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

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, fluores...

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Bibliographic Details
Published in:Molecular biology of the cell 2017-02, Vol.28 (4), p.524-534
Main Authors: Van Itallie, Christina M, Tietgens, Amber Jean, Anderson, James M
Format: Article
Language:English
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
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Summary: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.
ISSN:1059-1524
1939-4586
DOI:10.1091/mbc.e16-10-0698