Clinical Significance of Claudin Expression in Oral Squamous Cell Carcinoma

A change in claudin expression has been demonstrated in various tumors. The present study specifically compares claudin expression in oral squamous cell carcinoma (OSCC) with healthy oral epithelium from the same individual and analyzes the association between claudin expression and the clinically relevant course parameters. Our study includes tissue samples and clinically relevant follow-up data from 60 patients with primary and untreated OSCC. The oral mucosa was analyzed via Western blot for the expression of claudin-1, -2, -3, -4, -5, and -7. Importantly, the tumor and healthy tissues were obtained pairwise from patients, allowing for intraindividual comparisons. Both the healthy and tumor epithelium from the oral cavity did not express the claudin-3 protein. The intraindividual comparison revealed that, in OSCC, claudin-2 expression was higher, and the expression of claudin-4, -5, and -7 was lower than in healthy epithelium. An association was found between increased claudin-2 expression and shorter relapse-free survival. In addition, the reduced expression of claudin-4 had a negative impact on relapse-free survival. Furthermore, associations between the reduced expression of claudin-7 and the stage of a tumor, or the presence of lymph node metastases, were found. Thus, the expression level of claudin-2, -4, and -7 appears to be predictive of the diagnosis and prognosis of OSCC

Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells

Summary In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies

Structure and function of claudins

AbstractClaudins are tetraspan transmembrane proteins of tight junctions. They determine the barrier properties of this type of cell–cell contact existing between the plasma membranes of two neighbouring cells, such as occurring in endothelia or epithelia. Claudins can completely tighten the paracellular cleft for solutes, and they can form paracellular ion pores. It is assumed that the extracellular loops specify these claudin functions. It is hypothesised that the larger first extracellular loop is critical for determining the paracellular tightness and the selective ion permeability. The shorter second extracellular loop may cause narrowing of the paracellular cleft and have a holding function between the opposing cell membranes. Sequence analysis of claudins has led to differentiation into two groups, designated as classic claudins (1–10, 14, 15, 17, 19) and non-classic claudins (11–13, 16, 18, 20–24), according to their degree of sequence similarity. This is also reflected in the derived sequence-structure function relationships for extracellular loops 1 and 2. The concepts evolved from these findings and first tentative molecular models for homophilic interactions may explain the different functional contribution of the two extracellular loops at tight junctions

A strategy for enrichment of claudins based on their affinity to Clostridium perfringens enterotoxin

<p>Abstract</p> <p>Background</p> <p>Claudins, a family of protein localized in tight junctions, are essential for the control of paracellular permeation in epithelia and endothelia. The interaction of several claudins with <it>Clostridium perfringens </it>enterotoxin (CPE) has been exploited for an affinity-based enrichment of CPE-binding claudins from lysates of normal rat cholangiocytes.</p> <p>Results</p> <p>Immunoblotting and mass spectrometry (MS) experiments demonstrate strong enrichment of the CPE-binding claudins -3, -4 and -7, indicating specific association with glutathione-S-transferase (GST)-CPE_116–319fusion protein. In parallel, the co-elution of (non-CPE-binding) claudin-1 and claudin-5 was observed. The complete set of co-enriched proteins was identified by MS after electrophoretic separation. Relative mass spectrometric protein quantification with stable isotope labeling with amino acids in cell culture (SILAC) made it possible to discriminate specific binding from non-specific association to GST and/or matrix material.</p> <p>Conclusion</p> <p>CPE_116–319provides an efficient tool for single step enrichment of different claudins from cell lysates. Numerous proteins were shown to be co-enriched with the CPE-binding claudins, but there are no indications (except for claudins -1 and -5) for an association with tight junctions.</p

Brain endothelial tricellular junctions as novel sites for T cell diapedesis across the blood–brain barrier

The migration of activated T cells across the blood-brain barrier (BBB) is a critical step in central nervous system (CNS) immune surveillance and inflammation. Whereas T cell diapedesis across the intact BBB seems to occur preferentially through the BBB cellular junctions, impaired BBB integrity during neuroinflammation is accompanied by increased transcellular T cell diapedesis. The underlying mechanisms directing T cells to paracellular versus transcellular sites of diapedesis across the BBB remain to be explored. By combining in vitro live-cell imaging of T cell migration across primary mouse brain microvascular endothelial cells (pMBMECs) under physiological flow with serial block-face scanning electron microscopy (SBF-SEM), we have identified BBB tricellular junctions as novel sites for T cell diapedesis across the BBB. Downregulated expression of tricellular junctional proteins or protein-based targeting of their interactions in pMBMEC monolayers correlated with enhanced transcellular T cell diapedesis, and abluminal presence of chemokines increased T cell diapedesis through tricellular junctions. Our observations assign an entirely novel role to BBB tricellular junctions in regulating T cell entry into the CNS. This article has an associated First Person interview with the first author of the paper

Visualization and Quantitative Analysis of Reconstituted Tight Junctions Using Localization Microscopy

Tight Junctions (TJ) regulate paracellular permeability of tissue barriers. Claudins (Cld) form the backbone of TJ-strands. Pore-forming claudins determine the permeability for ions, whereas that for solutes and macromolecules is assumed to be crucially restricted by the strand morphology (i.e., density, branching and continuity). To investigate determinants of the morphology of TJ-strands we established a novel approach using localization microscopy

Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43.

Phosphorylation and lipidation provide posttranslational mechanisms that contribute to the distribution of cytosolic proteins in growing nerve cells. The growth-associated protein GAP43 is susceptible to both phosphorylation and S-palmitoylation and is enriched in the tips of extending neurites. However, how phosphorylation and lipidation interplay to mediate sorting of GAP43 is unclear. Using a combination of biochemical, genetic, and imaging approaches, we show that palmitoylation is required for membrane association and that phosphorylation at Ser-41 directs palmitoylated GAP43 to the plasma membrane. Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts. Sorting to the neuritic tip required palmitoylation and active transport and was increased by phosphorylation-mediated plasma membrane interaction. Vesicle tracking revealed transient association of a fraction of GAP43 with exocytic vesicles and motion at a fast axonal transport rate. Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones. Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells. Palmitoylation tags GAP43 for global sorting by piggybacking on exocytic vesicles, whereas phosphorylation locally regulates protein mobility and plasma membrane targeting of palmitoylated GAP43

Molecular organization of claudin-based tight junction strands and their modulation

Die parazelluläre Permeabilität für Solute und Wasser in Epithelien und Endothelien wird durch die Tight Junction (TJ) reguliert. TJs sind somit einerseits essentiell für die Bildung von Organschranken, anderseits limitieren sie die Wirkstoffaufnahme über Gewebebarrieren, z.B. die Bluthirnschranke. Die Ultrastruktur der TJ ist durch membranäre Stränge/Fibrillen im apikalen Bereich von Zell-Zellkontakten gekennzeichnet. Die Mitglieder der Claudin-Proteinfamilie (Cldn) bilden das Rückgrat der TJ- Stränge und deren gewebespezifische Claudin-Zusammensetzung bestimmt die gewebespezifischen Barriereeigenschaften der TJs maßgeblich. Allerdings ist unklar, wie TJ-Stränge auf molekularer Ebene organisiert sind und wie je nach Claudinzusammensetzung entweder parazelluläre Schranken oder Poren für Solute und Wasser gebildet werden. Im Rahmen der vorliegenden Habilitationsarbeit wurden molekularbiologische, mikroskopische, funktionelle und bioinformatische Methoden kombiniert, um Einsichten in den molekularen Mechanismus der Bildung barrierebildender TJ-Stränge und deren Modulierbarkeit zu erzielen. Es wurden erstmals Sequenzdeterminanten für die homophile Interaktion zwischen Claudinen identifiziert und verwendet, um zwei strukturell unterschiedliche Subgruppen (klassische und nicht-klassische Claudine) zu beschreiben. Für Cldn3 und Cldn5 wurden Interaktions-relevante Reste in Transmembransegment (TM) 3, der extrazellulären Schleife 2 (EZS2) und TM4 identifiziert. Z.B. wurde die Beteiligung von TM3-Resten an einer Claudinsubtyp-spezifischen cis- Dimerisierung und deren Bedeutung für die ultrastrukturelle Morphologie der TJ demonstriert. Die Interaktionsdeterminanten wurden zusammen mit Methoden der strukturellen Bioinformatik verwendet, um Homologiemodelle der unbekannten 3D- Struktur von Cldn3, Cldn5 und anderer Claudine zu erstellen und mechanistische Aspekte der Proteinfaltung und TJ-Strangbildung der Claudine zu erklären. Die kürzlich aufgeklärte Kristallstruktur von Cldn15 hat die zuvor von uns generierten Strukturmodelle in großen Teilen betätigt. In der Summe konnten wir z.B. zeigen, dass das unter klassischen Claudinen konservierte Motiv F, Y/F, x(9−10), E, L/I/M/F in der EZS2 eine Interaktion zwischen Claudinprotomeren vermittelt, die essentiell für die TJ-Strangbildung ist. Für Cldn5 wurde die grundlegende Bedeutung der EZS2 an der Abdichtung der TJ gegenüber Soluten in vitro in einem epithelialen Monolayer und in vivo im Zebrafischembryo demonstriert. Die homo- und heterophilen cis- und trans- Interaktionen zwischen Claudinen, die in zerebralen Schranken exprimiert werden, und deren Auswirkung auf die Mobilität dieser Proteine, auf deren Fähigkeit, TJ-Stränge zu bilden, und auf deren Strang-Morphologie wurden bestimmt. Erstmals wurden einzelne Claudinmoleküle direkt in TJ-Strängen visualisiert, von nichtpolymerisierten Molekülen abgegrenzt und gezeigt, dass Cldn3 und Cldn5 die gleiche relative Moleküldichte in den TJ-Strängen besitzen. Der molekulare Mechanismus, über den das Clostridium perfringens- Enterotoxin (CPE) an eine Untergruppe klassischer Claudine bindet, wurde weitgehend aufgeklärt und anschließend benutzt, um CPE-Varianten mit veränderten Claudin-Bindungseigenschaften zu generieren. Die erhaltenen Erkenntnisse zur Struktur und Organisation der TJ sowie zur CPE-Claudin- Interaktion können genutzt werden, um gezielt gewebsspezifische, größenabhängige und reversible Claudin-Modulatoren zu generieren. Diese TJ- Modulatoren könnten die parazelluläre Wirkstoffaufnahme z.B. über die Bluthirnschranke erheblich verbessern.The paracellular permeability for solutes and water in epithelia and endothelia is regulated by the tight junction (TJ). Hence, TJs are on the one hand essential for tissue barriers and on the other hand limit drug delivery across tissue barriers, e.g. the blood brain barrier. The members of the claudin protein family form the backbone of intramembranous TJ-strands. The tissue-specific claudin composition is the major determinant of the tissue- specific barrier properties of TJs. However, the molecular organization of the TJ-strands and the mechanism by which claudins form either paracellular barriers or pores for solutes and water is unclear. Here, molecular biological, microscopical, functional and bioinformatic methods were combined to gain insights in the molecular mechanism of TJ-strand formation and their modulation. Sequence determinants for interactions between claudins and for paracellular tightening were identified. Homology models of the 3D-structure of claudins were generated. It could be shown that a motif which is conserved among classic claudins (F, Y/F, x(9−10), E, L/I/M/F in the extracellular loop two) determines an interaction between claudin protomers which is essential for formation of TJ-strands. In addition the molecular mechanism of the interaction between the Clostridium perfringens-Enterotoxin (CPE) and a subgroup of classic claudins was elucidated and used to generate variants of the claudin binding domain of CPE with modified claudin binding properties for claudin modulation, e.g. to enhance paracellular drug delivery across the blood brain 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