Phosphatidylinositol polyphosphates (phosphoinositides, PIP[n]ns) are key signaling molecules in endo-and exocytosis, vesicular trafficking of proteins, transduction of extracellular signals, remodeling of the actin cytoskeleton, regulation of calcium flux, and apoptosis. A detailed understanding of the mechanisms by which phosphoinositides elicit their effects has been limited by the difficulty of getting PIP[n]s into cells; it has also been difficult to monitor changes in PIP[n] structure and localization when cell physiology is altered. To address this need, Echelon Research Laboratories (ERL) will first develop the Shuttle-PIP system, a simple and rapid method for delivery of PIP[n]s and their fluorescently-labeled analogs into living eukaryotic cells. Polyamine shuttle molecules would carry the polyanionic PIP[n] cargo into living animal, plant, and yeast cells. The process can be monitored using laser confocal or fluorescence microscopy by following the fluorescently-labeled, cell-permeable PIP[n]s, the fluorescent shuttle molecule, or both. Second, ERL will develop Shuttle-PIP kits for each of the eight PIP[n]s for delivery and subcellular visualization. Preliminary studies with NBD-PtdIns(4,5)P2 show that intracellular delivery is possible. Phase I will demonstrate feasibility for other PIP[n]s, optimize conditions for different cells, and optimize synthesis of the fluorescent shuttles and PIP[n]s.
Easy-to-use cell permeant preparations of fluorescently-labeled PIP[n]s would benefit basic and applied research. Changes of PIP[n]s in space and time can be monitored following extracellular stimulation, and agonist or antagonists for a given cellular process can be identified in cell-based assays. Direct observation of intracellular protein to specific PIP[n]s will be possible. These reagents can be employed in development of a multitude of high- throughput screening and genetic based assays.
