探討腺嘌呤核苷二磷酸核糖化相似因子ARL4D在犬腎上皮細胞之特性

Functional characterization of human ARF-like proteins, ARL4D in MDCK cells

陳佳欣(Chia-Hsin Chen)

指導教授 ： 李芳仁

國立臺灣大學/醫學院/分子醫學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.00836

摘要

腺嘌呤核苷二磷酸核醣化因子相似蛋白4D (ARL4D) 隸屬於Ras小分子G蛋白家族的成員之一。近年來的研究指出ARL4D參與在肌動蛋白的重組以及神經細胞的可塑性。為了探討更多ARL4D在生理上的意義，我們利用酵母菌雙雜合篩選法找出可能會與ARL4D作用的蛋白質α-catenin。Alpha-catenin是adherens junctions (AJ)的結構蛋白，負責強化細胞間黏附和維持AJ的穩定性，我們利用犬腎臟上皮細胞(MDCK)去研究ARL4D和α-catenin作用在生理上的功能，並探討ARL4D是否會影響細胞間的黏附作用。
　　我們利用Tet-off系統在MDCK細胞誘導ARL4D表現。當細胞培養在不含Doxcycline的培養液時，外生性的ARL4D會開始表現，此外，ARL4D的表現量會隨著藥物劑量及時間有所變化。在這篇研究當中，我們先利用pull down assay去驗證ARL4D可以和α-catenin有直接的作用，並在細胞免疫螢光染色觀察到ARL4D和α-catenin皆位於細胞膜。進一步，在細胞凝集的實驗中，我們發現ARL4D扮演著一個重要的角色去降低細胞間的黏附作用。另外，大量表現 ARL4D活化態的突變株時，會減弱細胞間黏附，並引起AJ另一個主要結構蛋白E-cadherin在細胞內累積的現象。而細胞黏附對於個體細胞要組裝成三度空間的組織結構是重要的。我們利用犬腎臟上皮細胞在三度空間培養下會長成囊胚的特性去觀察ARL4D是否會因為細胞黏附受損而影響到囊胚發育，初步結果顯示囊胚結構的AJ並沒有明顯的缺陷，然而ARL4D是否影響到囊胚形狀的發育仍需進一步觀察。總結來說，ARL4D和α-catenin作用可能會影響α-catenin的功能，讓E-cadherin在細胞中的位置有不正常的分佈，進而影響到細胞間的黏附作用。

關鍵字

腺嘌呤核苷二磷酸核醣化因子相似蛋白 ； 細胞黏附

並列摘要

ARL4D is a developmentally regulated member of the ADP-ribosylation factor-like (ARL) family, which belongs to Ras superfamily. Recent studies have shown that ARL4D is involved in actin remodeling and neural plasticity. In order to investigate more physiological function of ARL4D, we have identified a putative interacting protein, α-catenin, by using yeast-two hybrid system. Alpha-catenin is an intercellular adhesion protein that connects the E-cadherin-β-catenin complex to the actin cytoskeleton and strengthens cell-cell adhesiveness. In this study, we use Madin-Darby canine kidney (MDCK) cells as a model to explore the functional roles of ARL4D and α-catenin on the modulation of adherens junctions.
We used the inducible ARL4D expression in Tet-off system. In this system, the expression of exogenous ARL4D could be up-regulated by the omission of tetracycline derivative doxcycline. Besides, the expression of ARL4D was in a dosage dependent and time dependent manner. We first used pull down assay to verify whether ARL4D directly interacts with α-catenin in vitro. Immunofluorescence data showed that ARL4D colocalizes with α-catenin at plasma membrane. We further examined whether the function of α-catenin affected by ARL4D. In cell aggregation assay, we found that overexpression constitutively active form ARL4DQ80L weakened the cell-cell adhesion. In addition, and knockdown endogenous ARL4D in MDCK cells enhanced cell aggregation. ARL4D was required for diminishing the aggregation of MDCK cells. Because α-catenin was a key component to anchor E-cadherin at plasma membrane, we further observed the localization of E-cadherin. We found that ARL4DQ80L overexpression enhanced the accumulation of intracellular E-cadherin. These data implicated that ARL4D was involved in down-regulation of cell-cell adhesion.
Cell adhesion is crucial for the assembly of individual cells into the three- dimensional tissue. Thus, we provided three-dimensional culture as a more physiological relevant approach to address whether ARL4D affect tissue architecture. ARL4DQ80L overexpression did not affect the AJ in cysts. However, whether ARL4D affect the processes of individual cells into tissue structures will be examined in the future. Taken together, ARL4D might affect the function of α-catenin. In addition, ARL4D might be involved in regulation the localization of E-cadherin, and therefore modulated cell-cell adhesion.

並列關鍵字

ARL4D ； α-catenin ； adherens junction ； adhesion ； E-cadherin

參考文獻

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