Tight Junction Strands Research Articles

Claudin-4 polymerizes after a small extracellular claudin-3-like substitution

Tight junctions play a pivotal role in the functional integrity of the human body by forming barriers that compartmentalize tissues and protect the body from external threats. Essential components of tight junctions are the transmembrane claudin proteins, which can polymerize into tight junction strands and meshworks. This study delves into the structural determinants of claudin polymerization, utilizing the close homology yet strong difference in polymerization capacity between claudin-3 and claudin-4. Through a combination of sequence alignment and structural modeling, critical residues in the second extracellular segment are pinpointed. Molecular dynamics simulations provide insights into the interactions of and the conformational changes induced by the identified extracellular segment 2 residues. Live-STED imaging demonstrates that introduction of these residues from claudin-3 into claudin-4 significantly enhances polymerization in non-epithelial cells. In tight junction-deficient epithelial cells, mutated claudin-4 not only influences tight junction morphology but also partially restores barrier function. Understanding the structural basis of claudin polymerization is crucial, as it offers insights into the dynamic nature of tight junctions. This knowledge could be applied to targeted therapeutic interventions, offering insight to repair or prevent barrier defects associated with pathological conditions, or introduce temporary barrier openings during drug delivery.

CLDN18: Clinical, Pathological, and Genetic Signatures with Drug Screening in Gastric Adenocarcinoma.

The CLDN18 gene, encoding claudin 18.1 and claudin 18.2, is a key component of tight junction strands in epithelial cells that form a paracellular barrier that is critical in Stomach Adenocarcinoma (STAD). Our study included 1,095 patients with proven STAD, 415 from The Cancer Genome Atlas (TCGA) cohort and 680 from the Gene Expression Omnibus database. We applied various analyses, including gene set enrichment analysis, pathway analysis, and in vitro drug screening to evaluate survival, immune cells, and genes and gene sets associated with cancer progression, based on CLDN18 expression levels. Gradient boosting machine learning (70% for training, 15% for validation, and 15% for testing) was used to evaluate the impact of CLDN18 on survival and develop a survival prediction model. High CLDN18 expression correlated with worse survival in lymphocyte-poor STAD, accompanied by decreased helper T cells, altered metabolic genes, low necrosis-related gene expression, and increased tumor proliferation. CLDN18 expression showed associations with gene sets associated with various stomach, breast, ovarian, and esophageal cancers, while pathway analysis linked CLDN18 to immunity. Incorporating CLDN18 expression improved survival prediction in a machine learning model. Notably, nutlin-3a and niraparib effectively inhibited high CLDN18-expressing gastric cancer cells in drug screening. Our study provides a comprehensive understanding of the biological role of CLDN18-based bioinformatics and machine learning analysis in STAD, shedding light on its prognostic significance and potential therapeutic implications. To fully elucidate the molecular intricacies of CLDN18, further investigation is warranted, particularly through in vitro and in vivo studies.

TMEM25 is a Par3-binding protein that attenuates claudin assembly during tight junction development

The tight junction (TJ) in epithelial cells is formed by integral membrane proteins and cytoplasmic scaffolding proteins. The former contains the claudin family proteins with four transmembrane segments, while the latter includes Par3, a PDZ domain-containing adaptor that organizes TJ formation. Here we show the single membrane-spanning protein TMEM25 localizes to TJs in epithelial cells and binds to Par3 via a PDZ-mediated interaction with its C-terminal cytoplasmic tail. TJ development during epithelial cell polarization is accelerated by depletion of TMEM25, and delayed by overexpression of TMEM25 but not by that of a C-terminally deleted protein, indicating a regulatory role of TMEM25. TMEM25 associates via its N-terminal extracellular domain with claudin-1 and claudin-2 to suppress their cis- and trans-oligomerizations, both of which participate in TJ strand formation. Furthermore, Par3 attenuates TMEM25–claudin association via binding to TMEM25, implying its ability to affect claudin oligomerization. Thus, the TJ protein TMEM25 appears to negatively regulate claudin assembly in TJ formation, which regulation is modulated by its interaction with Par3.

Upregulation of SEMP1 Contributes to Improving the Biological Functions of Trophoblast via the PI3K/AKT Pathway in Preeclampsia.

Disturbance of extravillous trophoblast infiltration is associated with preeclampsia (PE), a severe condition of pregnancy characterized by hypertension and proteinuria. Senescence-associated epithelial membrane protein 1 (SEMP1), an integral membrane protein, is a vital component of tight junction strands in epithelial or endothelial cells, with no clear function reported in PE. Gene Expression Omnibus (GEO) datasets showed that SEMP1 expression was downregulated in the placental tissues of PE patients, which was confirmed by assessing SEMP1 levels in placental samples collected in our hospital. Furthermore, less SEMP1 was detected in cytokeratin 7 positive trophoblast cells in the spiral arteries of rat placentas post L-arginine methyl ester hydrochloride (L-NAME) treatment. Trophoblast cells acquired robust ability of proliferation, migration, and invasion when SEMP1 was overexpressed. Such capability was weakened in SEMP1-silenced cells. Trophoblast cells overexpressing SEMP1 secreted more vascular endothelial growth factor A (VEGFA), which facilitated the tube formation of human umbilical vein endothelial cells. Blockade of PI3K/AKT signaling transduction with LY294002 dampened the effects of SEMP1 on trophoblast cells. Collectively, we firstly indicated that SEMP1 inhibition is a potential driver for PE, which may be associated with the deactivation of the PI3K/AKT pathway.

Cholesterol-rich domain formation mediated by ZO proteins is essential for tight junction formation

Tight junctions (TJs) are cell-adhesion structures responsible for the epithelial barrier. We reported that accumulation of cholesterol at the apical junctions is required for TJ formation [K. Shigetomi, Y. Ono, T. Inai, J. Ikenouchi, J. Cell Biol. 217, 2373-2381 (2018)]. However, it is unclear how cholesterol accumulates and informs TJ formation-and whether cholesterol enrichment precedes or follows the assembly of claudins in the first place. Here, we established an epithelial cell line (claudin-null cells) that lacks TJs by knocking out claudins. Despite the lack of TJs, cholesterol normally accumulated in the vicinity of the apical junctions. Assembly of claudins at TJs is thought to require binding to zonula occludens (ZO) proteins; however, a claudin mutant that cannot bind to ZO proteins still formed TJ strands. ZO proteins were however necessary for cholesterol accumulation at the apical junctions through their effect on the junctional actomyosin cytoskeleton. We propose that ZO proteins not only function as scaffolds for claudins but also promote TJ formation of cholesterol-rich membrane domains at apical junctions.

Tight-junction strand networks and tightness of the epithelial barrier.

Tight junctions (TJs) are cell-cell junction structures critical for controlling paracellular permeability. On freeze-fracture replica electron microscopy, they appear as a continuous network of fibrils (TJ strands). TJ strands function as zippers that create a physical barrier against paracellular diffusion of molecules. The morphology of the TJ strand network varies greatly between tissues, and in recent years, studies have highlighted the mechanisms regulating the morphology of TJ strand networks and on their relevance to barrier function. In this review, we discuss evidence regarding the components of the TJ strand and the mechanisms for creating the TJ strand network. Furthermore, we discuss and hypothesize how its morphology contributes to the establishment of the epithelial barrier.

Claudin-10b cation channels in tight junction strands: Octameric-interlocked pore barrels constitute paracellular channels with low water permeability

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.

Molecular mechanism of claudin-15 strand flexibility: A computational study.

Claudins are one of the major components of tight junctions that play a key role in the formation and maintenance of the epithelial barrier function. Tight junction strands are dynamic and capable of adapting their structure in response to large-scale tissue rearrangement and cellular movement. Here, we present molecular dynamics simulations of claudin-15 strands of up to 225 nm in length in two parallel lipid membranes and characterize their mechanical properties. The persistence length of claudin-15 strands is comparable with those obtained from analyses of freeze-fracture electron microscopy. Our results indicate that lateral flexibility of claudin strands is due to an interplay of three sets of interfacial interaction networks between two antiparallel double rows of claudins in the membranes. In this model, claudins are assembled into interlocking tetrameric ion channels along the strand that slide with respect to each other as the strands curve over submicrometer-length scales. These results suggest a novel molecular mechanism underlying claudin-15 strand flexibility. It also sheds light on intermolecular interactions and their role in maintaining epithelial barrier function.

Unique structural features of claudin-5 and claudin-15 lead to functionally distinct tight junction strand architecture.

Members of the claudin family impart unique paracellular selectivity to tight junctions. However, the structure-function relationship between claudin's strand architecture and the paracellular charge- and size-selectivity is not well-understood. This work examines the molecular assembly of claudin-5, a barrier-forming protein, and claudin-15, a channel-forming protein, to determine their structural and functional properties. We adopt a bottom-up approach starting from claudin monomers to build the molecular architecture of the tight junction strands. First, we investigated the cis assembly of claudin-5 and -15 dimers using the Protein Association Energy Landscape method. Out of the millions of dimer conformations, we narrowed down key cis claudin-5 and -15 dimer conformations that were thermodynamically and kinetically stable. Second, we performed the trans assembly of dimers to identify the tetrameric building blocks that serve as the repeat units for strand formation. Finally, the strand assembly of the tetrameric repeat units showed fundamentally distinct strand architectures. In claudin-5, the cis and trans interactions seal the paracellular space, while in claudin-15, the gaps in the paracellular space lead to pore formation. This detailed study suggests that each member of the claudin family is unique and requires systematic molecular-level analysis for determining the strand architecture.

Effects of TAMP family on the tight junction strand network and barrier function in epithelial cells.

Occludin, tricellulin, and marvelD3 belong to the tight junction (TJ)-associated MARVEL protein family. Occludin and tricellulin jointly contribute to TJ strand branching point formation and epithelial barrier maintenance. However, whether marvelD3 has the same function remains unclear. Furthermore, the roles of the carboxy-terminal cytoplasmic tail, which is conserved in occludin and tricellulin, on the regulation of TJ strand morphology have not yet been explored in epithelial cells. We established tricellulin/occludin/marveld3 triple-gene knockout (tKO) MDCK II cells and evaluated the roles of marvelD3 in the TJ strand structure and barrier function using MDCK II cells and a mathematical model. The complexity of TJ strand networks and paracellular barrier did not change in tKO cells compared to that in tricellulin/occludin double-gene knockout (dKO) cells. Exogenous marvelD3 expression in dKO cells did not increase the complexity of TJ strand networks and epithelial barrier tightness. The expression of the carboxy-terminal truncation mutant of tricellulin restored the barrier function in the dKO cells, whereas occludin lacking the carboxy-terminal cytoplasmic tail was not expressed on the plasma membrane. These data suggest that marvelD3 does not affect the morphology of TJ strands and barrier function in MDCK II cells and that the carboxy-terminal cytoplasmic tail of tricellulin is dispensable for barrier improvement.

409 Corneoptosis, functional keratinocyte cell death, is tightly associated with spaciotemporal dynamics of epidermal tight junctions

Epidermis has two physical barrier components, stratum corneum (SC) and tight junction (TJ). Formation of SC or cornification is initiated by the cell death of the top layer (SG1) of stratum granulosum, termed ‘corneoptosis’ after prolonged intracellular Ca2+([Ca2+]i) elevation (∼60min), followed by rapid intracellular acidification. However, the exact timing of the onset of corneoptorsis remains to be clarified. In this study, we aimed to determine the spatiotemporal relationship between the turnover of TJs and corneoptosis. We performed intravital confocal microscopic imaging of mouse ear co-expressing ZO-1-Venus (TJ strand marker) and GCaMP3 (intracellular Ca2+ marker). Hoechst 33342 was intradermally injected to monitor the change of nuclear pH. Consistent with our previous observation, ZO-1-Venus signals showed two TJ strand-pattens in upper SG. According to the TJ turnover, three phases are observed; TJ-phase 1 which shows single old TJ strand formed in SG2 cells; TJ-phase 2 which shows double TJ strands; TJ-phase 3 which shows new single TJ strand. Time lapse imaging analysis revealed that the elevation of GCaMP3 signal was observed only in TJ-phase 2 which shows double TJs, but not in other TJ-phases. The elevation of [Ca2+]i lasted for approximately 60min. Subsequently, intracellular acidification was induced in these SG1 cells as detected by both the decrease of GCaMP3 signal and the increase of Hoechst 33342 signals. Simultaneously, ‘upper larger TJ strand’ of double-TJ strand in SG1 cells disappeared, resulting in change from TJ-phase 2 to TJ-phase 3. These findings indicated that the onset of corneoptosis is tightly associated with the spaciotemporal dynamics of TJs.

Computational Assessment of Different Structural Models for Claudin-5 Complexes in Blood-Brain Barrier Tight Junctions.

The blood-brain barrier (BBB) strictly regulates the exchange of ions and molecules between the blood and the central nervous system. Tight junctions (TJs) are multimeric structures that control the transport through the paracellular spaces between the adjacent brain endothelial cells of the BBB. Claudin-5 (Cldn5) proteins are essential for TJ formation and assemble into multiprotein complexes via cis-interactions within the same cell membrane and trans-interactions across two contiguous cells. Despite the relevant biological function of Cldn5 proteins and their role as targets of brain drug delivery strategies, the molecular details of their assembly within TJs are still unclear. Two different structural models have been recently introduced, in which Cldn5 dimers belonging to opposite cells join to generate paracellular pores. However, a comparison of these models in terms of ionic transport features is still lacking. In this work, we used molecular dynamics simulations and free energy (FE) calculations to assess the two Cldn5 pore models and investigate the thermodynamic properties of water and physiological ions permeating through them. Despite different FE profiles, both structures present single/multiple FE barriers to ionic permeation, while being permissive to water flux. These results reveal that both models are compatible with the physiological role of Cldn5 TJ strands. By identifying the protein-protein surface at the core of TJ Cldn5 assemblies, our computational investigation provides a basis for the rational design of synthetic peptides and other molecules capable of opening paracellular pores in the BBB.

Tight junction proteins occludin and ZO-1 as regulators of epithelial proliferation and survival.

Epithelial cells are the first line of mucosal defense. In the intestine, a single layer of epithelial cells must establish a selectively permeable barrier that supports nutrient absorption and waste secretion while preventing the leakage of potentially harmful luminal materials. Key to this is the tight junction, which seals the paracellular space and prevents unrestricted leakage. The tight junction is a protein complex established by interactions between members of the claudin, zonula occludens, and tight junction-associated MARVEL protein (TAMP) families. Claudins form the characteristic tight junction strands seen by freeze-fracture microscopy and create paracellular channels, but the functions of ZO-1 and occludin, founding members of the zonula occludens and TAMP families, respectively, are less well defined. Recent studies have revealed that these proteins have essential noncanonical (nonbarrier) functions that allow them to regulate epithelial apoptosis and proliferation, facilitate viral entry, and organize specialized epithelial structures. Surprisingly, neither is required for intestinal barrier function or overall health in the absence of exogenous stressors. Here, we provide a brief overview of ZO-1 and occludin canonical (barrier-related) functions, and a more detailed examination of their noncanonical functions.

Computational characterization of claudin-15 strand flexibility

Claudins self-polymerize into tight junction strand networks to seal the paracellular pathway between adjacent cells and form selective barriers to control the passive transport of water and small molecules. These anastomosing networks of claudin strands are flexible and dynamic, capable of maintaining barrier function throughout the cellular movement and large-scale tissue rearrangements. To understand the flexibility of tight junction strands, we performed molecular dynamics (MD) simulations of claudin-15 strands ranging from 27nm to 225 nm in two parallel lipid bilayers and characterized their mechanical properties.

Claudin-15 strand flexibility and its alteration in mutant strands

Tight junction claudins polymerize across two neighboring cell membranes to form a barrier for the transport of ions and small molecules in paracellular space. Functional properties of tight junction strands are dependent on the morphology, density, and composition of these strand networks. Although how claudins self-polymerize into flexible strands is still cloaked in mystery, computational studies have shed some light on the molecular characteristics of this flexibility. We carried out all-atom and hybrid molecular dynamics simulations of claudin-15 strands between 30 and 130 nm in two parallel lipid membranes and revealed that a mutation influencing the claudin subtype-specific structure restricts the architectural flexibility of claudin strands.

CLAUDIN-3 Protein Expression Relation with Dermatological Diseases: Review Article

Background: When it comes to barrier and pore creation in metazoans, especially vertebrates and tunicates. Claudins are an important family of transmembrane proteins. In epithelia and endothelia's apical junctional complex, chordate claudins are crucial architectural proteins for tight junction strands. Psoriasis patients often have some degree of subclinical intestinal irritation. In gastrointestinal mucosal biopsies, lymphocytic infiltration is found in about 60% of patients. Intestinal absorption is hampered by structural and functional changes in the colon caused by inflammatory cells. Those who have psoriasis have abnormalities in fecal fat excretion, decreased D-xylose absorption, and impaired lactase activity.Objective: To assess the relation between CLAUDIN-3 protein with dermatological diseases. Conclusion: Claudin-3 is a key component of tight junctions, which may serve as marker of gut barrier integrity. Claudin-3 level may be altered in patients with psoriasis.

Defective claudin-10 causes a novel variation of HELIX syndrome through compromised tight junction strand assembly

Formation of claudin-10 based tight junctions (TJs) is paramount to paracellular Na+ transport in multiple epithelia. Sequence variants in CLDN10 have been linked to HELIX syndrome, a salt-losing tubulopathy with altered handling of divalent cations accompanied by dysfunctional salivary, sweat, and lacrimal glands. Here, we investigate molecular basis and phenotypic consequences of a newly identified homozygous CLDN10 variant that translates into a single amino acid substitution within the fourth transmembrane helix of claudin-10. In addition to hypohidrosis (H), electrolyte (E) imbalance with impaired urine concentrating ability, and hypolacrimia (L), phenotypic findings include altered salivary electrolyte composition and amelogenesis imperfecta but neither ichthyosis (I) nor xerostomia (X). Employing cellular TJ reconstitution assays, we demonstrate perturbation of cis- and trans-interactions between mutant claudin-10 proteins. Ultrastructures of reconstituted TJ strands show disturbed continuity and reduced abundance in the mutant case. Throughout, both major isoforms, claudin-10a and claudin-10b, are differentially affected with claudin-10b showing more severe molecular alterations. However, expression of the mutant in renal epithelial cells with endogenous TJs results in wild-type-like ion selectivity and conductivity, indicating that aberrant claudin-10 is generally capable of forming functional paracellular channels. Thus, mutant proteins prove pathogenic by compromising claudin-10 TJ strand assembly. Additional ex vivo investigations indicate their insertion into TJs to occur in a tissue-specific manner.

Occludin and tricellulin facilitate formation of anastomosing tight-junction strand network to improve barrier function

Tight junctions (TJs) are composed of a claudin-based anastomosing network of TJ strands at which plasma membranes of adjacent epithelial cells are closely attached to regulate the paracellular permeability. Although the TJ proteins occludin and tricellulin have been known to be incorporated in the TJ strand network, their molecular functions remain unknown. Here, we established tricellulin/occludin-double knockout (dKO) MDCK II cells using a genome editing technique and evaluated the structure and barrier function of these cells. In freeze-fracture replica electron microscopy, the TJ strands of tricellulin/occludin-dKO cells had fewer branches and were less anastomosed compared with the controls. The paracellular permeability of ions and small tracers was increased in the dKO cells. A single KO of tricellulin or occludin had limited effects on the morphology and permeability of TJs. Mathematical simulation using a simplified TJ strand network model predicted that reduced cross-links in TJ strands lead to increased permeability of ions and small macromolecules. Furthermore, overexpression of occludin increased the complexity of TJ strand network and strengthened barrier function. Taken together, our data suggest that tricellulin and occludin mediate the formation and/or stabilization of TJ-strand branching points and contribute to the maintenance of epithelial barrier integrity.

Treatment with Atorvastatin During Vascular Remodeling Promotes Pericyte-Mediated Blood-Brain Barrier Maturation Following Ischemic Stroke.

We previously showed that newly formed vessels in ischemic rat brain have high blood-brain barrier (BBB) permeability at 3weeks after stroke due to a lack of major endothelial tight junction proteins (TJPs), which may exacerbate edema in stroke patients. Atorvastatin was suggested a dose-dependent pro-angiogenic effect and ameliorating BBB permeability beyond its cholesterol-lowering effects. This study examined our hypothesis that, during vascular remodeling after stroke, treatment with atorvastatin could facilitate BBB maturation in remodeling vasculature in ischemic brain. Adult spontaneously hypertensive rats underwent middle cerebral artery occlusion with reperfusion (MCAO/RP). Atorvastatin, at dose of 3mg/kg, was delivered daily starting at 14days after MCAO/RP onset for 7days. The rats were studied at multiple time points up to 8weeks with multimodal-MRI, behavior tests, immunohistochemistry, and biochemistry. The delayed treatment of atorvastatin significantly reduced infarct size and BBB permeability, restored cerebral blood flow, and improved the neurological outcome at 8weeks after MCAO/RP. Postmortem studies showed that atorvastatin promoted angiogenesis and stabilized the newly formed vessels in peri-infarct areas. Importantly, atorvastatin facilitated maturation of BBB properties in the new vessels by promoting endothelial tight junction (TJ) formation. Further in vivo and in vitro studies demonstrated that proliferating peri-vascular pericytes expressing neural-glial antigen 2 (NG2) mediated the role of atorvastatin on BBB maturation through regulating endothelial TJ strand formations. Our results suggested a therapeutic potential of atorvastatin in facilitating a full BBB integrity and functional stroke recovery, and an essential role for pericyte-mediated endothelial TJ formation in remodeling vasculature.

Weak Ultraviolet B Enhances the Mislocalization of Claudin-1 Mediated by Nitric Oxide and Peroxynitrite Production in Human Keratinocyte-Derived HaCaT Cells

A tight junction (TJ) makes a physical barrier in the epidermal cells of skin. Ultraviolet (UV) light may disrupt the TJ barrier, but the mechanism has not been well clarified. Weak UVB (5 mJ/cm2) caused mislocalization of claudin-1 (CLDN1), a component of the TJ strand, and disruption of TJ barrier in human keratinocyte-derived HaCaT cells. The UVB-induced mislocalization of CLDN1 was inhibited by monodansylcadaverine (MDC), a clathrin-dependent endocytosis inhibitor, suggesting that UVB enhances the internalization of CLDN1. Transepidermal electrical resistance and paracellular flux of lucifer yellow, a fluorescent hydrophilic marker, were rescued by MDC. UVB changed neither the total nor phosphorylation levels of CLDN1, but it increased both mono-ubiquitination and tyrosine nitration levels of CLDN1. Fluorescence measurements revealed that UVB increased intracellular free Ca2+, nitric oxide (NO), and peroxynitrite contents, which were inhibited by Opsin2 (OPN2) siRNA, suggesting that OPN2 functions as a UVB sensor. The effects of UVB were inhibited by an antagonist of transient receptor potential type vanilloid 1 (TRPV1) and Ca2+ chelator. Both NO donor and peroxynitrite donor induced the mislocalization of CLDN1 and disruption of TJ barrier, which were rescued by a NO synthase (NOS) inhibitor and a peroxynitrite scavenger. Weak UVB irradiation induced the disruption of TJ barrier mediated by mislocalization of CLDN1 in HaCaT cells. The OPN2/TRPV1/NOS signaling pathway may be a novel target for preventing destruction of the TJ barrier by UVB irradiation.