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© The Rockefeller University Press,
0021-9525/1999//831 $5.00
The Journal of Cell Biology, Volume 146, Number 4,
, 1999 831-842
Original Article |
Integrating the Actin and Vimentin Cytoskeletons
: Adhesion-Dependent Formation of Fimbrin–Vimentin Complexes in Macrophages
b Department of Biology and Division of Bioengineering and Environmental Health, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142
c Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60208
Whitehead Institute of Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142.(617) 258-6691(617) 258-5206
correia{at}wi.mit.edu
Cells adhere to the substratum through specialized structures that are linked to the actin cytoskeleton. Recent studies report that adhesion also involves the intermediate filament (IF) and microtubule cytoskeletons, although their mechanisms of interaction are unknown. Here we report evidence for a novel adhesion-dependent interaction between components of the actin and IF cytoskeletons. In biochemical fractionation experiments, fimbrin and vimentin coprecipitate from detergent extracts of macrophages using vimentin- or fimbrin-specific antisera. Fluorescence microscopy confirms the biochemical association. Both proteins colocalized to podosomes in the earliest stages of cell adhesion and spreading. The complex is also found in filopodia and retraction fibers. After detergent extraction, fimbrin and vimentin staining of podosomes, filopodia, and retraction fibers are lost, confirming that the complex is localized to these structures. A 1:4 stoichiometry of fimbrin binding to vimentin and a low percentage (1%) of the extracted vimentin suggest that fimbrin interacts with a vimentin subunit. A fimbrin-binding site was identified in the NH2-terminal domain of vimentin and the vimentin binding site at residues 143–188 in the CH1 domain of fimbrin. Based on these observations, we propose that a fimbrin–vimentin complex may be involved in directing the assembly of the vimentin cytoskeleton at cell adhesion sites.
Key Words: fimbrin • vimentin • cytoskeleton • adhesion • microfilaments
© 1999 The Rockefeller University Press
1.used in this paper: ABD, actin-binding domain; CH, calponin homology; F-actin, filamentous actin; GST, glutathione-S-transferase; IF, intermediate filament; MF, microfilament; MT, microtubule; PVDF, polyvinylidene difluoride
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