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Identification and characterication of novel proteins involved in epithelial morphogenesis

参与上皮形态发生的新型蛋白质的鉴定和表征

基本信息

批准号：
15390090
负责人：
FURUSE Mikio
金额：
$ 9.09万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15390090/
关键词：
epithelial cells cell polarity cell adhesion cytoskeletons GFP membrane domains

项目摘要

1.Improvement of the fluorescence localization-based retrovirus-mediated expression cloning for identification of novel proteins localized at epithelial cell structures.We constructed cDNA libraries of cultured epithelial cell lines MDCK and CSG using a mouse retrovirus expression vector. As host epithelial cells for retrovirus infection, MDCK cells overexpressing the retrovirus receptor were. In small-scale screenings, we confirmed that claudins, occludin, JAM, etc. can be cloned in this system. During several trials, we improved the protocol for effective screening.2.Identification of novel proteins concentrated in epithelial cell-cell junctions.In the middle-scale screening using the CSG cells-derived cDNA library, homer2, abLIM3, and a novel coiled-coil protein have been identified as novel proteins localizing at epithelial cell-cell junctions. We produced antibodies against these proteins and analyzed their tissue expression pattern. We also determined the domains required for junction allocalization for each protein. Among them, alLIM3 is a novel subtype of abLIM, which is known to function in neuron orretin a by interacting with actin filaments. We found that abLIM3 is localized at cell-cell junctions in endothelial cells in lung and muscle tissues, and interact with actin filaments in vitro. Tissue expression pattern of abLIM3 suggest some roles of this protein in cell-cell junctions in endothelial cells that receive extension and shrinkage.

1.荧光定位逆转录病毒介导的表达克隆技术的改进：利用小鼠逆转录病毒表达载体构建了MDCK和CSG两种上皮细胞系的cDNA文库。作为逆转录病毒感染的宿主上皮细胞，过表达逆转录病毒受体的MDCK细胞。在小规模筛选中，我们证实了claudins，occludin，JAM等可以在该系统中克隆。2.上皮细胞间连接蛋白的筛选在中规模筛选中，我们利用CSG细胞cDNA文库筛选到了homer 2、abLIM 3和一种新的卷曲螺旋蛋白，这些蛋白都是上皮细胞间连接蛋白。我们生产了针对这些蛋白质的抗体，并分析了它们的组织表达模式。我们还确定了每个蛋白质的连接allocation所需的域。其中，alLIM 3是abLIM的一种新亚型，已知其通过与肌动蛋白丝相互作用而在神经元orretin a中起作用。我们发现abLIM 3定位于肺和肌肉组织中内皮细胞的细胞-细胞连接处，并在体外与肌动蛋白丝相互作用。abLIM 3的组织表达模式表明该蛋白在接受延伸和收缩的内皮细胞中的细胞-细胞连接中的一些作用。