Phosphorylation of occludin correlates with occludin localization and function at the tight junction

1997 ◽  
Vol 273 (6) ◽  
pp. C1859-C1867 ◽  
Author(s):  
Vivian Wong
Keyword(s):  
Molecular Weight ◽  
Tight Junction ◽  
Tight Junctions ◽  
Growth Medium ◽  
Mdck Cells ◽  
Low Calcium ◽  
Phosphatase Treatment ◽  
And Function ◽  
Triton X ◽  
Mdck I

Multiple forms of occludin were found in Madin-Darby canine kidney (MDCK) cells. In the absence of cell-to-cell contacts, achieved by incubating cells in low-calcium growth medium, a cluster of lower-molecular-weight (LMW) occludin bands (∼65,000–68,000) was present in both MDCK I and II cells. On formation of tight junctions, achieved by changing the low-calcium growth medium to normal-calcium growth medium, a cluster of higher-molecular-weight (HMW) bands (∼72,000–75,000 for MDCK I cells and ∼70,000–73,000 for MDCK II cells) was also expressed. The HMW occludin bands could be eliminated by phosphatase treatment. Therefore, the HMW forms of occludin appeared to be the hyperphosphorylated product of the LMW forms. These HMW forms were Triton X-100 insoluble, which correlated with their localization at the tight junctions. Furthermore, depletion of tight junction-localized occludin by an occludin extracellular domian peptide (20) correlated with a decrease in the HMW forms of occludin. In conclusion, phosphorylation of occludin may be a mechanism by which occludin localization and function are regulated.

scholarly journals Connexin-Occludin Chimeras Containing the Zo-Binding Domain of Occludin Localize at Mdck Tight Junctions and Nrk Cell Contacts

1999 ◽  
Vol 146 (3) ◽  
pp. 683-693 ◽  
Author(s):  
Laura L. Mitic ◽  
Eveline E. Schneeberger ◽  
Alan S. Fanning ◽  
James Melvin Anderson
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Rat Kidney ◽  
Freeze Fracture ◽  
Binding Domain ◽  
Permeability Barrier ◽  
Cell Contacts ◽  
Cytoplasmic Proteins

Occludin is a transmembrane protein of the tight junction that functions in creating both an intercellular permeability barrier and an intramembrane diffusion barrier. Creation of the barrier requires the precise localization of occludin, and a distinct family of transmembrane proteins called claudins, into continuous linear fibrils visible by freeze-fracture microscopy. Conflicting evidence exists regarding the relative importance of the transmembrane and extracellular versus the cytoplasmic domains in localizing occludin in fibrils. To specifically address whether occludin's COOH-terminal cytoplasmic domain is sufficient to target it into tight junction fibrils, we created chimeras with the transmembrane portions of connexin 32. Despite the gap junction targeting information present in their transmembrane and extracellular domains, these connexin-occludin chimeras localized within fibrils when expressed in MDCK cells, as assessed by immunofluorescence and immunogold freeze-fracture imaging. Localization of chimeras at tight junctions depends on the COOH-terminal ZO-binding domain and not on the membrane proximal domain of occludin. Furthermore, neither endogenous occludin nor claudin is required for targeting to ZO-1–containing cell–cell contacts, since in normal rat kidney fibroblasts targeting of chimeras again required only the ZO-binding domain. These results suggest an important role for cytoplasmic proteins, presumably ZO-1, ZO-2, and ZO-3, in localizing occludin in tight junction fibrils. Such a scaffolding and cytoskeletal coupling function for ZO MAGUKs is analogous to that of other members of the MAGUK family.

scholarly journals ZO-1 Stabilizes the Tight Junction Solute Barrier through Coupling to the Perijunctional Cytoskeleton

2009 ◽  
Vol 20 (17) ◽  
pp. 3930-3940 ◽  
Author(s):  
Christina M. Van Itallie ◽  
Alan S. Fanning ◽  
Arlene Bridges ◽  
James M. Anderson
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Mdck Cells ◽  
Morphological Changes ◽  
Barrier Properties ◽  
Wild Type ◽  
Rock Inhibitor ◽  
Morphological Alterations ◽  
Solute Permeability ◽  
Cytoplasmic Proteins

ZO-1 binds numerous transmembrane and cytoplasmic proteins and is required for assembly of both adherens and tight junctions, but its role in defining barrier properties of an established tight junction is unknown. We depleted ZO-1 in MDCK cells using siRNA methods and observed specific defects in the barrier for large solutes, even though flux through the small claudin pores was unaffected. This permeability increase was accompanied by morphological alterations and reorganization of apical actin and myosin. The permeability defect, and to a lesser extent morphological changes, could be rescued by reexpression of either full-length ZO-1 or an N-terminal construct containing the PDZ, SH3, and GUK domains. ZO-2 knockdown did not replicate either the permeability or morphological phenotypes seen in the ZO-1 knockdown, suggesting that ZO-1 and -2 are not functionally redundant for these functions. Wild-type and knockdown MDCK cells had differing physiological and morphological responses to pharmacologic interventions targeting myosin activity. Use of the ROCK inhibitor Y27632 or myosin inhibitor blebbistatin increased TER in wild-type cells, whereas ZO-1 knockdown monolayers were either unaffected or changed in the opposite direction; paracellular flux and myosin localization were also differentially affected. These studies are the first direct evidence that ZO-1 limits solute permeability in established tight junctions, perhaps by forming a stabilizing link between the barrier and perijunctional actomyosin.

scholarly journals Production of ammonium by Helicobacter pylori mediates occludin processing and disruption of tight junctions in Caco-2 cells

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3267-3276 ◽  
Author(s):  
Simon D. Lytton ◽  
Wolfgang Fischer ◽  
Wolfram Nagel ◽  
Rainer Haas ◽  
Franz X. Beck
Keyword(s):  
Molecular Weight ◽  
Helicobacter Pylori ◽  
Tight Junction ◽  
Tight Junctions ◽  
Ammonium Salts ◽  
Gastric Mucosal Damage ◽  
Soluble Form ◽  
Low Molecular Weight ◽  
Partial Recovery ◽  
H Pylori

Tight junctions, paracellular permeability barriers that define epithelial cell polarity, play an essential role in transepithelial transport, cell–cell adhesion and lymphocyte transmigration. They are also important for the maintenance of innate immune defence and intestinal antigen uptake. Ammonium () is elevated in the gastric aspirates of Helicobacter pylori-infected patients and has been implicated in the disruption of tight-junction functional integrity and the induction of gastric mucosal damage during H. pylori infection. The precise mechanism of the effect of ammonium and the molecular targets of ammonium in host tissue are not yet identified. To study the effects of ammonium on epithelial tight junctions, the human colon carcinoma cell line Caco-2 was cultured on permeable supports and the transepithelial resistance (TER) was measured at different time intervals following exposure to ammonium salts or H. pylori-derived ammonium. A biphasic response to treatment with ammonium was found. Acute exposure to ammonium salts or NH3/ derived from urea metabolism by wild-type H. pylori resulted in a 20–30 % decrease in TER. After 24 h, the NH4Cl-treated cells showed a partial recovery of TER. In contrast, the control culture, or cultures that were exposed to supernatants derived from urease-deficient H. pylori, showed no significant decrease in TER. Occludin-specific immunoblots revealed the expression of a low-molecular-weight form of occludin of 42 kDa upon NH3/ exposure. The results indicate that modulation of tight-junction function by H. pylori is ammonium-dependent and linked to the accumulation of a low-molecular-weight and detergent-soluble form of occludin.

Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion

1998 ◽  
Vol 275 (3) ◽  
pp. C798-C809 ◽  
Author(s):  
Shobha Gopalakrishnan ◽  
Narayan Raman ◽  
Simon J. Atkinson ◽  
James A. Marrs
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Rho Gtpase ◽  
Mdck Cells ◽  
Atp Depletion ◽  
Cell Junctions ◽  
Mutant Proteins ◽  
Vitro Model ◽  
The Relationship

Tight junctions control paracellular permeability and cell polarity. Rho GTPase regulates tight junction assembly, and ATP depletion of Madin-Darby canine kidney (MDCK) cells (an in vitro model of renal ischemia) disrupts tight junctions. The relationship between Rho GTPase signaling and ATP depletion was examined. Rho inhibition resulted in decreased localization of zonula occludens-1 (ZO-1) and occludin at cell junctions; conversely, constitutive Rho signaling caused an accumulation of ZO-1 and occludin at cell junctions. Inhibiting Rho before ATP depletion resulted in more extensive loss of junctional components between transfected cells than control junctions, whereas cells expressing activated Rho better maintained junctions during ATP depletion than control cells. ATP depletion and Rho signaling altered phosphorylation signaling mechanisms. ZO-1 and occludin exhibited rapid decreases in phosphoamino acid content following ATP depletion, which was restored on recovery. Expression of Rho mutant proteins in MDCK cells also altered levels of occludin serine/threonine phosphorylation, indicating that occludin is a target for Rho signaling. We conclude that Rho GTPase signaling induces posttranslational effects on tight junction components. Our data also demonstrate that activating Rho signaling protects tight junctions from damage during ATP depletion.

Luminal nutrients alter tight-junction permeability in the rat jejunum: an in vivo perfusion model

1993 ◽  
Vol 71 (10-11) ◽  
pp. 835-839 ◽  
Author(s):  
D. C. Sadowski ◽  
J. B. Meddings
Keyword(s):  
Molecular Weight ◽  
Tight Junction ◽  
High Molecular Weight ◽  
Tight Junctions ◽  
Polyethylene Glycol 400 ◽  
Ussing Chambers ◽  
Perfusion Model ◽  
Tight Junction Permeability

The regulation of tight-junction permeability between enterocytes has been studied using in vitro perfused loops, Ussing chambers, and cultured cell monolayers. In this communication we demonstrate the ability of an in vivo perfusion model to monitor tight-junction permeability and respond appropriately to physiological luminal stimuli. By using the highly charged anionic ferrocyanide molecule, water flux could be accurately assessed in the rat, and the luminal clearance of high molecular weight dextrans could be used to probe the opening and closing of the paracellular pathway. By utilizing two different molecular weight dextrans markers simultaneously, each conjugated with a different fluorophore, we were able to calculate luminal clearances of these compounds by fluorometric techniques in the presence of luminal nutrients that have previously been demonstrated to open intercellular tight junctions. In the absence of luminal nutrients or the presence of a non-nutrient sugar such as mannitol, clearance of these compounds was negligible. However, with the addition of either D-glucose or L-alanine, clearance of both high molecular weight markers increased dramatically. Thus, opening of tight junctions between enterocytes appears to be a physiological event that occurs in vivo under conditions likely to be found in the lumen. Polyethylene glycol 400 (PEG-400) clearance did not correlate well with the clearance of either dextran marker, suggesting that this probe utilizes a different permeation pathway and may not be appropriate to quantify the nutrient-regulatable pathway observed with the former probes.Key words: intestinal permeability, glucose transport, paracellular transport.

scholarly journals The Small GTPase Rab13 Regulates Assembly of Functional Tight Junctions in Epithelial Cells

2002 ◽  
Vol 13 (6) ◽  
pp. 1819-1831 ◽  
Author(s):  
Anne-Marie Marzesco ◽  
Irene Dunia ◽  
Rudy Pandjaitan ◽  
Michel Recouvreur ◽  
Daniel Dauzonne ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Tight Junctions ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Freeze Fracture ◽  
Small Gtpase ◽  
Cell Contact ◽  
Junctional Complexes ◽  
And Function

Junctional complexes such as tight junctions (TJ) and adherens junctions are required for maintaining cell surface asymmetry and polarized transport in epithelial cells. We have shown that Rab13 is recruited to junctional complexes from a cytosolic pool after cell–cell contact formation. In this study, we investigate the role of Rab13 in modulating TJ structure and functions in epithelial MDCK cells. We generate stable MDCK cell lines expressing inactive (T22N mutant) and constitutively active (Q67L mutant) Rab13 as GFP-Rab13 chimeras. Expression of GFP-Rab13Q67L delayed the formation of electrically tight epithelial monolayers as monitored by transepithelial electrical resistance (TER) and induced the leakage of small nonionic tracers from the apical domain. It also disrupted the TJ fence diffusion barrier. Freeze-fracture EM analysis revealed that tight junctional structures did not form a continuous belt but rather a discontinuous series of stranded clusters. Immunofluorescence studies showed that the expression of Rab13Q67L delayed the localization of the TJ transmembrane protein, claudin1, at the cell surface. In contrast, the inactive Rab13T22N mutant did not disrupt TJ functions, TJ strand architecture nor claudin1 localization. Our data revealed that Rab13 plays an important role in regulating both the structure and function of tight junctions.

scholarly journals Occludin 1B, a Variant of the Tight Junction Protein Occludin

2000 ◽  
Vol 11 (2) ◽  
pp. 627-634 ◽  
Author(s):  
Zoia Muresan ◽  
David L. Paul ◽  
Daniel A. Goodenough
Keyword(s):  
Tight Junction ◽  
Base Pair ◽  
Mdck Cells ◽  
Human Colon ◽  
Reverse Transcription Pcr ◽  
Mouse Tissues ◽  
Junction Protein ◽  
Alternatively Spliced ◽  
Membrane Components ◽  
And Function

Occludin and claudin are the major integral membrane components of the mammalian tight junction. Although more than 11 distinct claudins have been identified, only 1 occludin transcript has been reported thus far. Therefore, we searched by reverse transcription–PCR for occludin-related sequences in Madin–Darby canine kidney (MDCK) mRNA and identified a transcript encoding an alternatively spliced form of occludin, designated occludin 1B. The occludin 1B transcript contained a 193-base pair insertion encoding a longer form of occludin with a unique N-terminal sequence of 56 amino acids. Analysis of the MDCK occludin gene revealed an exon containing the 193-base pair sequence between the exons encoding the original N terminus and the distal sequence, suggesting that occludin and occludin 1B arise from alternative splicing of one transcript. To assess the expression and distribution of occludin 1B, an antibody was raised against its unique N-terminal domain. Immunolabeling of occludin 1B in MDCK cells revealed a distribution indistinguishable from that of occludin. Furthermore, occludin 1B staining at cell-to-cell contacts was also found in cultured T84 human colon carcinoma cells and in frozen sections of mouse intestine. Immunoblots of various mouse tissues revealed broad coexpression of occludin 1B with occludin. The wide epithelial distribution and the conservation across species suggests a potentially important role for occludin 1B in the structure and function of the tight junction.

VAP-33 localizes to both an intracellular vesicle population and with occludin at the tight junction

1999 ◽  
Vol 112 (21) ◽  
pp. 3723-3732 ◽  
Author(s):  
L.A. Lapierre ◽  
P.L. Tuma ◽  
J. Navarre ◽  
J.R. Goldenring ◽  
J.M. Anderson
Keyword(s):  
Plasma Membrane ◽  
Tight Junction ◽  
Tight Junctions ◽  
Growth Regulation ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Subcellular Fractionation ◽  
Tissue Culture Cell ◽  
Cellular Functions ◽  
Vesicle Docking

Tight junctions create a regulated intercellular seal between epithelial and endothelial cells and also establish polarity between plasma membrane domains within the cell. Tight junctions have also been implicated in many other cellular functions, including cell signaling and growth regulation, but they have yet to be directly implicated in vesicle movement. Occludin is a transmembrane protein located at tight junctions and is known to interact with other tight junction proteins, including ZO-1. To investigate occludin's role in other cellular functions we performed a yeast two-hybrid screen using the cytoplasmic C terminus of occludin and a human liver cDNA library. From this screen we identified VAP-33 which was initially cloned from Aplysia by its ability to interact with VAMP/synaptobrevin and thus was implicated in vesicle docking/fusion. Extraction characteristics indicated that VAP-33 was an integral membrane protein. Antibodies to the human VAP-33 co-localized with occludin at the tight junction in many tissues and tissue culture cell lines. Subcellular fractionation of liver demonstrated that 83% of VAP-33 co-isolated with occludin and DPPIV in a plasma membrane fraction and 14% fractionated in a vesicular pool. Thus, both immunofluorescence and fractionation data suggest that VAP-33 is present in two distinct pools in the cells. In further support of this conclusion, a GFP-VAP-33 chimera also distributed to two sites within MDCK cells and interestingly shifted occludin's localization basally. Since VAP-33 has previously been implicated in vesicle docking/fusion, our results suggest that tight junctions may participate in vesicle targeting at the plasma membrane or alternatively VAP-33 may regulate the localization of occludin.

Rotavirus alters paracellular permeability and energy metabolism in Caco-2 cells

2000 ◽  
Vol 279 (4) ◽  
pp. G757-G766 ◽  
Author(s):  
Kathleen G. Dickman ◽  
Scott J. Hempson ◽  
Joseph Anderson ◽  
Scott Lippe ◽  
Liming Zhao ◽  
...  
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Intestinal Cells ◽  
Metabolic Dysfunction ◽  
Rotavirus Infection ◽  
Severe Diarrhea ◽  
Consistent Feature ◽  
And Function ◽  
Junction Structure

Rotaviruses infect epithelial cells of the small intestine, but the pathophysiology of the resulting severe diarrhea is incompletely understood. Histological damage to intestinal epithelium is not a consistent feature, and in vitro studies showed that intestinal cells did not undergo rapid death and lysis during viral replication. We show that rotavirus infection of Caco-2 cells caused disruption of tight junctions and loss of transepithelial resistance (TER) in the absence of cell death. TER declined from 300 to 22 Ω · cm2between 8 and 24 h after infection and was accompanied by increased transepithelial permeability to macromolecules of 478 and 4,000 Da. Distribution of tight junction proteins claudin-1, occludin, and ZO-1 was significantly altered during infection. Claudin-1 redistribution was notably apparent at the onset of the decline in TER. Infection was associated with increased production of lactate, decreased mitochondrial oxygen consumption, and reduced cellular ATP (60% of control at 24 h after infection), conditions known to reduce the integrity of epithelial tight junctions. In conclusion, these data show that rotavirus infection of Caco-2 intestinal cells altered tight junction structure and function, which may be a response to metabolic dysfunction.

Molecular Physiology and Pathophysiology of Tight Junctions III. Tight junction regulation by intracellular messengers: differences in response within and between epithelia

2000 ◽  
Vol 279 (4) ◽  
pp. G660-G665 ◽  
Author(s):  
Jurgen Karczewski ◽  
Jack Groot
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Imaging Techniques ◽  
Effector Proteins ◽  
Molecular Physiology ◽  
Intracellular Messengers ◽  
Tight Junction Regulation ◽  
Tight Junction Permeability ◽  
Permeability Studies ◽  
And Function

Tight junction permeability differs with the type of permeants, their size, and their charge. Selective changes in permeability do occur, and they illustrate the diversity in functional reactions of tight junctions. This suggests that special structures in the tight junctions are involved. More and more structural components of the tight junctions are becoming known. The divergence in behavior of native tissue and filter-grown epithelial monolayers with respect to the effects of intracellular messengers offers the possibility to relate structure and function. In addition to the tools for conventional permeability studies, probes have become available to detect changes in activation of intracellular effector proteins such as the protein kinase C isotypes, and with in situ imaging techniques the way is open for a functional approach in the study of tight junctions.

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