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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 歐展言(Chan-Yen Ou) | |
dc.contributor.author | Wai-Hou Tam | en |
dc.contributor.author | 譚偉濠 | zh_TW |
dc.date.accessioned | 2021-06-15T13:25:07Z | - |
dc.date.available | 2020-08-26 | |
dc.date.copyright | 2016-08-26 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-05-30 | |
dc.identifier.citation | Ahmari, S.E., Buchanan, J., and Smith, S.J. (2000). Assembly of presynaptic active zones from cytoplasmic transport packets. Nat. Neurosci. 3, 445-451.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51096 | - |
dc.description.abstract | 突觸(synapse)是神經細胞建立彼此聯結的特化結構。突觸活化區蛋白(active zone protein)和突觸小泡(synaptic vesicle)的這些突觸部件必需運送到應該形成突觸的區域組成一個有功能的結構讓神經進行訊息傳導。突觸部件的運送可藉由在微管(microtubule)上移動的分子馬達來達成。先前的研究指出,Gαs蛋白信號路徑中的蛋白激酶A(PKA)參與調控動力馬達(dynein)對於細胞內分子和胞器的運輸。然而PKA在突觸部件運輸和突觸形成中的作用尚不清楚。我的研究顯示,在線蟲的神經內Gαs信號調控突觸部件運送和突觸形成。活化Gαs路徑的突變體會有突觸小泡和活化區蛋白錯誤地分佈至樹突區域以及軸突突觸數目顯著的減少。我發現Gαs/GSA-1作用於DA9神經元內並且透過腺苷酸環化酶(adenylyl cyclase)和PKA來控制突觸形成。此外,我證明了PKA和PCTAIRE激酶1(PCT-1)在突觸小泡分佈和突觸形成的調控上相互拮抗,遺傳分析顯示PKA會作用在PCT-1的下游。我進一步的研究發現gsa-1(gf)的功能需要動力馬達的組件DHC-1以及其功能調節子NUD-2的參與。總結以上結果,Gαs信號調控負責逆向運輸的動力馬達去運送突觸小泡,以及該信號會被PCT-1激酶抑制以形成正確的突觸結構和神經迴路。 | zh_TW |
dc.description.abstract | Synapse requires synaptic vesicles and active zone proteins to conduct neurotransmission. Molecular motor proteins are responsible for synaptic component transport to synaptic regions on microtubules. Previous studies reported that protein kinase A (PKA), the component of Gαs protein signaling pathway, is implicated in regulating dynein-dependent cargo trafficking of intracellular molecules and organelles. However, the role of PKA in synaptic component transport and synapse formation remains unclear. Here, I showed that the Gαs signaling regulates the localization of synaptic components and synapse formation in the C. elegans. Gαs pathway activation mutants had the synaptic vesicles and active zone proteins mislocalized to the dendrites, and the number of axonal synapses was significantly reduced. I found that Gαs/GSA-1 functions cell-autonomously in DA9 neurons and acts through adenylyl cyclase and PKA to control synapse formation. Furthermore, I demonstrated that PKA and cyclin-dependent kinase PCT-1/PCTK1 antagonize each other in regulating synaptic vesicle distribution and synapse formation, and PKA likely functions downstream to PCT-1. Finally, I showed that the dynein motor component DHC-1 and its functional regulator NUD-2 are both required for gsa-1(gf) mutant phenotypes. Together, these results suggested that Gαs signaling requires dynein-mediated retrograde transport to localize synaptic vesicles to the dendrites, and this signaling is controlled by PCT-1 for correct synapse assembly and neural circuits. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T13:25:07Z (GMT). No. of bitstreams: 1 ntu-105-R02442034-1.pdf: 4622620 bytes, checksum: 92ba42d451e8235a3ff96b01dbace083 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 摘要…………………………………………………………………………….………...i
ABSTRACT………………………………………………………………………….....ii CONTENTS……………………………………………………………………………iv I. INTRODUCTION…………………………………………………………………….1 1.1 Synapse and neurotransmission……………………………………………...1 1.2 Motor protein for intracellular transport……………………………………….2 1.3 Axonal transport and neuronal disorders……………………………………..3 1.4 The role of protein kinase A in neuronal functions…………………………..4 II. MATERIALS AND METHODS…………………………………………………….7 2.1 Strains and Genetics…………………………………………………………....7 2.2 Cloning and Constructs…………………………………………………………7 2.3 Combination of different genotypes…………………………………………..8 2.4 Worm lysis for genomic DNA......................................................................9 2.5 Confocal imaging……………………………………………………………….9 2.6 Quantification…………………………………………………………………10 III. RESULTS…………………………………………………………………………13 3.1 Activation of Gαs signaling components disrupted synaptic vesicle localization and synapse formation…………………………….......……….......13 3.2 GSA-1 functioned cell-autonomously in DA9………………………...........16 3.3 The localization of other synaptic components UNC-10 and SNG-1 were also disrupted in gsa-1(gf) mutants……....……...........…………………..........17 3.4 gsa-1(gf) mutants displayed abnormal active zone structure………........18 3.5 Abnormal synaptic distribution was initiated from L3 stage in gsa-1(gf)..19 3.6 Gαs protein functioned through adenylyl cyclase and PKA……………....20 3.7 GSA-1 functioned in the same pathway with PCT-1 but not CDK-5….....21 3.8 PCT-1 antagonized GSA-1 signaling…………………………………….....23 3.9 The synaptic defects in pct-1 required ACY-1 and PKA……………….....24 3.10 Depletion of ACY-1 and PKA did not suppress cdk-5 mutant phenotype.......................................................................................................25 3.11 Gαs signaling inhibited PCT-1 by PKA phosphorylation at S316 residue............................................................................................................26 3.12 Overexpression of phosphomimetic KIN-2 rescued pct-1 mutant phenotype………………………………………………………………….............28 3.13 Dynein components DHC-1 and NUD-2 were required for Gαs signaling.........................................................................................................29 IV. DISCUSSION……………………………………………………………………..31 4.1 Gαs signaling regulates synaptic component distribution and locomotion......................................................................................................31 4.2 Gαs signaling is controlled by PCT-1 for correct synaptic vesicle localization…………………………………………...............…………………....32 4.3 Protein kinases may directly or indirectly regulates motor activity……....34 4.4 PKA may regulate synaptic transport through calcium signaling……......34 V. FIGURES………………………………………………………………………......36 Figure 1. Activation the components of Gαs signaling pathway disrupted synaptic vesicle localization and synapse formation………………….............36 Figure 2. GSA-1 acted cell-autonomously in DA9…………………………......39 Figure 3. The distribution of UNC-10 and SNG-1 was also disrupted in gsa- 1(gf)……………………………………………………………………………........41 Figure 4. gsa-1(gf) mutants displayed abnormal synapse structures…….....43 Figure 5. Abnormal synaptic vesicle localization in gsa-1(gf) was initiated at L3 stage………………..…………………………………………………………….....45 Figure 6. GSA-1 acted through ACY-1 and PKA…………………………........47 Figure 7. GSA-1 was in the same pathway with PCT-1 and not CDK-5….....50 Figure 8. Overexpression of PCT-1 antagonized Gαs signaling…………......53 Figure 9. Depletion ACY-1 or PKA suppressed pct-1 phenotype………........55 Figure 10. Ablation of ACY-1 and PKA did not suppress cdk-5 phenotype...58 Figure 11. Predicted PKA phosphorylation site in Pctaire kinase 1………....61 Figure 12. The rescue effect of PCT-1 in gsa-1(gf) depended on S316 residue............................................................................................................62 Figure 13. Predicted CDK phosphorylation site in PKA regulatory subunit…65 Figure 14. The synaptic defects in pct-1 mutants were rescued by overexpression of phosphomimetic KIN-2……………………………..............66 Figure 15. Gαs signaling requires dynein component DHC-1 and NUD-2......69 Figure 16. The model of synaptic component transport regulated by Gαs and CDK signaling……………………….………………………………………..........72 VI. REFERENCE………………………………………………………………….....73 VII. APPENDIX…………………………………………………………………….....81 7.1 Constructs and transgenic worms……………………………………..........81 7.2 Primers for genotyping and sequencing………………………………........83 7.3 Plasmid sequence and cDNAs……………………………………….........83 | |
dc.language.iso | en | |
dc.title | G蛋白信號路徑與PCT-1在調控突觸小泡定位的關係 | zh_TW |
dc.title | The relationship between G protein signaling pathway and PCT-1 in regulating synaptic vesicle localization | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林敬哲(Jing-Jer Lin),潘俊良(Chun-Liang Pan) | |
dc.subject.keyword | 突觸小泡定位,突觸形成,Gαs信號,蛋白激?A,PCTAIRE激?1, | zh_TW |
dc.subject.keyword | synaptic vesicle localization,synapse formation,Gαs signaling,PKA,PCT-1, | en |
dc.relation.page | 89 | |
dc.identifier.doi | 10.6342/NTU201600272 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-05-30 | |
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 |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
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ntu-105-1.pdf 目前未授權公開取用 | 4.51 MB | Adobe PDF |
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