Claudin-16 and claudin-19 interact and form a cation-selective tight junction complex.
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ABSTRACT:
SUBMITTER: Hou J
PROVIDER: S-EPMC2176193 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The molecular nature of tight junction architecture and permeability is a long-standing mystery. Here, by comprehensive biochemical, biophysical, genetic, and electron microscopic analyses of claudin-16 and -19 interactions--two claudins that play key polygenic roles in fatal human renal disease, FHHNC--we found that 1) claudin-16 and -19 form a stable dimer through cis association of transmembrane domains 3 and 4; 2) mutations disrupting the claudin-16 and -19 cis interaction increase tight junction ultrastructural complexity but reduce tight junction permeability; and 3) no claudin hemichannel or heterotypic channel made of claudin-16 and -19 trans interaction can exist. These principles can be used to artificially alter tight junction permeabilities in various epithelia by manipulating selective claudin interactions. Our study also emphasizes the use of a novel recording approach based on scanning ion conductance microscopy to resolve tight junction permeabilities with submicrometer precision.
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Project description:Claudin proteins constitute the backbone of tight junctions (TJs) regulating paracellular permeability for solutes and water. The molecular mechanism of claudin polymerization and paracellular channel formation is unclear. However, a joined double-rows architecture of claudin strands has been supported by experimental and modeling data. Here, we compared two variants of this architectural model for the related but functionally distinct cation channel-forming claudin-10b and claudin-15: tetrameric-locked-barrel vs octameric-interlocked-barrels model. Homology modeling and molecular dynamics simulations of double-membrane embedded dodecamers indicate that claudin-10b and claudin-15 share the same joined double-rows architecture of TJ-strands. For both, the results indicate octameric-interlocked-barrels: Sidewise unsealed tetrameric pore scaffolds interlocked with adjacent pores via the β1β2 loop of the extracellular segment (ECS) 1. This loop mediates hydrophobic clustering and, together with ECS2, cis- and trans-interaction between claudins of the adjacent tetrameric pore scaffolds. In addition, the β1β2 loop contributes to lining of the ion conduction pathway. The charge-distribution along the pore differs between claudin-10b and claudin-15 and is suggested to be a key determinant for the cation- and water permeabilities that differ between the two claudins. In the claudin-10b simulations, similar as for claudin-15, the conserved D56 in the pore center is the main cation interaction site. In contrast to claudin-15 channels, the claudin-10b-specific D36, K64 and E153 are suggested to cause jamming of cations that prevents efficient water passage. In sum, we provide novel mechanistic information about polymerization of classic claudins, formation of embedded channels and thus regulation of paracellular transport across epithelia.
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Project description:Claudins are tight junction integral membrane proteins that are key regulators of the paracellular pathway. Defects in claudin-16 (CLDN16) and CLDN19 function result in the inherited human renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). Previous studies showed that siRNA knockdown of CLDN16 in mice results in a mouse model for FHHNC. Here, we show that CLDN19-siRNA mice also developed the FHHNC symptoms of chronic renal wasting of magnesium and calcium together with defective renal salt handling. siRNA knockdown of CLDN19 caused a loss of CLDN16 from tight junctions in the thick ascending limb (TAL) without a decrease in CLDN16 expression level, whereas siRNA knockdown of CLDN16 produced a similar effect on CLDN19. In both mouse lines, CLDN10, CLDN18, occludin, and ZO-1, normal constituents of TAL tight junctions, remained correctly localized. CLDN16- and CLDN19-depleted tight junctions had normal barrier function but defective ion selectivity. These data, together with yeast two-hybrid binding studies, indicate that a heteromeric CLDN16 and CLDN19 interaction was required for assembling them into the tight junction structure and generating cation-selective paracellular channels.
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Project description:The extent of tight junction (TJ) formation is one of many factors that regulate motility, invasion, and metastasis. Claudins are required for the formation and maintenance of TJs. Claudin-3 (CLDN3) and claudin-4 (CLDN4) are highly expressed in the majority of ovarian cancers. We report here that CLDN3 and CLDN4 each serve to constrain the growth of human 2008 cancer xenografts and limit metastatic potential. Knockdown of CLDN3 increased in vivo growth rate by 2.3-fold and knockdown of CLDN4 by 3.7-fold in the absence of significant change in in vitro growth rate. Both types of tumors exhibited increase in birth rate as measured by Ki67 staining and decrease in death rate as reflected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Knockdown of either claudin did not alter expression of other TJ protein but did reduce TJ formation as measured by transepithelial resistance and paracellular flux of dextran, enhance migration and invasion in in vitro assays, and increase lung colonization following intravenous injection. Knockdown of CLDN3 and CLDN4 increased total lung metastatic burden by 1.7-fold and 2.4-fold, respectively. Loss of either CLDN3 or CLDN4 resulted in down-regulation of E-cadherin mRNA and protein, increased inhibitory phosphorylation of glycogen synthase kinase-3? (GSK-3?), and activation of ?-catenin pathway signaling as evidenced by increases in nuclear ?-catenin, the dephosphorylated form of the protein, and transcriptional activity of ?-catenin/T-cell factor (TCF). We conclude that both CLDN3 and CLDN4 mediate interactions with other cells in vivo that restrain growth and metastatic potential by sustaining expression of E-cadherin and limiting ?-catenin signaling.
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Project description:Tight junctions consist of a network of sealing strands that create selective ion permeability barriers between adjoining epithelial or endothelial cells. The current model for tight junction strands consists of paired rows of claudins (Cldn) coupled by a cis interface (X-1) derived from crystalline Cldn15. Here we show that tight junction strands exhibit a broad range of lateral bending, indicating diversity in cis interactions. By combining protein-protein docking, coevolutionary analysis, molecular dynamics, and a mutagenesis screen, we identify a new Cldn-Cldn cis interface (Cis-1) that shares interacting residues with X-1 but has an ~?17° lateral rotation between monomers. In addition, we found that a missense mutation in a Cldn14 that causes deafness and contributes stronger to Cis-1 than to X-1 prevents strand formation in cultured cells. Our results suggest that Cis-1 contributes to the inherent structural flexibility of tight junction strands and is required for maintaining permeability barrier function and hearing.
| S-EPMC6123731 | biostudies-literature
Project description:IQGAP1 is a scaffolding protein previously implicated in adherens junction formation. However, its role in the establishment or maintenance of tight junctions (TJs) has not been explored. We hypothesized that IQGAP1 could regulate TJ formation by modulating the expression and/or localization of junctional proteins, and we systematically tested this hypothesis in the model Madin-Darby canine kidney (MDCK) cell line. We find that IQGAP1 silencing enhances a transient increase in transepithelial electrical resistance (TER) observed during the early stages of TJ formation (Cereijido et al., 1978). Quantitative microscopy and biochemical experiments suggest that this effect of IQGAP1 on TJ assembly is accounted for by reduced expression and TJ recruitment of claudin 2, and increased TJ recruitment of claudin 4. Furthermore, we show that IQGAP1 also regulates TJ formation through its interactor CDC42, because IQGAP1 knockdown increases the activity of the CDC42 effector JNK and dominant-negative CDC42 prevents the increase in TER caused by IQGAP1 silencing. Hence, we provide evidence that IQGAP1 modulates TJ formation by a twofold mechanism: (1) controlling the expression and recruitment of claudin 2 and recruitment of claudin 4 to the TJ, and (2) transient inhibition of the CDC42-JNK pathway.
| S-EPMC4342576 | biostudies-literature
Project description:Claudin family tight junction proteins form charge- and size-selective paracellular channels that regulate epithelial barrier function. In the gastrointestinal tract, barrier heterogeneity is attributed to differential claudin expression. Here, we show that claudin-23 (CLDN23) is enriched in luminal intestinal epithelial cells where it strengthens the epithelial barrier. Complementary approaches reveal that CLDN23 regulates paracellular ion and macromolecule permeability by associating with CLDN3 and CLDN4 and regulating their distribution in tight junctions. Computational modeling suggests that CLDN23 forms heteromeric and heterotypic complexes with CLDN3 and CLDN4 that have unique pore architecture and overall net charge. These computational simulation analyses further suggest that pore properties are interaction-dependent, since differently organized complexes with the same claudin stoichiometry form pores with unique architecture. Our findings provide insight into tight junction organization and propose a model whereby different claudins combine to form multiple distinct complexes that modify epithelial barrier function by altering tight junction structure.
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Project description:Barrier function of hair follicles (HFs) is of great interest because they might be an entry port for allergens/pathogens, but could on the other hand be used for drug delivery or vaccination. Therefore we investigated tight junction (TJ) barrier function in human HFs. We show that there is a TJ barrier in the outermost living layer bordering to the environment from the infundibulum to the lower central part and between Henle's and Huxles layer of anagen HFs. In club hair typical for catagen and telogen HFs a TJ barrier is found surrounding the club. This demonstrates that there is a continuous TJ barrier along interfollicular epidermis and HFs in different phases of HF cycle. However, interestingly, in cell culture experiments we can show that barrier is less tight in HF keratinocytes compared to interfollicular keratinocytes. Knock-down of the TJ protein claudin-1, which we demonstrate here to be less expressed in HFs of lesional atopic dermatitis skin, results in impaired barrier function, decreased proliferation and increased apoptosis of hair keratinocytes. This is in line with a hair growth phenotype in claudin-1 deficient patients (NISCH syndrome) and corresponding knock-out mice and indicates an important role of claudin-1 in HF barrier function and growth.
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Project description:BackgroundThe blood-brain barrier (BBB) ensures central nervous system (CNS) homeostasis by strictly controlling the passage of molecules and solutes from the bloodstream into the CNS. Complex and continuous tight junctions (TJs) between brain endothelial cells block uncontrolled paracellular diffusion of molecules across the BBB, with claudin-5 being its dominant TJs protein. However, claudin-5 deficient mice still display ultrastructurally normal TJs, suggesting the contribution of other claudins or tight-junction associated proteins in establishing BBB junctional complexes. Expression of claudin-12 at the BBB has been reported, however the exact function and subcellular localization of this atypical claudin remains unknown.MethodsWe created claudin-12-lacZ-knock-in C57BL/6J mice to explore expression of claudin-12 and its role in establishing BBB TJs function during health and neuroinflammation. We furthermore performed a broad standardized phenotypic check-up of the mouse mutant.ResultsMaking use of the lacZ reporter allele, we found claudin-12 to be broadly expressed in numerous organs. In the CNS, expression of claudin-12 was detected in many cell types with very low expression in brain endothelium. Claudin-12lacZ/lacZ C57BL/6J mice lacking claudin-12 expression displayed an intact BBB and did not show any signs of BBB dysfunction or aggravated neuroinflammation in an animal model for multiple sclerosis. Determining the precise localization of claudin-12 at the BBB was prohibited by the fact that available anti-claudin-12 antibodies showed comparable detection and staining patterns in tissues from wild-type and claudin-12lacZ/lacZ C57BL/6J mice.ConclusionsOur present study thus shows that claudin-12 is not essential in establishing or maintaining BBB TJs integrity. Claudin-12 is rather expressed in cells that typically lack TJs suggesting that claudin-12 plays a role other than forming classical TJs. At the same time, in depth phenotypic screening of clinically relevant organ functions of claudin-12lacZ/lacZ C57BL/6J mice suggested the involvement of claudin-12 in some neurological but, more prominently, in cardiovascular functions.
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Project description:In the renal collecting duct, intercalated cells regulate acid-base balance by effluxing protons through the v-H+-ATPase, and bicarbonate via apical pendrin or the basolateral kidney anion exchanger 1 (kAE1). Additionally, collecting duct cells play an essential role in transepithelial absorption of sodium and chloride. Expression of kAE1 in polarized MDCK I cells was previously shown to decrease trans-epithelial electrical resistance (TEER), suggesting a novel role for kAE1 in paracellular permeability. In our study, we not only confirmed that inducible expression of kAE1 in mIMCD3 cells decreased TEER but we also observed (i) increased epithelial absolute permeability to both sodium and chloride, and (ii) that this effect was dependent on kAE1 activity. Further, kAE1 regulated tight junction properties through the tight junction protein claudin-4, a protein with which it physically interacts and colocalizes. These findings unveil a novel interaction between the junctional protein claudin-4 and the kidney anion exchanger, which may be relevant to ion and/or pH homeostasis.
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