The constitutive centromere component CENP-50 is required for recovery from spindle damage

We identified CENP-50 as a novel kinetochore component. We found that CENP-50 is a constitutive component of the centromere that colocalizes with CENP-A and CENP-H throughout the cell cycle in vertebrate cells. To determine the precise role of CENP-50, we examined its role in centromere function by generating a loss-of-function mutant in the chicken DT40 cell line. The CENP-50 knockout was not lethal; however, the growth rate of cells with this mutation was slower than that of wild-type cells. We observed that the time for CENP-50-deficient cells to complete mitosis was longer than that for wild-type cells. Centromeric localization of CENP-50 was abolished in both CENP-H- and CENP-I-deficient cells. Coimmunoprecipitation experiments revealed that CENP-50 interacted with the CENP-H/CENP-I complex in chicken DT40 cells. We also observed severe mitotic defects in CENP-50-deficient cells with apparent premature sister chromatid separation when the mitotic checkpoint was activated, indicating that CENP-50 is required for recovery from spindle damage.     

The ABCs of CENPs

Equal distribution of DNA in mitosis requires the assembly of a large proteinaceous ensemble onto the centromeric DNA, called the kinetochore. With few exceptions, kinetochore specification is independent of the DNA sequence and is determined epigenetically by deposition at the centromeric chromatin of special nucleosomes containing an H3-related histone, CENP-A. Onto centromeric CENP-A chromatin is assembled the so-called constitutive centromere-associated network (CCAN) of 16 proteins distributed in several functional groups as follows: CENP-C, CENP-H/CENP-I/CENP-K/, CENP-L/CENP-M/CENP-N, CENP-O/CENP-P/CENP-Q/CENP-R/CENP-U(50), CENP-T/CENP-W, and CENP-S/CENP-X. One role of the CCAN is to recruit outer kinetochore components further, such as KNL1, the Mis12 complex, and the Ndc80 complex (KMN network) to which attach the spindle microtubules with their structural and regulatory proteins. Among the CENPs in CCAN, CENP-C and CENP-T are required in parallel for operational kinetochore specification and spindle attachment. This review presents discussion of the latest structural and functional data on CENP-A and CENPs from the CCAN as well as their interaction with the KMN network.     

Whole-proteome genetic analysis of dependencies in assembly of a vertebrate kinetochore

Kinetochores orchestrate mitotic chromosome segregation. Here, we use quantitative mass spectrometry of mitotic chromosomes isolated from a comprehensive set of chicken DT40 mutants to examine the dependencies of 93 confirmed and putative kinetochore proteins for stable association with chromosomes. Clustering and network analysis reveal both known and unexpected aspects of coordinated behavior for members of kinetochore protein complexes. Surprisingly, CENP-T depends on CENP-N for chromosome localization. The Ndc80 complex exhibits robust correlations with all other complexes in a “core” kinetochore network. Ndc80 associated with CENP-T interacts with a cohort of Rod, zw10, and zwilch (RZZ)–interacting proteins that includes Spindly, Mad1, and CENP-E. This complex may coordinate microtubule binding with checkpoint signaling. Ndc80 associated with CENP-C forms the KMN (Knl1, Mis12, Ndc80) network and may be the microtubule-binding “workhorse” of the kinetochore. Our data also suggest that CENP-O and CENP-R may regulate the size of the inner kinetochore without influencing the assembly of the outer kinetochore.     

The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation

During cell division, kinetochores form the primary chromosomal attachment sites for spindle microtubules. We previously identified a network of 10 interacting kinetochore proteins conserved between Caenorhabditis elegans and humans. In this study, we investigate three proteins in the human network (hDsn1Q9H410, hNnf1PMF1, and hNsl1DC31). Using coexpression in bacteria and fractionation of mitotic extracts, we demonstrate that these proteins form a stable complex with the conserved kinetochore component hMis12. Human or chicken cells depleted of Mis12 complex subunits are delayed in mitosis with misaligned chromosomes and defects in chromosome biorientation. Aligned chromosomes exhibited reduced centromere stretch and diminished kinetochore microtubule bundles. Consistent with this, localization of the outer plate constituent Ndc80HEC1 was severely reduced. The checkpoint protein BubR1, the fibrous corona component centromere protein (CENP) E, and the inner kinetochore proteins CENP-A and CENP-H also failed to accumulate to wild-type levels in depleted cells. These results indicate that a four-subunit Mis12 complex plays an essential role in chromosome segregation in vertebrates and contributes to mitotic kinetochore assembly.     

The SUMO protease SENP6 is essential for inner kinetochore assembly

We have analyzed the mitotic function of SENP6, a small ubiquitin-like modifier (SUMO) protease that disassembles conjugated SUMO-2/3 chains. Cells lacking SENP6 showed defects in spindle assembly and metaphase chromosome congression. Analysis of kinetochore composition in these cells revealed that a subset of proteins became undetectable on inner kinetochores after SENP6 depletion, particularly the CENP-H/I/K complex, whereas other changes in kinetochore composition mimicked defects previously reported to result from CENP-H/I/K depletion. We further found that CENP-I is degraded through the action of RNF4, a ubiquitin ligase which targets polysumoylated proteins for proteasomal degradation, and that SENP6 stabilizes CENP-I by antagonizing RNF4. Together, these findings reveal a novel mechanism whereby the finely balanced activities of SENP6 and RNF4 control vertebrate kinetochore assembly through SUMO-targeted destabilization of inner plate components.     

The CENP-A NAC/CAD kinetochore complex controls chromosome congression and spindle bipolarity

Kinetochores are complex protein machines that link chromosomes to spindle microtubules and contain a structural core composed of two conserved protein-protein interaction networks: the well-characterized KMN (KNL1/MIND/NDC80) and the recently identified CENP-A NAC/CAD. Here we show that the CENP-A NAC/CAD subunits can be assigned to one of two different functional classes; depletion of Class I proteins (Mcm21R(CENP-O) and Fta1R(CENP-L)) causes a failure in bipolar spindle assembly. In contrast, depletion of Class II proteins (CENP-H, Chl4R(CENP-N), CENP-I and Sim4R(CENP-K)) prevents binding of Class I proteins and causes chromosome congression defects, but does not perturb spindle formation. Co-depletion of Class I and Class II proteins restores spindle bipolarity, suggesting that Class I proteins regulate or counteract the function of Class II proteins. We also demonstrate that CENP-A NAC/CAD and KMN regulate kinetochore-microtubule attachments independently, even though CENP-A NAC/CAD can modulate NDC80 levels at kinetochores. Based on our results, we propose that the cooperative action of CENP-A NAC/CAD subunits and the KMN network drives efficient chromosome segregation and bipolar spindle assembly during mitosis.     

CENP-A is required for accurate chromosome segregation and sustained kinetochore association of BubR1

CENP-A is an evolutionarily conserved, centromere-specific variant of histone H3 that is thought to play a central role in directing kinetochore assembly and in centromere function. Here, we have analyzed the consequences of disrupting the CENP-A gene in the chicken DT40 cell line. In CENP-A-depleted cells, kinetochore protein assembly is impaired, as indicated by mislocalization of the inner kinetochore proteins CENP-I, CENP-H, and CENP-C as well as the outer components Nuf2/Hec1, Mad2, and CENP-E. However, BubR1 and the inner centromere protein INCENP are efficiently recruited to kinetochores. Following CENP-A depletion, chromosomes are deficient in proper congression on the mitotic spindle and there is a transient delay in prometaphase. CENP-A-depleted cells further proceed through anaphase and cytokinesis with unequal chromosome segregation, suggesting that some kinetochore function remains following substantial depletion of CENP-A. We furthermore demonstrate that CENP-A-depleted cells exhibit a specific defect in maintaining kinetochore localization of the checkpoint protein BubR1 under conditions of checkpoint activation. Our data thus point to a specific role for CENP-A in assembly of kinetochores competent in the maintenance of mitotic checkpoint signaling.     

RNAi knockdown of human kinetochore protein CENP-H

The inner kinetochore protein complex binds to centromeres during the whole cell cycle. It serves as the basis for the binding of further kinetochore proteins during mitosis. CENP-H is one of the inner kinetochore proteins which is conserved amongst many eukaryotes. By specific RNAi knockdown, we reduced the CENP-H protein level in human HEp-2 cells down to less than 5% of its normal value. In these CENP-H knocked-down cells, we observed severe mitotic phenotypes like misaligned chromosomes and multipolar spindles, however, no mitotic arrest. Strong reduction of CENP-H resulted in a slightly reduced CENP-C level at the kinetochores and normal localisation of hBubR1, indicating a functional mitotic checkpoint at the hBubR1 protein level. In CENP-H knocked-down human cells, the misaligned chromosomes contained only reduced levels of CENP-E. Our data clearly indicate that CENP-H has an important impact on the architecture and function of the human kinetochore complex.     

The functional region of CENP-H interacts with the Nuf2 complex that localizes to centromere during mitosis

CENP-H is a constitutive centromere component that localizes to the centromere throughout the cell cycle. Because CENP-H is colocalized with CENP-A and CENP-C, it is thought to be an inner centromere protein. We previously generated a conditional loss-of-function mutant of CENP-H and showed that CENP-H is required for targeting of CENP-C to the centromere in chicken DT40 cells. In the present study, we used this mutant to identify the functional region of chicken CENP-H necessary for centromere targeting and cell viability. This region was found by yeast two-hybrid analysis to interact with Hec1, which is a member of the Nuf2 complex that transiently localizes to the centromere during mitosis. Coimmunoprecipitation experiments revealed that CENP-H interacts with the Nuf2 complex in chicken DT40 cells. Photobleaching experiments showed that both Hec1 and CENP-H form stable associations with the centromeres during mitosis, suggesting that Hec1 acts as a structural component of centromeres during mitosis. On the basis of these results and previously published data, we propose that the Nuf2 complex functions as a connector between the inner and outer kinetochores.     

Scc1/Rad21/Mcd1 is required for sister chromatid cohesion and kinetochore function in vertebrate cells.

Proteolytic cleavage of the cohesin subunit Scc1 is a consistent feature of anaphase onset, although temporal differences exist between eukaryotes in cohesin loss from chromosome arms, as distinct from centromeres. We describe the effects of genetic deletion of Scc1 in chicken DT40 cells. Scc1 loss caused premature sister chromatid separation but did not disrupt chromosome condensation. Scc1 mutants showed defective repair of spontaneous and induced DNA damage. Scc1-deficient cells frequently failed to complete metaphase chromosome alignment and showed chromosome segregation defects, suggesting aberrant kinetochore function. Notably, the chromosome passenger INCENP did not localize normally to centromeres, while the constitutive kinetochore proteins CENP-C and CENP-H behaved normally. These results suggest a role for Scc1 in mitotic regulation, along with cohesion.     

Drosophila CENP-A mutations cause a BubR1-dependent early mitotic delay without normal localization of kinetochore components

The centromere/kinetochore complex plays an essential role in cell and organismal viability by ensuring chromosome movements during mitosis and meiosis. The kinetochore also mediates the spindle attachment checkpoint (SAC), which delays anaphase initiation until all chromosomes have achieved bipolar attachment of kinetochores to the mitotic spindle. CENP-A proteins are centromere-specific chromatin components that provide both a structural and a functional foundation for kinetochore formation. Here we show that cells in Drosophila embryos homozygous for null mutations in CENP-A (CID) display an early mitotic delay. This mitotic delay is not suppressed by inactivation of the DNA damage checkpoint and is unlikely to be the result of DNA damage. Surprisingly, mutation of the SAC component BUBR1 partially suppresses this mitotic delay. Furthermore, cid mutants retain an intact SAC response to spindle disruption despite the inability of many kinetochore proteins, including SAC components, to target to kinetochores. We propose that SAC components are able to monitor spindle assembly and inhibit cell cycle progression in the absence of sustained kinetochore localization.     

Bub3p Facilitates Spindle Checkpoint Silencing in Fission Yeast

Although critical for spindle checkpoint signaling, the role kinetochores play in anaphase promoting complex (APC) inhibition remains unclear. Here we show that spindle checkpoint proteins are severely depleted from unattached kinetochores in fission yeast cells lacking Bub3p. Surprisingly, a robust mitotic arrest is maintained in the majority of bub3Δ cells, yet they die, suggesting that Bub3p is essential for successful checkpoint recovery. During recovery, two defects are observed: (1) cells mis-segregate chromosomes and (2) anaphase onset is significantly delayed. We show that Bub3p is required to activate the APC upon inhibition of Aurora kinase activity in checkpoint-arrested cells, suggesting that Bub3p is required for efficient checkpoint silencing downstream of Aurora kinase. Together, these results suggest that spindle checkpoint signals can be amplified in the nucleoplasm, yet kinetochore localization of spindle checkpoint components is required for proper recovery from a spindle checkpoint-dependent arrest.     

CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells

CENP-H has recently been discovered as a constitutive component of the centromere that co-localizes with CENP-A and CENP-C throughout the cell cycle. The precise function, however, remains poorly understood. We examined the role of CENP-H in centromere function and assembly by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-H, cell cycle arrest at metaphase, consistent with loss of centromere function, was observed. Immunocytochemical analysis of the CENP-H-deficient cells demonstrated that CENP-H is necessary for CENP-C, but not CENP-A, localization to the centromere. These findings indicate that centromere assembly in vertebrate cells proceeds in a hierarchical manner in which localization of the centromere-specific histone CENP-A is an early event that occurs independently of CENP-C and CENP-H.     

The CENP-H-I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres

In vertebrates, centromeres lack defined sequences and are thought to be propagated by epigenetic mechanisms involving the incorporation of specialized nucleosomes containing the histone H3 variant centromere protein (CENP)-A. However, the precise mechanisms that target CENP-A to centromeres remain poorly understood. Here, we isolated a multi-subunit complex, which includes the established inner kinetochore components CENP-H and CENP-I, and nine other proteins, from both human and chicken cells. Our analysis of these proteins demonstrates that the CENP-H-I complex can be divided into three functional sub-complexes, each of which is required for faithful chromosome segregation. Interestingly, newly expressed CENP-A is not efficiently incorporated into centromeres in knockout mutants of a subclass of CENP-H-I complex proteins, indicating that the CENP-H-I complex may function, in part, as a marker directing CENP-A deposition to centromeres.     

Mitotic Protein CSPP1 Interacts with CENP-H Protein to Coordinate Accurate Chromosome Oscillation in Mitosis

Mitotic chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochores. During chromosome alignment, kinetochore-bound microtubules undergo dynamic cycles between growth and shrinkage, leading to an oscillatory movement of chromosomes along the spindle axis. Although kinetochore protein CENP-H serves as a molecular control of kinetochore-microtubule dynamics, the mechanistic link between CENP-H and kinetochore microtubules (kMT) has remained less characterized. Here, we show that CSPP1 is a kinetochore protein essential for accurate chromosome movements in mitosis. CSPP1 binds to CENP-H in vitro and in vivo. Suppression of CSPP1 perturbs proper mitotic progression and compromises the satisfaction of spindle assembly checkpoint. In addition, chromosome oscillation is greatly attenuated in CSPP1-depleted cells, similar to what was observed in the CENP-H-depleted cells. Importantly, CSPP1 depletion enhances velocity of kinetochore movement, and overexpression of CSPP1 decreases the speed, suggesting that CSPP1 promotes kMT stability during cell division. Specific perturbation of CENP-H/CSPP1 interaction using a membrane-permeable competing peptide resulted in a transient mitotic arrest and chromosome segregation defect. Based on these findings, we propose that CSPP1 cooperates with CENP-H on kinetochores to serve as a novel regulator of kMT dynamics for accurate chromosome segregation.     

Differential requirements for the CENP-O complex reveal parallel PLK1 kinetochore recruitment pathways

Similar to other core biological processes, the vast majority of cell division components are essential for viability across human cell lines. However, recent genome-wide screens have identified a number of proteins that exhibit cell line–specific essentiality. Defining the behaviors of these proteins is critical to our understanding of complex biological processes. Here, we harness differential essentiality to reveal the contributions of the four-subunit centromere-localized CENP-O complex, whose precise function has been difficult to define. Our results support a model in which the CENP-O complex and BUB1 act in parallel pathways to recruit a threshold level of PLK1 to mitotic kinetochores, ensuring accurate chromosome segregation. We demonstrate that targeted changes to either pathway sensitizes cells to the loss of the other component, resulting in cell-state dependent requirements. This approach also highlights the advantage of comparing phenotypes across diverse cell lines to define critical functional contributions and behaviors that could be exploited for the targeted treatment of disease.     

Clathrin is spindle-associated but not essential for mitosis

Background

Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis.

Methodology/Principal Findings

Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression.

Conclusions/Significance

We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the localisation but not the impact of clathrin on mitosis appears to be robust in plants, mammalian and chicken B-cells.