Munc18 function in large dense-core vesicle exocytosis
Munc18 function in large dense-core vesicle exocytosis
by Attila Gulyas-Kovacs
Date of Examination:2005-01-26
Date of issue:2005-03-23
Advisor:Prof. Dr. Erwin Neher
Referee:Prof. Dr. Erwin Neher
Referee:Prof. Dr. Ralf Heinrich
Persistent Address:
http://dx.doi.org/10.53846/goediss-622
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Abstract
English
Neurotransmitter and hormon release is based on Ca2+ dependent exocytosis. The sequential steps of exocytosis are executed by proteins and protein complexes. Formation of the SNARE complex is essential in later steps of exocytosis. The Sec1/Munc18 proteins are also essential in exocytosis and other membrane trafficking processes but their molecular and cellular function is less clear than that of the SNARE proteins.In our work we studied the role of Munc18 in large dense-core vesicle exocytosis in adrenal chromaffin cells. We combined complementary techniques (electrophysiology, electronmicroscopy) to assay docking, priming and Ca2+ triggered fusion of the vesicles. First, we further confirmed the view that Munc18-1 is a positive regulator of large dense-core vesicle exocytosis. Second, we were interested the downstream molecular targets of Munc18-1 therefore we studied the physiological role of Munc18-1/syntaxin1 interaction. We showed that this interaction is highly important in functional vesicle docking but further experiments are needed to decide whether it is the only major factor or there are other essential downstream targets for Munc18-1. Third, we studied different Munc18 isoforms and our results imply that Munc18 may act not only at the docking but also at the priming step of large dense-core exocytosis. Although some publications argue for the regulatory role of Munc18 in single vesicle fusion kinetics, we found no sign for the direct involvement of Munc18 in Ca2+ triggered fusion. Fourth, we also focused on the upstream regulation of Munc18-1. We obtained opposing data to the hypothesis that Munc18-1 is an important element of the phorbol ester induced potentiation of catecholamine release in the diacylglycerol (DAG) route. The DAG pathway remains an important but poorly understood regulator of transmitter/hormon release, which probably has multiple branches targeting multiple molecules and exocytic steps. Taken together, our findings provided inspiring hints about many aspects of Munc18 function in Ca2+ dependent exocytosis. Perhaps the most important aspect is to match the consecutive steps of exocytosis with the molecular happenings along the Munc18/syntaxin complex SNARE complex axis. Our complementary assays combined with the gene knockout and rescue technique could promote adrenal chromaffin cells to become the first system where this match between cellular function and molecular structure is clarified.
Keywords: exocytosis; Munc18; chromaffin cells; exocytosis; Munc18; chromaffin cells
