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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15837完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 周子賓(Tze-Bin Chou) | |
| dc.contributor.author | Yu-Ching Lin | en |
| dc.contributor.author | 林于敬 | zh_TW |
| dc.date.accessioned | 2021-06-07T17:53:16Z | - |
| dc.date.copyright | 2012-08-27 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-17 | |
| dc.identifier.citation | Anderson KV, Nusslein-Volhard C (1984) Information for the dorsal--ventral pattern of the Drosophila embryo is stored as maternal mRNA. Nature 311: 223-227.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15837 | - |
| dc.description.abstract | 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。 | zh_TW |
| dc.description.abstract | 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. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-07T17:53:16Z (GMT). No. of bitstreams: 1 ntu-101-R99b43030-1.pdf: 8444294 bytes, checksum: 474a4be42337e06716bba90ae82f1db6 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 誌謝 i
中文摘要 ii Abstract iv List of tables xii List of figures xiii Abbreviations xv Introduction 1 I. Drosophila oogenesis 1 II. The dorsoventral axis formation in Drosophila embryonic development 3 1. The dorsal follicle cell fates are determined by the EGF receptor (Egfr) 4 2. An extracellular proteolytic cascade transmits the ventralising signal 5 3. The cytoplasmic events downsream of the Toll receptor 14 4. The generation of distinct cell types along the dorsoventral axis 15 III. Pipe sulfotransferase, a key determinant of embryonic DV polarity 16 1. Windbeutel (Wind; Wbl) is required for the function and localization of Pipe 18 2. Pipe activity in the ovary and the embryonic salivary gland does not require heparin sulfate glycosaminoglycans (HSPGs) 19 3. The vitelline membrane-like (VML) undergoes Pipe-dependent sulfation and affects DV pattern in hypomorphic pipe mutant background 20 IV. Heparan Sulfate Proteoglycans (HSPGs) modulation in Hedgehog (Hh) signaling pathway 21 1. Morphogens establish the positional information to regulate the development events 21 2. The graded signals establish extracellular morphogens 23 3. HSPGs regulate the distribution and signaling of morphogens 23 4. HSPGs is crucial for regulating morphogen behaviors 27 V. Rotini (Rti), the homolog of human GOLPH3 in Drosophila, involved in HSPGs biogenesis 28 1. Identification of rotini 28 2. Rti has no any conserved domains 28 3. Rti homologues function in Golgi trafficking 28 4. Rti regulates the retrograde trafficking of EXTs in HSPGs synthesis. 32 VI. The role of Rotini in the dorsal-ventral (DV) axis formation 34 1. rti GLC embryo displays weak dorsalized phenotype 34 2. Rti affects the graded nuclear location of Dorsal 35 3. Rti does not affect the distribution of Gurken 35 4. The linear relationship exists between Rti and Pipe 36 VII. The mechanism of versicle-mediated transport in the Golgi apparatus 37 VIII. The aim of this thesis 39 1. Fly stock 41 2. The autosomal FLP-DFS technique 41 3. Heat shock treatment 41 4. GAL4-UAS system 41 5. Generation of recombinant mutant clones in somatic tissue 21 6. Drosophila S2 cells maintenance 21 7. Transient transfection of S2 cell 21 8. Immunoprecipitation assay 21 9. Yeast two-hybrid assay 22 10. Cloning constructs 23 11. Fluorescence antibody staining of ovary 23 12. Western blotting analysis 24 13. Membrane stripping 24 14. Cuticle preparation 24 I. Rti does not act through HSPGs to affect dorsoventral axis formation in early embryos. 26 1. Ttv, Botv and Sotv are not detected during Drosophila oogenesis 26 2. The absence of Ttv, Botv and Sotv during oogenesis is further confirmed in ttv, botv and sotv mutant clone respectively. 26 3. EXT antibodies can recognize EXT proteins when EXTs are overexpressed in follicle cells. 27 II. In genetics, Rti has a genetic interaction with Pipe and acts upstream of Pipe. 27 1. Loss of VML expression increases the percentage of early-dead embryos in rti GLG 27 2. In genetics, Pipe is epistatic to Rti in ovarian follicle cells. 28 III. Rti is partially colocalized with Pipe in Drosophila ovarian follicle cells and interacts with Pipe in Drosophila S2 cells. 28 1. Rti is partially colocalized with Pipe in Drosophila ovarian follicle cells 29 2. Rti interacts with Pipe in Drosophila S2 cells with co-immunoprecipitation assay 29 3. Rti does not interact with Pipe in yeast two-hybrid assay 30 IV. Rti is capable to influence the subcellular localization of Pipe in the Golgi apparatus. 31 1. The subcellular localization of Pipe in the Golgi apparatus is altered in variant protein level of rti. 31 2. In statistical analysis, the value of colocalization percentage in rti overexpression or knockdown comparing to wild type is significantly different. 31 3. Pipe is not localizaed in ER when rti is knocked down 32 V. The mislocalized ratio of Pipe in rti knockdown condition is not affected when COPI or COPII is heterozygous mutated. 32 I. Rti affects the dorsoventral axis formation in a HSPGs-independent manner. 35 ttv and other GAG synthesis mutants show no defects in DV patterming. 35 ttv mutant in germ line does not affect the Dorsal nuclear uptake in early embryos. 35 GAG modifications are absent when DV axis is established. 36 Ttv, Botv and Sotv are not expressed during Drosophila oogenesis. 36 II. Rti derived from the follicle cells might participate in the DV axis formation by influencing the localization of Pipe in the Golgi. 36 Rti is expressed in both germ line and follicle cells during oogenesis. 36 The phenotype of dorsalized embryo is caused by rti GLC. 36 III. Rti might mediate the retrograde trafficking of Pipe in the Golgi complex. 37 IV. Future prospects 39 Reference 43 Table 76 Figure 78 | |
| dc.language.iso | en | |
| dc.subject | 硫基轉移酵素 | zh_TW |
| dc.subject | 高基氏體蛋白質GOLPH3 | zh_TW |
| dc.subject | 多醣蛋白HSPG | zh_TW |
| dc.subject | GAG醣鏈聚合酵素EXT | zh_TW |
| dc.subject | 背腹軸 | zh_TW |
| dc.subject | GOLPH3 | en |
| dc.subject | Sulfotransferases | en |
| dc.subject | Dorsoventral (DV) axis | en |
| dc.subject | Exostosins (EXT) | en |
| dc.subject | Heparan Sulfate Proteoglycans (HSPG) | en |
| dc.title | 果蠅GOLPH3(Rotini)影響Pipe在高基氏體的座落分布 | zh_TW |
| dc.title | Drosophila GOLPH3, Rotini, Affects the Subcellular Localization of Pipe in the Golgi Complex | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃偉邦,溫進德,董桂書 | |
| dc.subject.keyword | 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, | zh_TW |
| dc.subject.keyword | GOLPH3,Heparan Sulfate Proteoglycans (HSPG),Exostosins (EXT),Dorsoventral (DV) axis,Sulfotransferases, | en |
| dc.relation.page | 149 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2012-08-19 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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