Camille M H Clément1,2,3,4, Maika S Deffieu1,2, Cristina M Dorobantu3,4, Thomas F Baumert3,4,5, Nilda Vanesa Ayala-Nunez1,2,3,4, Yves Mély4,6, Philippe Ronde4,6, Raphael Gaudin1,2,3,4. 1. CNRS, IRIM Institut de Recherche en infectiologie de Montpellier, Montpellier, 34293, France. 2. Université de Montpellier, Montpellier, 34090, France. 3. Inserm U1110, Institut de Recherche sur les Maladies Virales et Hépatiques (IVH), Strasbourg, 67000, France. 4. Université de Strasbourg, Strasbourg, 67000, France. 5. Pole Hépato-digestif, Hôpitaux Universitaires de Strasbourg, Institut Hospitalo-universitaire, Strasbourg, 67000, France. 6. CNRS UMR 7021, Laboratoire de Bioimagerie et Pathologies, Faculté de pharmacie, Illkirch, 67401, France.
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.
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.
Authors: Yi-Ying Chou; Christian Cuevas; Margot Carocci; Sarah H Stubbs; Minghe Ma; David K Cureton; Luke Chao; Frances Evesson; Kangmin He; Priscilla L Yang; Sean P Whelan; Susan R Ross; Tom Kirchhausen; Raphaël Gaudin Journal: J Virol Date: 2016-04-14 Impact factor: 5.103
Authors: Nathan C Shaner; Michael Z Lin; Michael R McKeown; Paul A Steinbach; Kristin L Hazelwood; Michael W Davidson; Roger Y Tsien Journal: Nat Methods Date: 2008-05-04 Impact factor: 28.547
Authors: Matthew J Evans; Thomas von Hahn; Donna M Tscherne; Andrew J Syder; Maryline Panis; Benno Wölk; Theodora Hatziioannou; Jane A McKeating; Paul D Bieniasz; Charles M Rice Journal: Nature Date: 2007-02-25 Impact factor: 49.962