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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 | 劉雅雯(Ya-Wen Liu) | |
dc.contributor.author | Shan-Shan Lin | en |
dc.contributor.author | 林珊珊 | zh_TW |
dc.date.accessioned | 2021-06-17T07:02:27Z | - |
dc.date.available | 2021-02-23 | |
dc.date.copyright | 2021-02-23 | |
dc.date.issued | 2021 | |
dc.date.submitted | 2021-01-12 | |
dc.identifier.citation | Antonny, B., Burd, C., De Camilli, P., Chen, E., Daumke, O., Faelber, K., Ford, M., Frolov, V.A., Frost, A., Hinshaw, J.E., et al. (2016). Membrane fission by dynamin: what we know and what we need to know. The EMBO journal 35, 2270-2284. Au - Murray, L., Au - Gillingwater, T.H., and Au - Kothary, R. (2014). Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis. JoVE, e51162. Bashkirov, P.V., Akimov, S.A., Evseev, A.I., Schmid, S.L., Zimmerberg, J., and Frolov, V.A. (2008). GTPase cycle of dynamin is coupled to membrane squeeze and release, leading to spontaneous fission. Cell 135, 1276-1286. Bernadzki, K.M., Rojek, K.O., and Prószyński, T.J. (2014). Podosomes in muscle cells and their role in the remodeling of neuromuscular postsynaptic machinery. European Journal of Cell Biology 93, 478-485. Bezakova, G., and Ruegg, M.A. (2003). New insights into the roles of agrin. Nat Rev Mol Cell Biol 4, 295-308. 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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72638 | - |
dc.description.abstract | Dynamin是一種大型GTP水解酶,因其在胞吞過程中催化膜分裂而聞名。有越來越多研究發現dynamin的功能並不侷限於膜上,一些需要機動蛋白重塑的位置,例如足小體、侵襲體、以及板狀偽足上都可以見到dynamin的身影。儘管過去研究發現足小體可以調節神經肌肉接合處(neuromuscular junction, NMJ)的發育,且在這些足小體上可以看到dynamin的聚集,但dynamin是否以及如何調節NMJ的發育尚不清楚。在本篇研究中,我們從分子、細胞、及個體的角度來回答這個問題。我們發現廣泛存在於不同細胞的dynamin亞型:dynamin-2,具有能被GTP水解調控的肌動蛋白綑綁能力,他能聚集在足小體周圍來調控足小體的生長與功能。在動物實驗中,我們還發現dynamin-2會影響突觸後細胞骨架以及果蠅的電生理活性。總結來說,我們的研究證實dynamin-2能透過調節肌動蛋白骨架重塑,進而促進NMJ突觸後發育。 | zh_TW |
dc.description.abstract | Dynamin is a large GTPase most-known for catalyzing membrane fission during endocytosis. Growing evidence suggests that the function of dynamin is not restricted to membrane but also required at sites where endocytosis-independent actin remodeling occurs: including podosome, invadopodium, and lamellipodium. Although podosome has been shown to regulate NMJ development, and dynamin has been shown enriched at muscle podosomes. Whether and how dynamin regulates NMJ development remained unclear. In this study, we address this question from molecular, cellular, and organismal levels. We revealed that the ubiquitously expressed isoform, dynamin-2, is an actin-bundler with its activity regulated by GTP hydrolysis. It assembles around podosomes to regulate their growth, turnover, and function. On the organismal level, we uncover the impact of dynamin-2 in postsynaptic cytoskeleton and electrophysiological properties. To summarize, our study revealed a novel role of dynamin-2 in actin remodeling, which facilitates the process of postsynaptic NMJ development. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T07:02:27Z (GMT). No. of bitstreams: 1 U0001-1101202116271200.pdf: 15732981 bytes, checksum: 5d61a65a27be0c8ad4c9ac3ee609b61b (MD5) Previous issue date: 2021 | en |
dc.description.tableofcontents | Table of Contents 口試委員會審定書 i 致謝財團法人罕見疾病基金會 ii 致謝 iii 中文摘要 v Abstract vi Table of Contents vii Chapter 1-Introduction 1 1.1 Dynamin, a well-known catalyzer for membrane fission 1 1.2 The potential function of dynamin at postsynaptic NMJ 3 1.3 Podosome and its role during NMJ development 5 1.4 A tale of dynamin and actin filaments 6 Chapter 2-Material and Methods 8 2.1 Drosophila stocks 8 2.2 Dissecting of Drosophila larval body wall muscle 8 2.3 Cell culture 9 2.4 Transfection, lentiviral and adenoviral infection 10 2.5 Immunofluorescent staining 11 2.6 Fluorescence microscopy 12 2.7 Matrix degradation assay 13 2.8 TEM of Drosophila NMJs 14 2.9 Electrophysiology of Drosophila NMJ 16 2.10 Image analysis 17 2.11 Dynamin expression and purification 17 2.12 F-actin bundle sedimentation assay 18 2.13 Transmission electron microscopy 20 2.14 Quantification and statistical analysis 21 Chapter 3-Results 21 3.1 Dyn2 is enriched at postsynaptic NMJ 21 3.2 Dyn2 affects postsynaptic cytoskeletal organization 22 3.3 CNM-Dyn2 interfere electrophysiological activity of Drosophila larval NMJ 24 3.4 Dyn2 forms belt-shaped structures around the actin core of podosomes 26 3.5 Dyn2 is required for podosome growth 29 3.6 Dyn2 regulates podosome turnover 30 3.7 Actin binding, self-assembly, and GTP hydrolysis activity of Dyn2 are involved in podosome growth 33 3.8 Actin binding, self-assembly, and GTP hydrolysis activity of Dyn2 are involved in podosome turnover 34 3.9 Dyn2 is required for podosome-mediated ECM degradation 36 3.10 Dyn2 regulates AChR cluster perforation through podosome 37 3.11 Dyn2 remodels actin cytoskeleton through actin bundling activity 38 3.12 Actin bundling activity of Dyn2 is regulated by GTP hydrolysis 39 3.13 Dyn2 oligomers assemble around actin filaments 42 3.14 CNM-associated Dyn2 mutant proteins are insensitive to GTP hydrolysis 45 3.15 Dyn2 bundles both linear and branched actin filaments 46 Chapter 4-Discussion 47 4.1 Dyn2 regulates NMJ development 47 4.2 Dyn2 promotes podosome growth through its actin bundling activity 48 4.3 Dyn2, a novel regulator for podosome turnover 50 4.4 Dyn2 is a bundling protein for actin filaments 51 Chapter 5-Figures and Tables 53 Figure 1. Structure and assembly of Dyn 53 Figure 2. Structure of podosome 55 Figure 3. Dyn2 enriched at Drosophila postsynaptic NMJ 57 Figure 4. Dyn2 affects postsynaptic cytoskeletal organization in Drosophila 59 Figure 5. Effects of Dyn2 at Drosophila postsynaptic NMJ 61 Figure 6. Dyn2 affects postsynaptic SSR density in Drosophila 63 Figure 7. Dyn2 affects electrophysiological activities in Drosophila 65 Figure 8. Dyn2 did not affect GluRIIA distribution 67 Figure 9. Endogenous Dyn2 is enriched at mouse postsynaptic NMJ 69 Figure 10. AChR clustering and perforation in C2C12 myotubes on laminin-coated glass coverslips 71 Figure 11. AChR clustering and perforation in C2C12 myotubes 73 Figure 12. Dyn2 formed belt-shaped structure around some podosome core 75 Figure 13. Dyn2-belt localize in between podosome core and the radical actin cables 77 Figure 14. Level of Dyn2 enrichment correlates with podosome size 79 Figure 15. Dyn2 is required for podosome growth 81 Figure 16. Dyn2 forms belt-shaped structure as podosome matures 83 Figure 17. The correlation of Dyn2 enrichment and podosome lifespan 85 Figure 18. Dyn2 is required for podosome turnover 87 Figure 19. Dyn2 GTPase activity is involved in podosome growth and turnover 89 Figure 20. Mutations used in this study 91 Figure 21. Effects of mutant Dyn2 on podosome morphology in wild type myotubes 93 Figure 22. Rescuing effects of mutant Dyn2 on podosome morphology in Dyn2-depleted myotubes 95 Figure 23. Dyn2 is essential for podosome growth 97 Figure 24. Dyn2 is essential for podosome turnover 99 Figure 25. Dyn2 is essential for podosome growth in c-Src transformed NIH3T3 cells 101 Figure 26. Dyn2 is essential for podosome turnover in c-Src transformed NIH3T3 cells 103 Figure 27. Dyn2 regulates podosome lifespan in c-Src transformed NIH3T3 cells 105 Figure 28. Dyn2 regulates podosome-mediated matrix degradation 107 Figure 29. Expressing Dyn2 hyper self-assembly mutant perturbs matrix degradation activity in wild type myotubes 109 Figure 30. Dyn2 mediates AChR cluster organization 111 Figure 31. Expressing Dyn2 hyper self-assembly mutant perturbs AChR perforation 113 Figure 32. Dyn2 bundles in vitro reconstituted actin filaments 115 Figure 33. GTP, GDP, and GTPS addition reduced Dyn2-bundled actin 117 Figure 34. GTP hydrolysis reduced Dyn2-bundled actin 119 Figure 35. GTP hydrolysis induced disassembly of Dyn2-bundled actin 121 Figure 36. PRD domain of Dyn2 is not required for its actin bundling activity 123 Figure 37. Physical interruption failed to generate single actin bundles 125 Figure 38. Reducing temperature and Dyn2 concentration generated small actin bundles 127 Figure 39. Small actin bundles can be visualized under negative stain and CryoEM activity in wild type myotubes 129 Figure 40. Actin filaments packed inside the Dyn2 helix 131 Figure 41. Actin filaments bundled by hyper self-assembly Dyn2 mutant was less sensitive to GTP 133 Figure 42. Actin filaments bundled by CNM-Dyn2 mutant were less sensitive to GTP 135 Figure 43. Dyn2 bundles branched actin 137 Figure 44. GTP hydrolysis regulates disassembly of branched-actin bundles 139 Figure 45. branched-actin bundled by CNM-Dyn2 were less sensitive to GTP hydrolysis 141 Figure 46. Working model of Dyn2 in podosome turnover and its distinct role in pre- and postsynaptic NMJ 143 Table 1. Plasmids used in this study 145 Table 2. Antibodies used in this study 146 Chapter 6-References 147 | |
dc.language.iso | en | |
dc.title | Dynamin-2在神經肌肉接合處後端所扮演的角色 | zh_TW |
dc.title | The Role of Dynamin-2 in Postsynaptic Neuromuscular Junction | en |
dc.type | Thesis | |
dc.date.schoolyear | 109-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 李芳仁(Fang-Jen Lee),潘俊良(Chun-Liang Pan),姚季光(Chi-Kuang Yao),傅琪鈺(Chi-Yu Fu) | |
dc.subject.keyword | dynamin-2,神經肌肉接合處發育,足小體,肌動蛋白綑綁蛋白質, | zh_TW |
dc.subject.keyword | dynamin-2,NMJ morphogenesis,podosome,actin-bundling protein, | en |
dc.relation.page | 153 | |
dc.identifier.doi | 10.6342/NTU202100043 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2021-01-12 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
顯示於系所單位: | 分子醫學研究所 |
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