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

为了阐明细胞内膜系统在上皮细胞分化过程中信号整合的性质和机制，采用荧光定位逆转录病毒载体表达克隆技术（FL-REX）分离了特异的膜结构标记蛋白。利用三种不同上皮细胞系的mRNA构建egfp融合cDNA文库。用文库质粒转染MDCK细胞，选择并分离出具有EGFP在膜系统结构定位特征的细胞。从孤立的克隆体中。利用载体引物PCR回收整合cdna并测序。这些已鉴定的cdna包括：1)已被显著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，但其在细胞膜上的定位尚未被很好地表征。3)以前报道过定位的蛋白质，但在本研究中显示出意想不到的定位。4)功能和定位都未知的蛋白质。从这些类别中获得的细胞和序列信息对于进一步的研究将是有趣的。在这些过程中，获得了在特定膜结构上稳定表达egfp融合蛋白的MDCK克隆。利用这些细胞和许多技术，对获得的蛋白质进行了分析，蛋白质的细胞内定位和动力学变得更加清晰。这些新发现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过程中和分化后的信号转导膜动力学。此外，为了使用这些蛋白质作为功能导向分析的工具，利用细胞建立了一些这些蛋白质的RNA干扰程序。

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.