請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55495
標題: | 以10-N-壬基吖啶橙及含疊氮基心磷脂觀察細菌磷脂質功能域 Visualization of bacterial phospholipid domains by using 10-N-nonyl acridine orange and azide-containing cardiolipin |
作者: | Shih-Han Yan 顏示涵 |
指導教授: | 林俊宏(Chun-Hung Lin),史有伶(Yu-Ling Shih) |
關鍵字: | 大腸桿菌,心磷脂,細胞膜曲度,10-N-壬基?啶橙,點擊化學,脂質螢光標定, Escherichia coli.,cardiolipin (CL),cell membrane curvature,10-N-nonyl acridine orange (NAO),click chemistry,fluorescent lipid probes, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 心磷脂 (cardiolipin, CL) 為組成細胞膜與粒線體膜的磷脂質之一,其結構具有緊密的磷酸根頭基及四條自由擺盪的脂肪酸鏈,形成圓錐狀外型而易聚集於細胞膜中高曲度處之膜內側,如細菌細胞端點及分裂縊縮點。文獻指出CL功能域 (domain) 會影響某些蛋白質的定位、結構與功能如大腸桿菌中調節滲透壓的滲透轉運子 (osmosensory transporter) ProP、細胞分裂相關蛋白MinD。在真核生物中,CL在粒線體的分佈被認為能調控粒線體的型態及功能,故CL的分佈位置為一個重要的議題。
目前廣泛用來偵測CL的染劑為10-N-壬基吖啶橙 (10-N-nonyl acridine orange, NAO),本研究以NAO針對不同大腸桿菌菌株細胞膜中的CL分佈情形進行探討。 首先探討不同形態的菌體與細胞膜CL分佈的相關性,發現CL均分佈於高曲度的端點及縊縮處,我們進一步使用CL生合成途徑的酵素 (PgsA、PgpA、PgpB、Cls) 突變株探討CL在細胞膜的分佈情形,文獻指出PgpA、PgpB缺失雖降低磷脂醯甘油 (phosphatidylglycerol, PG) 的含量卻不影響下游產物CL的生成量,Cls缺失導致CL的生成量下降,然而CL在細胞膜的分佈卻尚未被探討。NAO染色結果顯示野生株與突變株的CL在細胞膜上的分佈情形有顯著差異,其中野生株的CL分佈多數呈現周邊一圈 (peripheral) 附加端點 (pole) 的情形;突變株則是單純周邊一圈 (peripheral-only) 比例最高。 此外,實驗過程中我們發現NAO的光漂白 (photobleach) 速度造成CL螢光成像實驗操作上的困難。因此本研究利用七步驟化學反應合成含疊氮基心磷脂 (azide-containing cardiolipin, N3-CL),並藉由點擊化學概念在銅離子催化下與含炔基螢光團 (AlexaFluor-594-alkyne) 進行1,3-偶極環加成 (1.3-dipolar cycloaddition) 反應,開發螢光心磷脂類似物 (fluorescent cardiolipin, F-CL) 並將F-CL送至菌體內並使之重新分佈。藉由比對NAO染色及F-CL餵養的兩種CL定位結果發現在野生株大腸桿菌中CL的分佈情形相似,皆以周邊一圈附加端點的螢光分佈比例最高,證明F-CL的可行性,期望此新工具能為CL的定位實驗開拓一條新的道路。 Cardiolipin (CL) belongs to the family of phospholipids. It is a conical-shaped lipid that has one head group and four acyl chains in the tail region. Because CL tends to aggregate in highly-curved membranes such as those at the polar and division sites of bacteria, it is classified as a high-curvature lipid. Previous studies indicated that CL domain affects localization, structure and function of proteins, such as osmosensory transporter ProP, cell division protein MinD in Escherichia coli. and fusion and fission-promoting protein in mitochondria, leading to an important issue in cell biology: how to detect the cellular localization of CL. 10-N-nonyl acridine orange (NAO) has been used as a fluorescent indicator for cardiolipin and is the only CL-specific dye to track its cellular localization currently. Here we visualize the bacterial phospholipid domains by using NAO to monitor CL distribution of cell membranes in different morphology of Escherichia coli. We found CL-enriched membrane domains located at cell poles and division sites. Next step we performed systematic analyses to compare the localization pattern of CL using NAO between wild-type and specific mutants that are defective on the phospholipid biosynthesis (pgsA, ΔpgpA, ΔpgpB, Δcls) of Escherichia coli. Data shows there are significant differences of CL distribution between wild-type and mutants. The major pattern of CL in wild-type is peripheral and pole while in mutants is peripheral-only. In experiment we found NAO suffer from easy photobleaching. Herein we first established a seven-step procedure to synthesize an azide-containing cardiolipin (N3-CL), which was shown to react with AlexaFluor-594-alkyne via 1,3-dipolar cycloaddition. The resulting fluorescent cardiolipin (F-CL) was successfully taken by Escherichia coli. We compare the CL distribution between NAO staining and F-CL positioning and found the similar result that CL occupy at peripheral and pole of cell membrane. It prove the feasibility of F-CL and we hope this usage will be of great promise to study bacterial phospholipid domains, which is applicable to various studies in cell imaging. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55495 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-103-1.pdf 目前未授權公開取用 | 11.81 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。