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    National Tsing Hua University Institutional Repository > 生命科學院  > 分子與細胞生物研究所 > 博碩士論文  >  在線蟲神經系統中探討 LIN-2, SYD-2 及 MAP1-A 對於 Kinesin-3 UNC-2015 移動機制之調節

    Please use this identifier to cite or link to this item: http://nthur.lib.nthu.edu.tw/dspace/handle/987654321/86821

    Title: 在線蟲神經系統中探討 LIN-2, SYD-2 及 MAP1-A 對於 Kinesin-3 UNC-2015 移動機制之調節
    Authors: 吳恭和
    Wu, Gong Her
    Description: GH009580820
    Date: 2015
    Keywords: UNC-2015 SYD-2 LIN-2 MAP-1A kinesin
    UNC-2015 SYD-2 LIN-2 MAP-1A kinesin
    Abstract: 線蟲之UNC-2015屬於kinesin-3 蛋白家族,是哺乳類KIF1A的同源基因。 UNC-2015 主要功能為參與神經系統中快速軸突突觸囊泡之運輸,並且對於神經細胞的生存與可塑性扮演相當重要的角色。目前,文獻中關於UNC-2015活性之調控機制細節並不詳盡,因此我專注於研究UNC-2015的活化以及不同蛋白質對於UNC-2015功能的影響。我在本論文中指出LIN-2 (CASK) 及SYD-2 (liprin-α) 與UNC-2015作用時,這兩個蛋白與UNC-2015的結合位置是相互重疊的。在本論文中,我藉由酵母菌雙雜合實驗(Yeast two-hybrid)及免疫沉澱分析(co-immunoprecipitation assays)歸納出UNC-2015/LIN-2, SYD-2/LIN-2 及 MAP-1A/LIN-2蛋白之間相互作用時彼此的結合區域。我更進一步利用雙分子螢光互補分析BiFC (bimolecular fluorescence complementation)研究上述蛋白質在線蟲中的結合作用以及結合後的蛋白質複合物在生物體中的位置。線蟲中UNC-2015移動速度分析實驗指出在lin-2 的突變株中,UNC-2015及其貨物(cargo)的標定蛋白SNB-1傳送速度會明顯的減慢,而此現象無法藉由SYD-2蛋白 大量表現彌補。 另外在lin-2 knock out /syd-2 knock down 的雙突變株中,UNC-2015減慢的現象並未因此加劇。這些實驗結果顯示,LIN-2 與SYD-2 是作用在相同UNC-2015調節路徑上,且LIN-2與SYD-2 需要同時存在才得以正確調控UNC-2015之功能。另外,本論文中也提及MAP-1A (microtubule-associate protein 1A) 也同樣由與LIN-2 的結合作用影響UNC-2015 與微管(microtubule)的接觸並進一步影響UNC-2015的移動速度。因此,我歸納出LIN-2為調控UNC-2015蛋白之運輸功能的樞紐。
    UNC-2015 (KIF1A) is a neuron-specific kinesin-3 which mainly involved in synaptic vesicles rapid axonal transport. While the function of this motor is of critical importance for neuronal viability and plasticity, little is known about how the motor is regulated. In this thesis, I identified a LIN-2 (CASK) binding site on UNC-2015 which overlaps with that of SYD-2 (liprin-α) binding domain. Yeast two-hybrid (Y2H) and co-immunoprecipitation (co-IP) assays reveal specific interaction domains between UNC-2015/LIN-2, SYD-2/LIN-2 and MAP-1A(microtubule-associate protein 1A)/LIN-2. These in vitro interactions were further confirmed by using in vivo BiFC (bimolecular fluorescence complementation) assay in living C. elegans. UNC-2015 motility, as well as transportation rate of UNC-2015 specific cargo, SNB-1 cargo, is significantly diminished in LIN-2 knockout (KO) worms, and this effect cannot be compensated by overexpressing UNC-2015’s activator protein SYD-2. Moreover, the diminishing effect on UNC-2015 motility can’t be further enhanced in LIN-2 KO/SYD-2 KD worm. These data reveal that LIN-2 and SYD-2 both act in the same UNC-2015 regulation pathway and both LIN-2 and SYD-2 are essential for orchestration on activating UNC-2015. Lastly, MAP-1A mediated UNC-2015 transport characteristics depending on LIN-2, suggesting that LIN-2 plays a central role in regulating kinesin-3 UNC-2015.
    URI: http://nthur.lib.nthu.edu.tw/dspace/handle/987654321/86821
    Source: http://thesis.nthu.edu.tw/cgi-bin/gs/hugsweb.cgi?o=dnthucdr&i=sGH009580820.id
    Appears in Collections:[分子與細胞生物研究所] 博碩士論文

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