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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王致恬 | |
| dc.contributor.author | Ning Chiang | en |
| dc.contributor.author | 姜寧 | zh_TW |
| dc.date.accessioned | 2021-06-16T23:36:47Z | - |
| dc.date.available | 2017-07-27 | |
| dc.date.copyright | 2012-07-27 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-07-26 | |
| dc.identifier.citation | Alberts, B. (1994). Molecular biology of the cell, 3rd edn (New York: Garland Pub.).
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Paralysis and early death in cysteine string protein mutants of Drosophila. Science 263, 977-980. Zinsmaier, K.E., Hofbauer, A., Heimbeck, G., Pflugfelder, G.O., Buchner, S., and Buchner, E. (1990). A cysteine-string protein is expressed in retina and brain of Drosophila. J Neurogenet 7, 15-29. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65328 | - |
| dc.description.abstract | 在可興奮性的細胞中，細胞欲釋放的物質會被包裹在囊泡裡，之後囊泡會被運送至靠近細胞膜處，當刺激訊號抵達而引起鈣離子流入細胞後，囊泡會和細胞膜融合，形成融合孔，釋放囊泡中所包裹的內容物，此種細胞釋放其內含物質的方式稱為鈣離子調控性胞吐作用。神經細胞利用此種鈣離子調控性胞吐作用的方式，釋放包裹在突觸囊泡或緻密核心囊泡中的神經傳導物質。半胱胺酸串鍊蛋白(CSP) 是一種囊泡蛋白質，已知可被多種激酶所磷酸化，前人研究也指出其與鈣離子感應蛋白 (Syt I) 有交互作用，因此 CSP 被視為可能影響鈣離子調控性胞吐作用、進而影響神經傳導物質釋放的候選因子。
為了研究 CSP 對胞吐作用的詳細機制，首先我們利用分子生物學技術，在 CSP 第十個絲胺酸的位置上製作點突變，以仿效 CSP 無法被磷酸化或 CSP 持續被磷酸化的狀態。接著，利用反轉錄定量聚合酶連鎖反應，我們確認了轉染至細胞中的 CSP 能夠被過量地表現。之後，運用免疫螢光染色方法，我們確定過量表現的 CSP 及其磷酸化的突變並不會改變 CSP在細胞中的位置。最後，我們使用氧化電流測定技術，檢驗了 CSP 及其磷酸化突變株對於融合孔動態的影響。 我們的實驗結果顯示， CSP 的磷酸化會增加囊泡的融合速率，並且促進融合孔的穩定性；因此我們得知，CSP 的磷酸化在鈣離子調控性胞吐作用中，對於融合孔扮演著重要的調控角色。 | zh_TW |
| dc.description.abstract | In excitable cells, vesicles are transported to fuse with the plasma membrane and release their contents through calcium-regulated exocytosis. Neurons tend to use this way to release their neurotransmitters packaged in synaptic vesicles or large dense-core vesicles. Cysteine string protein (CSP) is a vesicle-associated protein known to be phosphorylated by various protein kinases. Moreover, previous studies have shown that CSP can interact with synaptotagmin I (Syt I), which is the calcium sensor in calcium-regulated exocytosis. Therefore, CSP is thought to be a candidate modulating calcium-regulated exocytosis and thus neurotransmitter release.
In order to understand the detailed regulatory mechanisms of CSP on exocytosis, we first mutated the serine residue at the position of 10 in the amino acid sequence to make either phosphodeficient or phosphomimetic mutants. Subsequently, we used RT-qPCR to confirm the overexpression of CSP after transfection. By using immunofluorescence staining, we observed that overexpressed CSP would not change its subcellular localization. At last, we performed amperometry to investigate the effects of CSP phosphorylation on exocytosis in terms of kinetics of exocytosis and fusion pore dynamics. Our results suggest that CSP phosphorylation increases fusion rate and tends to stabilize exocytotic fusion pore. Therefore, CSP phosphorylation plays an important role in modulating fusion pore in calcium-regulated exocytosis. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T23:36:47Z (GMT). No. of bitstreams: 1 ntu-101-R99B43002-1.pdf: 3744864 bytes, checksum: f3f09fa8d13d6b8fa77c9126ec2d6929 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
致謝 iii 中文摘要 iv Abstract v Abbreviations vi Contents viii Chapter I: Introduction 1 1.1 Calcium-regulated exocytosis 1 1.2 Different secretory vesicles in calcium-regulated exocytosis 2 1.3 Fusion pore 2 1.4 Regulation of calcium-regulated exocytosis and fusion pore 3 1.4.1 SNARE complex 3 1.4.2 Synaptotagmin: the calcium sensor and fusion pore regulator 4 1.5 Cysteine string protein 4 1.5.1 Importance of CSP 5 1.5.2 Isoforms and structure 6 1.5.3 Function 6 1.5.4 Interaction with synaptotagmin I 7 1.5.5 Phosphorylation of CSP 7 1.6 Amperometry: the technique for studying exocytosis 8 1.7 Significance 9 1.8 Specific aims and experimental design 10 Chapter II: Materials and Methods 12 2.1 Subcloning and site-directed mutagenesis 12 2.2 Cell culture 13 2.3 Transient transfection 14 2.4 RNA extraction 15 2.5 Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) 16 2.6 Immunofluorescence staining 16 2.7 Amperometry 18 2.8 Statistics 19 Chapter III: Results 20 3.1 The expression levels of mRNA 20 3.1.1 Transient transfection of CSP increases CSP mRNA 20 3.1.2 Transient transfection of CSP does not change the mRNA levels of Syt I and SNARE proteins 21 3.2 Localization of CSP and SNARE proteins 22 3.2.1 Overexpression of CSP does not change the subcellular colocalization of CSP and Syt I 22 3.2.2 Overexpression of CSP does not change the distribution of CSP on synaptic vesicles and large dense-core vesicles 23 3.3 Effects of CSP phosphorylation on exocytosis 24 3.3.1 Phosphorylation of CSP increases fusion rate 24 3.3.2 Phosphorylation of CSP does not change the characteristics of spikes 26 3.4 Effects of phosphorylation of CSP on fusion pore dynamics 29 3.4.1 The duration of prespike foot is increased by the phosphorylation of CSP 29 3.4.2 Phosphorylation of CSP does not change the average height of prespike foot 30 3.5 Phosphorylation of CSP reduces the Kc and Kd of exocytosis 31 Chapter IV: Discussion 34 4.1 CSP-α is the most abundant CSP isoform in PC12 cells 34 4.2 The localization of CSP in calcium-regulated exocytosis 34 4.3 Interaction with Syt I and SNARE proteins 35 4.4 The effects on spike and fusion pore dynamics 36 4.5 Pros and cons of the present study 38 4.5.1 The expression level of mRNA 38 4.5.2 Distinguishing between phosphorylated and non-phosphorylated CSP 38 4.5.3 The plausibility of the resolution of confocal microscopy 38 4.5.4 The plausibility of cellular mean and fitting 39 4.6 Future direction 39 Chapter V: Conclusion 41 References 42 List of Figures 47 Figure 1. Constitutive and regulated exocytosis 47 Figure 2. Steps of calcium-regulated exocytosis 48 Figure 3. SNARE complex and Syt I 49 Figure 4. Full fusion and kiss-and-run in calcium-regulated exocytosis 50 Figure 5. Structure of CSP 51 Figure 6. Characteristics of spike and prespike foot 52 Figure 7. The mRNA expression levels of different CSP isoforms in PC12 cells. 53 Figure 8. The mRNA expression level of Syt I and SNARE proteins in PC12 cells. 55 Figure 9. The smaller view of subcellular localization of CSP and Syt I after overexpressing CSP in PC12 cells. 56 Figure 10. The larger view of subcellular localization of CSP and Syt I before and after overexpressing CSP in PC12 cells. 59 Figure 11. The smaller view of subcellular localization of CSP and synaptophysin after overexpressing CSP in PC12 cells. 60 Figure 12. The larger view of subcellular localization of CSP and synaptophysin before and after overexpressing CSP in PC12 cells. 63 Figure 13. The smaller view of subcellular localization of CSP and ChB after overexpressing CSP in PC12 cells. 64 Figure 14. The larger view of subcellular localization of CSP and ChB before and after overexpressing CSP in PC12 cells. 67 Figure 15. The effect of CSP phosphorylation on spike frequency. 68 Figure 16. The effect of CSP phosphorylation on spike characteristics. 70 Figure 17. The effect of CSP phosphorylation on prespike foot duration. 72 Figure 18. The effect of CSP phosphorylation on prespike foot characteristics. 74 Figure 19. The effect of CSP phosphorylation on rate constants of fusion pore kinetics. 75 Figure 20. The model of CSP phosphorylation modulating calcium-regulated exocytosis. 76 List of Tables 77 Table 1. Primers for subcloning and site-directed mutagenesis. 77 Table 2. Primers for RT-qPCR 78 Table 3. Antibodies used for immunofluorescence staining 80 Table 4. Values of factors in the model of fusion pore kinetics. 81 Table 5. Changes of values of factors in the model of fusion pore kinetics. 82 Table 6. Comparison of amperometric data to previous studies 83 Appendix 84 Appendix figure 1. The map to construct pIRES2EGFP-CSP 84 Appendix figure 2. Gel electrophoresis for site-directed mutagenesis 85 | |
| dc.language.iso | en | |
| dc.title | 半胱胺酸串鍊蛋白的磷酸化在鈣離子調控性胞吐作用中對於融合孔的影響 | zh_TW |
| dc.title | The Effects of Phosphorylation of Cysteine String Protein on Fusion Pores in Calcium-Regulated Exocytosis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 徐立中,盧主欽 | |
| dc.subject.keyword | 鈣離子調控性胞吐作用,融合孔,磷酸化,氧化電流測定技術,半胱胺酸串鍊蛋白, | zh_TW |
| dc.subject.keyword | calcium-regulated exocytosis,fusion pore,phosphorylation,amperometry,cysteine string protein, | en |
| dc.relation.page | 86 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-07-26 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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