Tight Junction Strands Research Articles

Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases.

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.

Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin.

Occludin is the only known integral membrane protein localizing at tight junctions (TJ), but recent targeted disruption analysis of the occludin gene indicated the existence of as yet unidentified integral membrane proteins in TJ. We therefore re-examined the isolated junction fraction from chicken liver, from which occludin was first identified. Among numerous components of this fraction, only a broad silver-stained band approximately 22 kD was detected with the occludin band through 4 M guanidine-HCl extraction as well as sonication followed by stepwise sucrose density gradient centrifugation. Two distinct peptide sequences were obtained from the lower and upper halves of the broad band, and similarity searches of databases allowed us to isolate two full-length cDNAs encoding related mouse 22-kD proteins consisting of 211 and 230 amino acids, respectively. Hydrophilicity analysis suggested that both bore four transmembrane domains, although they did not show any sequence similarity to occludin. Immunofluorescence and immunoelectron microscopy revealed that both proteins tagged with FLAG or GFP were targeted to and incorporated into the TJ strand itself. We designated them as "claudin-1" and "claudin-2", respectively. Although the precise structure/function relationship of the claudins to TJ still remains elusive, these findings indicated that multiple integral membrane proteins with four putative transmembrane domains, occludin and claudins, constitute TJ strands.

Test of a two-pathway model for small-solute exchange across the capillary wall.

We previously proposed a two-pathway model for solute and water transport across vascular endothelium (Fu, B. M., R. Tsay, F. E. Curry, and S. Weinbaum. J. Biomech. Eng. 116: 502-513, 1994) that hypothesized the existence of a continuous slit 2 nm wide along tight junction strands within the interendothelial cleft in parallel with 20 x 150-nm breaks in tight junctions. We tested this model by measuring capillary permeability coefficients (P) to a small solute (sodium fluorescein, radius 0.45 nm), assumed to permeate primarily the 2-nm small pore, and an intermediate-sized solute (FITC-alpha-lactalbumin, radius 2.01 nm) excluded from the small pore. Mean values of the paired diffusive permeability coefficients, Psodium fluorescein and PFITC-alpha-lactalbumin, were 34.4 and 2.9 x 10(-6) cm/s, respectively, after corrections for solvent drag and free dye (n = 26). These permeabilities were accounted for by transport through the large-break pathway without the additional capacity of the hypothetical 2-nm pathway. As a further test we examined the relative reductions of Psodium fluorescein and PFITC-alpha-lactalbumin produced by elevated intracellular cAMP. Within 20 min after the introduction of rolipram and forskolin, Psodium fluorescein and PFITC-alpha-lactalbumin decreased to 0.67 and 0.64 times their respective baseline values. These similar responses to permeability decrease were evidence that the two solutes were carried by a common pathway. Combined results in both control and reduced permeability states did not support the hypothesis that a separate pathway across tight junctions is available for solutes with a radius as large as 0.75 nm. If such a pathway is present, then its size must be smaller than that of sodium fluorescein.

Microvascular permeability and number of tight junctions are modulated by cAMP.

We tested the hypothesis that increased endothelial cell adenosine 3',5'-cyclic monophosphate (cAMP) decreases microvascular permeability in vivo. The effects of cAMP-specific phosphodiesterase type IV inhibition and adenylate cyclase activation on microvascular hydraulic conductivity (Lp) were investigated in intact individual capillaries and postcapillary venules in mesentery of pithed frogs (Rana pipiens). Treatment with rolipram (10 microM) and forskolin (5 microM) for 25 min decreased Lp to 37% of control. Rolipram alone also significantly decreased Lp. Isoproterenol (10 microM) decreased Lp to 27% of control within 20 min. A subgroup of eight vessels treated with rolipram and forskolin, in which mean Lp fell to 25% of control, was examined with transmission electron microscopy. The mean number of tight junctions in the treated vessels was 2.2 per cleft (303 clefts), significantly higher than in a matched control group (192 clefts), which was 1.7 per cleft. The results indicate that microvascular Lp can be modulated by intracellular cAMP and that one of the structural end points of stimulated cAMP levels is an increase in the mean number of tight-junction strands between endothelial cells.

Occludin-deficient embryonic stem cells can differentiate into polarized epithelial cells bearing tight junctions.

Occludin is the only known integral membrane protein of tight junctions (TJs), and is now believed to be directly involved in the barrier and fence functions of TJs. Occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occludin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from occludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TJ strands between wild-type and occludin-deficient epithelial cells. Furthermore, zonula occludens (ZO)-1, a TJ-associated peripheral membrane protein, was still exclusively concentrated at TJ in occludin-deficient epithelial cells. In good agreement with these morphological observations, TJ in occludin-deficient epithelial cells functioned as a primary barrier to the diffusion of a low molecular mass tracer through the paracellular pathway. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures, recruit ZO-1, and function as a barrier without occludin.

Potential interplay between luminal growth factors and increased tight junction permeability in epithelial carcinogenesis.

It has been generally accepted for many years that carcinogenesis proceeds in at least two distinct stages, the first being initiation, the second being tumor promotion (Boutwell, ’74). Initiation has been shown to be a heritable change imposed on DNA itself, whereas promotion is regarded as an extranuclear event, non heritable, and requiring chronic exposure to a tumor promoting agent (Diamond et al., ’80). One may think then of carcinogenesis as a series of dominoes which must fall in sequence in order to finally produce the condition which we term cancer. One can likewise conceive of it as a series of necessary and finally sufficient events, leading in the end to a tumor. The majority of lethal cancers are epithelial in origin (Fraumeni et al., ’89). The fact that epithelia line all of the major body fluid compartments places epithelial cells specifically in more pronounced contact with environmental carcinogenic agents than other somatic cells. In other words, if the process of carcinogenesis requires a tumor promotion phase entailing chronic uninterrupted exposure to an exogenous tumor promoting substance, epithelial cells in tissues such as lung and colon would be repetitive targets. There are two key characteristics of epithelia, two traits which are common among all of the various epithelial tissues. First, all epithelial cells are intrinsically polar, having an apical cell surface facing the luminal fluid compartment, and a basal-lateral membrane facing the interstitial fluid compartment. These two membrane domains contain not only different classes of membrane proteins, but distinct lipid compositions as well (Schneeberger and Lynch, ’92). The second characteristic common through all epithelial tissues is the most basic function of any epithelium, namely its ability to form a barrier between the two fluid compartments which the epithelium separates. The common thread between these two most fundamental characteristics is the tight junction or zonula occludens. This protein (or protein and lipid) circumferential band around the apical pole of each epithelial cell is first and foremost the selectively permeable diffusion barrier for the paracellular pathway. It prevents free flow of solutes from one fluid compartment to the other along the paracellular route between epithelial cells (Farquhar and Palade, ’65). The tight junction also has a role in maintaining the apical/ basal-lateral polarity of individual epithelial cells (Cereijido et al., ’89). When considering the scope of epithelial cancers across such a variety of tissues, and the fact that according to the two-stage carcinogenesis model an extranuclear change is necessary for epithelial cancer, the primacy of epithelial polarity and barrier function makes them key areas on which to focus attention. Evidence for altered permeability of tight junctions in transformed epithelia has been known for almost 30 years (Martinez-Palomo, ’70). A decrease in the number of tight junctional strands has been described in transitional carcinoma of the urinary bladder (Saito, ’84). Decreased transepithelial impedance has been recorded across the colons of mice treated with chemical carcinogens (Davies et al., ’89). Inflammatory bowel disease linked with increased cancer risk has itself been linked with increased tight junction permeability, not only in affected patients but in unaffected relatives as well (Hollander, ’88). On a molecular level, the tight junctional protein, ZO-1 (Stevenson et al., ’89), has been shown to possess significant sequence homology to a septate tumor suppressor protein of Drosophila, whose mutation leads to neoplastic growth of epithelia within larvae (Willott et al., ’93; Woods and Bryant, ’91). Finally, the interaction of the normal APC (adenomatous polyposis coli) colon cancer susceptibility gene product with the cell adhesion protein, β-catenin

A collagen substrate enhances the sealing capacity of tight junctions of A6 cell monolayers.

A6 cells, a kidney derived epithelial cell line, when cultured either on a collagen-coated substrate or on polycarbonate substrate without collagen form confluent monolayers that are similar in cell density and overall morphology. However, the transepithelial electrical resistance (TER) of monolayers grown on the collagen-coated substrate is ninefold higher than that of monolayers grown without collagen. A comparative freeze-fracture study showed that this large difference in TER is not related to the length or number of tight junction strands but to differences in the specific conductance of individual strands. This conductance was obtained considering the TER, the linear junctional density and the mean number of tight junction strands. We estimated the specific linear conductance of the tight junction strands to be 2.56 x 10(-7) S/cm for cells grown on collagen and 30.3 x 10(-7) S/cm for the cells grown without collagen. We also examined changes in distribution and phosphorylation states of the zonula occludens associated protein, ZO-1, during monolayer formation. Immunocytochemistry reveals that the distribution of ZO-1 follows a similar time course and pattern independent of the presence or absence of collagen. While the amount of ZO-1 expression is identical in cells grown on both substrates, this protein is phosphorylated to a greater extent during the initial stages of confluence in cells cultured on collagen. We suggest that the phosphorylation levels of ZO-1 in A6 cells at the early stages of monolayer formation may determine the final molecular structure and specific conductance of the tight junctions strands.

Dynamics of connexins, E-cadherin and alpha-catenin on cell membranes during gap junction formation.

We examined the dynamics of connexins, E-cadherin and alpha-catenin during gap-junction disassembly and assembly in regeneration hepatocytes by immunofluorescence microscopy, and immunogold-electron microscopy using SDS-digested freeze-replicas. The present findings suggest that during the disappearance of gap junctions most of the gap junction plaques are broken up into smaller aggregates, and then the gap junction proteins may be removed from the cell membrane, but some of the connexons or connexins remain dispersed in the plane of membrane as pure morphologically indistinguishable intramembrane proteins. Double-immunogold electron microscopy using a polyclonal antibody for connexins and a monoclonal antibody for E-cadherin or alpha-catenin revealed co-localization of these molecules at cell-to-cell contact sites during the reappearance of gap junction plaques. This implies that, at least in regenerating hepatocytes, the cadherin-catenin complex-mediated cell-to-cell contact sites act as foci for gap junction formation. In addition, connexin-immunoreactivity was also observed along tight junctional strands, suggesting that the gap junction may also form along the tight junctions.

Renal imaging techniques.

The ancient approach to obtain an image of the kidneys (and other internal organs) was 'section-inspection-imaging' by drawing, painting, sculpturing, and modelling. The present study follows chronologically the development and use of sectioning techniques from ancient (often forbidden) methods to modern microdissection and maceration of silicone-rubber-injected tubules. Inspection evolved from the use of the naked eye to magnifying lenses, microscopes and finally electron microscopy. Pertinent examples such as the description of the kidneys as the site of urine formation, the visualization of loop structures in the renal medulla and the imaging of tight junction strands are discussed. Inspection or visualization of renal structure and function has been revolutionized by modern noninvasive techniques, such as X-ray imaging, imaging by radioisotopes, ultrasound, computer tomography and nuclear magnetic resonance. Pertinent examples are given demonstrating the potency of the various techniques. The contribution of computerized data evaluation is discussed. The development of micropuncture and microperfusion techniques has opened the field for direct imaging not only of renal (sub)structural details but also of functional parameters such as transtubular reabsorption rates, single glomerular capillary filtration and conductance of the paracellular pathway. We focus particularly on techniques specifically designed to visualize renal hemodynamic and transport parameters.

Characteristics of the myelin sheath membrane of the spiral ganglion cell and the cochlear nerve of guinea pig.

The membrane structures of the myelin sheath of guinea pig spiral ganglion cell and cochlear nerve were examined using freeze-fracturing, rapid freezing and freeze substitution, and deep-etching techniques. The intermediate dense lines of the myelin sheath were observed only in the outermost several layers using thin and thick (1 micron) section techniques. The spaces between the two adjacent major dense lines at any layer of myelin sheath were occupied by flocculent materials in the freeze-substituted specimen. Periods between the two adjacent major dense lines were 13.3 nm in conventional fixation and 11.7 nm in rapid freezing followed by freeze substitution, respectively. A freeze-fracturing study showed no differences in membrane structures at any layer of myelin sheath. Developed tight-junction strands were observed parallel to the long axis of the myelin sheath in the deep-etched specimen. These findings may show the characteristics of myelin sheath membrane in the spiral ganglion cell and cochlear nerve of the guinea pig.

Development of blood-brain barrier tight junctions in the rat cortex

The structural equivalent of the blood-brain barrier are the complex tight junctions (TJs) between endothelial cells of brain capillaries. In this study, we have quantitatively investigated by the freeze-fracture technique the modulation of the fine structure of TJs in blood-brain barrier endothelial cells during development of the rat cerebral cortex. The complexity of the TJ network as defined by fractal dimension, the integrity of TJ strands and the degree of TJ particle association to the protoplasmic leaflet of the membrane bilayer in percent of total TJ length were evaluated at embryonic days (E) 13, 15, 18, postnatal day (P) 1 and adult. We observed that the overall complexity of the TJ network and P-face association of TJ particles are significantly increased between E18 and P1. The increase in both of these TJ parameters in combination with the completed particle insertion starting from E18 is likely to reflect the process of transition to the mature state of the blood-brain barrier, which is characterized by high complexity of TJs and predominance of P-face association of TJ particles and correlated tightly with previous physiological measurements, e.g. transendothelial electrical resistance. Two populations of TJs differing in TJ particle density were distinguishable at E15 and E18, which indicates a non-linear asynchronous mechanism of TJ assembly. At E13, particle-free membrane specializations arranged in a TJ-like pattern strongly resembled TJ specific grooves and ridges. Similar results were obtained from cultures of brain endothelial cells in the presence of low calcium conditions, which suggests the involvement of the cadherin/catenin complex in TJ regulation. The particle-free ‘TJ precursors’ strongly indicate an established TJ associated cytoskeletal network before the TJ particles are present in their intra-junctional location.

Development of blood-brain barrier tight junctions in the rat cortex

The structural equivalent of the blood-brain barrier are the complex tight junctions (TJs) between endothelial cells of brain capillaries. In this study, we have quantitatively investigated by the freeze-fracture technique the modulation of the fine structure of TJs in blood-brain barrier endothelial cells during development of the rat cerebral cortex. The complexity of the TJ network as defined by fractal dimension, the integrity of TJ strands and the degree of TJ particle association to the protoplasmic leaflet of the membrane bilayer in percent of total TJ length were evaluated at embryonic days (E) 13, 15, 18, postnatal day (P) 1 and adult. We observed that the overall complexity of the TJ network and P-face association of TJ particles are significantly increased between E18 and P1. The increase in both of these TJ parameters in combination with the completed particle insertion starting from E18 is likely to reflect the process of transition to the mature state of the blood-brain barrier, which is characterized by high complexity of TJs and predominance of P-face association of TJ particles and correlated tightly with previous physiological measurements, e.g. transendothelial electrical resistance. Two populations of TJs differing in TJ particle density were distinguishable at E15 and E18, which indicates a non-linear asynchronous mechanism of TJ assembly. At E13, particle-free membrane specializations arranged in a TJ-like pattern strongly resembled TJ specific grooves and ridges. Similar results were obtained from cultures of brain endothelial cells in the presence of low calcium conditions, which suggests the involvement of the cadherin/catenin complex in TJ regulation. The particle-free ‘TJ precursors’ strongly indicate an established TJ associated cytoskeletal network before the TJ particles are present in their intra-junctional location.

Occludin is a functional component of the tight junction.

Occludin's role in mammalian tight junction activity was examined by 'labeling' the occludin pool with immunologically detectable chick occludin. This was accomplished by first transfecting MDCK cell with the Lac repressor gene. HygR clones were then transfected with chick occludin cDNA inserted into a Lac operator construct. The resulting HygR/NeoR clones were plated on porous inserts and allowed to form tight junctions. Once steady state transepithelial electrical resistance was achieved, isopropyl- beta-D-thiogalactoside was added to induce chick occludin expression. Confocal laser scanning microscopy of monolayers immunolabeled with Oc-2 monoclonal antibody revealed that chick occludin localized precisely to the preformed tight junctions. When sparse cultures were maintained in low Ca2+ medium, chick occludin and canine ZO-1 co-localized to punctate sites in the cytoplasm suggesting their association within the same vesicular structures. In low calcium medium both proteins also co-localized to contact sites between occasional cell pairs, where a prominent bar was formed at the plasma membrane. Chick occludin was detectable by western blot within two hours of adding isopropyl- beta-D-thiogalactoside to monolayers that had previously achieved steady state transepithelial electrical resistance; this coincided with focal immunofluorescence staining for chick occludin at the cell membrane of some cells. A gradual rise in transepithelial electrical resistance, above control steady state values, began five hours after addition of the inducing agent reaching new steady state values, which were 30-40% above baseline, 31 hours later. Upon removal of isopropyl- beta-D-thiogalactoside chick occludin expression declined slowly until it was no longer detected in western blots 72 hours later; transepithelial electrical resistance also returned to baseline values during this time. While densitometric analysis of western blots indicated that the presence of chick occludin had no detectable effect on E-cadherin or ZO-1 expression, the possibility cannot be excluded that ZO-1 might be a limiting factor in the expression of chick occludin at the cell surface. To test whether expression of chick occludin affected the process of tight junction assembly, monolayers in low Ca2+ medium were treated with isopropyl- beta-D-thiogalactoside for 24 or 48 hours, before Ca2+ was added to stimulate tight junction assembly. Chick occludin did not alter the rate at which transepithelial electrical resistance developed, however, steady state values were 30-40% above control monolayers not supplemented with the inducing agent. By freeze fracture analysis, the number of parallel tight junction strands shifted from a mode of three in controls to four strands in cells expressing chick occludin and the mean width of the tight junction network increased from 175 +/- 11 nm to 248 +/- 16 nm. Two days after plating confluent monolayers that were induced to express chick occludin, mannitol flux was reduced to a variable degree relative to control monolayers. With continued incubation with the inducing agent, mannitol flux increased on day 11 to 50%, and TER rose to 45% above controls. Both of these changes were reversible upon removal of isopropyl- beta-D-thiogalactoside. These data are consistent with the notion that occludin contributes to the electrical barrier function of the tight junction and possibly to the formation of aqueous pores within tight junction strands.

Intestinal paracellular permeability during malnutrition in guinea pigs: effect of high dietary zinc.

Zinc has been shown to have beneficial effects in vitro on epithelial barrier function, and in vivo to reduce intestinal permeability in malnourished children with diarrhoea. To determine whether malnutrition alters intestinal paracellular permeability, and whether zinc prevents such alterations. Guinea pigs were fed a normal protein diet (NP group), a low protein diet (LP group), or a low protein diet enriched with 1800 ppm zinc (LPZn group) for three weeks. Intestinal permeability was measured on jejunal segments mounted in Ussing chambers by measuring ionic conductance and mucosal to serosal fluxes of 14C-mannitol, 22Na, and horseradish peroxidase. Tight junction morphology was assessed on cryofracture replicas. Mannitol and Na fluxes and ionic conductance increased in the LP group compared with the NP group but remained normal in the LPZn group. Accordingly, jejunal epithelia from the LP group, but not from the LPZn group, showed a small decrease in number of tight junctional strands compared with epithelia from the NP group. Neither malnutrition nor zinc treatment modified horseradish peroxidase fluxes. Malnutrition is associated with increased intestinal paracellular permeability to small molecules, and pharmacological doses of zinc prevent such functional abnormality.

Modulation of the junctional integrity by low or high concentrations of cytochalasin B and dihydrocytochalasin B is associated with distinct changes in F-actin and ZO-1.

In a study of Necturus gallbladder epithelium Benzel et al. (Benzel et al., 1980) found that low (0.2-1.2 microM) and higher concentrations (1.5 microM and more) of cytochalasin B (CB) caused an increase and decrease in the transepithelial electrical resistance (TER), respectively. Moreover, there were slight changes in the height and complexicity of tight junction (TJ) strands, as visualized by freeze-fracture and freeze-etching. To elucidate the mechanisms of these findings, we first demonstrated that the effect is also present in monolayers of Madin-Darby Canine Kidney strain 1 (MDCK-1) cells. Thus, a low concentration (0.1 ng/ml) cytochalasin B (CB) strengthened the permeability barrier, as evidenced quantitatively by increases in TER on transepithelial electrical measurements. Furthermore, indirect immunofluorescence and confocal microscopy demonstrated that this effect was paralleled with an accumulation of F-actin and the tight junction marker protein, ZO-1, at the level of TJ. Equimolar concentrations of dihydrocytochalasin B (dhCB), on the other hand, did not lead to a tightening of the epithelium. Confirming previous studies, there was a general decrease in epithelial resistance after treatment with high concentrations (1 microgram/ml) of CB and dhCB, which was accompanied by distinct changes in the F-actin network and distribution of ZO-1. We speculate that the divergent effects of CB and dhCB on the F-actin and ZO-1 organization might be due to specific effects on the transport of monosaccharides across the plasma membrane, or that CB and dhCB in distinct ways involve the turnover of phosphatidylinositols in the membrane, thereby modulating junctional permeability and F-actin structure.

THE INTER-SERTOLI CELL TIGHT JUNCTIONS IN GERM CELL-FREE SEMINIFEROUS TUBULES FROM PRENATALLY IRRADIATED RATS: A FREEZE-FRACTURE STUDY

The tight junctions between Sertoli cells were examined by freeze-fracture in 3-month-old prenatally irradiated rats, whose seminiferous tubules are devoid of germ cells. The replicas from irradiated tubules show elaborate interdigitations of the lateral membranes of Sertoli cells and very extensive tight junctions. These junctions are characterized by a great number of continuous parallel or complex interweaving strands of intramembranous particles, preferentially associated with E fracture faces. The presence of highly cross-linked tight junctional strands is compatible with an epithelium deprived of germ cells, with a reduced need for flexibility. Anomalous ectoplasmic specializations, consisting of groups of cisternae arranged perpendicularly to the lateral surface, are found in the irradiated tubules. These structures may be involved in a storage mechanism of redundant lateral membrane resulting from the elimination of germ cells. Typical gap junctions, intercalated between the tight junctional strands, are larger and more frequently found in treated animals than in controls. These findings indicate that a very tight permeability barrier seems to be established in the irradiated testis even in the absence of germ cells. Thus, the formation and maintenance of Sertoli tight junctions do not appear to be directly dependent on the presence of germ cells. Nevertheless, the alterations detected in the tight junction architecture and in the ectoplasmic specializations indicate that maturing germ cells probably contribute to the functional organization of the blood–testis barrier in the normal testis.

HOCl effects on the tight junctions of rabbit tracheal epithelium

We previously found that HOCl, produced from neutrophil products in infected airways, decreases electrical resistance (R) of rabbit tracheal epithelium. Interestingly, HOCl at 6 mM, a reasonable concentration in diseased airways, decreased R without apparent cell damage. This study sought to determine whether this noncytotoxic dose of HOCl causes morphological changes that correlate with the decrease in R. Excised rabbit tracheas were treated with 6 mM HOCl for 15 min, before and after which epithelial R was determined. Epithelial tissue was then fixed and prepared for transmission electron microscopy, immunofluorescent labeling of F-actin or the tight junction protein ZO-1, or freeze fracture to examine tight junction strands. HOCl treatment caused a 50% decrease in R. Electron micrographs showed no cell, cell membrane, or tight junction changes. By laser confocal microscopy, 6 mM HOCl did not affect the distribution of F-actin or ZO-1. However, morphometric analysis of freeze-fracture replicas showed that tight junction strand number was significantly decreased from 7.06 +/- 0.09 to 4.79 +/- 0.11 and junctional width was significantly decreased from 0.306 +/- 0.007 to 0.214 +/- 0.006 microns. These latter changes may have contributed to the observed decrease in epithelial R.

Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Application to the immunogold labeling of intercellular junctional complexes.

We propose a new electron microscopic method, the sodium dodecylsulphate (SDS)-digested freeze-fracture replica labeling technique, to study the two-dimensional distribution of integral membrane proteins in cellular membranes. Unfixed tissue slices were frozen with liquid helium, freeze-fractured, and replicated in a platinum/carbon evaporator. They were digested with 2.5% SDS to solubilize unfractured membranes and cytoplasm. While the detergent dissolved unfractured membranes and cytoplasm, it did not extract fractured membrane halves. After SDS-digestion, the platinum/carbon replicas, along with attached cytoplasmic and exoplasmic membrane halves, were processed for cytochemical labeling, followed by electron microscopic observation. As an initial screening, we applied this technique to the immunogold labeling of intercellular junction proteins: connexins (gap junction proteins), occludin (tight junction protein), desmoglein (desmosome protein), and E-cadherin (adherens junction protein). The immunogold labeling was seen superimposed on the image of a fracture face visualized by platinum/carbon shadowing. The immunoreaction was specific, and only the structures where the proteins were expected were labeled. For instance, anti-occludin immunogold complexes were observed immediately adjacent to the tight junction strands on the protoplasmic and exoplasmic fracture faces. No significant levels of gold label were associated with non-tight-junctional regions of plasma membranes. The procedures of the SDS-digested freeze-fracture replica labeling and its potential significance are discussed.

Linear gap and tight junctional assemblies between capillary endothelial cells in the eel rete mirabile.

Interendothelial tight junctions and gap junctions have been described in large blood vessels and in cultures of endothelium derived from large blood vessels. Transfer of microinjected small-molecular weight tracers between adjacent endothelial cells also has been demonstrated indicating the presence of gap junctional interendothelial communication. Similar transfer of tracers is evident between microvessel endothelial cells in culture and in microvessels in situ. However, gap junctions have not been detectable by electron microscopy of intact capillary systems. This may be due to limited sampling available in diffuse capillary systems and a small area of overlap between adjacent endothelial membranes. Thin slices of the parallel, tightly packed capillary bed of the eel rete mirabile were cryofixed and prepared for conventional TEM by freeze-substitution. Other samples were freeze-fractured and replicated for examination of endothelial junctional components. A novel tight-gap junctional complex between rete capillary endothelial cells is described. In freeze-fracture replicas of the membrane P face, rows of gap junction subunits are flanked on either side by linear depressions representing grooves previously occupied by tight junctional strands that partition to the E face. In thin sections, the junctions appear in profile as short lengths of closely apposed membranes characteristic of gap junctions. The tight junctional components imply a barrier to paracellular transport across the capillary wall between the endothelial cells. The gap junctional component may provide a mechanism for communication between endothelial cells along the length of the vessel wall.

Immunogold localization of actin in the testis and exocrine pancreas: spatial relationship with tight junctional strands.

The fracture-label technique was used in conjunction with a monoclonal antibody to actin and the phospholipase A2-colloidal gold (PLA2-CG) method to examine the spatial distribution of actin filaments in relation to the three-dimensional arrangement of tight junctional strands in rat testes and exocrine pancreatic acinar cells. The intimate association of actin filaments with tight junctional strands in the pancreas and testis was also illustrated by a double-labeling experiment in which freeze-fractured pancreas or testis was labeled with monoclonal antibody-protein A-gold (30 nm gold size) followed by incubation with a PLA2-CG complex (11 nm gold size). Freeze-fracture-exposed tight junctional strands in both testicular and exocrine pancreatic cells labeled by PLA2-CG complex, indicated the presence of phospholipids in these cylindrical membranous structures. Immunolabeling of freeze-fractured testes with a monoclonal antibody to actin revealed a narrow band of gold particles juxtaposed to the cytoplasmic aspect of the protoplasmic membrane halves decorated with parallel linear arrays of cylindrical tight junctional strands. Many of the gold particles representing actin antigenic sites were in direct contact with the cross-fractured tight junctional strands. Fracture-label preparations of exocrine pancreas labeled with the monoclonal anti-actin antibody also exhibited a similar labeling pattern at the apex of acinars cells where the tight junction complex is located. Double-labeling experiments revealed the simultaneous labeling of actin and phospholipids in the same fracture-label preparations. Digestion of testicular and pancreatic tissue samples in a free PLA2 solution prior to labeling with the monoclonal antibody or PLA2-CG complex removed not only the gold labeling previously seen over the tight junctional strands but also reduced drastically the immunolabeling for actin that was previously seen associated with the tight junction complex. Taken together, results of the present study showed that actin filaments are structural components of the tight junction strands and are connected to the cytoplasmic aspect of the latter structures. The interaction between this particular cytoskeletal element and the tight junction may be through the binding of a special domain of the actin filament to the phospholipids that partially make up the tight junctional complex.