ASSEMBLY OF THE TIGHT JUNCTION - THE ROLE OF DIACYLGLYCEROL

Extracellular Ca2+ triggers assembly and sealing of tight junctions (T Js) in MDCK cells. These events are modulated by G-proteins, phospholi pase 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 junctiona l fibrils observed in freeze-fracture replicas, decreased permeability of the intercellular space to [H-3]mannitol, and reorganization of ac tin filaments to the cell periphery, visualized by fluorescence micros copy 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 f ormation normally induced by adding Ca2+. diC8 counteracted this inhib ition, suggesting that PKC may be in the signaling pathway activated b y E-cadherin-mediated cell-cell adhesion. In addition, we found a nove l phosphoprotein of 130 kD which coimmunoprecipitated with the ZO-1/ZO -2 complex. Although the assembly and sealing of TJs may involve the a ctivation 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 transl ocation and assembly of preformed TJ subcomplexes.