Kinetochores play an essential role in chromosome segregation by forming dynamic connections with spindle microtubules. Here, we identify a set of 10 copurifying kinetochore proteins from Caenorhabditis elegans, seven of which were previously uncharacterized. Using in vivo assays to monitor chromosome segregation, kinetochore assembly, and the mechanical stability of chromosome-microtubule attachments, we show that this copurifying protein network plays a central role at the kinetochore-microtubule interface. In addition, our analysis suggests that the network is comprised of three groups of proteins that contribute in distinct ways to this interface: KNL proteins act after the assembly of centromeric chromatin to generate the core of the microtubule-binding interface, MIS proteins control the rate and extent of formation of this interface, and NDC proteins are necessary to sustain tension during interactions with spindle microtubules. We also purify a similar set of associated proteins from human cells that includes four novel proteins and has recognizable homologs from each functional class. Thus, this protein network is a conserved constituent of the outer kinetochore, and the functions defined by our analysis in C. elegans are likely to be widely relevant.[Keywords: Mitosis; centromere; microtubule; spindle; Caenorhabditis elegans] Supplemental material is available at http://www.genesdev.org. Accurate chromosome segregation in eukaryotes is achieved by dynamic interactions between chromosomes and spindle microtubules. As cells enter mitosis, each of the two sister chromatids that comprise a mitotic chromosome assembles a kinetochore, a specialized organelle that links the chromatids to spindle microtubules (for review, see Cleveland et al. 2003). Forces generated by kinetochores and the spindle drive chromosome alignment and, following dissolution of chromatid cohesion, segregation to opposite sides of the cell. The action of these forces is coupled to dynamic changes in the length of kinetochore-bound microtubule polymers. The magnitude of the forces acting on kinetochores is significantly greater than that required to generate chromosome movements (Nicklas 1988). The excess force is most likely used to generate tension across bipolar attachments that can be used to select correctly oriented chromosomes and prevent aneuploidy (for review, see Cheeseman and Desai 2004). Thus, kinetochores form attachments to spindle microtubules that couple dynamic polymer length changes to chromosome movement while sustaining significant forces.To understand how kinetochores generate a microtubule-binding interface with these properties, we are examining kinetochore function in the early Caenorhabditis elegans embryo. In contrast to the localized centromeres of vertebrates, C. elegans chromosomes are holocentric with diffuse kinetochores that form along their entire length. Despite this difference in chromosome architecture, the structure and molecular composition of C. elegans kinetochores is similar to that of other meta...

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2–associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain–containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.

Cytokinesis completes cell division and partitions the contents of one cell to the two daughter cells. Here we characterize CAR-1, a predicted RNA binding protein that is implicated in cytokinesis. CAR-1 localizes to germline-specific RNA-containing particles and copurifies with the essential RNA helicase, CGH-1, in an RNA-dependent fashion. The atypical Sm domain of CAR-1, which directly binds RNA, is dispensable for CAR-1 localization, but is critical for its function. Inhibition of CAR-1 by RNA-mediated depletion or mutation results in a specific defect in embryonic cytokinesis. This cytokinesis failure likely results from an anaphase spindle defect in which interzonal microtubule bundles that recruit Aurora B kinase and the kinesin, ZEN-4, fail to form between the separating chromosomes. Depletion of CGH-1 results in sterility, but partially depleted worms produce embryos that exhibit the CAR-1–depletion phenotype. Cumulatively, our results suggest that CAR-1 functions with CGH-1 to regulate a specific set of maternally loaded RNAs that is required for anaphase spindle structure and cytokinesis.