Studies Of Regulated Exocytosis From Neuroendocrine Cells

In this thesis we study cargo release and fusion pore dilation during calcium triggered exocytosis and the co-localization of calcium sensing proteins essential for exocytosis, in neuroendocrine cells. Pancreatic beta cells secrete several hormones, the most studied one being insulin. C-peptide is a protein which is co-stored with and secreted from the same vesicles as insulin. It is found in the soluble phase unlike insulin, which is found in the dense core. The pancreatic beta cells also secrete the Chromogranin B (CgB) which is mostly found in the dense cores of secretory vesicles. In chapter 1, we found that CgB, being a dense core protein, had slower release kinetics than C-peptide. These studies were carried out in the insulinomas cell line, Min6. Chromaffin cells are neuroendocrine cells which secrete cargo via LDCVs. Among the various hormones that it secretes, CgB and Tissue Plasminogen Activator (tPA) are of special interest as their functions are not fully understood. Both proteins constitute the dense core, though they have different molecular masses. In chapter 2, we compare the release of these proteins by tagging them to pH sensitive fluorophore, pHluorin to understand the fusion pore dilation in case of each protein. We find that the release kinetics of the larger tPA is significantly slower than that of the smaller CgB. Hence, tPA release requires more fusion pore dilation than CgB. Calcium triggered exocytosis, as the name suggests, is caused by the influx of calcium into neuronal and neuroendocrine cells. The calcium sensor present in these cells is the protein synaptotagmin (Syt). Chromaffin cells have only two isoforms of synaptotagmin, Syt-1 and Syt-7. These two isoforms differ in their function. In chapter 3, we investigate if they are sorted to different vesicles. Using confocal microscopy, we find that overexpressed Syt-1 and Syt-7 show 7.55% co-localization in chromaffin cells, indicating that they are indeed sorted into different vesicles