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 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.

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.

scholarly journals Structural and Functional Regulation of Tight Junctions by RhoA and Rac1 Small GTPases

1998 ◽  
Vol 142 (1) ◽  
pp. 101-115 ◽  
Author(s):  
Tzuu-Shuh Jou ◽  
Eveline E. Schneeberger ◽  
W. James Nelson
Keyword(s):  
Tight Junctions ◽  
Spatial Organization ◽  
Mdck Cells ◽  
Protein Localization ◽  
Freeze Fracture ◽  
Tracer Diffusion ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Solute Diffusion ◽  
Fence Function

Tight junctions (TJ) govern ion and solute diffusion through the paracellular space (gate function), and restrict mixing of membrane proteins and lipids between membrane domains (fence function) of polarized epithelial cells. We examined roles of the RhoA and Rac1 GTPases in regulating TJ structure and function in MDCK cells using the tetracycline repressible transactivator to regulate RhoAV14, RhoAN19, Rac1V12, and Rac1N17 expression. Both constitutively active and dominant negative RhoA or Rac1 perturbed TJ gate function (transepithelial electrical resistance, tracer diffusion) in a dose-dependent and reversible manner. Freeze-fracture EM and immunofluoresence microscopy revealed abnormal TJ strand morphology and protein (occludin, ZO-1) localization in RhoAV14 and Rac1V12 cells. However, TJ strand morphology and protein localization appeared normal in RhoAN19 and Rac1N17 cells. All mutant GTPases disrupted the fence function of the TJ (interdomain diffusion of a fluorescent lipid), but targeting and organization of a membrane protein in the apical membrane were unaffected. Expression levels and protein complexes of occludin and ZO-1 appeared normal in all mutant cells, although ZO-1 was more readily solubilized from RhoAV14-expressing cells with Triton X-100. These results show that RhoA and Rac1 regulate gate and fence functions of the TJ, and play a role in the spatial organization of TJ proteins at the apex of the lateral membrane.

scholarly journals The tight junction protein ZO-1 is concentrated along slit diaphragms of the glomerular epithelium.

1990 ◽  
Vol 111 (3) ◽  
pp. 1255-1263 ◽  
Author(s):  
E Schnabel ◽  
J M Anderson ◽  
M G Farquhar
Keyword(s):  
Tight Junction ◽  
Rat Kidney ◽  
Freeze Fracture ◽  
Kidney Tissue ◽  
Slit Diaphragm ◽  
Kidney Cortex ◽  
Adult Rats ◽  
Junction Protein ◽  
Collecting Tubules ◽  
Lateral Cell

The foot processes of glomerular epithelial cells of the mammalian kidney are firmly attached to one another by shallow intercellular junctions or slit diaphragms of unknown composition. We have investigated the molecular nature of these junctions using an antibody that recognizes ZO-1, a protein that is specific for the tight junction or zonula occludens. By immunoblotting the affinity purified anti-ZO-1 IgG recognizes a single 225-kD band in kidney cortex and in slit diaphragm-enriched fractions as in other tissues. When ZO-1 was localized by immunofluorescence in kidney tissue of adult rats, the protein was detected in epithelia of all segments of the nephron, but the glomerular epithelium was much more intensely stained than any other epithelium. Among tubule epithelia the signal for ZO-1 correlated with the known fibril content and physiologic tightness of the junctions, i.e., it was highest in distal and collecting tubules and lowest in the proximal tubule. By immunoelectron microscopy ZO-1 was found to be concentrated on the cytoplasmic surface of the tight junctional membrane. Within the glomerulus ZO-1 was localized predominantly in the epithelial foot processes where it was concentrated precisely at the points of insertion of the slit diaphragms into the lateral cell membrane. Its distribution appeared to be continuous along the continuous slit membrane junction. When ZO-1 was localized in differentiating glomeruli in the newborn rat kidney, it was present early in development when the apical junctional complexes between presumptive podocytes are composed of typical tight and adhering junctions. It remained associated with these junctions during the time they migrate down the lateral cell surface, disappear and are replaced by slit diaphragms. The distribution of ZO-1 and the close developmental relationship between the two junctions suggest that the slit diaphragm is a variant of the tight junction that shares with it at least one structural protein and the functional property of defining distinctive plasmalemmal domains. The glomerular epithelium is unique among renal epithelia in that ZO-1 is present, but the intercellular spaces are wide open and no fibrils are seen by freeze fracture. The presence of ZO-1 along slit membranes indicates that expression of ZO-1 alone does not lead to tight junction assembly.

Localization of a novel 210 kDa protein in Xenopus tight junctions

1997 ◽  
Vol 110 (8) ◽  
pp. 1005-1012 ◽  
Author(s):  
C.S. Merzdorf ◽  
D.A. Goodenough
Keyword(s):  
Monoclonal Antibody ◽  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Basolateral Membrane ◽  
Immunofluorescent Staining ◽  
Junctional Complex ◽  
Permeability Barrier ◽  
Membrane Domains ◽  
Kidney Liver

The tight junction is the most apical member of the intercellular junctional complex. It functions as a permeability barrier between epithelial cells and maintains the integrity of the apical and basolateral membrane domains. In order to study tight junctions in Xenopus laevis, a polyclonal antibody was raised which recognized Xenopus ZO-1. Monoclonal antibody 19B1 (mAb 19B1) was generated in rats using a crude membrane preparation from Xenopus lung as antigen. mAb 19B1 gave immunofluorescent staining patterns identical to those seen with anti-ZO-1 on monolayers of Xenopus A6 kidney epithelial cells and on frozen sections of Xenopus kidney, liver, and embryos. Electron microscopy showed that the 19B1 antigen colocalized with ZO-1 at the tight junction. Western blotting and immunoprecipitation demonstrated that ZO-1 is an approximately 220 kDa protein in Xenopus, while mAb 19B1 identified an approximately 210 kDa antigen on immunoblots. Immunoprecipitates of ZO-1 were not recognized by mAb 19B1 by western analysis. The solubility properties of the 19B1 antigen suggested that it is a peripheral membrane protein. Thus, the antigen recognized by the new monoclonal antibody 19B1 is not ZO-1 and represents a different Xenopus tight junction associated protein.

scholarly journals ZO-1 mRNA and protein expression during tight junction assembly in Caco-2 cells.

1989 ◽  
Vol 109 (3) ◽  
pp. 1047-1056 ◽  
Author(s):  
J M Anderson ◽  
C M Van Itallie ◽  
M D Peterson ◽  
B R Stevenson ◽  
E A Carew ◽  
...  
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Polyclonal Antibodies ◽  
Epithelial Cell Line ◽  
Cell Contact ◽  
Mrna Levels ◽  
Expression Library ◽  
Cell Contacts ◽  
Protein Levels ◽  
Cell Cell

We previously identified and characterized ZO-1 as a peripheral membrane protein specifically associated with the cytoplasmic surface of tight junctions. Here we describe the identification of partial cDNA sequences encoding rat and human ZO-1 and their use to study the assembly of tight junctions in the Caco-2 human intestinal epithelial cell line. A rat cDNA was isolated from a lambda-gtll expression library by screening with mAbs. Polyclonal antibodies were raised to cDNA-encoded fusion protein; several properties of these antibodies support this cDNA as encoding ZO-1. Expression of ZO-1 mRNA occurs in the rat and Caco-2 cells with a major transcript of approximately 7.5 kb. To disrupt tight junctions and study the subsequent process of assembly, Caco-2 cells were grown in suspension for 48 h in Ca++/Mg++-free spinner medium during which time they lose cell-cell contacts, become round, and by immunofluorescence microscopy show diffuse and speckled localization of ZO-1. Within hours of replating at confluent density in Ca++/Mg++-containing media, attached cells show discrete localization of ZO-1 at cell-cell contacts. Within 2 d, fully confluent monolayers form, and ZO-1 localizes in a continuous gasket-like fashion circumscribing all cells. ZO-1 mRNA levels are highest in cells in spinner culture, and upon replating rapidly fall and plateau at approximately 10% of initial levels after 2-3 wk in culture. ZO-1 protein levels are lowest in contact-free cells and rise five- to eightfold over the same period. In contrast, mRNA levels for sucrase-isomaltase, an apical membrane hydrolase, increase only after a confluent monolayer forms. Thus, in this model of contact-dependent assembly of the tight junction, there is both a rapid assembly beginning upon cell-cell contact, as well as a long-term modulation involving changes in expression of ZO-1 mRNA and protein levels.

Structure, biochemistry, and assembly of epithelial tight junctions

1987 ◽  
Vol 253 (6) ◽  
pp. C749-C758 ◽  
Author(s):  
B. Gumbiner
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Lateral Diffusion ◽  
Junctional Complex ◽  
Epithelial Cell Layer ◽  
Dependent Cell ◽  
Freeze Fracture Electron Microscopy ◽  
Dynamic Structures

The zonula occludens (ZO), also referred to as the tight junction, forms the barrier to the diffusion of molecules and ions across the epithelial cell layer through the paracellular space. The level of electrical resistance of the paracellular pathway seems to depend on the number of strands in the ZO observed by freeze-fracture electron microscopy (EM). The ZO also forms the boundary between the compositionally distinct apical and basolateral plasma membrane domains because it is a barrier to the lateral diffusion of lipids and membrane proteins that reside in the extracytoplasmic leaflet of the membrane bilayer. In contrast to its appearance in transmission EM, the tight junction is not a fusion between the outer membrane leaflets of neighboring cells. Rather it consists of protein molecules, including the newly discovered protein ZO-1 and probably others, which bring the plasma membranes into extremely close apposition so as to occlude the extracellular space. Very little is known about the assembly of tight junctions, but several kinds of evidence suggest that they are very dynamic structures. Other elements of the epithelial junctional complex including the zonula adherens (ZA), the Ca2+-dependent cell adhesion molecule uvomorulin, or L-CAM, and actin filaments of the cytoskeleton may participate in the assembly of the ZO.

scholarly journals CRB3 Binds Directly to Par6 and Regulates the Morphogenesis of the Tight Junctions in Mammalian Epithelial Cells

2004 ◽  
Vol 15 (3) ◽  
pp. 1324-1333 ◽  
Author(s):  
Céline Lemmers ◽  
Didier Michel ◽  
Lydie Lane-Guermonprez ◽  
Marie-Hélène Delgrossi ◽  
Emmanuelle Médina ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Transmembrane Protein ◽  
Direct Interaction ◽  
Extracellular Domain ◽  
Cytoplasmic Domain ◽  
Epithelial Morphogenesis ◽  
Neurotrophin Receptor ◽  
Tight Junction Formation

Crumbs is an apical transmembrane protein crucial for epithelial morphogenesis in Drosophila melanogaster embryos. A protein with all the characteristics for a Crumbs homologue has been identified from patients suffering from retinitis pigmentosa group 12, but this protein (CRB1) is only expressed in retina and some parts of the brain, both in human and mouse. Here, we describe CRB3, another Crumbs homologue that is preferentially expressed in epithelial tissues and skeletal muscles in human. CRB3 shares the conserved cytoplasmic domain with other Crumbs but exhibits a very short extracellular domain without the EGF- and laminin A-like G repeats present in the other Crumbs. CRB3 is localized to the apical and subapical area of epithelial cells from the mouse and human intestine, suggesting that it could play a role in epithelial morphogenesis. Indeed, expression of CRB3 or of a chimera containing the extracellular domain of the neurotrophin receptor p75NTR and the transmembrane and cytoplasmic domains of CRB3 led to a slower development of functional tight junctions in Madin-Darby canine kidney cells. This phenotype relied on the presence of CRB3 four last amino acids (ERLI) that are involved in a direct interaction with Par6, a regulator of epithelial polarity and tight junction formation. Thus, CRB3, through its cytoplasmic domain and its interactors, plays a role in apical membrane morphogenesis and tight junction regulation.

scholarly journals Continuous endocytic recycling of tight junction proteins: how and why?

2012 ◽  
Vol 53 ◽  
pp. 41-54 ◽  
Author(s):  
Andrew D. Chalmers ◽  
Paul Whitley
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Dynamic Behaviour ◽  
Tight Junction Proteins ◽  
Tissue Remodelling ◽  
Extracellular Signals ◽  
Junctional Proteins ◽  
Cytoplasmic Proteins ◽  
Dynamic Structures ◽  
Junction Proteins

Tight junctions consist of many proteins, including transmembrane and associated cytoplasmic proteins, which act to provide a barrier regulating transport across epithelial and endothelial tissues. These junctions are dynamic structures that are able to maintain barrier function during tissue remodelling and rapidly alter it in response to extracellular signals. Individual components of tight junctions also show dynamic behaviour, including migration within the junction and exchange in and out of the junctions. In addition, it is becoming clear that some tight junction proteins undergo continuous endocytosis and recycling back to the plasma membrane. Regulation of endocytic trafficking of junctional proteins may provide a way of rapidly remodelling junctions and will be the focus of this chapter.

Sign in / Sign up

Export Citation Format

Share Document