Cell, Vol. 102, 411–424, August 18, 2000, Copyright 2000 by Cell PressThe Xenopus Chromokinesin Xkid Is Essentialfor Metaphase Chromosome Alignment and Must BeDegraded to Allow Anaphase Chromosome MovementIn vertebrate cells, at least three microtubule-depen-dent forces influence chromosomemovementandposi-tioning: forces generated at the kinetochore, forces thatmove microtubules toward the spindle poles, and thepolar ejection force (Rieder et al., 1986), which pushesHironori Funabiki*†and Andrew W. Murray†Department of PhysiologyUniversity of California, San FranciscoSan Francisco, California 94143chromosome arms away from the spindle poles (re-viewed in Rieder and Salmon, 1994; Skibbens et al.,Summary1995). In tissue culture cells, individual kinetochores os-cillate between moving toward and away from the polesAt anaphase, the linkage between sister chromatidsto which they are attached (Skibbens et al., 1993; Khod-is dissolved and the separated sisters move towardjakov and Rieder, 1996; Waters et al., 1996). After biori-opposite poles of the spindle. We developed a methodented sister chromatid pairs congress, these move-to purify metaphase and anaphase chromosomes fromments oscillate about the metaphase plate. One modelpostulates that tension on the linkage between akineto-frog egg extracts and identified proteins that leavechore and a spindle pole regulatesthe direction ofchro-chromosomes at anaphase using a new form of ex-mosome movement; the greater the tension at the kinet-pression screening. This approach identified Xkid, aochore the more likely the kinetochore is to move awayXenopus homolog of human Kid (kinesin-like DNAfrom the pole (Salmon, 1989; Murray and Mitchison,binding protein) as a protein that is degraded in ana-1994). If the intensity of the polar ejection force de-phase by ubiquitin-mediated proteolysis. Immunode-creased with distance from the poles, this model couldpleting Xkid from egg extracts prevented normal chro-explain why bioriented chromosomes congress.mosome alignment on the metaphase spindle. AddingSeveralmicrotubule motors play roles inchromosomea mild excess of wild-type or nondegradable Xkid tomovement and positioning (Rieder and Salmon, 1998;egg extracts prevented the separated chromosomesHeald and Walczak, 1999). These include three mole-from moving toward the poles. We propose that Xkidcules found at the kinetochore: CENP-E, a plus-end-provides the metaphase force that pushes chromo-directed, kinesin-like motor (Yen et al., 1991), cyto-some arms toward the equator of the spindle and thatplasmic dynein, a minus-end-directed motor (Pfarr etits destruction is needed for anaphase chromosomeal., 1990; Steur et al., 1990), and XKCM1, a kinesin-likemovement.molecule that stimulates microtubule depolymerization(Desai et al., 1999a). CENP-E is required for metaphasechromosome alignment (Schaar et al., 1997; Wood etIntroductional., 1997), whereas dynein appears to capture the firstmicrotubules that interact with the kinetochore (RiederDuringcell division, interactions between chromosomesand Alexander, 1990; Skibbens et al., 1993). Chromo-and microtubules segregate the chromosomes into twosome bound motors (chromokinesins) appear to con-sets (Murray and Hunt, 1993). Errors in this processtribute to the polar ejection force, the bipolarity of thecausebirthdefectsandcontributetotumorprogression.spindle, and chromosome positioning on the spindleAtthebeginning of mitosis,thechromosomes condense(reviewed in Vernos and Karsenti, 1995) . Interfering withand microtubules form a bipolar spindle with a microtu-the Xenopus chromokinesin, Xklp1, alters the distribu-bule organizing center at each end. The microtubulestion of microtubules in the spindle and induces defectsare polar structures whose minus ends are located nearin metaphase chromosome alignment (Vernos et al.,the spindle pole and are moved poleward by microtu-1995).In femaleDrosophila meiosisI, thechromokinesinbule flux (Mitchison, 1989; Mitchison and Salmon, 1992;Nod is required for proper alignment of chromosomesDesai et al., 1998). As mitosis proceeds, the kineto-that have not recombined with each other (Theurkaufchores (the proteinaceous complex assembled on theand Hawley, 1992; Afshar et al., 1995). Mutants in an-centromeric DNA) attach to microtubules, the pairs ofother Drosophila chromokinesin, Klp38B, cause chro-replicatedsisterchromatidsalignwiththeirsisterkineto-mosome missegregation in meiosis and mitosis (Molinachores attached to opposite poles of the spindle (biori-et al., 1997; Ruden et al., 1997).entation), and migrate to the equator of the spindle,Cells trigger anaphase by dissolving the linkage be-equidistant from the two poles (congression). When alltween sister chromatids and segregating the separatedthe chromosomes have attached and congressed, thesisters to opposite poles of the spindle. Activating thecell is in metaphase and the chromosomes are said toanaphase promoting complex (APC, also known as thelie on the metaphase plate. During anaphase, the sistercyclosome, reviewed in Zachariae and Nasmyth, 1999)chromatids separate from each other and move towardinduces the destruction of several mitotic proteins.Sec-the spindle poles. Although the mechanical details ofurin degradation allows sister chromatids to separatemitosis are known, the molecular mechanisms of chro-(reviewed in Biggins and Murray, 1998), and mitoticmosome positioning and segregation are poorly under-cyclin destruction (Murray et al., 1989) inactivates thestood.mitosis inducing protein kinase, Cdk1 (cyclin dependentkinase 1, also known as Cdc2 or Cdc28) (reviewed inMorgan, 1997). Although sister separation requires securin*To whom correspondence should be addressed (e-mail: hironori@destruction, it does not require microtubule dependentmcb.harvard.edu).forces or the inactivation of Cdk1 (Rieder and Palazzo,†Present address: Department of Molecular and Cellular Biology,Harvard University, Cambridge, MA 02138.1992; Straight et al., 1996). Anaphase chromosomeCell412Figure 1. Purificationof Metaphaseand Ana-phase Chromosomes from Xenopus Egg Ex-tracts(A) The protocol to purify biotinylated chro-mosomes using streptavidin-coated mag-netic beads.(B) Hoechst 33258 staining of chromosomesfrom a metaphase (left) or an anaphase(right)extract after dilution into chromosome dilu-tion buffer. Bar, 10 ␮m.(C) Total extract proteins (lanes 4–6) or chro-mosomal proteins copurified with streptavidin-magnetic beads