The Conserved KMN Network Constitutes the Core Microtubule-Binding Site of the KinetochoreIntroductionResultsPartially Purified C. elegans KMN Protein Network Cosediments with MicrotubulesReconstitution of the KMN Network via Bacterial CoexpressionNDC-80 ComplexKNL-3 and the MIS ProteinsKNL-1 and KNL-1/MIS-12 ComplexReconstitution of the KMN Network from Its Constituent PartsTwo Distinct Microtubule-Binding and -Bundling Activities Are Present in the KMN NetworkReconstitution of the KMN Network Results in a Synergy between the Microtubule-Binding Activities of the NDC-80 Complex and KNL-1The KNL-1/MIS-12 Complex and the NDC-80 Complex Exhibit Cooperative Binding to MicrotubulesUltrastructural Analysis of NDC-80 Complexes Bound to MicrotubulesThe NDC-80 Complex Head Binds to Microtubules, but Not to KNL-1/MIS-12 ComplexThe Human Ndc80 Complex Head Domain Binds to and Bundles MicrotubulesAurora Kinase Phosphorylation of NDC-80 Regulates the Microtubule-Binding Activity of the NDC-80 ComplexDiscussionA Model for the Core Microtubule-Binding Site of the KinetochoreBuilding a Dynamic Interface with the Spindle: Intrinsic Affinity versus Valency of Interactions at the KinetochoreExperimental ProceduresProtein PurificationMicrotubule Cosedimentation AssaysElectron MicroscopySupplemental DataAcknowledgmentsReferencesThe Conserved KMN Network ConstitutestheCoreMicrotubule-BindingSite of the KinetochoreIain M. Cheeseman,1,*Joshua S. Chappie,2Elizabeth M. Wilson-Kubalek,2and Arshad Desai1,*1Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine (UCSD), CMM-East, Room 3052,La Jolla, CA 92093, USA2Center for Integrative Molecular Biosciences, Department of Cell Biology, The Scripps Research Institute,10550 North Torrey Pines Road, La Jolla, CA 92037, USA*Contact: [email protected] (I.M.C.), [email protected] (A.D.)DOI 10.1016/j.cell.2006.09.039SUMMARYThe microtubule-binding interface of the kineto-chore is of central importance in chromosomesegregation. Although kinetochore compo-nents that stabilize, translocate on, and affectthe polymeriz ation state of microtubules havebeen identified, none have proven essentialfor kinetochore-microtubule interactions. Here,we examined the conservedKNL-1/Mis12 com-plex/Ndc80 complex (KMN) network, which isessential for kinetochore-microtubule interac-tions in vivo. We identified two distinct microtu-bule-binding activities within the KMN network:one associated with the Ndc80/Nuf2 subunits ofthe Ndc80 complex, and a second in KNL-1. For-mation of the complete KMN network, whichadditionally requires the Mis12 complex andthe Spc24/Spc25 subunits of the Ndc80 com-plex, synergistically enhances microtubule-binding activity. Phosphorylation by Aurora B,which corrects improper kinetochore-microtu-bule connections in vivo, reduces the affinity ofthe Ndc80 complex for microtubules in vitro.Based on these findings, we propose that theconserved KMN network constitutes the coremicrotubule-binding site of the kinetochore.INTRODUCTIONIn the late nineteenth century, a differentiated chromo-somal region that connects to spindle fibers during mitosiswas first described (reviewed in Schrader, 1953). Electronmicroscopy provided a direct view of this region, termedthe kinetochore, and demonstrated that microtubuleplus ends directly embed in the outer kinetochore plate(reviewed in Rieder, 1982). However, these are not staticattachments, and chromosome movement is intimatelycoupled to changes in the polymerization dynamics ofkinetochore-bound microtubules (Maddox et al., 2003;Rieder and Salmon, 1998). More recently, the identifica-tion of numerous kinetochore proteins and elucidation oftheir in vivo loss-of-function phenotypes is providing amolecular view of this structure. Despite this progress,the identity of the core microtubule-binding site of the ki-netochore has remained elusive. Most significantly, com-ponents that both are essential in vivo for kinetochore-microtubule interactions and exhibit direct interactionswith microtubules in vitro have not been identified.A variety of approaches in different organisms haveidentified kinetochore components that interact with mi-crotubules (reviewed in Cleveland et al., 2003; Maiatoet al., 2004). These include the Dam1 ring complex, theplus end-tracking proteins CLASP, CLIP-170, and EB1,the minus end-directed microtubule motor dynein, the plusend-directed kinesin-7 (CENP-E), and the microtubule de-polymerizing kinesin-13 (Kin I) and kinesin-8 (Kip3). How-ever, while these proteins make important contributionsto chromosome movement, in vivo inhibitions indicatethat none of them are essential for the stable interactionof kinetochores with microtubules. Recently, the Dam1ring complex in budding yeast has received attention asa potential kinetochore-spindle connector. Although mu-tational inactivation of the Dam1 complex causes severechromosome missegregation, it does not eliminate all ki-netochore-spindle interactions (Cheeseman et al., 2001).In addition, this complex is genetically dispensable for via-bility in fission yeast (Sanchez-Perez et al., 2005) and is notdetectably conserved outside fungi. In metazoans, inhibi-tion of the two kinetochore motors (cytoplasmic dyneinand CENP-E) does not significantly alter chromosome-spindle interactions for the majority of chromosomes (Ho-well et al., 2001; Putkey et al., 2002). Inhibition of CLASPfamily proteins specifically inhibits microtubule polymeri-zation at kinetochores (Maiato et al., 2005) but does notprevent the formation of kinetochore-microtubule attach-ments. In total, the analysis of currently identified microtu-bule-binding proteins fails to account for the activity of thecore microtubule-binding site of the kinetochore.Cell 127, 983–997, December 1, 2006 ª2006 Elsevier Inc. 983We previously used a combination of biochemistry andfunctional genomics in C. elegans to identify a network of10 interacting proteins that plays a central role at the kinet-ochore-microtubule interface (Cheeseman et al., 2004;Desai et al., 2003). Phenotypic analysis by single-cell as-says partitioned the components of this network into threedistinct groups (see Figure 1A). Depletion of the ‘‘KNL’’proteins KNL-1 (termed Spc105 in budding yeast; Nekra-sov et al., 2003) or KNL-3 completely abolished kineto-chore-microtubule interactions. Depletion of the secondgroup, equivalent to the Mis12 complex in other organ-isms (De Wulf et al., 2003; Kline et al., 2006;