Characterisation of endogenous Claudin‐1 expression, motility and susceptibility to hepatitis C virus in CRISPR knock‐in cells. (20th February 2020)

• Record Type: Journal Article
• Authors: Clément, Camille M.H.; Deffieu, Maika S.; Dorobantu, Cristina M.; Baumert, Thomas F.; Ayala‐Nunez, Nilda Vanesa; Mély, Yves; Ronde, Philippe; Gaudin, Raphael
• Is Part Of: Biology of the cell. Volume 112:Number 5(2020)
• Page Start: 140
• Page End: 151
• Publication Date: 2020-02-20
• Subjects: CLDN1 -- FRAP -- Gene editing -- HCV -- Hepatocellular carcinoma; Cytology -- Periodicals; Electron microscopy -- Periodicals; 571.6
• DOI: 10.1111/boc.201900085
• Languages: English
• ISSNs: 0248-4900

Abstract

Abstract : Background Information: 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. Results: 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. Conclusions: 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. Significance: 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. Abstract : Research article : This study generated a new gene edited Huh7.5.1 cell line endogenously expressing the TagRFP‐T fluorescent protein fused to CLDN1. This cell line allows live measurement of CLDN1 expression and distribution.