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Tetracysteine Genetic Tags Complexed with Biarsenical Ligands as a Tool for Investigating Gap Junction Structure and Dynamics

机译:Tetracysteine遗传标记与双砷配体复合作为研究间隙连接结构和动力学的工具

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摘要

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.
机译:间隙连接(GJ)定义为两个相邻细胞之间的接触区域,其中包含数十到数千个紧密堆积的膜通道。细胞通过调节这些通道的合成,转运和周转来动态调节通过GJ的通讯。以前,我们通过将包含序列-Cys-Cys-Xaa-Xaa-Cys-Cys-的小的四半胱氨酸肽基序遗传附加到Cx43(Cx43-TC)的羧基末端来设计重组连接蛋白43(Cx43)(3)。 Cx43-TC在HeLa细胞中稳定表达,并通过将细胞暴露于可透过膜的非荧光配体(例如FlAsH(荧光素衍生物)和ReAsH(试卤灵衍生物))进行特异性标记。活细胞图像与高分辨率EM检测的直接相关性是可能的,因为结合的ReA​​sH不仅发荧光,而且还可以用于引发二氨基联苯胺(DAB)的光转化,从而引起不溶性亲油性沉淀的局部聚合,然后由EM可见。 Cx43-TC GJ可以用ReAsH标记并被光氧化以产生选择性染色的通道。在此,描述了与适当配体复合的这些四半胱氨酸标签的开发如何用于跨越从光学显微镜到电子断层扫描再到分子纯化和检测的分辨率范围的实验。

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