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J. Biol. Chem., Vol. 279, Issue 26, 27591-27598, June 25, 2004

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Two Protein 4.1 Domains Essential for Mitotic Spindle and Aster Microtubule Dynamics and Organization in Vitro^*

Sharon Wald Krauss, Gloria Lee, Joel Anne Chasis, Narla Mohandas¶, and Rebecca Heald||

From the Lawrence Berkeley National Laboratory, Life Sciences Division, University of California, Berkeley, 94720 California, the ^¶New York Blood Center, New York, New York 10021, and the ^||Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

Multifunctional structural proteins belonging to the 4.1 family are components of nuclei, spindles, and centrosomes in vertebrate cells. Here we report that 4.1 is critical for spindle assembly and the formation of centrosome-nucleated and motor-dependent self-organized microtubule asters in metaphase-arrested Xenopus egg extracts. Immunodepletion of 4.1 disrupted microtubule arrays and mislocalized the spindle pole protein NuMA. Remarkably, assembly was completely rescued by supplementation with a recombinant 4.1R isoform. We identified two 4.1 domains critical for its function in microtubule polymerization and organization utilizing dominant negative peptides. The 4.1 spectrin-actin binding domain or NuMA binding C-terminal domain peptides caused morphologically disorganized structures. Control peptides with low homology or variant spectrin-actin binding domain peptides that were incapable of binding actin had no deleterious effects. Unexpectedly, the addition of C-terminal domain peptides with reduced NuMA binding caused severe microtubule destabilization in extracts, dramatically inhibiting aster and spindle assembly and also depolymerizing preformed structures. However, the mutant C-terminal peptides did not directly inhibit or destabilize microtubule polymerization from pure tubulin in a microtubule pelleting assay. Our data showing that 4.1 is a crucial factor for assembly and maintenance of mitotic spindles and self-organized and centrosome-nucleated microtubule asters indicates that 4.1 is involved in regulating both microtubule dynamics and organization. These investigations underscore an important functional context for protein 4.1 in microtubule morphogenesis and highlight a previously unappreciated role for 4.1 in cell division.

Received for publication, March 12, 2004 , and in revised form, April 16, 2004.

^* This work was supported by Grants DK59079 (to S. W. K.), GM057839 (to R. H.), and DK32094 (to S. W. K., J. A. C., and N. M.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: University of California, Lawrence Berkeley National Lab, 1 Cycloton Rd, MS 74-157, Berkeley, CA 94720. Tel.: 510-486-4073; Fax: 510-486-6746; E-mail: sakrauss{at}lbl.gov.

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