果蠅去頭蓋蛋白質2調控卵母細胞的微管組成

Drosophila decapping protein 2, dDcp2,regulates microtubule organization in the oocyte

賴盈竹(Ying-Chu Lai)

指導教授 ： 周子賓

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.10076

摘要

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。
果蠅去頭蓋蛋白質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。
由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。
有絲分裂的初期，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。
根據研究，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。
本文推論，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。

關鍵字

去頭蓋蛋白質 ； 卵母細胞 ； 微管

並列摘要

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.
Since 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.
First, 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.
Second, 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.
Third, 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.
Finally, 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.
Several 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.
Our 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.

並列關鍵字

decapping protein ； microtubule

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