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標題: | 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 Interactions between microtubule-actin crosslinking factor 1 (MACF1) and Ca2+ during cancer cell migration |
作者: | Ting-Yu Lin 林庭宇 |
指導教授: | 蔡丰喬(Feng-Chiao Tsai) |
關鍵字: | 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, Ca2+ signaling,microtubule-actin crosslinking factor 1 (MACF1),cytoskeleton,cell adhesion,cell migration, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70222 |
DOI: | 10.6342/NTU201803580 |
全文授權: | 有償授權 |
顯示於系所單位: | 藥理學科所 |
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