論探詢果蠅卵中Dmoesin上游磷酸化調控因子以及果蠅去頭蓋蛋白質2和Cappuccino蛋白的關係

Investigate the upstream regulators for Dmoesin phosphorylation in the oocyte and the relationship between Drosophila decapping protein 2 and Cappuccino

蔡岳宏(Yue-Hong Tasi)

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(2017年)

https://doi.org/10.6342/NTU201700453

摘要

在果蠅卵細胞發育的過程中，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) 和生殖前驅細胞的發育。我們過去的研究發現降解體成員，果蠅去頭蓋蛋白質2 (Drosophila decapping protein 2,Dcp2) 與磷酸化的Dmoesin (Dmoe)形成anchor 幫助dDcp1-osk mRNP 坐落於後端。

我們提出假說：非磷酸化Dmoe 連接dDcp2，並且和dGe1-dDcp1-osk mRNP互動形成transporting complex 並傾向位於卵中央，當Dmoe 磷酸化改變，則p-Dmoe 與dDcp2 -dGe1-dDcp1-osk mRNP 形成anchorage complex 傾向坐落於後端。Transporting complex 和anchoring complex 的分配受Dmoe 磷酸化狀態決定。

本論文的第一部份: 尋找Dmoesin 磷酸化狀態上游的調控因子

我們證實在Drosophila Rho-kinase (Drok)缺失的卵中發現磷酸化Dmoe 表現降低，以及Osk 散落，顯示Drok 可能調控Dmoe 影響Osk 坐落。然而在卵中大量表現Drok，並無觀察到明顯的磷酸化Dmoe 和Osk 蛋白上升。關於Drok 是否藉調控Dmoe 來影響Osk 坐落需進一步分析。

另外，利用Protein Phosphatase 1 (PP1) RNAi 於卵內降解PP1 時，觀察到些許卵內Dmoe 被過度磷酸化，也產生了不正常的網狀結構。然而，Osk 蛋白質卻出現約十分之一的脫落和消失的性狀。顯示PP1 在卵內可能調控Dmoe 磷酸化，進而影響Osk 坐落。

本論文的第二部份：探討dDcp2 與actin nucleators 的互動關係。

osk mRNP 坐落需要F-肌動蛋白絲狀物(F-actin projections)。已知dDcp2 能促進 F- 肌動蛋白絲狀物產生， 但是機制不明。我們利用共同免疫沉澱(Co-immunoprecipitation)法，發現dDcp2 並不與actin nucleator Spire 互動，但與Capu 有微弱互動。dDcp2 與Capu 可能是間接互動關係。

關鍵字

奧斯卡蛋白

並列摘要

During Drosophila oogenesis, oskar (osk) mRNA anchoring to the posterior end of the Drosophila oocyte defines axial determination and development of the precursor of germ cells. We previously found that Drosophila decapping protein 2 (dDcp2) and phosphorylated Dmoesin (p-Dmoe) form the anchor which localizes dDcp1-osk mRNP at the posterior pole of the oocyte.

We proposed that non-phosphorylated Dmoe (nonp-Dmoe) interacts with dDcp2-dGe-1-dDcp1-osk mRNP to form the transporting complex which tends to reside in the ooplasm. p-Dmoe interacts with dDcp2-dGe1-dDcp1-osk mRNP to form the anchorage complex which tends to anchor at the posterior end of the oocyte. The phosphorylation status of Dmoe determines the allocation of both complexes.

The first part of the thesis：Investigate the upstream regulators for Dmoesin phosphorylation in the oocyte.

We found that the expression of p-Dmoe was reduced and Osk was mislocalized in Drosophila Rho-kinase (Drok) mutant oocytes. It indicates that Drok might affect Osk localization via regulating phosphorylation of Dmoe in the oocyte. However, overexpression of Drok cannot elevate the expression of p-Dmoe and promote the accumulation of Osk at the posterior end of the oocyte. Whether Drok regulates the phosphorylation status of Dmoe and consequently affects Osk behavior needs further experiments.

We expressed Protein Phosphatase 1 (PP1) RNAi to knock-down PP1 in oocytes and found hyper-phosphorylated Dmoe with a special reticular-like structure along oocyte cortex. Besides, Osk proteins mis-localized and disappeared from the posterior end of the oocyte. This suggested that PP1 might regulate the phosphorylation status of Dmoe and consequently affects Osk behavior in the oocyte.

The second part of this thesis： Examine the relationship between dDcp2 and Cappuccino.

Actin cytoskeleton is required for the localization of osk mRNA [1]. Our previous results found that dDcp2 could promote F-actin projections in the oocyte (Yi-Mei Lee, unpublished data), but the mechanism is not clear. Our
co-immunoprecipitation results showed that dDcp2 cannot interact with the actin nucleator, Spire but interacts ambiguously with Capu. This indicated that the interaction between dDcp2 and Capu might be indirect.

並列關鍵字

oskar protein

參考文獻

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