請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19856
標題: | NCS-1和Auxilin對牛腎上腺嗜鉻細胞胞吐作用的影響 Effects of NCS-1 and Auxilin on Exocytosis in Bovine Chromaffin Cells |
作者: | Yi-Ting Wang 王怡婷 |
指導教授: | 潘建源(Chien-Yuan Pan) |
關鍵字: | NCS-1,auxilin,囊泡循環,牛腎上腺嗜鉻細胞,安培紀錄法, NCS-1,auxilin,vesicle recycle,chromaffin cell,amperometry, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 在突觸前神經末梢,鈣離子透過結合蛋白啟動胞吐作用釋放神經傳導物質。Neuronal calcium sensor-1 (NCS-1) 含有EF-motif 結合鈣離子和N端荳蔻酸化修飾以嵌入細胞膜,屬於鈣離子結合蛋白的一員。已知NCS-1會調節鈣離子通道或結合胞吞作用相關蛋白,進而調控突觸前囊泡循環,但相關細節尚不清楚。Auxilin為一伴護蛋白,參與在胞吞作用中幫助網格蛋白脫離內吞囊泡表面。本實驗室先前以酵母菌雙雜合系統中證實NCS-1和Auxilin有相互作用,因此在本研究中想了解NCS-1和Auxilin在牛腎上腺嗜鉻細胞中,如何影響囊泡循環。透過安培紀錄法,以高鉀重複刺激後發現,NCS-1延遲胞吐作用發生,推論NCS-1 會減少可立即釋放的囊泡。NCS-1G2A點突變會延長囊泡膜與突觸膜之融合過程。另外,發現NCS-1E120Q點突變可能會降低神經傳導物質填入囊泡之效果。而利用螢光共振轉移和共軛焦顯微鏡影像技術,在NCS-1-EYFP和ECFP-Auxilin C half 共同表現的PC12細胞上,給予ATP刺激時會使EYFP螢光強度暫時下降。推測NCS-1和Auxilin受到ATP刺激會短暫分離後再次結合。本研究主題釐清了NCS-1和Auxilin在囊泡循環中扮演之功能,深入突觸傳遞之分子機制進行探討。 At the presynaptic terminals, calcium triggers the exocytosis mediated by various calcium binding proteins. Neuronal calcium sensor-1 (NCS-1) belongs to EF-hand superfamily of calcium binding proteins and has an N-terminal myristoylation for membrane association. NCS-1 does not only regulate exocytosis through Ca2+ channels but interact with endocyto-sis related protein. However, the detail how NCS-1 affect the vesicle recycling is not clear. Auxilin is a chaperone molecule involved in endocytosis, facilitating uncoating of clathrin and AP2 from endocytosed vesicle. We have previously verified that NCS-1 interacts with auxilin by yeast-two-hybridization. Therefore, we are interested in how NCS-1 and auxilin regulate the exo- and endocytosis using amperometry recording and calcium imaging. Re-petitive high K+ evoked exocytosis was investigated using bovine chromaffin cells express-ing NCS-1 and mutants. Amperometry recording shows that NCS-1 delayed the spike dis-tribution, suggesting that NCS-1 might decrease the size of the readily releasable pool. The prolonged halfwidth infers that NCS-1G2A might increase the fusion period. In addition, NCS-1E120Q diminished the quanta size, indicating NCS-1 might be involved in neurotrans-mitter filling. When NCS-1-EYFP and ECFP-auxilin C-half were coexpressed in PC12 cells, both Fluorescence resonance energy transfer (FRET) and confocal images revealed that YFP intensity dropped upon ATP stimulation, suggesting that NCS-1 and auxilin dissociate dur-ing stimulation. These results demonstrate NCS-1 regulate the kinetics of fusion pore during exocytosis and may interact with auxilin to regulate the vesicle recycling. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19856 |
全文授權: | 未授權 |
顯示於系所單位: | 腦與心智科學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-104-1.pdf 目前未授權公開取用 | 2.06 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。