The Journal of Cell Biology, Vol 126, 1231-1240, Copyright © 1994 by The Rockefeller University Press

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Characterization of an F-actin-binding domain in the cytoskeletal protein vinculin

AR Menkel, M Kroemker, P Bubeck, M Ronsiek, G Nikolai and BM Jockusch
Cell Biology Group, University of Bielefeld, Germany.

Vinculin, a major structural component of vertebrate cell-cell and cell- matrix adherens junctions, has been found to interact with several other junctional components. In this report, we have identified and characterized a binding site for filamentous actin. These results included studies with gizzard vinculin, its proteolytic head and tail fragments, and recombinant proteins containing various gizzard vinculin sequences fused to the maltose binding protein (MBP) of Escherichia coli. In cosedimentation assays, only the vinculin tail sequence mediated a direct interaction with actin filaments. The binding was saturable, with a dissociation constant value in the micromolar range. Experiments with deletion clones localized the actin-binding domain to a region confined by residues 893-1016 in the 170-residue-long carboxyterminal segment, while the proline-rich hinge connecting the globular head to the rodlike tail was not required for this interaction. In fixed and permeabilized cells (cell models), as well as after microinjection, proteins containing the actin-binding domain specifically decorated stress fibers and the cortical network of fibroblasts and epithelial cells, as well as of brush border type microvilli. These results corroborated the sedimentation experiments. Our data support and extend previous work showing that vinculin binds directly to actin filaments. They are consistent with a model suggesting that in adhesive cells, the NH2-terminal head piece of vinculin directs this molecule to the focal contact sites, while its tail segment causes bundling of the actin filament ends into the characteristic spear tip-shaped structures.
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