Fluorescent Biosensors for Multiplexed Imaging of Phosphoinositide Dynamics

Phosphoinositides constitute a critical family of lipids that regulate numerous cellular processes. Phosphatidylinositol 4,5-bisphosphate (PIP₂) is arguably the most important plasma membrane phosphoinositide and is involved in regulating diverse processes. It is also the precursor of phosphatidylinositol 3,4,5-trisphosphate (PIP₃), which is critical for growth factor signaling, as well as membrane polarization and dynamics. Studying these lipids remains challenging, because of their compartmentalized activities and location-dependent signaling profiles. Here, we introduce several new genetically encoded fluorescent biosensors, including FRET-based and dimerization-dependent fluorescent protein (ddFP)-based biosensors, that enable real-time monitoring of PIP₂ levels in live cells. In addition, we developed a red fluorescent biosensor for 3-phosphoinositides that can be co-imaged with the green PIP₂ indicator. Simultaneous visualization of the dynamics of PIP₂ and 3-phosphoinositides in the same cell shows that plasma membrane PIP₃ formation upon EGF stimulation is coupled to a decrease in the local pool of PIP₂.