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標題: | 果蠅卵母細胞中dDcp1-dDcp2A-dGe-1形成複合體以及dDcp2A影響肌動蛋白行為的功能分析 dDcp1-dDcp2A-dGe-1 form a complex and the analysis of dDcp2A function in F-actin behavior in the Drosophila oocyte |
作者: | Po-Hsun Chiang 江柏勳 |
指導教授: | 周子賓(Tze-Bin Chou) |
關鍵字: | dDcp2,dDcp1,dGe-1,果蠅卵母細胞,osk,肌動蛋白, dDcp2,dDcp1,dGe-1,Drosophila oocyte,osk,actin, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 中文摘要
在果蠅卵母細胞中,oskar (osk)訊息核醣核酸(mRNA)座落在卵母細胞後端以決定胚胎的體軸 (axial determination) 和極細胞(pole cells)的發育。 osk基因突變會造成果蠅胚胎產生腹節缺失的後端突變性狀(posterior group phenotype)以及極細胞缺失。 osk訊息核醣核酸複合體 (osk mRNP) 在護理細胞(nurse cells)生成,微管運輸蛋白Kinesin協助osk mRNP經微管運送到卵母細胞後端,形成新月狀(crescent)堆積; osk mRNP座落到卵母細胞後端之前,osk mRNA轉譯(translation)是被抑制且直到胚胎發育早期才進行mRNA降解(mRNA degradation)。 現今關於osk mRNP座落的假說中,轉譯後的Osk蛋白質在卵母細胞後端建立一個正向回饋(positive feedback)機制,經由F-肌動蛋白絲狀物(F-actin projections)的產生以固著更多的osk mRNP。 降解體(Processing body)是在細胞質中負責降解、儲存mRNA和抑制mRNA轉譯的蛋白質複合體,去頭蓋蛋白質1 ( decapping protein 1, Dcp1)是降解體的主要成員之一。已知果蠅的去頭蓋蛋白質1 (Drosophila decapping protein 1, dDcp1)是osk mRNP的成員並且參與osk mRNP座落到卵母細胞後端。降解體中的dDcp1出現在osk mRNP上的意義是我們研究的目標。 dDcp1 調控卵發育(oogenesis)中osk mRNP的座落,其他降解體的成員亦可能是osk mRNP座落所需。已知在人類細胞和植物細胞中Dcp1會跟另外兩個Processing body的主要成員:去頭蓋蛋白質2 (decapping protein 2, Dcp2)和人類促進去頭蓋大分子 (Human enhancer of decapping large subunit, Hedls,又稱Ge-1 (Gougerot syndrome)) 形成一個複合體,果蠅中dDcp2和dGe-1參與osk mRNP的座落機制是我們探討的目標。 果蠅dDcp2或dGe-1產生突變時,胚胎會有腹節缺失的後端突變性狀以及造成osk mRNP 在卵母細胞後端累積的新月狀減少或是脫落。 另外在dDcp2突變的卵中,dDcp1和dGe-1蛋白質會有散落或是減少的情況,顯示三者在卵母細胞中有遺傳相互作用 (genetic interaction),且dDcp1和dGe-1在卵母細胞的正常分布需要dDcp2。本論文以共同免疫沉澱(Co-immunoprecipitation)法第一次證實dDcp1、dDcp2A和dGe-1確實可以在卵母細胞中形成一個複合體。 在卵發育(oogenesis)第9~10b時期,dDcp2A和dGe-1分布在卵母細胞的細胞膜內緣(ooctye cortical layer)上,dDcp1-osk mRNP會貼附在dDcp2A和dGe-1旁邊。一個可能的假說是:在卵母細胞cortical layer上的dDcp2A- dGe-1複合體會與dDcp1作用以固著osk mRNP在卵母細胞後端。 根據此假說,原預期大量dDcp2A會促進更多的osk mRNP在卵母細胞後端堆積,然而大量產生dDcp2A在卵母細胞中並沒有明顯累積更多Osk蛋白質在後端,我們懷疑dDcp2A是否無法被大量生成在卵母細胞中。由dDcp2基因劑量(gene dosage)不會顯著影響卵母細胞中dDcp2A蛋白質數量的結果,本論文證實dDcp2A蛋白質數量在卵母細胞中是被嚴格調控。 dDcp2缺失(dDcp2 null)的卵母細胞和護理細胞中,肌動蛋白(actin)發生異常性狀且卵母細胞後端F-肌動蛋白絲狀物(F-actin projections)會消失,顯示dDcp2調控卵母細胞的肌動蛋白。由體外實驗(in vitro) 已知dDcp2A與肌動蛋白絲(actin filaments)有相互作用,且dDcp2A中間片段(dDcp2M)確實可以與肌動蛋白絲有相互作用;本論文進一步做了正反方向的連續截短dDcp2M蛋白質(sequential truncated dDcp2M protein),以尋找dDcp2A可以和肌動蛋白絲結合的最小區域。 為瞭解dDcp2M是否是主要參與肌動蛋白調控的區域,本論文將dDcp2M表現在dDcp2缺失的生殖細胞中,觀察到dDcp2M可以抑制部分肌動蛋白異常性狀,但是不能抑制在護理細胞中的肌動蛋白異常性狀。 結果說明dDcp2M雖然有和肌動蛋白絲的相互作用能力,但是並沒有完整的dDcp2A對肌動蛋白絲調控的功能。 本論文證實dDcp1、dDcp2A和dGe-1確實可以在卵母細胞中形成一個複合體,並且揭示dDcp2A中間片段dDcp2M與肌動蛋白絲有相互作用且可以部分抑制dDcp2 null在卵母細胞的肌動蛋白異常和osk mRNP異常性狀。 In the oocyte of Drosophila melanogaster, the localization of osk messenger RNA (mRNA) is involved in axial determination and pole cell development. The osk mutant embryos show lack of abdominal segmentation which is called posterior group phenotype and lack of pole cells. osk mRNA complexes (osk mRNP) are made in nurse cells. A Microtubule motor protein, Kinesin, mediates transport of osk mRNP by microtubule into the posterior end of the oocyte and osk mRNP are accumulated to form a crescent. Before the localization of osk mRNP to the posterior end of the oocyte, the translation of osk mRNA is repressed, and mRNA degradation is executed until early embryogenesis. Current hypothesis about osk mRNP localization is that after translation derepression, Osk proteins establish a positive feedback mechanism which promotes F-actin projections to anchor more osk mRNP in the posterior end of the oocyte. Processing body (P body) is a protein complex which is responsible for mRNA degradation, mRNA storage and mRNA translation repression in the cytoplasm. Decapping protein 1 (Dcp1) is a core component of osk mRNP. It is found that Dorsophila Dcp1 (dDcp1) directs the osk mRNP localization to the posterior end of the oocyte. The meaning for presence of dDcp1 on the osk mRNP is a goal of our study. dDcp1 regulates the osk mRNP localization in the oogenesis and other components of P body may also be required for osk mRNP localization. It is known that in the human cell and plant cell, Dcp1 can interact with other two core components of P body, decapping protein 2 (Dcp2) and Human enhancer of decapping large subunit (Hedls) which is also called Gougerot syndrome (Ge-1) to form a complex. Hence, we wonder whether Dorsophila Dcp2 (dDcp2) and Dorsophila Ge-1 (dGe-1) is involved in the osk mRNP localization. dDcp2 mutant or dGe-1 mutant embryos show a posterior group phenotype and both mutant oocytes show a reduced or detached crescent of the osk mRNP in the posterior end. In addition, dDcp1 and dGe-1 proteins are scattered or disappeared in the dDcp2 mutant oocyte. It is indicated that they have genetic interactions in the oocyte and the proper localizations of dDcp1 and dGe-1 are required for dDcp2 in the oocyte. In this thesis, the co- immunoprecipitation method first proves that dDcp1, dDcp2A and dGe-1 can form a complex in the oocyte. At stage 9~10b of the oogenesis, dDcp2A and dGe-1 distribute along the oocyte cortical layer, and dDcp1-osk mRNP juxtaposes the layer of dDcp2A and dGe-1. One possible hypothesis is that dDcp2 and dGe-1 form a complex on the oocyte cortical layer to interact with dDcp1 to anchor the osk mRNP in the posterior end of the oocyte. Based on this hypothesis, it is expected that the overexpression of dDcp2A can stimulate more accumulation of osk mRNP in the posterior end of the oocyte. However, the overexpression of dDcp2A does not stimulate more accumulation of osk mRNP in the posterior end of the oocyte. We suspect whether dDcp2A cannot be overexpressed in the oocyte. In this thesis, the result shows that dDcp2 gene dosage does not significantly affect the quantity of dDcp2A protein. It is confirmed that the quantity of dDcp2A protein is highly regulated in the oocyte. dDcp2 null oocyte and nurse cells show abnormal actin pattern. And dDcp2 null mutant results in absent F-actin projections in the posterior end of the oocyte. It is indicated that dDcp2 regulates the actin in the oocyte. Furthermore, dDcp2A can be associated with actin filaments in vitro and the middle region of dDcp2A (dDcp2M) can also be associated with actin filaments. In this thesis, in order to search the minimum region of dDcp2A which can be associated with actin filaments, sequential truncated dDcp2M proteins from 5' to 3' and 3' to 5' are constructed. To clarify whether dDcp2M is a core region of actin regulation of dDcp2A, dDcp2M is expressed in the dDcp2 null germ cell. The result shows that dDcp2M can partially suppress the abnormal actin pattern in the oocyte but is not sufficient to suppress the abnormal actin pattern in the nurse cell. It reveals that although dDcp2M can be associated with actin filaments, dDcp2M does not have the complete function of actin regulation of dDcp2A. In summary, this thesis proves that dDcp1, dDcp2A and dGe-1 can form a complex in the oocyte and reveals that the middle of dDcp2A, dDcp2M, can be associated with actin filaments and can partially suppress the abnormal actin pattern and osk mRNP in the dDcp2 null oocyte. |
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