果蠅去頭蓋蛋白質2影響濾泡細胞的微管表現形態

Drosophila decapping protein 2, dDcp2, affects the pattern of microtubule in follicle cells

蔡孟庭(Meng-Ting Tsai)

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(2010年)

https://doi.org/10.6342/NTU.2010.10030

摘要

細胞質中的P-body(Processing bodies)參與生物體內mRNA的代謝過程，包含 分解，儲存，和轉譯等。目前為止，大多數關於P-body的研究，多是以單細胞為實驗材料，而對於P-body在多細胞生物體發育過程中所扮演的角色與功能目前尚未有清楚的結論。我們以果蠅發育中的卵為研究材料，探討P-body成員影響細胞骨架(cytoskeleton)以及部份蛋白質分布狀態的關係。
先前的研究指出，在卵母細胞(oocyte)中，P-body成員之一，Drosophila decapping protein 2 (dDcp2，mRNA去頭蓋蛋白質2)，突變時會造成微管(microtubule)結構的表現下降至近似崩解；而由微管主導的下游行為，如細胞質流(ooplasmic streaming)也因而終止。
為探討在不同細胞中dDcp2和微管的關係，我們研究在包圍卵母細胞四周的濾泡細胞(follicle cells)內突變dDcp2後微管的表現行為及分布狀態。我們發現dDcp2在濾泡細胞和卵母細胞中調控微管的行為是相反的。在正常情況下，濾泡細胞的微管分布圍繞在細胞的周圍而且其分布方向與細胞周邊平行，底部-頂部 (apicobasal)的方向，不會往細胞質內生長。但在失去dDcp2的情況之下，微管的分布異常的增加， 並有多叢分枝往細胞質內伸長;而微絲(actin)的表現與分布亦有減少的趨勢。
而我們知道，在P-body中，dDcp2會結合其他成員去調控mRNA的代謝，因此我們觀察其它的P-body成員，像是 dDcp1(Drosophila decapping protein 1，果蠅去頭蓋蛋白質1)，Ge-1(或dHedls，the enhancer of mRNA-decapping large subunit，促進去頭蓋大單元蛋白)，Me31B(Maternal expression at 31B，decapping的增效蛋白)，和Pacman (核酸水解酵素)，所以我進一步研究在濾泡細胞中突變這些P-body成員後微管表現行為與分布狀態的。
在失去dDcp1，dGe-1，和Me31B後，微管的分布行為異常的增加且有額外的分枝往細胞質內延伸；此現象類似突變dDcp2。然而在失去Pacman後，微管跟正常細胞比較起，來並沒有明顯的差異。這顯示部分的P-body成員具有調控微管的表現型態的功能。由Pacman與其他成員的行為差異，這暗示P-body成員在調控微管時， 其組成員跟調控RNA代謝時的成員可能不完全相同；一種可能性為 P-body成員調控MT的表現行為並不是藉由調控mRNA代謝的這個方式而是各成員的另一蛋白質本身的功能。
進一步探討這些P-body成員在調控微管的表現行為時，是否也會去調控其它蛋白質的表現跟分布情況。我們發現，當失去這些P-body成員時，並不是只有單純的影響細胞骨架的分布，同時也會造成部分蛋白質的改變。像是，當濾泡細胞失去部分的P-body成員時,會造成微管相關蛋白質(Microtubule associating protein)- Stathmin的表現情形和分布狀況增加;另外，代表細胞極性的蛋白質Armadillo和Rho1的表現也有增加的趨勢而且改變了在細胞內分布的狀態;而與vesicle運送有關的蛋白質- Rab 7，也受到P-body成員消失的影響，造成Rab7在細胞內的表現和分布增加。這些結果顯示，MAP-stathmin， Armadillo， Rho1， 和Rab7，這些蛋白質的表現型態都會受到這些p-body成員的調控。根據這種現象，我們推測P-body影響微管的表現型態時，同時也伴隨著調控Stathmin， Armadillo， Rho1，和Rab 7的表現型態。
最後，基於我們的觀察，我們推測P-body可能具有新的功能,調控微管的表現型態,也會去調控其它的蛋白質的表現行為，像是， Stathmin， Armadillo， Rho1，和Rab 7，而這假說仍需未來進一步研究證實。

關鍵字

去頭蓋蛋白質2 ； 微管

並列摘要

P-bodies (mRNA processing bodies) are cytoplasmic granules that have been hypothesized to be sites of mRNA degradation, mRNA translational control, and mRNA storage. However, until recently, most studies of P-bodies are focus on single cells and it remained their regulation in multicellular system is unclear. Here we focus the relationship between P-body components and microtubule in developing Drosophila egg chambers.
Previous research indicates that dDcp2 (dacpping protein 2), as a component of P-bodies, may have a specific function in remodeling cytoskeleton organization in oocyte. The expression pattern of microtubules is disrupted in dDcp2 nutant oocyte. And the fast ooplasmic streaming, the behavior work by microtubule, is also reduced in dDcp2 mutant oocyte.
In the thesis, we focus on relationship between dDcp2 and the distribution of microtubule in different cells. We have investigated the expression pattern of microtubule by generating mutant clones in Drosophila follicle cells. Thus, loss of dDcp2 causes the different expression of microtubule between oocyte and follicle cells. In dDcp2 mutant follicle cells, we find that the expression pattern of microtubule is increased and additional microtubules branch toward cytoplasm. Moreover, w we know that dDcp2 associate with some proteins for regulating mRNA decay in cytoplasmic P-body granules, such as dDcp1 (dacpping protein 1), Ge-1 (or dHedls, Human enhancer of decapping large subunit), Me31B (an enhancer of dDcp2), and Pacman (the 5’-3’ exoribonuclease). Then, we investigates the relationship between P-body components and the expression of microtubules. In mutant dDcp1, Ge-1, and Me31B, mutant cells, the expression pattern of microtubule is increased dramaticly. However, in Pacman mutant cells, the expression of microtubules is normal. These results indicate that the partially or complete assembled P-bodies may has the function in regulating the expression of microtubules. And P-body components are different between in regulation of microtubule and in mRNA degradation. So, we suggest that the P-body does not use mRNA degradation to regulate the expression of microtubules.
When cells lost partial P-body components, dDcp2, dDcp1, Ge-1, and Me31B, it affected not only the expression of microtubule but also the expression of some proteins. First, in P-body components mutant cells, the expression pattern of stathmin, MAP (microtubule associating protein), is dramatically increased; Second, the expression pattern of Armadillo and Rho1, the marker of cell polarity, is increased and distribute over cytoplasm and nuclei in P-body components mutant cells; Third, interestingly, the expression pattern of Rab 7, a GTPase of vesicle, is increased in P-body components mutant cells. These results indicate that the expression of stathmin, Armadillo, Rho I, and Rab7 are increased in P-body components mutant cells. These suggest P-body components regulate not only the expression pattern of microtubule but also the expression pattern of some proteins.
　　According to our finding, we propose that the P-body components in follicle cells have the new function in regulating the expression pattern of microtubule, and some proteins- Stathmin, Armadillo, Rho I, and Rab7. However, the hypothesis need future works for confirming.

並列關鍵字

dDcp2 ； microtubule

參考文獻

Aizer, A., Brody, Y., Ler, L.W., Sonenberg, N., Singer, R.H., and Shav-Tal, Y. (2008). The dynamics of mammalian P body transport, assembly, and disassembly in vivo. Mol Biol Cell 19, 4154-4166.

Aizer, A., and Shav-Tal, Y. (2008). Intracellular trafficking and dynamics of P bodies. Prion 2, 131-134.

Anderson, P., and Kedersha, N. (2006). RNA granules. J Cell Biol 172, 803-808.

Balagopal, V., and Parker, R. (2009). Polysomes, P bodies and stress granules: states and fates of eukaryotic mRNAs. Curr Opin Cell Biol 21, 403-408.

Barr, F.A. (2009). Rab GTPase function in Golgi trafficking. Semin Cell Dev Biol 20, 780-783.