Riesen Franziska K, Rothen-Rutishauser Barbara, Wunderli-Allenspach Heidi
Biopharmacy, Department of Applied BioSciences, ETH Zurich, 8057 Zurich, Switzerland.
Histochem Cell Biol. 2002 Apr;117(4):307-15. doi: 10.1007/s00418-002-0398-y. Epub 2002 Mar 29.
To examine the dynamics of tight junctions (TJs) in living cells, chimera between the TJ-associated protein ZO-1 and green fluorescent protein (GFP) were constructed. If ZO-1 fused to the C-terminus of GFP (ZO1-CGFP) was stably expressed in MDCK cells, it was fully incorporated into TJs and colocalized with endogenous ZO-1. The GFP tag did not influence cell growth, transepithelial electrical resistance, and paracellular mannitol transport. The morphology of the transfected cells was unchanged. The ZO1-CGFP MDCK cell line thus represents an excellent tool to study TJ dynamics. The influence of the external calcium ion concentration on the formation and dynamics of TJs in living cells was thus explored. Upon opening of the TJs under short-term treatment with EGTA (up to 20 min), the localization of ZO1-CGFP at the membrane persisted. The rim-like pattern around the individual cells appeared fuzzier than in non-treated cells. Long-term calcium depletion resulted in the localization of ZO1-CGFP in the cytoplasm and the nucleus. After restoration of normal Ca(2+) concentrations, cell-cell contacts were restored and the localization of ZO1-CGFP was indistinguishable from the one in control cells kept at normal Ca(2+) concentrations. It remains open how the different localizations of ZO-1 correspond to changes in the signal transduction activity of the molecule.
为了研究活细胞中紧密连接（TJ）的动态变化，构建了TJ相关蛋白ZO-1与绿色荧光蛋白（GFP）的嵌合体。如果融合到GFP C末端的ZO-1（ZO1-CGFP）在MDCK细胞中稳定表达，它会完全整合到TJ中，并与内源性ZO-1共定位。GFP标签不影响细胞生长、跨上皮电阻和细胞旁甘露醇转运。转染细胞的形态没有改变。因此，ZO1-CGFP MDCK细胞系是研究TJ动态变化的优秀工具。由此探索了外部钙离子浓度对活细胞中TJ形成和动态变化的影响。在用EGTA短期处理（长达20分钟）导致TJ开放后，ZO1-CGFP在膜上的定位持续存在。单个细胞周围的边缘样模式比未处理的细胞显得更模糊。长期钙耗竭导致ZO1-CGFP在细胞质和细胞核中定位。恢复正常Ca(2+)浓度后，细胞间接触得以恢复，ZO1-CGFP的定位与保持在正常Ca(2+)浓度的对照细胞中的定位没有区别。ZO-1的不同定位如何与该分子信号转导活性的变化相对应仍不清楚。
