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研究生:陳資侑
研究生(外文):Tzu-You Chen
論文名稱:肌動蛋白去分枝因子GMF在黏菌細胞中發育及移動的探討
論文名稱(外文):The role of GMF, an actin debranching factor in Dictyostelium development and migration
指導教授:陳美瑜陳美瑜引用關係
指導教授(外文):Mei-Yu Chen
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:2014
畢業學年度:102
語文別:中文
論文頁數:52
中文關鍵詞:黏菌肌動蛋白去分枝因子
外文關鍵詞:Dictyosteliumactindebranching factor
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細胞移動參與在許多生理及病理過程,是個很重要的細胞行為。GMF蛋白質,屬於ADF/cofilinsuperfamily,會與Arp2/3 complex結合並且降低纖維肌動蛋白的分枝 (branching) 現象,也會使哺乳類細胞的隨機細胞移動速率降低。但是GMF是否會在in vivo中調控與細胞移動息息相關的纖維肌動蛋白以及它對於細胞移動時細胞偽足的影響為何仍然未知。黏菌是一個容易培養且操作的簡單真核生物,細胞移動對其生長發育也扮演重要腳色,因此我利用黏菌作為模型系統來探討GMF在細胞移動中的功能,策略是將gmfA基因剔除並觀察其對於黏菌的影響。首先發現gmfA-黏菌有發育缺陷,將gmfA送回gmfA-細胞則可以拯救發育缺陷。接著觀察隨機及有方向性的黏菌細胞移動,並分析其移動的速率和方向性,結果顯示gmfA-黏菌細胞相較於野生型黏菌細胞移動時的速率顯著降低,以外送質體回復gmfA-細胞中GMF的表現可以拯救其移動速率降低的表現型。進一步探討黏菌細胞內部纖維肌動蛋白與球狀肌動蛋白的平衡,以免疫螢光染色和Triton X-100 fractionation實驗分析,結果顯示纖維肌動蛋白與球狀肌動蛋白間的比例不受GMF存在與否的影響。接著再探討黏菌的纖維肌動蛋白動態是否因為GMF表現量不同而改變,由肌動蛋白聚合實驗結果發現過多或過少的GMF都可能會影響細胞纖維肌動蛋白的動態平衡。利用免疫螢光染色確認GMF與纖維肌動蛋白有局部的共同定位現象。並且,觀察纖維肌動蛋白在不同株黏菌細胞內的分佈,發現gmfA-細胞中的類絲狀偽足突起數目較野生型黏菌者減少;以放大倍率觀察細胞有方向性的爬行時的類絲狀偽足突起數目在gmfA-細胞中也是顯著減少。最後,為了探討上述所觀察到的現象是否與GMF和Arp2/3 複合體的結合有關係,將GMF中可能參與Arp2/3複合體結合的重要胺基酸做定點突變,發現突變的GMF無法拯救gmfA-細胞的發育缺陷,但尚需要進一步去證實這些點突變的確造成GMF與Arp2/3 複合體無法結合。綜合而論,本論文研究在黏菌系統中展示了GMF功能對於細胞偽足形成與細胞移動的重要性。
Cell migration is important in many physiological and pathogenic processes. It involves a series of molecular events including cell polarization, adhesion and force generation. Glia maturation factor (GMF), which belongs to the ADF/cofilin superfamily, is an actin debranching factor. Previous studies have indicated that GMF can directly interact with the Arp2/3 complex and inhibit its activity in actin nucleation. However, how GMF may regulate actin dynamics in vivo and affect the formation of membrane protrusions during cell migration is still unknown. In this thesis research, Dictyostelium discoideum was used as an experimental model system to elucidate the role of GMF in cell migration. The gmfA - null (gmfA-) cells formed smooth plaques on bacteria lawns,while produced slug when they were developed on the non-nutrient agar plate. Analysis of cell motility by random migration and micropipette assays revealed decreased speed of gmfA- cells, suggesting a function of GMF in regulating cell motility. Immunofluorescence staining showed that GMF partially colocalized with F-actin.Both gmfA- cells and gmfA- /pDM358-GMF cells (which overexpressed GMF) exhibited a slightly delayed actin polymerization peak in response to cAMP stimulation. Decreased filopodia-like protrusions formation was observed in gmfA- cells. Expressing gmfA eliminated abnormalities of gmfA- cells. To test if the the interaction between GMF and the Arp2/3 complex is important for the function of GMF, we generated three GMF mutants, each carrying substitution at an amino acid residue predicted to be involved in binding to the Arp2/3 complex; these three GMF mutants were not able to rescue the developmental defects of gmfA- cells. In summary, results from this thesis study have demonstrated the functional significance of GMF in pseudopod formation and cell migration in the Dictyostelium model system.
中文摘要 iv
Abstract vi
壹、 緒論 1
一、 Glia maturation factor 與 Arp2/3 complex 1
二、 細胞移動機轉與肌動蛋白細胞骨架重組的調控 2
三、 黏菌模型生物系統 3
四、 論文研究目標 4
貳、 實驗材料與方法 5
一、材料 5
二、方法 9
參、 實驗結果 19
一、gmfA剔除黏菌株的建立 19
二、觀察GMF對黏菌發育的影響 19
三、觀察GMF在不同黏菌發育階段的表現量 21
四、探討GMF與黏菌細胞移動的關係 21
五、探討GMF與黏菌細胞中肌動蛋白 (actin) 細胞骨架的關係 22
六、探討GMF對黏菌細胞偽足生成的影響 24
七、探討GMF蛋白中具功能重要性的位點 25
八、觀察表現哺乳類的GMF對黏菌細胞的影響 25
肆、 討論 27
一、GMF對纖維肌動蛋白動態的影響 27
二、 GMF影響黏菌細胞運動的可能機轉 27
三、 GMF突變黏菌細胞株與Arp2/3 複合體的交互作用 28
伍、 參考文獻 30
陸、附表 32
一、primer list 32
二、random migration analysis 33
三、micropipette assay 34
柒、附圖 35
一、gmfA基因剔除株的建立與鑑定 35
二、Ax2與候選gmfA剔除株細胞萃取物的西方墨點分析及純化的anti-GMF
免疫血清測試 36
三、gmfA- 突變株的發育表現型 37
四、表現GMF對gmfA-突變株發育表現行之影響 38
五、gmfA-突變株在不同黏菌發育階段的型態分析 39
六、GMF在黏菌不同發育階段的表現量 40
七、分析不同strains黏菌細胞移動路徑 41
八、分析不同strains黏菌細胞有方向性的移動路徑 42
九、Aggregation stage黏菌細胞GMF與F-actin的分佈位置 43
十、觀察AX2、gmfA-、gmfA- /pDM358-GMF黏菌細胞內部F-actin, G-actin
分佈 44
十一、觀察AX2、gmfA-、gmfA- /pDM358-GMF黏菌細胞內部F-actin, G-actin
分佈 45
十二、以Triton X-100 fractionation探討AX2、gmfA- 、gmfA- /pDM358-GMF
黏菌株F-actin, G-actin的比例 46
十三、Actin polymerization assay螢光讀值分析圖 47
十四、觀察AX2、gmfA-細胞內部纖維肌動蛋白分佈 48
十五、觀察AX2、gmfA-、gmfA- /pDM358-GMF filopodia-like protrusios數目
49
十六、site-directed mutagenesis將GMF位點突變,並且用西方墨點法確認
50
十七、利用Bacteria plate上的發育觀察發育現象 51
十八、西方墨點法確認GMF-beta在哺乳類細胞及黏菌細胞中的表現 52

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