A conserved protein network controls assembly of the outer kinetochore and its ability to sustain tension

Abstract

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...

Reconstruction of the Kinetochore during Meiosis in Fission Yeast Schizosaccharomyces pombe

“…NMS group proteins reassemble to the kinetochore during prophase and toward metaphase in meiosis, and subsequently DASH group proteins localize at the centromere during chromosome segregation. These groupings are generally consistent with the complex structures revealed by genetic interactions and proteomic analyses (De Wulf et al, 2003;Cheeseman et al, 2004;Obuse et al, 2004;Liu et al, 2005).…”

“…Biochemical analyses have revealed that the kinetochore complex is comprised of subcomplexes of proteins. Many of these proteins are conserved in other eukaryotes, from yeasts to humans (De Wulf et al, 2003;Nekrasov et al, 2003;Cheeseman et al, 2004;Obuse et al, 2004;reviewed in Meraldi et al, 2006).…”

A driving and coupling “Pac-Man” mechanism for chromosome poleward translocation in anaphase A

“…Moreover, the kinetochore structure in our model is specifically taken from the Dam1 ring complex in budding yeast (5)(6)(7). The kinetochore structure in other cells could be different from the ring-like geometry (35). In this aspect, this model could serve as a starting point for further investigations.…”

Keeping kinetochores on track

“…This approach identified seven novel orthologous kinetochore proteins in mammals, three novel proteins in D. melanogaster and two novel proteins in plants. These results implied that most kinetochore proteins are conserved in eukaryotes, a finding that was confirmed by biochemical and genetic studies identifying those same proteins as bona fide kinetochore proteins (Cheeseman et al, 2004;Foltz et al, 2006;Obuse et al, 2004;Okada et al, 2006;Schittenhelm et al, 2007). The overall conclusion of these studies was that the core of eukaryotic kinetochores is composed of two large conserved protein networks: on one side the KMN network, which consists of Knl-1, the hetero-tetrameric MIND/Mis12 subcomplex, and the hetero-tetrameric-NDC80 subcomplex, and on the other side the CCAN network (Constitutive Centromere Associated Network, which is also called CENP-A NAC/CAD or CENP-H/I complex), which consists of 15 subunits.…”