Cofilin mediates tight-junction opening by redistributing actin and tight-junction proteins

We previously found that capsaicin induces tight-junction (TJ) opening accompanied with cofilin dephosphorylation/activation in intestinal Caco-2 cells. Here, we examined the role of cofilin in TJ regulation, and analyzed the structural events that lead to TJ opening. We transfected Caco-2 cells with wild-type cofilin [cofilin(wt)] or its constitutively active mutant cofilin(S3A). We found that the decreases in transepithelial electrical resistance (TER) was slower in cofilin(wt) transfectants and faster in cofilin(S3A) mutants than in vector controls. Moreover, cofilin dephosphorylation corresponded to the rate of TER decrease. Capsaicin treatment changed the localization of TJ proteins and altered the F-actin structure, but in a manner different from those depend on myosin light chain kinase (MLCK). These results strongly support the importance of cofilin in TJ opening, suggesting cofilin as a target for TJ permeability regulation in epithelial cells.     

Tricellulin is a tight-junction protein necessary for hearing

The inner ear has fluid-filled compartments of different ionic compositions, including the endolymphatic and perilymphatic spaces of the organ of Corti; the separation from one another by epithelial barriers is required for normal hearing. TRIC encodes tricellulin, a recently discovered tight-junction (TJ) protein that contributes to the structure and function of tricellular contacts of neighboring cells in many epithelial tissues. We show that, in humans, four different recessive mutations of TRIC cause nonsyndromic deafness (DFNB49), a surprisingly limited phenotype, given the widespread tissue distribution of tricellulin in epithelial cells. In the inner ear, tricellulin is concentrated at the tricellular TJs in cochlear and vestibular epithelia, including the structurally complex and extensive junctions between supporting and hair cells. We also demonstrate that there are multiple alternatively spliced isoforms of TRIC in various tissues and that mutations of TRIC associated with hearing loss remove all or most of a conserved region in the cytosolic domain that binds to the cytosolic scaffolding protein ZO-1. A wild-type isoform of tricellulin, which lacks this conserved region, is unaffected by the mutant alleles and is hypothesized to be sufficient for structural and functional integrity of epithelial barriers outside the inner ear.     

Increases in guinea pig small intestinal transepithelial resistance induced by osmotic loads are accompanied by rapid alterations in absorptive-cell tight-junction structure

In some epithelia, mucosal exposure to osmotic loads produces an increase in transepithelial resistance that is presumed to relate to the collapse of the paracellular spaces. Since proximal small intestinal epithelium may transiently encounter osmotic loads during normal digestion, we examined the short-term effect of osmotic loads on resistance and on epithelial structure of mucosal sheets prepared from guinea pig jejunum using Ussing-chamber, thin-section electron- microscopic, and freeze-fracture techniques. After equilibration of mucosal sheets in chambers, mucosal buffer tonicity was increased to 600 mosM with mannitol. This resulted in a 64% increase in resistance within 20 min. Concomitantly, 600 mosM produced a decrease in tight- junction cation selectivity as judged from dilution potentials, collapse of paracellular spaces, decreased cytoplasmic electron density in 10-40% of absorptive cells, and focal absorptive-cell subjunctional lateral-membrane evaginations often associated with microfilament arrays. Freeze-fracture replicas of absorptive-cell tight junctions revealed significant increases in both strand count and depth. Preincubation with 5 micrograms/ml cytochalasin D reduced the 600 mosM resistance increase caused by 600 mosM exposure by 48% but did not prevent the collapse of paracellular spaces. Lowered temperatures that produced morphologic evidence consistent with a gel-phase transition of absorptive-cell lateral membranes prevented both the resistance response and the alterations in tight-junction structure. In conclusion, transient osmotic loads produce an increase in resistance in jejunal epithelium and alter both absorptive-cell tight-junction charge selectivity and structure. These responses, which may have physiologic implications, can be reduced by cytoskeletal inhibitors and ablated by conditions that restrict mobility of absorptive-cell lateral- membrane molecules.     

Thymic stromal lymphopoietin enhances tight-junction barrier function of human nasal epithelial cells

Epithelial-derived thymic stromal lymphopoietin (TSLP) triggers dendritic cell (DC)-mediated Th2-type inflammatory responses and is a master switch for allergic inflammatory diseases. In the present study, the expression and induction of TSLP and the effects of TSLP on the tight-junctional barrier of human nasal epithelial cells (HNECs) have been investigated in order to elucidate the role of TSLP in allergic rhinitis. We have found high expression of TSLP in the epithelium from patients with allergic rhinitis with recruitment and infiltration of DCs. In vitro, TSLP is significantly produced in HNECs after treatment with a toll-like receptor 2 (TLR2) ligand, Pam₃Cys-Ser-(Lys)₄, and a mixture of interleukin-1β and tumor necrosis factor-α. Treatment with TSLP rapidly enhances the barrier function of cultured HNECs, together with an increase of tight-junction proteins claudin-1, -4, -7, and occludin. The nasal-epithelial-derived TSLP thus not only activates DCs but also preserves the epithelial barrier via the upregulation of tight-junction proteins, thereby regulating antigen sensitization during the early stage of allergic rhinitis.     

Overexpression of the Xenopus tight-junction protein claudin causes randomization of the left-right body axis

This study presents Xenopus claudin (Xcla), a tight-junction protein that is abundantly expressed in eggs and neuroectodermal precursors during early development. It was isolated via a differential screen for mRNAs enriched in microsomes in the Xenopus blastula. The Xcla protein contains four transmembrane domains and a carboxy-terminal cytoplasmic region with a putative PDZ-binding site. We show that this PDZ-binding site of Xcla is critical for its correct localization on the cell membrane and that a truncated form leads to delocalization of the tight-junction protein ZO-1. Overexpression of Xcla causes changes in the cell adhesion properties of blastomeres and leads to visceral situs randomization. The results suggest that left-right axial patterning is very sensitive to changes in regulation of cell-cell interactions and implicate a tight-junction protein in the determination of left-right asymmetry.     

Occludin and claudins in tight-junction strands: leading or supporting players?

Tight junctions have attracted much interest from cell biologists, especially electron microscopists, since on freeze-fracture electron microscopy they appear as a well-developed network of continuous, anastomosing intramembranous strands (tight-junction strands). These strands might be directly involved in the 'barrier' as well as 'fence' functions in epithelial and endothelial cell sheets, but until recently little was known of their constituents. This review discusses current understanding of the molecular architecture of tight-junction strands, focusing on the recent discovery of two distinct types of tight-junction-specific integral membrane proteins, occludin and claudins.     

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.     

On macrotubule structure.

The number of protofilament pairs in macrotubules was calculated as a function of macrotubule diameter, protofilament angle to the long axis, and pair width. A comparison of these calculations with published observations suggests utilizasion of all 13 microtubule protofilaments in the formation of macrotubules in vivo.     

On gap junction structure

We have studied the stain distribution within rat liver gap junctions for specimens prepared by thin sectioning and negative staining. Pools of stain molecules exist in two specific locations with respect to the distinctive morphological units (connexons) of the junction. One pool of stain surrounds the connexons and is restricted to the extracellular space in the gap between the adjacent plasma membranes. The other pool of stain is located along in the central axis of each connexon, measures 1-2 nm in diameter and 4-5 nm in length, and is restricted to the gap region. On rare occasions, barely discernible linear densities seem to extend from this latter pool of stain and traverse the entire width of the junction. The data indicate the existence of a hydrophilic cavity along the central axis of te connexon which, in most instances, is restricted to the gap region. However, the precise depth to which this cavity may further extend along the connexon axis is still uncertain.     

Activation of AMP-activated protein kinase alleviates High-glucose-induced dysfunction of brain microvascular endothelial cell tight-junction dynamics

The blood-brain barrier, formed by specialized brain endothelial cells that are interconnected by tight junctions, strictly regulates paracellular permeability to maintain an optimal extracellular environment for brain homeostasis. Diabetes is known to compromise the blood-brain barrier, although the underlying mechanism remains unknown. The aim of this study was to elucidate the molecular mechanisms underlying disruption of the blood-brain barrier in diabetes and to determine whether activation of AMP-activated protein kinase prevents diabetes-induced blood-brain barrier dysfunction. Exposure of human brain microvascular endothelial cells to high glucose (25mmol/L d-glucose), but not to high osmotic conditions (20mmol/L l-glucose plus 5mmol/L d-glucose), for 2h to 1 week significantly increased the permeability of the blood-brain barrier in parallel with lowered expression levels of zonula occludens-1, occludin, and claudin-5, three proteins that are essential to maintaining endothelial cell tight junctions. In addition, high glucose significantly increased the generation of superoxide anions. Adenoviral overexpression of superoxide dismutase or catalase significantly attenuated the high-glucose-induced reduction of endothelial cell tight-junction proteins. Furthermore, administration of apocynin reversed the effects of high glucose on endothelial cell tight-junction proteins. Finally, activation of AMP-activated protein kinase with 5-amino-4-imidazole carboxamide riboside or adenoviral overexpression of constitutively active AMP-activated protein kinase mutants abolished both the induction of NAD(P)H oxidase-derived superoxide anions and the tight-junction protein degradation induced by high glucose. We conclude that high glucose increases blood-brain barrier dysfunction in diabetes through induction of superoxide anions and that the activation of AMP-activated protein kinase protects the integrity of the blood-brain barrier by suppressing the induction of NAD(P)H oxidase-derived superoxide anions.     

Behavior of tight-junction, adherens-junction and cell polarity proteins during HNF-4alpha-induced epithelial polarization

We previously reported that expression of tight-junction molecules occludin, claudin-6 and claudin-7, as well as establishment of epithelial polarity, was triggered in mouse F9 cells expressing hepatocyte nuclear factor (HNF)-4alpha [H. Chiba, T. Gotoh, T. Kojima, S. Satohisa, K. Kikuchi, M. Osanai, N. Sawada. Hepatocyte nuclear factor (HNF)-4alpha triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells, Exp. Cell Res. 286 (2003) 288-297]. Using these cells, we examined in the present study behavior of tight-junction, adherens-junction and cell polarity proteins and elucidated the molecular mechanism behind HNF-4alpha-initiated junction formation and epithelial polarization. We herein show that not only ZO-1 and ZO-2, but also ZO-3, junctional adhesion molecule (JAM)-B, JAM-C and cell polarity proteins PAR-3, PAR-6 and atypical protein kinase C (aPKC) accumulate at primordial adherens junctions in undifferentiated F9 cells. In contrast, CRB3, Pals1 and PATJ appeared to exhibit distinct subcellular localization in immature cells. Induced expression of HNF-4alpha led to translocation of these tight-junction and cell polarity proteins to beltlike tight junctions, where occludin, claudin-6 and claudin-7 were assembled, in differentiated cells. Interestingly, PAR-6, aPKC, CRB3 and Pals1, but not PAR-3 or PATJ, were also concentrated on the apical membranes in differentiated cells. These findings indicate that HNF-4alpha provokes not only expression of tight-junction adhesion molecules, but also modulation of subcellular distribution of junction and cell polarity proteins, resulting in junction formation and epithelial polarization.     

Bi-allelic variants in the ESAM tight-junction gene cause a neurodevelopmental disorder associated with fetal intracranial hemorrhage

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Capsaicin induces cofilin dephosphorylation in human intestinal cells: the triggering role of cofilin in tight-junction signaling

Previously, we demonstrated that capsaicin induces tight-junction (TJ) opening in human intestinal Caco-2 cells. In order to clarify the mechanism underlying the TJ opening action of capsaicin, we performed a proteomics study on capsaicin-treated Caco-2 cells. Phosphorylated cofilin was decreased significantly by capsaicin treatment. In addition, capsaicin induced Ca2+ influx in Caco-2 cells and there was a clear correlation between Ca2+) influx and cofilin dephosphorylation (activation). The Ca2+-chelating reagent EGTA blocked the cofilin dephosphorylation induced by both capsaicin and ionomycin, suggesting that the dephosphorylation was mediated by Ca2+ influx. Finally, transepithelial electrical resistance measurements showed that TJ opening accompanied cofilin dephosphorylation. Our data suggest that TJ opening is mediated by cofilin dephosphorylation, which is caused by capsaicin stimuli, including Ca2+ influx. This is the first report of capsaicin action via the dephosphorylation of cofilin in human intestinal cells.     

On the inflorescence structure ofAsclepiadaceae

Inflorescence structure in the familyAsclepiadaceae, particularly in the subfamilyAsclepiadoideae, is elucidated using the methodology and terminology of the school ofW. Troll. Asclepiadaceae inflorescences are principally thyrsoid systems, with variability resulting from different degrees of reduction of dichasial paracladia to bostryces, sciadioids, and, finally, to single flowers.     

On the structure of NADH-X

By following both the 285 nm Cotton effect and the increase in absorbance (ΔA₂₈₅) during the enzymic (glyceral-dehyde-3-phosphate dehydrogenase) and the non-enzymic (pyrophosphate buffer) formation of NADH-X, it is inferred that (i) the configuration at C-1′ is not changed, and (ii) the 285 nm Cotton effect in 6-hydroxy-1,4,5,6-tetrahydronicotinamide coenzymes arises not only from interaction with the ribose moiety but also from interaction with the C₆ chiral center.