Identification of adherens junction-associated GTPase activating proteins by the fluorescence localization-based expression cloning
Identification of adherens junction-associated GTPase activating proteins by the fluorescence localization-based expression cloning
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DOI：
10.1016/j.yexcr.2007.11.009
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发表时间：
2008-03-10
影响因子：
3.7
通讯作者：
Furuse, Mikio
中科院分区：
文献类型：
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作者：
Matsuda, Miho;Kobayashi, Yuka;Furuse, Mikio
包括紧密连接（TJs）、粘附连接（AJs）和桥粒在内的连接复合体在上皮细胞片的结构和功能中起着至关重要的作用。在这项研究中，我们评估了基于荧光定位的逆转录病毒介导的表达克隆（FL-REX）方法作为鉴定TJs和AJs新分子成分的方法。利用来自小鼠上皮细胞系mRNA的cdna - gfp融合表达文库，我们证实了各种已知的TJ-和aj -组分的cdna可以克隆到FL-REX中。此外，ARHGAP12和SPAL3的cdna被克隆为连接复合物的新组分，这两种被认为是小G蛋白的GTPase激活蛋白（GAPS）。使用内部产生的抗体进行免疫荧光染色表明，这些gap定位于各种小鼠组织的上皮细胞-细胞连接处，并且在共聚焦激光扫描显微镜下观察时对AJs具有特异性。这些数据表明FL-REX是鉴定定位于TJs和AJs的新蛋白的有力工具。(C) 2007爱思唯尔公司版权所有。
The junctional complex, including tight junctions (TJs), adherens junctions (AJs), and desmosomes, plays crucial roles in the structure and functions of epithelial cellular sheets. In this study, we evaluated the fluorescence localization-based retrovirus-mediated expression cloning (FL-REX) method as an approach to identify novel molecular components of TJs and AJs. Using an expression library of cDNA-GFP-fusions derived from mRNA of a mouse epithelial cell line, we confirmed that cDNAs for various known TJ- and AJ-components could be cloned in the FL-REX. Furthermore, cDNAs for ARHGAP12 and SPAL3, two putative GTPase activating proteins (GAPS) for small G proteins, were cloned as novel components of the junctional complex. Immunofluorescence staining using antibodies generated in-house demonstrated that these GAPs were localized at epithelial cell-cell junctions in various mouse tissues, and were specific to AJs when observed under confocal laser-scanning microscopy. These data suggest that FL-REX is a powerful tool to identify novel proteins localized at TJs and AJs. (C) 2007 Elsevier Inc. All rights reserved.