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.

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.

Inhibiting cadherin function by dominant mutant E-cadherin expression increases the extent of tight junction assembly

2000 ◽  
Vol 113 (6) ◽  
pp. 985-996 ◽  
Author(s):  
M.L. Troxell ◽  
S. Gopalakrishnan ◽  
J. McCormack ◽  
B.A. Poteat ◽  
J. Pennington ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Tight Junction ◽  
Mdck Cells ◽  
Freeze Fracture ◽  
Junctional Complex ◽  
Negative Effects ◽  
Tight Junction Formation ◽  
Freeze Fracture Electron Microscopy ◽  
E Cadherin ◽  
Cell Cell

Previous studies have shown that induction of cadherin-mediated cell-cell adhesion leads to tight junction formation, and that blocking cadherin-mediated cell-cell adhesion inhibits tight junction assembly. Here we report analysis of tight junction assembly in MDCK cells overexpressing a mutant E-cadherin protein that lacks an adhesive extracellular domain (T151 cells). Mutant E-cadherin overexpression caused a dramatic reduction in endogenous cadherin levels. Despite this, tight junction assembly was extensive. The number of tight junction strands observed by freeze-fracture electron microscopy significantly increased in T151 cells compared to that in control cells. Our data indicate that the hierarchical regulation of junctional complex assembly is not absolute, and that inhibition of cadherin function has both positive and negative effects on tight junction assembly.

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.

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.

Human zonulin, a potential modulator of intestinal tight junctions

2000 ◽  
Vol 113 (24) ◽  
pp. 4435-4440 ◽  
Author(s):  
W. Wang ◽  
S. Uzzau ◽  
S.E. Goldblum ◽  
A. Fasano
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Ussing Chamber ◽  
Intestinal Lumen ◽  
Epithelial Surface ◽  
Ussing Chambers ◽  
Zonula Occludens ◽  
Surface Receptor ◽  
Dynamic Structures ◽  
Zonula Occludens Toxin

Intercellular tight junctions are dynamic structures involved in vectorial transport of water and electrolytes across the intestinal epithelium. Zonula occludens toxin derived from Vibrio cholerae interacts with a specific intestinal epithelial surface receptor, with subsequent activation of a complex intracellular cascade of events that regulate tight junction permeability. We postulated that this toxin may mimic the effect of a functionally and immunologically related endogenous modulator of intestinal tight junctions. Affinity-purified anti-zonula occludens toxin antibodies and the Ussing chamber assay were used to screen for one or more mammalian zonula occludens toxin analogues in both fetal and adult human intestine. A novel protein, zonulin, was identified that induces tight junction disassembly in non-human primate intestinal epithelia mounted in Ussing chambers. Comparison of amino acids in the active zonula occludens toxin fragment and zonulin permitted the identification of the putative receptor binding domain within the N-terminal region of the two proteins. Zonulin likely plays a pivotal role in tight junction regulation during developmental, physiological, and pathological processes, including tissue morphogenesis, movement of fluid, macromolecules and leukocytes between the intestinal lumen and the interstitium, and inflammatory/autoimmune disorders.

scholarly journals Surface membrane vesicles from mononuclear cells stimulate erythroid stem cells to proliferate in culture

Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 583-594 ◽  
Author(s):  
N Dainiak ◽  
CM Cohen
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Mononuclear Cells ◽  
Surface Membrane ◽  
Freeze Fracture ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Target Cells ◽  
Freeze Fracture Electron Microscopy

Abstract In order to examine the contribution of cell surface materials to erythroid burst-promoting activity (BPA), we separated media conditioned by a variety of human cell types into pellets and supernatants by centrifugation. When added to serum-restricted cultures of nonadherent human marrow cells, pellets contained about half of the total stimulatory activity. Freeze-fracture electron microscopy of the pellets revealed the presence of unilamellar membrane vesicles ranging from 0.10 to 0.40 microM in diameter. The amount of BPA in culture increased with added vesicle concentration in a saturable fashion. Preparation of leukocyte conditioned medium (LCM) from 125I-wheat germ agglutinin labeled cells and studies comparing the glycoprotein composition of vesicles with that of leukocyte plasma membranes suggest that LCM-derived vesicles are of plasma membrane origin. Moreover, partially purified leukocyte plasma membrane preparations also contained BPA. While disruption of vesicles by freezing/thawing and hypotonic lysis did not alter BPA, heat, trypsin, or pronase treatment removed greater than 65% of BPA, implying that vesicle surface rather than intravesicular molecules express BPA. Results of BPA assays performed in two-layer clots indicated that proximity to target cells is required for vesicle BPA expression. We conclude that membrane vesicles spontaneously shed from cell surfaces may be important regulators of erythroid burst proliferation in vitro.

scholarly journals Erythrocyte membrane plaques from rats with magnesium deficiency

Blood ◽  
1977 ◽  
Vol 49 (4) ◽  
pp. 657-664 ◽  
Author(s):  
RJ Elin ◽  
HK Tan
Keyword(s):  
Erythrocyte Membrane ◽  
Plasma Membranes ◽  
Magnesium Deficiency ◽  
Freeze Fracture ◽  
Deficient Diet ◽  
White Rats ◽  
Dietary Magnesium ◽  
Freeze Fracture Electron Microscopy ◽  
And Control ◽  
Fracture Electron

Abstract This study investigated the anemia of dietary magnesium deficiency in inbred Fisher white rats using freeze-fracture electron microscopy. The plasma membranes of erythrocytes from animals receiving two different magnesium-deficient and control diets were observed at weekly or biweekly intervals for 6 wk. The earliest changes were small plaques on the external surface (ES) and fracture face (PF) of erythrocyte plasma membranes, which occurred after 2 wk of either magnesium-deficient diet. These plaques persisted and increased in size with progressive magnesium deficiency. When fully developed, the plaques consisted of round or oval elevations approximately 30–50 nm in diameter outlined by a narrow raised border. The surface of the plaques was smooth and devoid of intramembranous particles. Incubation of erythrocytes from magnesium-deficient rats in a physiologic solution containing 2 meq/liter magnesium for 1 hr at 37degrees C did not alter the appearance of the plaques. Erythrocytes from control rats, obtained during the same time periods, showed no plaques. Thus, a deficiency of magnesium in rats altered erythrocyte membrane structure.

scholarly journals VARIATIONS IN TIGHT AND GAP JUNCTIONS IN MAMMALIAN TISSUES

1972 ◽  
Vol 53 (3) ◽  
pp. 758-776 ◽  
Author(s):  
Daniel S. Friend ◽  
Norton B. Gilula
Keyword(s):  
Smooth Muscle ◽  
Gap Junctions ◽  
Tight Junctions ◽  
Adrenal Cortex ◽  
Freeze Fracture ◽  
Junctional Complex ◽  
Exocrine Glands ◽  
Rat Pancreas ◽  
Zonula Occludens ◽  
Mammalian Tissues

The fine structure and distribution of tight (zonula occludens) and gap junctions in epithelia of the rat pancreas, liver, adrenal cortex, epididymis, and duodenum, and in smooth muscle were examined in paraformaldehyde-glutaraldehyde-fixed, tracer-permeated (K-pyroantimonate and lanthanum), and freeze-fractured tissue preparations. While many pentalaminar and septilaminar foci seen in thin-section and tracer preparations can be recognized as corresponding to well-characterized freeze-fracture images of tight and gap junction membrane modifications, many others cannot be unequivocally categorized—nor can all freeze-etched aggregates of membrane particles. Generally, epithelia of exocrine glands (pancreas and liver) have moderate-sized tight junctions and large gap junctions, with many of their gap junctions basal to the junctional complex. In contrast, the adrenal cortex, a ductless gland, may not have a tight junction but does possess large gap junctions. Mucosal epithelia (epididymis and intestine) have extensive tight junctions, but their gap junctions are not as well developed as those of glandular tissue. Smooth muscle contains numerous small gap junctions The incidence, size, and configuration of the junctions we observed correlate well with the known functions of the junctions and of the tissues where they are found.

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.

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