The gap junction (GJ) is an aggregate of intercellular channels that facilitates cytoplasmic interchange of ions, second messengers, and other molecules of less than 1000 Da between cells. In excitable organs such as heart and brain, GJs configure extended intercellular pathways for stable and long-term propagation of action potential. In a previous study in adult rat heart, we have shown that the Drosophila disks-large related protein ZO-1 shows low to moderate colocalization at myocyte borders with the GJ protein Cx43. In the present study, we detail a protocol for characterizing the pattern and level of colocalization of ZO-1 with Cx43 in cultures of neonatal myocytes at the level of individual GJ plaques. The data indicate that ZO-1 shows on average a partial 26.6% overlap (SD = 11.3%) with Cx43 GJ plaques. There is a strong positive correlation between GJ plaque size and area of ZO-1 colocalization, indicating that the level of associated ZO-1 scales with the area of the GJ plaque. Qualitatively, the most prominent colocalization occurs at the plaque perimeter. These studies may provide insight into the presently unknown biological function of ZO-1 interaction with Cx43.

In order to improve our understanding of the host cell–parasite interactions in giardiasis, this study assessed the effects of Giardia lamblia on epithelial permeability and tight junctional ZO-1, determined whether epidermal growth factor (EGF) may affect Giardia-induced epithelial injury, and evaluated if EGF modulates epithelial colonization by live G. lamblia trophozoites. Permeability was assessed in assays of trans-epithelial fluxes of FITC-dextran, and ZO-1 integrity was characterized by confocal laser immunofluorescence microscopy in confluent epithelial cell monolayers. G. lamblia significantly increased paracellular permeability and disrupted tight-junctional ZO-1 of a novel non-transformed human small intestinal epithelial cell line (SCBN). Pre-treatment with EGF prevented the development of these abnormalities and significantly inhibited attachment of live trophozoites to the enterocytes, independently of a direct microbiocidal action. These findings demonstrate that G. lamblia may cause intestinal pathophysiology by disrupting tight junctional ZO-1 and increasing epithelial permeability. Apical administration of EGF prevents these abnormalities, and reduces epithelial colonization by the live parasites.