Abstract 1563 A novel FRET biosensor for visualizing membrane-mediated dimerization of PIP5K

The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of enzymes synthesize the majority of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) lipids in eukaryotes. Production of PI(4,5)P2 lipids is critical for the regulation of many cellular pathways including actin nucleation, endocytosis, Wnt signalosome formation, and ion channel gating. Despite the importance of PIP5K activity in eukaryotic cells, there remain open questions concerning the mechanisms that control PIP5K membrane localization and activity. Recently, we showed that PIP5K exist in a weak monomer-dimer equilibrium in solution but can shift to a dimeric state following membrane association. Dimerization potentiates PIP5K, increasing lipid kinase activity 20-fold. This provides a potential mechanism for rapid PI(4,5)P2 generation in cells. Additionally, blocking dimerization may be a key method cells leverage to disrupt PIP5K activity and PI(4,5)P2 production during cell signaling. Due to the transient nature of PIP5K dimerization, determining whether a protein or small molecule specifically blocks PIP5K dimerization is technically challenging. Here, we address this challenge by establishing a novel FÖrsterResonance Energy Transfer (FRET) biosensor to detect and quantify PIP5K dimerization on supported lipid bilayer technology using Total Internal Reflection Fluorescence Microscopy (TIRF-M). This FRET assay allows the frequency and duration of PIP5K dimerization to be quantified with high resolution. Using this FRET biosensor, we show that human PIP5K paralogs (α, β, and γ) can heterodimerize. The creation of this PIP5K dimerization FRET biosensor establishes a novel assay for examining how proteins and peptides modulate membrane-mediated dimerization of PIP5K, which will be critical for elucidating the mechanisms that control cellular PI(4,5)P2 lipid homeostasis in the future. We'd like to thank the NSF CAREER Award (S.D.H.), NIH biophysics training grant (B.R.D.), and the University of Oregon's John Keana Fellowship (B.R.D.) for supporting this research.