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系統識別號 U0026-2808201301514600
論文名稱(中文) 以影像呈現探究細胞中磷酸水解酶PP2A催化次單元及與其有交互作用的蛋白分子所形成的複合體
論文名稱(英文) Visualizing the Complexes Formed by Protein Phosphatase 2A Catalytic Subunit (PP2Ac) and its interacting protein in cells
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 101
學期 2
出版年 102
研究生(中文) 鄭侑玲
研究生(英文) Yu-Ling Cheng
學號 T16004027
學位類別 碩士
語文別 英文
論文頁數 82頁
口試委員 指導教授-蔣輯武
口試委員-張玲
口試委員-何中良
口試委員-鄭宏祺
中文關鍵字 蛋白質磷酸酶  雙分子螢光互補法  能量共振轉移法 
英文關鍵字 PP2A  MID1  Alpha4  TIP  BiFC  FRET 
學科別分類
中文摘要 蛋白質磷酸酶2A型(以下簡稱PP2A)屬於一種絲氨酸/蘇氨酸磷酸酶,參與了許多細胞內重要機制的調控,包含: 細胞週期的進行、細胞內訊息的傳遞及細胞骨架的動態等。典型的PP2A主要是由一個36-kDa的催化次單元(PP2Ac)及一個65-kDa的結構次單元(PP2A/A)作為核心,再加上具有調節功能的B次單元(PP2A/B)所組成的三元複合體。然而在細胞中除了A與B次單元外,PP2Ac已被發現會與其他蛋白,例如Alpha4 (α4)、TIP 和MID1等產生交互作用而形成非典型PP2A複合體,以對PP2A的活性進行嚴密的調控。在本研究中,我們首先運用了間接免疫染色法及直接於螢光顯微鏡下觀察螢光融合蛋白的方法觀察到在NIH3T3細胞中短暫表現PP2Ac時,PP2Ac在細胞各處皆有表現,但主要仍是以細胞質為最多。短暫表現α4時, 則可以觀察到α4在細胞中主要呈現均勻分佈。短暫表現TIP時,有較高的比例是分佈於細胞質當中。而MID1是微管結合蛋白(microtubule-associated protein),短暫表現於細胞中會呈現絲狀分佈。接著,我們運用雙分子螢光互補法(BiFC)的方式來探討PP2Ac在細胞中與α4、 TIP及MID1間的交互作用。由PP2Ac與α4所形成的複合體是呈現以細胞質及均勻分佈為主。而由PP2Ac與TIP所形成的複合體則是以均勻分佈為最多。由MID1與α4所形成的複合體會呈現兩種分佈型態:一為與微管相類似的絲狀分佈,或者會在細胞質中形成點狀聚集。此外,α4的存在可以促進PP2Ac與MID1間形成複合體,並且呈現點狀聚集分佈在細胞質中,而與PP2Ac結合有缺陷的α4突變型(α4MUT)則無法促進PP2Ac與MID1間的交互作用。有趣的是,我們也發現了MID1與TIP間會形成與MID1/α4類似表現型態的複合體。進一步我們也運用了免疫沉澱法驗證在BiFC方法中所觀察到的現象。最後,我們結合了BiFC與能量共振轉移法(FRET)觀測到PP2Ac、α4及MID1間確實會以三聚體的型式存在於細胞當中,並且是以點狀聚集的型態坐落在細胞質,而與PP2Ac結合有缺陷的α4突變型(α4MUT)就無法扮演轉接蛋白(adaptor protein)的角色,因此PP2Ac、α4MUT及MID1間就無法以三聚體型式存在。總結來說,我們成功運用了BiFC及BiFC結合FRET的系統來觀察細胞中非典型PP2A複合體的存在與分佈型態,同時這樣的系統也提供了我們將來能對於非典型PP2A複合體間的調節與動態變化進行更進一步的探討。
英文摘要 Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase involved in regulating essential cellular functions in eukaryotic cells. PP2A consists of a heterodimeric core enzyme, including a scaffolding subunit (PP2A/A) and a catalytic subunit (PP2Ac), and a variable regulatory subunit (PP2A/B). In addition to PP2A/A and PP2A/B subunits, several cellular proteins have been identified to interact with PP2Ac such as Alpha4 (α4), TIP and MID1, and have been shown to play an important role in regulating PP2A activity. By applying both indirect immunofluorescence (IF) and direct fluorescence microscopy, we showed that PP2Ac was mainly expressed in the cytoplasm, whereas α4 was distributed throughout the entire cells. In addition, TIP was located at cytoplasm, and MID1 was predominantly associated with microtubules. Next, we used bimolecular fluorescence complementation (BiFC) analysis to investigate the complexes formed by PP2Ac and its binding partners. Results of BiFC analysis showed that the distribution of PP2Ac/α4 complexes was either homogenous or mainly cytoplasmic, but the PP2Ac/TIP complexes were mainly homogenous. BiFC signals of the PP2Ac/MID1 complexes formed aggregations in the cytoplasm, which were only detected in the presence of wild-type α4, but not a mutant α4 defective in binding to PP2Ac. BiFC signals of the MID1/α4 complexes displayed both microtubule-like threads and cytoplasmic clumps. Intriguingly, we detected BiFC signals of the MID1/TIP complexes, which have not been shown to form complexes and displayed a distribution pattern similar to that of the MID1 and α4 complexes. The results of co-immunoprecipitation (Co-IP) analysis confirmed the interaction between MID1 and TIP. Next, by applying BiFC-based Fluorescence Resonance Energy Transfer (BiFC-based FRET) analysis, the trimeric complexes formed by PP2Ac, α4 and MID1 were successfully visualized in cells. However, the trimeric complexes of PP2Ac, MID1 and mutant α4 defective in binding to PP2Ac were not observed. In summary, we have successfully applied BiFC and BiFC-FRET to visualize the non-canonical complexes of PP2Ac and revealed novel MID1/TIP complexes in cells. Applications of the system for visualizing these PP2Ac-related complexes may provide more insights into the regulation and dynamics of these complexes in living cell.
論文目次 中文摘要
Abstract
List of contents .........................................................................................................................I
List of Figures .......................................................................................................................III
List of Tables ........................................................................................................................ V
List of Abbreviations......................................VI
Introduction .........................................................................................................................1
Reverse phosphorylation regulates cellular activities..................................................2
Protein phosphatase 2A (PP2A) .........................................................................................................................2
The canonical PP2A holoenzyme .........................................................................................................................2
The non-canonical PP2A subunit: Alpha4 (α4) ............................................................3
The ubiquitin E3 ligase:
MID1.....................................................................................................................4
The non-canonical PP2A subunit: TIP......................................................................................................................6
Bimolecular Fluorescence Complementation (BiFC) and Fluorescence Resonance Energy Transfer (FRET)...................................................................................................................6
Materials and Methods .........................................................................................................................9
Antibodies and sepharoses ........................................................................................................................10
Materials...............................................................................................................11
Western Blotting ........................................................................................................................11
Cell culture ........................................................................................................................13
Immunefluorescence......................................................................................................14
Inhibitors ........................................................................................................................14
Methods.................................................................................................................15
Cell culture.................................................................................................................15
Transfection by Lipofectamine 2000 (Invitrogen) ...........................................................15
Immunofluorescence (IF) ........................................................................................................................16
Immunopreicipitation (IP) ........................................................................................................................17
Establishing BiFC expression constructs and performing BiFC analysis...................................................17
Establishing Cyan Fluorescence Protein (CFP)-fused expression
constructs and performing BiFC-based FRET analysis ...........................................................18
Results ........................................................................................................................25
Investigating subcellular localization of PP2Ac and its binding proteins...........................................26
Investigating the interaction between α4, MID1 and PP2Ac by BiFC analysis..............................................28
Investigating the interaction between MID1 and TIP........................................................29
Visualizing association and localization of the ternary PP2Ac/α4/MID1
complex by BiFC based-FRET analysis ...........................................................30
Conclusion ........................................................................................................................31
Discussion ........................................................................................................................32
Visualization of the subcellular localization of non-canonical PP2A complexes
by BiFC and BiFC-based FRET assay ...........................................................33
Alpha4 protein, a scaffold of PP2Ac to interact with MID1.......................................................34
MID1/TIP, a novel complex? ...........................................................35
References ........................................................................................................................37
Figures ........................................................................................................................42
Tables ........................................................................................................................62
Appendix ........................................................................................................................66
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