Regulated exocytosis occurs in a number of cell types and has been studied in many of
these. There appears to be a commonality of function in the cells that exhibit regulated
exocytosis. Many proteins have been implicated in the process of regulated exocytosis
and a number of trigger signals have been identified. There are various means of
investigating regulated exocytosis and these include capacitance measurements,
amperometry and single-cell imaging. Reconstitution from defined components of the
steps leading to exocytosis has not yet been achieved, whereas the investigation of the
actual exocytotic event is well described. Access to the cytosolic components of the cell
is a requirement of any assay that attempts to investigate the process leading to the final
'fusion event' of regulated exocytosis.The aim of this research was to develop an in-vitro assay that would allow investigation
of the steps leading to regulated exocytosis on a plasma membrane. The method was
based on the use of ‘unroofed’ bovine adrenal chromaffin cells, viewed by total internal
reflection fluorescence microscopy. Development of the assay posed a number of
challenges, not all of which were overcome during the study. The basic platform for the
assay, a cell membrane patch with adhering secretory vesicles, was produced with some
degree of success. However the imaging method had a number of limitations and this
frustrated the method’s development.The results, although encouraging, were judged not to be robust enough to permit
development of a reconstituted system and a more reliable imaging procedure is
recommended for further stud
