Park Hyerim, Lee Sangkyu, Heo Won Do
Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea.
Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon, Republic of Korea.
Methods Mol Biol. 2016;1408:363-76. doi: 10.1007/978-1-4939-3512-3_25.
Optogenetic modules that use genetically encoded elements to control protein function in response to light allow for precise spatiotemporal modulation of signaling pathways. As one of optical approaches, LARIAT (Light-Activated Reversible Inhibition by Assembled Trap) is a unique light-inducible inhibition system that reversibly sequesters target proteins into clusters, generated by multimeric proteins and a blue light-induced heterodimerization module. Here we present a method based on LARIAT for optical inhibition of targets in living mammalian cells. In the protocol, we focus on the inhibition of proteins that modulate cytoskeleton and cell cycle, and describe how to transfect, conduct a photo-stimulation, and analyze the data.
利用基因编码元件响应光来控制蛋白质功能的光遗传学模块，能够对信号通路进行精确的时空调节。作为光学方法之一，LARIAT（光激活组装陷阱可逆抑制）是一种独特的光诱导抑制系统，它通过多聚体蛋白和蓝光诱导的异源二聚化模块，将靶蛋白可逆地隔离成簇。在此，我们介绍一种基于LARIAT的方法，用于对活的哺乳动物细胞中的靶标进行光学抑制。在该方案中，我们重点关注对调节细胞骨架和细胞周期的蛋白质的抑制，并描述转染、进行光刺激以及分析数据的方法。
