The long-term goal of our research plan is to understand the molecular mechanism of exocytosis. Using atomic force microscopy (AFM), a new group of plasma membrane structures called 'pits' 8and 'depressions', have been identified and implicated in exocytosis in live pancreatic acinar cells. Our immediate research goal is to characterize and determine the involvement of these structures in the exocytotic process. Impairment of the exocytotic process in cells is involved in a number of disease states. Understanding this vital cellular process will eventually lead to effective diagnosis and treatment of diseases resulting from secretory defects. Our studies demonstrate 'depressions' to dilate during secretion, returning to their resting size following completion of the process. Exposure of cells to cytochalasin B, a fungal toxin that inhibits actin polymerization, results in a decrease in size of 'depressions', accompanied by a loss in enzyme secretion. We therefore hypothesize 'depressions' to be the sites where vesicle may dock and fuse to release their contents. We seek to test this hypothesis and to gain further understanding of these structures and their biochemical composition. In view of this, our Specific Aims are (1) determine if secretory dock and fuse at 'depressions', (2) determine anatomy of 'pits' and 'depressions' at angstrom resolution, and (3) determine the biochemistry of 'pits' and 'depressions'. To determine if secretory vesicles dock and fuse at 'depressions', the capability of the AFM as a force spectroscope will be utilized. AFM tip disturbance at the cell surface as a consequence of expulsion of vesicular contents will be examined to determine if 'depressions' are secretory sites. To advance our understanding of the function of 'depressions', further AFM studies on the dynamics of its size will be carried out following exposure of cells to stimulators and inhibitors of secretion. Since fixatives are known to provide rigidity to biological samples, proper fixation that retain these PM structures, will enable angstrom resolution imaging by AFM and electron microscopy. To understand the biochemistry of 'pits' and 'depressions', the AFM cantilever will be used as a nanosurgical tool to isolate them for biochemical analysis using mass spectroscopy. Since actin regulates 'depression' morphology, actin antibody affinity columns will be used to isolate and characterize 'depressions' from solubilized acinar cell plasma membrane preparations. These studies will further our understanding of the exocytotic fusion machinery in pancreatic acinar cells, contributing to our understanding of this vital cellular process.
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