©2004 Nature Publishing Group .............................................................. A microtubule-binding myosin required for nuclear anchoring and spindle assembly Kari L. Weber 1 *, Anna M. Sokac 1,2 *, Jonathan S. Berg 3 , Richard E. Cheney 3 & William M. Bement 1,2 1 Department of Zoology, 2 Program in Cellular and Molecular Biology, University of Wisconsin, Madison, Madison, Wisconsin 53706, USA 3 Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, Chapel Hill, North Carolina 27599, USA * Present addresses: Department of Cell Biology, CB163, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA (K.L.W.); Department of Molecular Biology, Princeton University, Washington Road, Princeton, New Jersey 08544, USA (A.M.S.) ............................................................................................................................................................................. Proper spindle positioning and orientation are essential for asymmetric cell division and require microtubule–actin filament (F-actin) interactions in many systems 1,2 . Such interactions are particularly important in meiosis 3 , where they mediate nuclear anchoring 4–6 , as well as meiotic spindle assembly and rotation 7,8 , two processes required for asymmetric cell division. Myosin-10 proteins are phosphoinositide-binding 9 , actin-based motors that contain carboxy-terminal MyTH4 and FERM domains of unknown function 10 . Here we show that Xenopus laevis myo- sin-10 (Myo10) associates with microtubules in vitro and in vivo, and is concentrated at the point where the meiotic spindle contacts the F-actin-rich cortex. Microtubule association is mediated by the MyTH4-FERM domains, which bind directly to purified microtubules. Disruption of Myo10 function disrupts nuclear anchoring, spindle assembly and spindle–F-actin associ- ation. Thus, this myosin has a novel and critically important role during meiosis in integrating the F-actin and microtubule cytoskeletons. Microtubule–F-actin interactions are essential for a broad variety of basic biological processes that require polarized distribution of cellular components 2 . Mitotic spindle positioning in yeast, for example, is critically dependent on physical linkages between microtubules and cortical F-actin 1 . Microtubule–F-actin inter- actions are particularly important in oogenesis, which entails anchoring and translocation of microtubule-based structures within a comparatively large cell volume 3 . For example, the oocyte nucleus (germinal vesicle) is anchored by microtubules that extend from the nucleus to the F-actin-rich cortex 4–6 , whereas proper meiotic spindle assembly and/or targeting to the plasma membrane are F-actin dependent 3,7,8,11 . Further, the site of asymmetric spindle anchoring at the oocyte cortex is overlaid by a cap of high F-actin density in mammals 8,11 , amphibians 7 and invertebrates 12,13 ; this cap is thought to anchor the spindle at the cortex through interaction with microtubules 3,7,8,11 . At present, however, the molecular mecha- nisms that couple microtubules to F-actin during oogenesis are unknown. Xenopus Myo10 was identified in a polymerase chain reaction Figure 1 Myo10 associates with microtubules in vitro and in vivo. a, Domain organization of Myo10. b, Myo10 antibody recognizes single protein of ,230 kDa in egg extract immunoblot. c, Microtubule (P, pellet) and supernatant fractions (S) from egg extracts induced to (MT sample) or prevented from (Nocod sample) polymerizing microtubules (see Methods). Immunoblotting with antibodies to Myo10 (M10), tubulin (Tub) or actin (Actin) shows Myo10 cofractionates with microtubules but not actin filaments. d, Myo10 (M10, blue) immunolocalizes with aster microtubules (MT, red) but much less with F-actin (FA, green) in extracts. Scale bar, 30 mm. e, Myo10 also colocalizes with microtubules in intact eggs (arrowheads). Antibodies the same as in d. Scale bar, 20 mm. letters to nature NATURE | VOL 431 | 16 SEPTEMBER 2004 | www.nature.com/nature 325