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研究生:黃郁心
研究生(外文):Huang, Yu-Hsin
論文名稱:在線蟲神經系統中LIN-2與UNC-104的交互作用及其活化UNC-104運動能力的分子機制
論文名稱(外文):Molecular Mechanisms Underlying LIN-2's Ability to Interact with UNC-104 and to Activate Its Motility in the Nervous System of C. elegans
指導教授:王歐力
指導教授(外文):Oliver I. Wagner
口試委員:桑自剛潘俊良
口試日期:2011-3-28
學位類別:碩士
校院名稱:國立清華大學
系所名稱:分子與細胞生物研究所
學門:生命科學學門
學類:生物科技學類
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:56
中文關鍵詞:LIN-2蛋白微管分子馬達UNC-104/KIF1A神經軸突運輸秀麗隱桿線蟲
外文關鍵詞:LIN-2UNC-104/KIF1Aaxonal transportC. elegans
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UNC-104/KIF1A是一種具有神經專一性的分子馬達,於神經軸突的微管上移動,主要功能為運輸軸突中的突觸囊泡。然而,如何調控UNC-104其方向性運輸特性的分子機制尚未被完全了解。近期的研究指出UNC-104所攜帶的貨物(如突觸前驅物)能夠調控分子馬達的運動。例如active zone蛋白 SYD-2能夠促進UNC-104往anterograde的方向移動。SYD-2 會與UNC-104的特定區域coiled-coil結合,我們研究其他的文獻發現另一個蛋白LIN-2也會與這個區域結合,因此,我們推測LIN-2可能也會參與UNC-104的調控機制。LIN-2屬於一個蛋白質家族: membrane-associated guanylate kinase (MAGUK)家族,它在哺乳類中的同源蛋白為CASK,CASK是由許多特殊區堿構成的鷹架蛋白(multidomain scaffolding protein)且在神經中的表現量特別高。有趣的是,在之前的研究中發現UNC-104具有一個MAGUK蛋白的結合位(MAGUK binding stalk, MBS),而我們的酵母菌雙雜合實驗的確顯示UNC-104的MBS會與LIN-2的GUK區域結合。另一個有趣的發現為LIN-2ΔGUK雖然無法與UNC-104的MBS結合,卻會和UNC-104的FHA-CC2有很強的交互作用,有些研究發現FHA-CC2可能會負向地調控馬達的活性。此外,LIN-2的SH3-GUK被視為一個功能性單元,我們的實驗顯示SH3-GUK對於LIN-2 與UNC-104 的MBS結合是有抑制性的,然而,SH3-GUK卻與FHA-CC2有很強的交互作用。除了in vitro的探討,我們也發現在lin-2 基因被剔除的秀麗隱桿線蟲中,UNC-104往anterograde方向移動的能力受到影響 (anterograde移動路徑變短),而往retrograde方向的運輸(由另一個分子馬達dynein負責)卻被提昇了(retrograde速度變快)。這些數據顯示,在沒有LIN-2蛋白的情況下,UNC-104與dynein間類似拔河的競爭現象受到影響,UNC-104較容易輸給dynein,造成UNC-104往anterograde方向的運輸能力減弱,而由dynein負責往retrograde方向的運輸則被提昇。這些結果顯示了LIN-2所扮演的新角色:與UNC-104進行交互作用並且活化UNC-104的運動能力。
UNC-104/KIF1A is microtubule-dependent, neuron-specific kinesin responsible for fast axonal transport of synaptic vesicles. However, the molecular mechanism of regulating UNC-104 directional transport is not fully understood. Recent studies suggest that cargo as synaptic precursors could regulate the motor’s movements. For example, the active zone proteins SYD-2 was found to facilitate UNC-104’s motility in anterograde directions. SYD-2 binds to a specific domain at the motor’s coiled-coil region and reviewing the literature we found that another protein LIN-2 also binds to this region, therefore this protein might be also involved in motor regulation. LIN-2 is a member of the membrane-associated guanylate kinase (MAGUK) family, and its mammalian homologue of LIN-2 is CASK, multidomain scaffolding protein highly expressed in neurons. Interestingly, a previous study revealed that UNC-104 contains a MBS (MAGUK binding stalk) site, and indeed, our yeast two-hybrid assay showed that a MBS construct of UNC-104 interacts with LIN-2’s GUK domain. Interestingly, a LIN-2ΔGUK construct reveals reduced interaction with UNC-104’s MBS, but strong interaction with UNC-104’s FHA-CC2 domain, suggesting to negatively regulate the motor’s activity. In addition, the integrated SH3-GUK unit may be inhibitory for LIN-2’s interaction with the MBS while on the other hand it strongly interacts with the FHA-CC2 domain. Besides these in vitro investigations, we found that in lin-2 knockout worms, the anterograde transport of UNC-104 is reduced (displaying e.g. shorter run-lengths), while the retrograde transport (carried out by the dynein motor) is enhanced. These data implicate that loss-of-function of LIN-2 causes UNC-104 to suffer defeat in tug-of-war events between opposing motors, resulting in impaired anterograde but facilitated retrograde transport. Together, these data demonstrate a novel role for LIN-2 to directly interact with UNC-104 and to activate the motor’s motility.
1. Introduction 1
1.1 Molecular motors and axonal transport 1
1.2 Scaffolding proteins facilitate motor-cargo binding 2
1.3 Function and structure of C. elegans UNC-104 3
1.4 Regulation of processive motor activity of UNC-104/KIF1A 4
1.5 Bidirectional transport of cargo 5
1.6 C. elegans LIN-2 belongs to the MAGUK family 6
1.7 Multiple roles of CASK in mammalian nervous system 7
1.8 Hypothesis 8
2. Materials and methods 10
2.1 Plasmid cloning 10
2.2 Yeast two-hybrid assay 11
2.3 Strains and maintenance of C. elegans 12
2.4 Western blot analysis of LIN-2 protein expression 14
2.5 Microscopic transport assay: motility analysis and statistics 14
3. Results 16
3.1 A detailed sequence homology comparison between GAKIN’s MBS and UNC-104’s MBS 16
3.2 C. elegans LIN-2’s GUK domain binds to UNC-104’s MBS domain 16
3.3 An intramolecular SH3-GUK interaction within LIN-2 may regulate its binding to UNC-104 17
3.4 Loss-of-function of LIN-2 affects motor activity in C. elegans nervous system 18
4. Discussion 22
4.1 LIN-2 might employ more than one mode to interact with UNC-104 22
4.2 Proposed regulatory mechanisms mediated by intramolecular SH3-GUK interactions 23
4.3 LIN-2 may have the potential to compete with Liprin-α for binding to UNC-104 and perform different functions to regulate UNC-104 activity 25
4.4 Proposed function of LIN-2 in tug-of-war model 26
4.5 LIN-2 may be involved in regulating retrograde transport 27
5. Future perspective 29
6. References 30
7. Figures 35
8. Appendix 51

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1. Dynein和dynactin在突觸囊泡運輸以及在KIF1A/UNC-104的聚集與運輸中所扮演的角色
2. Visualization of interactions between molecular motors and their adaptors in the nervous system of C. elegans using the BiFC assay
3. 研究中間絲蛋白IFB-1在線蟲amphid神經中對粒線體運輸之影響
4. 探討磷酸激酶及磷酸酶影響線蟲神經纖毛生成及纖毛內運輸機制的可能性
5. 研究SNB-1和RAB-3在線蟲神經系統中的軸突運輸
6. 在線蟲神經系統中探討 LIN-2, SYD-2 及 MAP1-A 對於 Kinesin-3 UNC-104 移動機制之調節
7. 研究激酶GCK-2及PKG-1對線蟲神經纖毛生成及鞭毛內運輸機制之影響調控
8. 探討秀麗隱桿線蟲在dynein和dynactin變異下軸突中UNC-104的聚集與運輸
9. 透過細胞骨架與突觸前驅因子調節軸突的快速運輸
10. 第一章: 線蟲三聯複合體RAB-3-UNC-10-SYD-2之活性區調控驅動蛋白-3 UNC-104的作用角色 第二章: PKG-1與GCK-2 能調節線蟲感覺神經之纖毛長度與鞭毛內運輸 第三章: 線蟲中似神經絲蛋白TAG-63能促進神經軸突傳遞機制
11. 探討線蟲神經突觸缺失如何改變微管轉譯後修飾與軸突運輸
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