Occludin and tricellulin facilitate formation of anastomosing tight-junction strand network to improve barrier function

Abstract

Tight junctions (TJs) are composed of a claudin-based anastomosing network of TJ strands, at which plasma membranes of adjacent epithelial cells are closely attached to regulate the paracellular permeability. Although the TJ proteins occludin and tricellulin have been known to be incorporated in the TJ strand network, their molecular functions remain unknown. Here, we established tricellulin/occludin-double knockout (dKO) MDCK II cells using a genome editing technique, and evaluated the structure and barrier function of these cells. In freeze-fracture replica electron microscopy, the TJ strands of tricellulin/occludin-dKO cells had fewer branches and were less anastomosed compared with the controls. The paracellular permeability of ions and small tracers was increased in the dKO cells. A single KO of tricellulin or occludin had limited effects on the morphology and permeability of TJs. Mathematical simulation using a simplified TJ strand network model predicted that reduced cross-links in TJ strands lead to increased permeability of ions and small macromolecules. Furthermore, overexpression of occludin increased the complexity of TJ strand network and strengthened barrier function. Taken together, our data suggest that tricellulin and occludin mediate the formation and/or stabilization of TJ strand branching points, and contribute to the maintenance of epithelial barrier integrity. [Media: see text]

The Epithelial Cell Leak Pathway

“…The absence of ZO-1 and occludin in these aberrant strands might, however, alter the stability of the strands, thereby allowing permeation of large solutes. Consistent with this interpretation, a recent study demonstrated directly that the presence of occludin and tricellulin proteins in claudin-2 knockdown MDCK II cells, which express several other claudins, increased the complexity of the tight junction strands without changing the claudin protein expression pattern [ 79 ]. The cells with the less complex tight junction strand configuration exhibited decreased TER (increased Pore Pathway permeability) and increased permeability to macromolecules (Leak Pathway permeability).…”

“…Occludin: Occludin preferentially, but not exclusively, localizes to strand branch points which appear to be a major locus for strand breaks [ 62 ]. Occludin, along with tricellulin, increase strand complexity in MDCK II cells [ 79 ]. Thus, manipulation of occludin content or organization could modulate Leak Pathway permeability.…”

“…Since NHS-LC-biotin is a linear molecule, it is unclear which permeability pathway this solute would measure. Several groups have reported no effect of occludin knockdown on Leak Pathway permeability in MDCK II cells [ 79 , 130 , 131 , 132 ]. Occludin knockdown in Caco-2 cells was reported to increase Leak Pathway permeability [ 100 , 133 ], although this was later disputed [ 134 ].…”

“…In contrast, and as described above, both occludin and tricellulin appear to have roles in regulating Leak Pathway permeability. In most studies, knockdown of occludin does not alter Leak Pathway permeability [ 79 , 128 , 129 , 130 , 131 , 132 , 134 ]. This may reflect the fact that occludin knockdown or knockout induces a redistribution of tricellulin from the typical tricellular junction localization to bicellular junctions [ 79 , 200 ].…”

“…In most studies, knockdown of occludin does not alter Leak Pathway permeability [ 79 , 128 , 129 , 130 , 131 , 132 , 134 ]. This may reflect the fact that occludin knockdown or knockout induces a redistribution of tricellulin from the typical tricellular junction localization to bicellular junctions [ 79 , 200 ]. In MDCK II cells, which express low levels of tricellulin, tricellulin knockout did not alter Leak Pathway permeability [ 79 ], whereas, in HT-29/B6 cells, which express higher tricellulin levels, tricellulin knockdown increased permeability to 4 kDa and 10 kDa fluorescein-dextrans [ 111 ].…”