A Highlights from MBoC Selection: A complex of ZO-1 and the BAR-domain protein TOCA-1 regulates actin assembly at the tight junction |
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| Authors: | Christina M. Van Itallie Amber Jean Tietgens Evan Krystofiak Bechara Kachar James M. Anderson |
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| Affiliation: | University of Queensland;aLaboratory of Tight Junction Structure and Function, National Heart, Lung, and Blood Institute, Bethesda, MD 20892;bLaboratory of Cell Structure and Dynamics, National Institute of Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892 |
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| 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. |
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