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研究生:陳羽真
研究生(外文):Yu-Chen Chen
論文名稱:探討移動中骨癌細胞黏著斑組成變化之調控機制
論文名稱(外文):Characterization of the regulatory mechanism of focal adhesion organization in a migrating osteosarcoma cell
指導教授:郭津岑
指導教授(外文):Jean-Cheng Kuo
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:55
中文關鍵詞:黏著斑細胞移動酪氨酸激酶肌凝蛋白
外文關鍵詞:focal adhesioncell migrationtyrosine kinasemyosin
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本研究主要在探討負責細胞黏著的結構 - focal adhesions中蛋白組成如何改變以影響骨肉瘤細胞的移動。我們主要針對focal adhesions中具有SH2 domain的kinase – Src 、具有SH2 domain的phosphatases – SHP1及SHP2、和actin-binding protein - NMIIA,探討在細胞移動時其進入focal adhesions的機轉。我們發現Src及SHP2的SH2 domain是帶領其酵素進入focal adhesions的重要domain,而其中Src的SH2 domain會受到FAK活性及表現以調節其進入focal adhesions的程度。另外我們也進一步的發現抑制Src進入focal adhesion會影響Src下游的訊息傳遞而阻礙focal adhesion的形成,甚至是影響到細胞的移動力。在NMIIA的部分,更發現其進入focal adhesions是受到small GTPase Rac1的調控。由於我們先前已經證實其進入focal adhesions並不是透過Rac-mediated actin polymerization所造成,所以我們將更進一步的以蛋白質體的方式分析NMIIA上磷酸化的位點。
The dynamic organization of the cellular adhesive organelles, focal adhesions (FAs), is linked to drive constitutive cell migration. To determine how FA composition is dynamically organized, we focused on SH2 domain-containing kinase, Src, SH2-domain containing phosphatases, SHP1 and SHP2, and actin-binding protein, non-muscle myosin II heavy chain-A (NMIIA). In the studies of SH2 domain-containing kinases / phosphatases, we found that the SH2 domain of Src plays a key role in FA targeting of Src in a FAK-dependent manner. In addition, inhibition of targeting Src into FAs significantly down-regulates Src-mediated FA signaling, suppresses FA formation, and influences cell migratory behaviors. In the studies of NMIIA, we found that NMIIA is recruited to FAs in a Rac1-controlled manner, but it is not accompanied by Rac1-mediated actin polymerization. Therefore, we performed proteomic analysis to develop the phosphorylation profile of NMIIA and NMIIA’s interactome for further determination in the future.
CONTENTS
中文摘要 I
ABSTRACT II
CONTENTS III
INTRODUCTION 1
Cell Migration: A Physically Integrated Molecular Process 1
Enzyme modulates focal adhesion dynamics and turnover in migrating cells 2
Protein tyrosine phosphatase regulates FAs disassembly and migration 5
Non-muscle myosin II plays a critical role in FAs and cell migration 6
MATERIALS AND METHODS 8
RESULT 16
SHP2 NSH2 and Src SH2 domain are localizd in focal adhesions 16
FA abundance of Src is enhanced by FAK activity via its SH2 domain 17
The recruitment of Src SH2 to FAs is influenced by FAK activity, but not by Src activity 18
The localization of Src to FAs is disrupted by loss SH2 domain 19
Src and SrcY527F recruited to FAs are dependent on FAK activity 20
Src(SH2) protein turnover faster within FAs than SrcY527F 21
Src SH2 domain deletion mutant had significantly defect in cell mobility 21
Proteomic analysis to develop the phosphorylation profile of NMIIA on different FAs stage by mass spectrometry 22
DISCUSSION 24
Role of Src-FAK complex in focal adhesions 24
Role of Src SH2 domain in cell migration 25
REFERENCE 27
FIGURES AND LEGENDS 35
Figure 1. SHP2(NSH2) and Src(SH2) are co-localize with paxillin at focal adhesions. 35
Figure 2. FA abundance of Src is enhanced by FAK activity via its SH2 domain 37
Figure 3. The expression of GFP-tagged SH2 is not influenced by Src or FAK inhibitor. 39
Figure 4. The recruitment of Src(SH2) to focal adhesion is decrease in FAK-/- cells. 40
Figure 5. The recruitment of SHP2(NSH2) and Src(SH2) to focal adhesion is not influenced in Src-8T and SYF cells. 42
Figure 6. Src SH2 domain plays a critical role in directing Src to FAs. 43
Figure 7. The expression of GFP or GFP-tagged Src is not influenced by FAK inhibitor. 45
Figure 8. Loss of Src SH2 domain had significantly defect in cell mobility. 46
Figure 9. Proteomic analysis to develop the phosphorylation profile of NMIIA on different FAs stage by mass spectrometry. 48
TABLES 49
Table 1. Primary antibody information 49
Table 2. Primers for cloning 50
Table 3. Primers for cloning 51
Table 4. SH2 domain sequence 52
Table 5. Src sequence 53

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