周子賓臺灣大學:分子與細胞生物學研究所鄭仁和Cheng, Jen-HoJen-HoCheng2010-06-022018-07-062010-06-022018-07-062009U0001-2907200916440900http://ntur.lib.ntu.edu.tw//handle/246246/184754細胞質中的P-bodies (mRNA processing bodies) 參與調控mRNA的 分解，轉譯，以及儲存。從酵母菌到人類，P-body成員在分子結構上有高度的保守性，顯示它們在細胞生理上辦演重要的角色。除此之外，隨著代謝條件的改變，P-body也會產生組成結構組上的動態變異。然而，現今大多數關於P-body的研究，多是以單細胞為實驗材料。而其在多細胞生物體之間的調控，則尚未明瞭。此篇論文將以果蠅發育中的卵室(egg chamber)為材料，研究P-body在卵子發育(oogenesis)期間的動態形為及功能。性介紹卵源細胞(包含卵子, oocyte, 和護理細胞, nurse cells)中的P-bodies之後，內容將會集中在Ge-1(或Hedls，促進去頭蓋大單元蛋白)的研究上。Ge-1是P-body當中的結構蛋白，且在其N端預測出含有信號胜肽(signal peptide)。我們發現在包圍卵子的濾泡細胞(follicle cells)內，Ge-1可以藉由胞吞(endocytosis)的方式進入到卵子當中。此外，Ge-1還可以聚集卵子皮層部位(oocyte cortex)的dDcp1 (mRNA去頭蓋蛋白質1)，來形成P-body。此現象顯示這些P-body成員在發育卵子中的動態特性，且它們的組成可以來自不同的組織。此乃首次發現P-body成員(或至少Ge-1和dDcp1)，參與在非自發效應(non-autonomous effect)當中。下來將探討P-body與細胞骨架之間的關係。值得一提的是，dDcp1和Me31B (一個dDcp1的增效蛋白)，會在卵子內的細胞質流期間快速運動。根據推測，在卵子發育晚期時所啟動的細胞質流(ooplasmic steaming)，會加速Ge-1聚集其它P-body成員，促使它們組成功能性的P-bodies。此後，組合起來的P-body(可能只是部份或包含完整P-body成員)，將會被送入胚胎細胞的細胞質中，去調控mRNA。Abbreviations………………………………...…………………………………...p7文摘要………………………………...………………………………………....p8bstract………………………………...………………………………………......p9ntroduction………………………………...…………………………………....p11ection 1: P-Body (mRNA-processing body) components and their roles in mRNA degradation.1 Introduction to P-Body………………………………...…………………....p13.2 5’-3’ mRNA degradation in P-body……………………...………………....p13.3 mRNA decapping is catalyzed by the decapping complex…………………p14.4 Decapping protein 2, Dcp2, is the catalytic subunit of the decapping complex…………………………………………………………………………p15.5 Decapping protein 1, Dcp1, enhances Dcp2’s decapping activity………….p16.6 DExD/H-box helicase 1,Dhh1, the decapping activtor and translation repressor……………………………………………………………………...…p17.7 The Lsm1-7 complex and Pat1 recogize deadenylated mRNA and promote decapping……………………………………………………………………….p18.8 The P-body scaffold protein Edc3 assists the Dcp1-Dcp2 complex in mRNA decapping……………………………………………………………………….p19.9 Xrn1/Pacman exoribonuclease is responsible for the 5'' -3'' digestion after decapping……………………………………………………………………….p20.10 Ge-1, the enhancer of decapping large subunit……………………………p20ection 2: P-Bodies’ properties and thier dynamics under various cellular conditions.1 P-body harbors non-translating mRNA for its formation………………..…p21.2 mRNAs in P-bodies can be degraded or stored for return to translation…..p22.3 P-body components shuttle between the P-bodies and the cytoplasm……...p23.4 P-bodies fuction during cellular response to stress…………………………p24.5 P-bodies variation is correlative to the change in cellular condition……….p25ection 3: Using Drosophila Oogeneis as a model to study dynamic P-bodies.1 P-bodies resarch in Drosophila melanogaster………………………...……p26.2 Drosophila oogenesis as a modle to explore the dynamic nature of P-bodies in development…………………………………………………………….………p27.3 The purpose of this thesis……………………………………….…………..p28esultsection 1: Introduction of dynamic P-bodies in oogenesis.1 The distribution of dDcp1 and Me31B in the egg chamber………………...p33.2 dDcp1 and Dhh1/Me31B are dynamically linked at the oocyte cortex…….p34.3 Cortical dDcp1 aggregates in response to heat stress……………………….p35ection 2: Ge-1 from follicle cells recruits dDcp1 along the oocyte cortex through endocytosis pathway.1 Over-expressed Ge-1 recruits dDcp1 in follcle cells……………….………p36.2 Over-expressed Ge-1 in follicle cells can also recruit dDcp1 at the oocyte cortex……………………………………………………………………………p38.3 Ge-1 contains a putative signal peptide and colocolizes with Rab7 at the oocyte cortex in the stage 9 egg chamber……………………………………….p39.4 Ge-1 is released from Rab7 into ooplasm and recruits dDcp1 at stage 10.5 Ge-1 localizes posterior to dDcp1 and osk mRNP complex…….………….p41ection 3: Decapping proteins in follicle cells maintain the cytoarchitecture of egg chambers.1 Over-expressed Ge-1 recruits dDcp1 toward the basal side of follicle cells..p44.2 dDcp1 null mutation in follicle cells………………………………………..p45.3 dDcp1 mutation leads to Ge-1 accumulation in follicle cells…………...….p46.4 Decapping proteins are required to maintain cytoarchitecture in follicle cells………………………………………………………………………...……p47.5 dDcp1 and Ge-1 gain-of-function in follicle cells do not affect microtubules………………………………………………………………….....p49.6 dDcp1 mutation in follicle cells disrupts cortical actin skeleton in the oocyte………………………………………………………………………….p50.7 dDcp1 mutation in follicle cells does not alter the cortical polarity of oocyte………………………………………………………………………….p52ection 4: Cortical dDcp1 is dissociated from actin mesh and transported throughout the ooplasm by fast streaming.1 Cortical dDcp1 dissociates from actin mesh at the onset of ooplasmic streaming……………………………………………………………………….p53.2 The dissociation of cortical dDcp1 depends on the onset of ooplasmic but not on the developmental stages……………………………………………………p56.3 Cortical dDcp1 foci are subcortical to Capu network………………………p57.4 Fast streaming of dDcp1 and Me31B in the oocyte………………………...p58.5 Microtubule disruption leads to aggregation of dDcp1 in germline cells…..p59iscussion………………………………………………………………..……….p60. Cortical dDcp1 and Me31B is unlikely to engage in mRNA decapping/ degradation but may serve as a storage site for mRNAs during stress.. Follicle cells donated Ge-1 as a scaffold protein to initiate P-bodies assembly by recruiting cortical dDcp1 (and other P-body components) during oogenesis.. The connection between P-body components and microtubules.. Fast streming may accerlate the P-bodies assembly during late oogenesis. The FH1 domain of Capu is a potential ligand for the WH1 domian of Dcp1igures…………………………………………………………………………….p68eference…………………………………………………………………..……p104aterials and Methods………………………………………..…………….p121application/pdf29382335 bytesapplication/pdfen-US信號胜肽mRNA去頭蓋蛋白質1細胞質流P-bodiesoocytesignal peptidedDcp1Ge-1ooplasmic steaminghttp://ntur.lib.ntu.edu.tw/bitstream/246246/184754/1/ntu-98-R96b43007-1.pdf