| Summary: | 碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 100 === In excitable cells, vesicles are transported to fuse with the plasma membrane and release their contents through calcium-regulated exocytosis. Neurons tend to use this way to release their neurotransmitters packaged in synaptic vesicles or large dense-core vesicles. Cysteine string protein (CSP) is a vesicle-associated protein known to be phosphorylated by various protein kinases. Moreover, previous studies have shown that CSP can interact with synaptotagmin I (Syt I), which is the calcium sensor in calcium-regulated exocytosis. Therefore, CSP is thought to be a candidate modulating calcium-regulated exocytosis and thus neurotransmitter release.
In order to understand the detailed regulatory mechanisms of CSP on exocytosis, we first mutated the serine residue at the position of 10 in the amino acid sequence to make either phosphodeficient or phosphomimetic mutants. Subsequently, we used RT-qPCR to confirm the overexpression of CSP after transfection. By using immunofluorescence staining, we observed that overexpressed CSP would not change its subcellular localization. At last, we performed amperometry to investigate the effects of CSP phosphorylation on exocytosis in terms of kinetics of exocytosis and fusion pore dynamics.
Our results suggest that CSP phosphorylation increases fusion rate and tends to stabilize exocytotic fusion pore. Therefore, CSP phosphorylation plays an important role in modulating fusion pore in calcium-regulated exocytosis.
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