Detection of PIP₂ distributions in biological membranes using a peptide-based sensor

Organization and composition of the plasma membrane are important modulators of many cellular programs. Phosphatidylinositol phosphate (PIP) lipids are low-abundance membrane constituents with different arrangements of phosphate groups around an inositol head group, regulating many signaling pathways. Numerous strategies have been developed to detect and track PIP species to monitor their clustering, mobility, and interactions with binding partners. We implement a peptide-based, ratiometric sensor for the detection of PI(4,5)P₂ lipids in reconstituted membrane systems that permit absolute quantification of PI(4,5)P₂ densities down to physiological levels less than 4 mol per cent. The sensor is membrane-permeable and easily transferable to measurements in living cells. Application of calibrated sensors to cells expressing common mutations in the small GTPase, Ras, showed a reshaping of surface PI(4,5)P₂ levels and distributions in a mutation-specific manner. Brief treatment of G12C mutant Ras cells with the specific inhibitor, Sotorasib, resulted in alterations to surface PI(4,5)P₂ arrangements that resemble the wild-type (WT) Ras. Thus, the rapid redistribution of PI(4,5)P₂ lipids upon drug treatment emphasizes the tight coupling between membrane composition, organization, and downstream signaling outcomes. Tools and strategies to monitor membrane composition alongside cellular behaviors could provide pipelines to characterize therapeutics and improve the mechanistic understanding of how protein-lipid coupling drives cellular programs.