The most important aim of this grant application is to set up a cell-free system to study mechanisms of exocytosis at the single granule level in the pancreatic beta-cell. With this achieved, a powerful new method will be available to study docked granules and the fundamental mechanisms of insulin exocytosis. Initially it is proposed to develop and characterize this new method to study exocytosis on membrane sheets. beta-cells attached to coverslips will be """"""""unroofed"""""""" in the absence of Ca2+ to expose a flat layer of plasma membrane with """"""""docked"""""""" fluorescently labelled secretory granules. When stimulated by Ca2+, many of the granules undergo exocytosis that can be monitored by changes in fluorescent markers. Initially, the clonal beta-cell line, INS 832/13, a subclone of the INS-1 cells will be used. Subsequently, the study will be extended to primary rat beta-cells fluorescently labeled by an adenovirus approach. The system will be validated by measurement of the number of exocytotic events that occur when the membrane sheets are abruptly exposed to a stimulatory Ca2+ concentration, and when they are exposed to a stimulatory Ca2+ concentration after treatment with tetanus toxin (TTX) that blocks exocytosis. Similar studies will be performed in the presence of physiological inhibitors of secretion such as norepinephrine, and potentiators of exocytosis such as cyclic AMP and diacylglycerol. Validation will require that exocytosis is stimulated, inhibited and potentiated respectively by these conditions. Optimization of the system will be achieved by studying the effects of changes in sonication conditions, e.g. sonication intensity, time and buffer composition, and by changes in the buffer conditions under which exocytosis is stimulated. Subsequently, it will be possible to study 1. the mechanisms by which stimulation, inhibition and potentiation of exocytosis occur; 2. membrane-granule and protein/protein interactions important to the control of granule tethering, docking and exocytosis; and 3. physiological mechanisms such as time-dependent potentiation and biphasic insulin release. The method will also allow the study of some aspects of endocytosis.
