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Characterisation of endogenous Claudin-1 expression, motility and susceptibility to hepatitis C virus in CRISPR knock-in cells.

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.

journal_name

Biol Cell

journal_title

Biology of the cell

authors

Clément CMH,Deffieu MS,Dorobantu CM,Baumert TF,Ayala-Nunez NV,Mély Y,Ronde P,Gaudin R

doi

10.1111/boc.201900085

subject

Has Abstract

pub_date

2020-05-01 00:00:00

pages

140-151

issue

eissn

0248-4900

issn

1768-322X

journal_volume

112

pub_type

杂志文章

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Characterisation of endogenous Claudin-1 expression, motility and susceptibility to hepatitis C virus in CRISPR knock-in cells.