Tight Junction Strand Formation Research Articles

A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts.

Three integral membrane proteins, clau- din-1, -2, and occludin, are known to be components of tight junction (TJ) strands. To examine their ability to form TJ strands, their cDNAs were introduced into mouse L fibroblasts lacking TJs. Immunofluorescence microscopy revealed that both FLAG-tagged claudin-1 and -2 were highly concentrated at cell contact sites as planes through a homophilic interaction. In freeze-fracture replicas of these contact sites, well-developed networks of strands were identified that were similar to TJ strand networks in situ and were specifically labeled with anti-FLAG mAb. In glutaraldehyde-fixed samples, claudin-1-induced strands were largely associated with the protoplasmic (P) face as mostly continuous structures, whereas claudin-2-induced strands were discontinuous at the P face with complementary grooves at the extracellular (E) face which were occupied by chains of particles. Although occludin was also concentrated at cell contact sites as dots through its homophilic interaction, freeze-fracture replicas identified only a small number of short strands that were labeled with anti-occludin mAb. However, when occludin was cotransfected with claudin-1, it was concentrated at cell contact sites as planes to be incorporated into well- developed claudin-1-based strands. These findings suggested that claudin-1 and -2 are mainly responsible for TJ strand formation, and that occludin is an accessory protein in some function of TJ strands.

Effect of proteases and other treatments on the proliferative assembly of tight junction strands in the rat prostate tissue

As already observed with the freeze-fracture technique by Kachar and Pinto da Silva (1981) incubation of excised rat ventral prostate tissue at 37 degrees C results in proliferation of tight junction strands over the lateral membranes of the epithelial cells, an effect not occurring after incubation at 4 degrees C. Our investigations have shown that a pretreatment with protease inhibitor Contrykal at 4 degrees C suppresses the proliferation of tight junction strands at 37 degrees C. On the other hand the added proteases trypsin and pronase induce the proliferation already at 4 degrees C. The effect was lowered by higher concentrations of pronase. Incubation with solutions of the non-ionic detergent Triton X-100 at 4 degrees C could also induce tight junction proliferation, whereas this effect was nearly absent after an incubation at 4 degrees C with a hyperosmotic solution of arabinose and with 50% acetone. No relation was found between proliferation of tight junction strands and cluster formation of intramembraneous particles. In conclusion of the results a small protein or a peptide resembling fusions proteins is assumed as trigger for formation of tight junction strands, acting by destabilization of the bilayer arrangement of membrane lipids.