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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 周子賓(Tze-Bin Chou) | |
dc.contributor.author | Chin-Yu Teng | en |
dc.contributor.author | 鄧瑾瑜 | zh_TW |
dc.date.accessioned | 2021-06-16T09:37:27Z | - |
dc.date.available | 2020-02-17 | |
dc.date.copyright | 2017-02-17 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-02-10 | |
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Processing body components cooperate to assist the posterior localization of oskar mRNP complex in the oocyte. National Taiwan University, Taipei. Lu, C. C. (2014). The analysis of interactions between Drosophila processing-body components and Dmoe. National Taiwan University, Taipei. Chiang, P. C. (2014). dDcp1-dDcp2-dGe-1 form a complex and the analysis of dDcp2 function in F-actin behavior in the Drosophila. National Taiwan University, Taipei. Lee, Y. M. (2017). The oskar mRNA transporting and anchoring complexes in Drosophila oocyte. Unpublished doctoral dissertation, National Taiwan University, Taipei. We thank the excellent technical assistance of Technology Commons in College of Life Science and the Instrumentation Center sponsored by Ministry of Science and Technology, National Taiwan University (Taiwan)。 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59777 | - |
dc.description.abstract | osk mRNA坐落在果蠅卵細胞後端決定胚胎未來的體軸,以及影響生殖細胞形成與腹部發育,然而目前對osk mRNA坐落機制仍不清楚。文獻指出在khc突變或osk-K10中,osk mRNA坐落於卵細胞周圍,這暗示在此存在負責定錨osk mRNA的物質。我們先前許多研究結果指出dDcp2與Dmoe皆為osk坐落所需,且兩者形成複合體。因此我們認為dDcp2-Dmoe複合體可能擔任此角色。
本論文第一部份透過免疫螢光染色證實p-Dmoe自stage7/8開始將dDcp2從細胞質帶至卵細胞周圍,此dDcp2-Dmoe複合體能做為osk mRNA定錨根基。先前的研究指出當dDcp2與p-Dmoe大量表現時,卵細胞後端Osk增加;當dDcp2與nonp-Dmoe大量表現時,卵細胞質中產生一個明顯的異位Osk訊號點,dDcp2、nonp-Dmoe、dDcp1、dGe-1及Kinesin皆存在於相同的異位點。我們根據以上結果提出假說:Dmoe磷酸化狀態改變時,dDcp2-Dmoe複合體會改變Osk的坐落位置。當Dmoe磷酸化時,osk mRNP會被定錨在卵細胞後端形成定錨複合體(anchoring complex)。成功坐落的osk mRNA會轉譯出Osk。當Dmoe去磷酸化時,Osk則被送往卵細胞內部形成運輸複合體(transporting complex)。 論文第二部分為檢驗假說。首先證實了大量表現osk mRNA亦產生運輸複合體。接著在此背景下調控dDcp2、p-Dmoe及nonp-Dmoe表現量,並檢驗Osk在運輸複合體及定錨複合體之間的平衡變動。結果發現單獨提升dDcp2或p-Dmoe表現量皆能促進定錨複合體形成,而提升nonp-Dmoe表現量則促進運輸複合體形成。此外,同時提升dDcp2及total Dmoe表現量能使所有卵細胞產生定錨複合體。這些結果證實了我們的假說。然而,同時提升dDcp2及p-Dmoe表現量時卻使運輸複合體再度形成,目前仍無法解釋此現象。 論文第三部分為透過共同免疫沉澱法證實Osk和dDcp2具有交互作用。 總結,本論文證實Dmoe磷酸化狀態調控osk mRNP的定錨,Osk透過與dDcp2交互作用維持在卵細胞後端的坐落。 | zh_TW |
dc.description.abstract | The localization of osk mRNA at the posterior pole of Drosophila oocyte determines the posterior axis for embryo, and affects the germ cells formation and abdomen development; however, the mechanism remains unclear. Previous studies showed that in khc mutant or osk-K10 oocytes, osk mRNA is localized along oocyte cortex. It implies that there is a factor responsible for anchoring osk mRNP. Many of our previous studies showed that both dDcp2 and Dmoe are required for osk localization and they form a complex. Therefore, we assume that the dDcp2-Dmoe complex is a candidate of the cortical anchor for osk mRNP.
In the first part of this thesis, immunofluorescent staining confirms that since stage7/8, p-Dmoe recruits dDcp2 from the ooplasm to the cortex. Thus, the cortical dDcp2-Dmoe complex stands by osk mRNP and acts an anchor. Our previous studies showed that the posterior Osk is elevated when both dDcp2 and p-Dmoe are upregulated. Overexpressing both dDcp2 and nonp-Dmoe generates a significantly ectopic Osk dot in the ooplasm. And nonp-Dmoe, dDcp2, dDcp1, dGe-1 and Kinesin are all localized in the dot. Based on the above, we propose a hypothesis that dDcp2-Dmoe complex regulates osk localization through the change of Dmoe phosphorylation status. When Dmoe is phosphorylated, osk mRNP is anchored at the posterior pole, resulting in the translation of Osk and forming the anchoring complex. When Dmoe is dephosphorylated, Osk is moved inwardly and forms the transporting complex. The second part of this thesis is to confirm our hypothesis. Firstly, we prove that overexpressing osk mRNA generates the transporting complex in the oocyte. Next, under this condition, we regulate the expression of dDcp2, p-Dmoe or nonp-Dmoe to examine the change of Osk in the balance between the transporting complex and the anchoring complex. As a result, increasing the expression of dDcp2 or p-Dmoe alone promotes the formation of the anchoring complex while upregulating of nonp-Dmoe enhances the formation of the transporting complex. Moreover, overexpressing dDcp2 and total Dmoe simultaneously leads all oocytes to generate the anchoring complex. These results verify our hypothesis. However, overexpressing both dDcp2 and p-Dmoe generates the transporting complex again. We cannot interpret the phenomena. In the third part of this thesis, the co-immunoprecipitation assay indicates that Osk interacts with dDcp2 in the ovary. In conclusion, this thesis reveals that the phosphorylation status of Dmoe regulates osk mRNP anchoring and Osk maintains its posterior localization through the interaction with dDcp2. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T09:37:27Z (GMT). No. of bitstreams: 1 ntu-106-R01b43014-1.pdf: 4923819 bytes, checksum: b97941d03f5322cfd7411c16834e648c (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 致謝 I
中文摘要 II Abstract III Abbreviations V Table of contents VII Figure list X Introduction 1 1. The axis development of Drosophila 1 1.1. Oogenesis 1 1.2. Axis determination 2 2. The mechanism of osk localization 4 2.1. The function of osk during Drosophila oogenesis 4 2.2. Factors is required for the localization of osk during Drosophila oogenesis 5 2.3. The recent hypothesis of osk localization 12 3. Processing body 15 3.1. Processing body and its function 15 3.2. Evidence about the transportation of P bodies by cytoskeletons 17 4. Previous studies of dDcp2, dGe-1 and Dmoe 20 4.1. dDcp2 20 4.2. dGe-1 22 4.3. Dmoesin 25 5. Our working model for osk mRNP anchoring 28 6. The aims of this thesis 30 Materials and Methods 32 Drosophila stocks 32 Whole-mount ovary antibody staining 32 Whole-mount ovary in situ hybridization 33 Co-immunoprecipitation (Co-IP) 34 Western blot 35 Results 37 Part I. To further confirm the hypothesis that dDcp2-Dmoe complex forms the pre-localized anchor for osk mRNP 37 1. The distribution of phosphorylated and non-phosphorylated-Dmoe in egg chambers 38 2. The formation of the pre-localized dDcp2-Dmoe anchor for osk mRNP 39 Part II. The phosphorylation statuses of Dmoesin control the equilibrium of osk localization between the transporting and the anchoring complexes 41 1. Nonp-Dmoe and P body components reside in the transporting complex with Osk protein in UASp-osk oocytes 42 2. Overexpressing the osk mRNP anchorage systems could reduce Osk mislocalized phenotypes 43 3. Phosphorylation status of Dmoe plays a key role to regulate the localization of excess osk mRNA in the oocyte 46 Part III. The interaction between Osk protein and dDcp2 49 1. Long Osk forms an ectopic dot in UASp-osk 50 2. Ectopic long Oskar recruits dDcp2 to the anterior end of oocyte 51 3. Co-immunoprecipitation assay indicates that Osk interacts with dDcp2 in Drosophila ovary 52 Discussion 54 1. The two consecutive round of osk anchorage 54 1.1 The dDcp2-Dmoe complex is the pre-localized cortical anchor for osk mRNP 54 1.2 dDcp2 maintains the localization of Osk protein at the posterior pole 56 2. The equilibrium between the transporting and anchoring complex 57 2.1 The ectopic Osk dot in UASp-osk is the transporting complex 57 2.2 The possible biological function of the transporting complex 58 2.3 The regulation of transporting complex and anchoring complex 59 2.4 Dmoe needs to cooperate with dDcp2 for anchoring osk mRNP 60 2.5 Overexpression of both p-Dmoe and dDcp2 in UASp-osk causes the weird Osk pattern that is incompatible with our model 61 3. The combination of several mechanisms for osk localization 62 References 100 Figure 1. Oogenesis in an ovariole of Drosophila oocyte 64 Figure 2. The formation of AP and DV polarity in Drosophila 65 Figure 3. osk mRNA is transported from nurse cells to the oocyte 66 Figure 4. The localization of osk mRNP in the oocyte 67 Figure 5. A current model for osk mRNP and pole plasm anchorage at the posterior cortex of Drosophila oocyte 68 Figure 6. A model of osk mRNA localization during oogenesis of Drosophila 69 Figure 7. A cortical exclusion model for osk localization 70 Figure 8. A model for an osk-dependent positive-feedback loop 71 Figure 9. Our hypothesis of osk mRNP anchoring 72 Figure 10. The expression pattern of Dmoe in stage 3-10 egg chambers 73 Figure 11. Images of GFP fused Dmoe variants in living egg chambers 74 Figure 12. phosphorylated-Dmoe recruits dDcp2 to the cortex from stage 7/8 75 Figure 13. Osk phenotypes in UASp-osk oocytes 78 Figure 14. non-phospho-Dmoe resides in the ectopic Osk dot in the ooplasm 79 Figure 15. EGFP-dDcp2 forms an ectopic dot in the ooplasm 80 Figure 16. dGe-1-V5 aggregates in the ectopic Osk dot in the ooplasm 81 Figure 17. HA-dDcp1 involves in the ectopic Osk dot in the ooplasm 82 Figure 18. Overexpressing EGFP-dDcp2 could suppress mislocalized Osk 84 Figure 19. Overexpressing mcherry-dDcp2 could suppress mislocalized Osk 86 Figure 20. Overexpressing p-Dmoe could suppress mislocalized Osk 88 Figure 21. Both Dmoe, especially p-Dmoe, and dDcp2 are simultaneously needed for osk mRNP anchoring 90 Figure 22. Long Osk protein mislocalizes in UASp-osk oocytes 91 Figure 23. The ectopic expression of two Osk isoforms 93 Figure 24. Ectopic long Oskar recruits dDcp2 to the anterior end of oocyte 95 Figure 25. Osk interacts with dDcp2 in Drosophila ovary 96 Supplementary figure 1. The distribution pattern of the osk mRNP and P body components 98 | |
dc.language.iso | en | |
dc.title | Dmoesin調控果蠅卵母細胞中奧斯卡核糖核酸複合體的定錨 | zh_TW |
dc.title | Dmoesin regulates the anchorage of oskar mRNP complex in Drosophila oocyte | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 王致恬,溫進德 | |
dc.subject.keyword | 奧斯卡核糖核酸,果蠅去頭蓋蛋白質2, | zh_TW |
dc.subject.keyword | oskar mRNA,dDcp2,Dmoesin, | en |
dc.relation.page | 107 | |
dc.identifier.doi | 10.6342/NTU201700426 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-02-12 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
顯示於系所單位: | 分子與細胞生物學研究所 |
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