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 due to their compartmentalized activities and location-dependent signaling profiles. Here we introduce several new genetically encoded fluorescent biosensors that enable real-time monitoring of PIP2 levels in live cells, 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 dynamics of...