A fundamental question in cell and developmental biology is how epithelial cells construct the diffusion barrier allowing them to separate different body compartments. Formation of tight junction (TJ) strands, which are crucial for this barrier, involves the polymerization of claudins, TJ adhesion molecules, in temporal and spatial manners. ZO-1 and ZO-2 are major PDZ-domain-containing TJ proteins and bind directly to claudins, yet their functional roles are poorly understood. We established cultured epithelial cells (1(ko)/2(kd)) in which the expression of ZO-1/ZO-2 was suppressed by homologous recombination and RNA interference, respectively. These cells were well polarized, except for a complete lack of TJs. When exogenously expressed in 1(ko)/2(kd) cells, ZO-1 and ZO-2 were recruited to junctional areas where claudins were polymerized, but truncated ZO-1 (NZO-1) containing only domains PDZ1-3 was not. When NZO-1 was forcibly recruited to lateral membranes and dimerized, claudins were dramatically polymerized. These findings indicate that ZO-1 and ZO-2 can independently determine whether and where claudins are polymerized.

In well polarized epithelial cells, closely related ZO-1 and ZO-2 are thought to function as scaffold proteins at tight junctions (TJs). In epithelial cells at the initial phase of polarization, these proteins are recruited to cadherin-based spotlike adherens junctions (AJs). As a first step to clarify the function of ZO-1, we successfully generated mouse epithelial cell clones lacking ZO-1 expression (ZO-1؊/؊ cells) by homologous recombination. Unexpectedly, in confluent cultures, ZO-1؊/؊ cells were highly polarized with well organized AJs/TJs, which were indistinguishable from those in ZO-1؉/؉ cells by electron microscopy. In good agreement, by immunofluorescence microscopy, most TJ proteins including claudins and occludin appeared to be normally concentrated at TJs of ZO-1؊/؊ cells with the exception that a ZO-1 deficiency significantly up-or down-regulated the recruitment of ZO-2 and cingulin, another TJ scaffold protein, respectively, to TJs. When the polarization of ZO-1؊/؊ cells was initiated by a Ca 2؉ switch, the initial AJ formation did not appear to be affected; however, the subsequent TJ formation (recruitment of claudins/occludin to junctions and barrier establishment) was markedly retarded. This retardation as well as the disappearance of cingulin were rescued completely by exogenous ZO-1 but not by ZO-2 expression. Quantitative evaluation of ZO-1/ZO-2 expression levels led to the conclusion that ZO-1 and ZO-2 would function redundantly to some extent in junction formation/epithelial polarization but that they are not functionally identical. Finally, we discussed advantageous aspects of the gene knockout system with cultured epithelial cells in epithelial cell biology. The tight junction (TJ)1 is one type of cell-to-cell adhesion structure in epithelial cells. TJs constitute the epithelial junctional complex together with adherens junctions (AJs) and desmosomes and are located in the most apical part of the complex (1). TJs seal cells to create a primary barrier to the diffusion of solutes across the cellular sheet and also function as a boundary between the apical and basolateral membrane domains to produce their polarization (2-5). Therefore, TJs are essential structures for epithelial cells to exert their physiological functions.On ultrathin-section electron microscopy, TJs appear as a series of discrete sites of apparent fusion involving the outer leaflets of the plasma membranes of adjacent cells (1). On freeze-fracture electron microscopy, TJs appear as a set of continuous, anastomosing intramembranous particle strands (TJ strands) (6). TJ strands are mainly composed of linearly polymerized integral membrane proteins called claudins with molecular masses of ϳ23 kDa, which comprise a multigene family consisting of Ͼ20 members (5,7,8). Claudin molecules bear four transmembrane domains with both NH 2 and COOH termini located in the cytoplasm. In addition to claudins, two other types of integral membrane proteins have been reported to concentrate at TJs, occludin (9), and JAM (10).One of the inte...