Functional Study Of UNC-31 Domains In The C. Elegans Neuron Cell Exocytosis

Life activities of organisms is under strict control by a variety of regulation factors, in which the secretion of vesicle plays an important role and is the molecular basis of macro-control process. Observed by high resolution electron microscopy, vesicles can be divided into large dense core vesicles (LDCVs) and synaptic vesicles (SVs) categories. Large dense core vesicles mainly contain macromolecular protein and neuropeptides, and synaptic vesicles mainly carry neurotransmitter. With the rapid development of electrophysiological techniques and cellular imaging technology in recent years, we can directly monitor secretory vesicles in the high time resolution and high spatial resolution on the living cells, and to study the functional groups (such as proteins) interaction in the process and its mechanism.UNC-31 is discovered by S.Brenner in 1974 in the screening of EMS induced nematode mutation. Lack of UNC-31 in C. elegans leads to uncoordinated movement (UNC), unresponsive to stimulation, and defect in spawning. The mammalian homolog of CAPS (Ca2+-dependent activator protein for secretion) was found in 1992 in rat brain and has been confirmed one of indispensable protein in the process of vesicle secretion. Then It soon became a hot spot in filed of secretion study. This protein contains five predicted functional domains, from N-terminus to C terminus, they are dynactin 1 binding domain (DBD), C2 domain, PH domain, Munc-13 homology domain, and large dense core vesicle binding domain (DCVBD). Some oblique studies (such as sequence alignment, competitive overexpression) showed that these domains have their own features, but there is no direct evidence that these domains play roles in the UNC-31 functions in vesicle secretion process or only the none sense meaningless legacy of evolution. This thesis selects C. elegans as model organism, and combined of the traditional worm behavior and pharmacological assay, recently developed electrophysiological technique, total internal reflection fluorescence microscopy technique and in vivo fluorescence imaging to detect function of these domains. Our results demonstrate that these domains are all indispensible for the function of UNC-31 in exocytosis process, but the Munc-13 homology domain has shown some special properties. UNC-31 without this domain can still works in electrophysiological experiments, the neuron cells of C.elegans express a normal secretion, similar to wild type. In other experiments if knock out this domain from UNC-31 the worm shown phenotypes similar to the UNC-31 gene deletion mutants. We suspect that this special phenomenon may be due to extra high basis calcium concentration in electrophysiological experiments.