机会。
