Structural organization of the kinetochore-microtubule interface

Successful mitosis depends on the stable, yet regulated attachment of chromosomes to spindle microtubules. The kinetochore, a large macromolecular structure assembled at sites of centromeric heterochromatin, is responsible for generating and regulating these essential attachments. Over the last several years, concerted experimental efforts have brought the structural view of the kinetochore-microtubule interface more clearly into focus. Here, we review important recent advancements and discuss several unresolved questions regarding how kinetochores dynamically bridge mitotic chromosomes to spindle microtubules.     

Molecular analysis of kinetochore-microtubule attachment in budding yeast

The complex series of movements that mediates chromosome segregation during mitosis is dependent on the attachment of microtubules to kinetochores, DNA-protein complexes that assemble on centromeric DNA. We describe the use of live-cell imaging and chromatin immunoprecipitation in S. cerevisiae to identify ten kinetochore subunits, among which are yeast homologs of microtubule binding proteins in animal cells. By analyzing conditional mutations in several of these proteins, we show that they are required for the imposition of tension on paired sister kinetochores and for correct chromosome movement. The proteins include both molecular motors and microtubule associated proteins (MAPs), implying that motors and MAPs function together in binding chromosomes to spindle microtubules.     

Factors required for the binding of reassembled yeast kinetochores to microtubules in vitro

Kinetochores are structures that assemble on centromeric DNA and mediate the attachment of chromosomes to the microtubules of the mitotic spindle. The protein components of kinetochores are poorly understood, but the simplicity of the S. cerevisiae kinetochore makes it an attractive candidate for molecular dissection. Mutations in genes encoding CBF1 and CBF3, proteins that bind to yeast centromeres, interfere with chromosome segregation in vivo. To determine the roles played by these factors and by various regions of centromeric DNA in kinetochore function, we have developed a method to partially reassemble kinetochores on exogenous centromeric templates in vitro and to visualize the attachment of these reassembled kinetochore complexes to microtubules. In this assay, single reassembled complexes appear to mediate microtubule binding. We find that CBF3 is absolutely essential for this attachment but, contrary to previous reports (Hyman, A. A., K. Middleton, M. Centola, T.J. Mitchison, and J. Carbon. 1992. Microtubule- motor activity of a yeast centromere-binding protein complex. Nature (Lond.). 359:533-536) is not sufficient. Additional cellular factors interact with CBF3 to form active microtubule-binding complexes. This is mediated primarily by the CDEIII region of centromeric DNA but CDEII plays an essential modulatory role. Thus, the attachment of kinetochores to microtubules appears to involve a hierarchy of interactions by factors that assemble on a core complex consisting of DNA-bound CBF3.     

Kinetochore structure and function

The vertebrate kinetochore is a complex structure that specifies the attachments between the chromosomes and microtubules of the spindle and is thus essential for accurate chromosome segregation. Kinetochores are assembled on centromeric chromatin through complex pathways that are coordinated with the cell cycle. In the light of recent discoveries on how proteins assemble onto kinetochores and interact with each other, we review these findings in this article (which is part of the Chromosome Segregation and Aneuploidy series), and discuss their implications for the current mitotic checkpoint models - the template model and the two-step model. The template model proposes that Mad1-Mad2 at kinetochores acts as a template to change the conformation of another binding molecule of Mad2. This templated change in conformation is postulated as a mechanism for the amplification of the 'anaphase wait' signal. The two-step model proposes that the mitotic checkpoint complex (MCC) is the kinetochore-independent anaphase inhibitor, and the role of the unaligned kinetochore is to sensitize the anaphase-promoting complex/cyclosome (APC/C) to MCC-mediated inhibition.     

Polo-like kinase-1 regulates kinetochore-microtubule dynamics and spindle checkpoint silencing

Polo-like kinase-1 (Plk1) is a highly conserved kinase with multiple mitotic functions. Plk1 localizes to prometaphase kinetochores and is reduced at metaphase kinetochores, similar to many checkpoint signaling proteins, but Plk1 is not required for spindle checkpoint function. Plk1 is also implicated in stabilizing kinetochore-microtubule attachments, but these attachments are most stable when kinetochore Plk1 levels are low at metaphase. Therefore, it is unclear how Plk1 function at kinetochores can be understood in the context of its dynamic localization. In this paper, we show that Plk1 activity suppresses kinetochore-microtubule dynamics to stabilize initial attachments in prometaphase, and Plk1 removal from kinetochores is necessary to maintain dynamic microtubules in metaphase. Constitutively targeting Plk1 to kinetochores maintained high activity at metaphase, leading to reduced interkinetochore tension and intrakinetochore stretch, a checkpoint-dependent mitotic arrest, and accumulation of microtubule attachment errors. Together, our data show that Plk1 dynamics at kinetochores control two critical mitotic processes: initially establishing correct kinetochore-microtubule attachments and subsequently silencing the spindle checkpoint.     

Cyclin G-associated kinase promotes microtubule outgrowth from chromosomes during spindle assembly

During mitosis, all chromosomes must attach to microtubules of the mitotic spindle to ensure correct chromosome segregation. Microtubule attachment occurs at specialized structures at the centromeric region of chromosomes, called kinetochores. These kinetochores can generate microtubule attachments through capture of centrosome-derived microtubules, but in addition, they can generate microtubules themselves, which are subsequently integrated with centrosome-derived microtubules to form the mitotic spindle. Here, we have performed a large scale RNAi screen and identify cyclin G-associated kinase (GAK) as a novel regulator of microtubule generation at kinetochores/chromatin. This function of GAK requires its C-terminal J-domain, which is essential for clathrin recycling from endocytic vesicles. Consistently, cells lacking GAK show strongly reduced levels of clathrin on the mitotic spindle, and reduction of clathrin levels also inhibits microtubule generation at kinetochores/chromosomes. Finally, we present evidence that association of clathrin with the spindle is promoted by a signal coming from the chromosomes. These results identify a role for GAK and clathrin in microtubule outgrowth from kinetochores/chromosomes and suggest that GAK acts through clathrin to control microtubule outgrowth around chromosomes.     

hNuf2 inhibition blocks stable kinetochore-microtubule attachment and induces mitotic cell death in HeLa cells

Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in kinetochore-microtubule attachment and suggest that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends.     

Depletion of centromeric MCAK leads to chromosome congression and segregation defects due to improper kinetochore attachments

The complex behavior of chromosomes during mitosis is accomplished by precise binding and highly regulated polymerization dynamics of kinetochore microtubules. Previous studies have implicated Kin Is, unique kinesins that depolymerize microtubules, in regulating chromosome positioning. We have characterized the immunofluorescence localization of centromere-bound MCAK and found that MCAK localized to inner kinetochores during prophase but was predominantly centromeric by metaphase. Interestingly, MCAK accumulated at leading kinetochores during congression but not during segregation. We tested the consequences of MCAK disruption by injecting a centromere dominant-negative protein into prophase cells. Depletion of centromeric MCAK led to reduced centromere stretch, delayed chromosome congression, alignment defects, and severe missegregation of chromosomes. Rates of chromosome movement were unchanged, suggesting that the primary role of MCAK is not to move chromosomes. Furthermore, we found that disruption of MCAK leads to multiple kinetochore-microtubule attachment defects, including merotelic, syntelic, and combined merotelic-syntelic attachments. These findings reveal an essential role for Kin Is in prevention and/or correction of improper kinetochore-microtubule attachments.     

Xenopus Cep57 is a novel kinetochore component involved in microtubule attachment

For chromosomes to congress and segregate during cell division, kinetochores must form stable attachments with spindle microtubules. We find that the centrosome protein, xCep57, localizes to kinetochores and interacts with the kinetochore proteins Zwint, Mis12, and CLIP-170. Immunodepletion of xCep57 from egg extracts yields weakened and elongated bipolar spindles which fail to align chromosomes. In the absence of xCep57, tension is lost between sister kinetochores, and spindle microtubules are no longer resistant to low doses of nocodazole. xCep57 inhibition on isolated mitotic chromosomes inhibits kinetochore-microtubule binding in vitro. xCep57 also interacts with gamma-tubulin. In xCep57 immunodepleted extracts, sperm centrosomes nucleate with normal kinetics, but are unable maintain microtubule anchorage. This characterization places xCep57 in a novel class of proteins required for stable microtubule attachments at the kinetochore and at the centrosome and suggests that the mechanism of microtubule binding at these two places is mechanistically similar.     

Kinetochore-microtubule interactions: the means to the end

Kinetochores are proteinaceous complexes containing dozens of components; they are assembled at centromeric DNA regions and provide the major microtubule attachment site on chromosomes during cell division. Recent studies have defined the kinetochore components comprising the direct interface with spindle microtubules, primarily through structural and functional analysis of the Ndc80 and Dam1 complexes. These studies have facilitated our understanding of how kinetochores remain attached to the end of dynamic microtubules and how proper orientation of a kinetochore-microtubule attachment is promoted on the mitotic spindle. In this article, we review these recent studies and summarize their key findings.     

Loss of function of the Cik1/Kar3 motor complex results in chromosomes with syntelic attachment that are sensed by the tension checkpoint

The attachment of sister kinetochores by microtubules emanating from opposite spindle poles establishes chromosome bipolar attachment, which generates tension on chromosomes and is essential for sister-chromatid segregation. Syntelic attachment occurs when both sister kinetochores are attached by microtubules from the same spindle pole and this attachment is unable to generate tension on chromosomes, but a reliable method to induce syntelic attachments is not available in budding yeast. The spindle checkpoint can sense the lack of tension on chromosomes as well as detached kinetochores to prevent anaphase onset. In budding yeast Saccharomyces cerevisiae, tension checkpoint proteins Aurora/Ipl1 kinase and centromere-localized Sgo1 are required to sense the absence of tension but are dispensable for the checkpoint response to detached kinetochores. We have found that the loss of function of a motor protein complex Cik1/Kar3 in budding yeast leads to syntelic attachments. Inactivation of either the spindle or tension checkpoint enables premature anaphase entry in cells with dysfunctional Cik1/Kar3, resulting in co-segregation of sister chromatids. Moreover, the abolished Kar3-kinetochore interaction in cik1 mutants suggests that the Cik1/Kar3 complex mediates chromosome movement along microtubules, which could facilitate bipolar attachment. Therefore, we can induce syntelic attachments in budding yeast by inactivating the Cik1/Kar3 complex, and this approach will be very useful to study the checkpoint response to syntelic attachments.     

The Ndc80 complex uses a tripartite attachment point to couple microtubule depolymerization to chromosome movement

In kinetochores, the Ndc80 complex couples the energy in a depolymerizing microtubule to perform the work of moving chromosomes. The complex directly binds microtubules using an unstructured, positively charged N-terminal tail located on Hec1/Ndc80. Hec1/Ndc80 also contains a calponin homology domain (CHD) that increases its affinity for microtubules in vitro, yet whether it is required in cells and how the tail and CHD work together are critical unanswered questions. Human kinetochores containing Hec1/Ndc80 with point mutations in the CHD fail to align chromosomes or form productive microtubule attachments. Kinetochore architecture and spindle checkpoint protein recruitment are unaffected in these mutants, and the loss of CHD function cannot be rescued by removing Aurora B sites from the tail. The interaction between the Hec1/Ndc80 CHD and a microtubule is facilitated by positively charged amino acids on two separate regions of the CHD, and both are required for kinetochores to make stable attachments to microtubules. Chromosome congression in cells also requires positive charge on the Hec1 tail to facilitate microtubule contact. In vitro binding data suggest that charge on the tail regulates attachment by directly increasing microtubule affinity as well as driving cooperative binding of the CHD. These data argue that in vertebrates there is a tripartite attachment point facilitating the interaction between Hec1/Ndc80 and microtubules. We discuss how such a complex microtubule-binding interface may facilitate the coupling of depolymerization to chromosome movement.     

Phosphoregulation of Spc105 by Mps1 and PP1 regulates Bub1 localization to kinetochores

Kinetochores are the macromolecular complexes that interact with microtubules to mediate chromosome segregation. Accurate segregation requires that kinetochores make bioriented attachments to microtubules from opposite poles. Attachments between kinetochores and microtubules are monitored by the spindle checkpoint, a surveillance system that prevents anaphase until every pair of chromosomes makes proper bioriented attachments. Checkpoint activity is correlated with the recruitment of checkpoint proteins to the kinetochore. Mps1 is a conserved protein kinase that regulates segregation and the spindle checkpoint, but few of the targets that mediate its functions have been identified. Here, we show that Mps1 is the major kinase activity that copurifies with budding yeast kinetochore particles and identify the conserved Spc105/KNL-1/blinkin kinetochore protein as a substrate. Phosphorylation of conserved MELT motifs within Spc105 recruits the Bub1 protein to kinetochores, and this is reversed by protein phosphatase I (PP1). Spc105 mutants lacking Mps1 phosphorylation sites are defective in the spindle checkpoint and exhibit growth defects. Together, these data identify Spc105 as a key target of the Mps1 kinase and show that the opposing activities of Mps1 and PP1 regulate the kinetochore localization of the Bub1 protein.     

The spatial arrangement of chromosomes during prometaphase facilitates spindle assembly

Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposite spindle poles (amphitelic attachment). Exactly how amphitelic attachments are achieved during spindle assembly remains elusive. We employed photoactivatable GFP and high-resolution live-cell confocal microscopy to visualize complete 3D movements of individual kinetochores throughout mitosis in nontransformed human cells. Combined with electron microscopy, molecular perturbations, and immunofluorescence analyses, this approach reveals unexpected details of chromosome behavior. Our data demonstrate that unstable lateral interactions between kinetochores and microtubules dominate during early prometaphase. These transient interactions lead to the reproducible arrangement of chromosomes in an equatorial ring on the surface of the nascent spindle. A computational model predicts that this toroidal distribution of chromosomes exposes kinetochores to a high density of microtubules which facilitates subsequent formation of amphitelic attachments. Thus, spindle formation involves a previously overlooked stage of chromosome prepositioning which promotes formation of amphitelic attachments.     

Molecular architecture of a kinetochore-microtubule attachment site

Kinetochore attachment to spindle microtubule plus-ends is necessary for accurate chromosome segregation during cell division in all eukaryotes. The centromeric DNA of each chromosome is linked to microtubule plus-ends by eight structural-protein complexes. Knowing the copy number of each of these complexes at one kinetochore-microtubule attachment site is necessary to understand the molecular architecture of the complex, and to elucidate the mechanisms underlying kinetochore function. We have counted, with molecular accuracy, the number of structural protein complexes in a single kinetochore-microtubule attachment using quantitative fluorescence microscopy of GFP-tagged kinetochore proteins in the budding yeast Saccharomyces cerevisiae. We find that relative to the two Cse4p molecules in the centromeric histone, the copy number ranges from one or two for inner kinetochore proteins such as Mif2p, to 16 for the DAM-DASH complex at the kinetochore-microtubule interface. These counts allow us to visualize the overall arrangement of a kinetochore-microtubule attachment. As most of the budding yeast kinetochore proteins have homologues in higher eukaryotes, including humans, this molecular arrangement is likely to be replicated in more complex kinetochores that have multiple microtubule attachments.     

Aurora B kinase controls the targeting of the Astrin-SKAP complex to bioriented kinetochores

During mitosis, kinetochores play multiple roles to generate interactions with microtubules, and direct chromosome congression, biorientation, error correction, and anaphase segregation. However, it is unclear what changes at the kinetochore facilitate these distinct activities. Here, we describe a complex of the spindle- and kinetochore-associated protein Astrin, the small kinetochore-associated protein (SKAP), and the dynein light chain LC8. Although most dynein-associated proteins localize to unaligned kinetochores in an Aurora B-dependent manner, Astrin, SKAP, and LC8 localization is antagonized by Aurora B such that they target exclusively to bioriented kinetochores. Astrin-SKAP-depleted cells fail to maintain proper chromosome alignment, resulting in a spindle assembly checkpoint-dependent mitotic delay. Consistent with a role in stabilizing bioriented attachments, Astrin and SKAP bind directly to microtubules and are required for CLASP localization to kinetochores. In total, our results suggest that tension-dependent Aurora B phosphorylation can act to control outer kinetochore composition to provide distinct activities to prometaphase and metaphase kinetochores.     

The Cik1/Kar3 motor complex is required for the proper kinetochore-microtubule interaction after stressful DNA replication

In budding yeast Saccharomyces cerevisiae, kinetochores are attached by microtubules during most of the cell cycle, but the duplication of centromeric DNA disassembles kinetochores, which results in a brief dissociation of chromosomes from microtubules. Kinetochore assembly is delayed in the presence of hydroxyurea, a DNA synthesis inhibitor, presumably due to the longer time required for centromeric DNA duplication. Some kinetochore mutants are sensitive to stressful DNA replication as these kinetochore proteins become essential for the establishment of the kinetochore-microtubule interaction after treatment with hydroxyurea. To identify more genes required for the efficient kinetochore-microtubule interaction under stressful DNA replication conditions, we carried out a genome-wide screen for yeast mutants sensitive to hydroxyurea. From this screen, cik1 and kar3 mutants were isolated. Kar3 is the minus-end-directed motor protein; Cik1 binds to Kar3 and is required for its motor function. After exposure to hydroxyurea, cik1 and kar3 mutant cells exhibit normal DNA synthesis kinetics, but they display a significant anaphase entry delay. Our results indicate that cik1 cells exhibit a defect in the establishment of chromosome bipolar attachment in the presence of hydroxyurea. Since Kar3 has been shown to drive the poleward chromosome movement along microtubules, our data support the possibility that this chromosome movement promotes chromosome bipolar attachment after stressful DNA replication.     

Mitosis puts sisters in a strained relationship: force generation at the kinetochore

During mitosis, kinetochores couple chromosomes to the dynamic tips of spindle microtubules. These attachments convert chemical energy stored in the microtubule lattice into mechanical energy, generating force to move chromosomes. In addition to mediating robust microtubule attachments, kinetochores also integrate and respond to regulatory signals that ensure the accuracy of chromosome segregation during each cell division. Signals for corrective detachment act specifically on kinetochore-microtubule attachments that fail to generate normal levels of tension, although it is unclear how tension is sensed and how the attachments are released. In this review, we discuss the mechanisms by which kinetochore-microtubule attachments generate force during chromosome biorientation, and the pathways of maturation and regulation that lead to the formation of correct attachments.     

Binding of the essential Saccharomyces cerevisiae kinetochore protein Ndc10p to CDEII

Chromosome segregation at mitosis depends critically on the accurate assembly of kinetochores and their stable attachment to microtubules. Analysis of Saccharomyces cerevisiae kinetochores has shown that they are complex structures containing >/=50 protein components. Many of these yeast proteins have orthologs in animal cells, suggesting that key aspects of kinetochore structure have been conserved through evolution, despite the remarkable differences between the 125-base pair centromeres of budding yeast and the Mb centromeres of animal cells. We describe here an analysis of S. cerevisiae Ndc10p, one of the four protein components of the CBF3 complex. CBF3 binds to the CDEIII element of centromeric DNA and initiates kinetochore assembly. Whereas CDEIII binding by Ndc10p requires the other components of CBF3, Ndc10p can bind on its own to CDEII, a region of centromeric DNA with no known binding partners. Ndc10p-CDEII binding involves a dispersed set of sequence-selective and -nonselective contacts over approximately 80 base pairs of DNA, suggesting formation of a multimeric structure. CDEII-like sites, active in Ndc10p binding, are also present along chromosome arms. We propose that a polymeric Ndc10p complex formed on CDEII and CDEIII DNA is the foundation for recruiting microtubule attachment proteins to kinetochores. A similar type of polymeric structure on chromosome arms may mediate other chromosome-spindle interactions.     

The unstable F-box protein p58-Ctf13 forms the structural core of the CBF3 kinetochore complex.

Kinetochores are smaller and more accessible experimentally in budding yeast than in any other eukaryote. Believing that simple and complex kinetochores have important structural and functional properties in common, we characterized the structure of CBF3, the essential centromere-binding complex that initiates kinetochore formation in Saccharomyces cerevisiae. We find that the four subunits of CBF3 are multimeric in solution: p23(Skp1) and p58(Ctf13) form a heterodimer, and p64(Cep3) and p110(Ndc10) form homodimers. Subcomplexes involving p58 and each of the other CBF3 subunits can assemble in the absence of centromeric DNA. In these subcomplexes, p58 appears to function as a structural core mediating stable interactions among other CBF3 proteins. p58 has a short half-life in yeast, being subject to ubiquitin-dependent proteolysis, but we find that it is much more stable following association with p64. We propose that p23(Skp1)-p58-p64 complexes constitute the primary pool of active p58 in yeast cells. These complexes can either dissociate, reexposing p58 to the degradation pathway, or can bind to p110 and centromeric DNA, forming a functional CBF3 complex in which p58 is fully protected from degradation. This pathway may constitute an editing mechanism preventing the formation of ectopic kinetochores and ensuring the fidelity of chromosome segregation.