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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林俊宏(Chun-Hung Lin),史有伶(Yu-Ling Shih) | |
| dc.contributor.author | Shih-Han Yan | en |
| dc.contributor.author | 顏示涵 | zh_TW |
| dc.date.accessioned | 2021-06-16T04:05:41Z | - |
| dc.date.available | 2024-08-18 | |
| dc.date.copyright | 2014-09-15 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-18 | |
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Molecules 18, 7145-7159 (2013). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55495 | - |
| dc.description.abstract | 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。 | zh_TW |
| dc.description.abstract | 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. | en |
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| dc.description.tableofcontents | 縮寫對照表………………………………………………………………………………i
中文摘要………………………………………………………………………………..iii ABSTRACT...……………………………..…………………………………………….v 目錄…………………………………………………………………………………….vii 圖目錄…………………………………………………………………………………...x 表目錄………………………………………………………………………………….xii 流程目錄………………………………………………………………………………xiii 第一章 緒論 1 1.1 前言 1 1.2 大腸桿菌 2 1.2.1 大腸桿菌的簡介 2 1.2.2 大腸桿菌的生長與分裂機制 4 1.3 磷脂質 7 1.3.1 磷脂質的概述及生合成途徑 7 1.3.2 心磷脂 10 1.3.3 CL的螢光染劑：10-N-壬基吖啶橙 (10-N-nonyl acridine orange, NAO) 14 1.4 點擊化學 18 1.5 研究動機 20 第二章 材料與方法 21 2.1 材料 21 2.1.1 菌種 21 2.1.2 培養基 22 2.1.3 緩衝液 22 2.1.4 化學藥品及儀器 23 2.2 生物暨影像處理方法 25 2.2.1 10-N-壬基吖啶橙於非溫度敏感型之大腸桿菌細胞膜心磷脂染色之實驗 25 2.2.2 10-N-壬基吖啶橙於溫度敏感型之大腸桿菌細胞膜心磷脂染色之實驗 (低溫型，30 oC) 25 2.2.3 10-N-壬基吖啶橙於溫度敏感型之大腸桿菌細胞膜心磷脂染色之實驗 (高溫型，42 ℃) 26 2.2.4 細菌磷脂質之萃取 26 2.2.5 薄層色層分析 27 2.2.6 製備含AlexaFluor-594-CL之單層脂微粒 (small unilamellar vesicle, SUV) 溶液 27 2.2.7 含AlexaFluor-594-CL之單層脂微粒溶液併入勝任細胞膜上磷脂質 28 2.2.8 微分干涉差顯微鏡 (Differential interference contrast, DIC) 觀察菌體型態 29 2.2.9 螢光顯微鏡 (Fluorescent microscope) 觀察10-N-壬基吖啶橙於大腸桿菌細胞膜心磷脂染色之分佈情形 29 2.2.10 螢光顯微鏡觀察AlexaFluor-594-CL併入大腸桿菌細胞膜之分佈情形 30 2.2.11 影像分析 30 2.2.12 菌體分類暨統計 31 2.2.13 以Image J軟體針對心磷脂生合成突變體之心磷脂進行相對定量分析 34 2.3 合成方法與鑑定 35 2.3.1 合成含疊氮基心磷脂類似物 (N3-CL) 35 2.3.2 一價銅離子催化1,3-偶極環加成 (Copper(I)-catalyzed 1,3-dipolar cycloaddition) 40 第三章 實驗結果 41 3.1 細胞骨架及分裂相關蛋白突變體的心磷脂分佈 41 3.1.1 大腸桿菌野生株、∆minCDE及∆mreB突變株之型態觀察 41 3.1.2 大腸桿菌野生株、∆minCDE及∆mreB突變株之心磷脂分佈位置 43 3.2 磷脂質生合成相關酵素突變體的心磷脂分佈情形 46 3.2.1 磷酸醯磷酸生合成酵素：PgsA的突變 46 3.2.2 磷酸醯甘油及心磷脂生合成酵素：PgpA、PgpB、Cls的突變 48 3.3 合成含螢光團心磷脂類似物 62 3.3.1 合成含疊氮基心磷脂類似物 62 3.3.2 利用點擊化學合成含螢光心磷脂類似物 64 3.4 螢光心磷脂類似物於大腸桿菌細胞膜上的分佈 67 第四章 討論與總結 76 4.1 實驗結果討論 76 4.2 未來展望 81 4.3 實驗技術檢討與未來改進方向 83 第五章 參考文獻 85 第六章 附錄 95 6.1 磷脂質生合成相關酵素突變體的心磷脂分佈情形 (統計暨分析圖表) 95 6.2 螢光心磷脂類似物於大腸桿菌細胞膜的分佈情形 (統計暨分析圖表) 103 6.3 1H/13C NMR 光譜 108 | |
| dc.language.iso | zh-TW | |
| dc.subject | 大腸桿菌 | zh_TW |
| dc.subject | 心磷脂 | zh_TW |
| dc.subject | 細胞膜曲度 | zh_TW |
| dc.subject | 10-N-壬基?啶橙 | zh_TW |
| dc.subject | 點擊化學 | zh_TW |
| dc.subject | 脂質螢光標定 | zh_TW |
| dc.subject | Escherichia coli. | en |
| dc.subject | fluorescent lipid probes | en |
| dc.subject | click chemistry | en |
| dc.subject | 10-N-nonyl acridine orange (NAO) | en |
| dc.subject | cell membrane curvature | en |
| dc.subject | cardiolipin (CL) | en |
| dc.title | 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 | zh_TW |
| dc.title | Visualization of bacterial phospholipid domains by using 10-N-nonyl acridine orange and azide-containing cardiolipin | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳佩燁(Pei-Yeh Chen),徐尚德(Shang-Te Hsu) | |
| dc.subject.keyword | 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, | zh_TW |
| dc.subject.keyword | Escherichia coli.,cardiolipin (CL),cell membrane curvature,10-N-nonyl acridine orange (NAO),click chemistry,fluorescent lipid probes, | en |
| dc.relation.page | 120 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-08-19 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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