ZO-1 maintains its spatial distribution but dissociates from junctional fibrils during tight junction regulation

1993 ◽  
Vol 264 (5) ◽  
pp. C1096-C1101 ◽  
Author(s):  
J. L. Madara ◽  
S. Carlson ◽  
J. M. Anderson
Keyword(s):  
Spatial Distribution ◽  
Tight Junction ◽  
Tight Junctions ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Cell Junctions ◽  
Absorptive Cell ◽  
Physiological Regulation ◽  
Functional Relationships ◽  
Tight Junction Regulation

Tight junctions restrict diffusion of hydrophilic solutes through the paracellular pathways of columnar epithelia. It is now apparent that the barrier function of tight junctions is physiologically regulated. Current models of the tight junction envisage junctional subunits consisting of extracellular "kisses" between plasma membranes of adjacent cells, intramembrane components represented by freeze-fracture fibrils, and cytoplasmic elements of the cytoskeleton. Insights into functional relationships between these various components of tight junctions should be provided by mapping component interrelationships in states of altered junctional permeability. Here we define the spatial distribution of ZO-1 during a state of physiological regulation of intestinal absorptive cell tight junctions. Enhanced permeation of absorptive cell junctions in response to activation of apical membrane Na(+)-solute cotransporters does not lead to redistribution of the ZO-1 pool, as judged from quantitative ultrastructural immunolocalization studies employing two different ZO-1 antibodies. Surprisingly, ZO-1, which normally localizes under junctional kisses/fibrils, focally persists at sites where junctional kisses/fibrils are cleared. These findings suggest that 1) spatial redistribution of ZO-1 does not contribute to physiological regulation of junctions elicited by activation of Na(+)-solute cotransport and 2) ZO-1 and junctional fibrils may spatially dissociate during such regulated states.

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.

Effects of phlorizin and sodium on glucose-elicited alterations of cell junctions in intestinal epithelia

1990 ◽  
Vol 258 (1) ◽  
pp. C77-C85 ◽  
Author(s):  
K. Atisook ◽  
S. Carlson ◽  
J. L. Madara
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Intercellular Junctions ◽  
Glucose Absorption ◽  
Cell Junctions ◽  
Absorptive Cell ◽  
Analysis Model ◽  
Ussing Chambers ◽  
Perfusion Studies

Glucose alters absorptive cell tight junction structure and, as deduced from an impedance analysis model, diminishes tight junction resistance in the small intestine (J.R. Pappenheimer, J. Membr. Biol. 100: 137-148, 1987; and J.L. Madara and J.R. Pappenheimer, J. Membr. Biol. 100: 149-164, 1987). Here we provide further evidence in support of this hypothesis using the conventional approach of analysis of mucosal sheets mounted in Ussing chambers. This approach offers advantages for investigating underlying mechanisms, including the effects of ions and inhibitors on the regulation of intercellular junctions by glucose. We show that phlorizin blocks a resistance decrease elicited by glucose and demonstrate that substitution of choline for sodium also prevents the response. The dilatations in absorptive cell tight junctions that accompany this glucose-elicited response are similarly prevented by phlorizin exposure or sodium substitution. The effects of phlorizin on junctional permeability can also be demonstrated in vivo. Phlorizin reduces the transjunctional flux of creatinine in glucose-perfused intestines of anesthetized animals, even when account is taken of the reduction of fluid absorption caused by phlorizin. Last, in vivo perfusion studies suggest that although, at 25 mM luminal glucose, virtually all glucose absorption is transcellular, at a luminal glucose concentration of 125 mM approximately 30% of glucose absorption occurs paracellularly because of solvent drag across tight junctions of altered permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased Tyr phosphorylation of ZO-1 during modification of tight junctions between glomerular foot processes

1995 ◽  
Vol 268 (3) ◽  
pp. F514-F524 ◽  
Author(s):  
H. Kurihara ◽  
J. M. Anderson ◽  
M. G. Farquhar
Keyword(s):  
Tight Junction ◽  
Tyrosine Phosphorylation ◽  
Tight Junctions ◽  
Mesangial Cells ◽  
Basal Membrane ◽  
Cell Junctions ◽  
Phosphorylated Proteins ◽  
Cell Matrix ◽  
Rapid Changes ◽  
And Control

The slit diaphragms between the glomerular epithelial foot processes represent a variant of the tight junction that are rapidly replaced by typical tight junctions after perfusion with protamine sulfate (PS). To investigate the mechanism of signaling involved, tyrosine phosphorylation of glomerular proteins was analyzed in newborn, PS-treated, and control rats using antiphosphotyrosine immunoglobulin G. In glomeruli of normal adults, phosphotyrosine (Ptyr) staining was confined largely to mesangial cells by immunofluorescence, whereas in newborn and PS-treated rats, the Ptyr signal was dramatically increased in the glomerular epithelium. By immunogold labeling, it was found that newly phosphorylated proteins were concentrated along the newly formed tight junctions (cell-cell junctions) and the basal membrane of the foot processes (cell-matrix junctions). By immunoblotting, several prominent bands were detected with anti-Ptyr in glomerular lysates of controls; in PS-treated rats, additional bands were detected at 225, 180, and 100 kDa. The 225-kDa protein was identified as ZO-1 by immunoprecipitation with anti-ZO-1 followed by immunoblotting with anti-Ptyr. These findings indicate that ZO-1 is one of the targets for tyrosine phosphorylation after PS treatment. They indicate that phosphorylation of tight junction and other proteins occurs during the formation of tight junctions in glomeruli under circumstances where there are rapid changes in epithelial cell shape.

scholarly journals Rab13 regulates PKA signaling during tight junction assembly

2004 ◽  
Vol 165 (2) ◽  
pp. 175-180 ◽  
Author(s):  
Katja Köhler ◽  
Daniel Louvard ◽  
Ahmed Zahraoui
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Inhibitory Effect ◽  
Cell Junctions ◽  
Unknown Mechanism ◽  
Epithelial Tight Junctions ◽  
Actin Remodelling ◽  
Cell Cell

The GTPase Rab13 regulates the assembly of functional epithelial tight junctions (TJs) through a yet unknown mechanism. Here, we show that expression of the GTP-bound form of Rab13 inhibits PKA-dependent phosphorylation and TJ recruitment of the vasodilator-stimulated phosphoprotein, an actin remodelling protein. We demonstrate that Rab13GTP directly binds to PKA and inhibits its activity. Interestingly, activation of PKA abrogates the inhibitory effect of Rab13 on the recruitment of vasodilator-stimulated phosphoprotein, ZO-1, and claudin1 to cell–cell junctions. Rab13 is, therefore, the first GTPase that controls PKA activity and provides an unexpected link between PKA signaling and the dynamics of TJ assembly.

Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation

1997 ◽  
Vol 273 (4) ◽  
pp. C1378-C1385 ◽  
Author(s):  
Jerrold R. Turner ◽  
Brian K. Rill ◽  
Susan L. Carlson ◽  
Denise Carnes ◽  
Rachel Kerner ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Inhibitors ◽  
Tight Junction Regulation ◽  
Tight Junction Permeability ◽  
Mlc Phosphorylation

Tight junctions serve as the rate-limiting barrier to passive movement of hydrophilic solutes across intestinal epithelia. After activation of Na+-glucose cotransport, the permeability of intestinal tight junctions is increased. Because previous analyses of this physiological tight junction regulation have been restricted to intact mucosae, dissection of the mechanisms underlying this process has been limited. To characterize this process, we have developed a reductionist model consisting of Caco-2 intestinal epithelial cells transfected with the intestinal Na+-glucose cotransporter, SGLT1. Monolayers of SGLT1 transfectants demonstrate physiological Na+-glucose cotransport. Activation of SGLT1 results in a 22 ± 5% fall in transepithelial resistance (TER) ( P< 0.001). Similarly, inactivation of SGLT1 by addition of phloridzin increases TER by 24 ± 2% ( P < 0.001). The increased tight junction permeability is size selective, with increased flux of small nutrient-sized molecules, e.g., mannitol, but not of larger molecules, e.g., inulin. SGLT1-dependent increases in tight junction permeability are inhibited by myosin light-chain kinase inhibitors (20 μM ML-7 or 40 μM ML-9), suggesting that myosin regulatory light-chain (MLC) phosphorylation is involved in tight junction regulation. Analysis of MLC phosphorylation showed a 2.08-fold increase after activation of SGLT1 ( P< 0.01), which was inhibited by ML-9 ( P < 0.01). Thus monolayers incubated with glucose and myosin light-chain kinase inhibitors are comparable to monolayers incubated with phloridzin. ML-9 also inhibits SGLT1-mediated tight junction regulation in small intestinal mucosa ( P < 0.01). These data demonstrate that epithelial cells are the mediators of physiological tight junction regulation subsequent to SGLT1 activation. The intimate relationship between tight junction regulation and MLC phosphorylation suggests that a critical step in regulation of epithelial tight junction permeability may be myosin ATPase-mediated contraction of the perijunctional actomyosin ring and subsequent physical tension on the tight junction.

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 Cytochemical evidence for the presence of phospholipids in epithelial tight junction strands.

1993 ◽  
Vol 41 (5) ◽  
pp. 649-656 ◽  
Author(s):  
F W Kan
Keyword(s):  
Plasma Membrane ◽  
Tight Junction ◽  
Phospholipase A2 ◽  
Plasma Membranes ◽  
Membrane Lipids ◽  
Freeze Fracture ◽  
Fracture Face ◽  
Gold Particles ◽  
Pancreatic Cells ◽  
Specific Association

Previous freeze-fracture experiments using either glutaraldehyde-fixed and cryoprotected specimens or unfixed rapid-frozen samples led to the proposal that cylindrical strands of the tight junction (TJ) observed in freeze-fracture preparations are inverted cylindrical micelles made up of membrane lipids and, possibly, membrane proteins. However, no one has yet been able to directly label the structural fibrils of the TJ. To test the hypothesis that TJ strands observed on freeze-fracture preparations are composed at least partially of lipids, we have combined the phospholipase A2-gold and the fracture-label techniques for localization of phospholipids. Phospholipase A2, purified from bee venom, was adsorbed on gold particles and used for specific labeling of its substrate. Phospholipase A2-colloidal gold (PLA2-CG) complex was applied to freeze-fractured preparations of rat exocrine pancreatic cells and testicular Sertoli cells, both of which are known to have extensive TJ complexes on their plasma membranes. Fracture-label replicas of exocrine pancreatic cells revealed specific association of gold particles with TJ fibrils on the protoplasmic fracture-face of the plasma membrane. The majority of these gold particles were observed either directly on the top of the TJ fibrils or adjacent to these cylindrical structures. A high density of PLA2-CG labeling was also observed over the complementary exoplasmic fracture-face of the TJ complex. This intimate association of PLA2-CG labeling with the TJ is particularly evident in the Sertoli cell plasma membrane, where rows of gold particles were observed to be superimposed on parallel arrays of cylindrical strands of the TJ complex. The present findings provide direct cytochemical evidence to support the hypothesis that cylindrical TJ strands observed in freeze-fracture preparations contain phospholipids.

Tight junction regulation through vesicle trafficking: bringing cells together

2014 ◽  
Vol 42 (1) ◽  
pp. 195-200 ◽  
Author(s):  
Sarah J. Fletcher ◽  
Joshua Z. Rappoport
Keyword(s):  
Steady State ◽  
Tight Junction ◽  
Tight Junctions ◽  
Vesicle Trafficking ◽  
Present Review ◽  
Physiological Processes ◽  
Endosomal Recycling ◽  
Tight Junction Regulation ◽  
Epithelial Layers

Epithelial layers are integral for many physiological processes and are maintained by intercellular adhesive structures. During disease, these structures can disassemble, leading to breakdown of epithelia. TJs (tight junctions) are one type of intercellular adhesion. Loss of TJs has been linked to the pathogenesis of many diseases. The present review focuses on the role of vesicle trafficking in regulation of TJs, in particular trafficking of the TJ protein occludin. We examine how endocytosis and endosomal recycling modulate occludin localization under steady-state conditions and during stimulated TJ disassembly.

Osmotic water flow pathways across Necturus gallbladder: role of the tight junction

1994 ◽  
Vol 266 (4) ◽  
pp. G722-G730 ◽  
Author(s):  
K. Loeschke ◽  
C. J. Bentzel
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Water Flow ◽  
Freeze Fracture ◽  
Electrical Conductance ◽  
Leaky Epithelia ◽  
Necturus Gallbladder ◽  
Osmotic Water ◽  
Flow Pathways ◽  
Osmotic Water Flow

To explore the quantitative significance of passive water flow through tight junctions of leaky epithelia, transepithelial water flow rates were measured in Necturus gallbladder mounted in chambers. Osmotic flows generated by raffinose gradients were asymmetrical with the greater flow in the mucosal-to-serosal direction. In tissue fixed in situ, intercellular spaces were dilated during mucosal-to-serosal flow and closed during serosal-to-mucosal flow. Tight junctions were focally separated (blistered), which correlated with the magnitude of mucosal-to-serosal flow. Blisters were not observed during serosal-to-mucosal flow or in nontransporting gallbladders. In freeze-fracture replicas, blisters appeared as pockets between intramembranous strands. Protamine, which decreases electrical conductance and increases depth and complexity of the tight junction, reduced osmotic water flow by approximately 30% in the mucosal-to-serosal direction (100 mosmol/kg gradient) without altering serosal-to-mucosal flow. We suggest that in the steady state, at least 30% of osmotically driven water passes transjunctionally in the mucosal-to-serosal direction, but flow is transcellular in the serosal-to-mucosal direction. Directionally divergent pathways may account for flow asymmetry.

Further Observations on the Fine Structure of Freeze-Cleaved Tight Junctions

1973 ◽  
Vol 13 (3) ◽  
pp. 763-786 ◽  
Author(s):  
L. A. STAEHELIN
Keyword(s):  
Fine Structure ◽  
Tight Junction ◽  
Tight Junctions ◽  
Plasma Membranes ◽  
Central Area ◽  
Experimental Conditions ◽  
Cellular Space ◽  
Mesh Work ◽  
Definition Of ◽  
Sealing Elements

The fine structure of freeze-cleaved tight junctions has been examined in high-resolution replicas of rat small intestine. By carefully comparing the changes in the surface topography of the cleavage faces of specimens, which, before freezing, were either prefixed with glutaraldehyde and then infiltrated with glycerol, infiltrated with glycerol alone, or infiltrated with glycerol and then fixed with glutaraldehyde, a more precise definition of the tight-junction architecture at the supramolecular level has become possible. The results of this analysis suggest that the bilayer membranes contributing to a tight junction are held together along inter-connected lines of attachment that are arranged in the form of a continuous band-like mesh-work. Each line consists of 2 parallel rows, one in each membrane, of closely spaced adhesion particles. The sensitivity of these particles to glutaraldehyde and their cleaving behaviour under different experimental conditions indicate that they represent globular proteins which bridge the width of the adjoining membranes and are linked together in the plane of the inter-cellular space. Thus, the morphology of a tight-junction seal resembles a modified zipper with the locking units making head to head contact. Similarly, the presence of many open-ended sealing elements between crypt cells has been interpreted as suggesting that the formation of tight-junction seals could resemble a ‘zippering-up’ process. At the juncture of 3 cells the tight-junction meshwork is both modified and extended basally to produce a characteristic pattern. In the central area of such a triple junction, 3 parallel and very closely spaced vertical seals have been resolved, each joining a pair of adjacent plasma membranes. Small, regularly spaced, cross-bridging elements interlink pairs of central seals within the plane of each membrane. At different levels each individual central seal may further give rise to horizontal sealing elements which connect in a ladder-like fashion either to the major network or to vertical elements positioned at greater distances from the central axis. Evidence is presented suggesting that fragments of tight junctions can be internalized and broken down in lysosome-like vesicles.

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