CNRS, IRIM Institut de Recherche en infectiologie de Montpellier, Montpellier, 34293, France.
Université de Montpellier, Montpellier, 34090, France.
Biol Cell. 2020 May;112(5):140-151. doi: 10.1111/boc.201900085. Epub 2020 Feb 20.
Claudin-1 (CLDN1) is a four-span transmembrane protein localised at cell-cell tight junctions (TJs), playing an important role in epithelial impermeability and tissue homoeostasis under physiological conditions. Moreover, CLDN1 expression is up-regulated in several cancers, and the level of CLDN1 expression has been proposed as a prognostic marker of patient survival.
Here, we generated and characterised a novel reporter cell line expressing endogenous fluorescent levels of CLDN-1, allowing dynamic monitoring of CLDN-1 expression levels. Specifically, a hepatocellular carcinoma Huh7.5.1 monoclonal cell line was bioengineered using CRISPR/Cas9 to endogenously express a fluorescent TagRFP-T protein fused at the N-terminus of the CLDN1 protein. These cells were proved useful to measure CLDN1 expression and distribution in live cells. However, the cells were resistant to hepatitis C virus (HCV) infection, of which CLDN1 is a viral receptor, while retaining permissiveness to VSV-G-decorated pseudoparticles. Nonetheless, the TagRFP-CLDN1 cell line showed expected CLDN1 protein localisation at TJs and the cell monolayer had similar impermeability and polarisation features as its wild-type counterpart. Finally, using fluorescence recovery after photobleaching (FRAP) approaches, we measured that the majority of endogenous and overexpressed TagRFP-CLDN1 diffuses rapidly within the TJ, whereas half of the overexpressed EGFP-CLDN1 proteins were stalled at TJs.
The Huh7.5.1 TagRFP-CLDN1 edited cell line showed physiological features comparable to that of non-edited cells, but became resistant to HCV infection. Our data also highlight the important impact of the fluorescent protein chosen for endogenous tagging.
Although HCV-related studies may not be achieved with these cells, our work provides a novel tool to study the cell biology of TJ-associated proteins and a potential screening strategy measuring CLDN1 expression levels.
Claudin-1 (CLDN1) 是一种四跨膜蛋白，位于细胞-细胞紧密连接 (TJ) 处，在生理条件下对上皮细胞的通透性和组织稳态起着重要作用。此外，CLDN1 在几种癌症中表达上调，并且 CLDN1 表达水平被提出作为患者生存的预后标志物。
在这里，我们生成并表征了一种表达内源性荧光水平 CLDN-1 的新型报告细胞系，允许动态监测 CLDN1 表达水平。具体来说，使用 CRISPR/Cas9 对内源表达荧光 TagRFP-T 蛋白融合到 CLDN1 蛋白 N 端的肝细胞癌 Huh7.5.1 单克隆细胞系进行了生物工程改造。这些细胞可用于测量活细胞中 CLDN1 的表达和分布。然而，这些细胞对丙型肝炎病毒 (HCV) 的感染具有抗性，CLDN1 是 HCV 的病毒受体，同时保留对 VSV-G 修饰的假病毒颗粒的易感性。尽管如此，TagRFP-CLDN1 细胞系显示出预期的 TJ 处 CLDN1 蛋白定位，并且细胞单层具有与其野生型对应物相似的通透性和极化特征。最后，通过荧光恢复后光漂白 (FRAP) 方法，我们测量了大多数内源性和过表达的 TagRFP-CLDN1 在 TJ 内快速扩散，而过表达的 EGFP-CLDN1 蛋白的一半则停滞在 TJ 处。
Huh7.5.1 TagRFP-CLDN1 编辑细胞系表现出与未编辑细胞相当的生理特征，但对 HCV 感染具有抗性。我们的数据还突出了用于内源性标记选择的荧光蛋白的重要影响。
尽管这些细胞可能无法进行 HCV 相关研究，但我们的工作为研究 TJ 相关蛋白的细胞生物学提供了一种新工具，并提供了一种潜在的筛选策略来测量 CLDN1 表达水平。
