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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 歐展言 | |
| dc.contributor.author | Hao-Wen Lee | en |
| dc.contributor.author | 李顥文 | zh_TW |
| dc.date.accessioned | 2021-07-11T15:01:55Z | - |
| dc.date.available | 2024-08-28 | |
| dc.date.copyright | 2019-08-28 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-08-19 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78524 | - |
| dc.description.abstract | 神經為生物體中用來接收環境訊息並透過突觸（synapse）傳遞訊息的細胞。為了維持神經的正常運作，特定的蛋白質與囊泡必須被運送到正確的突觸區域以維持其功能。目前已知負責極性突觸運輸（polarized synaptic trafficking）的馬達蛋白（motor proteins）分為負責正向運輸（anterograde trafficking）的驅動蛋白（kinesin）UNC-116/kinesin-1與UNC-104/kinesin-3；以及逆向運輸（retrograde trafficking）的動力蛋白（dynein）。先前的研究指出在線蟲中有一類週期素的同系蛋白CYY-1會啟動它的下游激酶PCT-1來調控突觸運輸。在我們的研究中，我們發現一個鈣調素依賴性蛋白UNC-43/CaMKII會調控突觸運輸。缺少CYY-1會使得線蟲DA9神經中的突觸小泡被錯誤地送進樹突，而當UNC-43的突變被加入時，這個錯誤的運輸會被抑制。失去UNC-43並沒有辦法恢復UNC-104或UNC-116突變中突觸小泡的錯誤運輸，顯示這些驅動蛋白即使在缺乏UNC-43的狀況下仍能正常運作。透過曠時影像（timelapse imaging）我們發現在同時缺乏CYY-1與UNC-43的線蟲當中，逆向運輸的比例會大幅度下降。在同時缺乏CYY-1與UNC-43的線蟲當中使其DA9中過度表現UNC-43也無法逆轉UNC-43對於突觸運輸的救援效果，顯示UNC-43的調控可能不是一個細胞自主的反應。總而言之，UNC-43會透過抑制逆向運輸來調控突觸運輸，且這個運輸可能是跟驅動蛋白無關的。 | zh_TW |
| dc.description.abstract | Neurons are specialized cells that receive signals from environments and transmit signals to target cells through synapses. To maintain the function of synapses, proteins and vesicles, such as synaptic vesicle protein transport vesicles (STVs), must be precisely transported to synaptic region. Anterograde directed motors, UNC-116/kinesin-1, UNC-104/kinesin-3, and retrograde directed motor dynein regulate polarized synaptic trafficking. Previous study showed that a cyclin homolog CYY-1/cyclinc Y activates cyclin-dependent Pctaire kinase PCT-1 to regulate synaptic trafficking in C. elegans DA9 neuron. In our study, we identified a calmodulin-dependent kinase UNC-43/CaMKII necessary for regulating synaptic trafficking. When unc-43 was genetically ablated in cyy-1 mutant, mislocalized synaptic vesicles (SVs) in dendrites were reduced. Loss of UNC-43 was not able to rescue synaptic defect caused by loss of UNC-104 or UNC-116, indicating that these kinesins are still necessary in the absence of UNC-43. Results of timelapse imaging showed that retrograde transport was reduced in cyy-1; unc-43 double mutant. Overexpression of UNC-43 did not restore synaptic localization in cyy-1; unc-43(tm1605), indicating that UNC-43 might not function cell autonomously. Altogether, UNC-43 regulates synaptic trafficking by reducing retrograde transport events in an independent pathway with kinesin-mediated trafficking. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:01:55Z (GMT). No. of bitstreams: 1 ntu-108-R06442025-1.pdf: 5493920 bytes, checksum: e3e601b53f81251ee0e75b252f14c3a0 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 中文摘要 i
ABSTRACT ii TABLE OF CONTENTS iii 1. INTRODUCTION 1 1.1 Polarized trafficking in neuron 1 1.2 Functions and regulations of motor proteins 2 1.3 Axonal transport and neuronal disorders 4 1.4 The role of Ca2+ in neurons 5 2. MATERIALS AND METHODS 7 2.1 Strains and genetics 7 2.2 Cloning and constructs 8 2.3 Combination of different genotypes 9 2.4 Worm lysis for genomic DNA 10 2.5 Dynamic imaging 10 2.6 Functional assay of backward locomotion 10 2.7 Quantification 11 3. RESULTS 13 3.1 Loss of UNC-43/CaMKII suppresses synaptic defect caused by cyy-1 13 3.2 Gain of function of UNC-43 results in the similar phenotype to loss of function 15 3.3 UNC-43 seemed not to regulate kinesins directly 16 3.4 Retrograde transport was strongly suppressed in cyy-1; unc-43(tm1605) and cyy-1; unc-43(n498) 17 3.5 Loss of CDK-5 restored retrograde events in cyy-1; unc43(tm1605) 19 3.6 Loss of UNC-43 disrupted SVs localization in the absence of SYD-2 20 3.7 Depletion of UNC-43 suppressed the defect caused by loss of UNC-2 21 3.8 Overexpression of UNC-43 in DA9 did not restored SVs localization 21 3.9 Functional assay revealed that backward locomotion was damaged in cyy-1; unc-43 worms 22 3.10 CYY-1 and UNC-43 regulate mitochondria localization 23 3.11 Muscle arms were thinner but still existed in cyy-1; unc-43 (tm1605) 24 4. DISCUSSION 26 4.1 Synaptic targeting character of UNC-43 26 4.2 Dynamic Images revealed that retrograde transport was suppressed in the absence of UNC-43 27 4.3 Candidate screening of UNC-43 28 4.4 Overexpression of UNC-43 rescues synaptic formation but not trafficking 28 4.5 UNC-43 also regulate mitochondria and postsynaptic cell morphology 29 5. FIGURES 31 Figure 1 Loss of UNC-43 suppressed synaptic defect caused by losing CYY-1/PCY-1 but not CDK-5 32 Figure 2 Alternative allele of UNC-43 also suppressed cyy-1 defect 36 Figure 3 UNC-43 seemed not to regulate motor proteins directly 38 Figure 4 Loss of UNC-43 suppressed retrograde transport events 43 Figure 5 Loss of CDK-5 restored retrograde events that was suppressed in cyy-1; unc-43(tm1605) 46 Figure 6 UNC-43 seemed not to function through SYD-2-mediated synaptic formation and trafficking 48 Figure 7 Depletion of UNC-43 suppressed the defect caused by loss of UNC-2 49 Figure 8 Overexpression of UNC-43 could not restore the rescue effect in cyy-1; unc-43(tm1605) 53 Figure 9 Functional assay revealed that cyy-1; unc-43 worms were almost incapable of backward locomotion 55 Figure 10 UNC-43 regulated mitochondria localization with CYY-1 58 Figure 11 Loss of UNC-43 made both neurite and muscle arm thinner 59 6. REFERENCE 60 7 APPENDIX 80 7.1 Primers for genotyping and sequencing 80 7.2 Constructs and transgenic worms 83 7.3 Plasmid sequence and cDNA 84 8. SUPPLEMENTARY FIGURES 93 Figure S1 Genetic ablation of DNC-1 in cyy-1 mutant rescued synaptic defect 94 Figure S2 Kymographs with obvious events 99 | |
| dc.language.iso | en | |
| dc.subject | 動力蛋白 | zh_TW |
| dc.subject | 驅動蛋白 | zh_TW |
| dc.subject | 突觸小泡運輸 | zh_TW |
| dc.subject | UNC-43蛋白 | zh_TW |
| dc.subject | CYY-1蛋白 | zh_TW |
| dc.subject | synaptic vesicles trafficking | en |
| dc.subject | UNC-43 | en |
| dc.subject | CYY-1 | en |
| dc.subject | dynein complex | en |
| dc.subject | kinesin | en |
| dc.title | 研究UNC-43蛋白於神經運輸的角色 | zh_TW |
| dc.title | The characterization of UNC-43 in synaptic trafficking | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 簡正鼎,林敬哲,王歐力 | |
| dc.subject.keyword | 突觸小泡運輸,驅動蛋白,動力蛋白,CYY-1蛋白,UNC-43蛋白, | zh_TW |
| dc.subject.keyword | synaptic vesicles trafficking,kinesin,dynein complex,CYY-1,UNC-43, | en |
| dc.relation.page | 99 | |
| dc.identifier.doi | 10.6342/NTU201903855 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2019-08-19 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| dc.date.embargo-lift | 2024-08-28 | - |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
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