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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李芳仁 | |
| dc.contributor.author | Yi-Miao Chen | en |
| dc.contributor.author | 陳宜妙 | zh_TW |
| dc.date.accessioned | 2021-06-16T17:32:46Z | - |
| dc.date.available | 2017-09-18 | |
| dc.date.copyright | 2012-09-18 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-15 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64161 | - |
| dc.description.abstract | 腺嘌呤核苷二磷酸核醣化因子 (ADP- ribosylation factor , ARF) 隸屬於小分子G蛋白家族，主要的功能是參與在囊泡運輸與細胞骨架的調控，而腺嘌呤核苷二磷酸核醣化相似因子(ARL4A)是 ARF的子家族: 腺嘌呤核苷二磷酸核醣化相似蛋白 (ARL) 家族的其中一員。實驗室先前的研究指出，ARL4A 參與在細胞骨架的動態例如板狀偽足 (lamellipodia) 與細胞膜皺摺 (membrane ruffling)的形成。過去的研究利用犬腎上皮細胞株 (Madin-Darby canine kidney, MDCK)，使用DNA微陣列分析發現，ARL4A在肝細胞生長因子(HGF)的刺激下表現量會上升; 而先前的研究也指出HGF的刺激會造成細胞黏著連接 (Adherens junction, AJ)的拆解並且改變細胞的極性。這些研究給了我們一個暗示: ARL4A 可能會影響上皮細胞的型態，因此我們利用MDCK當作模型，想要探討ARL4A 是否會影響AJ的結構。
首先，我們在MDCK細胞裡表現ARL4A，發現野生型ARL4A 與ARL4A Q79L會導致細胞突出的形成並且使AJ的結構蛋白E-cadherin形成片狀的結構。為了更仔細觀察這個現象，我們建立了ARL4A的穩定細胞株，利用tet- off系統誘導ARL4A的表現，利用穩定細胞株表現ARL4A，我們觀察到了與在細胞株暫時性表現ARL4A一致的現象。 接著，我們發現表現ARL4A會影響已極化上皮細胞的E-cadherin結構但是不會影響細胞的極性，更進一步，我們發現ARL4A的表現會減少與細胞骨架結合的E-cadherin，顯示ARL4A可能會影響 E-cadherin複合蛋白的穩定性與E-cadherin和細胞骨架的結合。 最後，為了探討ARL4A影響E-cadherin結構可能的機制，我們挑選了ARNO和Pak兩個可能參與其中的蛋白，這兩個蛋白都被報導參與在細胞骨架的調控並且與ARL4A有直接的作用。ARNO 為ARF的鳥糞嘌呤核苷酸轉換因子(Guanine nucleotide exchange factor, GEF)，我們的實驗發現表現ARNO引起細胞突出的形成並且使E-cadherin形成片狀的結構，而降低內生性ARNO會減少由ARL4A所引起的細胞突出的形成，我們也發現若表現一個不會與ARL4A作用的突變型ARNO則不會產生細胞突出。反之，我們的實驗發現同時表現Pak與ARL4A不會增加細胞突出的形成，而降低內生性Pak也不影響由ARL4A所引起的細胞突出的形成。根據以上的結果，我們認為ARL4A與ARNO之間的交互作用對於細胞突出的產生是重要的，然而更詳細關於ARL4A如何影響E-cadherin的機制未來需要更進一步的探討。 | zh_TW |
| dc.description.abstract | The ADP- ribosylation factor (ARF) family of guanine- nucleotide- binding protein regulates vesicular trafficking and cytoskeleton remodeling. The ADP- ribosylation factor- like 4A (ARL4A) is a member of ARL subfamily. Our lab had found that ARL4A regulated cytoskeleton dynamics such as lamellipodia formation and membrane ruffling. Previous study showed that, by DNA microarray analysis, ARL4A gene expression in polarized Madin- Darby canine kidney (MDCK) cells would increase upon hepatocyte growth factor (HGF) treatment, which induced the disassembly of adhesion junction and altered the cell polarity. These studies provided us some hints that ARL4A may involve in the morphological changes of epithelial cells. Therefore, we used MDCK cell as a model to explore whether ARL4A overexpression would affect the structure of adherens junction.
Firstly, we found that ARL4A wild type and its constitutively active mutant (ARL4A Q79L) induced the formation of the cell protrusion and flat- like distribution of E-cadherin. To further investigate this phenotype, we established MDCK cell lines expressing ARL4A WT, ARL4A Q79L and ARL4A T34N (an ARL4A constitutively inactive mutant) in the tet-off system. After induction, we observed the formation of cell protrusion on ARL4A WT and ARL4A Q79L cells, which showed identical phenotype to the transient transfection. Next, we found that ARL4A affected the polarized E- cadherin structure, but did not affect the cell polarity. In addition, we found that ARL4A reduced levels of cytoskeleton- bound E- cadherin, which indicated that ARL4A affected the stability of E- cadherin complex and its association with the cytoskeleton. Finally, in order to find out the possible mechanism of how ARL4A affected the E-cadherin structure, we overexpressed two candidate proteins, ARF- GEF ARNO and p21- activated kinase Pak in MDCK- T23 cells to examine the phenotype. Both of them were well-known to regulate cytoskeleton dynamics and were proved to interact with ARL4A directly. We found that co- overexpressed ARNO and ARL4A increased the cell protrusion and flat- like distribution of E-cadherin. Knockdown of endogenous ARNO reduced the ARL4A- induced cell protrusion. Moreover, overexpressing the truncation mutant of ARNO, which was proved to loss the binding ability to ARL4A, diminished the cell protrusion. Otherwise, we found that co- overexpressed Pak1 and ARL4A did not increase the cell protrusion and knockdown of endogenous Pak1 did not affect the ARL4A- induced cell protrusion. Taken together, we suggested that the interaction between ARL4A and ARNO, but not Pak1, was required for the formation of cell protrusion. However, the detail mechanisms of how ARL4A affected the E-cadherin should be examined in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T17:32:46Z (GMT). No. of bitstreams: 1 ntu-101-R99448007-1.pdf: 3782914 bytes, checksum: 9ffd90a97373252ac191f63b5b8f672c (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | Table of content -------------------------------------------------------------------------------------i
口試委員審定書 ---------------------------------------------------------------------------------iii 中文摘要--------------------------------------------------------------------------------------------iv Abstract ---------------------------------------------------------------------------------------------vi Abbreviations ------------------------------------------------------------------------------------viii 1. Introduction --------------------------------------------------------------------------------------1 1.1. Small GTPases of Ras Superfamily -----------------------------------------------------1 1.2. ADP-Ribosylation Factor (Arf) family -------------------------------------------------2 1.3. ARL4 family proteins----------------------------------------------------------------------5 1.4. Adherens junctions ------------------------------------------------------------------------6 1.5. Cytohesin-2/ ARNO -----------------------------------------------------------------------7 1.6. p21- activated kinase (Pak)----------------------------------------------------------------9 2. Materials and methods ------------------------------------------------------------------------11 3. Results ------------------------------------------------------------------------------------------18 3.1. ARL4A WT and ARL4A Q79L induced the formation of cell protrusion--------18 3.2. The expression of ARL4A and its mutants in MDCK-T23 was doxycycline- dependent ---------------------------------------------------------------------------------20 3.3. Expression of ARL4A affected the polarized E-cadherin structure ---------------20 3.4. Expression of ARL4A did not affect the apical and tight junction-----------------21 3.5. ARL4A overexpression reduced the amount of actin cytoskeleton- associated E-cadherin in MDCK-T23 cells--------------------------------------------------------22 3.6. Co- expression of ARNO and ARL4A increased the cell protrusion--------------22 3.7. Depletion of ARNO reduced the formation of cell protrusion in ARL4A cells----------------------------------------------------------------------------------------23 3.8. ARL4A interacted with Pak1 103-112 in vivo----------------------------------------24 3.9. Pak1 mutants interacted with ARL4A by yeast two hybrid assays-----------------25 3.10. Co- expression of Pak1 and ARL4A did not enhance the cell protrusion-------25 3.11. Depletion of Pak1 did not affect the ARL4A- induced cell protrusion-----------26 4. Discussion --------------------------------------------------------------------------------------27 5. Tables -------------------------------------------------------------------------------------------30 Table 1. Antibodies used in this thesis ------------------------------------------------------30 Table 2. Oligonucleotides used in this thesis ----------------------------------------------32 6. Figures ------------------------------------------------------------------------------------------33 7. Supplementary Figures -----------------------------------------------------------------------58 8. Reference ---------------------------------------------------------------------------------------64 | |
| dc.language.iso | en | |
| dc.subject | 腺嘌呤核苷 | zh_TW |
| dc.subject | 二磷酸核醣化因子 | zh_TW |
| dc.subject | 細胞黏著連接 | zh_TW |
| dc.subject | Adherens junction | en |
| dc.subject | ARF | en |
| dc.subject | E-cadherin | en |
| dc.title | 探討腺嘌呤核苷二磷酸核糖化相似因子ARL4A影響犬腎上皮細胞型態之作用途徑 | zh_TW |
| dc.title | Characterization of ARL4A- induced morphological changes of MDCK cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張智芬,黃佩欣,周祖述 | |
| dc.subject.keyword | 腺嘌呤核苷,二磷酸核醣化因子,細胞黏著連接, | zh_TW |
| dc.subject.keyword | ARF,Adherens junction,E-cadherin, | en |
| dc.relation.page | 68 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-15 | |
| 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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