cCPE Fusion Proteins as Molecular Probes to Detect Claudins and Tight Junction Dysregulation in Gastrointestinal Cell Lines, Tissue Explants and Patient-Derived Organoids
Ayk Waldow,
Laura-Sophie Beier,
Janine Arndt,
Simon Schallenberg,
Claudia Vollbrecht,
Philip Bischoff,
Martí Farrera-Sal,
Florian N. Loch,
Christian Bojarski,
Michael Schumann,
Lars Winkler,
Carsten Kamphues,
Lukas Ehlen,
Jörg Piontek
Affiliations
Ayk Waldow
Clinical Physiology/Nutritional Medicine, Medical Department, Division of Gastroenterology, Infectiology, Rheumatology, Charité—Universitätsmedizin Berlin, 12203 Berlin, Germany
Laura-Sophie Beier
Clinical Physiology/Nutritional Medicine, Medical Department, Division of Gastroenterology, Infectiology, Rheumatology, Charité—Universitätsmedizin Berlin, 12203 Berlin, Germany
Janine Arndt
Berlin Institute of Health (BIH), Charité—Universitätsmedizin Berlin, BIH Center for Regenerative Therapies (BCRT), 13353 Berlin, Germany
Simon Schallenberg
Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Berlin Institute of Health, Institute of Pathology, 10117 Berlin, Germany
Claudia Vollbrecht
Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Berlin Institute of Health, Institute of Pathology, 10117 Berlin, Germany
Philip Bischoff
Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Berlin Institute of Health, Institute of Pathology, 10117 Berlin, Germany
Martí Farrera-Sal
Berlin Institute of Health (BIH), Charité—Universitätsmedizin Berlin, BIH Center for Regenerative Therapies (BCRT), 13353 Berlin, Germany
Florian N. Loch
Department of General and Visceral Surgery, Campus Benjamin Franklin, Charité—Universitätsmedizin Berlin, 12203 Berlin, Germany
Christian Bojarski
Medical Department, Division of Gastroenterology, Infectiology, Rheumatology, Charité—Universitätsmedizin Berlin, 12203 Berlin, Germany
Michael Schumann
Medical Department, Division of Gastroenterology, Infectiology, Rheumatology, Charité—Universitätsmedizin Berlin, 12203 Berlin, Germany
Claudins regulate paracellular permeability, contribute to epithelial polarization and are dysregulated during inflammation and carcinogenesis. Variants of the claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) are highly sensitive protein ligands for generic detection of a broad spectrum of claudins. Here, we investigated the preferential binding of YFP- or GST-cCPE fusion proteins to non-junctional claudin molecules. Plate reader assays, flow cytometry and microscopy were used to assess the binding of YFP- or GST-cCPE to non-junctional claudins in multiple in vitro and ex vivo models of human and rat gastrointestinal epithelia and to monitor formation of a tight junction barrier. Furthermore, YFP-cCPE was used to probe expression, polar localization and dysregulation of claudins in patient-derived organoids generated from gastric dysplasia and gastric cancer. Live-cell imaging and immunocytochemistry revealed cell polarity and presence of tight junctions in glandular organoids (originating from intestinal-type gastric cancer and gastric dysplasia) and, in contrast, a disrupted diffusion barrier for granular organoids (originating from discohesive tumor areas). In sum, we report the use of cCPE fusion proteins as molecular probes to specifically and efficiently detect claudin expression, localization and tight junction dysregulation in cell lines, tissue explants and patient-derived organoids of the gastrointestinal tract.
