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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡丰喬(Feng-Chiao Tsai) | |
| dc.contributor.author | Yen-Hsuan Chou | en |
| dc.contributor.author | 周彥萱 | zh_TW |
| dc.date.accessioned | 2022-11-24T03:21:23Z | - |
| dc.date.available | 2021-11-12 | |
| dc.date.available | 2022-11-24T03:21:23Z | - |
| dc.date.copyright | 2021-11-12 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-09-19 | |
| dc.identifier.citation | 1. Guan, J.-L., Cell migration: developmental methods and protocols. Vol. 294. 2005: Springer Science Business Media. 2. Vitorino, P. and T. Meyer, Modular control of endothelial sheet migration. Genes Dev, 2008. 22(23): p. 3268-81. 3. Simpson, K.J., et al., Identification of genes that regulate epithelial cell migration using an siRNA screening approach. Nat Cell Biol, 2008. 10(9): p. 1027-38. 4. Yamada, E., et al., Molecular cloning and characterization of a novel human STE20-like kinase, hSLK. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2000. 1495(3): p. 250-262. 5. Al-Zahrani, K.N., K.D. Baron, and L.A. Sabourin, Ste20-like kinase SLK, at the crossroads: a matter of life and death. Cell Adh Migr, 2013. 7(1): p. 1-10. 6. Storbeck, C.J., et al., The Ldb1 and Ldb2 transcriptional cofactors interact with the Ste20-like kinase SLK and regulate cell migration. Mol Biol Cell, 2009. 20(19): p. 4174-82. 7. Quizi, J.L., et al., SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration. Oncogene, 2013. 32(39): p. 4656-63. 8. Guilluy, C., et al., Ste20-related kinase SLK phosphorylates Ser188 of RhoA to induce vasodilation in response to angiotensin II Type 2 receptor activation. Circ Res, 2008. 102(10): p. 1265-74. 9. Wang, Y., R. Wang, and D.D. Tang, Ste20-like Kinase-mediated Control of Actin Polymerization Is a New Mechanism for Thin Filament-associated Regulation of Airway Smooth Muscle Contraction. Am J Respir Cell Mol Biol, 2020. 62(5): p. 645-656. 10. Machicoane, M., et al., SLK-dependent activation of ERMs controls LGN-NuMA localization and spindle orientation. J Cell Biol, 2014. 205(6): p. 791-9. 11. Zhapparova, O.N., et al., Ste20-like protein kinase SLK (LOSK) regulates microtubule organization by targeting dynactin to the centrosome. Mol Biol Cell, 2013. 24(20): p. 3205-14. 12. Johnson, T.M., R. Antrobus, and L.N. Johnson, Plk1 activation by Ste20-like kinase (Slk) phosphorylation and polo-box phosphopeptide binding assayed with the substrate translationally controlled tumor protein (TCTP). Biochemistry, 2008. 47(12): p. 3688-3696. 13. Ellinger‐Ziegelbauer, H., et al., Ste20‐like kinase (SLK), a regulatory kinase for polo‐like kinase (Plk) during the G2/M transition in somatic cells. Genes to Cells, 2000. 5(6): p. 491-498. 14. Hao, W., et al., Induction of apoptosis by the Ste20-like kinase SLK, a germinal center kinase that activates apoptosis signal-regulating kinase and p38. J Biol Chem, 2006. 281(6): p. 3075-84. 15. Cybulsky, A.V., et al., The Ste20-like kinase SLK promotes p53 transactivation and apoptosis. Am J Physiol Renal Physiol, 2009. 297(4): p. F971-80. 16. Sabourin, L.A. and M.A. Rudnicki, Induction of apoptosis by SLK, a Ste20-related kinase. Oncogene, 1999. 18(52): p. 7566-7575. 17. Wagner, S., et al., FAK/src-family dependent activation of the Ste20-like kinase SLK is required for microtubule-dependent focal adhesion turnover and cell migration. PLoS One, 2008. 3(4): p. e1868. 18. Baron, K.D., et al., Recruitment and activation of SLK at the leading edge of migrating cells requires Src family kinase activity and the LIM-only protein 4. Biochim Biophys Acta, 2015. 1853(7): p. 1683-92. 19. Bagci, H., et al., Mapping the proximity interaction network of the Rho-family GTPases reveals signalling pathways and regulatory mechanisms. Nat Cell Biol, 2020. 22(1): p. 120-134. 20. Raftopoulou, M. and A. Hall, Cell migration: Rho GTPases lead the way. Dev Biol, 2004. 265(1): p. 23-32. 21. Zaman, R., et al., Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain. J Cell Biol, 2021. 220(6). 22. Huveneers, S. and E.H. Danen, Adhesion signaling - crosstalk between integrins, Src and Rho. J Cell Sci, 2009. 122(Pt 8): p. 1059-69. 23. Bolado-Carrancio, A., et al., Periodic propagating waves coordinate RhoGTPase network dynamics at the leading and trailing edges during cell migration. Elife, 2020. 9. 24. Fehon, R.G., A.I. McClatchey, and A. Bretscher, Organizing the cell cortex: the role of ERM proteins. Nat Rev Mol Cell Biol, 2010. 11(4): p. 276-87. 25. Yu, L.-Y., et al., Synthetic dysmobility screen unveils an integrated STK40-YAP-MAPK system driving cell migration. Science Advances, 2021. 7(31): p. eabg2106. 26. Ridley, A.J., Membrane ruffling and signal transduction. Bioessays, 1994. 16(5): p. 321-327. 27. Nicholson-Dykstra, S., H.N. Higgs, and E.S. Harris, Actin dynamics: growth from dendritic branches. Current Biology, 2005. 15(9): p. R346-R357. 28. Berginski, M.E., et al., High-resolution quantification of focal adhesion spatiotemporal dynamics in living cells. PloS one, 2011. 6(7): p. e22025. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80906 | - |
| dc.description.abstract | 細胞遷移在許多生理程序中都扮演重要角色，因此研究細胞如何遷移成為一項重要的議題。我們實驗室先前利用「two-hit shRNA screening assay」，將119個與細胞遷移相關的基因個別敲減(knockdown)，並分別搭配給予三種訊息鏈的抑制劑。結果顯示ROCK抑制劑Y27632搭配敲減SLK基因，會大幅增加人類臍靜脈內皮細胞(HUVEC)的爬行速度。SLK(Ste20-like kinase)是一種絲氨酸/蘇氨酸蛋白激酶，目前已知會影響細胞週期、附著力、細胞凋亡與細胞骨架。然而其中詳細作用機轉還未被研究清楚。 我們實驗室先前利用人類口腔上皮癌細胞SAS研究，發現knockdown SLK會使細胞爬行速度及細胞爬行的方向性都降低；另外，有文獻指出SLK在細胞爬行的前緣會產生聚集的現象。因此我們提出假說：SLK聚集在細胞前緣的現象可能會調控爬行方向性。以免疫螢光染色、活細胞影像及膜蛋白分離技術，我們證實過度表現的SLK的確會聚集在遷移細胞的前緣。未來計畫利用CRIPSR knockin綠螢光蛋白，以觀察內生性SLK的分布位置。 另一方面我們也致力於釐清SLK與ROCK之間的交互作用，目前我們認為SLK與ROCK並無直接的調控關係。近來數篇報導提出。近年來數篇報導提出SLK與ROCK同為RhoA下游的effector蛋白，意味著兩者可能存在間接的競爭關係。而其中一篇報導更使我們注意到lymphocyte-oriented kinase(LOK)蛋白與SLK在演化上與功能上的高度相似性。目前我們以西方墨點法(western blot)與免疫螢光染色觀察過度表現或knockdown SLK時，搭配其他調控RhoA/ROCK訊息鏈的藥物，觀察應力纖維(stress fiber)及黏著斑(focal adhesion)訊號隨之產生的變化。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-24T03:21:23Z (GMT). No. of bitstreams: 1 U0001-1909202117110400.pdf: 4192321 bytes, checksum: ceaf1f0eb7ab8018bb1be03e02f93c78 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | 誌謝 1 摘要 2 Abstract 3 目錄 5 圖目錄 8 表目錄 10 第一章 介紹 11 1.1 細胞遷移的重要性及目前研究進展 11 1.2 找出相關基因與訊息傳遞間的交互作用：Two-hit shRNA screening 11 1.3 Ste20-like kinase(SLK) 13 1.4 SLK的特殊分布與細胞遷移方向性的關係 13 1.5 實驗室先前研究成果：SLK與ROCK可能無直接關聯 15 1.6 近來文獻報導顯示：SLK可能與RhoA產生交互作用 15 1.7 我們的研究目標 18 第二章 材料與方法 19 2.1 細胞培養與藥物處理 19 2.2 質體製備 19 2.3 包裝慢病毒Lentivirus package 20 2.4 活細胞影像Live cell image 20 2.5 膜質蛋白分離Membrane protein extraction 21 2.6 西方墨點法Western blot 21 2.7 免疫螢光染色Immunofluorescence 22 第三章 結果 23 3.1 SLK在細胞中的分布位置 23 3.1.1 過度表現之SLK有聚集在細胞邊緣現象 23 3.1.2 過度表現之SLK有聚集在遷移細胞前端的情形 25 3.1.3 內生性SLK在遷移細胞中的表現位置有待確認 32 3.2 SLK與RhoA/ROCK訊息鏈的關係 37 3.2.1 SLK與ROCK無直接關聯 37 3.2.2 以免疫螢光染色檢驗SLK在RhoA/ROCK訊息鏈中的影響層級 37 3.2.3 SLK與ROCK同為RhoA下游，兩者可能互為競爭關係 45 3.2.4 SLK可能能夠對ROCK下游訊息鏈產生直接的影響 52 第四章 討論 60 4.1 在SAS與HUVEC上調控SLK產生不同的影響 60 4.2 過度表現的SLK有聚集在細胞前緣現象 60 4.3 Endogenous SLK在遷移細胞中的位置有待釐清：關於SLK C-terminal domain的重要性與未來方向 61 4.4 驗證SLK是否透過RhoA對細胞遷移產生影響：以Rho抑制劑檢驗 62 4.5 驗證SLK是否透過ROCK對細胞遷移產生影響：穩定western blot實驗同時將LOK納入操控 63 4.6 大膽假設：SLK可能獨立影響pMLC 64 4.7 SLK與RhoA/ROCK活性在細胞中的分布可能相斥 66 4.8 結論 67 第五章 參考文獻 68 第六章 附錄 71 6.1 附圖 71 6.2 質體maps 76 6.3 Matlab程式稿 78 6.2.1 螢光版校正 78 6.2.2 前端/後端螢光比例分析 83 6.2.3 兩相鄰細胞核連線訊號分析 98 6.2.4 pMLC訊號分析 117 6.2.5 Focal adhesion分析 123 | |
| dc.language.iso | zh-TW | |
| dc.subject | ROCK | zh_TW |
| dc.subject | SLK | zh_TW |
| dc.subject | 細胞遷移 | zh_TW |
| dc.subject | 細胞前緣 | zh_TW |
| dc.subject | RhoA | zh_TW |
| dc.subject | SLK | en |
| dc.subject | ROCK | en |
| dc.subject | RhoA | en |
| dc.subject | leading edge | en |
| dc.subject | cell migration | en |
| dc.title | 探索SLK和ROCK在細胞遷移的拮抗作用 | zh_TW |
| dc.title | Investigation of SLK/ROCK antagonism in cell migration | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 賈景山(Hsin-Tsai Liu),曾炳輝(Chih-Yang Tseng),林耿慧 | |
| dc.subject.keyword | SLK,細胞遷移,細胞前緣,RhoA,ROCK, | zh_TW |
| dc.subject.keyword | SLK,cell migration,leading edge,RhoA,ROCK, | en |
| dc.relation.page | 139 | |
| dc.identifier.doi | 10.6342/NTU202103249 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-09-22 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 藥理學研究所 | zh_TW |
| 顯示於系所單位： | 藥理學科所 | |
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