Stephan Tetenborg, Elizabeth Martinez-Soler, John O Brien
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引用次数: 0
摘要
在瞬时转染细胞中过表达蛋白质是研究基本转运机制和潜在蛋白质相互作用的一种简单方法。虽然与体内实验相比,表达系统有明显的缺点,但它可以快速评估更多的保守功能,例如 ER 的输出或高尔基体中蛋白质的分拣。在之前的一项研究中,我们的研究小组描述了在转染的 HEK293T 细胞中为缝隙连接蛋白 Cx36 形成源自 ER 的清除囊泡的情况。这些清除囊泡因其同心结构而被称为 "轮状",是由未能逃离ER的Cx36通道形成的。在本文中,我们介绍了一种成像方案,该方案可用于确定在培养细胞中表达的 Cx36 是否存在ER滞留缺陷。我们在此提供的方案采用了常规共聚焦显微镜,其分辨率足以揭示ER轮的特征形状。
Characterizing ER Retention Defects of PDZ Binding Deficient Cx36 Mutants Using Confocal Microscopy.
Overexpression of proteins in transiently transfected cells is a simple way to study basic transport mechanisms and the underlying protein-protein interactions. While expression systems have obvious drawbacks compared to in vivo experiments, they allow a quick assessment of more conserved functions, for instance, ER export or sorting of proteins in the Golgi. In a previous study, our group described the formation of ER-derived removal vesicles for the gap junction protein Cx36 in transfected HEK293T cells. These removal vesicles, termed "whorls" because of their concentric structure, were formed by Cx36 channels that failed to escape the ER. In this article, we describe an imaging protocol that can be used to determine these ER retention defects for Cx36 expressed in cultured cells. The protocol we provide here employs regular confocal microscopy, which allows for sufficient resolution to reveal the characteristic shape of ER whorls.