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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李芳仁 | |
dc.contributor.author | Chieh-hsin Chen | en |
dc.contributor.author | 陳櫛昕 | zh_TW |
dc.date.accessioned | 2021-06-14T16:53:07Z | - |
dc.date.available | 2016-10-05 | |
dc.date.copyright | 2011-10-05 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-08-12 | |
dc.identifier.citation | Andrews, W. D., M. Barber, et al. (2007). 'Slit-Robo interactions during cortical development.' J Anat 211(2): 188-198.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/40609 | - |
dc.description.abstract | 中文摘要
腺嘌呤核苷二磷酸核醣化因子 (ADP-ribosylation factor, ARF) 是一群 GTPase 的集合,主要的功能是參與在囊泡傳遞與細胞骨架的調控。而腺嘌呤核苷二磷酸核糖化相似因子 4A (ARL4A) 則是 ARF 的子家族:腺嘌呤核苷二磷酸核醣化因子相似蛋白 (ARL) 家族的其中一員,先前的研究指出:在胚胎發育的過程中,ARL4A 的 mRNA 會表現在大腦分區、體節形成之區域,因此推測 ARL4A 可能與神經系統的發育有關,但是與 ARL4A 相關的生理機制目前仍是未知。為了找出 ARL4A 可能的功能,我們運用酵母菌雙雜交系統 (Yeast Two‐Hybrid System)以 ARL4A為誘餌來篩選人類胚胎時期腦的 cDNA library ,在這些被篩選出的 ARL4A 結合蛋白中,我們挑選了 Robo1 來進行後續的研究。 Robo1 是一個會表現在神經軸突生長端的一個蛋白質受器,他會接收他的受質:slit2 的訊息,進而去調控神經細胞軸突的生長方向;而 slit2 與 Robo1 的訊息機制也被發現到會去影響其他種類的細胞,並抑制細胞的移動與爬行 (例如:白血球細胞、乳癌細胞與肺癌細胞等等) 。而我們研究的主題,便是在探討 ARL4A 與Robo1 之間的作用與功能。 首先我們製備了抗 Robo1 的抗體,在免疫染色中,這個抗體能夠辨認到外生性的 Robo1。在酵母菌雙雜交系統中,我們發現到 Robo1 的其中一段位於 C 端的小片段(1394- 1454) 即足夠能與 ARL4A 有交互作用,且相較於野生型的 ARL4A 或 ARL4A‐Q79L,Robo1 比較偏好於與ARL4A-T34N 進行作用。進一步地,我們在這段片段中利用突變的方法搜尋主要與 ARL4A 結合的胺基酸,我們找到了其中兩個位置突變的 Robo1 (Robo1-A1, Robo1-A2) 無法與 ARL4A 作用。我們同時也用pull down assay 來確認酵母菌雙雜交系統中所看到的這些結果,結果亦顯示:Robo1 是利用他的 C 端與 ARL4A 作用,而上述兩個突變的 Robo1 的確失去了與 ARL4A 作用的能力。然而不同的是,在 pull down assay 的結果中,相較於 ARL4A-T34N , Robo1 比較偏好於跟野生型 ARL4A 與 ARL4A-Q79L 作用。在之後的研究中,我們會使用免疫共沉澱的方式來解決這個問題。 接著,我們想要了解 Robo1 與 ARL4A 在細胞生理功能上的相關性,我們發現到:外生性的 Robo1 表現時,會對細胞的移動進行抑制,不過當我們表現無法與 ARL4A 作用的 Robo1 ( Robo1-A1 與 Robo1-A2 ) 時,卻看不到這樣的抑制現象。由這些結果,我們推測 ARL4A 在 Robo1 所調控的細胞行動抑制中,扮演著重要的角色。此外,我們同時也表現並純化了Robo1 的受質:slit2,以便在未來能更進一步的探討ARL4A 與Robo1 之間的交互作用與關聯。 | zh_TW |
dc.description.abstract | Abstract
ADP-ribosylation factors (ARFs) are small GTPase involved in vesicular trafficking and cytoskeleton remodeling. The ADP-ribosylation factor-like 4A (ARL4A) is a member of the ARL subfamily in the ARF family. Our lab previously showed that ARL4A is expressed in neurogensis and somitogenesis during mouse embryonic development, however the biological functions of ARL4A remain unclear. To explore its functions, our lab screened a human fetal brain cDNA library for putative interacting proteins of ARL4A and identified a candidate effector, the growth cone receptor Robo1. Robo1 is guided by its ligand, SLIT2 in neurons and regulates neural migration. In nonneural cells, SLIT2 inhibits chemotaxis of leukocytes and cell migration in lung and breast cancers. My study focuses on functional characterization of ARL 4A–Robo interaction. Firstly, we raised polyclonal antisera against a recombinant protein of intracellular domain of Robo1 in rabbits, and the antisera recognized endogenous ROBO1 through immunoblot analysis. Next, we characterized the interaction region of ARL4A and Robo1. By using yeast two hybrid, we found that Robo1 interacted with ARL4A through the C-terminal of intracellular domain. The residues 1394- 1454 of C-terminal ROBO1 were sufficient for ARL4A interaction. We also examined the binding preference of ROBO1 for wide type ARL4A (ARL4A WT) and two mutants, constitutively-active form (ARL4A Q79L), and inactive form (ARL4A T34N)ARL4. We found that ARL4A T34N was much preferable to interact with Robo. We used “alanine scanning” to identify two mutation constructs of Robo1, Robo1-A1 and Robo1-A2 that lost the binding ability with ARL4A. By using GST pull-down assays, we showed the same results that Robo directly interacted with ARL4A through its C-terminal and the two Robo1 mutants lost the binding ability with ARL4A; while differently with yeast two hybrid, ARL4A WT and ARL4A Q79L are more preferable for the interaction. In the future, we will perform in vivo immunoprecipitation to verify these results. Next, we wanted to address whether Robo1 are functionally associated with ARL4A. We found that the cell motility was inhibited in Robo-overexpressed cells, while the Robo-A1 and RoboA2-overexpressing cells did not. These data suggested that ARL4A may enhance Robo-regulated inhibition of cell motility. Finally, we generated and purified slit2 proteins, ligand of Robo1, to further study the interplay of these two molecules in cell migration. | en |
dc.description.provenance | Made available in DSpace on 2021-06-14T16:53:07Z (GMT). No. of bitstreams: 1 ntu-100-R98448007-1.pdf: 2828281 bytes, checksum: 9de8c955b124a8a038482cdb0d93480e (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | Table of content ------------------------------------------------------------------------------------ i中文摘要------------------------------------------------------------------------------------------- iv Abstract -------------------------------------------------------------------------------------------- vi Abbreviations ----------------------------------------------------------------------------------- viii 1. Introduction --------------------------------------------------------------------------------------1
1.1. Small GTPase of Ras Superfamily ------------------------------------------------------1 1.2. Arf Family of GTPases -------------------------------------------------------------------2 1.3. ARL4 family protein-----------------------------------------------------------------------4 1.4 Slit-Robo signaling pathway --------------------------------------------------------------5 1.5 Downstream signaling pathway of Robo1 ----------------------------------------------6 1.6 CL1-0 and CL1-5 ---------------------------------------------------------------------------8 2. Materials and methods -------------------------------------------------------------------------9 3. Results ------------------------------------------------------------------------------------------19 3.1. Antiserum against Robo1 is able to detect endogenous Robo1 --------------------19 3.2. Robo1 interacts with wild type ARL4A, ARL4A‐Q79L, and ARL4A-T34N --20 3.3. Identification of specific ARL4A‐interacting region of Robo1 -------------------20 3.4. Robo1 (a.a. 1342-1651) binds to wile type ARL4A, ARL4A-Q79L and ARL4A-T34N mutant in vitro ----------------------------------------------------------21 3.5. Lower expression of slit2, Robo1, and ARL4A in CL1-5 --------------------------22 3.6. Overexpression of Robo1 in CL1-0 inhibits the cell migration --------------------23 3.7. Treatment of purified slit2 protein inhibits the migration ability of CL1-5 ------24 4. Discussion --------------------------------------------------------------------------------------26 5. Tables -------------------------------------------------------------------------------------------30 Table 1. Antibodies used in this thesis ------------------------------------------------------30 Table 2. Oligonucleotides used in this thesis ----------------------------------------------31 Table 3. A brief summary of vectors used in this thesis ----------------------------------34 6. Figures ------------------------------------------------------------------------------------------35 Figure 1. Specificity of the antiserum against Robo1 ------------------------------------35 Figure 2. Localization of the exogenous FLAG‐Robo1 ---------------------------------39 Figure 3. Robo1 (a.a 1342-1475) retained specific interaction with wild type ARL4A , ARL4A‐Q79L, and ARL4A-T34N ---------------------------------------------37 Figure 4. Robo1 (a.a 1394-1454) retained specific interaction with wild type ARL4A, ARL4A‐Q79L, and ARL4A-T34N ----------------------------------------------39 Figure 5. Robo1 (a.a1370-1475) mutants could not interact with ARL4A ------------41 Figure 6. Robo1 C3 fragment (a.a. 1342-1651) preferentially binds to ARL4A-WT and ARL4A‐Q79L mutant in vitro ------------------------------------------43 Figure 7. Robo1 mutants could not interact with ARL4A in vitro ----------------------44 Figure 8. Expression level of slit2, Robo1, and ARL4A in cell lines ------------------45 Figure 9. Robo1 mutants lost the inhibiting ability on cell migration ------------------46 Figure 10. The localization of Robo1, Robo1-A1, and Robo1-A2 in CL1-0----------48 Figure 11. Overexpression and Purification of slit2 proteins ----------------------------49 Figure 12. Slit2 proteins inhibit the migration ability of CL1-5 in dosage- dependent ---------------------------------------------------------------------------------------51 7. Supplementary Figures -----------------------------------------------------------------------53 Figure S1. Domain organizations of human Robo1 and Robo4 ------------------------53 Figure S2. Slit/Robo signaling events ------------------------------------------------------54 Figure S3. A Model for the signaling pathway mediating slit repulsion ---------------55 Figure S4. Amino acid sequence and mutants of human ARL4A -----------------------56 Figure S5. Interaction of hARL4 and mutants with Robo in yeast two-hybrid -------58 Figure S6. Interaction of hARL4 and mutants with Robo mutants in yeast two-hybrid --------------------------------------------------------------------------------------59 Figure S7. The localization of Robo1 in ARL4A-knockdown 293T cells -------------60 Figure S8. The phenotype of ARL4A overexpressd Robo1-knockdown 293T cells -61 8. Reference ---------------------------------------------------------------------------------------62 | |
dc.language.iso | en | |
dc.title | 人類四A 腺嘌呤核苷二磷酸核糖化相似因子
與其結合蛋白之特性探討 | zh_TW |
dc.title | Functional characterization of human ARF-like protein, ARL4A and its interacting protein | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 周祖述,黃佩欣,李秀香 | |
dc.subject.keyword | 腺嘌呤核苷,二磷酸核糖化相似因子,酵母菌雙雜交系統,囊泡傳遞,細胞骨架, | zh_TW |
dc.subject.keyword | Arf,Arl,Robo,slit2,cell migration, | en |
dc.relation.page | 65 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-08-12 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
顯示於系所單位: | 分子醫學研究所 |
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