Biogenesis and trafficking of regulated secretory granules

Regulated secretion of neuropeptides is essential for maintaining homeostasis within the endocrine and nervous systems. Secretion requires not only a highly regulated exocytotic mechanism, but also necessitates coordinated synthesis of the large dense core vesicles (LDCVs, also referred to as secretory granules) in which peptides are stored. Peptides are synthesized in the ER as inactive precursors and transported to the trans-Golgi network (TGN) where they are packaged into immature secretory granules. These granules go through a series of maturation steps, transforming them into regulated exocytotic carriers. During this maturation process, condensation of soluble contents and selective removal of membranes occurs. Since LDCVs cannot be refilled locally after their contents are secreted, it is particularly important to understand how neurons support regulated release of peptides. Therefore, the overall aim of these studies was to investigate the mechanisms by which these trafficking events occur and are regulated.^ To investigate the trafficking of secretory granule proteins, fluorescently tagged granule proteins were visualized in living cultured cells by confocal microscopy. The sorting, localization, and secretion of fluorescent cargo proteins were evaluated relative to that of endogenous hormones and enzymes in endocrine cell lines. When expressed in AtT-20 or GH3 cells, the GFP fusion protein partitioned from endogenous hormone into separate secretory granule pools. A simple self-aggregation default theory may explain this little acknowledged, but commonly observed, tendency for both naturally occurring and exogenous content proteins to segregate from each other into distinct secretory granules. Although luminal acidification was not essential for soluble content protein segregation, this work revealed a pH independent role for the vacuolar H+ ATPase in secretory granule biogenesis. Inhibition of the V-ATPase resulted in rapid loss of regulated secretion and formation of large multilamellar organelles, with components of newly formed secretory granules and lysosomes mixed together. Finally, the dynamic behavior of secretory granules in peptidergic trigeminal ganglion neurons was investigated. Our findings that transport of peptidergic granules was regulated by neuronal stimulation suggest that control of mobility is important for sustaining the secretory response.