Paracellular Diffusion Barrier Research Articles

Cell-intrinsic and -extrinsic functions of the ESCRT-III component shrub in cytokinetic abscission of Drosophila sensory organ precursor.

Although the molecular mechanisms governing abscission of isolated cells are largely decrypted, those of epithelial progenitors surrounded by epidermal cells (ECs) connected via cellular junctions remain largely unexplored. Here, we investigated the remodeling of the paracellular diffusion barrier ensured by septate junctions (SJs) during cytokinesis of Drosophila sensory organ precursor (SOP). We report that SOP cytokinesis involves the coordinated, polarized assembly and remodeling of SJs in the dividing cell and its neighbors, which remain connected to the former via membrane protrusions pointing towards the SOP midbody. SJs assembly and midbody basal displacement occur faster in SOPs than in ECs, leading to a quicker disentanglement of neighboring cells membrane protrusions prior to midbody release. As reported in isolated cells, the endosomal sorting complex required for transport-III component Shrub/CHMP4B is recruited at the midbody and cell-autonomously regulates abscission. In addition, Shrub is recruited to membrane protrusions, is required for SJ integrity, and alteration of SJ integrity leads to premature abscission. Our study uncovers cell- intrinsic and -extrinsic functions of Shrub in coordinating remodeling of the SJs and SOP abscission.

Molecular mechanisms of molecular transfer across the blood-brain barrier as a target for pharmacological action Part 1. Structure, function and pathology of the BBB

Biological barriers play a key role in maintaining the integrity and functioning of the body at all levels of its organization. The barrier function at the cellular level is based on the hydrophobic properties of the cytoplasmic membrane, which provide selective permeability for various substances, depending on their chemical properties. At higher levels of organization, from tissue to organism, the barrier function is based on intercellular protein complexes of the plasma membrane, which form paracellular diffusion barriers and separate internal and external fluid media, which is a necessary condition for the development and functioning of each organ. The blood-brain barrier (BBB) ​​plays an important role in maintaining the function of the brain. The review reflects the main stages in the embryonic development of the BBB, as well as presents current data on the morphological and functional features of the organization of the BBB, including molecular mechanisms that mediate the barrier function due to the complex participation of vascular cells of cerebral microvessels, as well as gene expression of enzyme complexes, active and passive substance transport mechanisms through the BBB. The high transport selectivity of the BBB is an urgent problem for the delivery of drugs to the brain. At the same time, it is equally important to improve the principles of pharmacotherapy for the correction of impaired BBB functions in various types of pathology of the nervous and other body systems. This review aims to convey to the developers of modern targeted drugs new information about the molecular genetic mechanisms of the transport of substances through the BBB, as well as to draw the attention of specialists in the field of precision medicine to the problem of violations of the barrier function of cerebral vessels in neurological and other diseases of a modern person.

Recent advances in understanding tight junctions

Tight junctions (TJs) are one type of cell–cell junction in epithelial cell types in vertebrates. They form a paracellular diffusion barrier and create the boundary between the apical and basolateral plasma membrane domains. The molecular constituents of TJs have mostly been identified, and now their cell biology has shifted to understanding of their formation, dynamics, and functional regulation as well as their relationship to the organization of epithelial cells. Accumulating novel findings are supported by new methods, including super-resolution microscopy, quantitative microscopy, biophysical measurements, and genome editing-mediated gene manipulation. As a conceptual breakthrough, liquid-liquid phase separation seems to be involved in the formation of TJs as super-molecular complexes. This short article summarizes seminal studies in the cell biology of TJs from the last three years.

Ultrastructural differentiation of plasma membrane and cell junctions in the hindgut cells is synchronized with key developmental transitions in Porcellio scaber

Differentiation of transporting epithelial cells during development of animal organisms includes remodelling of apical and basal plasma membranes to increase the available surface for transport and formation of occluding junctions, which maintain a paracellular diffusion barrier. This study provides a detailed ultrastructural analysis of apical and basal plasma membrane remodelling and cell junction formation in hindgut cells during late embryonic and early postembryonic development of the crustacean Porcellio scaber. Hindgut cells in late-stage embryos are columnar with flat apical and basal plasma membranes. In early-stage marsupial mancae the hindgut cells begin to acquire their characteristic dome shape, the first apical membrane folding is evident and the septate junctions expand considerably, all changes being probably associated with the onset of active feeding. In postmarsupial mancae the apical labyrinth is further elaborated and the septate junctions are expanded. This coincides with the transition to an external environment and food sources. First basal infoldings appear in the anterior chamber of early-stage marsupial mancae, but in the papillate region they are mostly formed in postmarsupial mancae. In molting late-stage marsupial mancae, the plasma membrane acquires a topology characteristic of cuticle-producing arthropod epithelia and the septate junctions are considerably reduced.

Inner Blood-Retinal Barrier Regulation in Retinopathies.

The neural retina is protected from the blood circulation by the presence of a highly selective inner blood-retinal barrier (iBRB). The presence of sophisticated tight junctions (TJs) between the endothelial cells (ECs) of the iBRB helps mediate the very low passive permeability of the tissue, permitting entry of nutrients into the retina but excluding harmful toxic material and inflammatory cells. The most highly enriched TJ protein is claudin-5, which is critical in mediating the passive paracellular diffusion barrier properties of the iBRB. In numerous retinal degeneration pathologies, TJ disruption is observed, and a more refined understanding of this disruption could be used for therapeutic benefit.

Process of tight junction recovery in the injured vocal fold epithelium: Morphological and paracellular permeability analysis

The vocal fold epithelium that includes tight junction (TJ)-based barrier function protects underlying connective tissues from external insults. TJs play an important role to control paracellular permeability of not only solutes but also ions, and preserve the vocal fold homeostasis. However, the distribution of TJs and paracellular diffusion barrier across the entire vocal fold epithelium are still unknown. The aim of this study was to identify the distribution of TJs in the vocal fold epithelium and to characterize the recovery process of TJ-based paracellular diffusion barrier in a rat model of vocal fold injury. Animal experiments with controls. Normal and vocal fold-injured rats were used. Larynges were harvested for immunohistochemical examination of TJ proteins. For functional analysis, a tracer permeability assay was performed using EZ-Link Sulfo-NHS-LC-Biotin. TJ proteins occludin and zonula occludens 1 signals were localized to the junctional regions of the most luminal cell layers of the vocal fold epithelium. The injured region had been recovered with epithelium at 5 days postinjury, but the paracellular diffusion barrier assays revealed that biotinylation reagents diffused into the lamina propria at 5 days postinjury, and were blocked at the epithelium at 14 and 28 days postinjury. It was strongly suggested that TJs in the vocal fold epithelium exist at the junctional regions of the first layer of stratified squamous epithelium. TJ-based paracellular diffusion barrier following vocal fold injury is recovered by 14 days postinjury, and this period corresponds with the time course of structural changes in the regenerating epithelium layer. NA. Laryngoscope, 128:E150-E156, 2018.

The response of claudin-like transmembrane septate junction proteins to altered environmental ion levels in the larval mosquito Aedes aegypti.

Septate junctions (SJs) occlude the paracellular pathway and function as paracellular diffusion barriers within invertebrate epithelia. However, integral components of SJs and their contribution to barrier properties have received considerably less attention than those of vertebrate occluding junctions. In arthropods, SJ proteins have only been identified in Drosophila and among these are three integral claudin-like proteins, Megatrachea (Mega), Sinuous (Sinu) and Kune-kune (Kune), as well as a receptor-like transmembrane SJ protein known as Neurexin IV (Nrx IV). In this study, mega, sinu, kune and nrx IV are identified and characterized in aquatic larvae of the mosquito Aedes aegypti and a role for these proteins in ionoregulatory homeostasis is considered. Transcripts encoding Mega, Sinu, Kune and Nrx IV were found in iono/osmoregulatory tissues such as the midgut, Malpighian tubules, hindgut and anal papillae, but abundance was greater in the hindgut and anal papillae. Using immunohistochemical and western blot analysis it was found that Kune localized to the regions of intercellular contact between epithelial cells of the rectum and posterior midgut and in the apical membrane domain of the syncytial epithelium of anal papillae. To investigate a potential role for integral SJ proteins in larval A. aegypti iono/osmoregulation, abundance was examined in animals reared in freshwater or brackish water (30% seawater). In iono/osmoregulatory epithelia, larvae exhibited tissue-specific alterations in mega mRNA and Kune protein abundance, but not sinu or nrx IV mRNA. These studies provide a first look at the potential contribution of integral SJ components to iono/osmoregulatory homeostasis in an aquatic invertebrate.

Different effects of ZO-1, ZO-2 and ZO-3 silencing on kidney collecting duct principal cell proliferation and adhesion

Coordinated cell proliferation and ability to form intercellular seals are essential features of epithelial tissue function. Tight junctions (TJs) classically act as paracellular diffusion barriers. More recently, their role in regulating epithelial cell proliferation in conjunction with scaffolding zonula occludens (ZO) proteins has come to light. The kidney collecting duct (CD) is a model of tight epithelium that displays intense proliferation during embryogenesis followed by very low cell turnover in the adult kidney. Here, we examined the influence of each ZO protein (ZO-1, -2 and -3) on CD cell proliferation. We show that all 3 ZO proteins are strongly expressed in native CD and are present at both intercellular junctions and nuclei of cultured CD principal cells (mCCDcl1). Suppression of either ZO-1 or ZO-2 resulted in increased G0/G1 retention in mCCDcl1 cells. ZO-2 suppression decreased cyclin D1 abundance while ZO-1 suppression was accompanied by increased nuclear p21 localization, the depletion of which restored cell cycle progression. Contrary to ZO-1 and ZO-2, ZO-3 expression at intercellular junctions dramatically increased with cell density and relied on the presence of ZO-1. ZO-3 depletion did not affect cell cycle progression but increased cell detachment. This latter event partly relied on increased nuclear cyclin D1 abundance and was associated with altered β1-integrin subcellular distribution and decreased occludin expression at intercellular junctions. These data reveal diverging, but interconnected, roles for each ZO protein in mCCDcl1 proliferation. While ZO-1 and ZO-2 participate in cell cycle progression, ZO-3 is an important component of cell adhesion.

Signalling at tight junctions during epithelial differentiation and microbial pathogenesis.

Tight junctions are a component of the epithelial junctional complex, and they form the paracellular diffusion barrier that enables epithelial cells to create cellular sheets that separate compartments with different compositions. The assembly and function of tight junctions are intimately linked to the actomyosin cytoskeleton and, hence, are under the control of signalling mechanisms that regulate cytoskeletal dynamics. Tight junctions not only receive signals that guide their assembly and function, but transmit information to the cell interior to regulate cell proliferation, migration and survival. As a crucial component of the epithelial barrier, they are often targeted by pathogenic viruses and bacteria, aiding infection and the development of disease. In this Commentary, we review recent progress in the understanding of the molecular signalling mechanisms that drive junction assembly and function, and the signalling processes by which tight junctions regulate cell behaviour and survival. We also discuss the way in which junctional components are exploited by pathogenic viruses and bacteria, and how this might affect junctional signalling mechanisms.

SnapShot: Epithelial Tight Junctions

Tight junctions form a morphological and functional border between the apical and basolateral cell surface domains that serves as a paracellular diffusion barrier, enabling epithelial cells to separate compartments of different composition. Tight junctions also contribute to the generation and maintenance of cell polarity and regulate signaling mechanisms that guide cell behavior, shape, and gene expression. This SnapShot illustrates their components, organization, and functions.

Functional tight junction barrier localizes in the second layer of the stratum granulosum of human epidermis

BackgroundMammalian epidermis has two diffusion barriers, the stratum corneum (SC) and tight junctions (TJs). We reported previously that a single living cell layer exists between the SC and TJ-forming keratinocytes in mice; however, the exact location of the TJ barrier in human epidermis has not been defined. ObjectiveTo investigate the precise distribution of epidermal TJs in relation to various cell–cell junction proteins and the SC and to clarify the barrier function of TJs against macromolecules in human skin. MethodsThe localization of various junctional proteins was investigated in human skin sections and in the roofs of bullae formed by ex vivo exfoliative toxin (ET) treatment in three dimensions. ET and single-chain variable fragments (scFv) against desmoglein 1 were used as large diffusion probes. ResultsHuman stratum granulosum (SG) cells have a distinct distribution of TJ, adherens junction, and desmosome proteins in the uppermost three layers (SG1–SG3 from the surface inward). Ex vivo injection of ET or scFv demonstrated that only SG2–SG2 junctions function as a TJ barrier, limiting the inside-out diffusion of these proteins. The roofs of bullae formed by ex vivo ET treatment consisted of SC, SG1 cells, and TJ-forming SG2 cells, probably mimicking bulla formation in bullous impetigo. ConclusionHuman epidermis has three SG cell layers with distinct properties just beneath the SC, of which only SG2 cells have functional TJs. Our results suggest that human epidermal TJs between SG2 cells form a paracellular diffusion barrier against soluble proteins, including immunoglobulins and bacterial toxins.

A Mechanism for Localized Lignin Deposition in the Endodermis

The precise localization of extracellular matrix and cell wall components is of critical importance for multicellular organisms. Lignin is a major cell wall modification that often forms intricate subcellular patterns that are central to cellular function. Yet the mechanisms of lignin polymerization and the subcellular precision of its formation remain enigmatic. Here, we show that the Casparian strip, a lignin-based, paracellular diffusion barrier in plants, forms as a precise, median ring by the concerted action of a specific, localized NADPH oxidase, brought into proximity of localized peroxidases through the action of Casparian strip domain proteins (CASPs). Our findings in Arabidopsis provide a simple mechanistic model of how plant cells regulate lignin formation with subcellular precision. We speculate that scaffolding of NADPH oxidases to the downstream targets of the reactive oxygen species (ROS) that they produce might be a widespread mechanism to ensure specificity and subcellular precision of ROS action within the extracellular matrix.

Tight junction protein claudin 3 expression in glioma

Among the brain tumors, Glioma is the most malignant disease that causes death and the prognosis of patients remains poor. Tight junctions (TJs) are important in formation of endothelial cells of the blood-brain barrier (BBB) and acts as a restrictive paracellular diffusion barrier. Claudins are imp components of TJs, and expression of these proteins were altered selectively in cerebral microvessels of human GBM and other pathological conditions, resulting in brain edema. The aim of this study was to determine the pattern of expression of tight junction associated protein claudin 3 in low grade and high grade glioma cell lines C6, U373, U118, T98 and U87MG using Real Time PCR and western blot methods. Interestingly quantitative Real Time PCR and western blot results have shown down regulation of claudin 3 in high grade glioma to low grade glioma. Our results concluded that decreased expression of claudin 3 was correlated with disease progression.

Crooked, Coiled and Crimpled are three Ly6-like proteins required for proper localization of septate junction components

Cellular junction formation is an elaborate process that is dependent on the regulated synthesis, assembly and membrane targeting of constituting components. Here, we report on three Drosophila Ly6-like proteins essential for septate junction (SJ) formation. SJs provide a paracellular diffusion barrier and appear molecularly and structurally similar to vertebrate paranodal septate junctions. We show that Crooked (Crok), a small GPI-anchored Ly6-like protein, is required for septa formation and barrier functions. In embryos that lack Crok, SJ components are produced but fail to accumulate at the plasma membrane. Crok is detected in intracellular puncta and acts tissue-autonomously, which suggests that it resides in intracellular vesicles to assist the cell surface localization of SJ components. In addition, we demonstrate that two related Ly6 proteins, Coiled (Cold) and Crimpled (Crim), are required for SJ formation and function in a tissue-autonomous manner, and that Cold also localizes to intracellular vesicles. Specifically, Crok and Cold are required for correct membrane trafficking of Neurexin IV, a central SJ component. The non-redundant requirement for Crok, Cold, Crim and Boudin (Bou; another Ly6 protein that was recently shown to be involved in SJ formation) suggests that members of this conserved family of proteins cooperate in the assembly of SJ components, possibly by promoting core SJ complex formation in intracellular compartments associated with membrane trafficking.

Epithelial Polarity Proteins Regulate Drosophila Tracheal Tube Size in Parallel to the Luminal Matrix Pathway

Regulation of epithelial tube size is critical for organ function. However, the mechanisms of tube size control remain poorly understood. In the Drosophila trachea, tube dimensions are regulated by a luminal extracellular matrix (ECM). ECM organization requires apical (luminal) secretion of the protein Vermiform (Verm), which depends on the basolateral septate junction (SJ). Here, we show that apical and basolateral epithelial polarity proteins interact to control tracheal tube size independently of the Verm pathway. Mutations in yurt (yrt) and scribble (scrib), which encode SJ-associated polarity proteins, cause an expansion of tracheal tubes but do not disrupt Verm secretion. Reducing activity of the apical polarity protein Crumbs (Crb) suppresses the length defects in yrt but not scrib mutants, suggesting that Yrt acts by negatively regulating Crb. Conversely, Crb overexpression increases tracheal tube dimensions. Reducing crb dosage also rescues tracheal size defects caused by mutations in coracle (cora), which encodes an SJ-associated polarity protein. In addition, crb mutations suppress cora length defects without restoring Verm secretion. Together, these data indicate that Yrt, Cora, Crb, and Scrib operate independently of the Verm pathway. Our data support a model in which Cora and Yrt act through Crb to regulate epithelial tube size.

Grainy head promotes expression of septate junction proteins and influences epithelial morphogenesis

Transcription factors of the Grainy head (Grh) family are required in epithelia to generate the impermeable apical layer that protects against the external environment. This function is conserved in vertebrates and invertebrates, despite the differing molecular composition of the protective barrier. Epithelial cells also have junctions that create a paracellular diffusion barrier (tight or septate junctions). To examine whether Grh has a role in regulating such characteristics, we used an epidermal layer in the Drosophila embryo that has no endogenous Grh and lacks septate junctions, the amnioserosa. Expression of Grh in the amnioserosa caused severe defects in dorsal closure, a process similar to wound closure, and induced robust expression of the septate junction proteins Coracle, Fasciclin 3 and Sinuous. Grh-binding sites are present within the genes encoding these proteins, consistent with them being direct targets. Removal of Grh from imaginal disc cells caused a reduction in Fasciclin 3 and Coracle levels, suggesting that Grh normally fine tunes their epithelial expression and hence contributes to barrier properties. The fact that ectopic Grh arrests dorsal closure also suggests that this dynamic process relies on epithelia having distinct adhesive properties conferred by differential deployment of Grh.

Spectrins and AnkyrinB Constitute a Specialized Paranodal Cytoskeleton

Paranodal junctions of myelinated nerve fibers are important for saltatory conduction and function as paracellular and membrane protein diffusion barriers flanking nodes of Ranvier. The formation of these specialized axoglial contacts depends on the presence of three cell adhesion molecules: neurofascin 155 on the glial membrane and a complex of Caspr and contactin on the axon. We isolated axonal and glial membranes highly enriched in these paranodal proteins and then used mass spectrometry to identify additional proteins associated with the paranodal axoglial junction. This strategy led to the identification of three novel components of the paranodal cytoskeleton: ankyrinB, alphaII spectrin, and betaII spectrin. Biochemical and immunohistochemical analyses revealed that these proteins associate with protein 4.1B in a macromolecular complex that is concentrated at central and peripheral paranodal junctions in the adult and during early myelination. Furthermore, we show that the paranodal localization of ankyrinB is disrupted in Caspr-null mice with aberrant paranodal junctions, demonstrating that paranodal neuron-glia interactions regulate the organization of the underlying cytoskeleton. In contrast, genetic disruption of the juxtaparanodal protein Caspr2 or the nodal cytoskeletal protein betaIV spectrin did not alter the paranodal cytoskeleton. Our results demonstrate that the paranodal junction contains specialized cytoskeletal components that may be important to stabilize axon-glia interactions and contribute to the membrane protein diffusion barrier found at paranodes.

Effect of estrogen on the expression of occludin in ovariectomized mouse brain

The blood–brain barrier (BBB) and blood–cerebrospinal fluid barrier (BCSFB), formed by interactions of tight junctions (TJs) in the endothelia or the choroids plexus, respectively, are important diffusion barriers between systemic circulation and neural tissue of the central nervous system (CNS). Epidemiological incidence of degenerative changes in the CNS is significantly higher in postmenopausal women than in men, suggesting an important role of ovarian steroids for CNS integrity in women. To elucidate the role estrogen in the maintenance of paracellular diffusion barriers in the CNS, changes in the expression of occludin, a TJ protein, following ovariectomy (OVX), as well as the effect of estrogen on the expression of occludin in OVX female brain, were examined in mice. Immunoreactivity of occludin was found in brain endothelial capillaries, choroids plexus, ependyma, and aqueduct. In OVX brain, expression of occludin mRNA and protein was decreased a little but significantly relative to a sham control brain at the estrous stage. Estrogen (17β estradiol) significantly up-regulated occludin mRNA levels in OVX mice. Together, these results suggest that occludin is an important structural element in both the BBB and BCSFB, and that expression of occludin in female brain tissue is sensitive to estrogen levels.

JRAB/MICAL-L2 Is a Junctional Rab13-binding Protein Mediating the Endocytic Recycling of Occludin

The dynamic turnover of tight junctions (TJs) is essential for epithelial-mesenchymal transitions and/or mesenchymal-epithelial transitions during epithelial morphogenesis. We previously demonstrated that Rab13 specifically mediates the endocytic recycling of occludin. Here, we identified MICAL-L2 (molecule interacting with CasL-like 2) as a novel Rab13-binding protein. Immunoprecipitation and immunofluorescence microscopy showed that MICAL-L2 specifically bound to the GTP-bound form of Rab13 via its C terminus, which contained a coiled-coil domain, and localized at TJs in epithelial MTD-1A cells. Recycling assay demonstrated that a MICAL-L2 mutant lacking the Rab13-binding domain (MICAL-L2-N) specifically inhibited the endocytic recycling of occludin but not transferrin receptor. Ca2+ switch assay further revealed that MICAL-L2-N as well as Rab13 Q67L inhibited the recruitment of occludin to the plasma membrane, the development of transepithelial electrical resistance, and the formation of a paracellular diffusion barrier. MICAL-L2 was displaced from TJs upon actin depolymerization and was distributed along radiating actin cables and stress fibers in Ca2+-depleted MTD-1A and fibroblastic NIH3T3 cells, respectively. These results suggest that MICAL-L2 mediates the endocytic recycling of occludin and the formation of functional TJs by linking Rab13 to actin cytoskeleton. We rename MICAL-L2 as JRAB (junctional Rab13-binding protein).

Astrocyte mediated modulation of blood-brain barrier permeability does not correlate with a loss of tight junction proteins from the cellular contacts.

In the central nervous system (CNS) complex endothelial tight junctions (TJs) form a restrictive paracellular diffusion barrier, the blood-brain barrier (BBB). Pathogenic changes within the CNS are frequently accompanied by the loss of BBB properties, resulting in brain edema. In order to investigate whether BBB leakiness can be monitored by a loss of TJ proteins from cellular borders, we used an in vitro BBB model where brain endothelial cells in co-culture with astrocytes form a tight permeability barrier for 3H-inulin and 14C-sucrose. Removal of astrocytes from the co-culture resulted in an increased permeability to small tracers across the brain endothelial cell monolayer and an opening of the TJs to horseradish peroxidase as detected by electron microscopy. Strikingly, opening of the endothelial TJs was not accompanied by any visible change in the molecular composition of endothelial TJs as junctional localization of the TJ-associated proteins claudin-3, claudin-5, occludin, ZO-1 or ZO-2 or the adherens junction-associated proteins beta-catenin or p120cas did not change. Thus, opening of BBB TJs is not readily accompanied by the complete loss of the junctional localization of TJ proteins.