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標題: | 果蠅GOLPH3(Rotini)影響Pipe在高基氏體的座落分布 Drosophila GOLPH3, Rotini, Affects the Subcellular Localization of Pipe in the Golgi Complex |
作者: | Yu-Ching Lin 林于敬 |
指導教授: | 周子賓(Tze-Bin Chou) |
關鍵字: | 高基氏體蛋白質GOLPH3,多醣蛋白HSPG,GAG醣鏈聚合酵素EXT,背腹軸,硫基轉移酵素, GOLPH3,Heparan Sulfate Proteoglycans (HSPG),Exostosins (EXT),Dorsoventral (DV) axis,Sulfotransferases, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | 果蠅胚胎發育過程中,背腹軸(dorsal-ventral polarity)的形成主要由一系列絲胺酸蛋白酶(serine proteases)的連鎖反應調控,在胚胎腹面可以活化Toll接受體(receptor)的配體(ligand)。然而,早在蛋腹部發育時, Pipe硫基轉移酶(sulfotransferase)則調控了這一系列絲胺酸蛋白酶的活化。在功能上而言,Pipe調控的受質硫化,在發育中背腹軸的形成扮演著很重要的角色。
Rotini(Rti)是人類GOLPH3蛋白的同源物,先前研究顯示Rti蛋白參與在多醣蛋白(HSPGs;Heparan Sulfate Proteoglycans) 合成多醣鏈(GAG;Glycoamino- glycans)之生成當中。此外,當果蠅生殖細胞(germ-line cells)發生Rti缺失時,造成胚胎扭曲及Dorsal蛋白不正常分布,這些都指向Rti參與在背腹軸形成的過程當中。本論文主要目的為,詳細了解Rti在背腹軸形成過程中所扮演的角色。 本論文先排除了Rti藉由調控多醣蛋白HSPGs的機制去影響背腹軸的形成。此外,在果蠅卵巢濾泡細胞(follicle cells)裡,Rti和Pipe兩者的分佈有部分重疊。在果蠅S2細胞中,被證實Rti會和Pipe有交互作用。這些結果顯示,Rti與Pipe是參與在背腹軸形成過程中兩個直接作用的成員。 正常情況下,Pipe和cis-Golgi標記有50.3%面積重疊。在統計分析上,當Rti表現量上升時,Pipe和cis-Golgi標記重疊部分顯著上升(58.5%),而當Rti表現量下降時,Pipe和cis-Golgi標記重疊部分顯著下降(35.8%)。在遺傳實驗中,證實Rti作用於Pipe上游,這和Rti去調控Pipe分布的關係相符合。這些資料顯示,Rti有能力去影響Pipe在高基氏體的座落分布。 根據這些結果推論,Rti是一個參與在背腹軸形成過程中的新成員,藉由調控Pipe在高基氏體的座落分布。之後,將利用外被蛋白I(COPI;coat protein complex I)或外被蛋白II(COPII)的突變,去觀察Rti是否參與在高基氏體的正向運輸或逆向運輸去調控Pipe的座落。 The dorsal-ventral (DV) polarity of the Drosophila embryo is controlled by the serine protease cascade, which generates the activated ligand for Toll receptor on the ventral site of the embryo. Pipe sulfotransferase, a homolog of vertebrate glycosaminoglycan-modifying enzymes, directs the ventral activation of the serine proteolytic cascade. Functionally, Pipe-mediated sulfation provides a spatial cue for dorsoventral axis formation in the developing egg chamber. Previous studies have shown that Rotini (Rti), a Drosophila homologe of human GOLPH3 protein, is required for the synthesis of heparan sulfate proteoglycans (HSPGs) glycosaminoglycan (GAG) chains. In addition, we have found that Rti also participates in dorovental axis formation, since rti loss-of-function in germ line exhibits a twisted embryonic phenotype and abnormal distribution of Dorsal protein. This thesis is aimed to understand how Rti plays the role in DV axis determination. Here, we showed that Rti influences DV axis through a mechanism not responsible for the synthesis of HSPGs. Moreover, we observed that Rti is partially colocalized with Pipe in Drosophila ovarian follicle cells and coimmunoprecipitates with Pipe from Drosophila S2 cells. These results suggest that Rti and Pipe are the two interacting components in DV determination. In addition, we observed 50.3% of Pipe-cis-Golgi marker colocalization area in wild-type follicle cells. In a statistically significant manner, the percentage of Pipe-cis-Golgi marker colocalization area is significantly increased (58.5%) in rti overexpression and is significantly decreased (35.8%) in rti knockdown. Genetically, Rti acts upstream of Pipe, which is consistent with its role in influening the distribution of Pipe. These results show that Rti is capable to affect the subcellular localization of Pipe in the Golgi. Based in these finding, we propose that Rti is a new factor involved in DV axis formation by affecting the subcellular localization of Pipe. In the future, we will use the mutants of COPI (coat protein complex I), mediating intra-Golgi and Golgi to ER vesicle trafficking, and COPII (coat protein complex II), mediating ER to Golgi vesicle trafficking, to examine whether Rti participates in the forward or retrograde trafficking of Pipe in the Golgi. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15837 |
全文授權: | 未授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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