Focal Tight Junction Research Articles

Campylobacter fetus impairs barrier function in HT-29/B6 cells through focal tight junction alterations and leaks.

Infections by Campylobacter species are the most common foodborne zoonotic disease worldwide. Campylobacter jejuni and C. coli are isolated most frequently from human stool samples, but severe infections by C. fetus (Cf), which can cause gastroenteritis, septicemia, and abortion, are also found. This study aims at the characterization of pathological changes in Cf infection using an intestinal epithelial cell model. The Cf-induced epithelial barrier defects appeared earlier than those of avian Campylobacter species like C. jejuni/C. coli. Two-path impedance spectroscopy (2PI) distinguished transcellular and paracellular resistance contributions to the overall epithelial barrier impairment. Both transcellular and paracellular resistance of Cf-infected HT-29/B6 monolayers were reduced. The latter was attributed to activation of active anion secretion. Western blot analysis showed no decrease in tight junction (TJ) protein expression (claudin-1, -2, -3, and -4) but showed redistribution of claudin-1 off the TJ domain. In addition, Cf induced epithelial cell death, cell detachment, and lesions (focal leaks), as the result of which macromolecule flux (10-kDa dextran) was increased in Cf-invaded cell monolayers. In conclusion, barrier dysfunction from Cf infection was due to TJ protein redistribution, cell death induction, and leak formation, resulting in bacterial translocation, ion leak flux, and antigen uptake (leaky gut).

Electron microscope observations on the formation of primitive villi in rat small intestine with special reference to intercellular junctions.

Formation and fusion of intraepithelial cavities have long been considered an essential process in the histogenesis of the intestinal mucosa. By electron microscope observation of thin sections and freeze-fracture replicas of the small intestine of rat fetuses, we first demonstrated the initial steps in the formation of intraepithelial cavities: A focal tight junction (macula occludens) was formed in the abluminal part of the epithelium, after which the membrane of an intracellular cavity was fused with that of the focal tight junction to form an intercellular (intraepithelial) cavity enclosed by a zonula occludens. The present study also revealed that gap junctions appeared and enlarged simultaneously with the formation of primitive villi and differentiation of absorptive cells. These gap junctions gradually came to be confined in the epithelium of intervillous regions where proliferation and differentiation of epithelial cells took place. Absorptive cells in villi rarely had gap junctions. These results suggest that tight and gap junctions play important roles in the histogenesis of the intestinal mucosa, and in the proliferation and differentiation of epithelial cells.

Histogenesis of the mucosa of the descending colon in mouse fetuses.

The present study clarify intraepithelial events occurring during the formation of intestinal crypts in the descending colon. In early stage, the mucosa is lined with a simple columnar epithelium consisting of undifferentiated cells which are mutually joined by the junctional complex surrounding the intestinal lumen. The epithelium becomes thickened and stratified, and secondary junctional complexes are formed in the abluminal part of the epithelium. An intracellular cavity opens to the focal tight junction of the secondary junctional complex to form an intraepithelial (intercellular) cavity surrounded by the tight junction. The intraepithelial cavity grows by the fusion of intracellular cavities and vesicles as well as by the division of surrounding cells. Intraepithelial cavities fuse with each other and with the main intestinal lumen. This, together with mesenchymal invasion, results in the formation of intestinal crypts lined with a simple columnar epithelium, where various cell types begin to differentiate. These processes seem essentially similar to those occurring in the developing small intestine except that villi, not crypts, are formed in the small intestine. It is suggested that the formation site and elongation of the tight junction provide for the location and fusion of intraepithelial cavities and eventually result in the formation of villi or crypts, alternatively.

Ultrastructural comparative study on lymphatic capillaries of the subendocardium, myocardium, and subepicardium of the heart left ventricle

A comparative study, with quantitative analysis, was done on the fine structure of the lymphatic capillaries in the subepicardium, myocardium, and subendocardium of the left ventricle from dog hearts. In the three layers, the vesicular system of lymphatic capillaries was quite similar. More than 63% of the plasmalemmal vesicles are in contact with the abluminal or luminal endothelial membrane of cardiac lymphatic capillaries. Volume density measurements showed that plasmalemmal vesicles occupied between 8.0 and 9.3% of the endothelial cytoplasm. Overlapping and interdigitation contacts were frequent, whereas end to end contacts were scarce. The frequency of open junctions wider than 50 nm was inferior to 1%. The intercellular cleft was often obstructed by a focal tight junction (macula occludens). Intercellular spaces without specialized junctions were more frequent with subepicardial and myocardial lymphatic capillaries as compared with subendocardial vessels. Important quantitative variations of the fine structure were encountered on the abluminal side of the endothelial wall. In the subendocardium, the basement membrane covered 33% of the lymphatic endothelium whereas for the subepicardium and myocardium lymphatic capillaries, the values were reduced significantly to 18% ( P < 0.05) and 10% ( P < 0.001), respectively. An inverse relation was observed between the proportion of endothelium covered by basement membrane and the frequency of abluminal endothelial projections associated with the endothelial wall. Endothelial projections (EP) decreased in the following order: myocardium (19 EP/100 μm), subepicardium (15 EP/100 μm), and subendocardium (10 EP/100 μm). Quantitative morphological variations in the fine structure of lymphatic capillaries are believed to result from physiological (muscular movements, metabolic activity) and morphological (thickness of the connective tissue support surrounding lymphatic capillaries) differences existing between the three cardiac layers.

Tight junction contact events and temporary gap junctions in the sciatic nerve fibres of the chicken during Wallerian degeneration and subsequent regeneration.

Tight and gap junctions are described on the basis of freeze-fractures in normal chicken sciatic nerves as well as during Wallerian degeneration and subsequent regeneration. 1. Small calibre nerve fibres display a fairly continuous tight junction contact zone in the membranes of the mesaxons, paranodal loops and Schmidt-Lanterman incisures. Large fibres with more than 40 lamellae have only focal tight junction contacts in the mesaxonal membranes. 2. With the onset of Wallerian degeneration (days 2-4 post-crush, distal stump) myelinic tight junctions become arranged as maculae composed of one circular or several polygonally oriented strands that are criss-crossed by other tight junctional strands. These maculae are subsequently found in the membranes of cytoplasmic vacuoles of the Schwann cells, indicating an endocytotic mode of uptake. Tight junctions are not found between the 5th and 6th day after crush. 3. During the proliferation phase of the Schwann cells and the arrangement of these cells into Büngner cell bands (2 to 8 days post-crush) gap junctions appear between the Schwann cells of the bands. These junctions then disappear with the onset of remyelination (8 days post-crush). 4. With the onset of remyelination (from the 8th day onwards) short focal tight junctions appear in the membranes of the outer mesaxons. Shortly thereafter, when the sheaths possess 4 to 8 lamellae, tight junctions also appear in the membranes of the inner mesaxons, the paranodal loops and the cytoplasmic inclusions. The characteristic differences of tight junction elaboration in small versus large nerve fibres are re-established after three months of regeneration. The elaborated tight junctions in small and early remyelinating fibres point to a specific function; in small fibres (versus large fibres) the tight junctions might effect a separation of the intramyelinic extracellular space as a single compartment. The tight junction contacts in early remyelinating fibres support the hypothesis that myelin growth occurs within the myelin spiral and not by a free rotation and elongation of the Schwann cell tongues. It is assumed that the gap junctions between the Schwann cells contribute to the co-ordination of the Schwann cell band formation, which is involved in the guidance of sprouting axons.

The development of cell junction during nephrogenesis.

The differentiation and distribution of intercellular junctions especially during the early development stages of the rabbit nephron was studied by freeze-fracture electron microscopy. Metanephrogenic cells were found to be connected by sporadic focal tight junctions. During the formation of the renal vesicle similar tight junctions occurred on the periphery as well as near the developing lumen. These focal tight junction increased in size and coalesced to broad zonulae occludentes lining the vesicular lumen at a later stage. Broad occluding junctions were also observed in the different nephron segments of the S-shaped stage. Ultrastructurally, these early maculae and zonulae occludentes consisted of beaded rows of particles. As development progressed, continuous tight junctions formed, whereas the number of strands decreased with the exception of the distal tubule. In contrast to the parietal glomerular epithelium, the initial occluding zonules of the visceral glomerular cells were gradually reduced to maculae occludentes, and finally disappeared. These results suggest that zonulae occludentes appear synchronously with the establishing lumen; the ultrastructural differentiation of tight junction strands seems to be completed with the onset of glomerular filtration.

The effects of calcium deficiency on the formation of the zonula occludens and blastocoel in the mouse embryo

The mechanism of fluid transport in the developing preimplantation mouse embryo has been studied in vitro by inhibiting zonular tight junction formation. Compaction, the morphogenetic process permitting zonular blastomere adhesions at the 8-cell stage, was suppressed by lowering extracellular calcium (Ca). The Ca threshold required for compaction is 0.04–0.06 m M, and in concentrations above the threshold, the rate of compaction is concentration dependent, whereas the rate of blastocyst formation is not and proceeds normally. At 0.02 m M Ca, both compaction and blastocyst development are completely prevented. Although focal tight and gap junctions are present, zonular tight junctions do not develop. We conclude that Ca is required for the maximization of cell-cell contact, but not for focal tight junction and gap junction formation. When early morulae are cultured in 0.02 m M Ca, small trophoblastic vesicles develop frequently with intracellular fluid vacuoles. If early 8-cell embryos are similarly cultured, cell division continues and many blastomeres acquire small intracellular membrane-bounded vaculoes. These coalesce, the cell volume increases, and the nucleus becomes eccentrically positioned, resulting in a giant vacuolated blastomere reminiscent of a miniaturized blastocyst. We propose that (1) vacuole formation may be an exaggeration of an intermediate intracellular step in fluid transport and (2) normal cell polarity established by zonular tight junctions is required for transcellular fluid transport.

Development of cell-to-cell relationships in the thyroid gland of the chick embryo

Thyroid glands of 7 to 21 day-old chick embryos were examined by electron microscopy using freeze-fracture and thin-section preparations. The primitive follicle lumen first appears between two adjacent epithelial cells in 8 day-old embryos, and is formed in the region of a focal tight junction (macula occludens). The focal tight junction develops into the zonula occludens when the primitive follicle lumen first forms. The zonula occludens is, at first, composed of 4.6 +/- 2.45 strands, but with increasing embryonic age the number of strands increases to 5.9 +/- 1.41 in 13 day-old, and 8.0 +/- 1.75 in 19 day-old embryos. Thyroglobulin stored within the embryonic gland lumina is isolated from the mesenchymal tissue even at the first appearance of these follicle cavities. Well developed gap junctions already occur in the thyroid gland of the 7 day-old embryo, so that an intimate relationship and communication between these cells already exists at the time of their functional differentiation.