The role of phosphoinositides in mast cell exocytosis
The phosphoinositides (Pin) are a family of phospholipids that contain myo inositol as their headgroup. Despite being present in eukaryotic membranes with low abundance, their high rates of metabolic turnover allow them to control a plethora of cellular functions. In particular, phosphatidylinositol 4,5-&wphosphate (PtdIns(4,5)P2) regulates several processes, including preparing secretory organelles to undergo fusion with the plasma membrane in response to a stimulus (regulated exocytosis), budding and fission of vesicular cargo from the plasma membrane (endocytosis), and controlling the cortical actin cytoskeleton. In this thesis, the role of Pin in regulated exocytosis is examined in mast cells, since these undergo an acute, massive and rapid exocytosis, without any immediate endocytosis. Using a reconstitution approach, it was not possible to define which Pin are involved in exocytosis, although it was concluded that at least one Pin that is not PtdIns(4,5)P2 is required. In order to study PtdIns(4,5)P2 dynamics in primary mast cells, a novel quantitative immunofluoescence technique for Pin was established. Using this technique, PtdIns(4,5)P2 was identified at the plasma membrane of mast cells, but was depleted almost entirely during exocytosis the latter observation was confirmed using biochemical approaches. This depletion was blocked by inhibitors of phospholipase C (PLC), an enzyme that breaks down PtdIns(4,5)P2 into diacylglycerol (DAG) and the calcium mobilising messenger, inositol 1,4,5-frisphosphate (Ins(l,4,5)P3). Although PLC activity was required for initiation of calcium signalling in mast cells, experiments whereby the Ins(l,4,5)P3/calcium pathway was bypassed demonstrated further requirements for PLC activity. These were not precisely defined, but simple elimination of plasma membrane PtdIns(4,5)P2 or production of DAG were not sufficient. Both events may be required in conjunction, however. A model is proposed whereby elimination of plasma membrane PtdIns(4,5)P2 together with production of DAG may activate the protein machinery for membrane fusion during exocytosis.