This project examines the biophysical process involved in the process of secretion of adrenalin-containing granules from chromaffin cells. The overall hypothesis is that chromaffin granule motion toward the plasma membrane is essential for a sustained secretory response, and that regulation of this motion provides the cell with a means to control secretion. In addition to testing that hypothesis, this project also has an overall major goal of introducing and demonstrating new optical microscopy techniques that should find general utility in cell biology. The results should have significance not only to understanding how the adrenal medulla gland secretes a major hormone that affects the whole body, but also for (a) understanding the mechanisms of secretion in other cells with chemical transmitters (e.g., cells in the neurological system) and (b) proving the utility of novel fluorescence microscope techniques that selectively probe membrane-proximal processes. This project proposes to use several new variations of a technique pioneered in the PI's lab: total internal reflection fluorescence (TIRF) microscopy - to simultaneously provide both real time imaging and sufficiently thin optical sectioning. The expertise with TIRF is combined with the co-investigator's and collaborator's expertise in fluorescence-marking the granules by transient transfection to express a granule-specific green fluorescent protein (GFP) fusion protein.
The specific aims are grouped into five related areas. (a) Granule motions (on the scale of several nanometer) near the plasma membrane (within tens of nanometers) will be characterized with respect to speed, distance, unidirectionality, lateral motion, and population classes among granules. (b) These motion parameters will be compared between the basal and the secretory state of the cells. In particular the role of specific proteins of the secretory pathway (SNAREs and Rab3a) in granule motion and docking will be examined in premeabilized ells. (c) Submicroscopic morphological changes in the plasma membrane during secretion will be examined by new and sensitive optical techniques. (d) The effective viscosity of the inside of the granule will be examined by a polarized TIR fluorescence photobleaching technique. If the granule is internally gel-like, then tumbling rates of the whole granule will be measured as a means to distinguish free from docked granules. (e) Changes in filamentous proteins and in local Ca2+ transients coincident with granule fusion will be examined in a preliminary study.
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