Green genetically encoded IP3 biosensor for hierarchical analysis of its signaling network

ABSTRACT Inositol 1,4,5-trisphosphate (IP 3 ) is a key second messenger that regulates diverse physiological processes. Visualization of IP 3 dynamics in living cells is therefore important for understanding its signaling processes. In this study, we developed genetically encoded green fluorescent IP 3 biosensors named Green iPenguins with distinct half-maximal effective concentrations (EC 50 ) for IP 3 , enabling detection of IP 3 signals over a range of concentrations. The biosensors displayed more than a 4-fold increase in fluorescence intensity upon IP 3 and showed high specificity for IP 3 over structurally related molecules. When expressed in HEK293T cells, the biosensors enabled visualization of IP 3 dynamics involved in different signaling pathways. They were also compatible with dual-color imaging, allowing simultaneous monitoring of IP 3 together with cAMP or Ca 2+ signals. In addition, the hierarchical relationship between IP 3 and Ca 2+ signaling was visualized, providing insight into the temporal relationship between these two second messengers. The biosensors are expected to facilitate future studies of physiological processes involving IP 3 signaling networks.