The Roles Of Synaptotagmin I And IX In Secretory Regulation

Synaptotagmin(Syt) is a large protein family which are mainly existes on secretory vesicles and granules in neuron, neuroendocrine and endocrine cells. As the Ca2+ sensor in Ca2+-dependent neurotransmitter and hormone release, Synaptotagmins trigger and regulate the fusion process between vesicle membrane and target membrane, play important role in the secretory regulation in neuron, neurorndocrine and endocrine cells; and regulate the transfer of proteins and membranes to destination. There are 16 isoforms of Synaptotagmin in mammalian, as the different location and different construction, each one has different role in secretory regulation. Though the interaction between the isoform of Synaptotagmin and the different factors, Synaptotagmins are involved in the exocytosis and recycling of secretory vesicles. Calcium increase in cell is the precondition to trigger the exocytosis in regulative exocytosis. As the calcium sensor, Synaptotagmins involve in most of membrane fusion events in neuron, neuroendocrine and endocrine cells.Because Synaptotagmin is a large family including many isoforms, it is necessary to find the useful and efficient methods to identify the role of each isoform, and explain the relationship between structure and function. Our research focuses on two isoforms, I and IX, in INS-1 and PC12 cells to establish a efficient assay system to identify roles of different isoforms in protein family.RNAi, RNA interfering, is a technique for silencing the gene expression at the step of post-transcription. The major advantage, in comparison with knockout and antisense technologies, is that this method doesn't affect the gene construction and has relative high efficacy of silencing of target protein expression. In our experiment, expression of Synaptotagmin I and IX were reducing to 10-20% in comparison with native cells by Syt-sh. And the silencing efficiency of siRNA and shRNA was compared in this research. PC12 cells and INS-1 cells were chosen as the modes for neuroendocrine and endocrine cells, membrane capacitance measurement and carbon fibre amperometry were used to detect the function changes between native cells and Syt gene-silenced cells. Qdots, a new fluorescence probe, was used in immonolabeling and tracing of endocytotic vesicles cycling in live cells.The results showed that siRNA and shRNA had equivalent efficiency in Synaptotagmni I and IX gene silencing in PC12 cells. The use of design method by Ui-Tei, et al showed same efficiency for siRNA and shRNA. Immunocytochemistry assay show that SytI colocalized with insulin granulesin INS-1 cells and LDCV in PC12 cells.When INS-1 cells were transfected with SytI-sh and SytIX-sh, in comparison with control (cells without transfected with shRNA encoded plasmid), the results taken by membrane capacitate measurement and amperometry showed that burst components of Ca2+-dependent exocytosis were reduced significantly as the result of Ca2+ sensitivity was lower than control. In addition, kinetics of exocytosis was altered significantly, because the rate constants of burst were markedly deceased in SytI-sh cells. And the dilation of fusion pore was inhibited in SytI-sh cells showed by amperometry. The membrane capacitance measurement also showed that Syt I involved in the fast endocytosis in INS-1 cells. Although Syt IX was abundant in INS-1 cells, it had no compensation to knockdown of Syt I expression. The results were different in PC12 cells. Silencing of Syt I in PC12 cells could not affect the Ca2+-depandent exocytosis of LDCV. Meanwhile, silencing of Syt IX didn't make any changes in comparison with control.Quantum dots (Qdots) not only was used in immunocytochemistry assay to localize SytI in cells, but also used to track the endocrine vesicles in PC12 cells. The the characteristics of fluorescence granules movements in consistent with the kinetic characteristics of LDCV in PC12 cells.On the bases of experiments, we conclude:1) siRNA and shRNA showed equivalent silencing efficiency for Syt expression . siRNA can be used as a tool for high thoughput screen in gene function assay. Analysis of western blot bands could calibrate the relative efficacy of silence.2) The results meant that Syt I was the major Ca2+ sensor in insulin granules release and play an important role in fast endocytosis followed exocyrosis. And Syt IX didn't compensate the lost of Syt I in INS-1 cells. Meanwhile, knockdown of the Syt I expression in PC12 cells didn't affect the exocytosis of LDCV. The results show the difference roles of SytI and SytIX between endocrine cells and neuroendorine cells.3) Qdots are powerful tool in vesicles or granules tracing in live cells and in immunolabeling assay.