Connexin-Occludin Chimeras Containing the ZO-Binding Domain of Occludin Localize at MDCK Tight Junctions and NRK Cell Contacts

• Published in: The Journal of cell biology 1999-08, Vol.146 (3), p.683-693
• Main Authors: Mitic, Laura L., Schneeberger, Eveline E., Fanning, Alan S., Anderson, James Melvin
• Format: Article
• Language: English
• Subjects: Animals; Cell Line; Cell Membrane - enzymology; Cell Membrane - metabolism; Cell Membrane - ultrastructure; Cells; Chimeras; Claudin-1; Connexins; Connexins - genetics; Connexins - metabolism; Dogs; Endothelial cells; Epithelial cells; Fibroblasts - cytology; Fibroblasts - enzymology; Fibroblasts - metabolism; Fibroblasts - ultrastructure; Fluorescent Antibody Technique; Freeze Fracturing; Gap Junction beta-1 Protein; Gap junctions; Gap Junctions - metabolism; Gap Junctions - ultrastructure; Gene Deletion; Guanylate Kinases; Humans; Intercellular junctions; Intercellular Junctions - metabolism; Intercellular Junctions - ultrastructure; Kidney - cytology; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Membranes; Microscopy, Electron; Nucleoside-Phosphate Kinase - metabolism; Occludin; Original; Phosphoproteins - metabolism; Proteins; Rats; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Solar fibrils; Stem cells; Tight junctions; Tight Junctions - metabolism; Tight Junctions - ultrastructure; Transfection; Zonula Occludens-1 Protein
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.146.3.683

Occludin is a transmembrane protein of the tight junction that functions in creating both an intercellular permeability barrier and an intramembrane diffusion barrier. Creation of the barrier requires the precise localization of occludin, and a distinct family of transmembrane proteins called claudins, into continuous linear fibrils visible by freeze-fracture microscopy. Conflicting evidence exists regarding the relative importance of the transmembrane and extracellular versus the cytoplasmic domains in localizing occludin in fibrils. To specifically address whether occludin's COOH-terminal cytoplasmic domain is sufficient to target it into tight junction fibrils, we created chimeras with the transmembrane portions of connexin 32. Despite the gap junction targeting information present in their transmembrane and extracellular domains, these connexin-occludin chimeras localized within fibrils when expressed in MDCK cells, as assessed by immunofluorescence and immunogold freeze-fracture imaging. Localization of chimeras at tight junctions depends on the COOH-terminal ZO-binding domain and not on the membrane proximal domain of occludin. Furthermore, neither endogenous occludin nor claudin is required for targeting to ZO-1-containing cell-cell contacts, since in normal rat kidney fibroblasts targeting of chimeras again required only the ZO-binding domain. These results suggest an important role for cytoplasmic proteins, presumably ZO-1, ZO-2, and ZO-3, in localizing occludin in tight junction fibrils. Such a scaffolding and cytoskeletal coupling function for ZO MAGUKs is analogous to that of other members of the MAGUK family.