Control of membrane fusion dynamics during regulated exocytosis

The study of regulated exocytosis uniquely allows the direct measurement of intracellular membrane fusion events in real time. We have exploited this to examine factors that regulate not only the extent but also the dynamics of single fusion/release events. The general strategy used has been to asse ss exocytosis in transiently transfected PC12 or adrenal chromaffin cells. We aimed to design mutant constructs based on in vitro biochemistry in some cases informed by knowledge of protein structure. Using this approach we h ave demonstrated an inhibitory role for the putative Rab3 effector Noc2 tha t requires interaction with Rab3. Using carbon-fibre amperometry on adrenal chromaffin cells, we have demonstrated regulation of the kinetics of singl e granule release events consistent with changes in fusion pore dynamics an d switches between full fusion and 'kiss-and-run' fusion. These studies hav e demonstrated a late role for cysteine string protein in exocytosis. In ad dition, they have focused attention on a key role for Munc18 in the regulat ion of post-fusion events that affect fusion pore dynamics.