ACS Chem. Biol. 15, 33–38 (2020)

Phosphoinositides (PIs) such as PI(4,5)P2 are important phospholipids that act as anchoring molecules for signaling proteins at the plasma membrane (PM) and as second messengers downstream of growth factor receptors, as is the case for PI(3,4,5)P3. To expand the PI biosensor toolbox, Hertel et al. generated PlcR, whose sensing unit, which is flanked by FRET donor and acceptor fluorophores, is based on a PI(4,5)P2-binding PH domain and a negatively charged pseudoligand that can bind to the PI-binding pocket of this domain. Displacement of the pseudoligand by PI(4,5)P2 induces a conformational change that the authors could monitor by real-time FRET changes. Using the same sensing unit, they next generated dPlcR, which has at its ends a dimerization-dependent green fluorescent protein pair that increases fluorescence in the presence of PI(4,5)P2. Similarly, the authors generated dInPAkt with a dimerization-dependent red fluorescent protein pair to monitor 3-phosphoinositides, including PI(3,4)P2 and PI(3,4,5)P3, generated by PI3K downstream of growth factor receptors. The combination of dlnPAkt with dPlcR led to the conclusion that PI(3,4,5)P3 production is correlated with PI(4,5)P2 depletion and that a substantial fraction of PI(4,5)P2 is unavailable to PI3K. These tools should prove useful for uncovering real-time PI signaling dynamics in living cells.