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Title: | 果蠅GOLPH3(Rotini)調控Pipe的逆向運輸並影響背腹軸的發育 Drosophila GOLPH3, Rotini, mediates the retrograde trafficking of Pipe and influences the dorsal-ventral polarity |
Authors: | Po-Lin Chen 陳柏霖 |
Advisor: | 周子賓(Tze-Bin Chou) |
Keyword: | 高基氏體蛋白質GOLPH3,多醣蛋白,背腹軸,硫基轉移酶,逆向運輸, GOLPH3,Heparan Sulfate Proteoglycans (HSPGs),Dorsal-Ventral polarity (DV),Sulfotransferase,Retrograde, |
Publication Year : | 2013 |
Degree: | 碩士 |
Abstract: | 在果蠅的胚胎發育中,背腹軸(dorsal-ventral axis)的生成主要由兩條訊息傳遞鏈決定,分別是決定背部發育的EGFR訊息鏈以及決定腹面發育的一系列絲胺酸蛋白酶(serine protease)。Pipe為硫基轉移酶,作用在濾泡細胞中的高基氏體,調控這一系列絲胺酸蛋白酶的活化,最後轉錄因子Dorsal入核決定胚胎腹面發育。當pipe缺失時會造成胚胎背部化(dorsalized)。
Rotini(rti)是人類GOLPH3蛋白同源物,除了已知rti參與多醣蛋白(HSPGs, Heparan Sulfate proteoglycan)的生合成外,也發現當rti在果蠅生殖細胞缺失時,會造成胚胎背部化,並影響轉錄因子Dorsal分佈,此外,濾泡細胞的rti缺失時,則會影響腹面齒狀帶的發育,這些證據指出表現於生殖及濾泡細胞的rti皆會影響背腹軸的發育。在果蠅S2細胞株中也證明Rti與Pipe有交互作用,且濾泡細胞中的Rti表現量會影響Pipe的座落。因此,濾泡細胞中的rti是透過何種機制調控Pipe的座落,進而影響背腹軸發育,則是本文探討的主題。 本文利用果蠅幼蟲的翅碟作為實驗材料,其原因為基因型容易操控,細胞數多,且具典型高基氏體形態,適合於電子顯微鏡下研究Pipe的座落。實驗結果首先確定Pipe能正確在翅碟表現,且在rti量上升時,Pipe與cis-Golgi重疊面積增加,rti下降時,重疊面積減少,證明Pipe在翅碟的分佈與濾泡細胞一致,也暗示rti可能藉由高基氏體內的逆向運輸(retrograde)調控Pipe的座落。在外殼蛋白I或Ⅱ(COPI,Ⅱ; coat protein complex I,Ⅱ)缺失時,會影響Pipe的座落分佈,將rti補回便能救回此兩種突變背景所造成的Pipe錯位,證明Rti可能同時參與正向(anterograde)與逆向運輸。進一步利用電子顯微鏡觀察,正常情況下Pipe分佈於整個高基氏體,當rti大量上升時,會造成Pipe累積在mid-及cis-Golgi;rti下降時則使Pipe離開高基氏體,並分佈在細胞質、多泡體(multivesicular body)及溶酶體(lysosome)。而在rti下降時,Pipe並未回到內質網,表示Rti可能參與非COPⅡ調控的正向運輸。此外,免疫沉澱實驗證明Pipe與COPI及COPⅡ皆有交互作用,然而Rti只與COPI有交互作用。證明rti會與外殼蛋白I合作,透過逆向運輸調控Pipe的座落。且此種逆向運輸效應受到rti劑量所調控。 根據以上結果,推論表現於濾泡細胞的rti會藉由與COPI的交互作用,調控Pipe的逆向運輸,並影響果蠅胚胎背腹軸的發育。 The dorsal-ventral (D/V) axis of Drosophila embryo is determined by two signaling pathways: the dorsal side of embryo is regulated by EGFR pathway in follicle cells, and ventral side is mediated by the serine protease cascade. Pipe encodes a sulfotransferase that functions in Golgi of follicle cells to activate the serine protease cascade. Finally, the transcription factor, Dorsal, enters the nucleus to determine the development of ventral side of embryo. The pipe mutants show a dorsalized embryo. Rotini (rti), a homologue of human GOLPH3 protein, has been known to be involved in HSPGs modification. In addition, rti germline clone (GLC) shows a dorsalized embryo and affects the distribution of Dorsal, and rti follicle cell clone causes denticle belts disappeared. It suggests both rti in germline and follicle cells can influence the D/V polarity. Previous studies reveal rti interacts with Pipe in Drosophila S2 cells, and Pipe subcellular localization is altered with rti dosage in follicle cells. The thesis is aimed to investigate how rti of follicle cells regulate Pipe trafficking to affect the D/V polarity. The wing imaginal discs of Drosophila are utilized because of controllable genotype, large cell numbers, and its typical Golgi morphology is suitable to study Pipe under electron microscope. The results show Pipe is localized accurately in wing disc cells, and the overlapping area with cis-Golgi marker increases when rti is overexpressed, but decreases when rti is knocked down. It indicates that subcellular distribution of Pipe in disc cells is consistent with follicle cells, and suggests rti may regulate the retrograde trafficking of Pipe in Golgi. Pipe subcellular localization is affected in COPI or COPII mutants, and rti overexpression can suppress Pipe mislocalization in COPI and COPII mutants, representing that Rti is involved in both anterograde and retrograde trafficking. Under electron microscope, Pipe is distributed in the whole Golgi, but accumulated in mid- and cis-Golgi when rti is overexpressed. However, Pipe is distributed in cytosol, multivesicular bodies and lysosomes when rti is knocked down. It is further found Pipe is not accumulated in ER when rti is knocked down, suggesting Rti may be involved in a COPII-independent anterograde pathway. In co-immunoprecipitation assay, Pipe can interact with COPI and COPII, but Rti only interact with COPI. In conclusion, rti can interact with COPI to mediate retrograde trafficking of Pipe. In addition, the retrograde degree is dependent on Rti dosage. Based on these results, rti in follicle cells can interact with COPI to mediate Pipe subcellular localization by retrograde and influence D/V polarity of Drosophila embryo. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61040 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 分子與細胞生物學研究所 |
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