Tetenborg Stephan, Martinez-Soler Elizabeth, O Brien John
College of Optometry, University of Houston, Houston, TX, USA.
Bio Protoc. 2024 Jul 20;14(14):e5034. doi: 10.21769/BioProtoc.5034.
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.
在瞬时转染细胞中过表达蛋白质是研究基本转运机制以及潜在蛋白质-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，但它们能快速评估更保守的功能，例如内质网输出或蛋白质在高尔基体中的分选。在先前的一项研究中，我们团队描述了在转染的HEK293T细胞中形成的用于间隙连接蛋白Cx36的内质网衍生清除囊泡。这些清除囊泡因其同心结构而被称为“漩涡”，由未能逃离内质网的Cx36通道形成。在本文中，我们描述了一种成像方案，可用于确定在培养细胞中表达的Cx36的这些内质网滞留缺陷。我们在此提供的方案采用常规共聚焦显微镜，其具有足够的分辨率来揭示内质网漩涡的特征形状。
