Detection of the tight junction-associated protein ZO-1 in astrocytes and other nonepithelial cell types

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Abstract
ZO-1 is a high molecular mass phosphoprotein peripherally associated with the cytoplasmic surface of tight junctions in epithelial and endothelial cells. We report here that ZO-1 is also present in several nonepithelial cell types in vitro that are not believed to form tight junctions, including primary cultures of astrocytes, Schwann cells, and dermal fibroblasts and the C6 glioma, S-180 (sarcoma), and P3 myeloma cell lines. Immunoblots of cell extracts probed with a ZO-1-specific monoclonal antibody reveal a single band that comigrates with ZO-1 from rodent epithelial cells at 225 kDa. In addition, these cells contain a single mRNA species of identical size to that previously reported for ZO-1 in epithelial tissues, as determined by Northern blots probed with a partial ZO-1 cDNA. Immunofluorescence microscopy demonstrates diverse ZO-1 distributions in these cells. In astrocytes, identified by the presence of glial fibrillary acidic protein, ZO-1 is localized at discrete sites of cell-cell contact as well as within the cell cytoplasm. In contrast, S-180 cells display diffuse staining at the cell periphery and within the cytoplasm. Dermal fibroblasts show no staining above background, although ZO-1 was detected on immunoblots of fibroblast cell extracts. Immunofluorescence staining of frozen sections of mouse brain demonstrates no detectable ZO-1 immunoreactivity outside blood vessels where endothelial cell tight junctions of the blood-brain barrier are located. These studies suggest that, although ZO-1 is found to be associated with the tight junction, it has a broader distribution than previously recognized.