title: Visualizing the dynamic coupling of claudin strands to the actin cytoskeleton through ZO-1.
abstract: The organization and integrity of epithelial tight junctions depend on interactions between claudins, ZO scaffolding proteins, and the cytoskeleton. However, although binding between claudins and ZO-1/2/3 and between ZO-1/2/3 and numerous cytoskeletal proteins has been demonstrated in vitro, fluorescence recovery after photobleaching analysis suggests interactions in vivo are likely highly dynamic. Here we use superresolution live-cell imaging in a model fibroblast system to examine relationships between claudins, ZO-1, occludin, and actin. We find that GFP claudins make easily visualized dynamic strand patches between two fibroblasts; strand dynamics is constrained by ZO-1 binding. Claudin association with actin is also dependent on ZO-1, but colocalization demonstrates intermittent rather than continuous association between claudin, ZO-1, and actin. Independent of interaction with ZO-1 or actin, claudin strands break and reanneal; pulse-chase-pulse analysis using SNAP-tagged claudins showed preferential incorporation of newly synthesized claudins into break sites. Although claudin strand behavior in fibroblasts may not fully recapitulate that of epithelial tight junction strands, this is the first direct demonstration of the ability of ZO-1 to stabilize claudin strands. We speculate that intermittent tethering of claudins to actin may allow for accommodation of the paracellular seal to physiological or pathological alterations in cell shape or movement.
authors.affiliation: Laboratory of Tight Junction Structure and Function, National Heart, Lung, and Blood Institute, National Institutes of Health, 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, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Amber Jean
authors.initials: AJ
authors.lastname: Tietgens
authors.affiliation: Laboratory of Tight Junction Structure and Function, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
authors.forename: James M
authors.initials: JM
authors.lastname: Anderson
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D062445:Claudin-1 / Q000378:metabolism; D057167:Claudins / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D057054:Molecular Imaging / Q000379:methods; D062793:Occludin / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D051381:Rats; D019108:Tight Junctions / Q000378:metabolism; D062826:Zonula Occludens-1 Protein / Q000378:metabolism* / Q000502:physiology*
chemical_list: D000199:Actins; D062445:Claudin-1; D057167:Claudins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D062793:Occludin; D010750:Phosphoproteins; D062826:Zonula Occludens-1 Protein