? Phospholipids (PL) including phosphoinositides (PIPn) are crucial structural and signaling molecules for proper inter- and intra-cellular communication in an organism. The disruption of lipid homeostasis is associated with numerous human pathologies. High levels of LPA, LPC, PI (3,4,5) P3, PI (3) P and PI (4,5) P2 are linked to specific pathologies such as cancer, myotubular myopathy and Lowe's syndrome. The inter-conversions of phospholipids are mediated by the interplay of phospholipases, kinases, phosphatases and other lipid binding proteins. It is important to analyze and quantify cellular and extra-cellular phospholipids to measure enzyme activities associated with specific pathologies. We propose to develop a fluorogenic assay based on chemically or genetically modified proteins that selectively recognize PLs and PIPns. The modification will be site-specific and will incorporate novel light sensitive entities. These reagents will be applied in Phase II to develop high throughput screening methods for rapid and automated assay of lipid kinases and phosphatases. Such reagents can also be used in the development of cell-based assays. ? ?
