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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡丰喬(Feng-Chiao Tsai) | |
| dc.contributor.author | Ting-Yu Lin | en |
| dc.contributor.author | 林庭宇 | zh_TW |
| dc.date.accessioned | 2021-06-17T04:24:27Z | - |
| dc.date.available | 2023-10-03 | |
| dc.date.copyright | 2018-10-03 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-15 | |
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Walsh, and H.J. Vogel, Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs. Biochem J, 2007. 405(2): p. 199-221. 33. Yap, K.L., et al., Diversity of conformational states and changes within the EF-hand protein superfamily. Proteins, 1999. 37(3): p. 499-507. 34. Zhang, J., J. Yue, and X. Wu, Spectraplakin family proteins - cytoskeletal crosslinkers with versatile roles. J Cell Sci, 2017. 130(15): p. 2447-2457. 35. Kapur, M., et al., Calcium tips the balance: a microtubule plus end to lattice binding switch operates in the carboxyl terminus of BPAG1n4. EMBO Rep, 2012. 13(11): p. 1021-9. 36. Applewhite, D.A., et al., The actin-microtubule cross-linking activity of Drosophila Short stop is regulated by intramolecular inhibition. Mol Biol Cell, 2013. 24(18): p. 2885-93. 37. Lane, T.R., E. Fuchs, and K.C. Slep, Structure of the ACF7 EF-Hand-GAR Module and Delineation of Microtubule Binding Determinants. Structure, 2017. 25(7): p. 1130-1138.e6. 38. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70222 | - |
| dc.description.abstract | 近年來的研究已發現局部波動性鈣離子訊號在細胞遷移和癌症轉移中都扮演了重要的角色，但鈣離子影響細胞遷移與癌症進展的機制目前還不清楚。我們因此想藉由對微小管和肌動蛋白交聯因子1 (microtubule-actin crosslinking factor 1, MACF1)的研究來探討鈣離子的作用。MACF1在結構上會連接微小管(microtubule)與肌動蛋白(actin)，因此能維持細胞型態，並在細胞爬行的過程中串聯微小管與黏著分子(focal adhesion)幫助細胞具方向性的移動。因為MACF1結構上具有能夠與鈣離子結合的EF hand片段，使我們推測鈣離子可能藉由與MACF1結合進而影響到MACF1對細胞遷移與黏著分子的調控作用，因此我們想要研究鈣離子與MACF1在癌細胞遷移時的交互作用。
首先我們發現口腔鱗狀上皮細胞癌細胞(SAS)中的MACF1表現量需被抑制達九成才會明顯降低細胞遷移的速度，而同時抑制MACF1與CTNNA1、Rac1、paxillin 和MLCK其中一種肌動蛋白調節因子的功能，也凸顯了MACF1連接微小管與肌動蛋白，對於細胞遷移與細胞骨架重塑的重要性。接下來我們經由調控MACF1的表現量與改變細胞內外的鈣離子濃度，證實鈣離子會藉由MACF1影響細胞遷移。因為MACF1過大使轉染效率極差，因此我們製作了MACF1 C端包含EF hand至與微小管及微小管正端追蹤蛋白EB1結合區域的質體，並觀察在細胞內鈣離子濃度增加的情況下，MACF1片段分布位置的變化，同時我們也將其上兩段EF hand分別突變進行觀察，並且提出了MACF1與鈣離子交互作用的模型。 我們綜合先前研究與實驗結果，推測MACF1原先捲曲關閉並與EB1結合的構型，在鈣離子結合後會解開變成開放的構型並且轉而接上微小管，而其中鈣離子與EF1片段的結合會幫助MACF1結合到微小管上。EF1及EF2片段則可能調節MACF1形成二聚體(dimer)或低聚物(oligomer)的功用，鈣離子結合到EF hand片段會抑制MACF1形成二聚體或低聚物，進一步過度表現MACF1 C端進行細胞遷移實驗的結果也符合我們的假設。我們目前正進一步驗證鈣離子與MACF1交互作用模型的正確性，同時期許在釐清鈣離子與MACF1之間的交互作用對癌細胞遷移的影響後，未來能藉由操縱MACF1橋樑的角色用於臨床治療。 | zh_TW |
| dc.description.abstract | Ca2+ signaling plays an important role in cell migration and cancer metastasis, but how Ca2+ coordinates with other structural components to regulate cell migration machinery and cancer progression remains unclear. Searching for novel Ca2+ regulatory molecules and discovering the interaction between Ca2+ and these molecules may help us understand how Ca2+ controls proper cell motility. The microtubule-actin crosslinking factor 1 (MACF1), also called actin cross linking factor 7 (ACF7), bridges microtubule and actin to shape the morphology of cells and sustain directional cell movement. The existence of Ca2+ binding EF hand motif in MACF1 suggests that Ca2+ regulates MACF1 to control cell migration. We therefore study how Ca2+ interacts with MACF1 to regulate cell migration and cancer metastasis.
To validate the effect of MACF1 on cell migration, we suppressed MACF1 in SAS, a head and neck squamous cell carcinoma cell line, and conducted scratch wound healing assays. We first noticed that MACF1 knock down decreased cell motility only when it was almost completely lost. We next investigated how MACF1 changed cell migration machinery by inhibiting both MACF1 and one of the actin- based structure modulating molecules α-catenin, Rac1, paxillin and myosin-light chain kinase. These double knockdown experiments revealed that MACF1 was linked to all above actin modulating molecules, indicating the importance of actin-microtubule binding on cytoskeletal remodeling. We confirmed that Ca2+ regulates MACF1 to control cell migration by changing the cytosolic Ca2+ concentration. Then we constructed and overexpressed the C-terminus of MACF1 and its EF hand mutant and observed the localization change in the presence of high Ca2+. Based on the previous studies and our results, we purposed the model that, in the absence of Ca2+, MACF1 is in the closed conformation which binds to EB1 and other MACF1 to form dimer or oligomer. After Ca2+ binding, MACF1 turn into the open conformation binding to microtubule and loses the ability to bind to other MACF1. We are currently validating the model of interaction between Ca2+ and MACF1, with the hope to develop new Ca2+-MACF1 targeting therapies against cancer metastasis. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T04:24:27Z (GMT). No. of bitstreams: 1 ntu-107-R05443010-1.pdf: 3280828 bytes, checksum: fb19c8fe4229aa6651a2fa5d736b5867 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 誌謝 1
中文摘要 2 ABSTRACT 3 目錄 5 圖目錄 7 表目錄 8 第一章、 緒論 9 1.1 微小管和肌動蛋白交聯因子1 (microtubule and actin cross-linking factor 1， MACF1)會在細胞爬行過程中串聯微小管與黏著分子 9 1.2 鈣離子也會影響細胞爬行和黏著分子的動態變化 12 1.3 鈣離子可能透過結合到MACF1上的EF hand而影響它的功能 13 第二章、 材料與方法 14 2.1 細胞培養 14 2.2 RNA與cDNA製備 14 2.3 定量即時聚合酶鏈鎖反應(Quantitative real time polymerase chain reaction，qPCR) 15 2.4 細胞遷移實驗 (Migration assay) 15 2.5 質體製備 16 2.6 轉染(Transfection) 16 2.7 慢病毒(Lentivirus)製作及感染 16 2.8 活細胞攝影與ionomycin處理 17 第三章、 結果 18 3.1 人類口腔鱗狀細胞癌細胞株(SAS) MACF1表現量高 18 3.2 MACF1對SAS細胞遷移速度的影響 20 當MACF1被抑制達九成時會顯著降低細胞遷移的速度 20 MACF1轉染效率差尚無法釐清過度表現對細胞遷移的影響 23 3.3 MACF1連接肌動蛋白與微小管在細胞骨架重塑的重要性 25 3.4 鈣離子會透過MACF1影響細胞遷移 28 抑制MACF1使EGTA增加細胞遷移速度的效果消失 28 雙重調控MACF1與STIM1顯示鈣離子經由MACF1影響細胞遷移 28 抑制MACF1使EGTA AM降低細胞遷移速度的效果減弱 29 抑制MACF1不會改變thapsigargin的效果 29 3.5 鈣離子的結合改變MACF1與MACF1 C-terminus的功能 32 製作MACF1 C-terminus與其EF hand mutant的質體 32 鈣離子改變MACF1 與MACF1 C-terminus的分布位置 35 過度表現EGFP-MACF1tail增加細胞遷移速度 38 鈣離子與MACF1交互作用的模式圖 38 第四章、 討論 40 4.1 MACF1的抑制效率多寡影響細胞遷移實驗的結果 40 4.2 雙重抑制MACF1與CTNNA1、Paxillin與Rac1的效率 40 4.3 藥物或基因調控改變鈣離子濃度對MACF1的影響 41 4.4 局部鈣離子訊號與MACF1相關細胞骨架組成物質的動態變化 43 4.5 驗證鈣離子與MACF1交互作用模式圖 45 附錄 46 Matlab scripts 48 參考資料 74 | |
| dc.language.iso | zh-TW | |
| dc.title | 微小管和肌動蛋白交聯因子1(MACF1)與鈣離子在癌細胞遷移時的交互作用 | zh_TW |
| dc.title | Interactions between microtubule-actin crosslinking factor 1 (MACF1) and Ca2+ during cancer cell migration | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 湯銘哲(Ming-Jer Tang),張智芬(Zee-Fen Chang),陳壁彰(Bi-Chang Chen) | |
| dc.subject.keyword | 鈣離子訊息傳遞,微小管和肌動蛋白交聯因子1 (MACF1),細胞骨骼,細胞黏著,細胞遷移, | zh_TW |
| dc.subject.keyword | Ca2+ signaling,microtubule-actin crosslinking factor 1 (MACF1),cytoskeleton,cell adhesion,cell migration, | en |
| dc.relation.page | 76 | |
| dc.identifier.doi | 10.6342/NTU201803580 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-08-15 | |
| 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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