Zonula occludens-1 function in the assembly of tight junctions in Madin-Darby canine kidney epithelial cells

Mol Biol Cell. 2006 Apr;17(4):1922-32. doi: 10.1091/mbc.e05-07-0650. Epub 2006 Jan 25.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Bacterial Proteins / analysis
  • Bacterial Proteins / genetics
  • Calcium / metabolism
  • Dogs
  • Epithelial Cells / metabolism*
  • Genetic Complementation Test
  • Kidney / cytology
  • Luminescent Proteins / analysis
  • Luminescent Proteins / genetics
  • Membrane Proteins / antagonists & inhibitors
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Membrane Proteins / physiology*
  • Mice
  • Mutation
  • Occludin
  • Phosphoproteins / antagonists & inhibitors
  • Phosphoproteins / genetics
  • Phosphoproteins / physiology*
  • Point Mutation
  • Protein Structure, Tertiary / genetics
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / pharmacology
  • Tight Junctions / drug effects
  • Tight Junctions / genetics*
  • Tight Junctions / metabolism
  • Zonula Occludens-1 Protein
  • Zonula Occludens-2 Protein


  • Bacterial Proteins
  • Luminescent Proteins
  • Membrane Proteins
  • Occludin
  • Ocln protein, mouse
  • Phosphoproteins
  • RNA, Small Interfering
  • Tjp1 protein, mouse
  • Tjp2 protein, mouse
  • Zonula Occludens-1 Protein
  • Zonula Occludens-2 Protein
  • yellow fluorescent protein, Bacteria
  • Calcium