周子賓Chou, Tze-Bin臺灣大學:分子與細胞生物學研究所賴盈竹Lai, Ying-ChuYing-ChuLai2010-06-022018-07-062010-06-022018-07-062009U0001-0408200923474100http://ntur.lib.ntu.edu.tw//handle/246246/184718在果蠅胚胎發育的過程(oogenesis)中, 母系訊息核醣核酸 (maternal mRNA)在卵母細胞內特定位置的分布與定位對於胚胎發育扮演非常重要的角色。其中，oskar(osk)訊息核醣核酸在卵母細胞後端的定位，決定胚胎腹部與極細胞(pole cell)的發育；纖維性肌動蛋白(F-actin)與微管(microtubule) 皆參與osk訊息核醣核酸的運送和卵母細胞後端的定位。蠅去頭蓋蛋白質2 (Drosophila decapping protein 2,dDcp2) 已知參與調控 osk mRNA往卵母細胞後端運送的過程。在完全缺失果蠅去頭蓋蛋白質2的背景下，Osk呈現表現錯位 (mislocalized)的不正常型態，異於正常胚胎中會在後端呈現的新月狀。此外，在果蠅去頭蓋蛋白質2突變的卵母細胞中，有不正常的纖維性肌動蛋白累積與結塊狀的情形；更且卵母細胞的胞質流動(ooplasmic streaming)也產生停滯的現象。於母系訊息核醣核酸的運送與細胞胞質的流動是透過驅動蛋白(kinesin)在微管上的運送而產生。本論文確定，當 dDcp2 突變時卵母細胞內的微管動態結構受損且無法形成平行的微管束(microtubule bundles)，而組成微管結構的單體微管蛋白 (tubulin)的表現量也因為dDcp2 突變而下降。dDcp2 可能是藉由調控微管與微管蛋白之間的動態平衡，進而影響驅動蛋白的分布與細胞內物質運送以及細胞質流的發生。絲分裂的初期，細胞核中的染色質開始凝聚並且中心粒往細胞的兩端移動，並延伸出許多絲狀的物質，其中一部份絲狀物就構成紡錘體。在 dDcp2 突變的胚胎中可發現染色質無法完全成功的凝聚，另外紡錘體的表現也下降或者消失；紡錘體應該執行的功能如分離染色分體以抵達細胞的兩極也都無法同步的執行。本論文證實由於dDcp2 對動態結構的微管與微管蛋白的影響，在dDcp2突變中，因此造成紡錘體的消失以及衍生的有絲分裂的變異。據研究，核醣核酸裂解體(Processing bodies)與特定的核醣核酸結合蛋白(RNA-binding protein)例如Staufen可沿著微管所構築的運輸軌道進行方向性的移動。另外，若破壞了微管結構也會造成裂解體重組與分布改變。根據先前我們發現裂解體中的成員可能扮演調控微管的角色，故我們研究兩者之間的相互關係。最後，證實了在果蠅的訊息核醣核酸裂解系統中的去頭蓋蛋白質1 (Drosophila decapping protein 1,dDcp1) 和dDcp2以及(Human enhancer of decapping large subunit ,dHedls/dGe-1) 附著在果蠅的S2 細胞株內的微管結構。文推論，dDcp2附著在微管結構上並且參予微管與微管蛋白之間組成與分解的動態平衡，導致微管動態組成受損並消失，進而影響了細胞內物質的運送以及有絲分裂的過程。Microtubules and actin filaments are required for the selective accumulation of oskar(osk) mRNA at the posterior end in the Drosophila oocyte. Our previous research uncovered that Drosophila decapping protein 2, dDcp2, is necessary for the proper posterior location of osk mRNP complex. The posterior determinants— Osk, Staufen and Vasa display a mislocalized pattern in dDcp2 deletion mutant comparing with the posterior crescent pattern in wild type oocyte. More strikingly, an abnormal actin accumulation clumps and a halted ooplasmic streaming were found in the mid and late stage of the dDcp2 mutant oocytes. ince microtubules dominants the localization of maternal determinants and the onset of ooplasmic streaming, we suspect that dDcp2 may modulate the intracellular microtubule network during oogenesis. This working hypothesis is supported by several observations. irst, dDcp2 mutants show a disorganization of the oocyte microtubule cytoskeleton. Comparing with wild type, dDcp2 mutants is defected in the dynamic reorientation of microtubule at stage 9. Moreover, microtubule bundles can’t form a typical parallel array during stage 10B in dDcp2de21, dDcp2RAf, and dDcp2NF mutant GLC oocytes. Microtubules are less abundant in dDcp2 mutants, showing that a key function of dDcp2 in vivo is maintenance of the microtubule cytoskeleton.econd, α-tubulin protein expression level is reduced in dDcp2 and dGe-1/dHedls mutant larvae, respectively; similarly, the dynamic tyrosinated tubulin protein expression level is also decreased in the dDcp2 de21 mutant GLC oocytes, and this might explain the effect on the microtubule cytoskeleton.hird, dDcp2 is required for successful completion for the early embryo development in Drosophila. The loss-of-function of dDcp2 results in defects in the proper alignment of the dynamic spindles and chromosomal motions in early embryos.inally, dDcp1 and dDcp2 are partially colocalized in Drosophila S2 cells. We further showed that components of processing bodies such as dDcp1, dDcp2 and dGe-1/dHedls are able to adhere to microtubule filaments.everal findings suggest that a relationship exists between Processing bodies and microtubules. The disruption of microtubule by drug can stimulate the accumulation of P-bodies in yeast and mammalian cells, whereas the 5’-3’ exonuclease Xrn1 is able to promote the assembly of tubulin into microtubule in vitro. Further, the loss-of-function of GW182 protein can lead to the aggregation of tubulin filamentous in Drosophila embryos.ur findings indicate that dDcp2, as a component of P-bodies, may have a specific function in remodeling microtubule organization which appears unrelated toits RNA decapping function in vegetative cells. Therefore, we suspect that in dDcp2 mutation the disruption of microtubule may in turn affect the organization of actin filament and the proper chromosome segregation during mitosis.INTRODUCTION…………………………………………………………1 . Drosophila oogenesis and Cytoskeleton…………………………..1.icrotubules structure and organization…………………………………………..1.he posttranslational modification on the tubulin…………………………………3.rganization of microtubule bundles by MAPs…………………………...………4.he cellular functions of microtubule in Drosophila development- Microtubule dependent transport…………………………...………………………….………..6.he cellular functions of microtubule in Drosophila development- mitotic spindle formation during the early embryogenesis……………………………….………14I . Processing bodies and decapping proteins……………….…..21.he overall of mRNA degradation ………………. ……………………...…...…21.he decapping proteins ……………….……………………………………....…22II. Intracellular trafficking and dynamics of P bodies………31.rocessing bodies are dynamic structures ……………….………………………31.he interaction between cytoskeleton and Processing bodies……………………32V.resent studies on Drosophila decapping protein 2………34.rosophila decapping protein 2……………….……………………………....…34.he aim of this thesis……………….………………………………………....…37aterials and Methods……………….……………………………….38.rosophila keeping and maintenance……………….………………………..….38.erm-line clone generation……………….……………………..……………….39.hole-mount ovary antibody staining……….……………………..……...…….42.isible microtubule staining……….……………………..………………...…….43.rosophila S2 Cell culture……….……………………..………………...…..….43.luorescence antibody staining of S2 cells……….……………………..…….…44.urification of dDcp2 in BL21(DE3)-pLysS expression system……………..….45esults……………….………………………………. ………………………... 46.Dcp2 is required for microtubule organization in the oocyte……………….…………………………………………………......46.utation in dDcp2de21 disrupts microtubule organization in the oocyte…... 46.icrotubule organization is disrupted in the dDcp2 RAf GLC oocytes……...49.icrotubule organization is disrupted in the dDcp2NF mutant GLC oocytes ……………….…………………………………………………......50.icrotubule organization is abolished in the dGe-1/HedlsH159mutant GLC oocytes……………….…………………………………………………......52.he dDcp1-foci aggregates in dDcp2 and dGe-1/dHedls mutants is independent of the disruption of microtubule organization………………...55I.Dcp2 and dGe-1/dHedls are involved in maintaining tubulin pools……………….…………………………………………...57.he expression level of α-tubulin protein is decreased in the dDcp2 and dGe-1/dHedls mutants……………….……………………………………..58.he expression level of dynamic tubulin protein is decreased in the dDcp2de21 mutants……………….……………………….……..……………………..60.he salivary gland displays a abnormal cytoskeleton structure in the dDcp2RAf homozygous mutants…………………….……..………...………61II.Dcp2 is required for proper cell division..………...……...…63.he loss-of-function of dDcp2 reduced the microtubule organizing centerssembly…………….……………………….……..…………………….....64.he loss-of-function of dDcp2 affects the proper alignment of the dynamic spindles and the chromosomal motions.……..…………….…………….....65.Dcp2 BG325JΔA affects the proper behavior of mitosis …….…………….....68V.omponents of processing bodies are adhered to microtubule filaments in Drosophila S2 cells………….....71.Dcp1 and dDcp2 are partially colocalized in Drosophila S2 cells………….....71.Dcp1, dDcp2 and dGe-1/dHedls are able to anchor to microtubule filaments...72.rocessing bodies are dynamic structure during cell division…….…………….74iscussion.Dcp2 is required for dynamic microtubule organization and several microtubule-dependent processes including the localization of maternal determinants and the ooplasmic streaming…….………….………………….77Dcp2 affects the localization of oskar mRNA…….………….………………...77Dcp2 is required for microtubule organization in the oocyte….………………..79Dcp2 is required for cytoplasmic streaming…….………….…………………...80.oth dDcp2 and dGe-1/Hedls mutants disrupt the organization of microtubule cytoskeleton in the oocyte….………….……….………….….….82.Dcp2 is involved in maintaining the equilibrium between microtubule filaments and tubulin pools…….………….……….………….….…….……...84.-bodies components affect Oskar posterior transport…….….….……….....89he function of dDcp1 in Oskar posterior transport….………….….…………...89he function of dGe-1/dHedls in Oskar transport and microtubule organzation...91.cp2 is required for dynamic mitotic spindle formation…….….…………...92.ntracellular trafficking and dynamics of P bodies…….….………………....94ntracellular trafficking and mRNA transport…….….………………………......95he dynamic P-bodies assembly in dDcp2 and dGe-1/dHedls mutants is independent of the disruption of microtubule organization…………………......99ynamics of dDcp1 and dDcp2 in the cytoplasm…….….……………………..101eference…….….………………………….….…………………….….……103ist of figuresigure 1. Microtubule structure and dynamic organization…………..……….……113igure 2. Dynamic interaction between P-Bodies and transport mRNP Particles on the microtubule………….……….……………. ……….…………….……….………..115igure3. Microtubule reorganization and kinesin-dependent cytoplasmic streaming in the oocyte………….……….……………. ……….…………….……….…………116igure 4.The mechanism of mitotic spindle formation and mitotic spindle in early embryo………….……….……………. ……….…………….……….………….…118igure 5. Overall of early embryogenesis and cytoskeleton rearrangement………..120igure 6. Spindle morphogenesis and centrosome-directed amphiastral …………..121igure 7. General mRNA degradation pathway and decapping proteins …………..122igure 8. Four predicted transcripts of dDcp2 and alignment of Dcp2 proteins from different organisms ..…...….……………. ……….…………….……….………….124igure 9. Distribution of dDcp2 in the oocytes…….…………….……….………...126igure 10. dDcp2 colocalized with dDcp1,Me31B and dHedls….……….………...128igure 11. A genomic map of dDcp2 and rescued genomic fragments….………....129igure 12. The microtubule organization in the wild-type oocytes…….…………..131igure 13. Mutant in dDcp2 de21 disrupts microtubule organization in the oocytes...133igure 14. Microtubule organization is disrupted in the dDcp2RAf GLC oocytes….135igure 15. Microtubule organization is disrupted in the dDcp2 NF GLC oocytes…..137igure 16. Microtubule organization is abolished in the dHedlsH159 mutant GLC oocytes..…...….……………………….…………….……….……………………...139igure 17.The dynamic P-bodies assembly via dDcp2 and dGe-1/dHedls mutants is independent of the disruption of microtubule organization …….……….………....140igure 18. The 　-tubulin protein expression level is decreased in the dDcp2 and dHedls mutants ..…...….……………. ……….…………….…….………………...141igure 19. The formation and migration of centrosomes is not affected by dDcp2de21 homozygous embryos.….……………. ……….……………..………………..........142igure 20. The loss-of-function of dDcp2 affects the proper alignment of the chromosomal congression……………. ……….……………..………………….....143igure 21. dDcp2 is required for proper cell division and dynamic spindles formation during mitosis..…...….…………….……….………….….………….….………….144igure 22. Abnormal mitosis can be observed in dDcp2 BG325JA　 GLC egg chambers…………………………………………………145igure 23. dDcp1 and dDcp2 are partially colocalized in Drosophila S2 cells……46igure 24.Components of processing bodies are adhered to microtubule filaments in S2 cells………………………………147igure 25. PBs are distinct cytoplasmic entities with their number and size varying during the cell cycle………………148ppendix………………………………………………………………149application/pdf13948061 bytesapplication/pdfen-US去頭蓋蛋白質卵母細胞微管decapping proteinmicrotubulehttp://ntur.lib.ntu.edu.tw/bitstream/246246/184718/1/ntu-98-R96b43008-1.pdf