在線蟲神經系統中LIN-2與UNC-104的交互作用及其活化UNC ...

... 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 ... 資料載入處理中... 跳到主要內容 臺灣博碩士論文加值系統 ::: 網站導覽| 首頁| 關於本站| 聯絡我們| 國圖首頁| 常見問題| 操作說明 English |FB專頁 |Mobile 免費會員 登入| 註冊 功能切換導覽列 (159.65.11.210)您好！臺灣時間：2022/08/1312:01 字體大小：       ::: 詳目顯示 recordfocus 第1筆/ 共1筆  /1頁 論文基本資料 摘要 外文摘要 目次 參考文獻 電子全文 紙本論文 論文連結 QRCode 本論文永久網址: 複製永久網址Twitter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):MolecularMechanismsUnderlyingLIN-2'sAbilitytoInteractwithUNC-104andtoActivateItsMotilityintheNervousSystemofC.elegans指導教授:王歐力指導教授(外文):OliverI.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-2、UNC-104/KIF1A、axonaltransport、C.elegans相關次數: 被引用:0點閱:261評分:下載:20書目收藏:0 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activezone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-associatedguanylatekinase(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(multidomainscaffoldingprotein)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(MAGUKbindingstalk,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invitro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/KIF1Aismicrotubule-dependent,neuron-specifickinesinresponsibleforfastaxonaltransportofsynapticvesicles.However,themolecularmechanismofregulatingUNC-104directionaltransportisnotfullyunderstood.Recentstudiessuggestthatcargoassynapticprecursorscouldregulatethemotor’smovements.Forexample,theactivezoneproteinsSYD-2wasfoundtofacilitateUNC-104’smotilityinanterogradedirections.SYD-2bindstoaspecificdomainatthemotor’scoiled-coilregionandreviewingtheliteraturewefoundthatanotherproteinLIN-2alsobindstothisregion,thereforethisproteinmightbealsoinvolvedinmotorregulation.LIN-2isamemberofthemembrane-associatedguanylatekinase(MAGUK)family,anditsmammalianhomologueofLIN-2isCASK,multidomainscaffoldingproteinhighlyexpressedinneurons.Interestingly,apreviousstudyrevealedthatUNC-104containsaMBS(MAGUKbindingstalk)site,andindeed,ouryeasttwo-hybridassayshowedthataMBSconstructofUNC-104interactswithLIN-2’sGUKdomain.Interestingly,aLIN-2ΔGUKconstructrevealsreducedinteractionwithUNC-104’sMBS,butstronginteractionwithUNC-104’sFHA-CC2domain,suggestingtonegativelyregulatethemotor’sactivity.Inaddition,theintegratedSH3-GUKunitmaybeinhibitoryforLIN-2’sinteractionwiththeMBSwhileontheotherhanditstronglyinteractswiththeFHA-CC2domain.Besidestheseinvitroinvestigations,wefoundthatinlin-2knockoutworms,theanterogradetransportofUNC-104isreduced(displayinge.g.shorterrun-lengths),whiletheretrogradetransport(carriedoutbythedyneinmotor)isenhanced.Thesedataimplicatethatloss-of-functionofLIN-2causesUNC-104tosufferdefeatintug-of-wareventsbetweenopposingmotors,resultinginimpairedanterogradebutfacilitatedretrogradetransport.Together,thesedatademonstrateanovelroleforLIN-2todirectlyinteractwithUNC-104andtoactivatethemotor’smotility. 1.Introduction11.1Molecularmotorsandaxonaltransport11.2Scaffoldingproteinsfacilitatemotor-cargobinding21.3FunctionandstructureofC.elegansUNC-10431.4RegulationofprocessivemotoractivityofUNC-104/KIF1A41.5Bidirectionaltransportofcargo51.6C.elegansLIN-2belongstotheMAGUKfamily61.7MultiplerolesofCASKinmammaliannervoussystem71.8Hypothesis82.Materialsandmethods102.1Plasmidcloning102.2Yeasttwo-hybridassay112.3StrainsandmaintenanceofC.elegans122.4WesternblotanalysisofLIN-2proteinexpression142.5Microscopictransportassay:motilityanalysisandstatistics143.Results163.1AdetailedsequencehomologycomparisonbetweenGAKIN’sMBSandUNC-104’sMBS163.2C.elegansLIN-2’sGUKdomainbindstoUNC-104’sMBSdomain163.3AnintramolecularSH3-GUKinteractionwithinLIN-2mayregulateitsbindingtoUNC-104173.4Loss-of-functionofLIN-2affectsmotoractivityinC.elegansnervoussystem184.Discussion224.1LIN-2mightemploymorethanonemodetointeractwithUNC-104224.2ProposedregulatorymechanismsmediatedbyintramolecularSH3-GUKinteractions234.3LIN-2mayhavethepotentialtocompetewithLiprin-αforbindingtoUNC-104andperformdifferentfunctionstoregulateUNC-104activity254.4ProposedfunctionofLIN-2intug-of-warmodel264.5LIN-2maybeinvolvedinregulatingretrogradetransport275.Futureperspective296.References307.Figures358.Appendix51 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 電子全文  國圖紙本論文 連結至畢業學校之論文網頁點我開啟連結註:此連結為研究生畢業學校所提供，不一定有電子全文可供下載，若連結有誤，請點選上方之〝勘誤回報〞功能，我們會盡快修正，謝謝！ 推文 網路書籤 推薦 評分 引用網址 轉寄                                                                                                                                                                                                                    top 相關論文 相關期刊 熱門點閱論文 1. 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 2. 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