Abstract. Tight junctions, the most apical of the intercellular junctions that connect individual cells in a epithelial sheet, are thought to form a seal that restricts paracellular and intramembrane diffusion. To analyze the functioning of tight junctions, we generated stable MDCK strain 2 cell lines expressing either full-length or COOH-terminally truncated chicken occludin, the only known transmembrane component of tight junctions. Confocal immunofluorescence and immunoelectron microscopy demonstrated that mutant occludin was incorporated into tight junctions but, in contrast to full-length chicken occludin, exhibited a discontinuous junctional staining pattern and also disrupted the continuous junctional ring formed by endogenous occludin. This rearrangement of occludin was not paralleled by apparent changes in the junctional morphology as seen by thin section electron microscopy nor apparent discontinuities of the junctional strands observed by freeze-fracture. Nevertheless, expression of both wildtype and mutant occludin induced increased transepithelial electrical resistance (TER). In contrast to TER, particularly the expression of COOH-terminally truncated occludin led to a severalfold increase in paracellular flux of small molecular weight tracers. Since the selectivity for size or different types of cations was unchanged, expression of wild-type and mutant occludin appears to have activated an existing mechanism that allows selective paracellular flux in the presence of electrically sealed tight junctions. Occludin is also involved in the formation of the apical/basolateral intramembrane diffusion barrier, since expression of the COOH-terminally truncated occludin was found to render MDCK cells incapable of maintaining a fluorescent lipid in a specifically labeled cell surface domain.

An epithelial cell line (MDCK) was used to prepare monolayers which, in vitro, develop properties of transporting epithelia. Monolayers were formed by plating cells at high densities (106 cells/cm") on collagen-coated nylon cloth disks to saturate the area available for attachment, thus avoiding the need for cell division.An electrical resistance developed within 4-6 h after plating and achieved a steady-state value of 104 _ 1.8 l).cm z after 24 h. Mature monolayers were morphologically and functionally polarized. They contained junctional complexes composed of desmosomes and tight junctions with properties similar to those of "leaky" epithelia. Monolayers were capable of maintaining a spontaneous electrical potential sensitive to amiloride, produced a net water flux from the apical to basal side, and discriminated between Na § and CI-ions.The MDCK permeability barrier behaves as a "thin" membrane with negatively charged sites. It has: (a) a linear conductance/concentration relationship; (b) an asymmetric instantaneous current/voltage relationship; (c) a reduced ability to discriminate between Na § and C1-caused by lowering the pH; and (d) a characteristic pattern of ionic selectivity which suggests that the negatively charged sites are highly hydrated and of medium field strength.Measurements of Na § permeability by electrical and tracer methods ruled out exchange diffusion as a mechanism for ion permeation and the lack of current saturation in the I/A~ curves does not support the involvement of carriers. The discrimination between Na § and CI-was severely but reversibly decreased at low pH. suggesting that Na § channels which exclude C1-contain acidic groups dissociated at neutral pH.Bound Ca § ions are involved in maintaining the integrity of the junctions in MDCK monolayers as was shown by a reversible drop of resistance and opening of the junctions in Ca++-free medium containing EGTA.Several other epithelial cell lines are capable of developing a significant resistance under the conditions used to obtain MDCK monolayers.J. CELL BIOLOGY 9 The Rockefeller University Press 9

The experimental opening and resealing of occluding junctions in monolayers of cultured MDCK cells (epithelioid of renal origin) was explored by measuring changes in the electrical resistance across the monolayer and by freeze-fracture electron microscopy . As in natural epithelia, the function of occluding junctions as permeability barriers specifically depends on extracellular Ca" concentration and fails if this ion is replaced by Mg`or Ba ++ . The removal of Ca" and the addition of EGTA to the bathing medium opened the junctions and reduced the transepithelial resistance . Resealing was achieved within 10-15 min by restoring Ca" . Quantitative freeze-fracture electron microscopy showed that junctional opening, caused by lack of Ca", was accompanied by simplification of the pattern of the membrane strands of the occluding junction without disassembly or displacement of the junctional components . Resealing of the cellular contacts involved the gradual return to a normal junctional pattern estimated as the average number of strands constituting the junction . The occluding junctions were also opened by the addition of the ionophore A23187, suggesting that the sealing of the contacts requires high Ca" on the extracellular side and low Ca'c oncentration of the cytoplasmic compartment. The opening process could be blocked by low temperature (7 .5°C) . Resealing did not depend on serum factors and did not require protein synthesis; therefore, it seems to be caused by reassembly of preexisting membrane junctional components . The restoration of the junctions occurred simultaneously with the establishment of ion-selective channels ; the Na'/Cl -and the cation/cation selectivity were recovered with the same time-course as the electrical resistance . The role of the cytoskeleton in the process of junctional reassembly is reported in the companion article (Meza et al ., 1980, f. Cell Biol ., 87 : 746-754.) . intermixed with "leaky" ones (7).As in the case of leaky epithelia, the monolayer has the ability to discriminate Na' from Cl -with a 9:1 permeability ratio, and the monovalent cations of the series IA follow Eisenman's 6th pattern (corresponding to negative sites with a medium force field) .The occluding junctions of MDCK monolayers can be opened by removing Ca" and adding EGTA to the bathing medium . Restoration of Ca" reseals the junction (5). functional resealing, which is completed in 2-3 h, offers the opportunity to study the factors that participate in the reassembly process of the cellular contacts by monitoring changes in transmural electrical resistance and by modifications of the freeze-fracture electron microscopy pattern of the junctions.

Synthesis and assembly of tight junctions are studied in monolayers of MDCK cells plated at a density sufficient for confluence, allowed to attach for 1 hr, and transferred to fresh media without cells containing or not Ca2+. 20 hr later, while monolayers with Ca2+ have fully developed junctions that confer an electrical resistance across of 346 +/- 51 omega cm2, those without Ca2+ have a negligible resistance. If at this time Ca2+ is added, junctions assemble and seal with a fast kinetics, that can be followed through the development of electrical resistance, penetration of ruthenium red, and electron microscopy. Drugs that impair synthesis, maturation and transport of proteins (cycloheximide, tunicamycin, monensin) indicate that protein components are synthesized early upon plating, do not seem to require N-glycosylation, and are stored in the Golgi compartment. Upon addition of Ca2+ they are transferred to the membrane with the participation of microfilaments but not of microtubules. These components seem to insert directly in the position they occupy in the strands, and the cell circles its perimeter with one strand as early as 15 min, even if in some segments it only consists of a row of particles. New strands develop in association with previous ones, and the pattern completes in 4 to 6 hr. Ca2+ is required for the maintenance of the assembly and also for the sealing with neighboring cells. These processes cannot occur below 25 degrees C. Serum is not required. Polarized distribution of intramembrane particles (IMP) in apical and basolateral regions follows the same time course as junction formation, in spite of the fence constituted by those strands that are already assembled. This suggests that IMP do not redistribute by lateral displacements in the plane of the membrane, but by removal and insertion in the apical and basolateral domains.

Abstract. Extracellular Ca 2÷ triggers assembly and sealing of tight junctions (TJs) in MDCK cells. These events are modulated by G-proteins, phospholipase C, protein kinase C (PKC), and calmodulin. In the present work we observed that 1,2-dioctanoylglycerol (diC8) promotes the assembly of TJ in low extracellular Ca 2÷, as evidenced by translocation of the TJassociated protein ZO-1 to the plasma membrane, formation of junctional fibrils observed in freeze-fracture replicas, decreased permeability of the intercellular space to [3H]mannitol, and reorganization of actin illaments to the cell periphery, visualized by fluorescence microscopy using rhodamine-phalloidin. In contrast, diC8 in low Ca 2÷ did not induce redistribution of the Ca-dependent adhesion protein E-cadherin (uvomorulin). Extracellular antibodies to E-cadherin block junction formation normally induced by adding Ca 2+. die8 counteracted this inhibition, suggesting that PKC may be in the signaling pathway activated by E-cadherinmediated cell-cell adhesion.In addition, we found a novel phosphoprotein of 130 kD which coimmunoprecipitated with the ZO-1/ZO-2 complex. Although the assembly and sealing of TJs may involve the activation of PKC, the level of phosphorylation of ZO-1, ZO-2, and the 130-kD protein did not change after adding Ca 2+ or a PKC agonist. The complex of these three proteins was present even in low extracellular Ca 2+, suggesting that the addition of Ca 2+ or diC8 triggers the translocation and assembly of preformed TJ subcomplexes.