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The kinetochore is a large protein complex that ensures accurate chromosome segregation during mitosis by connecting the centromere and spindle microtubules. One of the kinetochore sub-complexes, the constitutive centromere-associated network (CCAN), associates with the centromere and recruits another sub-complex, the KMN (KNL1, Mis12, and Ndc80 complexes) network (KMN), which binds to spindle microtubules. The CCAN-KMN interaction is mediated by two parallel pathways (CENP-C- and CENP-T-pathways) in the kinetochore, which bridge the centromere and microtubules. Here, we discuss dynamic protein-interaction changes in the two pathways that couple the centromere with spindle microtubules during mitotic progression.

Unlike other organisms that have evolved distinct H2A variants for different functions, Drosophila melanogaster has just one variant which is capable of filling many roles. This protein, H2A.V, combines the features of the conserved variants H2A.Z and H2A.X in transcriptional control/heterochromatin assembly and DNA damage response, respectively. Here we show that mutations in the gene encoding H2A.V affect chromatin compaction and perturb chromosome segregation in Drosophila mitotic cells. A microtubule (MT) regrowth assay after cold exposure revealed that loss of H2A.V impairs the formation of kinetochore-driven (k) fibers, which can account for defects in chromosome segregation. All defects are rescued by a transgene encoding H2A.V that lacks the H2A.X function in the DNA damage response, suggesting that the H2A.Z (but not H2A.X) functionality of H2A.V is required for chromosome segregation. We also found that loss of H2A.V weakens HP1 localization, specifically at the pericentric heterochromatin of metaphase chromosomes. Interestingly, loss of HP1 yielded not only telomeric fusions but also mitotic defects similar to those seen in H2A.V null mutants, suggesting a role for HP1 in chromosome segregation. We also show that H2A.V precipitates HP1 from larval brain extracts indicating that both proteins are part of the same complex. Moreover, we found that the overexpression of HP1 rescues chromosome missegregation and defects in the kinetochore-driven k-fiber regrowth of H2A.V mutants indicating that both phenotypes are influenced by unbalanced levels of HP1. Collectively, our results suggest that H2A.V and HP1 work in concert to ensure kinetochore-driven MT growth.

Chromosome bipolar attachment is achieved when sister kinetochores are attached by microtubules emanating from opposite spindle poles, and this process is essential for faithful chromosome segregation during anaphase. A fundamental question in cell biology is how cells ensure that chromosome segregation only occurs after bipolar attachment. It is well documented that unattached kinetochores activate the spindle assembly checkpoint (SAC) to delay chromosome segregation. Therefore, the silencing of the SAC is thought to trigger anaphase onset, but how correct chromosome attachment is coupled with SAC silencing and the subsequent anaphase onset is poorly understood. The establishment of chromosome bipolar attachment not only results in the occupancy of kinetochores by microtubules but also applies tension on sister kinetochores. A long-standing debate is whether the kinetochore attachment (occupancy) or the tension silences the SAC. Recent work in budding yeast reveals the SAC silencing network SSN that prevents SAC silencing prior to tension generation at kinetochores. Therefore, this signaling pathway ensures that SAC silencing and the subsequent anaphase onset occur only after chromosome bipolar attachment applies tension on chromosomes. This review will summarize the recent advances in the understanding of the SAC silencing process.

Spindly is a coiled-coil domain containing protein, also known as CCDC99, that is an important regulator of the mitotic checkpoint. It belongs to a functionally conserved family of proteins that controls the localization and function of cytoplasmic dynein at kinetochores. At kinetochores Spindly interacts with the RZZ (Rod/ZW10/Zwilch) complex and is required for the recruitment of cytoplasmic dynein to kinetochores. Loss of Spindly results in loss of kinetochore dynein functions, which impairs poleward chromosome movements and mitotic checkpoint inactivation. In addition, Spindly regulates the establishment of stable microtubule attachments via regulation of the RZZ complex. Thus, Spindly couples dynein-mediated poleward chromosome movements to the establishment of stable microtubule attachments and subsequent mitotic checkpoint inactivation and provides an important switch for the control of mitosis.

More than two decades of research has resulted in the identification of some 60 microtubule motor proteins, several of which have been implicated in mitosis. Although some kinesin super-family proteins function as microtubule depolymerases at kinetochores, such as Kinesin-8 and -13, it is now appreciated that there are only two force-producing kinetochore associated motors, the plus end-directed microtubule motor CENP-E and the minus end-directed microtubule motor cytoplasmic dynein. Defining their roles at kinetochores has been hampered by the complexity of mitosis itself, and a multiplicity of mitotic roles, at least for cytoplasmic dynein. Nonetheless, recent advances have served to define the primary roles of the two kinetochore motors in detail.

The spindle assembly checkpoint (SAC) is a mechanism that prevents premature chromosome segregation in anaphase before all chromosomes are correctly attached to the mitotic spindle. Errors in chromosome segregation lead to aneuploidy, which may be causally involved in tumorgenesis. Kinetochore complexes are the structural components of the SAC, which are tightly regulated by various mechanisms including phosphorylation and ubiquitin-dependent proteolysis. Recent studies shed new light on the regulatory pathways of the ubiquitin proteasome system involved in SAC signaling. Here we present evidence that a Cul3-based E3 ubiquitin-ligase is required to maintain SAC signaling in human cells. Inactivation of the Cul3/KLHL9/KLHL13 ligase leads to premature degradation of Cyclin B and exit from the mitotic state in the presence of microtubule poisons. We discuss possible mechanisms how Cul3 may be required to maintain SAC activity by ubiquitination of the chromosomal passenger protein Aurora B.

Faithful distribution of the genome to daughter cells requires that replicated chromatids are compacted and resolved from one another, and that functional kinetochores assemble at their centromeres to mediate spindle interactions. A molecular and mechanistic understanding of these critical processes is far from complete. We discovered that linker histone H1, a major chromatin component, is required for longitudinal compaction of replicated sperm chromosome arms in Xenopus laevis egg extracts that recapitulate the cell cycle in vitro. Due to their elongated structure, H1-depleted chromosomes could not be aligned or segregated by the spindle despite apparently normal kinetochore assembly. Interestingly, enrichment of H1 onto chromatin required passage through interphase, when DNA replication takes place. Here we show that interphase-dependent loading is also a feature of kinetochore components including Ndc80. Thus, linker histone H1 contributes to chromosome condensation in vertebrates, and important molecular determinants of mitotic chromosome and kinetochore structure and function are established during interphase.

The vast accumulation of knowledge from genome sequencing projects and studies with model organisms has presented a remarkable challenge to biologists: to understand the functions of thousands of highly conserved genes and how they work together to regulate fundamental cellular processes. This challenge is compounded by the inescapable reality that most genes are 'buffered' by other genes that contribute to the same biological processes, limiting the impact of phenotypic studies with single mutants. In budding yeast, functional genomic methods have been developed for the systematic application of established genetic techniques. In particular, the Synthetic Genetic Array (SGA) method allows genome-wide synthetic lethal (SL) and synthetic dosage lethal (SDL) screens thus enabling an unbiased survey of genetic interactions. We have used genes encoding components of the yeast kinetochore as a biological testbed for assaying the utility of SGA-based SL and SDL screens for revealing new pathways and genes involved in chromosome segregation. We identified 211 nonessential deletion mutants that were unable to tolerate either overexpression or loss of function of kinetochore genes. Our study uncovered a wealth of relationships between gene products that functionally interact with the kinetochore, and also highlighted the value of performing genome-wide screens with both hypomorphic and hypermorphic alleles of query genes. Here, we will highlight our recent kinetochore SGA genomic screens, in the broader context of applying complementary genetic screening approaches in the systematic exploration of biological pathways or functional complexes.

The kinetochore, a multi-protein complex assembled on centromeric chromatin in mitosis, is essential for sister chromosome segregation. We show here that inhibition of histone deacetylation blocks mitotic progression at prometaphase in two human tumor cell lines by interfering with kinetochore assembly. Decreased amounts of hBUB1, CENP-F and the motor protein CENP-E were present on kinetochores of treated cells. These kinetochores failed to nucleate and inefficiently captured microtubules, resulting in activation of the mitotic checkpoint. Addition of histone deacetylase inhibitors prior to the end of S-phase resulted in decreased HP1-beta on pericentromeric heterochromatin in S-phase and G(2), decreased pericentromeric targeting of Aurora B kinase, resulting in decreased premitotic phosphorylation of pericentromeric histone H3(S10) in G(2), followed by assembly of deficient kinetochores in M-phase. HP1-beta, Aurora B and the affected kinetochore proteins all were present at normal levels in treated cells; thus, effects of the inhibitors on mitotic progression do not seem to reflect changes in gene expression. In vitro kinase activity of Aurora B isolated from treated cells was unaffected. We propose that the increased presence in pericentromeric heterochromatin of histone H3 acetylated at K9 is responsible for the mitotic defects resulting from inhibition of histone deacetylation.

Accurate partition of duplicated genetic material to the daughter cells during mitosis relies on the maintenance of the physical linkage (cohesion) between sister chromatids until their bipolar attachment to the mitotic spindle. In response to a single straying chromatid within a cell, a surveillance mechanism called the spindle checkpoint blocks the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C), stabilizes securin (an APC/C substrate and an inhibitor of separase), and delays the activation of separase. This in turn prevents cleavage of cohesin by separase, preserves sister chromatid cohesion, and delays the onset of anaphase. The protein kinase, Bub1, is a key component of the spindle checkpoint. Bub1 has an upstream function in regulating the kinetochore localization of Mad2 and other downstream checkpoint components. In addition, recent biochemical studies have shown that Bub1 directly phosphorylates the APC/C activator, Cdc20, and inhibits APC/C. Finally, Bub1 has a noncheckpoint function at the kinetochores and preserves centromeric cohesion through the MEI-S332/shugoshin family of proteins. Therefore, Bub1 performs multiple tasks in mitosis that ensure the proper inheritance of chromosomes.

The mitotic spindle checkpoint arrests cells in metaphase until all kinetochores are correctly attached to spindle fibres. In a search for new components of the spindle checkpoint, based on protein interaction, we have identified mouse Telomeric Repeat Binding Factor 1 (Trf1) as a protein that interacts directly with the spindle checkpoint protein Mad1 and the mitotic kinase Nek2. Trf1 has a role in the regulation of telomere length, but has also been implicated in cell cycle regulation. These interactions now provide a direct link between telomere length regulation and the control of mitotic progression.

Rho GTPases including Rho, Rac and Cdc42 are involved in cell morphogenesis by inducing specific types of actin cytoskeleton and alignment and stabilization of microtubules. Previous studies suggest that they also regulate cell cycle progression; Rho, Rac and Cdc42 regulate the G(1)-S progression and Rho controls cytokinesis. However, a role of Rho GTPases in nuclear division has not been definitely shown. We have recently found that Cdc42 and its downstream effector mDia3 are involved in bi-orientation and stabilization of spindle microtubules attachment to kinetochores and regulate chromosome alignment and segregation. Here, we discuss how this is coordinated with other events in mitosis, particularly, with the action of Rho in cytokinesis and how attachment of microtubules to kinetochores is achieved and stabilized. We also discuss redundancy of dc42 and Cdc42-related GTPase(s) and potential mechanisms of chromosome instability in cancer.

Anaphase A chromatid-to-pole motion is fundamental for proper chromosome segregation in most systems. During the past several decades, two models for the mechanism of anaphase A have come to prominence. The Pacman model posits that chromatids induce the depolymerization of microtubule plus-ends embedded in kinetochores, thereby 'chewing' their way poleward. Alternatively, the Poleward-flux model posits that chromatids are 'reeled-in' to poles by the continual depolymerization of the minus-ends of kinetochore-associated microtubules, which are focused at spindle poles. In a recent study, we reported that anaphase A in Drosophila requires the depolymerization of both ends of kinetochore-associated microtubules, simultaneously. This is driven by two members of the Kin I subfamily of kinesins, termed KLP59C and KLP10A, which target specifically to chromatids and spindle poles, respectively. We have termed this hybrid of Pacman and Poleward flux the Kin I-dependent Pacman-flux mechanism for anaphase A. Here, we discuss the implications of these findings and explore potential additional components required to drive chromatid-to-pole motion by such a mechanism.

The spindle checkpoint in the yeast Saccharomyces cerevisiae is an intracellular signal transduction pathway comprised of two branches that inhibit two different mitotic transitions in cells treated with benzimidazole drugs such as nocodazole. The kinetochore is an integral component of the MAD2 branch of the spindle checkpoint pathway. Current models propose that the kinetochore is required for both the establishment and maintenance of the spindle checkpoint but a role for the kinetochore in the maintenance of spindle checkpoint in yeast has never been directly tested. We used a temperature sensitive ndc10-1 mutant to inactivate kinetochores before and after arresting cells in mitosis to determine the role of kinetochores in the establishment and maintenance of the spindle checkpoint. We show that both establishment and maintenance requires kinetochore function in response to spindle damage induced by benzimidazole drugs. Excess expression of the Mps1 protein kinase causes wild type cells and ndc10-1 cells to arrest in mitosis. Unlike the spindle checkpoint arrest activated by benzimidazoles, this arrest can be maintained independently of kinetochores. The arrest induced by excess Mps1p is independent of BUB2. Therefore, mitotic arrest induced by excess Mps1p expression is due to the action of the MAD2 branch of the spindle checkpoint pathway and excess Mps1p acts downstream of the kinetochore.