Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, Republic of Korea.
Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon, 34126, Republic of Korea.
Nat Commun. 2019 Jan 14;10(1):211. doi: 10.1038/s41467-018-08217-3.
Ras and Rho small GTPases are critical for numerous cellular processes including cell division, migration, and intercellular communication. Despite extensive efforts to visualize the spatiotemporal activity of these proteins, achieving the sensitivity and dynamic range necessary for in vivo application has been challenging. Here, we present highly sensitive intensiometric small GTPase biosensors visualizing the activity of multiple small GTPases in single cells in vivo. Red-shifted sensors combined with blue light-controllable optogenetic modules achieved simultaneous monitoring and manipulation of protein activities in a highly spatiotemporal manner. Our biosensors revealed spatial dynamics of Cdc42 and Ras activities upon structural plasticity of single dendritic spines, as well as a broad range of subcellular Ras activities in the brains of freely behaving mice. Thus, these intensiometric small GTPase sensors enable the spatiotemporal dissection of complex protein signaling networks in live animals.
Ras 和 Rho 小 GTPases 对于许多细胞过程至关重要，包括细胞分裂、迁移和细胞间通讯。尽管已经做出了大量努力来可视化这些蛋白质的时空活性，但要实现体内应用所需的灵敏度和动态范围仍然具有挑战性。在这里，我们提出了高灵敏度的强度测定法小 GTPase 生物传感器，可在体内单个细胞中可视化多种小 GTPase 的活性。红移传感器与蓝光可控光遗传学模块相结合，以高度时空方式实现了蛋白质活性的同时监测和操纵。我们的生物传感器揭示了单个树突棘结构可塑性时 Cdc42 和 Ras 活性的空间动力学，以及自由活动小鼠大脑中广泛的亚细胞 Ras 活性。因此，这些强度测定法小 GTPase 传感器能够在活体动物中对复杂蛋白质信号网络进行时空剖析。
