Claudins create charge-selective channels in the paracellular pathway between epithelial cells

2002 ◽  
Vol 283 (1) ◽  
pp. C142-C147 ◽  
Author(s):  
Oscar R. Colegio ◽  
Christina M. Van Itallie ◽  
Heather J. McCrea ◽  
Christoph Rahner ◽  
James Melvin Anderson
Keyword(s):  
Electrical Resistance ◽  
Freeze Fracture ◽  
Extracellular Domain ◽  
Inducible Promoter ◽  
Site Directed Mutagenesis ◽  
Solute Flux ◽  
Ionic Charge ◽  
Cell Monolayers ◽  
Charge Selectivity ◽  
Freeze Fracture Electron Microscopy

Epithelia separate tissue spaces by regulating the passage of ions, solutes, and water through both the transcellular and paracellular pathways. Paracellular permeability is defined by intercellular tight junctions, which vary widely among tissues with respect to solute flux, electrical resistance, and ionic charge selectivity. To test the hypothesis that members of the claudin family of tight junction proteins create charge selectivity, we assessed the effect of reversing the charge of selected extracellular amino acids in two claudins using site-directed mutagenesis. Claudins were expressed in cultured Madin-Darby canine kidney cell monolayers under an inducible promoter, and clones were compared with and without induction for transmonolayer electrical resistance and dilution potentials. Expression and localization of claudins were determined by immunoblotting, immunofluorescence microscopy, and freeze-fracture electron microscopy. We observed that substituting a negative for a positive charge at position 65 in the first extracellular domain of claudin-4 increased paracellular Na+ permeability. Conversely, substituting positive for negative charges at three positions in the first extracellular domain of claudin-15, singly and in combination, reversed paracellular charge selectivity from a preference for Na+ to Cl−. These results support a model where claudins create charge-selective channels in the paracellular space.

Occluding junctions in cultured epithelial monolayers

1981 ◽  
Vol 240 (3) ◽  
pp. C96-C102 ◽  
Author(s):  
M. Cereijido ◽  
I. Meza ◽  
A. Martinez-Palomo
Keyword(s):  
Electrical Resistance ◽  
Mdck Cells ◽  
Freeze Fracture ◽  
Structural Components ◽  
Epithelial Monolayers ◽  
Cation Selectivity ◽  
Occluding Junctions ◽  
Freeze Fracture Electron Microscopy ◽  
Macromolecular Tracers ◽  
Fracture Electron

When MDCK cells are cultured in monolayers, they synthesize, assemble, and seal occluding junctions that limit the paracellular route. These processes may be impaired by inhibitors of the protein synthesis but not by inhibitors of the synthesis of RNA. Once established, the occluding junctions confer to the monolayer an overall electrical resistance of 80–600 omega . cm2. At the microscopical level, the resistance of individual junctions have large variations along the perimeter of a given cell. This agrees with the images of freeze-fracture electron microscopy where the network of the junction varies abruptly from 1 to 10 strands. The junctions are impermeable to macromolecular tracers, have a 9 to 1 Na+/Cl- discrimination, and a cation selectivity following the order: K+ greater than Na+ greater than Rb+ greater than Cs+ greater than Li+. Sealing requires extracellular Ca2+, but the junctions open when the concentration of Ca2+ in the cytoplasm increases. The structural components of the cytoskeleton (microtubules and microfilaments) seem to be involved in the junctional events as revealed by staining with immunofluorescent specific antibodies. If the cells are treated with cytochalasin B, actin microfilaments disorganize, the junctions open, and the electrical resistance across the monolayers falls. The resealing of the tight junction is inhibited by this drug.

scholarly journals Experimental modulation of occluding junctions in a cultured transporting epithelium.

1980 ◽  
Vol 87 (3) ◽  
pp. 736-745 ◽  
Author(s):  
A Martinez-Palomo ◽  
I Meza ◽  
G Beaty ◽  
M Cereijido
Keyword(s):  
Electron Microscopy ◽  
Electrical Resistance ◽  
Freeze Fracture ◽  
Ionophore A23187 ◽  
Bathing Medium ◽  
Serum Factors ◽  
Cation Selectivity ◽  
Occluding Junctions ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

The experimental opening and resealing of occluding junctions in monolayers of cultured MDCK cells (epithelioid of renal origin) was explored by measuring changes in the electrical resistance across the monolayer and by freeze-fracture electron microscopy. As in natural epithelia, the function of occluding junctions as permeability barriers specifically depends on extracellular Ca++ concentration and fails if this ion is replaced by Mg++ or Ba++. The removal of Ca++ and the addition of EGTA to the bathing medium opened the junctions and reduced the transepithelial resistance. Resealing was achieved within 10-15 min by restoring Ca++. Quantitative freeze-fracture electron microscopy showed that junctional opening, caused by lack of Ca++, was accompanied by simplification of the pattern of the membrane strands of the occluding junction without disassembly or displacement of the junctional components. Resealing of the cellular contacts involved the gradual return to a normal junctional pattern estimated as the average number of strands constituting the junction. The occluding junctions were also opened by the addition of the ionophore A23187, suggesting that the sealing of the contacts requires high Ca++ on the extracellular side and low Ca++ concentration of the cytoplasmic compartment. The opening process could be blocked by low temperature (7.5 degrees C). Resealing did not depend on serum factors and did not require protein synthesis; therefore, it seems to be caused by reassembly of preexisting membrane junctional components. The restoration of the junctions occurred simultaneously with the establishment of ion-selective channels; the Na+/Cl- and the cation/cation selectivity were recovered with the same time-course as the electrical resistance. The role of the cytoskeleton in the process of junctional reassembly is reported in the companion article.

scholarly journals Occluding junctions and paracellular pathways studied in monolayers of MDCK cells

1983 ◽  
Vol 106 (1) ◽  
pp. 205-215 ◽  
Author(s):  
M. Cereijido ◽  
L. Gonzalez-Mariscal ◽  
L. Borboa
Keyword(s):  
Electrical Resistance ◽  
Intercellular Space ◽  
Mdck Cells ◽  
Freeze Fracture ◽  
Apical Surface ◽  
Leaky Epithelia ◽  
Occluding Junctions ◽  
Freeze Fracture Electron Microscopy ◽  
Current Flows ◽  
High Conductance

MDCK cells (epithelioid, derived from the kidney of a normal dog) cultured in monolayers on a permeable support, exhibit properties of natural transporting epithelia. Comparisons of the electrical resistance across the plasma membrane of MDCK cells (as studied with microelectrodes) and the resistance across the whole monolayer, (mounted as a flat sheet between two chambers) indicate that most of the current flows through an extracellular pathway. Scanning of the electrical field over the apical surface shows that this pathway is located at the intercellular space. Yet conductance is not evenly distributed along the intercellular space as in leaky epithelia, but is restricted to sites scattered irregularly along the intercellular space. Studies of freeze fracture electron microscopy indicate that the number of strands of the junctions is also distributed irregularly, varying from 1 to 10 in a few nanometers. This suggests that regions with few strands would correspond to spots with high conductance and vice versa. However, in this preparation the sealing property of the junction bears little relationship to its structure. Thus by changing the temperature from 37 to 3 degrees C and back, the electrical resistance increases reversibly by 306%, while the number and arrangement of the strands show no significant modification. The resistance of the monolayer varies also with the age of the cells, suggesting that sealing and ion-permeating components of the junction may be dynamic entities that are not permanently installed, but can be accommodated to the requirements of the tissue.

A dominant mutant of occludin disrupts tight junction structure and function

1999 ◽  
Vol 112 (12) ◽  
pp. 1879-1888 ◽  
Author(s):  
S.D. Bamforth ◽  
U. Kniesel ◽  
H. Wolburg ◽  
B. Engelhardt ◽  
W. Risau
Keyword(s):  
Tight Junction ◽  
Freeze Fracture ◽  
Integral Membrane Protein ◽  
Epithelial Cell Line ◽  
Permeability Barrier ◽  
Cell Monolayers ◽  
Junction Protein ◽  
N Terminus ◽  
Freeze Fracture Electron Microscopy ◽  
And Function

The tight junction is the most apical intercellular junction of epithelial cells and forms a diffusion barrier between individual cells. Occludin is an integral membrane protein specifically associated with the tight junction which may contribute to the function or regulation of this intercellular seal. In order to elucidate the role of occludin at the tight junction, a full length and an N-terminally truncated murine occludin construct, both FLAG-tagged at the N terminus, were stably introduced into the murine epithelial cell line CSG 120/7. Both constructs were correctly targeted to the tight junction, as defined by colocalization with another tight junction protein, ZO-1. The construct lacking the N terminus and extracellular domains of occludin was found to exert a dramatic effect on tight junction integrity. Cell monolayers failed to develop an efficient permeability barrier, as demonstrated by low transcellular electrical resistance values and an increased paracellular flux to small molecular mass tracers. Furthermore, gaps were found to have been induced in the P-face associated tight junction strands, as visualized by freeze-fracture electron microscopy. These findings demonstrate an important role for the N-terminal half of occludin in tight junction assembly and maintaining the barrier function of the tight junction.

The Molecular Physiology of Tight Junction Pores

Physiology ◽  
2004 ◽  
Vol 19 (6) ◽  
pp. 331-338 ◽  
Author(s):  
Christina M. Van Itallie ◽  
James Melvin Anderson
Keyword(s):  
Cell Adhesion ◽  
Caenorhabditis Elegans ◽  
Tight Junction ◽  
Electrical Resistance ◽  
Large Family ◽  
Paracellular Transport ◽  
Molecular Physiology ◽  
Ionic Charge ◽  
Charge Selectivity ◽  
Cell Adhesion Proteins

Tight junctions form selective barriers that regulate paracellular transport across epithelia. A large family of tetraspanning cell-cell adhesion proteins called claudins create the barrier and regulate electrical resistance, size, and ionic charge selectivity. Study of inherited human claudin diseases and the outcome of the genetic manupulation of claudins in mice, Drosophila, and Caenorhabditis elegans are furthering our understanding of paracellular physiology.

Image Analysis of Intramembrane Particles in Freeze-Fractured Biomembranes Using Computer Simulation Techniques

1975 ◽  
Vol 33 ◽  
pp. 214-215
Author(s):  
D.J. Benefiel ◽  
R.S. Weinstein
Keyword(s):  
Membrane Proteins ◽  
Freeze Fracture ◽  
Integral Membrane Proteins ◽  
Systematic Evaluation ◽  
Intramembrane Particles ◽  
Modeling Methods ◽  
Simulation Techniques ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron ◽  
Computer Simulation Techniques

Intramembrane particles (IMP or MAP) are components of most biomembranes. They are visualized by freeze-fracture electron microscopy, and they probably represent replicas of integral membrane proteins. The presence of MAP in biomembranes has been extensively investigated but their detailed ultrastructure has been largely ignored. In this study, we have attempted to lay groundwork for a systematic evaluation of MAP ultrastructure. Using mathematical modeling methods, we have simulated the electron optical appearances of idealized globular proteins as they might be expected to appear in replicas under defined conditions. By comparing these images with the apearances of MAPs in replicas, we have attempted to evaluate dimensional and shape distortions that may be introduced by the freeze-fracture technique and further to deduce the actual shapes of integral membrane proteins from their freezefracture images.

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