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研究生:鄭鎌杰
研究生(外文):Cheng, Lien-Chieh
論文名稱:細胞黏著分子Echinoid蛋白質功能之研究
論文名稱(外文):Functional Dissection of Cell Adhesion Molecule Echinoid
指導教授:徐瑞洲
指導教授(外文):Hsu, Jui-Chou
口試委員:陳令儀白麗美
口試日期:2011-7-12
學位類別:碩士
校院名稱:國立清華大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:英文
論文頁數:47
中文關鍵詞:Echinoid
外文關鍵詞:Echinoid
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Echinoid(Ed)是一細胞黏著分子中的跨膜蛋白,其在細胞外的結構包含了7個免疫球蛋白結構區域和二個纖維蛋白結構區域,透過跨膜區域可表現在細胞膜上。細胞內結構是315個未完全了解其功能的胺肌酸序列,最後4個胺肌酸是一非典型的PDZ-Binding Motif,透過細胞外的結構Ed可和鄰近細胞上的Ed做Homophilic Interaction,而細胞內則是利用PDB-binding Motif 透過Canoe/Bazooka 和Actin Filaments交互作用,因此,當Ed和DE-Cadherin共同表現於細胞的Adherens Junctions (AJs)上時,透過細胞內外不同的結構對細胞的發育形態造成影響。當Ed發生突變的時候,我們可以在果蠅三齡幼蟲翅膀的Imaginal Disc上觀察到,失去Ed的細胞相對於正常細胞會產生一個細胞分選 (Cell Sorting)的現象,並伴隨著細胞頂端表面積收縮(Apical Constriction),而在正常細胞和Ed缺失的細胞之間,Ed因失去Homophilic Interaction而消失在交界處,並且在正常細胞中Ed消失的一端產生Actin Cable。在本篇報告中,我們希望可在短時間內透過即時影像的方式,觀察ed Mutant Clone的形成,設計一可受Rho-4作用的Cleavage Site在Ed的Transmembrane上,觀察細胞在Ed失去Homophilic Interaction時細胞發展的過程。此外,細胞間的Ed透過Extracellular Domain進行Homophilic Interaction,為探討其形成的方式及結構,透過將不同部分的Extracellular Domain表現至ed Mutant Clone中,觀察Wild-Type Ed是否表現回交界處,以了解Ed對Homophilic Interaction重要的區域,進而了解Ed上對 Homophilic Interaction的重要區域。最後,在包圍 ed Mutant Clone交界上的正常細胞中,Ed intracellular domain 不平均的分布和Actin Cable產生在交界處有關,因此透過對Ed intracellular domain不同程度的切除,我們能了解Ed intracellular domain對Actin Cable產生時重要的區域,以進一步探討其和Actin Cable之間的關連性。
Echinoid (Ed), a component of adherens junctions (AJs), is classified in immunoglobulin superfamily of cell adhesion molecules (CAMs). As a transmembrane protein, Ed is divided into two main domains: the extracellular domain and the intracellular domain. Ed demonstrates the homophilic interaction between adjacent cells by the extracellular domain, composed of seven immunoglobulin (IG) domains and two fibronectin type III (FnIII) domains, and has a non-typical PDZ-binding motif in the intracellular domain interacting with actin filaments through Canoe or Bazooka. In previous reports, ed mutant clone strongly segregated from wild-type cells and formed smooth and round borders without Ed at the interface. The apical surface of clones decreased accompanying with actin cable which was formed at the clonal side of wild type cells.

To see a process of ed mutant clone formation, a cleavage site activated by rho-4 has been designed on Ed trasmembrane domain. As Ed extracellular domain cleaved, we could see a process of ed mutant clone formation in short time by losing homophilic interaction. In addition, by combining ed-RNAi with overexpression experiments in this report, functional dissection of Ed can be performed. We found a certain part of Ed extracellular domain can be homophilic interaction and recruit WT-Ed at the interface. This could help to investigate Ed homophilic interaction. Finally, recent data showed that actin cable formation came from unevenly distribution of Ed intracellular domain in wild type cells surrounding ed mutant clone. A certain part of Ed intracellular domain influencing actin cable formation could be found by Ed intracellular truncations.

Chapters
1. Introduction P01

2. Materials and Methods P04

3. Results
3.1 A Process of ed Mutant Clone Formation P08
3.2 Homophilic Interaction of Ed Extracellular Domain P12
3.3 Ed Intracellular Domain and Actin Cable Formation P17

4. Discussion
4.1 A Live Imaging Performed by Two Expression Systems P18
4.2 Homophilic Interaction of Ed Extracellular Domain P19
4.3 Differential Expression of EdICD and Actin Cable Formation P21

Figures P24
Tables P44
References P45

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Escudero, L. M., Wei, S. Y., Chiu, W. H., Modolell, J., & Hsu, J. C. (2003). Echinoid synergizes with the Notch signaling pathway in Drosophila mesothorax bristle patterning. Development, 130(25), 6305-6316.
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