ZO-1 stabilizes the tight junction solute barrier through coupling to the perijunctional cytoskeleton.
ZO-1 binds numerous transmembrane and cytoplasmic proteins and is required for assembly of both adherens and tight junctions, but its role in defining barrier properties of an established tight junction is unknown. We depleted ZO-1 in MDCK cells using siRNA methods and observed specific defects in the barrier for large solutes, even though flux through the small claudin pores was unaffected. This permeability increase was accompanied by morphological alterations and reorganization of apical actin and myosin. The permeability defect, and to a lesser extent morphological changes, could be rescued by reexpression of either full-length ZO-1 or an N-terminal construct containing the PDZ, SH3, and GUK domains. ZO-2 knockdown did not replicate either the permeability or morphological phenotypes seen in the ZO-1 knockdown, suggesting that ZO-1 and -2 are not functionally redundant for these functions. Wild-type and knockdown MDCK cells had differing physiological and morphological responses to pharmacologic interventions targeting myosin activity. Use of the ROCK inhibitor Y27632 or myosin inhibitor blebbistatin increased TER in wild-type cells, whereas ZO-1 knockdown monolayers were either unaffected or changed in the opposite direction; paracellular flux and myosin localization were also differentially affected. These studies are the first direct evidence that ZO-1 limits solute permeability in established tight junctions, perhaps by forming a stabilizing link between the barrier and perijunctional actomyosin.
Van Itallie
Christina M
CM
Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. vitallie@med.unc.edu
Fanning
Alan S
AS
Bridges
Arlene
A
Anderson
James M
JM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002463:Cell Membrane Permeability; D048430:Cell Shape; D015638:Cytochalasin D / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D004285:Dogs; D055785:Gene Knockdown Techniques; D006576:Heterocyclic Compounds, 4 or More Rings / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D019384:Nucleic Acid Synthesis Inhibitors / Q000378:metabolism; D010641:Phenotype; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D019108:Tight Junctions / Q000378:metabolism*; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D006576:Heterocyclic Compounds, 4 or More Rings; D008565:Membrane Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D010750:Phosphoproteins; D034741:RNA, Small Interfering; C569042:TJP1 protein, human; C569046:TJP2 protein, human; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; C472645:blebbistatin; D015638:Cytochalasin D; D054460:rho-Associated Kinases; D002118:Calcium