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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17868完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王致恬(Chih-Tien Wang) | |
| dc.contributor.author | Yu-Tien Hsiao | en |
| dc.contributor.author | 蕭愉恬 | zh_TW |
| dc.date.accessioned | 2021-06-08T00:45:05Z | - |
| dc.date.copyright | 2015-09-02 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-08-05 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17868 | - |
| dc.description.abstract | SNARE複合體可調控囊泡融合。在三個SNARE蛋白質中，僅SNAP-25（SN25）可被蛋白質激酶A（protein kinase A，PKA）和蛋白質激酶C（protein kinase C，PKC）磷酸化，且磷酸化位置分別在SN25的T138和S187。以往的研究顯示，PKA或PKC磷酸化的SN25可藉由控制囊泡池（vesicle pool）的大小或補充囊泡池來調控囊泡的分泌量。然而，目前仍不清楚（1）SN25的磷酸化如何調控胞吐作用的動態;（2）此調控是否可以顯著影響大規模的網路活動。為了解決第一個問題，我們利用可測得單一囊泡釋放的氧化電流技術（single-event amperometry），在PC12細胞內研究SN25b的磷酸化對胞吐動態的影響，尤其是對融合孔（fusion pore）動態的影響。此研究中，我們使用兩種不同secretogogues在短時間內(約1分鐘)誘發鈣離子依賴性胞吐作用（Ca2+-dependent exocytosis），包括高濃度氯化鉀（KCl）、高濃度氯化鉀與福斯克林（Forskolin，一種腺苷酸環化酶的激活劑；FSK）的混合液（KCl FSK）。與對照組相比，KCl降低過量表現SN25b細胞的囊泡釋放速率，但KCl FSK更為顯著降低過量表現SN25b細胞的囊泡釋放速率，藉由免疫沉澱實驗顯示KCl FSK可使SN25b被PKA磷酸化，降低鈣離子依賴性胞吐作用的速率。此外， 和SN25b相比，過量表現SN25b突變株（SN25b-T138A或SN25b-S187A）的細胞，其囊泡釋放速率增加，顯示經由被PKA或PKC磷酸化的SN25b會負向調控囊泡釋放速率。有趣的是，在過量表現SN25b-T138A的細胞中，KCl FSK會減少其融合孔開啟的時間，顯示被PKA磷酸化的SN25b可穩定融合孔，但SN25b-S187A則否；因此，SN25b可在短時間內藉由PKA的磷酸化來調控胞吐作用的動態，此現象可能與提高SN25b與Stx1的交互作用有關。為了探討SN25b的磷酸化是否能進一步影響大規模的網路活動，我們檢測發育中大鼠視網膜內的模式化、自發性放電的現象（稱為視網膜波，此現象伴隨著週期性PKA和PKC活性的上升）。我們在突觸前神經元（星狀無軸突細胞，starburst amacrine cells，SACs）中過量表現SN25b或其突變株，利用即時鈣離子影像偵測在視網膜節神經元(retinal ganglion cells) 內鈣離子濃度瞬間的變化。在SACs過量表現SN25b，會降低視網膜波的頻率；而SACs過量表現SN25b-T138A或SN25b-S187A ，不會改變視網膜波的頻率。這些結果顯示被PKA或PKC磷酸化的SN25b可負向調控突觸前神經元神經傳導物質的釋放，進而可影響突觸後神經元的大規模網路活動。總而言之，我們的研究結果證明， SN25的單一氨基酸上的轉譯後修飾（post-translational modification），可調控神經傳導物質釋放的動態以及突觸後神經元大規模的網路活動；故細胞內訊息傳遞分子的即時變化，足以在短時間內造成神經傳導的多樣性。 | zh_TW |
| dc.description.abstract | The SNARE complex mediates vesicle fusion. Among three SNARE proteins, only SN25 can be phosphorylated by protein kinase A (PKA) at the residue of T138 and by protein kinase C (PKC) at the residue of S187. Previous studies showed that SN25 phosphorylation by PKA or PKC can regulate secretion via controlling the size of vesicle pool or recruiting vesicles, respectively. However, it remains unclear (1) how SN25 phosphorylation regulates the kinetics of exocytosis and (2) whether this regulation can cause a significant effect on the large-scale network activity. To address the first question, we performed single-event amperometry in PC12 cells to study the effects of SN25b phosphorylation on the exocytotic kinetics, with a special focus on the dynamics of fusion pore, reflected by foot signals preceding amperometry spikes (prespike foot, PSF). Two different secretogogues were applied to trigger Ca2+-dependent exocytosis, including KCl alone and KCl with forskolin. KCl alone reduced the secretion rate in cells overexpressing SN25b compared to control, but KCl with forskolin even more reduced the secretion rate in cells overexpressing SN25 compared to control, suggesting that SN25b may down-regulate calcium-dependent exocytosis via PKA phosphorylation. Furthermore, the secretion rate was increased in cells overexpressing the SN25b phosphodeficient mutant (SN25b-T138A or SN25b-S187A) compared to SN25b, confirming that SN25b down-regulates the secretion rate via the PKA- or PKC-phosphorylation site. Moreover, KCl with forskolin reduced PSF lifetime in cells overexpressing SN25b-T138A, but not SN25b-S187A, suggesting that SN25b PKA-phosphodeficiency destabilizes the fusion pore. Taken together, SN25b phosphorylation may regulate the kinetics of exocytosis in secretory cells. To address whether SN25b phosphorylation can cause a significant effect on the large-scale network activity, the patterned spontaneous, correlated activity (termed retinal waves) was detected in the developing rat retina where the PKA and PKC activities remain high. Live calcium imaging was subsequently performed to monitor the wave-associated calcium transients after molecular manipulation in the wave-initiating neurons (starburst amacrine cells, SACs). The frequency of retinal waves was reduced by overexpressing SN25b in SACs, whereas SN25b-T138A or SN25b-S187A in SACs did not change the wave frequency, suggesting that SN25b phosphorylation by PKA or PKC may down-regulate the wave activity from presynaptic neurons. Together, our results suggest that post-translation modification at a single residue of SN25 is sufficient to serve as a molecular regulator in modulating neurotransmitter release and the large-scale network activity. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T00:45:05Z (GMT). No. of bitstreams: 1 ntu-104-F96B43010-1.pdf: 8495388 bytes, checksum: 19973df9ce202e8b6662b50b645c5ff5 (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 口試委員會審定書 i 致謝 ii 中文摘要 iv Abstract vi Abbreviations viii Contents 1 CHAPTER I BACKGROUND AND SIGNIFICANCE 7 1 Characteristics of Ca2+-dependent exocytosis 7 1.1 Calcium influx via calcium channels 7 1.2 Vesicle cycle during Ca2+-dependent exocytosis 8 2 Proteins involved in Ca2+-dependent exocytosis: SNARE complex and synaptotagmins 9 2.1 SNARE complex the membrane fusion machinery 10 2.2 Synaptotagmins (Syts) calcium sensors 10 3 Fusion pore 11 3.1 Structure of fusion pores 11 3.2 Modes of fusion pores 12 3.3 Kinetics of fusion pores 12 4 SNAP-25 (SN25) 13 4.1 Structure of SN25 13 4.2 Isoforms of SN25 13 4.3 Function of SN25 14 5 Neural development 15 5.1 patterned spontaneous activity during neural circuit refinement 16 6 Visual system 16 6.1 Retinal architecture 16 6.2 Visual transduction 17 6.3 Patterned spontaneous activity in retinas - Retinal waves 18 6.5 SN25 in the developing retina 19 7 Real-time techniques to monitor cellular physiology 20 7.1 Single-event amperometry 20 7.2 Fluorescence resonance energy transfer (FRET) 20 7.3 Calcium imaging 21 7.4 Patch-clamp 22 8 Objectives for this study 22 9 Significance 23 CHAPTER II MATERIALS AND METHODS 25 1 Site-directed mutagenesis 25 2 Vector construction and subcloning 26 3 Cell culture 26 4 Animals 27 5 Primary retinal explants culture 27 6 Transient transfection 28 7 Single-event amperometry 29 8 RNA extraction 30 9 Reverse transcriptase-quantitative real-time PCR (RT-qPCR) 31 10 Fluorescence resonance energy transfer (FRET) 32 11 Live calcium imaging 33 12 Whole-cell voltage-clamp 34 13 Statistics 35 CHAPTER III RESULTS 37 1 Effects of PKA-mediated SN25b phosphorylation 37 Overexpression of SN25b or SN25b-T138A does not change the expression of essential exocytotic proteins or the SN25 cellular localization in PC12 cells 37 Acute application of FSK enhances PKA activity and increases the level of SN25 phosphorylation. 38 PKA-mediated SN25b phosphorylation decreases secretion rate via the residue T138 40 PKA-mediated SN25 phosphorylation decreases full-fusion frequency without altering the fraction of kiss-and-run events 41 SN25b PKA-phosphodeficiency destabilizes the open fusion pore leading to dilation 43 PKA-mediated SN25b phosphorylation promotes the open fusion pore towards dilating or closing. 45 PKA-mediated SN25b phosphorylation does not alter the voltage-gated Ca2+ influxes and the intracellular calcium levels 47 Acute application of FSK does not induce gene expression of Syt4 and SN25 isoforms. 48 PKA-mediated SN25b phosphorylation enhances the interaction of Stx1 48 PKA-mediated SN25b phosphorylation reduces the frequency of wave-associated Ca2+ transients 49 PKA-mediated SN25b phosphorylation does not alter the spatial correlation of wave-associated Ca2+ transients 51 2 Effects of PKC-mediated SN25b phosphorylation 51 Overexpression of SN25b-S187A does not change the expression of essential exocytotic proteins in PC12 cells 51 Acute application of KCl enhances PKC activity 52 PKC-mediated SN25b phosphorylation decreases secretion rate via the residue S187 53 PKC-mediated SN25b phosphorylation decreases full-fusion frequency without altering the fraction of kiss-and-run events 54 PKC-mediated SN25b phosphorylation does not regulate the life time of fusion pores 56 The SN25b PKC-phosphodeficiency promotes the open fusion pore towards closing with additional FSK application 56 PKC-mediated SN25b phosphorylation does not alter the voltage-gated Ca2+ influxes and the intracellular calcium levels 58 PKC-mediated SN25b phosphorylation enhances the interaction of Stx1 58 PKC-mediated SN25b phosphorylation reduces the frequency of wave-associated Ca2+ transients 59 PKC-mediated SN25b phosphorylation does not alter the spatial correlation of wave-associated Ca2+ transients 60 CHAPTER IV DISCUSSION 62 CHAPTER V CONCLUSION 70 References 71 List of figures Figure 1. Ca2+-dependent exocytosis and fusion machinery. 78 Figure 2. The changes of expression levels after transfection with the SN25 PKA-phosphodeficient mutant. 79 Figure 3. The real-time changes in PKA activity by secretagogues. 81 Figure 4. The secretion rate is down-regulated by SN25b, but not by SN25b-T138A, upon applying KCl with FSK. 83 Figure 5. Kiss-and-run events and full-fusion events in cells overexpressing the SN25b-PKA-phosphodeficient mutant. 85 Figure 6. The distributions of mean amplitude for KR events and prespike feet (PSF) in the cells overexpressing SN25b-T138A. 87 Figure 7. The mean amplitude of KR events does not be changed with time in cells overexpressing SN25b-T138A. 89 Figure 8. Spike characteristics in cells overexpressing SN25b-T138A. 91 Figure 9. PSF duration is reduced by SN25b-T138A upon application of KCl with FSK. 93 Figure 10. The SN25b PKA-phosphodeficient mutant regulates fusion pore kinetics. 95 Figure 11. The changes in whole-cell Ca2+ currents and intracellular Ca2+ concentration in cells overexpressing SN25b-T138A. 97 Figure 12. FSK does not induce Syt4 gene expression in PC12 cells during the recording time of this study. 98 Figure 13. SACs overexpressing SN25b, but not SN25b-T138A, show a decrease in Ca2+ transient frequency. 100 Figure 14. The size and spatial correlation of Ca2+ transients are not changed in the SACs overexpressing SN25b or SN25b-T138A. 102 Figure 15. The changes of expression levels after transient transfection with the SN25 PKC-phosphodeficient mutant. 103 Figure 16. The real-time changes in PKC activity by secretagogues. 105 Figure 17. The secretion rate is down-regulated by SN25b, and the SN25b PKC-phosphodeficient mutant upon applying KCl with FSK. 107 Figure 18. The distributions of mean amplitude for KR events and PSF in the cells overexpressing SN25b-S187A. 109 Figure 19. The mean amplitude of KR events is not changed over time in cells overexpressing SN25b-S187A. 111 Figure 20. Kiss-and-run events and full-fusion events in cells overexpressing SN25b-PKC-phosphodeficient mutant. 113 Figure 21. Spike characteristics in cells overexpressing SN25b-S187A. 115 Figure 22. PKC-mediated SN25b phosphorylation does not affect PSF duration. 117 Figure 23. The SN25b PKC-phosphodeficiency promotes the open fusion pore towards closing. 119 Figure 24. The changes in whole-cell Ca2+ currents and intracellular Ca2+ concentration in cells overexpressing SN25b-S187A. 121 Figure 25. SACs overexpressing SN25b, but not SN25b-S187A, show a decrease in the Ca2+ transient frequency. 122 Figure 26. The size and spatial correlation of Ca2+ transients are not changed in SACs overexpressing SN25b or SN25b-S187A. 124 Figure 27. The model of PKA- or PKC-mediated SN25b phosphorylatioin in Ca2+-dependent exocytosis. 126 List of tables Table 1. Primers for subcloning and site-directed mutagenesis. 127 Table 2. Primers for RT-qPCR. 128 Appendix 129 Figure 1. The changes of expression levels after transient transfection. 130 Figure 2. The subcellular localization of SN25 in transfected PC12 cells. 132 Figure 3. The SN25-Stx1 binding is decreased in SN25b PKA-phosphodeficient mutants. 134 Figure 4. The subcellular localization of SN25 in the transfected retina. 135 Figure 5. The SN25-Stx1 binding is decreased in the SN25b PKC-phosphodeficient mutant. 136 Figure 6. The subcellular localization of SN25 in transfected PC12 cells. 137 The 39th Annual Meeting of the Society for Neuroscience (17-21 October 2009, Chicago, IL, U.S.A.): abstract and poster 138 The 40th Annual Meeting of the Society for Neuroscience (13-17 November 2010, San Diego, CA, U.S.A.): abstract and poster 140 The 41st Annual Meeting of the Society for Neuroscience (12-16 November 2011, Washington, DC, U.S.A.): abstract and poster 142 The 42nd Annual Meeting of the Society for Neuroscience (13-17 October 2012, New Orleans, LA, U.S.A.): abstract and poster 145 The 44th Annual Meeting of the Society for Neuroscience (15-19 November 2014, Washington, DC, U.S.A.): abstract and poster 147 2014 Institute of Molecular and Cellular Biology Poster Contest: abstract and poster 150 | |
| dc.language.iso | en | |
| dc.title | SNAP-25b的磷酸化調控分泌細胞的胞吐作用動態及發育中大鼠的視網膜波 | zh_TW |
| dc.title | SNAP-25b phosphorylation modulates the exocytotic kinetics in secretory cells and retinal waves in the developing rat retina | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 徐立中(Li-Chung Hsu),焦傳金,溫進德,盧主欽,陳示國 | |
| dc.subject.keyword | SNAP-25,磷酸化,鈣離子調控的胞吐作用,融合孔動態,氧化電流測定術,kiss and run,prespike foot,全融合,視網膜波,星狀無軸突細胞, | zh_TW |
| dc.subject.keyword | SNAP-25,phosphorylation,Ca2+-dependent exocytosis,fusion pore kinetics,amperometry,kiss and run,full fusion,prespike foot,retinal waves,starburst amacrine cells, | en |
| dc.relation.page | 152 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2015-08-05 | |
| 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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