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J Cell Biol. 1993 October 2; 123(2): 293–302.
PMCID: PMC2119828

Assembly of the tight junction: the role of diacylglycerol

Abstract

Extracellular Ca2+ triggers assembly and sealing of tight junctions (TJs) in MDCK cells. These events are modulated by G-proteins, phospholipase C, protein kinase C (PKC), and calmodulin. In the present work we observed that 1,2-dioctanoylglycerol (diC8) promotes the assembly of TJ in low extracellular Ca2+, as evidenced by translocation of the TJ-associated protein ZO-1 to the plasma membrane, formation of junctional fibrils observed in freeze-fracture replicas, decreased permeability of the intercellular space to [3H]mannitol, and reorganization of actin filaments to the cell periphery, visualized by fluorescence microscopy using rhodamine-phalloidin. In contrast, diC8 in low Ca2+ did not induce redistribution of the Ca-dependent adhesion protein E-cadherin (uvomorulin). Extracellular antibodies to E-cadherin block junction formation normally induced by adding Ca2+. diC8 counteracted this inhibition, suggesting that PKC may be in the signaling pathway activated by E-cadherin-mediated cell-cell adhesion. In addition, we found a novel phosphoprotein of 130 kD which coimmunoprecipitated with the ZO-1/ZO-2 complex. Although the assembly and sealing of TJs may involve the activation of PKC, the level of phosphorylation of ZO-1, ZO-2, and the 130-kD protein did not change after adding Ca2+ or a PKC agonist. The complex of these three proteins was present even in low extracellular Ca2+, suggesting that the addition of Ca2+ or diC8 triggers the translocation and assembly of preformed TJ subcomplexes.

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Selected References

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