Abstract
Polyphosphoinositides (PPIs) are universal lipid regulators that control a great variety of cellular function serving as docking sites and conformational switches for a large number of signaling proteins associated with cellular membranes. PPIs are produced from phosphatidylinositol (PtdIns) that also is a structural lipid of biological membranes. PtdIns synthesis is believed to occur primarily in the ER, although it is debated whether it also happens at the plasma membrane (PM). Considerable progress has been made to determine the localization and dynamic changes in PPIs both in live and fixed cells, yet little has been done to map the distribution and possible functional diversity of the PtdIns pools in mammalian cells. Here we devised a strategy to address this question by using a PtdIns specific PLC enzyme isolated from Listeria monocytogenes together with a highly sensitive diacylglycerol sensor to determine the distribution and also to alter the level of PtdIns in living cells. Our studies reveal that a significant metabolically highly active PtdIns pool exists associated with tiny mobile structures within the cytoplasm in addition to the known ER and PM PtdIns pools. We will show our most recent data on the consequences of PtdIns depletion within the various PtdIns pools and on PPI production and will also discuss principles of PPI modulation of multiple effectors.
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