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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 | 楊東霖 | zh_TW |
dc.contributor.advisor | Tony Yang | en |
dc.contributor.author | 卓曼婷 | zh_TW |
dc.contributor.author | MAN TENG CHEOK | en |
dc.date.accessioned | 2023-10-03T17:20:44Z | - |
dc.date.available | 2023-11-10 | - |
dc.date.copyright | 2023-10-03 | - |
dc.date.issued | 2023 | - |
dc.date.submitted | 2023-08-02 | - |
dc.identifier.citation | Anvarian, Z., Mykytyn, K., Mukhopadhyay, S., Pedersen, L. B., & Christensen, S. T. (2019). Cellular signalling by primary cilia in development, organ function and disease. Nat Rev Nephrol, 15(4), 199-219. https://doi.org/10.1038/s41581-019-0116-9
DeCaen, P. G., Delling, M., Vien, T. N., & Clapham, D. E. (2013). Direct recording and molecular identification of the calcium channel of primary cilia. Nature, 504(7479), 315-318. https://doi.org/10.1038/nature12832 Dodgson, J., Chessel, A., Cox, S., & Carazo Salas, R. E. (2015). Super-Resolution Microscopy: SIM, STED and Localization Microscopy. In T. E. S. Dahms & K. J. Czymmek (Eds.), Advanced Microscopy in Mycology (pp. 47-60). Springer International Publishing. https://doi.org/10.1007/978-3-319-22437-4_3 Endesfelder, U., & Heilemann, M. (2015). Direct stochastic optical reconstruction microscopy (dSTORM). Methods Mol Biol, 1251, 263-276. https://doi.org/10.1007/978-1-4939-2080-8_14 Galbraith, C. G., & Galbraith, J. A. (2011). Super-resolution microscopy at a glance. Journal of cell science, 124(10), 1607-1611. Gallagher, B. R., & Zhao, Y. (2021). Expansion microscopy: A powerful nanoscale imaging tool for neuroscientists. Neurobiol Dis, 154, 105362. https://doi.org/10.1016/j.nbd.2021.105362 Gelderblom, H. R., & Krüger, D. H. (2014). 1 - Helmut Ruska (1908–1973): His Role in the Evolution of Electron Microscopy in the Life Sciences, and Especially Virology. In P. W. Hawkes (Ed.), Advances in Imaging and Electron Physics (Vol. 182, pp. 1-94). Elsevier. https://doi.org/https://doi.org/10.1016/B978-0-12-800146-2.00001-1 Goetz, S. C., & Anderson, K. V. (2010). The primary cilium: a signalling centre during vertebrate development. Nat Rev Genet, 11(5), 331-344. https://doi.org/10.1038/nrg2774 Heintzmann, R., & Ficz, G. (2006). Breaking the resolution limit in light microscopy. Briefings in Functional Genomics, 5(4), 289-301. Karagiannis, E. D., & Boyden, E. S. (2018). Expansion microscopy: development and neuroscience applications. Curr Opin Neurobiol, 50, 56-63. https://doi.org/10.1016/j.conb.2017.12.012 Kiesel, P., Alvarez Viar, G., Tsoy, N., Maraspini, R., Gorilak, P., Varga, V., Honigmann, A., & Pigino, G. (2020). The molecular structure of mammalian primary cilia revealed by cryo-electron tomography. Nat Struct Mol Biol, 27(12), 1115-1124. https://doi.org/10.1038/s41594-020-0507-4 Kiprilov, E. N., Awan, A., Desprat, R., Velho, M., Clement, C. A., Byskov, A. G., Andersen, C. Y., Satir, P., Bouhassira, E. E., Christensen, S. T., & Hirsch, R. E. (2008). Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery. J Cell Biol, 180(5), 897-904. https://doi.org/10.1083/jcb.200706028 Mazo, G., Soplop, N., Wang, W. J., Uryu, K., & Tsou, M. F. (2016). Spatial Control of Primary Ciliogenesis by Subdistal Appendages Alters Sensation-Associated Properties of Cilia. Dev Cell, 39(4), 424-437. https://doi.org/10.1016/j.devcel.2016.10.006 Orhon, I., Dupont, N., Pampliega, O., Cuervo, A. M., & Codogno, P. (2015). Autophagy and regulation of cilia function and assembly. Cell Death & Differentiation, 22(3), 389-397. https://doi.org/10.1038/cdd.2014.171 Partridge, R., Conlisk, N., & Davies, J. A. (2012). In-lab three-dimensional printing: an inexpensive tool for experimentation and visualization for the field of organogenesis. Organogenesis, 8(1), 22-27. https://doi.org/10.4161/org.20173 Prevo, B., Scholey, J. M., & Peterman, E. J. G. (2017). Intraflagellar transport: mechanisms of motor action, cooperation, and cargo delivery. Febs j, 284(18), 2905-2931. https://doi.org/10.1111/febs.14068 Qin, H., Diener, D. R., Geimer, S., Cole, D. G., & Rosenbaum, J. L. (2004). Intraflagellar transport (IFT) cargo: IFT transports flagellar precursors to the tip and turnover products to the cell body. J Cell Biol, 164(2), 255-266. https://doi.org/10.1083/jcb.200308132 Rust, M. J., Bates, M., & Zhuang, X. (2006). Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods, 3(10), 793-796. https://doi.org/10.1038/nmeth929 Samper, E., Flores, J. M., & Blasco, M. A. (2001). Restoration of telomerase activity rescues chromosomal instability and premature aging in Terc-/- mice with short telomeres. EMBO Rep, 2(9), 800-807. https://doi.org/10.1093/embo-reports/kve174 Sanderson, M. J., Smith, I., Parker, I., & Bootman, M. D. (2014). Fluorescence microscopy. Cold Spring Harb Protoc, 2014(10), pdb.top071795. https://doi.org/10.1101/pdb.top071795 Valente, E. M., Rosti, R. O., Gibbs, E., & Gleeson, J. G. (2014). Primary cilia in neurodevelopmental disorders. Nat Rev Neurol, 10(1), 27-36. https://doi.org/10.1038/nrneurol.2013.247 Veland, I. R., Awan, A., Pedersen, L. B., Yoder, B. K., & Christensen, S. T. (2009). Primary cilia and signaling pathways in mammalian development, health and disease. Nephron Physiol, 111(3), p39-53. https://doi.org/10.1159/000208212 Wang, W., Jack, B. M., Wang, H. H., Kavanaugh, M. A., Maser, R. L., & Tran, P. V. (2021). Intraflagellar Transport Proteins as Regulators of Primary Cilia Length. Front Cell Dev Biol, 9, 661350. https://doi.org/10.3389/fcell.2021.661350 Waters, A. M., & Beales, P. L. (2011). Ciliopathies: an expanding disease spectrum. Pediatr Nephrol, 26(7), 1039-1056. https://doi.org/10.1007/s00467-010-1731-7 Winey, M., Meehl, J. B., O'Toole, E. T., & Giddings, T. H., Jr. (2014). Conventional transmission electron microscopy. Mol Biol Cell, 25(3), 319-323. https://doi.org/10.1091/mbc.E12-12-0863 Wong, S. Y., & Reiter, J. F. (2008). The primary cilium at the crossroads of mammalian hedgehog signaling. Curr Top Dev Biol, 85, 225-260. https://doi.org/10.1016/s0070-2153(08)00809-0 Xu, J., Ma, H., & Liu, Y. (2017). Stochastic Optical Reconstruction Microscopy (STORM). Curr Protoc Cytom, 81, 12.46.11-12.46.27. https://doi.org/10.1002/cpcy.23 Yamanaka, M., Smith, N. I., & Fujita, K. (2014). Introduction to super-resolution microscopy. Microscopy, 63(3), 177-192. Yoder, B. K. (2007). Role of primary cilia in the pathogenesis of polycystic kidney disease. J Am Soc Nephrol, 18(5), 1381-1388. https://doi.org/10.1681/asn.2006111215 Yoon, J., Comerci, C. J., Weiss, L. E., Milenkovic, L., Stearns, T., & Moerner, W. E. (2019). Revealing Nanoscale Morphology of the Primary Cilium Using Super-Resolution Fluorescence Microscopy. Biophys J, 116(2), 319-329. https://doi.org/10.1016/j.bpj.2018.11.3136 | - |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90724 | - |
dc.description.abstract | 初級纖毛(Primary cilia)是脊椎動物細胞中用於感覺信號及細胞外環境偵測的細胞胞器。纖毛內運輸作用蛋白(Intraflagellar Transport Protein,IFT protein)負責初級纖毛的生成組裝、維持以及細胞之間訊號傳遞的溝通工作。過去的研究指出初級纖毛缺陷和鞭毛內運輸作用蛋白缺失會引發多種纖毛相關疾病,包括神經發育遲緩以及生理系統功能障礙等。然而,導致初級纖毛缺陷的生長、組裝和拆除機制,以及訊號傳遞的方式仍未完全解析。
在本研究中,我們使用了次五奈米超解析多色光學顯微技術 (EX-dSTORM),對纖毛內運輸作用蛋白的結構進行了全面的解析。透過結合dSTORM (Direct stochastic optical reconstruction microscopy)和EX(Expansion microscopy) 實現5納米解析求的生物分子結構影像,闡釋纖毛的結構模型和IFT蛋白在方式纖毛上的運輸模型。這項研究的結果將有助於更新我們對纖毛,不同的纖毛內運輸作用蛋白共同合作結構的認識,並更深入瞭解初級纖毛的生成和維護過程。冀望未來可以藉由這個纖毛結構模型更好地了解初級纖毛的結構和IFT 轉運機制,並根據其調控機制開發出針對纖毛類疾病的療法。 | zh_TW |
dc.description.abstract | Primary cilia are microtubule-based organelles essential for mediating mechano- /photo-/chemo-sensations in mammalian cells. Intraflagellar transport (IFT) proteins are crucial components to support ciliary assembly, maintenance, and cellular signaling. The ciliary microtubule doublets of the ‘9+0’ arrangement serve as tracks for molecular motor-based transport along cilia. Although the tapering structure of primary cilia has been examined by electron microscopy, the spatial correlation between the IFT pattern and axonemal structure remains elusive due to insufficient light resolution. Here, we integrated single-molecule localization microscopy and sample expansion techniques achieving two-color imaging with effective localization precision of sub-5 nm to explore the axonemal ultrastructure and molecular organization of IFT and IFT-associated proteins in retinal pigment epithelial cilia. Our results revealed the step-wise reduction of ciliary diameter, different from the well-known paradigm showing a gradual change in the width of axonemal bundles. Surprisingly, our data showed that the IFT proteins congested near the end of the microtubule doubles within the ciliary compartment. In addition, we resolved the molecular architecture of IFT complexes, cargo, and motor proteins to characterize their relative localizations. We found that IFT proteins presented distinct distributions, including isolated units, paired configurations, or alternating arrangements in IFT trains. Together, our ultrastructural light nanoscopic imaging enables us to perform morphological analysis of primary cilia, which guides the localization of IFT complexes at segmented sites of cilia, suggesting the asynchronous axonemal growth of microtubule doubles. | en |
dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-10-03T17:20:44Z No. of bitstreams: 0 | en |
dc.description.provenance | Made available in DSpace on 2023-10-03T17:20:44Z (GMT). No. of bitstreams: 0 | en |
dc.description.tableofcontents | 口試委員會審定書 i
誌謝 ii 中文摘要 iii ABSTRACT iv LIST OF FIGURES vi LIST OF TABLES viii Chapter1 Introduction 1 1.1 Background 1 1.2 Literature review 3 1.2.1 Resolution limitation of optical microscopy 3 1.2.2 Super-resolution methods 4 1.2.3 dSTORM microscopy 6 1.2.4 Expansion microscopy 8 1.2.5 Primary cilia 9 1.2.6 Intraflagellar Transport Protein 12 1.2.7 Primary cilia and human ciliopathies 14 1.3 Research motivation 16 1.4 Thesis structure 18 Chapter2 Experimental Design 19 2.1 Combination of dSTORM and post-labelling expansion microscopy 21 2.2 Expansion microscopy benefits for immunostaining 24 2.3 The replacement of tradition fiducial marker in EX-dSTORM 25 Chapter3 Materials and Methods 27 3.1 Cell culture 27 3.2 Antibodies 28 3.3 Expansion sample preparation and immunofluorescence 29 3.4 EX-dSTORM imaging 32 3.5 Drift correction 35 3.6 Image analysis 37 3.7 Protein structure model 38 Chapter4 Results and Discussions 39 4.1 Subvert the traditionally considered cilia ‘9+0’ structure. 39 4.1.1 Structural differences of primary cilia under different image technologies 39 4.1.2 Dynamic changes in axonemal mammalian primary cilia width insights from Ex-dSTORM 42 4.1.3 Differences in width reduction phenomenon for ciliary membrane 47 4.2 Schematic diagram of primary cilia and the definition of cilia groups 50 4.2.1 Schematic diagram of primary cilia and cilia groups 50 4.2.2 Normalization of primary cilia length revealing the position of discontinuous points and width reduction 52 4.3 Molecular-resolution imaging enables the reconstruction of the 3D computational model of the ciliary axoneme. 58 4.4 Ex-dSTORM discloses the correlation between the discontinuous points and IFT88 molecules. 62 4.5 High-resolution imaging reveals the IFT train pattern and different distribution of IFT particles and motor on cilla axoneme 69 4.6 Motor and BBSome do not continuously integrate with the IFT complex during cilia transportation 74 Chapter5 Discussion and conclusion 80 Chapter6 Reference 85 | - |
dc.language.iso | en | - |
dc.title | 以次五奈米超解析多色光學顯微技術定位鞭毛內運輸作用蛋白的結構及訊號傳導方式 | zh_TW |
dc.title | Molecular resolution imaging reveals step-wise reduction of axonemal diameter and ultrastructural architecture of intraflagellar transport proteins | en |
dc.type | Thesis | - |
dc.date.schoolyear | 111-2 | - |
dc.description.degree | 碩士 | - |
dc.contributor.oralexamcommittee | 黃念祖;陳奕帆 | zh_TW |
dc.contributor.oralexamcommittee | Nien-Tsu Huang;Yih-Fan Chen | en |
dc.subject.keyword | 初級纖毛,纖毛內運輸作用蛋白,超解析影像,擴展顯微鏡,多色成像, | zh_TW |
dc.subject.keyword | Ex-dSTORM,dSTORM,Expansion microscopy,Super-resolution,Primary cilia,IFT, | en |
dc.relation.page | 88 | - |
dc.identifier.doi | 10.6342/NTU202302562 | - |
dc.rights.note | 未授權 | - |
dc.date.accepted | 2023-08-04 | - |
dc.contributor.author-college | 電機資訊學院 | - |
dc.contributor.author-dept | 生醫電子與資訊學研究所 | - |
顯示於系所單位: | 生醫電子與資訊學研究所 |
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