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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 蔡丰喬 | |
dc.contributor.author | Cheng-Yu Fan | en |
dc.contributor.author | 范誠祐 | zh_TW |
dc.date.accessioned | 2021-06-17T06:37:44Z | - |
dc.date.available | 2023-10-03 | |
dc.date.copyright | 2018-10-03 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-08-15 | |
dc.identifier.citation | 1. Kaylene J. Simpson, L.M.S., James Bui, Angela Reynolds, Devin Leake, Anastasia Khvorova, Joan S. Brugge, Identification of genes that regulate epithelial cell migration using an siRNA screening approach. Nature cell biology 2008.
2. Simona Wagner, C.J.S., Kristin Roovers, Ziad Y. Chaar, Piotr Kolodziej, Marlene McKay, Luc A. Sabourin, 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. Erik S. Welf, H.E.J., and Jason M. Haugh, Bidirectional coupling between integrin-mediated signaling and actomyosin mechanics explains matrix-dependent intermittency of leading-edge motility. Mol Biol Cell. , 2013. 4. Bonneton C, S.J., Thiery JP., Relationship Between Cell Migration and Cell Cycle During the Initiation of Epithelial to Fibroblastoid Transition. Cell Motil Cytoskeleton. , 1999. 5. Hicklin, D.J. and L.M. Ellis, Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol, 2005. 23(5): p. 1011-27. 6. Muraki, K. and K. Tanigaki, Neuronal migration abnormalities and its possible implications for schizophrenia. Front Neurosci, 2015. 9: p. 74. 7. Louis SF, Z.P., Vascular smooth muscle cell motility: From migration to invasion. Exp Clin Cardiol., 2010. 8. Philip Vitorino, T.M., Modular control of endothelial sheet migration. GENES & DEVELOPMENT 2008. 9. Eitaro Yamada, K.T., Susumu Itoh, Yo-ichiro Kameda, Yasuhiro Kohama, Hiroshi Yamamoto, Molecular cloning and characterization of a novel human STE20-like kinase, hSLK. Biochimica et Biophysica Acta, 1999. 10. LUC A. SABOURIN, P.S., JULIAN WAGNER, AND MICHAEL A. RUDNICKI, Caspase 3 Cleavage of the Ste20-Related Kinase SLK Releases and Activates an Apoptosis-Inducing Kinase Domain and an Actin-Disassembling Region. MOLECULAR AND CELLULAR BIOLOGY, 1999. 11. Rudnicki, L.A.S.a.M.A., Induction of apoptosis by SLK, a Ste20-related kinase. Oncogene, 1999. 12. Simona Wagner, T.A.F., Paul O’Reilly, Karri Hume, and Luc A. Sabourin, Association of the Ste20-like Kinase (SLK) with the Microtubule. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 2002. 13. JL Quizi1, K Baron1,2, KN Al-Zahrani1,2, P O’Reilly1, RK Sriram1,2, J Conway2, A-A Laurin2 and LA Sabourin1,2, SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration. Oncogene, 2013. 14. Ziad Chaar‡1, P.O.R., Irwin Gelman¶, and Luc A. Sabourin‡§2, v-Src-dependent Down-regulation of the Ste20-like Kinase SLK by Casein Kinase II*. THEJOURNALOFBIOLOGICALCHEMISTRY 2006. 15. Chris J. Storbeck, S.W., * Paul O’Reilly,* Marlene McKay,† Robin J. Parks,† Heiner Westphal,‡ and Luc A. Sabourin*†, The Ldb1 and Ldb2 Transcriptional Cofactors Interact with the Ste20-like Kinase SLK and Regulate Cell Migration. Molecular Biology of the Cell, 2009. 16. Olga N. Zhapparovaa, A.I.F., Nadezhda E. Vorobyevab, Sofia A. Bryantsevaa, and c. and Elena S. Nadezhdinaa, Ste20-like protein kinase SLK (LOSK) regulates microtubule organization by targeting dynactin to the centrosome. Molecular Biology of the Cell, 2013. 17. Christophe Guilluy, M.R.-D., Laurent Loufrani, Anne Bourge ́, Daniel Henrion, Luc Sabourin, Gervaise Loirand, Pierre Pacaud, Ste20-Related Kinase SLK Phosphorylates Ser188 of RhoA to Induce Vasodilation in Response to Angiotensin II Type 2 Receptor Activation. Circulation research, 2008. 18. Kyla D. Baron a, K.A.-Z.a., Jillian Conway a, Cédrik Labrèche a, Christopher J. Storbeck a, Jane E. Visvader c, Luc A. Sabourin a,b,⁎, Recruitment and activation of SLK at the leading edge of migrating cells requires Src family kinase activity and the LIM-only protein 4. Biochimica et Biophysica Acta, 2015. 19. K.Rottner, A.H., J.V.Small, <Interplay between Rac and Rho in the control of substrate contact dynamics.pdf>. Current Biology, 1999. 20. Zaidel-Bar, R., et al., Early molecular events in the assembly of matrix adhesions at the leading edge of migrating cells. J Cell Sci, 2003. 116(Pt 22): p. 4605-13. 21. Hotulainen, P. and P. Lappalainen, Stress fibers are generated by two distinct actin assembly mechanisms in motile cells. J Cell Biol, 2006. 173(3): p. 383-94. 22. Taya, S., et al., Direct interaction of insulin-like growth factor-1 receptor with leukemia-associated RhoGEF. J Cell Biol, 2001. 155(5): p. 809-20. 23. Kozasa T, J.X., Hart MJ, Sternweis PM, Singer WD, Gilman AG, Bollag G, Sternweis PC <Guanine nucleotide exchange factor GEF115 specifically mediates activation of Rho and serum response factor by the G protein α subunit Gα13.pdf>. Science, 1998. 24. Kozasa T, J.X., Hart MJ, Sternweis PM, Singer WD, Gilman AG, Bollag G, Sternweis PC <p115 RhoGEF, a GTPase Activating Protein for Gα12 and Gα13.pdf>. Science, 1998. 25. Shamah SM, L.M., Goldberg JL, Estrach S, Sahin M, Hu L, Bazalakova M, Neve RL, Corfas G, Debant A, Greenberg ME <EphA Receptors Regulate Growth Cone Dynamics through the Novel Guanine Nucleotide Exchange Factor Ephexin.pdf>. Cell, 2001. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72362 | - |
dc.description.abstract | 細胞遷移是一個相當基礎且重要的過程,在胚胎發育、組織修復與癌症轉移中都扮演著重要角色。先前的研究找出了不少調控細胞遷移的基因,但這些基因間如何互相作用則仍然不是很清楚,我們因此進行了一個”two-hit” shRNA screen:同時處理shRNA與小分子訊息抑制藥物,並在內皮細胞上使用傷口癒合試驗以探索這些基因在細胞遷移過程中可能的加乘與拮抗作用。從試驗結果我們找到了Ste20-like kinase (SLK)—一個絲氨酸/蘇氨酸蛋白激活酶,SLK在同時knockdown與加上ROCK抑制劑Y27632時顯著地提升了HUVEC細胞的爬行,因此我們推測SLK與ROCK訊息鏈在細胞爬行中具有交互作用。
為了測試SLK-ROCK交互作用是否為一在各種細胞間普遍存在的現象,我們以同樣平台測試了頭頸癌細胞SAS細胞株。與HUVEC結果不同的是:在癌細胞SAS中進行SLK knockdown,會降低爬行速度,暗示著SLK主要不是藉由改變細胞活動力來影響細胞遷移。因此我們進行了單一細胞追蹤法分析傷口癒合試驗中的每一顆獨立的移動細胞,發現SLK knockdown破壞、而SLK over-expression適度地提升了SAS細胞的移動極性。而我們的觀察與先前文獻也指出SLK會累積在細胞移動前緣,因此我們推測:細胞爬行時SLK會累積在移動前緣並且調控細胞極性。 我們更一步地以單一細胞追蹤法探索了SLK與ROCK訊息鏈之間的交互作用。十分有趣地,ROCK抑制反轉了SLK knockdown所造成的細胞活動力降低,支持了兩分子間具有交互作用的想法。另外我們也發現SLK knockdown能夠反轉RhoA knockdown對細胞動力與黏著斑(focal adhesion)的效果、並且ROCK抑制完全地消除了黏著斑的生成,這些結果顯示在細胞遷移中,SLK對RhoA-ROCK訊息軸線具有重大影響力。此外我們發現ROCK抑制顯著地減少了細胞協調性,此過程可能是透過對黏著小帶(adheren junction) 的調控來達成的。因此SLK與ROCK在細胞遷移的交互作用,也有可能是透過不同的遷移機制調控加成所造成的 (SLK控制細胞極性,而ROCK影響了細胞間的協調性)。 總結以上,我們的結果顯示(1) SLK會累積在細胞移動前緣,調控細胞的移動極性,(2) SLK調控了RhoA-ROCK訊息軸線在細胞遷移中的效果,(3)在細胞遷移上SLK與ROCK可能經由不同機制加成進行交互作用(細胞極性與協調性),更進一步的機轉探討仍然在進行中。藉由這些研究我們將能對SLK與ROCK訊息鏈在細胞遷移上的交互作用有更多認識,並可以此發展嶄新的藥理方針來治療細胞遷移相關的疾病。 | zh_TW |
dc.description.abstract | Cell migration is a fundamental process in embryonic development, tissue repair and cancer metastasis. Previous studies have identified many genes regulating cell migration, but how these genes interact with each other during cell migration remains unclear. We therefore conducted a “two-hit” screen using shRNAs combined with small molecule inhibitors and scratch wound healing assays in endothelial cells, to identify potential synergistic or antagonistic effects among signaling molecules during cell migration. The screen identified Ste20-like kinase (SLK), a serine-threonine kinase. Its knockdown together with ROCK inhibitor Y27632 drastically increased HUVEC migration. We therefore hypothesized that SLK interacts with ROCK signaling during cell migration.
To test if the SLK-ROCK interaction is an universal phenomenon across cell types, we tested the head and neck cancer cells (SAS cell line) on the above platform. In contrast to HUVEC cells, knockdown of SLK in cancer cell line SAS decreased its migration speed, suggesting that SLK might not primarily affect cell motility to change cell migration. We thus performed single-cell tracing techniques to analyze traces of individual moving cell in scratch wound healing assays, showing that SLK knockdown disrupted the migration polarity of SAS cells while SLK over-expression moderately enhanced cell polarity. Together with our observation and previous reports that SLK was accumulated at the migrating cell front, we propose that SLK accumulates at the leading edge of the migrating cell to regulate cell polarity. We further explored the interaction between SLK and ROCK using single-cell tracing. Interestingly, ROCK inhibition reversed the decreased motility caused by SLK knockdown, supporting the idea of interaction between these two molecules. We also noticed that SLK knockdown reversed the effect of RhoA knockdown on cell motility and focal adhesion, and that ROCK inhibition totally abolished focal adhesion formation. All above indicate a pivotal role of SLK on the RhoA-ROCK axis during cell migration. Moreover, we found that ROCK inhibition significantly reduced cell coordination, which probably proceed through the regulation of adherens junctions during cell migration. Hence, SLK and ROCK may also interact with each other by regulating different cell migration modules (polarity vs. coordination.) Taken together, our results indicate that (1) SLK accumulates at the leading edge of migrating cells regulating cell migration polarity, that (2) SLK regulates the RhoA-ROCK axis during cell migration, and that (3) SLK may also interact with ROCK via modular collaboration (polarity vs. coordination.) Detailed mechanistic investigation is still under the way. Through these work we will elucidate how SLK interacts with ROCK signaling during cell migration and develop new pharmacological strategies to treat cell migration-related diseases. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T06:37:44Z (GMT). No. of bitstreams: 1 ntu-107-R05443017-1.pdf: 14092129 bytes, checksum: bbee20e8ad8d858595f695a10b9400df (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 誌謝 1
中文摘要 3 ABSTRACT 5 目錄 7 圖目錄 11 表目錄 13 Chapter 1 介紹 Introduction 14 1.1 細胞爬行之重要性與疾病發展 14 1.2 利用RNA interfering方式找尋細胞爬行之調控者 14 1.3 “Two-hit” shRNA screen: 找尋細胞爬行調控者與訊息傳遞鍊之關聯 14 1.4 SLK與ROCK signaling在細胞爬行上有趣的加乘作用 15 1.5 Ste20-like Serine/Threonine Kinase (SLK) 17 1.6 SLK與ROCK訊息傳遞鍊之關聯 19 1.7 研究目標: 探討SLK如何調控細胞爬行與SLK如何與ROCK訊息傳遞鍊在細胞爬行之交互作用 19 Chapter 2 材料與方法 Material and Methods 20 2.1 細胞培養 Cell culture 20 2.2 質體 Plasmid Constructs 20 2.3 脂質體轉染 Liposomal Transfection 22 2.4 慢病毒製造 Lentivirus Production 22 2.5 MTT試驗 22 2.6 Quantitative Polymerase Chain Reaction (QPCR) 22 2.7 西方墨點法 Western Blot 23 2.8 細胞遷移試驗 Migration Assay 23 2.9 免疫螢光染色 Immunofluorescence Assay 25 2.10 計算細胞核/膜 YFP訊號比值 25 Chapter 3 實驗結果 Results 26 3.1 SLK在不同細胞中的表現量 26 3.2 SLK knockdown使細胞爬行變慢 28 3.3 SLK over-expression對細胞爬行速度沒有顯著影響 32 3.4 細胞遷移的參數Fig. 8 32 3.5 SLK影響細胞極性使其失去方向 35 3.6 假說:SLK在細胞爬行時堆積在細胞前緣,適量增加提升極性、沒有SLK則使細胞喪失方向 39 3.6.1 重複免疫螢光染色試驗判斷是否SLK堆積在細胞前緣 39 3.6.2 以融合質體SLK-YFP fusion plasmid驗證假說 41 3.6.3 SLK-YFP與YFP-SLK兩種融合質體在細胞中出現不同分佈 43 3.6.4 裁減YFP-SLK融合質體中SLK C端60個氨基酸減少了YFP在膜的訊號累積 43 3.7 探討SLK與ROCK訊息鍊之交互作用 45 3.7.1 在SAS細胞中ROCK訊息抑制救回了SLK knockdown所造成的速度減少 45 3.7.2 ROCK訊息抑制減少細胞與細胞間的協調性 45 3.8 串連SLK與ROCK:SLK磷酸化RhoA再磷酸化下游ROCK? 49 3.8.1 我們未能抓到理想的p-RhoA西方墨點法條件 49 3.8.2 RhoA knockdown如同ROCK inhibition降低了細胞間的協調性 51 3.8.3 雙重knockdown RhoA與SLK:shSLK反而抵銷了shRhoA效果 51 3.9 修正ROCK與SLK互動假說:RhoA抑制SLK、SLK抑制ROCK調控細胞協調性 53 3.9.1 ROCK透過α-catenin訊息鍊影響細胞協調性 53 3.9.2 ROCK訊息抑制減少細胞邊緣focal adhesion 55 3.9.3 再次地shSLK抵銷了shRhoA的效果這次在focal adhesion上 56 Chapter 4 討論 Discussion 59 4.1 SLK堆積領導前緣引導細胞方向性假說:不同程度over-expression對方向性的影響 59 4.2 以SLK-YFP融合質體產生的YFP訊號位置強度驗證SLK調控細胞極性的假說 60 4.3 分析裁切版YFP-SLK-C2質體減少膜上YFP訊號累積之統計差異 60 4.4 找尋SLK domain、分佈位置與細胞遷移表現之關聯 61 4.5 傷口癒合試驗(wound-healing assay)與隨機細胞遷移試驗(random-migration assay)之差異與目的 61 4.6 有刮傷口與無傷口試驗對細胞遷移結果的影響 62 4.7 以SAS口腔鱗狀癌細胞株作為初步探索細胞遷移平台 62 4.8 SLK knockdown與ROCK抑制在不同細胞上具有不同效果 62 4.9 爬行速度並非SLK主要影響的參數 65 4.10 SLK over-expression時SLK量是否有真的被提昇 65 4.11 兩種SLK over-expression分析方法:純分析YFP表現細胞與整體細胞分析 65 4.12 不理想的phospho-RhoA西方墨點法條件 68 4.13 SLK為可能的RhoA/ROCK共同作用者 68 4.14 以SLK level rescue實驗檢驗SLK對細胞遷移效果 68 4.15 Focal ahesions之動態形成與異質性(heterogeneity) 69 4.16 ROCK抑制阻止focal adhesion生成、SLK knockdown破壞focal adhesion消失 69 4.17 RhoA knockdown與ROCK抑制對focal adhesion結果差異甚大 69 4.18 SLK, RhoA-ROCK對focal adhesion大小的影響 70 4.19 RhoA knockdown產生微小且為數眾多的focal complexes 70 4.20 RhoA之調控方式 71 4.21 使用全內角反射螢光顯微鏡(tirf)拍攝focal adhesion 71 4.22 SLK與ROCK細胞遷移之交互作用可能來自獨立的效果加乘 71 4.23 結論 72 Chapter 5 表格 Tables 73 Chapter 6 參考文獻 Reference 76 Chapter 7 附錄Appendix 79 7.1 Calcnuclei: 計算細胞核數目 79 7.2 Tracenuclei: 追蹤細胞爬行路徑 80 7.3 Getcellparameters: 計算細胞遷移參數 80 7.4 Calc_shRNA_speed: 將分析結果畫成bar圖 82 7.5 GetCTnum: 計算qPCR CT數 85 7.6 Getlevel: 畫出qPCR各組mRNA level 86 7.7 Analyze_cell_border: 手動圈細胞分析YFP在膜/質比 87 | |
dc.language.iso | zh-TW | |
dc.title | 利用單一細胞追蹤技術探索SLK與ROCK訊息鏈
在癌細胞遷移過程之交互作用 | zh_TW |
dc.title | Using single-cell tracing technique to elucidate SLK-ROCK interaction in cancer cell migration | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 賈景山,林耿慧,曾炳輝 | |
dc.subject.keyword | 細胞遷移,SLK,ROCK訊息傳遞鍊, | zh_TW |
dc.subject.keyword | Cell migration,SLK,ROCK signaling, | en |
dc.relation.page | 89 | |
dc.identifier.doi | 10.6342/NTU201803690 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-08-16 | |
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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