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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.