The roles of endomembranes and intracellular domians in the regulation of signaling in epithelial cell differentiation

内膜和细胞内结构域在上皮细胞分化信号传导调节中的作用

基本信息

批准号：
16570166
负责人：
OHASHI Masato
金额：
$ 2.43万
依托单位：
National Institute of Natural Sciences, Okazaki Research Facilities
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

In order to elucidate the nature and the mechanism of signal integrations by cellular endomembrane system in epithelial cell differentiation, specific marker proteins of emdomembrane structures were isolated by FL-REX (fluorescence localization-based retrovirus-mediaetd expression cloning) technique. mRNA from three different epithelial cell lines was used to construct EGFP-fused cDNA libraries.MDCK cells were transfected with the library plasmids, and those cells that exhibited EGFP localization characteristic of structures in endomembrane system was selected and isolated. From the isolated clones. integrated cDNAs were recovered by PCR using the vector primers and were sequenced. These identified cDNAs included the following: 1)proteins that have been significantly characterized and known to localize to specific membranes in the endomembrane sysem. 2)Known proteins but whose localization on the endomembrane has not been well-characterized. 3)Proteins whose localization has previously been reported but in the present study showed unexpected localilzation. 4)Proteins neither of whose functions or localization has been known. Cells and sequence information obtained from any of these categories would be interesting for further studies.During these processes, MDCK clones that stably express EGFP-fused proteins at specific endomembrane structures were obtained. Using these cells and a number of techniques, analyses on the obtained proteins were made and the intracellular localizations and dynamics of the proteins have been becoming clearer. These newly developed marker proteins and cells stably expressing them would be useful for analyzing membrane dynamics in signal transduction during and after the epithelial cell differentiation. In addition, in order to use these proteins as tools for functionally-oriented analyses, RNA interference procedures for some these proteins were established using the cells.

为了阐明上皮细胞分化过程中细胞内膜系统信号整合的性质和机制，通过FL-REX（基于荧光定位的逆转录病毒介导的表达克隆）技术分离了内膜结构的特异性标记蛋白。使用来自三种不同上皮细胞系的mRNA构建EGFP融合的cDNA文库。用文库质粒转染MDCK细胞，选择并分离那些表现出EGFP内膜系统结构定位特征的细胞。来自分离的克隆。使用载体引物通过PCR回收整合的cDNA并进行测序。这些鉴定的 cDNA 包括以下内容：1) 已被显着表征并已知定位于内膜系统中特定膜的蛋白质。 2）已知的蛋白质，但其在内膜上的定位尚未得到很好的表征。 3)先前已报道过定位但在本研究中显示出意想不到的定位的蛋白质。 4) 其功能或定位均未知的蛋白质。从这些类别中的任何一个获得的细胞和序列信息对于进一步的研究来说都是有趣的。在这些过程中，获得了在特定内膜结构稳定表达 EGFP 融合蛋白的 MDCK 克隆。使用这些细胞和多种技术，对获得的蛋白质进行了分析，蛋白质的细胞内定位和动态变得更加清晰。这些新开发的标记蛋白和稳定表达它们的细胞将有助于分析上皮细胞分化期间和之后信号转导的膜动力学。此外，为了使用这些蛋白质作为功能导向分析的工具，使用细胞建立了一些蛋白质的RNA干扰程序。