A dominant mutant of occludin disrupts tight junction structure and function.
The tight junction is the most apical intercellular junction of epithelial cells and forms a diffusion barrier between individual cells. Occludin is an integral membrane protein specifically associated with the tight junction which may contribute to the function or regulation of this intercellular seal. In order to elucidate the role of occludin at the tight junction, a full length and an N-terminally truncated murine occludin construct, both FLAG-tagged at the N terminus, were stably introduced into the murine epithelial cell line CSG 120/7. Both constructs were correctly targeted to the tight junction, as defined by colocalization with another tight junction protein, ZO-1. The construct lacking the N terminus and extracellular domains of occludin was found to exert a dramatic effect on tight junction integrity. Cell monolayers failed to develop an efficient permeability barrier, as demonstrated by low transcellular electrical resistance values and an increased paracellular flux to small molecular mass tracers. Furthermore, gaps were found to have been induced in the P-face associated tight junction strands, as visualized by freeze-fracture electron microscopy. These findings demonstrate an important role for the N-terminal half of occludin in tight junction assembly and maintaining the barrier function of the tight junction.
Bamforth
S D
SD
Max Planck Institute for Physiological and Clinical Research, Department of Molecular Cell Biology, Germany.
Kniesel
U
U
Wolburg
H
H
Engelhardt
B
B
Risau
W
W
D000818:Animals; D002460:Cell Line; D017097:Electric Impedance; D004847:Epithelial Cells / Q000502:physiology; D005614:Freeze Fracturing; D005799:Genes, Dominant; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods; D062793:Occludin; D012313:RNA / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013329:Structure-Activity Relationship; D019108:Tight Junctions / Q000502:physiology*; D014407:Tumor Cells, Cultured
D008565:Membrane Proteins; D062793:Occludin; C568986:Ocln protein, mouse; D012313:RNA