Two classes of tight junctions are revealed by ZO-1 isoforms

1993 ◽  
Vol 264 (4) ◽  
pp. C918-C924 ◽  
Author(s):  
M. S. Balda ◽  
J. M. Anderson
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Rna Splicing ◽  
Seminiferous Tubules ◽  
Junction Protein ◽  
Acid Domain ◽  
Ribonuclease Protection ◽  
Tissue Compartments ◽  
Junctional Resistance ◽  
Amino Acid Domain

The tight junction forms the intercellular barrier separating tissue compartments. The characteristics of this barrier are remarkably diverse among different epithelia and endothelia and are not explained by our limited knowledge of its molecular composition. Two isoforms of the 220-kDa tight junction protein ZO-1 result from alternative RNA splicing and differ by an internal 80-amino acid domain, termed alpha (E. Willott, M. S. Balda, M. Heintzman, B. Jameson, and J. M. Anderson. Am. J. Physiol. 262 (Cell Physiol. 31): C1119-C1124, 1992). Using antibodies specific for each isoform and double-labeled immunofluorescence microscopy, we observed that the ZO-1 alpha- isoform is restricted to junctions of endothelial cells and highly specialized epithelial cells of both seminiferous tubules (Sertoli cells) and renal glomeruli (podocytes); in contrast, the ZO-1 alpha+ isoform is expressed in cells of all other epithelia examined. Both immunoblotting and ribonuclease protection analysis confirmed this pattern of expression. This distribution does not correlate with differences in junctional resistance or ultrastructural complexity. Instead, we observe a correlation with junctional plasticity; ZO-1 alpha- is expressed in structurally dynamic junctions, whereas ZO-1 alpha+ is expressed in those which are less dynamic. This is the first molecular distinction among tight junctions and reveals a fundamental dichotomy with implications for how the paracellular barriers of endothelia and epithelia are regulated.

GENETIC SUSCEPTIBILITY TO IBD OPERATES VIA CONTROL OF APICAL JUNCTIONAL COMPLEXES

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S30-S30
Author(s):  
Isabelle Hébert-Milette ◽  
Chloé Lévesque ◽  
Guy Charron ◽  
John Rioux
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Intestinal Inflammation ◽  
Protein Localization ◽  
Junctional Complex ◽  
Epithelial Permeability ◽  
3D Cultures ◽  
Apical Junctional Complex ◽  
Junction Protein ◽  
The Impact

Abstract Introduction Intestinal permeability is increased in unaffected 1st degree relatives of patients with inflammatory bowel disease (IBD), and is considered a risk factor for the development of IBD, likely increasing the interactions between intestinal microorganisms and the immune system. We recently reported that C1orf106, a gene located within a genomic region associated with IBD, regulates epithelial permeability. We further demonstrated that a rare coding variant within C1orf106 (p.Y333F) decreases protein stability and that lower levels of C1orf106 protein leads altered stability of adherens junctions (AJ) and to an increase in epithelial permeability. Hypothesis In addition to altering AJ, we believe that C1orf106 is also involved in the regulation of tight junction (TJ) formation, which also impacts epithelial permeability. Objectives The objectives of the project are to (a) validate the impact of C1orf106 on tight junctions and (b) verify the impact of C1orf106 IBD-associated variants on intestinal barrier integrity. Results We observed that knocking down the expression of C1orf106 in Caco-2 cells leads to a number of phenotypes in human epithelial monolayer (2D) and spheroid (3D) cultures that are associated with alterations in TJs. Specifically, when studying the dynamic reformation of TJ in 2D cultures after transient withdrawal of calcium, which is required for TJ stability, we observed that lower levels of C1orf106 resulted in (1) decreased recovery of barrier function as measured by transepithelial electrical resistance (TEER); (2) an alteration of tight junction protein localization; and (3) thickening of the circumferential actin belt. Moreover, in 3D cultures, we observed an altered spheroid formation associated with impaired epithelial polarization. In addition, our preliminary studies of human induced pluripotent stem cell (hiPSC)-derived epithelial cultures support that Y333F heterozygotes also have altered structure and function of their tight junctions. Conclusion Our observations indicate an important role of C1orf106 in apical junctional complex (AJC) formation likely mediated by a regulation of the circumferential actin belt. This can affect other functions of AJC, like the establishment of cell polarity. AJC formation is important for epithelial repair after an injury and its dysregulation impairs the formation of an impermeable epithelial barrier, which likely facilitates the passage of microorganisms and the induction and maintenance of intestinal inflammation.

scholarly journals Changes in Tight Junctional Resistance of the Cervical Epithelium Are Associated with Modulation of Content and Phosphorylation of Occludin 65-Kilodalton and 50-Kilodalton Forms

Endocrinology ◽  
2006 ◽  
Vol 147 (2) ◽  
pp. 977-989 ◽  
Author(s):  
Ling Zhu ◽  
Xin Li ◽  
Robin Zeng ◽  
George I. Gorodeski
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tight Junction ◽  
Tyrosine Phosphorylation ◽  
Tight Junctions ◽  
Early Stage ◽  
Cervical Epithelium ◽  
Kinase C ◽  
Low Levels ◽  
Junctional Resistance

Treatment of human cervical epithelial CaSki cells with ATP or with the diacylglyceride sn-1,2-dioctanoyl diglyceride (diC8) induced a staurosporine-sensitive transient increase, followed by a late decrease, in tight-junctional resistance (RTJ). CaSki cells express two immunoreactive forms of occludin, 65 and 50 kDa. Treatments with ATP and diC8 decreased the density of the 65-kDa form and increased the density of the 50-kDa form. ATP also decreased threonine phosphorylation of the 65-kDa form and increased threonine phosphorylation of the 50-kDa form and tyrosine phosphorylation of the 65- and 50-kDa forms. Staurosporine decreased acutely threonine and tyrosine phosphorylation of the two isoforms and in cells pretreated with staurosporine ATP increased acutely the density of the 65-kDa form and threonine phosphorylation of the 65-kDa form. Treatment with N-acetyl-leucinyl-leucinyl-norleucinal increased the densities of the 65- and 50-kDa forms. Pretreatment with N-acetyl-leucinyl-leucinyl-norleucinal attenuated the late decreases in RTJ induced by ATP and diC8 and the decrease in the 65-kDa and increase in the 50-kDa forms induced by ATP. Correlation analyses showed that high levels of RTJ correlated with the 65-kDa form, whereas low levels of RTJ correlated negatively with the 65-kDa form and positively with the 50-kDa form. The results suggest that in CaSki cells 1) occludin determines gating of the tight junctions, 2) changes in occludin phosphorylation status and composition regulate the RTJ, 3) protein kinase-C-mediated, threonine dephosphorylation of the 65-kDa occludin form increases the resistance of assembled tight junctions, 4) the early stage of tight junction disassembly involves calpain-mediated breakdown of occludin 65-kDa form to the 50-kDa form, and 5) increased levels of the 50-kDa form interfere with occludin gating of the tight junctions.

Campylobacter jejuni inhibits the absorptive transport functions of Caco-2 cells and disrupts cellular tight junctions

Microbiology ◽  
2005 ◽  
Vol 151 (7) ◽  
pp. 2451-2458 ◽  
Author(s):  
Amanda MacCallum ◽  
Simon P. Hardy ◽  
Paul H. Everest
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Campylobacter Jejuni ◽  
Fluid Transport ◽  
Cell Function ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Ussing Chambers ◽  
Cell Monolayers ◽  
Junction Protein

Caco-2 cells are models of absorptive enterocytes. The net transport of fluid from apical to basolateral surfaces results in ‘domes' forming in differentiated monolayers. Here, the effect of Campylobacter jejuni on this process has been examined. C. jejuni caused no changes in short-circuit current upon infection of Caco-2 cell monolayers in Ussing chambers. Thus, no active secretory events could be demonstrated using this model. It was therefore hypothesized that C. jejuni could inhibit the absorptive function of enterocytes and that this may contribute to diarrhoeal disease. C. jejuni infection of fluid-transporting (‘doming’) Caco-2 cells resulted in a significant reduction in dome number, which correlated with a decrease in tight junction integrity in infected monolayers, when measured as transepithelial electrical resistance. Defined mutants of C. jejuni also reduced dome numbers in infected monolayers. C. jejuni also altered the distribution of the tight junction protein occludin within cell monolayers. The addition to monolayers of extracellular gentamicin prevented these changes, indicating the contribution of extracellular bacteria to this process. Thus, tight junction integrity is required for fluid transport in Caco-2 cell monolayers as leaky tight junctions cannot maintain support of transported fluid at the basolateral surface of infected cell monolayers. Inhibition of absorptive cell function, changes in epithelial resistance and rearrangement of tight junctional proteins such as occludin represent a potential diarrhoeal mechanism of C. jejuni.

Localization and differential expression of two isoforms of the tight junction protein ZO-1

1992 ◽  
Vol 262 (5) ◽  
pp. C1119-C1124 ◽  
Author(s):  
E. Willott ◽  
M. S. Balda ◽  
M. Heintzelman ◽  
B. Jameson ◽  
J. M. Anderson
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Cell Lines ◽  
Dna Amplification ◽  
Immunoblot Analysis ◽  
Protein Isoforms ◽  
Variable Expression ◽  
Junction Protein ◽  
Epithelial Cell Lines ◽  
Normal Rat

ZO-1 is a peripheral membrane protein of approximately 225 kDa located on the cytoplasmic side of all tight junctions. ZO-1 cDNA sequencing disclosed the presence of a 240-bp sequence in only some of the ZO-1 cDNAs studied. This 240-bp region encoded an inframe insertion of 80 amino acids, named motif-alpha. Expression of the predicted transcripts in normal rat and human tissues and in human epithelial cell lines (Caco-2, T84, Hep G2) was shown by reverse transcription of RNA and then DNA amplification. Immunoblot analysis showed both protein isoforms were present; however, in different cell lines, their amounts differed markedly relative to each other. Immunolocalization at light and ultrastructural levels, using antibodies generated against motif-alpha or shared sequences flanking it, indicated both forms localized indistinguishably to tight junctions. These observations demonstrate the existence and variable expression of ZO-1 isoforms and raise the question whether these isoforms contribute to tight junction diversity in different epithelia.

scholarly journals Potential Regulatory Effects of Corticotropin-Releasing Factor on Tight Junction-Related Intestinal Epithelial Permeability are Partially Mediated by CK8 Upregulation

2017 ◽  
Vol 44 (3) ◽  
pp. 1161-1173 ◽  
Author(s):  
Hu Yue ◽  
Lu  Bin ◽  
Chen Chaoying ◽  
Zhang Meng ◽  
Li Meng ◽  
...  
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Intestinal Permeability ◽  
Corticotropin Releasing Factor ◽  
Intestinal Epithelial ◽  
Epithelial Permeability ◽  
Expression Levels ◽  
Rhoa Activity ◽  
Junction Protein ◽  
Regulatory Effects

Background/Aims: Intestinal permeability and stress have been implicated in the pathophysiology of irritable bowel syndrome (IBS). Cytokeratin 8 (CK8), for the first time, has been shown to mediate corticotropin-releasing factor (CRF)-induced changes in intestinal permeability in animal models of IBS. In this study, we investigated the regulatory effects of CRF on the permeability of human intestinal epithelial cells through the CK8-mediated tight junction. Methods: The expression levels of corticotropin-releasing factor receptor 1 (CRFR1) and corticotropin-releasing factor receptor 2 (CRFR2) on the HT29 cell surface were determined by immunofluorescence, RT-PCR, and Western blotting. After treatment with 100 nM CRF for 72 h, the translocation of FITC-labelled dextran was measured in a transwell chamber; the structural changes of tight junctions were observed under transmission electron microscopy; the expression levels of CK8, F-actin and tight junction proteins ZO-1, claudin-1, and occludin were detected by immunoblotting and immunofluorescence. The activity of RhoA was detected by immunoprecipitation. Furthermore, the effects of CRF on intestinal epithelial permeability were examined in CK8-silenced HT29 cells, which were constructed by shRNA interference. Results: CRF treatment increased FITC-labelled dextran permeability, caused the opening of tight junctions, induced increased fluorescence intensity of CK8 and decreased the intensities of ZO-1, claudin-1, and occludin, together with structural disruption. The expression levels of F-actin, occludin, claudin-1, and ZO-1 were downregulated. RhoA activity peaked at 30 min after CRF treatment. CRF-induced increased permeability, and downregulation of claudin-1 and occludin were not blocked by CK8 silencing. Nevertheless, CK8 silencing blocked the effects of CRF regarding the decrease in the expression levels of F-action and ZO-1 and increase in RhoA activity. Conclusion: CRF may increase intestinal epithelial permeability by upregulating CK8 expression, activating the RhoA signalling pathway, promoting intestinal epithelial actin remodelling, and decreasing the expression of the tight junction protein ZO-1. Other CK8-independent pathways may be involved in the downregulation of claudin-1 and occludin, which might also contribute to increased intestinal epithelial permeability.

scholarly journals Blood-brain barrier dysfunction in ischemic stroke: targeting tight junctions and transporters for vascular protection

2018 ◽  
Vol 315 (3) ◽  
pp. C343-C356 ◽  
Author(s):  
Wazir Abdullahi ◽  
Dinesh Tripathi ◽  
Patrick T. Ronaldson
Keyword(s):  
Endothelial Cells ◽  
Ischemic Stroke ◽  
Tight Junction ◽  
Tight Junctions ◽  
Blood Brain Barrier ◽  
Functional Expression ◽  
Brain Barrier ◽  
Vascular Protection ◽  
Blood Brain ◽  
Junction Protein

The blood-brain barrier (BBB) is a physical and biochemical barrier that precisely controls cerebral homeostasis. It also plays a central role in the regulation of blood-to-brain flux of endogenous and exogenous xenobiotics and associated metabolites. This is accomplished by molecular characteristics of brain microvessel endothelial cells such as tight junction protein complexes and functional expression of influx and efflux transporters. One of the pathophysiological features of ischemic stroke is disruption of the BBB, which significantly contributes to development of brain injury and subsequent neurological impairment. Biochemical characteristics of BBB damage include decreased expression and altered organization of tight junction constituent proteins as well as modulation of functional expression of endogenous BBB transporters. Therefore, there is a critical need for development of novel therapeutic strategies that can protect against BBB dysfunction (i.e., vascular protection) in the setting of ischemic stroke. Such strategies include targeting tight junctions to ensure that they maintain their correct structure or targeting transporters to control flux of physiological substrates for protection of endothelial homeostasis. In this review, we will describe the pathophysiological mechanisms in cerebral microvascular endothelial cells that lead to BBB dysfunction following onset of stroke. Additionally, we will utilize this state-of-the-art knowledge to provide insights on novel pharmacological strategies that can be developed to confer BBB protection in the setting of ischemic stroke.

scholarly journals Distinct claudins and associated PDZ proteins form different autotypic tight junctions in myelinating Schwann cells

2002 ◽  
Vol 159 (2) ◽  
pp. 361-372 ◽  
Author(s):  
Sebastian Poliak ◽  
Sean Matlis ◽  
Christoph Ullmer ◽  
Steven S. Scherer ◽  
Elior Peles
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Schwann Cells ◽  
Pdz Domain ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Guanylate Kinase ◽  
Pdz Proteins ◽  
Junction Protein ◽  
Zona Occludens

The apposed membranes of myelinating Schwann cells are joined by several types of junctional specializations known as autotypic or reflexive junctions. These include tight, gap, and adherens junctions, all of which are found in regions of noncompact myelin: the paranodal loops, incisures of Schmidt-Lanterman, and mesaxons. The molecular components of autotypic tight junctions have not been established. Here we report that two homologues of Discs Lost–multi PDZ domain protein (MUPP)1, and Pals-associated tight junction protein (PATJ), are differentially localized in myelinating Schwann cells and associated with different claudins. PATJ is mainly found at the paranodal loops, where it colocalized with claudin-1. MUPP1 and claudin-5 colocalized in the incisures, and the COOH-terminal region of claudin-5 interacts with MUPP1 in a PSD-95/Disc Large/zona occludens (ZO)-1 (PDZ)-dependent manner. In developing nerves, claudin-5 and MUPP1 appear together in incisures during the first postnatal week, suggesting that they coassemble during myelination. Finally, we show that the incisures also contain four other PDZ proteins that are found in epithelial tight junctions, including three membrane-associated guanylate-kinase proteins (membrane-associated guanylate-kinase inverted-2, ZO-1, and ZO-2) and the adaptor protein Par-3. The presence of these different tight junction proteins in regions of noncompact myelin may be required to maintain the intricate cytoarchitecture of myelinating Schwann cells.

scholarly journals Angiomotin family proteins are novel activators of the LATS2 kinase tumor suppressor

2011 ◽  
Vol 22 (19) ◽  
pp. 3725-3733 ◽  
Author(s):  
Murugan Paramasivam ◽  
Ali Sarkeshik ◽  
John R. Yates ◽  
Maria J. G. Fernandes ◽  
Dannel McCollum
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Cell Density ◽  
Hippo Pathway ◽  
The Other ◽  
Transcriptional Coactivator ◽  
Inhibition Of Growth ◽  
Conserved Domain ◽  
Junction Protein ◽  
The Hippo Pathway

LATS2 kinase functions as part of the Hippo pathway to promote contact inhibition of growth and tumor suppression by phosphorylating and inhibiting the transcriptional coactivator YAP. LATS2 is activated by the MST2 kinase. How LATS2 is activated by MST2 in response to changes in cell density is unknown. Here we identify the angiomotin-family tight junction protein AMOTL2 as a novel activator of LATS2. Like AMOTL2, the other angiomotin-family proteins AMOT and AMOTL1 also activate LATS2 through a novel conserved domain that binds and activates LATS2. AMOTL2 binds MST2, LATS2, and YAP, suggesting that AMOTL2 might serve as a scaffold protein. We show that LATS2, AMOTL2, and YAP all localize to tight junctions, raising the possibility that clustering of Hippo pathway components at tight junctions might function to trigger LATS2 activation and growth inhibition in response to increased cell density.

scholarly journals Tricellulin Forms a Barrier to Macromolecules in Tricellular Tight Junctions without Affecting Ion Permeability

2009 ◽  
Vol 20 (16) ◽  
pp. 3713-3724 ◽  
Author(s):  
Susanne M. Krug ◽  
Salah Amasheh ◽  
Jan F. Richter ◽  
Susanne Milatz ◽  
Dorothee Günzel ◽  
...  
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Electrical Resistance ◽  
Tight Junction Protein ◽  
Ion Permeability ◽  
Barrier Functions ◽  
Low Level ◽  
Central Tube ◽  
Junction Protein

Tricellulin is a tight junction protein localized in tricellular tight junctions (tTJs), the meeting points of three cells, but also in bicellular tight junctions (bTJs). To investigate its specific barrier functions in bTJs and tTJs, TRIC-a was expressed in low-level tricellulin–expressing cells, and MDCK II, either in all TJs or only in tTJs. When expressed in all TJs, tricellulin increased paracellular electrical resistance and decreased permeability to ions and larger solutes, which are associated with enhanced ultrastructural integrity of bTJs toward enhanced strand linearity. In tTJs in contrast, ultrastructure was unchanged and tricellulin minimized permeability to macromolecules but not to ions. This paradox is explained by properties of the tTJ central tube which is wide enough for passage of macromolecules, but too rare to contribute significantly to ion permeability. In conclusion, at low tricellulin expression the tTJ central tube forms a pathway for macromolecules. At higher expression, tricellulin forms a barrier in tTJs effective only for macromolecules and in bTJs for solutes of all sizes.

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