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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 | 蔡丰喬(Feng-Chiao Tsai) | |
dc.contributor.author | TIEN-TSAI LIN | en |
dc.contributor.author | 林天財 | zh_TW |
dc.date.accessioned | 2021-06-17T03:20:28Z | - |
dc.date.available | 2025-08-18 | |
dc.date.copyright | 2020-09-03 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-08-18 | |
dc.identifier.citation | 參考資料 1. Yang, Y., et al., Integrators of the cytoskeleton that stabilize microtubules. Cell, 1999. 98(2): p. 229-38. 2. Byers, T.J., et al., Novel actin crosslinker superfamily member identified by a two step degenerate PCR procedure. FEBS Lett, 1995. 368(3): p. 500-4. 3. Mridu Kapur., etal., Calcium tips the balance: a microtubule plus end to lattice binding switch operates in the carboxyl terminus of BPAG1n4. EMBO Rep., 2012 Nov 6;13(11):1021-9 4. Thomas R Lane., etal., Structure of the ACF7 EF-Hand-GAR Module and Delineation of Microtubule Binding Determinants. Structure, 2017 Jul 5;25(7):1130-1138.e6. 5. Jessica L. Maiers etal., ZO-1 recruitment to α-catenin – a novel mechanism for coupling the assembly of tight junctions to adherens junctions. Journal of Cell Science, 2013 Sep 1;126(Pt 17):3904-15. 6. Yanlei Ma etal., ACF7 regulates inflammatory colitis and intestinal wound response by orchestrating tight junction dynamics. Nature Communications 2017 May 25;8:15375. 7. Dongming Sun et al., Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): Identification of a novel group of microtubule associated proteins. Journal of Cell Science, 2001 Jan;114(Pt 1):161-172. 8. HAIXIA LI etal., Scutellarin inhibits cell migration by regulating production of αvβ6 integrin and E-cadherin in human tongue cancer cells. ONCOLOGY REPORTS, 2010 Nov;24(5):1153-60. 9. Kazuaki Umeda etal., ZO-1 and ZO-2 independently determine where claudins are polymerized in tight-junction strand formation. Cell, 2006 Aug 25;126(4):741-54. 10. Le Shen etal., Actin depolymerization disrupts tight junctions via caveolae-mediated endocytosis. Molecular Biology of the Cell, 2005 Sep;16(9):3919-36. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/69594 | - |
dc.description.abstract | 微管-肌動蛋白交聯因子1(MACF1)是與F-肌動蛋白和微管相互作用的細胞骨架交聯蛋白。在其N 端有兩個與Ca2+結合的EF hand,是否Ca2+會調控MACF1 與Microtubule 結合的相互作用仍然是未解的疑問。因此,我們探討細胞內和細胞外Ca2+如何分別調節細胞骨架重塑和細胞粘附。在細胞內,我們使用GFP 標籤和N 末端截短MACF1 蛋白MACF1 Tail(其EF hand 有各種突變或缺失)的螢光活細胞成像瞭解Ca2+如何調節MACF1 及微管間相互作用。EFhand 缺失的蛋白顯示MACF1 對微管尖端EB1 結合不須鈣離子調控。排除內生性MACF1 影響後,我們發現相較於微管lattice,MACF1 對微管尖端結構EB1具有較高親和力。在不同的Ca2+濃度下,MACF1 Tail 顯現微管結構間的結合轉移。在細胞外部分,隨機爬行試驗表明敲除癌細胞中的MACF1 除了能降低速度外,還可以增強細胞間的協調性。我們發現MACF1 藉由αE-catenin 影響細胞間的協調性。而粘附連接adherens junctions 在MACF1 調節細胞間的協調性卻並非主要的角色。連同文獻報導,抑制MACF1 能藉由微管調控影響緊密連接蛋白Tight Junction 的動態變化,我們假設MACF1 直接透過細胞骨架影響Tight Junction 的動態變化達到調節細胞間的協調性。通過這些方法,我們將闡明MACF1 如何調節細胞骨架和細胞黏附,希望在不久的將來開發出針對EMT和/或黏附相關疾病的新穎治療策略。 | zh_TW |
dc.description.abstract | Interactions between microtubule-actin crosslinking factor 1 (MACF1) and cell junction during cancer cell migration Microtubule-actin crosslinking factor 1 (MACF1) is a cytoskeletal crosslinking protein that interacts with F-actin and microtubules. Although there are two putative Ca2+-binding EF hands at its C-terminus, whether and how Ca2+ interacts with MACF1 remains elusive. We thus explored how intracellular and extracellular Ca2+ regulated cytoskeletal remodeling and cell adhesion respectively. In the intracellular aspect, we studied how Ca2+ regulated MACF1-microtubule interaction using GFPtagged N terminus truncated MACF1 proteins with various mutations or deletions on their EF hands. SAS cells expressing MACF1 tail with or without EF-truncation showed similar distribution patterns in microtubule lattice and microtubule tips, indicating that EF-hand might not be mandatory for the binding of MACF1 to microtubule tip. However, affinities of MACF1 to microtubule tips might be higher than that to microtubule lattices. Moreover, the transfer of MACF1 binding from microtubule tips to microtubule lattices might depend on Ca2+. In the extracellular part, our random migration assays demonstrated that MACF1 knockdown in cancer cells increased cell-cell coordination in addition to their speed reduction. Interestingly, α-Ecatenin knockdown abolished above effect of MACF1 on cell-cell coordination. Our MACF1-CDH1 double knockdown experiment revealed that adherens junctions might not contribute to the effect of MACF1 on cell-cell coordination. Together with literature reports that MACF1 knockdown reduced tight junction dynamics through microtubule, we hypothesized that MACF1 induced tight junction internalization via directly switching the balance from cell-cell adhesion to cell-matrix adhesion. To verify this hypothesis, we are currently using immunofluorescence to examine how MACF1 alters the dynamics of cell-cell adhesion. With these approaches we will elucidate how MACF1 regulate cytoskeletons and cell adhesion, with the hope to develop novel therapeutic strategies against EMT and/ or adhesion-related diseases in the near future. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T03:20:28Z (GMT). No. of bitstreams: 1 U0001-1808202011164300.pdf: 5355120 bytes, checksum: 99358f718bec58a1b5b5591ce808da7d (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | 致謝................................................................................................................................ 1 中文摘要........................................................................................................................ 2 Abstract .......................................................................................................................... 3 圖目錄............................................................................................................................ 6 第一章、緒論................................................................................................................ 7 1.1微管-肌動蛋白交聯因子1(microtubule-actin crosslinking factor 1,MACF1)在細胞爬行中串聯微小管與黏著分子 ................................................. 7 1.2 鈣離子可能透過MACF1上EF hand影響功能 ............................................. 8 第二章、實驗動機........................................................................................................ 9 2.1 MACF1中EF hand受鈣離子調控對Microtubule結合的影響 ..................... 9 2.2 MACF1對細胞爬行的影響 ............................................................................ 10 當MACF1被抑制時,細胞爬行速度下降 .................................................. 10 第三章、材料與方法.................................................................................................. 14 3.1 細胞培養 ........................................................................................................ 14 3.2 細胞遷移試驗(Cell Migration Random Migration) ....................................... 14 3.3 質體製備 ........................................................................................................ 14 3.4 轉染(Transfection) ......................................................................................... 15 3.5 活細胞拍攝 .................................................................................................... 16 3.6 包病毒測MOI ................................................................................................ 17 第四章、 結果.......................................................................................................... 18 4.1 MACF1結合上Microtubule Tip EB1不需EF hand協助 .............................. 18 MACF1會結合上Microtubule的EB1 .......................................................... 18 MACF1 Tail with EF Truncate會結合上Microtubule的EB1 ....................... 18 4.2 MACF1 Tail對EB1結合力較Microtubule lattice高 .................................... 22 MACF1 Tail依表現量高低有兩種型態 ......................................................... 22 MACF1 Tail對Microtubule EB1結合力較Microtubule lattice高 ............... 23 4.3 Full-length MACF1在細胞內鈣離子提升後會從EB1轉移Microtubule lattice ..................................................................................................................... 27 MACF1在細胞內鈣離子提升後會從EB1轉移Microtubule lattice ........... 27 MACF1 Tail在細胞內鈣離子提升後不會從EB1轉移Microtubule lattice . 31 MACF1 tail加入ionomycin與CaCl2提高細胞內鈣離子後,相較於加藥前可見細胞邊緣處Microtubule Tip端有comet,加藥後在Tip端仍有Comet,經計算Microtubule Tip末端Tip與定量結果,藍色是加藥前,紅色是加藥後,加藥前後Tip端的Comet沒有明顯變化(圖15)。 ......... 31 MACF1 Tail在細胞內鈣離子下降後結合EB1更明顯 ................................. 35 4.4 MACF1藉由α-Ecatenin調控細胞爬行 ......................................................... 36 shCTNNA1抑制α-Ecatenin ........................................................................... 36 shCTNNA1有效減少shMACF1時所增加的Cell-Cell Coordination ............ 36 4.5 MACF1調控α-Ecatenin蛋白質表現 ............................................................. 40 shMACF1抑制α-Ecatenin distribution in Immunofluorescence .................. 40 shMACF1不影響α-Ecatenin mRNA in qPCR ................................................. 42 shMACF1減少α-Ecatenin的蛋白質量 ........................................................ 42 4.6 MACF1並非藉由adherens junction調控細胞Coordination....................... 43 shCDH1抑制E-cadherin ................................................................................ 44 shCDH1無法有效減少shMACF1時所增加的Cell-Cell Coordination ......... 44 shCTNNB1抑制β-catenin .............................................................................. 46 shCTNNB1無法有效減少shMACF1時所增加的Cell-Cell Coordination .... 47 4.7 MACF1可能藉由Tight Junction調控細胞Coordination ............................. 49 shTJP1抑制ZO-1 ........................................................................................... 49 第五章、討論.............................................................................................................. 51 5.1 MACF1與Microtubule結合關係是否受鈣離子調控 .................................. 51 5.2 MACF1 Tail對EB1結合力較Microtubule lattice高 .................................... 52 5.3 MACF1對Coordination調控並非由Adherens Junction影響 ..................... 52 5.4 MACF1是否藉由Tight Junction調控Cell-Cell Coordination ....................... 53 5.5 MACF1對ZO-1調控是否藉由Actin影響 .................................................... 54 5.6 去除α-Ecatenin是否完全破壞Cell-Cell Junction影響coordination ......... 54 5.7 Full-length MACF1與MACF1 Tail型態不同.................................................. 54 附錄.............................................................................................................................. 55 Construct Map ....................................................................................................... 55 Map 1、 EGFP-MACF1tail ............................................................................. 55 Map 2、EGFP-MACF1tail EF hand truncate ................................................... 56 Map 3、 mCherry-MACF1 Cas9 gDNA .......................................................... 56 Map 4、 mCherry-MACF1 Donor template gDNA ........................................ 57 表 2、shRNA序列。 ........................................................................................... 58 Script ............................................................................................................................ 60 參考資料...................................................................................................................... 67 | |
dc.language.iso | zh-TW | |
dc.title | 微小管和動肌蛋白交聯因子(MACF1)在細胞爬行中與細胞動態連結的交互作用 | zh_TW |
dc.title | Interactions between microtubule-actin crosslinking factor 1 (MACF1) and cell junction during cancer cell migration | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳壁彰(Bi-Chang Chen),蔡金吾(Jin-Wu Tsai),張智芬(Zee-Fen Chang) | |
dc.subject.keyword | 微小管和動肌蛋白交聯因子(MACF1), | zh_TW |
dc.subject.keyword | microtubule-actin crosslinking factor 1 (MACF1), | en |
dc.relation.page | 67 | |
dc.identifier.doi | 10.6342/NTU202003935 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2020-08-19 | |
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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