Visualizing the dynamic coupling of claudin strands to the actin cytoskeleton through ZO-1

• Published in: Molecular biology of the cell Vol. 28; no. 4; pp. 524 - 534
• Main Authors: Van Itallie, Christina M, Tietgens, Amber Jean, Anderson, James M
• Format: Journal Article
• Language: English
• Published: United States The American Society for Cell Biology 15-02-2017
• Series: A Highlights from
• Subjects: Actin Cytoskeleton - metabolism; Actins - metabolism; Animals; Cell Line; Claudin-1 - metabolism; Claudins - metabolism; Cytoskeletal Proteins - metabolism; Cytoskeleton - physiology; HEK293 Cells; Humans; Membrane Proteins - metabolism; Molecular Imaging - methods; Occludin - metabolism; Phosphoproteins - metabolism; Rats; Tight Junctions - metabolism; Zonula Occludens-1 Protein - metabolism; Zonula Occludens-1 Protein - physiology
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.e16-10-0698

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.