Kinesin Is a Candidate for Cross-bridging Microtubules and Intermediate Filaments
SELECTIVE BINDING OF KINESIN TO DETYROSINATED TUBULIN AND VIMENTIN*
- From the Departments of ‡Anatomy and Cell Biology and¶Pathology, Columbia University, New York, New York 10032
Abstract
We showed previously that stable, detyrosinated (Glu) microtubules function to localize vimentin intermediate filaments in fibroblasts (Gurland, G., and Gundersen, G. G. (1995)J. Cell Biol. 131, 1275–1290). To identify candidate proteins that mediate the Glu microtubule-vimentin interaction, we incubated microtubules with microtubule-interacting proteins and saturating levels of antibodies to Glu or tyrosinated (Tyr) tubulin. Antibodies to Glu tubulin prevented the microtubule binding of kinesin obtained from fibroblast or brain extracts more effectively than antibodies to Tyr tubulin. Scatchard plot analysis showed that kinesin heads bound to Glu microtubules with an ∼2.8-fold higher affinity than to Tyr microtubules. Purified brain kinesin cosedimented with vimentin, but not with neurofilaments, indicating that kinesin specifically associates with vimentin without accessory molecules. Kinesin binding to vimentin was not sensitive to ATP, and kinesin heads failed to bind to vimentin. By SDS-polyacrylamide gel electrophoresis, a kinesin heavy chain of ∼120 kDa and a light chain of ∼64 kDa were detected in vimentin/kinesin pellets. The light chain reacted with a general kinesin light chain antibody, but not with two other antibodies that recognize the two known isoforms of kinesin light chain in brain, suggesting that the kinesin involved in binding to vimentin may be a specific one. These results demonstrate a kinesin-based mechanism for the preferential interaction of vimentin with detyrosinated microtubules.
Footnotes
-
↵* This work was supported in part by National Institutes of Health Grant GM 42026 (to G. G. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵§ Supported by National Institutes of Health Training Grant NIAAC 00189.
-
↵‖ To whom correspondence should be addressed: Dept. of Anatomy and Cell Biology, Columbia University, 630 W. 168th St., New York, NY 10032. Tel.: 212-305-3708; Fax: 212-305-3970.
-
↵1 The abbreviations used are: MTs, microtubules; Glu MTs, detyrosinated microtubules; Tyr MTs, tyrosinated microtubules; IF, intermediate filament; mAb, monoclonal antibody; Pipes, 1,4-piperazinediethanesulfonic acid; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; PAGE, polyacrylamide gel electrophoresis; MAP, microtubule-associated protein.
-
↵2 G. Kreitzer and G. G. Gundersen, submitted for publication.
-
↵3 G. Liao and G. G. Gundersen, unpublished observations.
-
- Received December 2, 1997.
- Revision received February 10, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
