cells, transfection of high-resistance MDCK-C7 cells withCldn10b dramatically decreased transepithelial resistance, increased cation permeability, and changed monovalent cation selectivity from Eisenman sequence IV to X, indicating the presence of a high field-strength binding site that almost completely removes the hydration shell of the permeating cations. The extent of all these effects strongly depended on the endogenous claudins of the transfected cells. Supplementary material available online at

ZO-1, ZO-2, and ZO-3 are closely related scaffolding proteins that link tight junction (TJ) transmembrane proteins such as claudins, junctional adhesion molecules, and occludin to the actin cytoskeleton. Even though the zonula occludens (ZO) proteins are among the first TJ proteins to have been identified and have undergone extensive biochemical analysis, little is known about the physiological roles of individual ZO proteins in different tissues or during vertebrate development. Here, we show that ZO-3 knockout mice lack an obvious phenotype. In contrast, embryos deficient for ZO-2 die shortly after implantation due to an arrest in early gastrulation. ZO-2 ؊/؊ embryos show decreased proliferation at embryonic day 6.5 (E6.5) and increased apoptosis at E7.5 compared to wild-type embryos. The asymmetric distribution of prominin and E-cadherin to the apical and lateral plasma membrane domains, respectively, is maintained in cells of ZO-2 ؊/؊ embryos. However, the architecture of the apical junctional complex is altered, and paracellular permeability of a low-molecular-weight tracer is increased in ZO-2 ؊/؊ embryos. Leaky TJs and, given the association of ZO-2 with connexins and several transcription factors, effects on gap junctions and gene expression, respectively, are likely causes for embryonic lethality. Thus, ZO-2 is required for mouse embryonic development, but ZO-3 is dispensable. This is to our knowledge the first report showing that an individual ZO protein plays a nonredundant and critical role in mammalian development.Tight junctions (TJs) are regions of intimate contact between the plasma membrane domains of adjoining cells and are predominantly found in columnar epithelial and endothelial cells, where they are part of the apical junctional complex (reviewed in reference 47). TJs are also found in hepatocytes (13), keratinocytes (13), and Schwann cells (40).TJs contain integral membrane proteins, notably claudins, occludin, and junctional adhesion molecules, which engage in homotypic and heterotypic interactions through their extracellular domains with corresponding proteins on adjoining cells (reviewed in reference 3 and 52). On the cytoplasmic side, adaptor or scaffolding molecules tether the integral membrane TJ proteins to the actin cytoskeleton. The best-characterized adaptors that directly link transmembrane TJ proteins to the cytoskeleton are ZO-1, ZO-2, and ZO-3, three closely related proteins that are widely expressed in different tissues and organs (reviewed in reference 21). Zonula occludens (ZO) proteins carry three PDZ (PSD-95/Dlg/ZO-1) domains, an Src homology 3 domain, and a guanylate kinase-like domain, and hence belong to the membrane-associated guanylate kinaselike superfamily of proteins (reviewed in references 20 and 21). A growing number of both structural and regulatory proteins that associate with the different domains of ZO proteins have been identified (reviewed in reference 21). Some of these interacting partners bind selectively to an individual ZO protein; others bind to two...

Claudin-16 (Cldn16) is selectively expressed at tight junctions (TJs) of renal epithelial cells of the thick ascending limb of Henle's loop, where it plays a central role in the reabsorption of divalent cations. Over 20 different mutations in the CLDN16 gene have been identified in patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), a disease of excessive renal Mg2+ and Ca2+ excretion. Here we show that disease-causing mutations can lead to the intracellular retention of Cldn16 or affect its capacity to facilitate paracellular Mg2+ transport. Nine of the 21 Cldn16 mutants we characterized were retained in the endoplasmic reticulum, where they underwent proteasomal degradation. Three mutants accumulated in the Golgi complex. Two mutants were efficiently delivered to lysosomes, one via clathrin-mediated endocytosis following transport to the cell surface and the other without appearing on the plasma membrane. The remaining 7 mutants localized to TJs, and 4 were found to be defective in paracellular Mg2+ transport. We demonstrate that pharmacological chaperones rescued surface expression of several retained Cldn16 mutants. We conclude that FHHNC can result from mutations in Cldn16 that affect intracellular trafficking or paracellular Mg2+ permeability. Knowledge of the molecular defects associated with disease-causing Cldn16 mutations may open new venues for therapeutic intervention.

Autosomal recessive polycystic kidney disease (ARPKD), usually considered to be a genetically homogeneous disease caused by mutations in PKHD1, has been associated with ciliary dysfunction. Here, we describe mutations in the DAZ interacting protein 1-like (DZIP1L) gene in patients with ARPKD, findings we have further validated by loss-of-function studies in mice and zebrafish. DZIP1L localizes to centrioles and at the distal end of basal bodies, and interacts with septin2, a protein implicated in maintenance of the periciliary diffusion barrier at the ciliary transition zone. Consistent with a defect in the diffusion barrier, we found that the ciliary membrane translocation of the PKD proteins, polycystin-1 and −2, is compromised in DZIP1L mutant cells. Together, these data provide the first conclusive evidence that ARPKD is not a homogeneous disorder, and establishes DZIP1L as a second gene involved in its pathogenesis.