Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.

Abstract

Abstract. Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full-length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOHterminal ,~150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.

Tight junction properties of the immortalized human bronchial epithelial cell lines Calu‐3 and 16HBE14o‐

“…By using 2PMEM, which suffers from only minimal chromatic aberration and which allows simultaneous excitation of multiple fluorochromes, the authors were able to provide morphological evidence in support of the suspected close association between these proteins. Previously, the morphological association between occludin and ZO-1 has been inferred from images obtained by nonconfocal or single photon confocal microscopy in which testing for colocalization can be problematic [9,29].…”

Trichomonas vaginalis perturbs the junctional complex in epithelial cells

“…Both proteins are found associated with the peripheral plasma membrane protein ZO-1, at the cytosolic face. Occludin binds directly through its carboxyl terminus to ZO-1 [31] and it is also associated with ZO-3 [32]. ZO-1 is, in turn, associated with ZO-2 [33] and AF-6 [34].…”

Involvement of Cdc42 small G protein in cell-cell adhesion, migration and morphology of MDCK cells

“…Tight junction also plays an important role in the formation of cell polarity and barrier (for reviews, see Schneeberger and Lynch, 1992;Gumbiner, 1993;Anderson and Van Itallie, 1995). Claudin and occludin, transmembrane proteins that interact with each other at the extracellular surface, play important roles in the formation of tight junction (Furuse et al, 1994(Furuse et al, , 1998. Several peripheral membrane proteins, including ZO-1, ZO-2, cingulin, the 7H6 antigen, Rab13 small G protein, and symplekin, are localized at tight junction (Citi et al, 1988;Woods and Bryant, 1993;Zhong et al, 1993;Jesaitis and Goodenough, 1994;Zahraoui et al, 1994;Keon et al, 1996).…”