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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 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.