通過誘變表徵新型微管分支因子 SSNA1

、線上人數：33

、訪客IP：18.224.44.21

姓名	普秧卡(Priyanka Vinothkumar) 查詢紙本館藏	畢業系所	生命科學系
論文名稱	通過誘變表徵新型微管分支因子 SSNA1 (Characterization of the novel microtubule-branching factor SSNA1 by mutagenesis)
檔案	[Endnote RIS 格式] [Bibtex 格式] [相關文章] [文章引用] [完整記錄] [館藏目錄] 至系統瀏覽論文 (2026-9-1以後開放)
摘要(中)	Sjögren 綜合徵核自身抗原 1 (SSNA1) 最初被確定為 Sjögren 綜合徵患者自身抗體的主要靶點，它影響了西方世界估計 0.5% 的人口，但其生物學功能和醫學相關性在很大程度上是未知的。除了其核定位外，SSNA1 還定位於幾個基於微管的結構，包括中心體和中體，因此可能調節細胞分裂。最近，據報導，除了中心體之外，SSNA1 還存在於神經元中，並促進軸突生長、伸長和分支。此外，最近的一份報告表明，SSNA1 重組蛋白自組裝成小簇和纖維，促進體外微管成核和分支。為了闡明 SSNA1 在微管重塑中的作用，我們開始在體外和細胞中系統地研究哺乳動物 SSNA1。首先，我們觀察到當在細胞中表達時，SSNA1 會自我相互作用並形成液滴狀結構，這讓人想起液液相分離 (LLPS) 現象。由於 LLPS 是由蛋白質內在無序區域賦予的多價相互作用介導的，我們使用 IUPred2A 和 PONDR 等生物信息學軟件分析了 SSNA1 的一級氨基酸序列，以預測疾病傾向。我們發現 SSNA1 帶有本質上無序的 N 和 C 末端。為了進一步確定液滴形成所需的基本殘基或基序，我們構建了一個 SSNA1 突變體文庫，其中包括一系列丙氨酸掃描和截短突變體以及幾種可能受有絲分裂和/或中心體定位激酶調節的抗磷酸化和模擬突變體.通過在體外和體內表徵這些突變體，我們現在已經確定了控制 SSNA1 自組裝的關鍵基序和磷酸化依賴性殘基。
摘要(英)	Sjögren syndrome nuclear autoantigen 1 (SSNA1) was originally identified as a major target of autoantibodies in patients with Sjögren syndrome, which affects an estimated 0.5% of the population of the western world, but its biological function and medical relevance are largely unknown. In addition to its nuclear localization, SSNA1 has been localized to several microtubule-based structures including centrosomes and the mid-body and may thus regulate cell division. Recently SSNA1 was also reported to be present in neurons apart from centrosomes and promotes axon growth, elongation and branching. Moreover, a recent report demonstrated that SSNA1 recombinant proteins self-assemble into small clusters and fibrils that promote microtubule nucleation and branching in vitro. To elucidate the role of SSNA1 in microtubule remodeling, we set out to systematically investigate mammalian SSNA1 in vitro and in cells. First, we observed that when expressed in cells, SSNA1 self-interacts and forms droplet-like structures, which is reminiscent of the liquid-liquid phase separation (LLPS) phenomenon. Since LLPS is mediated by multivalent interactions conferred by the intrinsically disordered regions of the proteins, we analyzed the primary amino acid sequence of SSNA1 using bioinformatics softwares such as IUPred2A and PONDR to predict disorder propensity. We found that SSNA1 carries intrinsically disordered N and C-termini. To further identify essential residues or motifs required for droplet formation, we constructed a SSNA1 mutant library which includes a series of alanine-scan and truncated mutants as well as several phosphorylation-resistant and mimicking mutants potentially regulated by mitotic and/or centrosome-localized kinases. By characterizing these mutants in vitro and in vivo, we have identified key motifs and phosphorylation-dependent residues that control SSNA1 self-assembly.
關鍵字(中)	★ SSNA1 ★ 誘變	關鍵字(英)	★ SSNA1 ★ Mutagenesis
論文目次	Table of Contents 1 Introduction 1.1 Microtubules and microtubule-based cellular structures………………………. 1 1.1.1 Mitotic spindle and midbody in dividing cells…………………………… 2 1.1.2 Primary cilia in resting cells………………………………………………. 2 1.1.3 Axons in primary neurons…………………………………………………. 3 1.2 Localization and interacting partners of SSNA1 at microtubule-based structures... 4 1.2.1 SSNA1 was identified as a nuclear autoantigen……………………………. 4 1.2.2 SSNA1 localizes to spindle poles and the midbody during cell division…… 4 1.2.3 SSNA1 localizes to centrosomes/basal bodies during ciliogenesis………… 5 1.2.4 SSNA1 localizes to axonal branching sites during neuronal morphogenesis…6 1.2.5 SSNA1 self-assembles into fibrils…………………………………………. 6 1.3 Liquid-liquid phase separation (LLPS) in biological systems……………………. 7 1.3.1 Driving force of liquid-liquid phase separation……………………………. 7 1.3.2 Examples of phase-separated centrosomal proteins……………………....... 8 1.3.3 Examples of phase-separated microtubule-associated proteins……………. 8 2 Specific Aim…………………………………………………………….. 10 3 Materials and methods…………………………………………………. 11 4 Results…………………………………………………………………... 15 3.1 SSNA1 sequence alignment among different species………………………… 15 3.2 SSNA1 contains a coiled-coil domain and intrinsically disordered regions (IDRs) 3.3 SSNA1 forms higher-order oligomers in vitro…………………………………… 15 3.4 SSNA1 forms droplets in vivo………………………………………………………. 16 3.5 SSNA1 truncated variants induce droplet and/or filament formation………… 16 3.6 Alanine scan mutagenesis identifies key residues for droplet formation in vivo.. 17 3.7 Hydrophobic but not electrostatic interaction is required for droplet formation.. 18 3.8 Disulfide bonds are required for droplet formation……………………………. 18 3.9 Phospho-mimicking mutants disrupt droplet formation………………………. 19 5 Discussion……………………………………………………………....... 20 6 References……………………………………………………………….. 22 7 Figures………………………………………………………………….. 27 8 Appendixes……………………………………………………………… 41
參考文獻	1. Aki, T., Funakoshi, T., Nishida-Kitayama, J., & Mizukami, Y. (2008). TPRA40/GPR175 regulates early mouse embryogenesis through functional membrane transport by Sjögren’s syndrome-associated protein NA14. Journal of Cellular Physiology, 217(1), 194–206. https://doi.org/10.1002/jcp.21492 2. Basnet, N., Nedozralova, H., Crevenna, A. H., Bodakuntla, S., Schlichthaerle, T., Taschner, M., Cardone, G., Janke, C., Jungmann, R., Magiera, M. M., Biertümpfel, C., & Mizuno, N. (2018). Direct induction of microtubule branching by microtubule nucleation factor SSNA1. Nature Cell Biology, 20(10), 1172–1180. https://doi.org/10.1038/s41556-018-0199-8 3. Chabin-Brion, K., Me Marceiller, J., Perez, F., Settegrana, C., Drechou, A., Ve Durand, G., & Poü, C. (2001). The Golgi Complex Is a Microtubule-organizing Organelle. In Molecular Biology of the Cell (Vol. 12). 4. Conde, C., & Cáceres, A. (2009). Microtubule assembly, organization and dynamics in axons and dendrites. Nature Reviews Neuroscience, 10(5), 319–332. https://doi.org/10.1038/nrn2631 5. Efimov, A., Kharitonov, A., Efimova, N., Loncarek, J., Paul, M., Andreyeva, N., Gleeson, P., Galjart, N., Maia, A. R. R., Ian, X., Yates, J. R., Maiato, H., Khodjakov, A., Akhmanova, A., & Kaverina, I. (2009). NIH Public Access. 12(6), 917–930. https://doi.org/10.1016/j.devcel.2007.04.002.Asymmetric 6. Errico, A., Claudiani, P., D’Addio, M., & Rugarli, E. I. (2004). Spastin interacts with the centrosomal protein NA14, and is enriched in the spindle pole, the midbody and the distal axon. Human Molecular Genetics, 13(18), 2121–2132. https://doi.org/10.1093/hmg/ddh223 7. Etienne-Manneville, S. (2013). Microtubules in cell migration. Annual Review of Cell and Developmental Biology, 29, 471–499. https://doi.org/10.1146/annurev-cellbio-101011-155711 8. Franker, M. A. M., & Hoogenraad, C. C. (2013). Microtubule-based transport -basic mechanisms, traffic rules and role in neurological pathogenesis. Journal of Cell Science, 126(11), 2319–2329. https://doi.org/10.1242/jcs.115030 9. Ganguly, S., Williams, L. S., Palacios, I. M., & Goldstein, R. E. (2012). Cytoplasmic streaming in Drosophila oocytes varies with kinesin activity and correlates with the microtubule cytoskeleton architecture. Proceedings of the National Academy of Sciences of the United States of America, 109(38), 15109–15114. https://doi.org/10.1073/pnas.1203575109 10. Goodson, H. V., & Jonasson, E. M. (2018). Microtubules and microtubule-associated proteins. Cold Spring Harbor Perspectives in Biology, 10(6). https://doi.org/10.1101/cshperspect.a022608 11. Goodson, H. V, & Jonasson, E. M. (2021). Microtubules and Microtubule-Associated Proteins. 12. Goyal, U., Renvoisé, B., Chang, J., & Blackstone, C. (2014). Spastin-interacting protein NA14/SSNA1 functions in cytokinesis and axon development. PLoS ONE, 9(11). https://doi.org/10.1371/journal.pone.0112428 13. Hodges, M. E., Scheumann, N., Wickstead, B., Langdale, J. A., & Gull, K. (2010). Reconstructing the evolutionary history of the centriole from protein components. Journal of Cell Science, 123(9), 1407–1413. https://doi.org/10.1242/jcs.064873 14. Ishihara, K., Nguyen, P. A., Groen, A. C., Field, C. M., & Mitchison, T. J. (2014). Microtubule nucleation remote from centrosomes may explain how asters span large cells. Proceedings of the National Academy of Sciences of the United States of America, 111(50), 17715–17722. https://doi.org/10.1073/pnas.1418796111 15. Kapitein, L. C., & Hoogenraad, C. C. (2015). Building the Neuronal Microtubule Cytoskeleton. In Neuron (Vol. 87, Issue 3, pp. 492–506). Cell Press. https://doi.org/10.1016/j.neuron.2015.05.046 16. Lai, C. K., Gupta, N., Wen, X., Rangell, L., Chih, B., Peterson, A. S., Bazan, J. F., Li, L., & Scales, S. J. (2011). Functional characterization of putative cilia genes by high-content analysis. Molecular Biology of the Cell, 22(7), 1104–1119. https://doi.org/10.1091/mbc.E10-07-0596 17. Lévêque, M. F., Berry, L., & Besteiro, S. (2016). An evolutionarily conserved SSNA1/DIP13 homologue is a component of both basal and apical complexes of Toxoplasma gondii. Scientific Reports, 6. https://doi.org/10.1038/srep27809 18. Lin, Y. H., Forman-Kay, J. D., & Chan, H. S. (2018). Theories for Sequence-Dependent Phase Behaviors of Biomolecular Condensates. Biochemistry, 57(17), 2499–2508. https://doi.org/10.1021/acs.biochem.8b00058 19. Maiato, H., DeLuca, J., Salmon, E. D., & Earnshaw, W. C. (2004). The dynamic kinetochore-microtubule interface. In Journal of Cell Science (Vol. 117, Issue 23, pp. 5461–5477). https://doi.org/10.1242/jcs.01536 20. Meunier, S., & Vernos, I. (2016). Acentrosomal Microtubule Assembly in Mitosis: The Where, When, and How. In Trends in Cell Biology (Vol. 26, Issue 2, pp. 80–87). Elsevier Ltd. https://doi.org/10.1016/j.tcb.2015.09.001 21. Mitrea, D. M., & Kriwacki, R. W. (2016). Phase separation in biology; Functional organization of a higher order Short linear motifs - The unexplored frontier of the eukaryotic proteome. Cell Communication and Signaling, 14(1), 1–20. https://doi.org/10.1186/s12964-015-0125-7 22. Mogensen, M. M., & Tucker, J. B. (1987). Evidence for microtubule nucleation at plasma membrane-associated sites in Drosophila. 23. Murata, T., Sonobe, S., Baskin, T. I., Hyodo, S., Hasezawa, S., Nagata, T., Horio, T., & Hasebe, M. (2005). Microtubule-dependent microtubule nucleation based on recruitment of γ-tubulin in higher plants. Nature Cell Biology, 7(10), 961–968. https://doi.org/10.1038/ncb1306 24. Petry, S., Groen, A. C., Ishihara, K., Mitchison, T. J., & Vale, R. D. (2013). Branching microtubule nucleation in xenopus egg extracts mediated by augmin and TPX2. Cell, 152(4), 768–777. https://doi.org/10.1016/j.cell.2012.12.044 25. Petry, S., & Vale, R. D. (2015a). Microtubule nucleation at the centrosome and beyond. Nature Cell Biology, 17(9), 1089–1093. https://doi.org/10.1038/ncb3220 26. Petry, S., & Vale, R. D. (2015b). Microtubule nucleation at the centrosome and beyond. In Nature Cell Biology (Vol. 17, Issue 9, pp. 1089–1093). Nature Publishing Group. https://doi.org/10.1038/ncb3220 27. Pfannenschmid, F., Wimmer, V. C., Rios, R. M., Geimer, S., Kröckel, U., Leiherer, A., Haller, K., Nemcová, Y., & Mages, W. (2003a). Chlamydomonas DIP13 and human NA14: A new class of proteins associated with microtubule structures is involved in cell division. Journal of Cell Science, 116(8), 1449–1462. https://doi.org/10.1242/jcs.00337 28. Phair, R. D., & Misteli, T. (2000). 35007077. 404(April). 29. Pollard, T. D., & Goldman, R. D. (2018). Overview of the cytoskeleton from an evolutionary perspective. Cold Spring Harbor Perspectives in Biology, 10(7). https://doi.org/10.1101/cshperspect.a030288 30. Price, H. P., Hodgkinson, M. R., Curwen, R. S., MacLean, L. M., Brannigan, J. A., Carrington, M., Smith, B. A., Ashford, D. A., Stark, M., & Smith, D. F. (2012). The orthologue of Sjögren’s Syndrome nuclear autoantigen 1 (SSNA1) in trypanosoma brucei is an immunogenic self-assembling molecule. PLoS ONE, 7(2). https://doi.org/10.1371/journal.pone.0031842 31. Ramos-Morales, F., Infante, C., Fedriani, C., Bornens, M., & Rios, R. M. (1998). NA14 is a novel nuclear autoantigen with a coiled-coil domain. Journal of Biological Chemistry, 273(3), 1634–1639. https://doi.org/10.1074/jbc.273.3.1634 32. Reiner, O., & Sapir, T. (2013). LIS1 functions in normal development and disease. In Current Opinion in Neurobiology (Vol. 23, Issue 6, pp. 951–956). https://doi.org/10.1016/j.conb.2013.08.001 33. Rodrguez-Rodrguez, M., Trevio, M. A., Laurents, D. V., Arranz, R., Valpuesta, J. M., Rico, M., Bruix, M., & Jimnez, M. A. (2011). Characterization of the structure and self-recognition of the human centrosomal protein NA14: Implications for stability and function. Protein Engineering, Design and Selection, 24(12), 883–892. https://doi.org/10.1093/protein/gzr050 34. Sánchez-Huertas, C., Freixo, F., Viais, R., Lacasa, C., Soriano, E., & Lüders, J. (2016). Non-centrosomal nucleation mediated by augmin organizes microtubules in post-mitotic neurons and controls axonal microtubule polarity. Nature Communications, 7. https://doi.org/10.1038/ncomms12187 35. Tassin, A.-M., Maro, B., & Bornens, M. (n.d.). Fate of Microtubule-organizing Centers during Myogenesis in Vitro. 36. Tischfield, M. A., Cederquist, G. Y., Gupta, M. L., & Engle, E. C. (2011). Phenotypic spectrum of the tubulin-related disorders and functional implications of disease-causing mutations. Current Opinion in Genetics and Development, 21(3), 286–294. https://doi.org/10.1016/j.gde.2011.01.003 37. Uversky, V. N. (2017). Intrinsically disordered proteins in overcrowded milieu: Membrane-less organelles, phase separation, and intrinsic disorder. Current Opinion in Structural Biology, 44, 18–30. https://doi.org/10.1016/j.sbi.2016.10.015 38. Weidemann, M., Schuster-Gossler, K., Stauber, M., Wrede, C., Hegermann, J., Ott, T., Boldt, K., Beyer, T., Serth, K., Kremmer, E., Blum, M., Ueffing, M., & Gossler, A. (2016). CFAP157 is a murine downstream effector of FOXJ1 that is specifically required for flagellum morphogenesis and sperm motility. Development (Cambridge), 143(24), 4736–4748. https://doi.org/10.1242/dev.139626 39. Ahn, Jong Il et al. 2020. “Phase Separation of the Cep63•Cep152 Complex Underlies the Formation of Dynamic Supramolecular Self-Assemblies at Human Centrosomes.” Cell Cycle 19(24): 3437–57. https://doi.org/10.1080/15384101.2020.1843777 40. Ambadipudi, Susmitha et al. 2017. “Liquid-Liquid Phase Separation of the Microtubule-Binding Repeats of the Alzheimer-Related Protein Tau.” Nature Communications 8(1): 1–13. http://dx.doi.org/10.1038/s41467-017-00480-0 41. Hoffmann, Ingrid. 2021. “Centrosomes in Mitotic Spindle Assembly and Orientation.” Current Opinion in Structural Biology 66: 193–98. https://doi.org/10.1016/j.sbi.2020.11.003 42. Hu, Chi Kuo, Margaret Coughlin, and Timothy J. Mitchison. 2012. “Midbody Assembly and Its Regulation during Cytokinesis.” Molecular Biology of the Cell 23(6): 1024–34. 43. King, Matthew R., and Sabine Petry. 2020. “Phase Separation of TPX2 Enhances and Spatially Coordinates Microtubule Nucleation.” Nature Communications 11(1): 1–13. http://dx.doi.org/10.1038/s41467-019-14087-0 44. Plotnikova, Olga V., Elena N. Pugacheva, and Erica A. Golemis. 2009. 94 Methods in cell biology Primary Cilia and the Cell Cycle. First edition. Elsevier. http://dx.doi.org/10.1016/S0091-679X(08)94007-3 45. Ukmar-Godec, Tina, Susanne Wegmann, and Markus Zweckstetter. 2020. “Biomolecular Condensation of the Microtubule-Associated Protein Tau.” Seminars in Cell and Developmental Biology 99(June): 202–14. https://doi.org/10.1016/j.semcdb.2019.06.007
指導教授	魏妊亘高永旭鄭惠春(Dr. Jen-Hsuan Wei Dr. Yung-Hsi Kao Dr. Hui-Chun Cheng)	審核日期	2021-8-11
推文	facebook plurk twitter funp google live udn HD myshare reddit netvibes friend youpush delicious baidu
網路書籤	Google bookmarks del.icio.us hemidemi myshare

博碩士論文 108821606 詳細資訊