title: A complex of ZO-1 and the BAR-domain protein TOCA-1 regulates actin assembly at the tight junction.
abstract: Assembly and sealing of the tight junction barrier are critically dependent on the perijunctional actin cytoskeleton, yet little is known about physical and functional links between barrier-forming proteins and actin. Here we identify a novel functional complex of the junction scaffolding protein ZO-1 and the F-BAR-domain protein TOCA-1. Using MDCK epithelial cells, we show that an alternative splice of TOCA-1 adds a PDZ-binding motif, which binds ZO-1, targeting TOCA-1 to barrier contacts. This isoform of TOCA-1 recruits the actin nucleation-promoting factor N-WASP to tight junctions. CRISPR-Cas9-mediated knockout of TOCA-1 results in increased paracellular flux and delayed recovery in a calcium switch assay. Knockout of TOCA-1 does not alter FRAP kinetics of GFP ZO-1 or occludin, but longer term (12 h) time-lapse microscopy reveals strikingly decreased tight junction membrane contact dynamics in knockout cells compared with controls. Reexpression of TOCA-1 with, but not without, the PDZ-binding motif rescues both altered flux and membrane contact dynamics. Ultrastructural analysis shows actin accumulation at the adherens junction in TOCA-1-knockout cells but unaltered freeze-fracture fibril morphology. Identification of the ZO-1/TOCA-1 complex provides novel insights into the underappreciated dependence of the barrier on the dynamic nature of cell-to-cell contacts and perijunctional actin.
authors.affiliation: Laboratory of Tight Junction Structure and Function, National Heart, Lung, and Blood Institute, Bethesda, MD 20892 Christina.vanitallie@nih.gov.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Van Itallie
authors.affiliation: Laboratory of Tight Junction Structure and Function, National Heart, Lung, and Blood Institute, Bethesda, MD 20892.
authors.forename: Amber Jean
authors.initials: AJ
authors.lastname: Tietgens
authors.affiliation: Laboratory of Cell Structure and Dynamics, National Institute of Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Evan
authors.initials: E
authors.lastname: Krystofiak
authors.affiliation: Laboratory of Cell Structure and Dynamics, National Institute of Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Bechara
authors.initials: B
authors.lastname: Kachar
authors.affiliation: Laboratory of Tight Junction Structure and Function, National Heart, Lung, and Blood Institute, Bethesda, MD 20892.
authors.forename: James M
authors.initials: JM
authors.lastname: Anderson
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D022005:Adherens Junctions / Q000378:metabolism*; D000818:Animals; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004285:Dogs; D055786:Gene Knockout Techniques; D057809:HEK293 Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D017434:Protein Structure, Tertiary; D019108:Tight Junctions / Q000378:metabolism*; D062826:Zonula Occludens-1 Protein / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; C482527:FNBP1L protein, human; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein