Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia.

TLDR

Tight junctions are targeted by immunogens derived from junction‑enriched membrane fractions to generate specific monoclonal antisera. Hybridomas were screened for immunoblot reactivity and for localization to the junctional complex in frozen sections of mouse liver. The R26.4C hybridoma produces an antibody that detects a ~225‑kDa polypeptide, ZO‑1, localized to tight junctions in liver, kidney, colon, testis, arterial endothelium, and MDCK cells, indicating it is a ubiquitous tight‑junction component.

Abstract

A tight junction-enriched membrane fraction has been used as immunogen to generate a monoclonal antiserum specific for this intercellular junction. Hybridomas were screened for their ability to both react on an immunoblot and localize to the junctional complex region on frozen sections of unfixed mouse liver. A stable hybridoma line has been isolated that secretes an antibody (R26.4C) that localizes in thin section images of isolated mouse liver plasma membranes to the points of membrane contact at the tight junction. This antibody recognizes a polypeptide of approximately 225,000 D, detectable in whole liver homogenates as well as in the tight junction-enriched membrane fraction. R26.4C localizes to the junctional complex region of a number of other epithelia, including colon, kidney, and testis, and to arterial endothelium, as assayed by immunofluorescent staining of cryostat sections of whole tissue. This antibody also stains the junctional complex region in confluent monolayers of the Madin-Darby canine kidney epithelial cell line. Immunoblot analysis of Madin-Darby canine kidney cells demonstrates the presence of a polypeptide similar in molecular weight to that detected in liver, suggesting that this protein is potentially a ubiquitous component of all mammalian tight junctions. The 225-kD tight junction-associated polypeptide is termed "ZO-1."