Abstract
Tricellulin is an important component of tricellular tight junctions (TJs) and is involved in the formation of tricellular contacts. However, little is known about its regulation during the assembly and disassembly of tricellular TJs. By using the well-differentiated pancreatic cancer cell line HPAC, which highly expresses tricellulin at tricellular contacts, we have investigated changes in the localization, expression and phosphorylation of tricellulin and in its TJ functions as a barrier and fence during the destruction and formation of TJs induced by changes in the extracellular calcium concentration. During both extracellular Ca2+ depletion caused by EGTA treatment and Ca2+ repletion after Ca2+ starvation, the expression of tricellulin increased in whole lysates and in Triton-X-100-insoluble fractions without any change in its mRNA. The increases in immunoreactivity revealed by Western blotting were prevented by alkaline phosphatase treatment. Immunoprecipitation assays showed that tricellulin was phosphorylated on threonine residues when it increased after Ca2+ depletion and repletion. In the early stage after Ca2+ repletion, tricellulin was expressed not only at tricellular contacts but also in the cytoplasm and at bicellular borders. In confocal laser microscopy, tricellulin was observed at the apical-most regions and basolateral membranes of tricellular contacts after Ca2+ repletion. Knockdown of tricellulin delayed the recovery of the barrier and fence functions after Ca2+ repletion. Thus, the dynamic behavior of tricellulin during the destruction and formation of TJs under various extracellular calcium conditions seems to be closely associated with the barrier and fence functions of TJs.Electronic supplementary materialThe online version of this article (doi:10.1007/s00441-012-1512-7) contains supplementary material, which is available to authorized users.
Highlights
Tight junctions are the most apical components of intercellular junctional complexes
When the membranes of tricellular contacts began to detach, gaps formed at these regions from 5 min after EGTA treatment but tricellulin was initially maintained at the membranes around the gap formation (Fig. 1b)
Ca2+ depletion caused by EGTA treatment induced the disruption of tight junctions and displaced the tricellulin from tricellular contacts to bicellular junctions and to the cytoplasm; this was correlated with a significant reduction in Transepithelial electrical resistance (TER) (Figs. 1, 2)
Summary
Tight junctions are the most apical components of intercellular junctional complexes They inhibit solute and water flow through the paracellular space (termed the “barrier” function; Schneeberger and Lynch 1992; Gumbiner 1993). They separate the apical from the basolateral cell surface domains, thereby establishing cell polarity (termed the “fence” function; van Meer and Simons 1986; Cereijido et al 1998). The lipolysis-stimulated lipoprotein receptor (LSR), originally identified and analyzed as a receptor for the uptake of triacylglyceride-rich lipoproteins (Yen et al 1999), has been defined as an integral membrane protein localized at tricellular tight junctions and the cytoplasmic domain of LSR has been reported to be responsible for the recruitment of tricellulin (Masuda et al 2011)
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