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研究生:杜育銘
研究生(外文):Yu-Ming Tu
論文名稱:研究Min蛋白引導脂質雙層膜中物質產生二維疏密波之現象
論文名稱(外文):Study of Min Protein-Induced Membrane Species Waves in vitro
指導教授:趙玲趙玲引用關係
口試委員:史有伶謝之真林資榕
口試日期:2015-07-08
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學工程學研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:57
中文關鍵詞:Min 蛋白系統自組裝支撐式脂雙層膜平台動態圖形
外文關鍵詞:Min systemself-organizationsupported lipid bilayersdynamic pattern formation
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在大腸桿菌中,Min蛋白藉由在細胞兩極來回的動態振盪,能調控細胞分裂的位置。目前也已發現Min蛋白之自發性構成之動態波現象,可以藉由將Min蛋白中的MinD,MinE和腺苷三磷酸 (Adenosine triphosphate)(ATP)加入以大腸桿菌的細胞膜所形成之支撐式脂雙層膜平台,而在體外重現。然而,Min蛋白如何與大腸桿菌中的脂質雙層膜作用,以及如何影響到其它膜中生物分子之機制依舊不甚清楚。在本論文中,我們假設Min蛋白嵌入膜和離開膜之動態情況會影響脂質雙層膜外層脂質膜中頭基區域的空間密度,進而間接促使頭基較一般脂質分子為大之膜中生物分子,會較喜好填補頭基部分較為寬鬆的區域,因而在密度寬鬆的區域造成累積,而在密度較為緊緻的區域則有減少的現象,進而能產生其隨時間空間變化的濃度分佈。在本系統中,我們利用大腸桿菌的細胞膜所形成之支撐式脂質雙層膜來在體外重建生物體內的系統,並從大腸桿菌內純化出Min蛋白,來系統性地討論Min蛋白對脂質雙層膜之影響。我們觀察到脂質雙層膜在加入MinD,MinE和腺苷三磷酸後,會形成動態的螺旋濃度分佈波形。螢光漂白回復測試法和影像處理分析進一步指出脂質雙層膜上的濃度分佈動態波形與Min蛋白形成的表面波形有不同的特性,顯示膜中物質之濃度分佈並非源自於與Min蛋白有直接之鍵結。我們以帶有螢光分子的數種膜中物質作為生物分子模型,並將之鑲嵌進脂質雙層膜的系統,在加入Min蛋白後,的確發現Min蛋白的動態震盪現象只會影響頭基部分較大之膜中生物分子。此實驗觀察符合我們的假設: Min蛋白的動態現象會影響到脂質雙層膜上的生物分子,並可能具有選擇性的影響特定生物分子。此結果也提供了一個 Min蛋白如何與其他膜中分子作用的可能機制。

In the bacterium Escherichia coli, the proper placement of the division site selection is regulated in part by the pole-to-pole oscillations of Min proteins. The oscillation dynamics emerges from the self-organization of MinD, MinE and ATP in vivo, which also forms planar surface waves on supported lipid bilayer in vitro. However, it is still unclear how the Min proteins affect the E. coli lipid membrane and interact with the other E. coli membrane proteins, even though how Min proteins interact with the membrane surface to generate the in vivo oscillations is a subject of intense study. We hypothesized that the spatial oscillations of Min protein systems could play a crucial role in changing lipid membrane dynamics and therefore indirectly influence the dynamics of other membrane associated species. We reconstituted the system on supported E. coli lipid bilayer platforms in order to systematically explore the underlying mechanism between lipid membranes and Min proteins. We observed fluorescently labeled lipids in the E. coli membranes moving in spiral waves after the addition of unlabeled MinD, MinE and ATP to the membranes. Fluorescence recovery after photobleaching (FRAP) and kymograph analyses showed that dynamics of the labeled lipid membrane waves had different characteristics from those of the Min protein waves. In the future, some E. coli signaling membrane proteins could be incorporated into the membrane to examine whether their movement or clustering can be influenced by the Min proteins-induced membrane waves. The result could provide us insights into the long-standing question about other possible function of the E. coli Min protein oscillation phenomenon.

Table of Content
Acknowledgement i
摘要 ii
Abstract iii
Table of Content V
Table Captions VII
Figure Captions VII
1. Introduction 1
1.1. Supported Lipid Bilayer 5
1.2. Min Protein Oscillation Cycle in vivo 6
1.3. Min Protein Self-Organization Surface Waves in vitro 8
2. Materials and Methods 11
2.1. Materials 11
2.2. Apparatus 15
2.2.1. Apparatus for Min Protein Over-expression 15
2.2.2. Apparatus for Min Protein Purification 15
2.3. Overexpression and Purification of MinE 17
2.4. Overexpression and Purification of MinD 18
2.5. ATPase assay 20
2.6. Lipid Preparation 20
2.7. Large Unilamellar Vesicles (LUVs) Preparation 21
2.8. Polydimethylsiloxane (PDMS) Chamber Preparation 22
2.9. Supported Lipid Bilayers (SLBs) Preparation 23
2.10. Observation of Membrane Species Waves. 23
2.11. Fluorescence Microscopy Images and Fluorescence Recovery after Photobleaching (FRAP) 24
2.12. Image Analysis by Image J and the kymograph Tool 25
3. Results 26
3.1. Response of the E. coli Supported Lipid Bilayer Containing Texas-Red DHPE to the Addition of MinD, MinE and ATP 26
3.2. Response of the E. coli Supported Lipid Bilayer Containing Other Types of Fluorescently Labeled Membrane Species to the Addition of MinD, MinE and ATP 28
3.3. Response of the E. coli Supported Lipid Bilayer Containing Both Texas-Red DHPE and BODIPY-HPC to the Addition of MinD, MinE and ATP 31
3.4. Hypothesized Mechanism of How Min Protein Dynamics Can Cause the Concentration Waves of Membrane Species 33
3.5. The Concentration Waves of the Membrane Species Have Different Characteristics as the Labeled Min Protein Oscillation Waves do. 36
4. Discussions 39
4.1. Phenomena Other Than Membrane Species Concentration Waves Different Formation Membrane Species Patterns Induced by after the Addition of MinD, MinE and ATP 39
4.2. Improvement of Min Protein Purification 42
4.2.1. Creating Imidazole Concentration Gradient in a HisTrap affinity Column to Increase MinD Purity 43
4.2.3. ATPase Assay of Min Protein Hydrolysis 48
4.2.4. Summary of Comparison of Two MinD Purification Methods and Some Improved Process 50
4.3. The Potential Physiological Function of Min Protein Oscillation 51
5. Conclusions 52
6. References 53


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