Optogenetic phospholipase C for measuring phosphoinositide turnover and DAG generation

Phospholipase C plays a pivotal role in cellular signaling by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PIP₂) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃), thereby initiating lipid- and calcium-mediated signaling cascades. Conventional approaches to studying PLC activity often rely on receptor activation or pharmacological stimulation, which can introduce variability and unintended signaling crosstalk. To improve these limitations, this chapter presents a dual-imaging strategy using opto-PLCβ, a light-activated PLC variant, enabling precise temporal control of phosphoinositide turnover in live cells. By integrating optogenetic stimulation with DAG- and PIP₂-specific fluorescent biosensors and confocal microscopy, this method allows real-time, quantitative analysis of lipid turnover kinetics and spatial dynamics, offering enhanced resolution and reliability compared to conventional techniques.