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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 | Yi-Ting Huang | en |
dc.contributor.author | 黃一婷 | zh_TW |
dc.date.accessioned | 2021-06-08T00:46:22Z | - |
dc.date.copyright | 2015-09-02 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-07-29 | |
dc.identifier.citation | Albillos, A., G. Dernick, H. Horstmann, W. Almers, G. Alvarez de Toledo and M. Lindau (1997). 'The exocytotic event in chromaffin cells revealed by patch amperometry.' Nature 389(6650): 509-512. Ales, E., L. Tabares, J. M. Poyato, V. Valero, M. Lindau and G. Alvarez de Toledo (1999). 'High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism.' Nat Cell Biol 1(1): 40-44. Alvarez de Toledo, G., R. Fernandez-Chacon and J. M. Fernandez (1993). 'Release of secretory products during transient vesicle fusion.' Nature 363(6429): 554-558. Bai, J., W. C. Tucker and E. R. Chapman (2004). 'PIP2 increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane.' Nat Struct Mol Biol 11(1): 36-44. Bai, J., C. T. Wang, D. A. Richards, M. B. Jackson and E. R. Chapman (2004). 'Fusion pore dynamics are regulated by synaptotagmin*t-SNARE interactions.' Neuron 41(6): 929-942. Benesch, S., S. Lommel, A. Steffen, T. E. B. Stradal, N. Scaplehorn, M. Way, J. Wehland and K. Rottner (2002). 'Phosphatidylinositol 4,5-biphosphate (PIP2)-induced vesicle movement depends on N-WASP and involves Nck, WIP, and Grb2.' Journal of Biological Chemistry 277(40): 37771-37776. Berton, F., C. Iborra, J. A. Boudier, M. J. Seagar and B. Marqueze (1997). 'Developmental regulation of synaptotagmin I, II, III, and IV mRNAs in the rat CNS.' J Neurosci 17(4): 1206-1216. Betz, A., P. Thakur, H. J. Junge, U. Ashery, J. S. Rhee, V. Scheuss, C. Rosenmund, J. Rettig and N. Brose (2001). 'Functional interaction of the active zone proteins Munc13-1 and RIM1 in synaptic vesicle priming.' Neuron 30(1): 183-196. Bhalla, A., M. C. Chicka and E. R. Chapman (2008). 'Analysis of the synaptotagmin family during reconstituted membrane fusion. Uncovering a class of inhibitory isoforms.' J Biol Chem 283(31): 21799-21807. Bhalla, A., M. C. Chicka, W. C. Tucker and E. R. Chapman (2006). 'Ca(2+)-synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion.' Nat Struct Mol Biol 13(4): 323-330. Brodin, L., P. Low, H. Gad, J. Gustafsson, V. A. Pieribone and O. Shupliakov (1997). 'Sustained neurotransmitter release: new molecular clues.' Eur J Neurosci 9(12): 2503-2511. Brose, N., A. G. Petrenko, T. C. Sudhof and R. Jahn (1992). 'Synaptotagmin: a calcium sensor on the synaptic vesicle surface.' Science 256(5059): 1021-1025. Bruns, D. and R. Jahn (1995). 'Real-time measurement of transmitter release from single synaptic vesicles.' Nature 377(6544): 62-65. Butz, S., R. Fernandez-Chacon, F. Schmitz, R. Jahn and T. C. Sudhof (1999). 'The subcellular localizations of atypical synaptotagmins III and VI. Synaptotagmin III is enriched in synapses and synaptic plasma membranes but not in synaptic vesicles.' J Biol Chem 274(26): 18290-18296. Campagna, J. A., D. Prevette, R. W. Oppenheim and J. L. Bixby (1997). 'Target contact regulates expression of synaptotagmin genes in spinal motor neurons in vivo.' Mol Cell Neurosci 8(6): 377-388. Chapman, E. R. (2002). 'Synaptotagmin: a Ca(2+) sensor that triggers exocytosis?' Nat Rev Mol Cell Biol 3(7): 498-508. Chen, F., L. Ma, M. C. Parrini, X. Mao, M. Lopez, C. Wu, P. W. Marks, L. Davidson, D. J. Kwiatkowski, T. Kirchhausen, S. H. Orkin, F. S. Rosen, B. J. Mayer, M. W. Kirschner and F. W. Alt (2000). 'Cdc42 is required for PIP(2)-induced actin polymerization and early development but not for cell viability.' Curr Biol 10(13): 758-765. Chiang, C. W., Y. C. Chen, J. C. Lu, Y. T. Hsiao, C. W. Chang, P. C. Huang, Y. T. Chang, P. Y. Chang and C. T. Wang (2012). 'Synaptotagmin I regulates patterned spontaneous activity in the developing rat retina via calcium binding to the C2AB domains.' PLoS One 7(10): e47465. Chiang, N., Y. T. Hsiao, H. J. Yang, Y. C. Lin, J. C. Lu and C. T. Wang (2014). 'Phosphomimetic mutation of cysteine string protein-alpha increases the rate of regulated exocytosis by modulating fusion pore dynamics in PC12 cells.' PLoS One 9(6): e99180. Chow, R. H., L. von Ruden and E. Neher (1992). 'Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells.' Nature 356(6364): 60-63. Colquhoun, D. and A. G. Hawkes (1982). 'On the stochastic properties of bursts of single ion channel openings and of clusters of bursts.' Philos Trans R Soc Lond B Biol Sci 300(1098): 1-59. Craxton, M. (2004). 'Synaptotagmin gene content of the sequenced genomes.' BMC Genomics 5(1): 43. Davis, A. F., J. Bai, D. Fasshauer, M. J. Wolowick, J. L. Lewis and E. R. Chapman (1999). 'Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes.' Neuron 24(2): 363-376. DePina, A. S. and G. M. Langford (1999). 'Vesicle transport: the role of actin filaments and myosin motors.' Microsc Res Tech 47(2): 93-106. Elmqvist, D., W. W. Hofmann, J. Kugelberg and D. M. Quastel (1964). 'An Electrophysiological Investigation of Neuromuscular Transmission in Myasthenia Gravis.' J Physiol 174: 417-434. Elmqvist, D. and D. M. Quastel (1965). 'A quantitative study of end-plate potentials in isolated human muscle.' J Physiol 178(3): 505-529. Fukuda, M., J. A. Kowalchyk, X. Zhang, T. F. Martin and K. Mikoshiba (2002). 'Synaptotagmin IX regulates Ca2+-dependent secretion in PC12 cells.' J Biol Chem 277(7): 4601-4604. Gauthier, B. R. and C. B. Wollheim (2008). 'Synaptotagmins bind calcium to release insulin.' Am J Physiol Endocrinol Metab 295(6): E1279-1286. Greengard, P., F. Valtorta, A. J. Czernik and F. Benfenati (1993). 'Synaptic vesicle phosphoproteins and regulation of synaptic function.' Science 259(5096): 780-785. Grimberg, E., Z. Peng, I. Hammel and R. Sagi-Eisenberg (2003). 'Synaptotagmin III is a critical factor for the formation of the perinuclear endocytic recycling compartment and determination of secretory granules size.' J Cell Sci 116(Pt 1): 145-154. Hilbush, B. S. and J. I. Morgan (1994). 'A third synaptotagmin gene, Syt3, in the mouse.' Proc Natl Acad Sci U S A 91(17): 8195-8199. Jackson, M. B. (1992). 'Ion channels. Single-channel analysis.' Methods Enzymol 207: 729-746. Jackson, M. B. (2010). 'SNARE complex zipping as a driving force in the dilation of proteinaceous fusion pores.' J Membr Biol 235(2): 89-100. Jackson, M. B. and E. R. Chapman (2006). 'Fusion pores and fusion machines in Ca2+-triggered exocytosis.' Annu Rev Biophys Biomol Struct 35: 135-160. Jackson, M. B. and E. R. Chapman (2008). 'The fusion pores of Ca2+ -triggered exocytosis.' Nat Struct Mol Biol 15(7): 684-689. Jarvis, S. E. and G. W. Zamponi (2005). 'Masters or slaves? Vesicle release machinery and the regulation of presynaptic calcium channels.' Cell Calcium 37(5): 483-488. Kabachinski, G., M. Yamaga, D. M. Kielar-Grevstad, S. Bruinsma and T. F. Martin (2014). 'CAPS and Munc13 utilize distinct PIP2-linked mechanisms to promote vesicle exocytosis.' Mol Biol Cell 25(4): 508-521. Klyachko, V. A. and M. B. Jackson (2002). 'Capacitance steps and fusion pores of small and large-dense-core vesicles in nerve terminals.' Nature 418(6893): 89-92. Kozlov, M. M., S. L. Leikin, L. V. Chernomordik, V. S. Markin and Y. A. Chizmadzhev (1989). 'Stalk mechanism of vesicle fusion. Intermixing of aqueous contents.' Eur Biophys J 17(3): 121-129. Li, C., B. Ullrich, J. Z. Zhang, R. G. Anderson, N. Brose and T. C. Sudhof (1995). 'Ca(2+)-dependent and -independent activities of neural and non-neural synaptotagmins.' Nature 375(6532): 594-599. Lin, R. C. and R. H. Scheller (1997). 'Structural organization of the synaptic exocytosis core complex.' Neuron 19(5): 1087-1094. Littleton, J. T., J. Bai, B. Vyas, R. Desai, A. E. Baltus, M. B. Garment, S. D. Carlson, B. Ganetzky and E. R. Chapman (2001). 'synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo.' J Neurosci 21(5): 1421-1433. Littleton, J. T., H. J. Bellen and M. S. Perin (1993). 'Expression of synaptotagmin in Drosophila reveals transport and localization of synaptic vesicles to the synapse.' Development 118(4): 1077-1088. Littleton, J. T., M. Stern, K. Schulze, M. Perin and H. J. Bellen (1993). 'Mutational analysis of Drosophila synaptotagmin demonstrates its essential role in Ca(2+)-activated neurotransmitter release.' Cell 74(6): 1125-1134. Marqueze, B., J. A. Boudier, M. Mizuta, N. Inagaki, S. Seino and M. Seagar (1995). 'Cellular localization of synaptotagmin I, II, and III mRNAs in the central nervous system and pituitary and adrenal glands of the rat.' J Neurosci 15(7 Pt 1): 4906-4917. Martin, T. F. (1994). 'The molecular machinery for fast and slow neurosecretion.' Curr Opin Neurobiol 4(5): 626-632. Mizuta, M., N. Inagaki, Y. Nemoto, S. Matsukura, M. Takahashi and S. Seino (1994). 'Synaptotagmin III is a novel isoform of rat synaptotagmin expressed in endocrine and neuronal cells.' J Biol Chem 269(16): 11675-11678. Neher, E. and A. Marty (1982). 'Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells.' Proc Natl Acad Sci U S A 79(21): 6712-6716. Nonet, M. L., K. Grundahl, B. J. Meyer and J. B. Rand (1993). 'Synaptic function is impaired but not eliminated in C. elegans mutants lacking synaptotagmin.' Cell 73(7): 1291-1305. Rettig, J. and E. Neher (2002). 'Emerging roles of presynaptic proteins in Ca++-triggered exocytosis.' Science 298(5594): 781-785. Richards, D. A., C. Guatimosim and W. J. Betz (2000). 'Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals.' Neuron 27(3): 551-559. Richards, D. A., C. Guatimosim, S. O. Rizzoli and W. J. Betz (2003). 'Synaptic vesicle pools at the frog neuromuscular junction.' Neuron 39(3): 529-541. Richmond, J. (2005). 'Synaptic function.' WormBook: 1-14. Rizzoli, S. O. and W. J. Betz (2005). 'Synaptic vesicle pools.' Nat Rev Neurosci 6(1): 57-69. Sagi-Eisenberg, R. (2007). 'The mast cell: where endocytosis and regulated exocytosis meet.' Immunol Rev 217: 292-303. Schiavo, G., G. Stenbeck, J. E. Rothman and T. H. Sollner (1997). 'Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25, can explain docked vesicles at neurotoxin-treated synapses.' Proc Natl Acad Sci U S A 94(3): 997-1001. Schuh, M. (2011). 'An actin-dependent mechanism for long-range vesicle transport.' Nat Cell Biol 13(12): 1431-1436. Segovia, M., E. Ales, M. A. Montes, I. Bonifas, I. Jemal, M. Lindau, A. Maximov, T. C. Sudhof and G. Alvarez de Toledo (2010). 'Push-and-pull regulation of the fusion pore by synaptotagmin-7.' Proc Natl Acad Sci U S A 107(44): 19032-19037. Shoji-Kasai, Y., M. Itakura, M. Kataoka, S. Yamamori and M. Takahashi (2002). 'Protein kinase C-mediated translocation of secretory vesicles to plasma membrane and enhancement of neurotransmitter release from PC12 cells.' Eur J Neurosci 15(8): 1390-1394. Sollner, T., M. K. Bennett, S. W. Whiteheart, R. H. Scheller and J. E. Rothman (1993). 'A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion.' Cell 75(3): 409-418. Sollner, T., S. W. Whiteheart, M. Brunner, H. Erdjument-Bromage, S. Geromanos, P. Tempst and J. E. Rothman (1993). 'SNAP receptors implicated in vesicle targeting and fusion.' Nature 362(6418): 318-324. Sorensen, J. B. (2005). 'SNARE complexes prepare for membrane fusion.' Trends Neurosci 28(9): 453-455. Stenmark, H. (2009). 'Rab GTPases as coordinators of vesicle traffic.' Nature Reviews Molecular Cell Biology 10(8): 513-525. Sugita, S., O. H. Shin, W. Han, Y. Lao and T. C. Sudhof (2002). 'Synaptotagmins form a hierarchy of exocytotic Ca(2+) sensors with distinct Ca(2+) affinities.' EMBO J 21(3): 270-280. Sutton, R. B., B. A. Davletov, A. M. Berghuis, T. C. Sudhof and S. R. Sprang (1995). 'Structure of the first C2 domain of synaptotagmin I: a novel Ca2+/phospholipid-binding fold.' Cell 80(6): 929-938. Sutton, R. B., J. A. Ernst and A. T. Brunger (1999). 'Crystal structure of the cytosolic C2A-C2B domains of synaptotagmin III. Implications for Ca(+2)-independent snare complex interaction.' J Cell Biol 147(3): 589-598. Thapa, N. and R. A. Anderson (2012). 'PIP2 signaling, an integrator of cell polarity and vesicle trafficking in directionally migrating cells.' Cell Adh Migr 6(5): 409-412. Tucker, W. C., J. M. Edwardson, J. Bai, H. J. Kim, T. F. Martin and E. R. Chapman (2003). 'Identification of synaptotagmin effectors via acute inhibition of secretion from cracked PC12 cells.' J Cell Biol 162(2): 199-209. Ullrich, B., C. Li, J. Z. Zhang, H. McMahon, R. G. Anderson, M. Geppert and T. C. Sudhof (1994). 'Functional properties of multiple synaptotagmins in brain.' Neuron 13(6): 1281-1291. Verderio, C., D. Pozzi, E. Pravettoni, F. Inverardi, U. Schenk, S. Coco, V. Proux-Gillardeaux, T. Galli, O. Rossetto, C. Frassoni and M. Matteoli (2004). 'SNAP-25 modulation of calcium dynamics underlies differences in GABAergic and glutamatergic responsiveness to depolarization.' Neuron 41(4): 599-610. Voets, T., T. Moser, P. E. Lund, R. H. Chow, M. Geppert, T. C. Sudhof and E. Neher (2001). 'Intracellular calcium dependence of large dense-core vesicle exocytosis in the absence of synaptotagmin I.' Proc Natl Acad Sci U S A 98(20): 11680-11685. Vrljic, M., P. Strop, J. A. Ernst, R. B. Sutton, S. Chu and A. T. Brunger (2010). 'Molecular mechanism of the synaptotagmin-SNARE interaction in Ca2+-triggered vesicle fusion.' Nat Struct Mol Biol 17(3): 325-331. Wang, C. T., J. Bai, P. Y. Chang, E. R. Chapman and M. B. Jackson (2006). 'Synaptotagmin-Ca2+ triggers two sequential steps in regulated exocytosis in rat PC12 cells: fusion pore opening and fusion pore dilation.' J Physiol 570(Pt 2): 295-307. Wang, C. T., R. Grishanin, C. A. Earles, P. Y. Chang, T. F. Martin, E. R. Chapman and M. B. Jackson (2001). 'Synaptotagmin modulation of fusion pore kinetics in regulated exocytosis of dense-core vesicles.' Science 294(5544): 1111-1115. Wang, C. T., J. C. Lu, J. Bai, P. Y. Chang, T. F. Martin, E. R. Chapman and M. B. Jackson (2003). 'Different domains of synaptotagmin control the choice between kiss-and-run and full fusion.' Nature 424(6951): 943-947. Wightman, R. M. and C. L. Haynes (2004). 'Synaptic vesicles really do kiss and run.' Nat Neurosci 7(4): 321-322. Zhang, X., M. J. Kim-Miller, M. Fukuda, J. A. Kowalchyk and T. F. Martin (2002). 'Ca2+-dependent synaptotagmin binding to SNAP-25 is essential for Ca2+-triggered exocytosis.' Neuron 34(4): 599-611. Zhang, X., J. Rizo and T. C. Sudhof (1998). 'Mechanism of phospholipid binding by the C2A-domain of synaptotagmin I.' Biochemistry 37(36): 12395-12403. Zhang, Z., E. Hui, E. R. Chapman and M. B. Jackson (2009). 'Phosphatidylserine regulation of Ca2+-triggered exocytosis and fusion pores in PC12 cells.' Mol Biol Cell 20(24): 5086-5095. Zhang, Z., E. Hui, E. R. Chapman and M. B. Jackson (2010). 'Regulation of exocytosis and fusion pores by synaptotagmin-effector interactions.' Mol Biol Cell 21(16): 2821-2831. Zhang, Z. and M. B. Jackson (2008). 'Temperature dependence of fusion kinetics and fusion pores in Ca2+-triggered exocytosis from PC12 cells.' J Gen Physiol 131(2): 117-124. Zucker, R. S. (1999). 'Calcium- and activity-dependent synaptic plasticity.' Curr Opin Neurobiol 9(3): 305-313. Zucker, R. S. and W. G. Regehr (2002). 'Short-term synaptic plasticity.' Annu Rev Physiol 64: 355-405. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17927 | - |
dc.description.abstract | Synaptotagmin (Syt) 是一個至少擁有17種成員的蛋白家族,他們被視為一種感應鈣離子的蛋白。在神經細胞中,Syt可以藉由他們的C2A與C2B和鈣離子結合,進而影響神經傳導物質的釋放。先前已經有研究指出,Syt I能夠藉由與鈣離子結合來調控由鈣離子調控胞吐作用(Ca2+-dependent exocytosis)的動態變化。在本論文中我們將探討Syt III在鈣離子調控胞吐作用的動態變化中所扮演的角色。因為我們先前發現,Syt III蛋白在囓齒動物視網膜發育過程中的關鍵時期(P4-P6)的視網膜節細胞與視神經中大量表現,而此時期視網膜會經由自發性的放電反應來調控視野分離,由此推測Syt III在發育過程中可能會藉由調控胞吐作用來影響神經傳導素的釋放並影響發育中雙眼動物的視野分離;然而,目前Syt III如何調控作用的詳細機制仍然尚未明瞭。本論文中,我們將Syt III第386和520個胺基酸利用點突變的方式以降低Syt III其C2A與C2B和鈣離子結合的能力,為了進一步的了解對胞吐作用產生的影響,我們利用單一囊泡安培測定法(Amperometry)直接偵測在神經內分泌細胞(neuroendocrine cell)中神經傳導素的釋放,並且結合了免疫螢光染色及免疫沉澱法來探討Syt III在緻密核心囊泡胞吐作用中的分子機制。 我們的研究結果顯示,Syt III蛋白的主要表現於細胞膜上而非緻密核心囊泡,並且Syt III有增加總胞吐作用的趨勢,而在刺激發生後的中、後期紀錄中,我們發現胞吐作用的釋放頻率明顯高於控制組及突變組(Syt III-C2AB*),並且,Syt III可增加full fusion在刺激發生的中、後期紀錄中發生的頻率,而在突變的組別則有著和Syt III相反的結果。除此之外,Syt III明顯減少融合孔(fusion pore)的開啟時間,而突變組則會增加融合孔(fusion pore)的開啟時間。在我們提出的胞吐作用動態的模型中,我們發現Syt III會同時增加速率常速kc與kd,表示Syt III會藉由促進緻密核心囊泡離開fusion pore state (O)往dilation state (D)及close state (C)來降低融合孔(fusion pore)的開啟時間,而Syt III-C2AB*則藉由抑制緻密核心囊泡離開fusion pore state (O)往dilation state (D)及close state (C)來增加融合孔(fusion pore)的開啟時間。利用免疫沉澱的方式,我們發現Syt III 在沒有與鈣離子結合的情況下能夠與SN25交互作用,但在有鈣離子的情況下,Syt III與鈣離子結合後會加強和SN25的交互作用。 綜合上述,我們可以得知Syt III能夠藉由其C2A與C2B和鈣離子結合來影響胞吐作用的動態變化,除此之外,Syt III可能會藉由和鈣離子結合來促進緻密核心囊泡往細胞膜的方向移動,藉此來增加刺激發生後的中、後期胞吐作用的發生,並且我們推測Syt III 會藉由與鈣離子結合來增加與SN25的交互作用去減少融合孔(fusion pore)的開啟時間。因此,Syt III可能作為一個細胞膜上的感應蛋白,來調控密核心囊泡胞吐作用的動態變化。 | zh_TW |
dc.description.abstract | Synaptotagmin (Syt) protein family consists of at least seventeen isoforms. Most of them have been reported as vesicular Ca2+ sensors by Ca2+ binding to their C2AB domains. For example, Syt I plays a role in regulating the kinetics of vesicular exocytosis. However, the functions of other isoforms remain to be identified. Previously, we found that Syt III is upregulated in retinal output neurons (retinal ganglion cells, RGCs) and optic nerves during a critical period (postnatal day P6 in rats) of visual circuit development. However, how Syt III functions in regulated exocytosis remains unclear. Here, we investigated if Syt III involves in Ca2+-regulated exocytosis by Ca2+ binding to its C2AB domains. We used PC12 cells as the model to examine Syt III’s function in regulated exocytosis, by combining molecular perturbation, immunofluorescence staining, and amperometry (a single-vesicle technique to measure real-time exocytosis). By mutating aspartate to asparagine at the site 386 and 520 of Syt III, we blocked the Ca2+ binding ability in the C2AB domains (D386, 520N, designated Syt III-C2AB*). After overexpressing control vector, wild-type Syt III, or Syt III-C2AB* in PC12 cells, we analyzed single exocytotic events by amperometry. We found that Syt III was poorly colocalized with dense-core vesicles (DCVs) but localized to plasma membrane. Moreover, overexpressing Syt III increased secretion rate after long-time stimulus compared to control or Syt III-C2AB*, suggesting that Syt III may involve in vesicle recruitment by Ca2+ binding to its C2AB domains. From co-immunoprecipitation, we found that binding to Ca2+ can enhance the interaction between Syt III and SN25, accounting for a decrease in the prespike foot (PSF) duration. These results suggested that in contrast to the conventional role of Syt family, Syt III functions as a “plasma membrane” Ca2+ sensor in modulating the kinetics of regulated exocytosis, via Ca2+ binding to its C2AB domains. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T00:46:22Z (GMT). No. of bitstreams: 1 ntu-104-R02b43008-1.pdf: 6814649 bytes, checksum: e4b5bbb46b576fe2ffd5524c3fedfdb4 (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 口試委員審定書……………………………………………..……………………..i 致謝............................................................................................................................ii 中文摘要….…………………………………………………….....………………..iv Abstract…………………………………………………………………..…...…….vi Abbreviations………………………………………….……..………......……….viii Chapter I Introduction 1 1.1 Calcium-dependent exocytosis 1 1.2 Different types of vesicles in neurotransmitter release 2 1.3 Different vesicle pools 2 1.4 Fusion pores 3 1.5 The mechanism of membrane fusion 4 1.6 Synaptotagmin (Syt) 5 1.7 Amperometry 8 1.8 Significance 9 1.9 Specific aims and experimental design 10 Chapter II Materials and Methods 12 2.1 Site-directed mutagenesis 12 2.2 Competent cells 12 2.3 Cell culture 13 2.4 Transfection 14 2.5 Immunofluorescence staining 15 2.6 Coimmunoprecipitation and immunoblotting analysis 16 2.7 Amperometry 18 2.8 RNA extraction 19 2.9 Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) 20 2.10 Statistics 21 Chapter III Results 22 3.1 The mRNA expression levels by RT-qPCR 22 3.2 The localization of Syt III 24 3.3 Effects of Syt III and its C2AB mutant (Syt III-C2AB) in Ca2+-dependent exocytosis 26 3.4 The mechanism of Syt III and its C2AB mutant in regulating Ca2+-dependent exocytosis 36 Chapter IV Discussion 41 4.1 The localization of Syt III and the regulation of DCV exocytosis 42 4.2 The effect of Syt III and its C2AB mutant on secretion rate 43 4.3 The effects of Syt III and its C2AB mutant on PSF duration. 45 4.4 The effect of Syt III and its C2AB mutant on fusion kinetic model 48 4.5 Future directions 49 Chapter V Conclusion 52 References.............................................................................................................53 List of figures Figure 1. Full fusion and kiss-and-run events in calcium-dependent exocytosis 61 Figure 2. Different vesicle pools in exocytosis 63 Figure 3. The process of calcium-dependent exocytosis 65 Figure 4. SNARE complex and Syt I 67 Figure 5. Diagram of the domain structure of synaptotagmin 69 Figure 6. Calcium binding sites in synaptotagmin III 71 Figure 7. The mRNA expression levels of Syt isoforms and SNARE proteins 73 Figure 8. Subcellular localization of Syt III and HA in transfected PC12 cells. 75 Figure 9. Subcellular localization of Syt III and ChB in transfected PC12 cells. 77 Figure 10. Subcellular localization of Syt III and SN25 in transfected PC12 cells. 79 Figure 11. The effects of Syt III and the C2AB mutant on secretion rate. 81 Figure 12. The effects of Syt III and the C2AB mutant on full fusion frequency. 83 Figure 13. The effects of Syt III and the C2AB mutant on the fraction of KR events. 85 Figure 14. The effects of Syt III and the C2AB mutant on the spike characteristics. 87 Figure 15. The effects of Syt III and the C2AB mutant on the duration, mean amplitude, and area of the prespike foot. 89 Figure 16. The effects of Syt III and the CAB mutant on fusion pore kinetics. 91 Figure 17. The protein expression level of Syt III SNARE proteins post transfection. 93 Figure 18. The interactions between Syt III and SNARE proteins. 95 Figure 19. The kinetic model of Ca2+ binding to Syt III or its C2AB mutant. 97 List of tables Table 1. List of primers used in this study...............................................................98 Table 2. Characteristics of spikes in cells overexpressing Syt III and its C2AB mutant...................................................................................................................99 Table 3.The list of primary and secondary antibodies............................................100 Table 4. Comparison of the effects caused by Syt III and its C2AB domains.......101 Appendix Appendix 1. The SN25 localization in cells overexpressing SN25 and its phosphomutants. 104 Appendix 2. The SN25 localization in cells overexpressing SN25 and its phosphomutants. 106 Appendix 3. 2014 Annual meeting of the Society for Neuroscience (Washington, D.C., U.S.A. 11/15-19/2014): Abstract and poster................................................107 Appendix 3. The 2015 Poster competition in the Institute of Molecular and Cellular Biology at National Taiwan University, Taipei, Taiwan (6/5 2015): Abstract and Poster.....................................................................................................................109 | |
dc.language.iso | en | |
dc.title | Synaptotagmin III在PC12中 藉由鈣離子結合至C2AB區塊調控胞吐作用的動態變化 | zh_TW |
dc.title | Synaptotagmin III modulates the kinetics of regulated exocytosis by Ca2+ binding to the C2AB domains in PC12 cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 徐立中(Li-Chung Hsu),盧主欽(Juu-Chin Lu),陳示國(Shih-Kuo Chen) | |
dc.subject.keyword | Sytnaptotagmin,單一囊泡安培測定法,緻密核心囊泡,鈣離子調控的胞吐作用,融合孔的動態, | zh_TW |
dc.subject.keyword | Synaptotagmin III,dense-core vesicle,Ca2+-regulated exocytosis,amperometry,secretion rate,fusion pore dynamics, | en |
dc.relation.page | 110 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2015-07-30 | |
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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