Localization of phosphatidylinositol 4,5-P(2) important in exocytosis and a quantitative analysis of chromaffin granule motion adjacent to the plasma membrane.

A slow ATP-dependent priming step precedes a rapid, Ca(2+)-dependent triggering step in exocytosis in chromaffin cells and in most, if not all, differentiated secretory cells. A major component of ATP-dependent secretion in permeabilized cells reflects the maintenance of the polyphosphoinositides, especially PtdIns-4,5-P2. Here we summarize recent experiments with PH-GFP (binds to PtdIns-4,5-P2) that indicate that PtdIns-4,5-P2 is localized primarily on the plasma membrane in chromaffin cells, and that it is this pool that plays a role in exocytosis. It is demonstrated that transiently expressed PH-GFP inhibits secretion in subsequently permeabilized cells. Recent studies using total internal reflection fluorescent microscopy (TIRFM) to measure chromaffin granule motion adjacent to the plasma membrane are also summarized. The quantitative analysis indicates that chromaffin granule motion is highly restricted and suggests that chromaffin granules are caged or tethered immediately adjacent to the plasma membrane.