半胱胺酸串鍊蛋白的磷酸化在鈣離子調控性胞吐作用中對於融合孔的影響

Abstract

在可興奮性的細胞中，細胞欲釋放的物質會被包裹在囊泡裡，之後囊泡會被運送至靠近細胞膜處，當刺激訊號抵達而引起鈣離子流入細胞後，囊泡會和細胞膜融合，形成融合孔，釋放囊泡中所包裹的內容物，此種細胞釋放其內含物質的方式稱為鈣離子調控性胞吐作用。神經細胞利用此種鈣離子調控性胞吐作用的方式，釋放包裹在突觸囊泡或緻密核心囊泡中的神經傳導物質。半胱胺酸串鍊蛋白(CSP) 是一種囊泡蛋白質，已知可被多種激酶所磷酸化，前人研究也指出其與鈣離子感應蛋白 (Syt I) 有交互作用，因此 CSP 被視為可能影響鈣離子調控性胞吐作用、進而影響神經傳導物質釋放的候選因子。 為了研究 CSP 對胞吐作用的詳細機制，首先我們利用分子生物學技術，在 CSP 第十個絲胺酸的位置上製作點突變，以仿效 CSP 無法被磷酸化或 CSP 持續被磷酸化的狀態。接著，利用反轉錄定量聚合酶連鎖反應，我們確認了轉染至細胞中的 CSP 能夠被過量地表現。之後，運用免疫螢光染色方法，我們確定過量表現的 CSP 及其磷酸化的突變並不會改變 CSP在細胞中的位置。最後，我們使用氧化電流測定技術，檢驗了 CSP 及其磷酸化突變株對於融合孔動態的影響。 我們的實驗結果顯示， CSP 的磷酸化會增加囊泡的融合速率，並且促進融合孔的穩定性；因此我們得知，CSP 的磷酸化在鈣離子調控性胞吐作用中，對於融合孔扮演著重要的調控角色。

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.