Reversible Optogenetic Control of Subcellular Protein Localization in a Live Vertebrate Embryo.
Reversible Optogenetic Control of Subcellular Protein Localization in a Live Vertebrate Embryo.
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DOI：
10.1016/j.devcel.2015.12.011
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发表时间：
2016-01-11
影响因子：
11.8
通讯作者：
Clarke JDW
中科院分区：
文献类型：
--
作者：
Buckley CE;Moore RE;Reade A;Goldberg AR;Weiner OD;Clarke JDW
我们证明了光敏色素系统的效用，可以快速和可逆地将蛋白质招募到活的脊椎动物胚胎中特定细胞内的特定亚细胞区域。利用光敏色素系统的光诱导异二聚化已被用作分析培养中单细胞的信号通路的有力工具，但尚未被用于可逆地操纵多细胞生物体中蛋白质的精确亚细胞位置。在这里，我们报告了使用该系统在体内操纵蛋白质所需的实验条件。作为原理的证明，我们证明了我们可以在神经管和胚胎的包膜层上皮细胞中以高时间和空间精度操纵顶端极性蛋白PARD3的定位。我们对光遗传成分表达和发色团纯化和传递的优化应该会显著降低在其他多细胞生物体中建立这个强大的光遗传系统的障碍。光敏色素系统已经被优化用于多细胞生物体中，蛋白质募集可以被严格控制在特定的亚细胞区域，蛋白质募集发生具有高结合和逆转动力学，顶端极性蛋白PARD3的亚细胞定位是被操纵的，光敏色素系统是用于操纵蛋白质定位的有力工具。在这里，巴克利等人。优化活斑马鱼胚胎中的光敏色素系统，并以高空间和时间分辨率展示快速和可逆的蛋白质招募到特定细胞内的特定亚细胞区域。
We demonstrate the utility of the phytochrome system to rapidly and reversibly recruit proteins to specific subcellular regions within specific cells in a living vertebrate embryo. Light-induced heterodimerization using the phytochrome system has previously been used as a powerful tool to dissect signaling pathways for single cells in culture but has not previously been used to reversibly manipulate the precise subcellular location of proteins in multicellular organisms. Here we report the experimental conditions necessary to use this system to manipulate proteins in vivo. As proof of principle, we demonstrate that we can manipulate the localization of the apical polarity protein Pard3 with high temporal and spatial precision in both the neural tube and the embryo’s enveloping layer epithelium. Our optimizations of optogenetic component expression and chromophore purification and delivery should significantly lower the barrier for establishing this powerful optogenetic system in other multicellular organisms. The phytochrome system has been optimized for use within multicellular organisms Protein recruitment can be tightly controlled to a specific subcellular region Protein recruitment occurs with high binding and reversal kinetics The subcellular localization of the apical polarity protein Pard3 is manipulated The phytochrome system is a powerful tool that is used to manipulate protein localization. Here, Buckley et al. optimize the phytochrome system in live zebrafish embryos and demonstrate rapid and reversible protein recruitment to specific subcellular regions within specific cells, at a high spatial and temporal resolution.