Assembly of the tight junction: the role of diacylglycerol.
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.
Balda
M S
MS
Department of Internal Medicine, Yale University, School of Medicine, New Haven, Connecticut 06510.
Gonzalez-Mariscal
L
L
Matter
K
K
Cereijido
M
M
Anderson
J M
JM
D000199:Actins / Q000032:analysis / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000502:physiology; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D004075:Diglycerides / Q000494:pharmacology / Q000502:physiology*; D004285:Dogs; D005614:Freeze Fracturing; D019204:GTP-Binding Proteins / Q000502:physiology; D007365:Intercellular Junctions / Q000378:metabolism* / Q000648:ultrastructure; D007668:Kidney / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D008353:Mannitol / Q000493:pharmacokinetics; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000502:physiology; D010738:Type C Phospholipases / Q000494:pharmacology; D062826:Zonula Occludens-1 Protein
D000199:Actins; D002147:Calmodulin; D004075:Diglycerides; D008565:Membrane Proteins; D010750:Phosphoproteins; D062826:Zonula Occludens-1 Protein; C044295:1,2-dioctanoylglycerol; D008353:Mannitol; D011493:Protein Kinase C; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium