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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 周子賓 | |
dc.contributor.author | Shu-Chuan Chang | en |
dc.contributor.author | 張淑娟 | zh_TW |
dc.date.accessioned | 2021-06-08T06:05:37Z | - |
dc.date.copyright | 2011-08-08 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-08-05 | |
dc.identifier.citation | Reference
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/25219 | - |
dc.description.abstract | 果蠅發育過程中背-腹軸(dorsal/ventral axis)的建立是由三大訊號來調控,分別為背部的濾泡細胞(follicle cells)中Egfr訊號傳遞、腹部的濾泡細胞中Pipe表現,活化一系列絲胺酸蛋白酶(Serine proteases)的連鎖反應以及胚胎細胞內Toll的訊號傳遞。
Rotini (Rti)是人類GOLPH3蛋白的同源物,Rti參與硫醣蛋白(HSPGs; Heparan Sulfate Proteoglycans)之多醣鏈生成所必需,先前的研究已知Rti 協助Exostosin蛋白聚合酵素在高基氏體的傳送運輸,使多醣鏈的聚合反應得以進行。當rti缺失時會使硫醣蛋白生成異常,導致Hedgehog型態決定素的不正常表現和缺損的訊號傳遞。 此外,當Rotini (Rti)在生殖細胞(germ cells)內缺失會造成胚胎扭曲,而本論文證實Dorsal 入核的表現也受到影響而確立Rti參與了背腹軸的建立;然而,Rti並非藉由調控硫醣蛋白來影響果蠅卵子與胚胎背腹軸的形成。 卵子發育過程中,早期階段Rti主要表現在生殖細胞,但到了晚期階段則只表現於濾泡細胞內,當Rti在生殖細胞內缺失時會造成濾泡細胞內的Rti產生不正常的入核現象,本論文推測生殖細胞和濾泡細胞間存在有嚴密的互動。 在果蠅S2 細胞株裡, Rti明顯地影響Pipe蛋白在細胞內的表現情形。Pipe在高基氏體內協助醣蛋白質(proteoglycan)的硫修飾;基於Rti調控硫醣蛋白合成所需的聚合酵素在高基氏體內的運輸, 本論文推測Rti或參與Pipe蛋白酵素在高基氏體內的運送。 我們觀察背-腹軸建立所需之一系列相關蛋白的行為,發現到當Rti缺失時會造成Snk (Snake)蛋白無法正常坐落於卵母細胞(oocyte)表層,並在卵母細胞質中形成累積塊;此結果與抑制胞吐作用(exocytosis)時造成Snk蛋白的累積性狀是一致的。類似的,Rti 缺失亦會降低Gd (Gastrulation defective) 蛋白在卵母細胞表層(cortex)的表現量,這再次顯示Rti在卵母細胞內應也參與了分泌物質的運送。 Rti及其同源物與4-磷脂醯肌醇(phosphatidylinositol 4-phosphate)有直接的相互作用,且Rti之同源物皆參與了細胞內運輸。 已知4-磷脂醯肌醇大量存留於高基氏體的膜上且為細胞內分泌性囊泡運輸所必需,Rti或是以輔助者的角色來協助許多物質的運送。由Rti影響背腹軸決定分子的輸送行為,本論文推測Rti除了可以在高基氏體內協助酵素的傳送外,也可能藉由和4-磷脂醯肌醇的鍵結參與了許多高基氏體外的分泌性運輸。 | zh_TW |
dc.description.abstract | The dorsal-ventral axis formation in Drosophila development is regulated by three major signaling: Egfr signaling pathway in dorsal follicle cells, the expression of Pipe in ventral follicle cells that actives the serine proteases cascade, and the Toll pathway in embryo.
Previous studies have proved that Rotini (Rti), a Drosophila homologue of human GOLPH3, is necessary for the biogenesis of Heparan Sulfate Proteoglycans (HSPGs) GAG chains. Rti is involved in the trafficking of these polymerases, EXT proteins, in Golgi complex to regulate the polymerization of GAG chains. As a result, mutated rti will cause the defects of HSPGs biosynthesis and Hedgehog (Hg) signaling. rti loss-of-function in germline will display twisted embryos and affect the distribution of Dorsal proteins. Additionally, these results indicate Rti participates in D/V axis formation. But we definitively rule out the possiblity of Rti modulates the synthesis of HSPGs to establish the D/V axis. During Drosophila oogenesis, Rti is expressed in germline at early stages, but majorly in follicle cells when developing egg chambers enter later stages. Losing function of rti in germline would result in abnormal expression pattern of Rti in follicle cells. That reveals there is a dynamic interaction between germline and follicle cells. In Drosophila S2 cells, Rti obviously affects the expression of Pipe proteins. Pipe is a sulfotransferase and works in Goigi complex. Additionally, we conjecture that Rti may assist the transport of Pipe and the mechanism is like the one which that Rti regulates the trafficking of the EXTs in Golgi. We observed a series of behaviors of D/V related proteins in Drosophila oogenesis and found rti loss-of-function in germline reduces the localized stability of Snake (Snk) proteins at the cortex and clump over the space of oocyte. This irregular distribution of Snk in rti1164A4 GLC background is similar to the result when the exocytosis is blocked. Similarly, rti loss-of-function in germline also affects the distribution of Gastrulation defective (Gd) proteins. These results suggest Rti may participate in secreted transport in oocyte. Related published papers have proved that there is a direct interaction between Rti and phosphatidylinositol 4-phosphate (PtdIn4P). Furthermore, GOLPH3 homologues are involved in different trafficking pathways. PtdIn4P is specifically enriched on Golgi apparatus in human and performs an essential role in secretory pathway regulating exit of cargo from TGN. Additionally, based on our findings, we conjecture that Rti may be an auxiliary and participate in multiple trafficking pathways for different protein effectors. In other words, Rti not only mediates the trafficking of the specific enzymes in Golgi, but also collaborates with PtdIn4P in the sorting of proteins into transport vesicles for exocytosis or delivery to the endosomal system. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T06:05:37Z (GMT). No. of bitstreams: 1 ntu-100-R98b43025-1.pdf: 4327234 bytes, checksum: d3b8fcc98581e6fe73534b6c401f7679 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | Table of contents
碩士學位論文口試委員會審定書 致謝…………………………………………………………………i 中文摘要……………………………………………………………ii Abstract…………………………………………………………..... iv List of figures………………………………………………………xiii Abbreviations……………………………………………………….xv Introduction………………………………………...……………………1 I. Overview of Drosophila oogenesis……………………………...………1 II. Dorsoventral axis formation in Drosophila embryo….….….….………3 1. Asymmetrically localized Gurken activates the EGF receptor (Egfr) and determines dorsal follicle cell fates……………………………….……3 2. The ventralising cascade of Drosophila…………...……………………4 3. The cytoplasmic events downstream of the Toll receptor.…..……………13 4. The generation of distinct cell types along the dorsoventral axis……...….14 III. HSPGs are required for morphogens distribution and signaling..……15 1. Development events are governed by positional information established by morphogens……….…..…………………………….………………15 2. Formation of secreted morphogen gradient………….…………………16 3. HSPGs modulate morphogen distribution and signaling………….…..…17 4. HSPGs play an important role in regulating morphogen behaviors…...……20 IV. Rotini, the Golgi protein GOLPH3 in human, is required for HSPGs biogenesis……..………………………………………………………21 1. Rotini does not have any conserved domain……….……………………22 2. Rotini and related homozugues function in Golgi trafficking…......………22 3. Rotini is required for Hh expression and signaling……...………….……25 4. Rotini mediates the HSPGs biogenesis……....…………………………26 V. PtdIns(4)P performs an essential role in the secretory pathway.………27 1. The spatial distributions of phosphoinositide lipids are tightly regulated.…….…..…………………………………………………27 2. PtdIns(4)P is involved in membrane biosynthetic and vesicle budding machineries to coordinate the Golgi functions.…………………………28 VI. The aim of this thesis.…………………………………………………34 Materials and Methods…..……………………………………………36 1. Fly stocks and maintenance……..……………………………………36 2. The autosomal FLP-DFS technique……...……………………………36 3. Heat shock treatment……...…………………………………………38 4. GAL4-UAS system………..…………………………………………39 5. Drosophila S2 cells maintenance……...………………………………39 6. Transient transfection of S2 cells………………………………...……41 7. rotini dsRNA synthesis…..…..………………………………………42 8. Cloning constructs……..……………………………………………45 9. Immunohistochemistry……..……………………………..…………47 10. Western blotting analysis……..………………………………………49 11. Membrane stripping….....……………………………………………50 Results……...……………………………………………………………52 I. rti loss-of-function in germline affects dorsoventral axis formation in Drosophila embryo…..………………………..………………………53 1. rtiGLC embryos display weak dorsalized phenotype…….………………53 2. Rti affects the graded nuclear location of Dorsal….….…………………54 II. Ttv-dependent HSPGs play no role in dorsoventral axis formation…..55 III. The expression pattern of Rotini in Drosophila oogenesis and embryogenesis…...……………………………………………………57 1. The production of Rotini antibody…….………………………………57 2. Rti is expressed in both germline and follicle cells………………………58 3. Rti is ubiquitously expressed in embryo……..…………………………59 IV. Rti can enter follicle cell nuclei in rti GLC ovary.......…………………59 V. Rti does not affect the distribution of Gurken...………………………60 VI. During oogenesis, the expressions of Windbeutel (Wind) and Nudel (Ndl) are not affected when rti is mutated………...…………………………61 1. rti loss-of-function does not affect the expression of Wind.………………62 2. The behavior of Ndl is not affected when Rti function is lost…..…….65 VII. Rti regulates the expressions of Windbeutel (Wind), Slalom (Sll) and PipST2 proteins in Drosophila S2 cells….……………………………67 1. Wind, Sll and PipST2 proteins are down regulated in S2 cells whose Rti expression is suppressed by RNAi…....………………………………68 2. PipST2 is obviously up regulated in S2 cells whose Rti is over expressed…..………………………………………………………69 3. The linear relationship between the proteins of PipST2 and Rti….…..……70 VIII. Rti may be involved in transporting Gastrulation defective (Gd) and Snake (Snk) from oocyte to perivitelline space…………………71 1. The distribution of Gd is affected by rti GLC mutation…….……………72 2. Rti is necessary to mediate the behavior of Snk in oocyte….….…………73 3. The novel function of Rti may involve in secreted transport…….……......74 Discussion......……………………………………………………….77 1. Rti plays a role in dorsoventral axis formation….…………………78 2. Rti does not act through HSPGs to influence D/V axis formation…...……………………………………………………79 Missing the functions of HSPGs biosynthetic enzymes does not affect D/V patterning…...……………...………………………………………79 HSPGs are not produced when D/V axis is established….………………80 The absence activity of ttv does not affect the expression of Dorsal…….…80 3. Rti functions in both germline and follicle cells…….………….…81 Rti has no Nuclear localization signal (NLS)...………………………....82 4. Rti function in follicle cells may influence the dorsoventral axis formation..……………………………………………………….83 The possibility of Rti regulates the behavior of PipST2 during oogenesis….………………………………………………………84 The depletion of both functions of Rti in germline and follicle cells will elucidate more….…….……………………………………………85 5. Rti may be involved in exocytotic trafficking…....………………86 Rti obviously affects the distributions of Gd and Snk……...……………87 Rti may transport these serine proteases from oocyte to perivitelline space…….…………………………………………………………89 The putative mechanism of Rti and PtdIn4P collaborate in secreted transport….…….………………………………………………….90 6. Future prospects…………………………………………………92 Reference……....……………………………………………………95 Table…………………………………………………………………….120 Figures……...…………………………………………………………..121 | |
dc.language.iso | en | |
dc.title | 果蠅GOLPH3(Rotini)調控背腹軸形成的探討 | zh_TW |
dc.title | The Approach of Drosophila GOLPH3, Rotini,
in Modulating Dorsal-Ventral Axis Formation | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 黃偉邦,顆逢春,王致恬,溫進德 | |
dc.subject.keyword | 背-腹軸,硫醣蛋白,4-磷脂醯肌醇,胞吐作用,分泌性運輸, | zh_TW |
dc.subject.keyword | dorsal/ventral axis,HSPGs,phosphatidylinositol 4-phosphate,exocytosis,secreted transport, | en |
dc.relation.page | 151 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2011-08-05 | |
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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