The Study Of SEC-10's Function In The Intracellular Transport In C.elegans

Exocyst complex, which is composed of SEC-3, SEC-5, SEC-6, SEC-8, SEC-10, SEC-15, EXO-70 and EXO-84, plays an important role in the intracellular transport. To date, it is mainly considered that exocyst complex functions in the terthering process and the transport from Golgi apparatus to plasma membrane. However, the mechanism still needs to be uncovered. Besides, it is also reported that SEC-5 and SEC-8 may also function in the endocytosis. In order to clarify the mechanism and find out if there are other pathways which exocyst complex may involve in, we study the SEC-10.C.elegans, a powerful model organism, has lots of advantages, so we choose it to be our research platform. First, we construct a worm-mutant library, which is the first one in China. After screening the library, we obtain the sec-10 gene knockout mutant. The sec-10 gene knockout induces a lot of defective phenotypes, and we find that the sec-10 gene is expressed widely in C.elegans. Then we focus on three aspects: the neuromuscular junction, the coelomocyte and the egg-laying.The electrophysiology data show that sec-10 gene knockout has no effect on the three ionotropic receptors reported already, and the minis is also unaffected. However, the drug assay deduced the postsynaptic receptors are hypersensitive, which indicates some other kinds of receptors other than the ionotropic receptors are affected in sec-10 mutant. Based on the GFP endocytosis, the coelomocyte uptake defect is found to be obvious in sec-10 mutant, but the GFP secreted by body-wall muscle is normal. The confocol images show sec-10 gene knockout disrupts the moleculars involved in the Clathrin-dependent endocytosis. On the other hand, the endosomes, lysosome and golgi apparatus are unaffected. Besides, the rhodamin-dextran behaves normally after absorbed into the coelomocyte. We speculate that the Clathrin-dependent endocytosis is blocked while the intracellular transport does not changed. At last, we performed several assays to find which step induces the egg-laying defect. We found the YP170::GFP secreted by intestine is normal, however, there is obvious difference between sec-10 mutant and wild type when the oocytes absorb the YP170::GFP, the YP170::GFP is necessary for the later development, which indicated the egg-laying defect is led by this reason.