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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 潘建源(Chien-Yuan Pan) | |
| dc.contributor.author | Wee-Shin Lim | en |
| dc.contributor.author | 林偉莘 | zh_TW |
| dc.date.accessioned | 2021-06-17T02:42:06Z | - |
| dc.date.available | 2020-08-24 | |
| dc.date.copyright | 2017-08-24 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-08-16 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68919 | - |
| dc.description.abstract | 最近的一些研究表示，Auxilin的突變與早發性的帕金森症有關。 Auxilin被認為會協助Hsc70把細胞胞吞後形成的網格蛋白（Clathrin）鞘膜解除。另一方面，Auxilin會調節和結合AP2 以及 Dynamin形成網格蛋白的囊泡，促進早期細胞胞吞作用。 Auxilin參與在形成網格蛋白的胞吞作用過程中的調控機制尚不清楚，我們之前的Yeast Two Hybrid實驗顯示NCS-1會和Auxilin 產生交互作用。我們在這篇文章假設Auxilin可能與NCS-1相互作用，NCS-1是一種在胞吐過程中調節PI4P的蛋白質。Auxilin和NCS-1 都參與在PIP2脂質激酶途徑中並都會和一些細胞膜蛋白結合，這兩種蛋白質在囊泡回收期間會有很高的機會進行交互作用。本研究旨在透過Calcium Imaging了解Auxilin及其Auxilin突變蛋白在神經元突觸前囊泡再循環中的作用。第二個目的是透過記錄螢光成像Fluorescent Resonance Energy Transfer (FRET)的變化來研究NCS-1和Auxilin 還有突變的Auxilin之間的相互作用。我們將Auxilin和突變的Auxilin質體轉染到介於DIV10-12的神經元細胞，研究突觸小泡回收中的作用。為了監測胞吞作用，我們使用Flou-2鈣螯合劑染料來檢測高濃度鉀觸發細胞鈣濃pin度的變化。我們的研究結果顯示，Auxilin的表現可能增強突觸前神經元的囊泡回收，然而突變Auxilin的表現似乎阻礙了突觸前神經元的胞吞作用。我們還共同轉染NCS-1-EYFP和ECFP-Auxilin到HEK293中觀察蛋白質之間的相互作用，用4-bromo calcium ionophore A-23187刺激細胞和記錄FRET熒光的變化。 FRET結果表示，Auxilin和NCS-1在A23187刺激後具有相互作用，但交互作用不會發生在具有點突變的Auxilin H874Q蛋白上。這些結果表明，NCS-1和Auxilin的作用對於囊泡回收是重要的，因為突變的Auxilin可能會干擾細胞胞吞作用並導致神經退化性疾病。 雖然NCS-1和Auxilin相互作用帶來的功能仍然是未知的，但是是一個有趣的課題作為我們下一步進一步的研究。 | zh_TW |
| dc.description.abstract | Recent studies indicate that the mutant forms of auxilin are linked to early onset Parkinson’s disease. Auxilin was known as a cofactor that facilitates with Hsc70 to uncoat clathrin complex from newly formed vesicles in clathrin-mediated endocytosis (CME). On the other hand, auxilin also binds and regulates AP2, Dynamin, and proteins involved in the formation of CME. From previous study in our lab, we observed that NCS-1 can interact with auxilin in a yeast two-hybrid screen in which NCS-1 was used as a bait. In this study, we hypothesize that NCS-1 may act as a calcium binding protein and recruit auxilin during early stage of CME. NCS-1 is a protein that regulates PI4P and promotes synapse vesicles exocytosis. These two proteins both regulating membrane proteins and PIP2 lipid kinase pathways have a high chance to interact with each other during vesicle recycling. This study aims to understand the effect of auxilin and its mutant variants on synapse vesicles recycling via Ca2+ imaging. The second aim is to study the physical interaction between NCS-1 and mutant auxilin by using Förster Resonance Energy Transfer (FRET) in fluorescent imaging. We show that overexpression of auxilin may enhance the vesicle recycling on the pre-synapse neuron and results in increased neurotransmission. Our FRET results show that auxilin can interact with NCS-1 after A23187 induced intracellular calcium elevation. However, auxilin H874Q, a mutant that cannot bind with Hsc70 protein, fails to interact with NCS-1 under the same condition. These results indicated that the roles of NCS-1 and auxilin are important on vesicle recycling as deformed auxilin might disturb the cell endocytosis and might link to neurodegeneration diseases. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T02:42:06Z (GMT). No. of bitstreams: 1 ntu-106-R04b21002-1.pdf: 980091 bytes, checksum: 24df121ee68e434ee5715ff0a66db98d (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 口試委員審定書 2
Acknowledgement 4 中文摘要 5 Abstract 6 1. Introduction 7 1.1 Synaptic vesicle recycling 7 1.2 Clathrin-coated endocytosis and the roles of human auxilin-1. 9 1.3 Neuronal calcium sensor protien-1 and its role in exocytosis. 10 1.4 The importance roles of calcium binding proteins in synapse vesicle recycling. 11 1.5 Regulation of synapse vesicle recycling and neurodegeneration diseases. 12 Aim 14 2. Methodology and Material 2.1 Chemicals 15 2.2 Primary culture of rat embryonic cortical neuron 15 2.3 Culture of HEK293T cell lines 16 2.4 Cell transfection by lipofectamine 3000 17 2.5 Förster resonance energy transfer (FRET) 18 2.6 Ca2+ imaging study of neurotransmission 18 2.7 E coli. Transformation 19 2.8 Plasmid extraction 19 2.9 Western blots 20 2.10 Data analysis 21 3. Results 3.1 Overexpression of pcDNA3.1-Auxilin in HEK 293T 22 3.2 Stationary FRET show close distance between ECFP-Auxilin and NCS1-YFP. 22 3.3 ECFP-Auxilin and NCS1-YFP have interaction after puffing A23187. 23 3.4 Transfection in DIV10-12 neurons did not had effect on neurotransmission 24 3.5 Overexpression of Auxilin in DIV12 neuron enhance neurotransmission 25 3.6 Addition results and supplementary results 26 4. Discussion and conclusion 4.1 The interaction of Auxilin and NCS-1 28 4.2 Auxilin enhance neurotransmission and the relationship with vesicle recycling. 29 5. References 30 6. Figures 33 | |
| dc.language.iso | en | |
| dc.subject | 網格蛋白 | zh_TW |
| dc.subject | 蛋白質交互作用 | zh_TW |
| dc.subject | 突觸小泡回收 | zh_TW |
| dc.subject | 胞吞作用 | zh_TW |
| dc.subject | 神經傳導 | zh_TW |
| dc.subject | calcium binding protein | en |
| dc.subject | synapse vesicles recycling | en |
| dc.subject | clathrin-mediated endocytosis | en |
| dc.subject | FRET | en |
| dc.subject | Calcium Imaging | en |
| dc.subject | Protein interaction | en |
| dc.subject | Neurotransmission | en |
| dc.subject | NCS-1 | en |
| dc.subject | Auxilin | en |
| dc.title | Auxilin 強化神經傳導和NCS-1有交互作用 | zh_TW |
| dc.title | Auxilin Enhance Neurotransmission and Interacts with NCS-1 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 謝秀梅(Hsiu-Mei Hsieh),梁淑鈴(Shu-Ling Liang),姚季光(Chi-Kuang Yao) | |
| dc.subject.keyword | 神經傳導,蛋白質交互作用,網格蛋白,胞吞作用,突觸小泡回收, | zh_TW |
| dc.subject.keyword | Auxilin,NCS-1,Neurotransmission,Protein interaction,Calcium Imaging,FRET,clathrin-mediated endocytosis,calcium binding protein,synapse vesicles recycling, | en |
| dc.relation.page | 51 | |
| dc.identifier.doi | 10.6342/NTU201703437 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-08-16 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生命科學系 | zh_TW |
| 顯示於系所單位： | 生命科學系 | |
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