Abstract

The dynamic composition of the cell membrane lipid bilayer is critical for numerous cellular processes, including signal transduction, membrane trafficking, and cytoskeletal organization. Monitoring the spatio-temporal dynamics of specific lipid species, such as phosphatidylinositol phosphates (PIPs) and phosphatidic acid, remains a significant challenge in live-cell imaging. This study addresses this need by developing a panel of novel **recombinant protein biosensors** designed for high-specificity, real-time detection of key cell membrane lipids. We engineered genetically-encoded lipid-binding domains, derived from established signaling proteins, and fused them to bright, photostable fluorescent proteins. Through rigorous *in vitro* binding assays and live-cell microscopy, we demonstrate that these biosensors exhibit superior affinity and selectivity for their target lipids compared to existing probes. Key findings include the visualization of rapid, localized changes in PI(4,5)P₂ and PI(3,4,5)P₃ concentrations at the plasma membrane following growth factor stimulation, revealing previously uncharacterized kinetic details of lipid turnover. Furthermore, the biosensors successfully tracked lipid reorganization during endocytosis and cell migration. These robust, non-invasive tools provide a powerful new methodology for dissecting the complex roles of membrane lipids in cellular physiology and pathology, offering a valuable resource for cell biology and drug discovery research.