跳到主要內容

臺灣博碩士論文加值系統

(35.172.136.29) 您好!臺灣時間:2021/07/29 07:50
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:魏淑宜
研究生(外文):Shu-Yi Wei
論文名稱:細胞粘著分子Echinoid在果蠅發育過程多重功能之研究
論文名稱(外文):Echinoid, an essential component of adherens junction, has multiple functions in Drosophila development
指導教授:徐瑞洲
指導教授(外文):Jui-Chou Hsu
學位類別:博士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:114
中文關鍵詞:細胞粘著分子EGFR訊號傳遞Notch訊號傳遞
外文關鍵詞:Cell adhesion moleculesEGFR signal pathwayNotch signal pathway
相關次數:
  • 被引用被引用:0
  • 點閱點閱:141
  • 評分評分:
  • 下載下載:7
  • 收藏至我的研究室書目清單書目收藏:0
Echinoid (Ed) 是一類似於細胞黏著分子(CAM)的蛋白,具有7個immunoglobulin (Ig) domains、2個fibronectin type III (Fn III) domains、一個transmembrane (TM) domain以及C端315個胺基酸和其他分子在結構上及功能上無相似性的區域,其最後四個胺基酸(EIIV)為非典型的PDZ-binding motif。
已極化的表皮細胞會形成單層結構,由表皮間隙的分子群將表皮與側皮以及基皮區隔開來,這些分子開啟了細胞與細胞之間的粘著性,進而使特定的分子送達到特定區域以維持細胞的極性以及完整性。Adherens junctions (AJs)主要有DE-cadherin、β-catenin/Armadillo (Arm)、α-catenin等分子分布,DE-cadherin其分子細胞外的區域會與鄰近細胞的DE-cadherin結合,而細胞內的區域會與Arm、α-catenin連結,DE-cadherin complex將actin polymers連接到細胞膜上的受器,以維持細胞和細胞之間的粘著性。
我們於果蠅翅盤進行遺傳混合性同型合子分析以及免疫化學染色以釐清Ed在作為細胞粘著分子所扮演的角色。結果顯示Ed與DE-cadherin以及Arm共同表現在AJs,Ed-細胞群聚成平滑的圓形,意味這些細胞被區隔開來。失去Ed造成細胞表面積變小,細胞黏著分子Arm、DE-cadherin和Actin大量聚積於AJs將Ed-細胞緊緊捉在一起。以上結果顯示Ed-和Ed-之間的親和力大於Ed-和Ed+之間。失去DE-cadherin有類似ed mutant的表現型,DE-cadherin-細胞表面積縮小,於AJs Ed與含有PDZ區域的分子Bazooka(Baz)大量增加,yeast two-hybrid analysis以及GST pull down assay結果得知Baz藉由PDZ區域與Ed、Arm結合,透過兩種不同的機制將Baz連結到AJs。綜合以上結果,Ed為一基本的細胞粘著分子與DE-cadherin協同調控細胞的粘著度。
Ed在果蠅發育過程中扮演了多重角色。在過去的研究發現Ed定義了一個全新的途徑,於果蠅眼盤透過抑制不適當的EGFR訊號以避免過多的第八號感光細胞(R8)被選出,使果蠅複眼具有正確數目的感光細胞以及錐細胞(cone cell)。藉由遺傳混合性同型合子分析(genetic mosaic analysis)得知Ed分子是透過Ed-Ed蛋白之間的作用將訊號傳遞給鄰近細胞。於本研究,在果蠅眼盤大量表現Ed以及另一細胞粘著分子Neuroglian (Nrg),Ed和Nrg具高度協同作用以抑制EGFR訊號,此效應需要Ed蛋白之細胞內區域而非Nrg。細胞凝聚測試(cell aggregation test)和免疫沉澱結果顯示Ed不僅會與Ed結合也會與Nrg結合,此外,Ed和Nrg在眼盤表現位置一致。綜合以上結果得知Nrg為Ed之異型合子配體,活化Ed之後進而拮抗EGFR的作用。
除了在複眼發育扮演了角色,Ed亦參予果蠅背板剛毛的發育。失去ed導致背板有多餘的大型剛毛、小剛毛密度增加,混合性同型合子分析指出多餘的感受器官前驅物(SOP)是源自於有achaete-scute表現的細胞群,為細胞自主性(cell-autonomous)的效應,ed mutant胚胎有過多神經細胞生成的表現型,這些結果指出ed和Notch (N)訊號有關聯。事實上,ed mutant會使前神經細胞群的E(Spl)m8表現量降低,ed mutant和N途徑上的分子有強大協同的作用,造成嚴重的神經性剛毛表現型。因此,在剛毛發育過程Ed可能會促進Notch訊號,與其在複眼發育抑制EGFR機制截然不同。然而,EGFR訊號亦作用在剛毛的發育,Ed透過加強N訊號或者直接抑制EGFR訊號去拮抗EGFR訊號促進剛毛發育的作用。
細胞粘著分子E-cadherin同時亦為抑制人類腫瘤形成的蛋白,E-cadherin調節細胞粘著性以維持細胞的完整度,失去E-cadherin腫瘤細胞易發生轉移,失去Ed與DE-cadherin有相似的效應,Ed調控細胞的粘著性意味其在腫瘤生成以及轉移過程是否扮演角色?此外,ed mutant胚胎有過多的中樞以及周邊神經細胞生成,Ed亦參予在果蠅複眼發育以及背板剛毛的形成過程,果蠅複眼感光細胞以及剛毛均為周邊神經細胞,顯示Ed對於神經細胞生成具有某種程度的影響。
Ed is a putative cell-adhesion molecule (CAM), which contains 7 immunoglobulin (Ig) domains, 2 fibronectin type III (Fn III) domains, and a transmembrane (TM) domain, followed by a 315 amino acid intracellular domain with no identifiable structural or functional amino acid motif whose last four amino acids (EIIV) are responsible for non-typical PDZ binding.
Polarized epithelial cells form a monolayer in which the apical is separated with the lateral and basal membrane by the apical junctional complex. Apical junctional comeplex first initiates cell-cell adhesion which in turn activates protein-sorting mechanism for establishing cell polarity and maintaining cell integrity. Adherens junctions (AJs) are mainly targeted by of DE-cadherin, β-catenin/Armadillo (arm), and α-catenin. DE-cadherin form homophilic binding via its extracellular Ig domains while its cytoplasmic region.bind with Arm and α-catenin linking membrane receptors to actin polymers. DE-cadherin complex therefore regulates cell-cell adhesion.
To investigate the canonical role of Ed as a CAM, we performed the mosaic analysis and immunochemistry staining in Drosophila wing imaginal disc. The results showed that Ed is colocalized with DE-cadherin, and Arm at the AJs and ed clones exhibit rounded and smooth countour indicating Ed- cells are sorted out. Removing of Ed from AJs will cause wing disc cells to have greatly reduced apical surface and highly accumulation of DE-cadherin, Arm, and actin at AJs to hold the Ed- cells together. Based on above results, it was shown that the affinity between Ed- and Ed- is higher than Ed- and Ed+. Similarly, removing of DE-cadherin will also produce cells with smaller apical surface but elevated Ed and the PDZ-containing protein Bazooka (Baz) at the position of AJs. Yeast two-hybrid analyses and GST pull down assay illustrate that Baz can both bind with Ed and Arm via its PDZ domain, thereby locating to AJs by Ed- or Arm-dependant mechanism. Together, these results indicate that Ed is an essential component of AJs and cooperates with DE-cadherin to mediate cell-cell adhesion.
Ed has multiple functions during Drosophila development. Previous studies found that Ed defines a novel pathway which limits R8 photoreceptor specification by inhibiting inappropriate EGFR signaling within R8 equivalence groups. Consequently, flies possess exact number of the photoreceptor and cone cells. Genetic mosaic analysis shows that Ed exerts its function by homophilic binding. In this study, co-expression of Ed and Neuroglian (Nrg), a L1-type CAM, in eye exhibits a strong genetic synergy in inhibiting EGFR signaling. This synergistic effect requires the intracellular domain of Ed, but not that of Nrg. Cell aggregation test and immunoprecipitation results show that Ed not only form homophilic binding but also engages heterophilic trans-interaction with Nrg. Furthermore, Ed co-localizes with Nrg in eye imaginal disc. Together, our results suggest a model in which Nrg acts as a heterophilic ligand and activator of Ed which in turn antagonizes EGFR signaling.
Besides ommatidium specification, Ed also involves in mesothoracic bristle development. Loss-of-function of ed leads to the formation of extra macrochaetae near the extant ones and increases the density of microchaetae. Analysis of ed mosaics indicates that extra sensory organ precursors (SOPs) arise from proneural clusters of achaete-scute expression in a cell-autonomous way. ed embryos also exhibit a neurogenic phenotype. These phenotypes suggest a functional relation between ed and the Notch (N) pathway. Indeed, mutation of ed reduces the expression level of the N pathway effector E(spl)m8 in proneural clusters. Moreover, combinations of moderate loss of function for ed and for different components of N pathway show clear synergistic effects on neurogenic bristles phenotypes. We conclude that Ed facilitates N pathway in mesothoracic bristle development, but not like its inhibiting EGFR signal in eye development. However, EGFR also acts in bristle development. We present that Ed antagonizes the bristle promoting activity of the Egfr pathway, either by enhancement of N pathway or , similar to the eye, by more direct effect on EGFR signal.
E-cadherin is also a tumor suppressor gene in human. Disruption of E-cadherin-meditated adhesion is a key step in progression toward invasive phase of carcinoma. Loss-of-function conditions for ed and DE-cadherin cause silimar phenotypes. Ed involves in cell-cell adhesion that means Ed also play a role in tumorigenesis and metastasis? Furthermore, ed embryo exhibit extra C.N.S. and P.N.S. neurons. Ed also serves in Drosophila eye development and mesothoracic bristle development. Photoreceptor cells of the ommatidium and bristles are sensory nerves. We suggest that Ed might meditate the development of the nervous tissues.
Contents 1
Publication List 4
Abbreviations 5
中文摘要 7
Abstract 9
Chapter 1
Background
1.1 Cellullar junctions 11
1.2 Cell-adhesion molecules 13
1.3 Echinoid 14
1.4 Friend of Echinoid 15
1.5 Neuronglian 16
1.6 Drosophila eye development 16
1.7 Drosophila mesothoracic bristle development 21
1.8 EGF receptor pathway 25
1.9 Notch pathway 29
1.10 Specific Aims 31

Chapter 2
Echinoid, an essential component of adherens junction, cooperates with DE- cadherin to mediate cell-cell adhesion
2.1 Abstract 32
2.2 Introduction 33
2.3 Materials and Methods
Drosophila stocks and Mosaic analysis 35
Histochemistry 35
Molecular biology 36
Protein interaction assays 36
2.4 Results
Echinoid and Bazooka are co-localized to adherens junctions of Drosophila wing imaginal disc 37
ed1x5 mosaic clones exhibit round and smooth contours and
highly accumulation of DE-cadherin, Armadillo and associated actin 37
Loss of Ed mainly affects levels of AJs molecules and the reduced apical surface continues to Gap junctions 39
loss-of- function of DE-cadherin causes silimar phenotypes as ed clones 40
Bazooka binds with Ed and Arm 40
2.5 Discussion
Ed co-operates with DE-cadherin to meditate cell-cell adhesion 41
The reduced apical surface continues to Gap junctions in Ed- cells 42
Does DE-cadherin/Arm signals increase in Ed- cells? 42
2.6 Figures 44

Chapter 3
Neuroglian activates Echinoid to antagonize the Drosophila EGF receptor signaling pathway
3.1 Abstract 49
3.2 Introduction 50
3.3 Materials and Methods
Drosophila stocks 51
Molecular Biology 52
Histology 52
Transfection and maintenance of S2 cells 52
Cell aggregation assays 53
SDS-polyacrylamide gel electrophoresis and Western blot analysis 53
Co-immunoprecipitation procedure 53
3.4 Results
Over-expression of ed and nrg results in a loss of photoreceptor and cone cellsin the developing Drosophila imaginal eye disc 54
Ed is co-localized with Nrg 55
Ed acts as a homophilic adhesion protein 55
Ed and Nrg engage in a robust heterophilic trans-interaction 56
Ed is co-immunoprecipitated with Nrg 56
Ed acts as receptor in the signal-receiving cells 57
3.5 Discussion
Nrg is a heterologous ligand of Ed 58
Nrg is an autonomous activator of RTK 59
Autonomous vs. non-autonomous effects of ed on EGFR signaling 59
3.6 Tables 60
3.7 Figures 62

Chapter 4
Echinoid synergizes with the Notch signaling pathway in Drosophila mesothorax bristle patterning
4.1 Abstract 70
4.2 Introduction 71
4.3 Materials and Methods
Drosophila stocks 73
Molecular biology 73
Mosaic analysis 73
Histochemistry 74
4.4 Results
LOF mutations at the ed locus promote development of extra bristles 74
Overexpression of ed 76
Generation of a dominant negative form of Ed 76
Effects on ac/sc and E(spl)m8 expression 77
Interactions between ed and N signaling in bristle development 77
Ed colocalizes with N at the zonula adherens of wing imaginal disc cells 80
ed produces a moderate neurogenic phenotype in the embryo 80
Antagonistic activities between Ed and EGFR pathway 81
4.5 Discussion
ed facilitates N signaling 81
Interaction of ed with the N signaling pathway 82
Ed and EGFR signaling 84
ed and fred 85
4.6 Tables 87
4.7 Figures 88

Chapter 5
Conclusion
5.1 Conclusions 96
5.2 References 98
Ahmed, A., Chandra, S., Magarinos, M. and Vaessin, H. (2003). echinoid mutants exhibit neurogenic phenotypes and show synergistic interactions with the Notch signaling pathway. Development 130, 6295-6304.
Anderson, D. J. and Blobel, G. (1983). Immunoprecipitation of proteins from cell-free translations. Methods Enzymol. 96, 111-20.
Artavanis-Tsakonas, S., Matsuno, K. and Fortini, M. E. (1995). Notch signaling. Science 268, 225-232.
Artavanis-Tsakonas, S., Rand, M. D. and Lake, R. J. (1999). Notch signaling: cell fate control and signal integration in development. Science 284, 770-776.
Artero, R. D., Castanon, I. and Baylies, M. K. (2001). The immunoglobulin-like protein Hibris functions as a dose-dependent regulator of myoblast fusion and is differentially controlled by Ras and Notch signaling. Development 128, 4251-4264.
Bachmann, A., Schneider, M., Theilenberg, E., Grawe, F. and Knust, E. (2001). Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity. Nature 414, 638-643.
Bai, J., Chiu, W., Wang, J., Tzeng, T., Perrimon, N and Hsu J.(2001). The cell adhesion molecule Echinoid defines a new pathway that antagonizes the Drosophila EGF receptor signaling pathway. Development 128, 591-601.
Bailey, A. M. and Posakony, J. W. (1995). Suppressor of Hairless directly activates transcription of Enhancer of split Complex genes in response to Notch receptor activity. Genes Dev. 9, 2609-2622.
Baker, N. E. and Yu, S. Y. (1997). Proneural function of neurogenic genes in the developing Drosophila eye. Curr. Biol. 7, 122-132.
Baker, N. E. and Yu, S. Y. (2001). The Egf receptor defines domains of cell cycle progression and survival to regulate cell number in the developing Drosophila eye. Cell 104, 699-708.
Banerjee, U., Renfranz, P. J., Hinton, D. R., Rabin, B. A. and Benzer, S. (1987). The sevenless+ protein is expressed apically in cell membranes of developing Drosophila retina; it is not restricted to cell R7. Cell 51, 151-158.
Banerjee, U., Renfranz, P. J., Pollock, J .A. and Benzer, S. (1987a). Molecular characterization and expression of sevenless, a gene involved in neuronal pattern formation in the Drosophila eye. Cell 49, 281-291.
Bang, A. G., Hartenstein, V. and Posakony, J. W. (1991). Hairless is required for the development of adult sensory organ precursor cells in Drosophila. Development 111, 89-104.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關論文