Zonula occludens-1 function in the assembly of tight junctions in Madin-Darby canine kidney epithelial cells.
Zonula occludens (ZO)-1 was the first tight junction protein to be cloned and has been implicated as an important scaffold protein. It contains multiple domains that bind a diverse set of junction proteins. However, the molecular functions of ZO-1 and related proteins such as ZO-2 and ZO-3 have remained unclear. We now show that gene silencing of ZO-1 causes a delay of approximately 3 h in tight junction formation in Madin-Darby canine kidney (MDCK) epithelial cells, but mature junctions seem functionally normal even in the continuing absence of ZO-1. Depletion of ZO-2, cingulin, or occludin, proteins that can interact with ZO-1, had no discernible effects on tight junctions. Rescue of junction assembly using murine ZO-1 mutants demonstrated that the ZO-1 C terminus is neither necessary nor sufficient for normal assembly. Moreover, mutation of the PDZ1 domain did not block rescue. However, point mutations in the Src homology 3 (SH3) domain almost completely prevented rescue. Surprisingly, the isolated SH3 domain of ZO-1 could also rescue junction assembly. These data reveal an unexpected function for the SH3 domain of ZO-1 in regulating tight junction assembly in epithelial cells and show that cingulin, occludin, or ZO-2 are not limiting for junction assembly in MDCK monolayers.
McNeil
Elizabeth
E
Center for Cell Signaling, Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908-0577.
Capaldo
Christopher T
CT
Macara
Ian G
IG
D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism*; D005816:Genetic Complementation Test; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D009154:Mutation; D062793:Occludin; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D017354:Point Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D019108:Tight Junctions / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D062793:Occludin; C568986:Ocln protein, mouse; D010750:Phosphoproteins; D034741:RNA, Small Interfering; C569043:Tjp1 protein, mouse; C569047:Tjp2 protein, mouse; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; C054562:yellow fluorescent protein, Bacteria; D002118:Calcium