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

点击翻译按钮获取中文摘要

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.