Intensiometric biosensors visualize the activity of multiple small GTPases in vivo

Abstract

Discovery and engineering of fluorescent proteins have largely contributed to the technologies that trace dynamic changes of intracellular protein activities. Such effort is remarkable since isolating the function of a protein in the homeostasized nature of signaling network is critical when tracking back abnormal signaling processes in the disease state. 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 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 novel intensiometric small GTPase sensors enable the spatiotemporal dissection of complex protein signaling networks in live animals.