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標題: | LMBD1蛋白質參與小鼠胚胎纖維母細胞遷移之探討 Roles of LMBD1 protein in cell migration of mouse embryonic fibroblasts |
作者: | 李有容 Yu-Jung Lee |
指導教授: | 張明富 Ming-Fu Chang |
關鍵字: | Lmbrd1基因,細胞遷移,點狀黏著,小鼠胚胎纖維母細胞,纖網蛋白, Lmbrd1 gene,cell migration,focal adhesion,mouse embryonic fibroblast,fibronectin, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 細胞遷移對於形態發生、傷口癒合以及原腸胚形成 (gastrulation)非常重要。由Lmbrd1基因所表現之LMBD1蛋白質在小鼠胚胎發育過程中的原腸胚形成時期是必須的。本實驗室先前研究發現在小鼠神經母細胞瘤細胞株 (N2a)中,LMBD1蛋白質過度表現會造成細胞移動能力下降,並且在人類非小細胞肺癌細胞株 (H1299)中也發現LMBRD1基因被剔弱後可以促進細胞移動。細胞遷移過程中點狀黏著 (focal adhesion)的形成與瓦解是非常重要的步驟,而FAK以及SHP-2蛋白質的活性對於點狀黏著的成熟與瓦解非常重要。實驗室先前的研究結果發現將N2a與H1299細胞株中LMBRD1基因剔弱後,其FAK-Y397的磷酸化程度有提升的現象。除此之外,在N2a細胞株中Lmbrd1基因被剔弱會使LMBD1/FAK/SHP-2的蛋白質複合體瓦解。有研究指出LMBD1基因缺失的胚胎有中胚層形成異常的情形,與FAK及SHP-2基因缺失之胚胎有一樣的現象,並且FAK與SHP-2基因缺失的纖維母細胞其移動能力有下降的現象。這些現象指出LMBD1蛋白質可能在原腸胚形成的細胞遷移過程中扮演調控角色。本研究主要探討LMBD1蛋白質在小鼠胚胎纖維母細胞移動過程中所扮演的角色。結果顯示Lmbrd1+/-的細胞移動能力上升,並且也觀察到FAK與PI3K/Akt的訊息傳遞路徑受到活化。Lmbrd1+/-細胞初期貼附在poly-L-lysine與fibronectin細胞基質的能力優於野生型細胞,在fibronectin上的移動能力也較好。本研究為了進一步分析LMBD1與點狀黏著相關蛋白質之交互作用,利用GST-pull down assay進行分析。結果顯示LMBD1可藉由胺基酸位置220-300與FAK以及paxillin有交互作用。免疫螢光分析的結果也發現LMBD1與F-actin和paxillin有部份共位現象。而在免疫沉澱分析結果發現Lmbrd1+/-細胞中LMBD1/FAK蛋白質複合體的形成有下降的趨勢。綜合上述實驗結果推測LMBD1蛋白質可能作為支架蛋白調控點狀黏著的FAK與SHP-2之間的動態平衡,並且進一步調控細胞遷移。 Cell migration is important for morphogenesis, wound healing and gastrulation. The LMBD1 protein which is encoded by the limb region 1 (LMBR1) domain containing 1 gene (Lmbrd1) has been suggested to be essential for the gastrulation of mouse embryogenesis. Previous studies in our laboratory demonstrated that the cell migration of mouse neuroblastoma Neuro2a (N2a) cells was interfered with overexpression of LMBD1. In addition, the ability of cell migration was increased in human non-small cell lung carcinoma H1299 cells with LMBRD1 knockdown. Focal adhesion assembly/disassembly is critical for cell migration, and the activity of focal adhesion kinase (FAK) and SH2 domain-containig protein tyrosine phosphatase (SHP-2) is important for focal adhesion maturation and turnover. Our futher studies showed that phosphorylation level of FAK-Y397 was elevated in N2a and H1299 cells with LMBRD1 knockdown. Furthermore, the protein complex of LMBD1/FAK/SHP-2 was dissociated in N2a cells with Lmbrd1 knockdown. Others also showed aberrant mesoderm formation in LMBD1-deficient embryos similar to those FAK and SHP-2 knockout embryos. In addition, FAK-deficient and SHP-2-deficient fibroblasts showed retardation in cell motility. These suggest that LMBD1 protein plays a role in the regulation of cell migration during gastrulation. In this study, the role of LMBD1 protein involved in the cell migration of mouse embryonic fibroblasts (MEFs) was investigated. Results showed that the rate of cell migration was elevated in Lmbrd1+/- MEFs, and the activity of FAK and PI3K/Akt were also elevated. Lmbrd1+/- MEFs showed a better ability of initial adhesion on poly-L-lysine and fibronectin and increased the cell migration on fibronectin. To further analyze potential interactions between LMBD1 and focal adhesion associated proteins, GST-pull down assay was performed. Results demonstrated that LMBD1 interacted with FAK and paxillin and mainly through the domain from amino acid residues 220 to 300. Immunofluorscence staining showed that LMBD1 partially colocalized with F-actin and paxillin. In addition, immunoprecipitation data showed that the formation of LMBD1/FAK protein complex was decreased in Lmbrd1+/- MEFs. These results suggest that LMBD1 functions as a scaffold protein to regulate the dynamic of SHP-2 and FAK complex at focal adhesion site, and further, to regulate cell migration. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78964 |
DOI: | 10.6342/NTU201803448 |
全文授權: | 未授權 |
電子全文公開日期: | 2023-10-09 |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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