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

Vesicular trafficking is of critical importance for a variety of life functions, and tightly related to some diseases. It consists of exocystosis and endocytosis, which are regulated by many molecules. Studies in different model organisms have revealed lots of components involved in vesicular trafficking, but the mechanism of some components is still unclear. In this paper, we dedicate to exploring the molecular mechnism of SEC-10 and Synaptotagminâ…¦(Sytâ…¦) in exocytosis and endocytosis respectively. Especially, the study of SEC-10 in Caenorhabditis elegans (C. elegans) combined the cellular traffic with behavior characteristic, which indicates the new role of exocyst in endocytic pathway.In this study, we investigated the roles of Syt VII on exocytosis of large dense core vesicles (LDCVs) in PC12 cells. Syt VII, which has a higher Ca2+ affinity and slower disassembly kinetics with lipid than Syt I and Syt IX, was regarded as being uninvolved in synaptic vesicle (SV) exocytosis but Instead possibly as a calcium sensor for the slower kinetic phase of dense core vesicles (DCVs) release. But at present the localization and function of Syt VII remain unclear. Using TIRFM, our results showed that Syt VII is predominantly located on DCVs in PC12 cells. By using high temporal resolution capacitance and amperometry measurements, it was demonstrated that the knockdown of endogenous Syt VII attenuated the fusion of DCV with the plasma membrane, reduced the amplitude of the exocytotic burst of the Ca2+-triggered DCV release without affecting the slope of the sustained component, and blocked the fusion pore expansion. This suggests that Syt VII is the Ca2+ sensor of DCV fusion machinery and is an essential factor for the establishment and maintenance of the pool size of releasable DCVs in PC 12 cells.The researches are taken on C. elegans to reveal the roles of SEC-10 in worm behaviors and kinds of life affairs. Especially, we study the molecular mechanism of SEC-10 in endocytotic pathway of kinds of cells. Sec10 is one component of exocyst (Sec3,Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, Exo84), which is strongly implicated in cellular traffics and vesicle-tethering events. Recent years several studies suggested the roles of exocyst in endocytosis, but the detailed molecular mechanism is still unclear. Here, we obtained the first C. elegans mutant library in China, and isolated a C-terminal truncated mutation of sec-10 by screening this library. Results from analysis of phenotype and behavior in sec-10 mutants indicat that SEC-10 may be functional in several tissues and cells. And the embryonic lethality in sec-10 mutant, which is consistent with the conclusions in mice and drosophila, suggests that exocyst may be required for development. In addition, the pulse-chase assays of endocytosis and observation of distribution and morphology of endocytosis-related-organelles in polarized intestinal cells and nonpolarized coelomocyte cells and oocytes indicate that exocyst may be required for the endocytic pathways in the different kinds of cells.The study in coelomocytes indicates that:1. The clathrin mediated endocytosis (CME) and amiloride-sensitive macropinocytosis are required for coelomocyte endocytosis. For small-sized fluid-phase object, either of them could be used for efficient uptake. For large-sized fluid-phase object, macropinocytosis is the dominating pathway; 2. In sec-10 mutant coelomocytes, the clathrin mediated pinocytosis and amiloride-sensitive macropinocytosis are severely disrupted. The subcellular distribution of several molecules involved in endocytosis are abnormal. sec-10 mutant worms show obvious Cup phenotype because of blockage at early step of endocytosis; 3. The distribution and morphology of endocytic organelles are abnormal. However, postendocytic trafficking is not impaired, which suggesting that the defect of endocytic organelles is attributed to the block of early step in endocytosis.The study on sec-10 mutant intestinal cells indicates that:1. SEC-10 is expressed in intestine throughout development from embryo to adulthood. Sec-10 mutation results in defect in defecation; 2. SEC-10 is required for basolateral postendocytic trafficking but not apical; 3. The transport of plasm membrane receptor protein (clathrin-dependent or-indepent) from early endosomes to recycling endosomes is impaired by sec-10 mutation; 4. In vivo pulse-chase experiments and fluorescent protein labelling experiments show that loss of SEC-10 function change the destination of endocytosed molecules, in which the molecules are transported to lysosomes instead of recycling endosomes.In conclusion, the researches on sec-10 mutant strain including not only the analysis of phenotype and behavior characteristic, but also the studying of endocytotic pathways both in polarized cell and in nonpolarized cell, describe the function of SEC-10 in endocytosis. Our results indicate that exocyst involves in some other vesicle traffics besides exocytotic pathway. The importance of SEC-10 for endocytosis in different cells of C. elegans shed new light on functional study of exocyst in mammalian.