果蠅去頭蓋蛋白質2調控卵母細胞內胞質的流動

Abstract

根據實驗室先前的研究，已知果蠅去頭蓋蛋白質1 (Drosophila decapping protein 1, dDcp1)是osk訊息核醣核酸(osk mRNA)輸送複合體的成員，並且為osk訊息核醣核酸往卵母細胞後端運送的過程所需。加上根據對其他物種的研究，指出去頭蓋蛋白質1與去頭蓋蛋白質2會形成一去頭蓋完全酵素體，執行分解訊息核醣核酸的功能。因此，我們想繼續探討︰是否在果蠅個體中去頭蓋蛋白質2(Drosophila decapping protein 2,dDcp2)會同様伴隨去頭蓋蛋白質1，共同參與osk訊息核醣核酸複合體(osk mRNP complex)的成員，並且負責 osk往卵母細胞後端運送的過程。 果蠅去頭蓋蛋白質已知有兩種蛋白表現形，分別是去頭蓋蛋白質A(dDcp2-PA)與去頭蓋蛋白質B(dDcp2-PB)。根據去頭蓋蛋白質2在其他物種中的序列比對，有三個保守性區域分別是區塊A(Box A), 區塊B(Box B), 以及與執行去頭蓋功能相關區域( Nudix motif)，在這兩種表現型中皆具有這三個保守性區域。為了研究果蠅去頭蓋蛋白質2在發育過程中所扮演的角色，我們建立了幾個果蠅去頭蓋蛋白質2基因轉殖突變株，並在完全缺失果蠅去頭蓋蛋白質2的背景下分析這些轉殖突變株的個別功能。此篇論文中，我們把重點放在去頭蓋酵素功能的研究上。根據其他研究指出，在Nudix motif中的兩個重要胺基酸的突變，即會造成果蠅去頭蓋酵素功能的重大缺陷。因此希望透過所建立的dDcp2E133/134A，這個在去頭蓋功能相關區域中含有兩個胺基酸突變的轉殖品系，與dDcp2de21，這個果蠅去頭蓋蛋白質2完全缺失的品系的比較，進一步了解去頭蓋酵素功能對於發育遺傳上的重要性。 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。所以，果蠅去頭蓋蛋白質1突變的胚胎，呈現一個典型的後端性狀--胚胎的腹部是完全缺失的；然而大多數果蠅去頭蓋蛋白質2突變的胚胎，卻呈現只有部分腹部體節缺失的特徵。進一步對果蠅去頭蓋蛋白質2突變的卵母細胞做觀察，除了Osk，Staufen 以及 Vasa同樣對於胚胎後端發育重要的蛋白，三種皆呈現一表現錯位的不正常型態，異於正常胚胎中會在後端呈現的新月狀。此外，果蠅去頭蓋蛋白質2突變的母體所下的蛋，頭部呼吸觸角(dorsal appendages)的發育是有缺陷的。這些突變的性狀類似於與纖維性肌動蛋白(F-actin)合成相關的三個基因-- chic、spire、capu突變的性狀。又加上在中晚期果蠅去頭蓋蛋白質2突變的卵母細胞中，很特別的，有不正常纖維性肌動蛋白累積與塊狀結構的情形。根據以上這些特徵，皆暗示果蠅去頭蓋蛋白質2扮演著與那三個與調控纖維性肌動蛋白合成相關基因相似的角色。 卵母細胞內細胞胞質的流動(ooplasmic streaming)，是細胞胞器或囊泡透過驅動蛋白(Kinesin)在微管(microtubule)上運送所造成細胞質移動的現象。根據其他研究指出：Capu與Spire共同調控之路徑控制了卵母細胞中胞質前後時期不同流動狀態的轉換；在卵母細胞發育至第8期到第10A期時間中，透過Capu與Spire所合成的纖維性肌動蛋白網狀構造，會抑制動驅動蛋白的運動使得微管呈現極性梯度型態，細胞質在此時期的卵母細胞中是呈現些微抖動的現象；而在接下來的第10B期到第11期的時間，纖維性肌動蛋白網狀構造會瓦解，如此一來，驅動蛋白失去了約束力，在自由運動的狀況下使細胞質產生了流動，這種流動也因此順帶將微管沖刷成在細胞膜表面下的平行陣狀構造，此時，卵母細胞細胞質呈現快速流動現象。 在果蠅去頭蓋蛋白質2突變下，原本期望有一個卵母細胞胞質提早快速流動的突變性狀，因為這個性狀是chic、 spire 與 capu基因突變中皆具有的一個獨特特徵。但不如期望地，反而得到一個卵母細胞胞質停止快速流動的結果。為了探究原因，接著檢驗纖維性肌動蛋白網狀構造與驅動蛋白的行為，是否因果蠅去頭蓋蛋白質2突變被影響，進而導致卵母細胞胞質停止了快速流動。然而，纖維性肌動蛋白網狀構造一如正常個體中在恰當時序瓦解，不過驅動蛋白倒是受到影響呈現散開或是沒在後端出現的型態。因此，被抑制的卵母細胞胞質流動，可由被影響的驅動蛋白此證據，得到支持。 此外，在果蠅去頭蓋蛋白質2完全缺失的品系(dDcp2de21)，以及在其背景下放入含有在果蠅去頭蓋蛋白質2中兩個對於酵素功能重要的胺基酸突變基因片段(dDcp2E133/134A)的果蠅中，都觀察到在卵母細胞中，不正常纖維性肌動蛋白塊狀結構以及細胞質停止流動的情形，所以我們假定這些現象與果蠅去頭蓋蛋白質2的酵素功能相關，而球狀肌動蛋白(G-actin)基因的訊息核醣核酸(mRNA)，則被視為是要被果蠅去頭蓋蛋白質2分解的對象之ㄧ。在這個假設中，過量表現的球狀肌動蛋白造成卵母細胞胞質快速流動時期的停止現象。透過球狀肌動蛋白過量表現的果蠅品系的觀察以模擬假設的情況，得到許多結果與果蠅去頭蓋蛋白質2完全缺失的品系所呈現的突變性狀相似，其中一項即是卵母細胞胞質快速流動時期的停止，此項結果提供假設一個支持證據。 最後，基於卵母細胞胞質流動的停止，與纖維性肌動蛋白網狀結構的正常分解，我們提出了果蠅去頭蓋蛋白質2沒有參與Spir與Capu所調控的路徑的假論。而卵母細胞胞質流動的停止的現象似乎與肌動蛋白的過量表現相關，然而是否果蠅去頭蓋蛋白質2的突變會造成球狀肌動蛋白的過量表現，則需進一步研究以待證實。

Drosophila decapping protein 1, dDcp1, is reported to be a component of osk mRNP complex and directs its posterior localization in the oocyte. According to others’ report that Dcp1 and Dcp2 function together as a decapping holoenzyme, and we attempt to study whether Drosophila decapping protein 2, dDcp2, also involves in the osk mRNP complex and being required for its posterior transportation process. There are two dDcp2 isoforms, dDcp2-PA and dDcp2-PB. Three evolutionally conserved domains—Box A, Box B, and Nudix motif are found in dDcp2 isoforms. To study the developmental role of dDcp2, we established several mutant transgenic lines and analyzed their functions in dDcp2del21 null background. It has been reported that two amino acids in the Nuidix fold being important for decapping activity mutations would abolish decapping enzymatic activity. To investigate the importance of decapping function we emphasized our study in dDcp2E133/134A transgenic line, which contains two amino acids mutations in the Nuidix fold and comparing with dDcp2de21. The localization of osk at the posterior of the oocyte is critical for both the development of abdomen and pole cells. Although GLC embryos of dDcp1 mutation display a typical posterior phenotype with abdominal deletion, most of the dDcp2 mutant GLC embryos present mild abdominal segmentation defect. In these dDcp2 mutant GLC oocytes, the posterior determinants-- Osk, Staufen and Vasa proteins display a mislocalized pattern comparing with a posterior crescent pattern in wild type oocytes. Besides, eggs laid by the dDcp2 mutant females display dorsal appendages defect. These mutant characteristics are similar to mutations of chic, spire and capu which regulate F-actin polymerization. In addition, an abnormal F-actin accumulation and clumps formed in mid and late stages of the dDcp2 mutant GLC oocytes. These findings suggest that dDcp2 may play a similar role with these actin regulators. Cytoplasmic streaming of the oocyte is named ooplasmic streaming. The flow is thought to be generated by kinesin-dependent transport of organelles or vesicles on microtubule. It is reported that the Capu/Spire pathway controls alternative states of the oocyte cytoplasm. During stages 8-10A, they assemble an actin mesh that suppresses KinesinⅠ motility to maintain a polarized microtubule cytoskeleton gradient and ooplasm is just seething in this time. During stages 10B-11, actin mesh disassembles, unrestrained KinesinⅠ movement generates flows that wash microtubules to parallel arrays below the surface forms fast ooplasmic streaming. At first, a premature fast ooplasmic streaming which is a distinct phenotype in chic, spire and capu mutations is expected in dDcp2 mutant. But unexpectedly, a halted ooplasmic fast streaming phenotype is found in dDcp2 mutant GLC oocytes. Next, actin mesh and KinesinⅠ behaviors were further examined if they are affected to inhibit ooplasmic streaming in dDcp2 mutation. However, actin mesh disassembles as wild-type schedule in the dDcp2 mutation. Nevertheless, KinesinⅠ displays a scattered or absent phenotype in the dDcp2 mutation. Hence, the blocked streaming phenotype is supported by the disrupted KinesinⅠ behavior. Moreover, since both dDcp2de21 and dDcp2E133/134A GLCs present abnormal actin clumps in the oocyte and halted ooplasmic streaming, it is proposed the halted fast ooplasmic streaming has correlation to the disrupted dDcp2 enzymatic activity and actin mRNA is therefore to be though as the substrate of dDcp2. To verify this hypothesis, ectopic actin expression lines thought to mimic overexpressed G-actin were used for observation and get several similar results to dDcp2 mutants, especially the halted fast ooplasmic streaming. Therefore, the part of overexpressed actin would cause halted fast ooplasmic streaming is supported. Finally, we propose that the dDcp2 may not involve in Spir/Capu pathway because a halted fast ooplasmic streaming and normal disassembled actin mesh structure. The halted fast ooplasmic streaming is related to the overexpressed actin but whether overexpressed actin is really the case in the dDcp2 mutants need future works for confirmation.