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Tight junctions are cell-cell adhesion structures in epithelial cell sheets that surround organ compartments in multicellular organisms and regulate the permeation of ions through the intercellular space. Claudins are the major constituents of tight junctions and form strands that mediate cell adhesion and function as paracellular barriers. We report the structure of mammalian claudin-15 at a resolution of 2.4 angstroms. The structure reveals a characteristic β-sheet fold comprising two extracellular segments, which is anchored to a transmembrane four-helix bundle by a consensus motif. Our analyses suggest potential paracellular pathways with distinctive charges on the extracellular surface, providing insight into the molecular basis of ion homeostasis across tight junctions.

Claudins (Cldn) are essential membrane proteins of tight junctions (TJs), which form the paracellular permselective barrier. They are produced by a multi-gene family of 24 reported members in mouse and human. Based on a comprehensive search combined with phylogenetic analyses, we identified three novel claudins (claudin-25, -26, and -27). Quantitative RT-PCR revealed that the three novel claudins were expressed in a tissue- and/or developmental stage-dependent manner. Claudins-25 and -26, but not claudin-27, were immunofluorescently localized to TJs when exogenously expressed in cultured MDCK and Eph epithelial cell lines. These findings expand the claudin family to include at least 27 members.

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Claudins are main cell-cell adhesion molecules of tight junctions (TJs) between cells in epithelial sheets that form tight barriers that separate the apical from the basolateral space but also contain paracellular channels that regulate the flow of ions and solutes in between these intercellular spaces. Recently, the first crystal structure of a claudin was determined, that of claudin-15, which indicated the parts of the large extracellular domains that likely form the pore-lining surfaces of the paracellular channels. However, the crystal structure did not show how claudin molecules are arranged in the cell membrane to form the backbone of TJ strands and to mediate interactions between adjacent cells, information that is essential to understand how the paracellular channels in TJs function. Here, we propose that TJ strands consist of claudin protomers that assemble into antiparallel double rows. This model is based on cysteine crosslinking experiments that show claudin-15 to dimerize face to face through interactions between the edges of the extracellular β-sheets. Strands observed by freeze-fracture electron microscopy of TJs also show that their width is consistent with the dimensions of a claudin dimer. Furthermore, we propose that extracellular variable regions are responsible for head-to-head interactions of TJ strands in adjoining cells, thus resulting in the formation of paracellular channels. Our model of the TJ architecture provides a basis to discuss structural mechanisms underlying the selective ion permeability and barrier properties of TJs.

The ezrin-radixin-moesin (ERM) family of proteins crosslink actin filaments and integral membrane proteins. Radixin (encoded by Rdx) is the dominant ERM protein in the liver of wildtype mice and is concentrated at bile canalicular membranes (BCMs). Here we show that Rdx(-/-) mice are normal at birth, but their serum concentrations of conjugated bilirubin begin to increase gradually around 4 weeks, and they show mild liver injury after 8 weeks. This phenotype is similar to human conjugated hyperbilirubinemia in Dubin-Johnson syndrome, which is caused by mutations in the multidrug resistance protein 2 (MRP2, gene symbol ABCC2), although this syndrome is not associated with overt liver injury. In wildtype mice, Mrp2 concentrates at BCMs to secrete conjugated bilirubin into bile. In the BCMs of Rdx(-/-) mice, Mrp2 is decreased compared with other BCM proteins such as dipeptidyl peptidase IV (CD26) and P-glycoproteins. In vitro binding studies show that radixin associates directly with the carboxy-terminal cytoplasmic domain of human MRP2. These findings indicate that radixin is required for secretion of conjugated bilirubin through its support of Mrp2 localization at BCMs.

Coordinated beating of cilia in the trachea generates a directional flow of mucus required to clear the airways. Each cilium originates from a barrel-shaped basal body, from the side of which protrudes a structure known as the basal foot. We generated mice in which exons 6 and 7 of Odf2, encoding a basal body and centrosome-associated protein Odf2/cenexin, are disrupted. Although Odf2(ΔEx6,7/ΔEx6,7) mice form cilia, ciliary beating is uncoordinated, and the mice display a coughing/sneezing phenotype. Whereas residual expression of the C-terminal region of Odf2 in these mice is sufficient for ciliogenesis, the resulting basal bodies lack basal feet. Loss of basal feet in ciliated epithelia disrupted the polarized organization of apical microtubule lattice without affecting planar cell polarity. The requirement for Odf2 in basal foot formation, therefore, reveals a crucial role of this structure in the polarized alignment of basal bodies and coordinated ciliary beating.

Zonula occludens (ZO)-1/2/3 are the members of the TJ-MAGUK family of membrane-associated guanylate kinases associated with tight junctions. To investigate the role of ZO-1 (encoded by Tjp1) in vivo, ZO-1 knockout (Tjp1 ؊/؊ ) mice were generated by gene targeting. Although heterozygous mice showed normal development and fertility, delayed growth and development were evident from E8.5 onward in Tjp1 ؊/؊ embryos, and no viable Tjp1 ؊/؊ embryos were observed beyond E11.5. Tjp1 ؊/؊ embryos exhibited massive apoptosis in the notochord, neural tube area, and allantois at embryonic day (E)9.5. In the yolk sac, the ZO-1 deficiency induced defects in vascular development, with impaired formation of vascular trees, along with defective chorioallantoic fusion. Immunostaining of wild-type embryos at E8.5 for ZO-1/2/3 revealed that ZO-1/2 were expressed in almost all embryonic cells, showing tight junction-localizing patterns, with or without ZO-3, which was confined to the epithelial cells. ZO-1 deficiency depleted ZO-1-expression without influence on ZO-2/3 expression. In Tjp1 ؉/؉ yolk sac extraembryonic mesoderm, ZO-1 was dominant without ZO-2/3 expression. Thus, ZO-1 deficiency resulted in mesoderms with no ZO-1/2/3, associated with mislocalization of endothelial junctional adhesion molecules. As a result, angiogenesis was defected in Tjp1 ؊/؊ yolk sac, although differentiation of endothelial cells seemed to be normal. In conclusion, ZO-1 may be functionally important for cell remodeling and tissue organization in both the embryonic and extraembryonic regions, thus playing an essential role in embryonic development. INTRODUCTIONIn multicellular organisms, cell-cell adhesion is critical for development and morphogenesis. Various types of cell-cell adhesion-related molecules have been identified, and evidence has accumulated that their expression and functions are critically regulated in a spatiotemporally highly organized way (Tsukita et al., 2001;Halbleib and Nelson, 2006). In general, cell-cell adhesion molecules are integral membrane proteins that associate with peripheral membrane proteins to regulate and integrate cell-cell adhesion-related phenomena. Zonula occludens (ZO)-1 is a founding member of membrane-associated guanylate kinase (MAGUK) family proteins of tight junctions (TJs), composed of three postsynaptic density 95/disc-large/ZO-1 (PDZ) domains, a Src homology 3 domain, a guanylate kinase (GUK) domain, an acidic domain, and an actin binding region (Itoh et al., 1993; Willott et al., 1993;Jesaitis and Goodenough, 1996;Haskins et al., 1998). It was first defined as an antigen for monoclonal antibodies that recognized TJs in epithelial cells, and it was subsequently revealed as a peripheral membrane protein with a molecular mass of 220 kDa, which underlied the cytoplasmic surface of plasma membranes of TJs (Stevenson et al., 1986). Evidence has accumulated that in epithelial cells, ZO-1 is exclusively located at TJs, but when TJs are not formed, it is concentrated in adherens junctions (AJs) (Itoh et al., 199...