Differential Contribution Of Synaptotagmin Isoforms To Different Exocytotic Components In LβT2 Cells

Synaptotagmin (Syt) is a large protein family existed 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 over 16 isoforms of synaptotagmin in mammalian, because 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.The anterior pituitary gonadotropes secret pituitary gonadotropins and serve as a control center for integration of hormonal signals in the reproductive axis, deficiency in hormone secretion from gonadotrops will impact development and mature of sex. The study on the mechanism of secretion and its regulation is important to further understand the essence of secretion and to treat secretion-relevant deseases. In the present study, we systemic investigated on molecule mechanism of Syt regulated exocytosis in single gonadotrope cell line, LβT2.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. 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. Our experiments were performed by fast release of Ca2+ from NP-EGTA using strong UV-illumination. Membrane capacitance and Ca2+ concentration were monitored simultaneously during the stimulation. We inserted Syt IV and Syt VII fragments into the enhanced green fluorescent protein (EGFP) expression vector pEGFP-N1 to form recombined plasmid pSyt-IV-EGFP-N1 and pSyt VII-EGFP-N1. We used the total internal reflection fluorescence microscopy (TIRFM) to observe locolization of Syts in LβT2 cells. Though the experiments, we concluded:(1) In our experiment, expression of synaptotagmin I, IV, VII and IX in syt gene-silenced cells were reducing to 27%, 36%, 32% and 25% in comparison with native cells.(2) Syt I gene silencing cells have no significant effect on each component. Syt IX gene silencing cells selectively reduces the fast burst and sustained component, without significant changes in slow burst component. Syt VII gene silencing cells selectively reduces the slow burst component, without significant changes in other kinetic parameters.(3) Syt IX colocalized with LDCV in LβT2 cells. Syt VII colocalized with lysosome in LβT2 cells.We speculated that different Syt isoform might regulate the fast burst component and slow burst component respectively. Syt VII is localized on the lysosome, which indicates that the slow burst component may be partly composed of exocytotic lysosome. Syt IX is a Ca2+ sensor for fast exocytosis. The gene silence of Syt IX significantly reduces the fast burst component of secretion. The result also displays that Syt IX participates in the process of vesicle activation and reduces the vesicle deactivation.