Abstract 2361 Modulation and surveillance of phosphatidylinositol 3,5-bisphosphate in live cells using novel molecular tools

Phosphatidylinositol 3,5-bisphosphate PI(3,5)P2 is a minor phosphoinositide lipid produced from phosphatidylinositol 3-phosphate (PI3P) by the conserved lipid kinase, PIKfyve (Fab1 in yeast). PIKfyve is part of a larger molecular complex that is formed with two additional proteins, the inositol lipid phosphatase, Sac3 (Fig4 in yeast), and the scaffolding protein, ArPIKfyve (Vac14 in yeast). Mutations in the proteins forming this PIKfyve/ArPIKfyve/Sac3 complex are associated with various human diseases, including COVID-SARS2. At the steady-state, PI(3,5)P2 is one of the least abundant inositol lipids, but its levels show a prominent increase after hyperosmotic shock, which was first described in yeast, but also subsequently observed in plant and mammalian cells. In order to better understand the role of PI(3,5)P2 in the control of cellular functions, we developed new molecular tools that allow for the rapid generation of PI(3,5)P2 within specific subcellular membrane compartments. In this presentation, we will show an engineered minimal catalytic fragment of PIKfyve and demonstrate its robust catalytic activity, which allows for the rapid localized conversion of resident PI3P to PI(3,5)P2 when recruited to Rab5 endosomes. Utilizing the recently identified PI(3,5)P2-sensitive PX domain of the Dictyostelium protein, SNXA, we were able to monitor the real-time generation of PI(3,5)P2 in live HEK293A cells using both confocal microscopy and bioluminescence resonance energy transfer (BRET)-based kinetic analyses. Overall, these unique molecular tools will allow us to identify novel molecular targets and biological processes that are controlled by PI(3,5)P2 production and turnover.