The N and C termini of ZO-1 are surrounded by distinct proteins and functional protein networks.

BACKGROUND: Biotin ligase tagging with ZO-1 was applied to identify a more complete tight junction proteome. RESULTS: Identical but also different proteins and functional networks were identified near the N and C ends of ZO-1. CONCLUSION: The ends of ZO-1 are embedded in different functional subcompartments of the tight junction. SIGNIFICANCE: Biotin tagging with ZO-1 expands the tight junction proteome and defines subcompartments of the junction. The proteins and functional protein networks of the tight junction remain incompletely defined. Among the currently known proteins are barrier-forming proteins like occludin and the claudin family; scaffolding proteins like ZO-1; and some cytoskeletal, signaling, and cell polarity proteins. To define a more complete list of proteins and infer their functional implications, we identified the proteins that are within molecular dimensions of ZO-1 by fusing biotin ligase to either its N or C terminus, expressing these fusion proteins in Madin-Darby canine kidney epithelial cells, and purifying and identifying the resulting biotinylated proteins by mass spectrometry. Of a predicted proteome of ∼9000, we identified more than 400 proteins tagged by biotin ligase fused to ZO-1, with both identical and distinct proteins near the N- and C-terminal ends. Those proximal to the N terminus were enriched in transmembrane tight junction proteins, and those proximal to the C terminus were enriched in cytoskeletal proteins. We also identified many unexpected but easily rationalized proteins and verified partial colocalization of three of these proteins with ZO-1 as examples. In addition, functional networks of interacting proteins were tagged, such as the basolateral but not apical polarity network. These results provide a rich inventory of proteins and potential novel insights into functions and protein networks that should catalyze further understanding of tight junction biology. Unexpectedly, the technique demonstrates high spatial resolution, which could be generally applied to defining other subcellular protein compartmentalization.

Setting up a selective barrier at the apical junction complex.

Across the animal kingdom the apical junction complex of epithelial cells creates both a permeability barrier and cell polarity. Although based on overlapping and evolutionarily conserved proteins, the cell-cell contacts of nematodes, flies and mammals appear to differ in morphology and functional organization. Emerging evidence shows that the selective pore-like properties of vertebrate and invertebrate barriers are created by the claudin family. Similarly, assembly of the barriers requires a conserved set of polarity-generating protein complexes, particularly the PAR protein complexes.

Newly synthesized claudins but not occludin are added to the basal side of the tight junction.

A network of claudin strands creates continuous cell-cell contacts to form the intercellular tight junction barrier; a second protein, occludin, is associated along these strands. The physiological barrier remains stable despite protein turnover, which involves removal and replacement of claudins both in the steady state and during junction remodeling. Here we use a pulse-block-pulse labeling protocol with fluorescent ligands to label SNAP/CLIP-tags fused to claudins and occludin to identify their spatial trafficking pathways and kinetics in Madin-Darby canine kidney monolayers. We find that claudins are first delivered to the lateral membrane and, over time, enter the junction strand network from the basal side; this is followed by slow replacement of older claudins in the strands. In contrast, even at early times, newly synthesized occludin is found throughout the network. Taking the results together with our previous documentation of the mechanism for claudin strand assembly in a fibroblast model, we speculate that newly synthesized claudins are added at strand breaks and free ends; these are most common in the basalmost edge of the junction. In contrast, occludin can be added directly within the strand network. We further demonstrate that claudin trafficking and half-life depend on carboxy-terminal sequences and that different claudins compete for tight junction localization.

Isolation and functional characterization of the actin binding region in the tight junction protein ZO-1.

Zonula occludens (ZO)-1 is a member of the MAGUK (membrane-associated guanylate kinase homologs) family of membrane-associated signaling molecules that binds directly to both cytosolic and transmembrane components of the tight junction and is believed to organize these proteins within the apical junctional complex. It also binds directly to F-actin, although the functional relevance of this interaction is unknown. To address this issue, we have used VSVG-tagged transgenes to dissect ZO-1 and have identified a 220 amino acid region of ZO-1 that is necessary for its association with F-actin in MDCK cell pull-down assays. A GST fusion expressing this region can bind directly to F-actin in vitro, whereas a GFP fusion expressing this domain decorates actin stress fibers when expressed in MDCK cells. These results indicate that this actin-binding region (ABR) is both necessary and sufficient for binding to F-actin in vitro and in vivo. VSVG-tagged transgenes that lack the ABR still accumulate at both early and late cell-cell contacts in MDCK cells, suggesting that the ABR is not required for tight junction localization. However, accumulation of constructs lacking the ABR is markedly reduced at tight junctions in confluent cells, suggesting that the ABR does play an important role in the localization of ZO-1 at junctions. Furthermore, the ABR is required for localization to a novel actin-rich pool of ZO-1 that accumulates in puncta at the free edge of cells before initiation of cell-cell contact. We conclude that direct interactions between ZO-1 and F-actin play a role in several different steps of junction assembly.

Expression, solubilization, and biochemical characterization of the tight junction transmembrane protein claudin-4.

The tight junction tetraspan protein claudin-4 creates a charge-selective pore in the paracellular pathway across epithelia. The structure of the pore is unknown, but is presumed to result from transcellular adhesive contacts between claudin's extracellular loops. Here we report the expression of claudin-4 by baculovirus infection of Sf9 cells and describe the biochemical analysis suggesting it has a hexameric quaternary configuration. We show the detergent perfluoro-octanoic acid is able to maintain oligomeric claudin species. Sucrose velocity centrifugation and laser light scattering are also used to investigate the oligomeric state of claudin-4. In contrast to proteins of similar topology, such as gap junction family connexins, the oligomeric state of claudins appears more dynamic. These data suggest the structural organization of claudins in tight junction pores is unique.

Claudin interactions in and out of the tight junction.

Claudins form the paracellular tight junction seal in epithelial tissues. Although there is still limited information on how these proteins are organized at the junction, a number of recent studies have provided useful insights both into claudin-claudin interactions and into interactions between claudins and other proteins. The focus of this review is to summarize recent information about claudin interactions and to identify critical unanswered questions about claudin organization and tight junction structure which will be required to understand claudin function.

The life and work of Shoichiro Tsukita.

We dedicate the 2008 Berlin conference and this collection of scientific manuscripts to the memory of our colleague Shoichiro Tsukita. His seminal scientific contributions and impact on the field of tight junctions is substantial. Shoichiro was a professor at Kyoto University and one the world's most influential biologists when he passed away on December 11, 2005 at the age of 52 from complications of pancreatic cancer. He was a pioneer in several areas of cell biology; most particularly he will be remembered as the founding father of the molecular study of tight junctions.

Claudin-2-dependent changes in noncharged solute flux are mediated by the extracellular domains and require attachment to the PDZ-scaffold.

Paracellular transport through the tight junction shows selectivity for both ionic charge and solute size. It is known that charged residues on the extracellular loops of claudins control charge selectivity. It is also known that inducible expression of claudin-2, but not claudin-4, will selectively increase the permeability for polyethylene glycol (PEG) molecules which are <0.4 A in radius, but it is not known whether permeability is controlled by the same regions of claudins which control charge selectivity. Using inducible expression of chimeras of claudin-2 and claudin-4 in monolayers of MDCK II cells we show that the extracellular loops alone are responsible for controlling the permeability for noncharged PEGs as well as for charge selectivity. Further, the cytoplasmic C-terminal PDZ-binding motif is required for wild-type claudin-2 to control permeability, suggesting a requirement for attachment to the PDZ scaffold in order to form pores. These observations support a model where the loops form pores controlling permeability for both charged and noncharged solutes which are smaller than 0.4 A. They leave unanswered why both claudin-2 and -4 can influence electrical properties while only -2 can selectively increase permeability for small PEGs.

The molecular physiology of tight junction pores.

Tight junctions form selective barriers that regulate paracellular transport across epithelia. A large family of tetraspanning cell-cell adhesion proteins called claudins create the barrier and regulate electrical resistance, size, and ionic charge selectivity. Study of inherited human claudin diseases and the outcome of the genetic manupulation of claudins in mice, Drosophila, and Caenorhabditis elegans are furthering our understanding of paracellular physiology.

Claudin extracellular domains determine paracellular charge selectivity and resistance but not tight junction fibril architecture.

Tight junctions (TJs) regulate paracellular permeability across epithelia and vary widely in their transepithelial electrical resistance (TER) and charge selectivity. The claudin family of transmembrane proteins influences these properties. We previously reported that claudin-4 increased TER approximately 300% when expressed in low-resistance Madin-Darby canine kidney (MDCK) II cells and decreased the paracellular permeability for Na(+) more than Cl(-) (Van Itallie C, Rahner C, and Anderson JM. J Clin Invest 107: 1319-1327, 2001). In comparison, we report here that expression of claudin-2 increases TER by only approximately 20% and does not change the ionic selectivity of MDCK II cells from their cation-selective background. To test whether the extracellular domains of claudins-4 and -2 determine their unique paracellular properties, we determined the effects of interchanging these domains between claudins-4 and -2. Inducible expression of wild-type claudins and extracellular domain chimeras increased both the number and depth of fibrils, but the characteristic fibril morphologies of claudin-4 or -2 were not altered by switching extracellular domains. Like claudin-4, chimeras expressing the first or both extracellular domains of claudin-4 on claudin-2 increased TER severalfold and profoundly decreased the permeability of Na(+) relative to Cl(-). In contrast, chimeras expressing the first or both extracellular domains of claudin-2 on claudin-4 increased the TER by only approximately 60 and approximately 40%, respectively, and only modestly altered charge selectivity. These results support a model in which the claudins create paracellular channels and the first extracellular domain is sufficient to determine both paracellular charge selectivity and TER.

Claudins create charge-selective channels in the paracellular pathway between epithelial cells.

Epithelia separate tissue spaces by regulating the passage of ions, solutes, and water through both the transcellular and paracellular pathways. Paracellular permeability is defined by intercellular tight junctions, which vary widely among tissues with respect to solute flux, electrical resistance, and ionic charge selectivity. To test the hypothesis that members of the claudin family of tight junction proteins create charge selectivity, we assessed the effect of reversing the charge of selected extracellular amino acids in two claudins using site-directed mutagenesis. Claudins were expressed in cultured Madin-Darby canine kidney cell monolayers under an inducible promoter, and clones were compared with and without induction for transmonolayer electrical resistance and dilution potentials. Expression and localization of claudins were determined by immunoblotting, immunofluorescence microscopy, and freeze-fracture electron microscopy. We observed that substituting a negative for a positive charge at position 65 in the first extracellular domain of claudin-4 increased paracellular Na+ permeability. Conversely, substituting positive for negative charges at three positions in the first extracellular domain of claudin-15, singly and in combination, reversed paracellular charge selectivity from a preference for Na+ to Cl-. These results support a model where claudins create charge-selective channels in the paracellular space.