Functional Study On Secretory Regulatory Proteins Of Dense Core Vesicles

Vesicular exocytosis and endocytosis in neurons and neuroendocrine cells constitutes the basis for neurotransmission and hormone signaling. Many components involved in the trafficking of synaptic vesicles (SVs) do not differ in core aspects of their fusion machinery. The many differences in the properties of DCVs and SVs suggest that these two classes of secretory organelles employ different molecules in secretion. The study on DCVs secretion will be very helpful to the progress towards comprehensive understanding of the molecular mechanism of the coordinated vesicular trafficking process.The nematode C. elegans provides a powerful model system for exploring the molecular basis of neurotransmission. Yet, the lack of direct functional assays of release process has largely prevented in depth understanding of the mechanism of vesicular exocytosis and endocytosis. We address this technical limitation by developing direct electrophysiological assays including membrane capacitance and amperometry measurements in primary C. elegans neurons. In addition, we have succeeded in monitoring the docking of single dense core vesicles (DCV) employing total internal reflection fluorescence microscopy. With these approaches and mutant perturbation analysis, we provide direct evidence that UNC-31 is required for DCV exocytosis. We propose that UNC-31 functions in concert with UNC-18 in the initiation and stabilization of docking.Synaptotagmin I (Syt I) is a Ca2+ sensor for triggering fast synchronized release of neurotransmitters. However, controversy remains whether Syt I is also obligatory for the exocytosis and endocytosis of larger dense core vesicles (LDCVs) in endocrine cells. In this study, we used a short hairpin RNA (shRNA) to silence the expression of Syt I, and investigated the roles of Syt I on exocytosis and endocytosis in INS-1 cells. Our results demonstrated that expression of Syt I is remarkably reduced by the Syt I gene targeting shRNA. Using high-time resolution capacitance measurement, we found that the silence of Syt I decreased the calcium sensitivity of fusion of insulin granules and therefore reduced the exocytotic burst triggered by step-like [Ca2+]i elevation. In addition, the occurrence frequency and amplitude of fast endocytosis were remarkably reduced in the silenced cells. We conclude that Syt I not only participates in the Ca2+-sensing of LDCV fusion with plasmalemma, but also plays a crucial role in fast endocytosis in INS-1 cells.Many cells utilize a GTP-dependent pathway to trigger exocytosis in addition to Ca2+-triggered exocytosis. However, little is known about the mechanism by which GTP triggers exocytosis independent of Ca2+. We used dual-color evanescent field microscopy to compare the motion and fusion of large dense core vesicles stimulated by either mastoparan in Ca2+-free conditions or high K+ in the presence of Ca2+. We demonstrate that mastoparan is hardly effective in triggering the fusion of the pre-docked vesicles, but predominantly mobilizes cytosolic vesicles. In contrast, Ca2+-dependent exocytosis is largely due to pre-docked vesicles. Fusion kinetics analysis and carbon-fibre amperometry reveal that mastoparan induces a brief'kiss-and-run'fusion and releases only a small amount of the cargo, whereas Ca2+ stimulates a more persistent opening of the fusion pore and larger release of the contents. Furthermore, we show that mastoparan-released vesicles require a much shorter time to reach fusion competence once they approach the plasma membrane. Our data suggest the involvement of different mechanisms not only in triggering and fusion, but also in the docking and priming process for Ca2+- and GTP-dependent exocytosis.