Chmp4c is required for stable kinetochore-microtubule attachments

Formation of stable kinetochore-microtubule attachments is essential for accurate chromosome segregation in human cells and depends on the NDC80 complex. We recently showed that Chmp4c, an endosomal sorting complex required for transport protein involved in membrane remodelling, localises to prometaphase kinetochores and promotes cold-stable kinetochore microtubules, faithful chromosome alignment and segregation. In the present study, we show that Chmp4c associates with the NDC80 components Hec1 and Nuf2 and is required for optimal NDC80 stability and Hec1-Nuf2 localisation to kinetochores in prometaphase. However, Chmp4c-depletion does not cause a gross disassembly of outer or inner kinetochore complexes. Conversely, Nuf2 is required for Chmp4c kinetochore targeting. Constitutive Chmp4c kinetochore tethering partially rescues cold-stable microtubule polymers in cells depleted of the endogenous Nuf2, showing that Chmp4c also contributes to kinetochore-microtubule stability independently of regulating Hec1 and Nuf2 localisation. Chmp4c interacts with tubulin in cell extracts, and binds and bundles microtubules in vitro through its highly basic N-terminal region (amino acids 1–77). Furthermore, the N-terminal region of Chmp4c is required for cold-stable kinetochore microtubules and efficient chromosome alignment. We propose that Chmp4c promotes stable kinetochore-microtubule attachments by regulating Hec1–Nuf2 localisation to kinetochores in prometaphase and by binding to spindle microtubules. These results identify Chmp4c as a novel protein that regulates kinetochore-microtubule interactions to promote accurate chromosome segregation in human cells.     

HURP is a Ran-importin beta-regulated protein that stabilizes kinetochore microtubules in the vicinity of chromosomes

BACKGROUND: Formation of a bipolar mitotic spindle in somatic cells requires the cooperation of two assembly pathways, one based on kinetochore capture by centrosomal microtubules, the other on RanGTP-mediated microtubule organization in the vicinity of chromosomes. How RanGTP regulates kinetochore-microtubule (K-fiber) formation is not presently understood. RESULTS: Here we identify the mitotic spindle protein HURP as a novel target of RanGTP. We show that HURP is a direct cargo of importin beta and that in interphase cells, it shuttles between cytoplasm and nucleus. During mitosis, HURP localizes predominantly to kinetochore microtubules in the vicinity of chromosomes. Overexpression of importin beta or RanT24N (resulting in low RanGTP) negatively regulates its spindle localization, whereas overexpression of RanQ69L (mimicking high RanGTP) enhances HURP association with the spindle. Thus, RanGTP levels control HURP localization to the mitotic spindle in vivo, a conclusion supported by the analysis of tsBN2 cells (mutant in RCC1). Upon depletion of HURP, K-fiber stabilization is impaired and chromosome congression is delayed. Nevertheless, cells eventually align their chromosomes, progress into anaphase, and exit mitosis. HURP is able to bundle microtubules and, in vitro, this function is abolished upon complex formation with importin beta and regulated by Ran. These data indicate that HURP stabilizes K-fibers by virtue of its ability to bind and bundle microtubules. CONCLUSIONS: Our study identifies HURP as a novel component of the Ran-importin beta-regulated spindle assembly pathway, supporting the conclusion that K-fiber formation and stabilization involves both the centrosome-dependent microtubule search and capture mechanism and the RanGTP pathway.     

Coordination of Chromosome Alignment and Mitotic Progression Chromosome-Based Ran Signal

Mitotic spindle assembly and chromosome segregation are controlled by the cell cycle machinery and by the guanosine triphosphatase Ran (RanGTPase). We developed a spatial model that allows us to simulate RanGTP production with different degrees of chromosome alignment in mitosis. Aided by this model, we defined three factors that modulate mitotic RanGTP gradients and mitotic progression in somatic cells. First, the concentration of RanGTPtransport-receptor (represented by RanGTP-importin β) and its spatial distribution are very sensitive to the level of RanBP1. Reduction of RanBP1 leads to an elevated RanGTP-transport receptor concentration throughout the cell, which disrupts spindle assembly and weakens spindle checkpoint control. Second, the completion of chromosome alignment at the metaphase plategenerates highest local RanGTP concentrations on chromosomes that could lead to spindle checkpoint silencing and metaphase-anaphase transition. Finally, chromosomal RanGTP production could be dampened by a reduction of RCC1 phosphorylation in mitosis. Our spatialsimulation of RanGTP production using individual chromosomes should provide means to further understand how the Ran system and the cell cycle machinery coordinately regulate mitosis.     

Dynein prevents erroneous kinetochore-microtubule attachments in mitosis

Equal distribution of the genetic material during cell division relies on efficient congression of chromosomes to the metaphase plate. Prior to their alignment, the Dynein motor recruited to kinetochores transports a fraction of laterally-attached chromosomes along microtubules toward the spindle poles. By doing that, Dynein not only contributes to chromosome movements, but also prevents premature stabilization of end-on kinetochore-microtubule attachments. This is achieved by 2 parallel mechanisms: 1) Dynein-mediated poleward movement of chromosomes counteracts opposite polar-ejection forces (PEFs) on chromosome arms by the microtubule plus-end-directed motors chromokinesins. Otherwise, they could stabilize erroneous syntelic kinetochore-microtubule attachments and lead to the random ejection of chromosomes away from the spindle poles; and 2) By transporting chromosomes to the spindle poles, Dynein brings the former to the zone of highest Aurora A kinase activity, further destabilizing kinetochore-microtubule attachments. Thus, Dynein plays an important role in keeping chromosome segregation error-free by preventing premature stabilization of kinetochore-microtubule attachments near the spindle poles.     

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.     

CENP-E kinesin interacts with SKAP protein to orchestrate accurate chromosome segregation in mitosis

Mitotic chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochore. Although previous studies show that the mitotic kinesin CENP-E forms a link between attachment of the spindle microtubule to the kinetochore and the mitotic checkpoint signaling cascade, the molecular mechanism underlying dynamic kinetochore-microtubule interactions in mammalian cells remains elusive. Here, we identify a novel interaction between CENP-E and SKAP that functions synergistically in governing dynamic kinetochore-microtubule interactions. SKAP binds to the C-terminal tail of CENP-E in vitro and is essential for an accurate kinetochore-microtubule attachment in vivo. Immunoelectron microscopic analysis indicates that SKAP is a constituent of the kinetochore corona fibers of mammalian centromeres. Depletion of SKAP or CENP-E by RNA interference results in a dramatic reduction of inter-kinetochore tension, which causes chromosome mis-segregation with a prolonged delay in achieving metaphase alignment. Importantly, SKAP binds to microtubules in vitro, and this interaction is synergized by CENP-E. Based on these findings, we propose that SKAP cooperates with CENP-E to orchestrate dynamic kinetochore-microtubule interaction for faithful chromosome segregation.     

CENP-U cooperates with Hec1 to orchestrate kinetochore-microtubule attachment

Mitosis is an orchestration of dynamic interaction between chromosomes and spindle microtubules by which genomic materials are equally distributed into two daughter cells. Previous studies showed that CENP-U is a constitutive centromere component essential for proper chromosome segregation. However, the precise molecular mechanism has remained elusive. Here, we identified CENP-U as a novel interacting partner of Hec1, an evolutionarily conserved kinetochore core component essential for chromosome plasticity. Suppression of CENP-U by shRNA resulted in mitotic defects with an impaired kinetochore-microtubule attachment. Interestingly, CENP-U not only binds microtubules directly but also displays a cooperative microtubule binding activity with Hec1 in vitro. Furthermore, we showed that CENP-U is a substrate of Aurora-B. Importantly, phosphorylation of CENP-U leads to reduced kinetochore-microtubule interaction, which contributes to the error-correcting function of Aurora-B. Taken together, our results indicate that CENP-U is a novel microtubule binding protein and plays an important role in kinetochore-microtubule attachment through its interaction with Hec1.     

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.     

Live cell imaging reveals plant aurora kinase has dual roles during mitosis

The proper segregation of chromosomes during mitosis is required for accurate distribution of genetic information by two daughter cells. Here, we used live cell imaging of microtubules and kinetochores after treatment with an Aurora kinase inhibitor, hesperadin, in tobacco BY-2 cells to analyze the function of plant Aurora kinase during mitosis. Hesperadin treatment induced the delay of CenH3 alignment on the spindle equator. Furthermore, two types of dynamics of lagging CenH3s were observed during chromosome segregation. The findings indicate that the plant Aurora kinase has dual roles; correction of aberrant kinetochore-microtubule attachment and dissociation of cohesin during chromosome alignment and segregation.     

Complete kinetochore tracking reveals error-prone homologous chromosome biorientation in mammalian oocytes

Chromosomes must establish stable biorientation prior to anaphase to achieve faithful segregation during cell division. The detailed process by which chromosomes are bioriented and how biorientation is coordinated with spindle assembly and chromosome congression remain unclear. Here, we provide complete 3D kinetochore-tracking datasets throughout cell division by high-resolution imaging of meiosis I in live mouse oocytes. We show that in acentrosomal oocytes, chromosome congression forms an intermediate chromosome configuration, the prometaphase belt, which precedes biorientation. Chromosomes then invade the elongating spindle center to form the metaphase plate and start biorienting. Close to 90% of all chromosomes undergo one or more rounds of error correction of their kinetochore-microtubule attachments before achieving correct biorientation. This process depends on Aurora kinase activity. Our analysis reveals the error-prone nature of homologous chromosome biorientation, providing a possible explanation for the high incidence of aneuploid eggs observed in mammals, including humans.     

Kinetochores' gripping feat: conformational wave or biased diffusion?

Climbing up a cliff while the rope unravels underneath your fingers does not sound like a well-planned adventure. Yet chromosomes face a similar challenge during each cell division. Their alignment and accurate segregation depends on staying attached to the assembling and disassembling tips of microtubule fibers. This coupling is mediated by kinetochores, intricate machines that attach chromosomes to an ever-changing microtubule substrate. Two models for kinetochore-microtubule coupling were proposed a quarter century ago: conformational wave and biased diffusion. These models differ in their predictions for how coupling is performed and regulated. The availability of purified kinetochore proteins has enabled biochemical and biophysical analyses of the kinetochore-microtubule interface. Here, we discuss what these studies reveal about the contributions of each model.     

Formation of stable attachments between kinetochores and microtubules depends on the B56-PP2A phosphatase

Error-free chromosome segregation depends on the precise regulation of phosphorylation to stabilize kinetochore-microtubule attachments (K-fibres) on sister chromatids that have attached to opposite spindle poles (bi-oriented). In many instances, phosphorylation correlates with K-fibre destabilization. Consistent with this, multiple kinases, including Aurora B and Plk1, are enriched at kinetochores of mal-oriented chromosomes when compared with bi-oriented chromosomes, which have stable attachments. Paradoxically, however, these kinases also target to prometaphase chromosomes that have not yet established spindle attachments and it is therefore unclear how kinetochore-microtubule interactions can be stabilized when kinase levels are high. Here we show that the generation of stable K-fibres depends on the B56-PP2A phosphatase, which is enriched at centromeres/kinetochores of unattached chromosomes. When B56-PP2A is depleted, K-fibres are destabilized and chromosomes fail to align at the spindle equator. Strikingly, B56-PP2A depletion increases the level of phosphorylation of Aurora B and Plk1 kinetochore substrates as well as Plk1 recruitment to kinetochores. Consistent with increased substrate phosphorylation, we find that chemical inhibition of Aurora or Plk1 restores K-fibres in B56-PP2A-depleted cells. Our findings reveal that PP2A, an essential tumour suppressor, tunes the balance of phosphorylation to promote chromosome-spindle interactions during cell division.     

Plk1 phosphorylates sgt1 at the kinetochores to promote timely kinetochore-microtubule attachment

Accurate chromosome segregation during cell division maintains genomic integrity and requires the proper establishment of kinetochore-microtubule attachment in mitosis. As a key regulator of mitosis, Polo-like kinase 1 (Plk1) is essential for this attachment process, but the molecular mechanism remains elusive. Here we identify Sgt1, a cochaperone for Hsp90, as a novel Plk1 substrate during mitosis. We show that Sgt1 dynamically localizes at the kinetochores, which lack microtubule attachments during prometaphase. Plk1 is required for the kinetochore localization of Sgt1 and phosphorylates serine 331 of Sgt1 at the kinetochores. This phosphorylation event enhances the association of the Hsp90-Sgt1 chaperone with the MIS12 complex to stabilize this complex at the kinetochores and thus coordinates the recruitment of the NDC80 complex to form efficient microtubule-binding sites. Disruption of Sgt1 phosphorylation reduces the MIS12 and NDC80 complexes at the kinetochores, impairs stable microtubule attachment, and eventually results in chromosome misalignment to delay the anaphase onset. Our results demonstrate a mechanism for Plk1 in promoting kinetochore-microtubule attachment to ensure chromosome stability.     

A dual role for Bub1 in the spindle checkpoint and chromosome congression

The spindle checkpoint ensures faithful chromosome segregation by linking the onset of anaphase to the establishment of bipolar kinetochore-microtubule attachment. The checkpoint is mediated by a signal transduction system comprised of conserved Mad, Bub and other proteins. In this study, we use live-cell imaging coupled with RNA interference to investigate the functions of human Bub1. We find that Bub1 is essential for checkpoint control and for correct chromosome congression. Bub1 depletion leads to the accumulation of misaligned chromatids in which both sister kinetochores are linked to microtubules in an abnormal fashion, a phenotype that is unique among Mad and Bub depletions. Bub1 is similar to the Aurora B/Ipl1p kinase in having roles in both the checkpoint and microtubule binding. However, human Bub1 and Aurora B are recruited to kinetochores independently of each other and have an additive effect when depleted simultaneously. Thus, Bub1 and Aurora B appear to function in parallel pathways that promote formation of stable bipolar kinetochore-microtubule attachments.     

A mechanism of coupling RCC1 mobility to RanGTP production on the chromatin in vivo

The RanGTP gradient across the interphase nuclear envelope and on the condensed mitotic chromosomes is essential for many cellular processes, including nucleocytoplasmic transport and spindle assembly. Although the chromosome-associated enzyme RCC1 is responsible for RanGTP production, the mechanism of generating and maintaining the RanGTP gradient in vivo remains unknown. Here, we report that regulator of chromosome condensation (RCC1) rapidly associates and dissociates with both interphase and mitotic chromosomes in living cells, and that this mobility is regulated during the cell cycle. Our kinetic modeling suggests that RCC1 couples its catalytic activity to chromosome binding to generate a RanGTP gradient. Indeed, we have demonstrated experimentally that the interaction of RCC1 with the chromatin is coupled to the nucleotide exchange on Ran in vivo. The coupling is due to the stable binding of the binary complex of RCC1-Ran to chromatin. Successful nucleotide exchange dissociates the binary complex, permitting the release of RCC1 and RanGTP from the chromatin and the production of RanGTP on the chromatin surface.     

Lateral and End-On Kinetochore Attachments Are Coordinated to Achieve Bi-orientation in Drosophila Oocytes

In oocytes, where centrosomes are absent, the chromosomes direct the assembly of a bipolar spindle. Interactions between chromosomes and microtubules are essential for both spindle formation and chromosome segregation, but the nature and function of these interactions is not clear. We have examined oocytes lacking two kinetochore proteins, NDC80 and SPC105R, and a centromere-associated motor protein, CENP-E, to characterize the impact of kinetochore-microtubule attachments on spindle assembly and chromosome segregation in Drosophila oocytes. We found that the initiation of spindle assembly results from chromosome-microtubule interactions that are kinetochore-independent. Stabilization of the spindle, however, depends on both central spindle and kinetochore components. This stabilization coincides with changes in kinetochore-microtubule attachments and bi-orientation of homologs. We propose that the bi-orientation process begins with the kinetochores moving laterally along central spindle microtubules towards their minus ends. This movement depends on SPC105R, can occur in the absence of NDC80, and is antagonized by plus-end directed forces from the CENP-E motor. End-on kinetochore-microtubule attachments that depend on NDC80 are required to stabilize bi-orientation of homologs. A surprising finding was that SPC105R but not NDC80 is required for co-orientation of sister centromeres at meiosis I. Together, these results demonstrate that, in oocytes, kinetochore-dependent and -independent chromosome-microtubule attachments work together to promote the accurate segregation of chromosomes.     

Abnormal kinetochore-generated pulling forces from expressing a N-terminally modified Hec1

Background

Highly Expressed in Cancer protein 1 (Hec1) is a constituent of the Ndc80 complex, a kinetochore component that has been shown to have a fundamental role in stable kinetochore-microtubule attachment, chromosome alignment and spindle checkpoint activation at mitosis. HEC1 RNA is found up-regulated in several cancer cells, suggesting a role for HEC1 deregulation in cancer. In light of this, we have investigated the consequences of experimentally-driven Hec1 expression on mitosis and chromosome segregation in an inducible expression system from human cells.

Methodology/Principal Findings

Overexpression of Hec1 could never be obtained in HeLa clones inducibly expressing C-terminally tagged Hec1 or untagged Hec1, suggesting that Hec1 cellular levels are tightly controlled. On the contrary, a chimeric protein with an EGFP tag fused to the Hec1 N-terminus accumulated in cells and disrupted mitotic division. EGFP- Hec1 cells underwent altered chromosome segregation within multipolar spindles that originated from centriole splitting. We found that EGFP-Hec1 assembled a mutant Ndc80 complex that was unable to rescue the mitotic phenotypes of Hec1 depletion. Kinetochores harboring EGFP-Hec1 formed persisting lateral microtubule-kinetochore interactions that recruited the plus-end depolymerase MCAK and the microtubule stabilizing protein HURP on K-fibers. In these conditions the plus-end kinesin CENP-E was preferentially retained at kinetochores. RNAi-mediated CENP-E depletion further demonstrated that CENP-E function was required for multipolar spindle formation in EGFP-Hec1 expressing cells.

Conclusions/Significance

Our study suggests that modifications on Hec1 N-terminal tail can alter kinetochore-microtubule attachment stability and influence Ndc80 complex function independently from the intracellular levels of the protein. N-terminally modified Hec1 promotes spindle pole fragmentation by CENP-E-mediated plus-end directed kinetochore pulling forces that disrupt the fine balance of kinetochore- and centrosome-associated forces regulating spindle bipolarity. Overall, our findings support a model in which centrosome integrity is influenced by the pathways regulating kinetochore-microtubule attachment stability.     

Virtual breakdown of the nuclear envelope in fission yeast meiosis

Asymmetric localization of Ran regulators (RanGAP1 and RanGEF/RCC1) produces a gradient of RanGTP across the nuclear envelope. In higher eukaryotes, the nuclear envelope breaks down as the cell enters mitosis (designated "open" mitosis). This nuclear envelope breakdown (NEBD) leads to collapse of the RanGTP gradient and the diffusion of nuclear and cytoplasmic macromolecules in the cell, resulting in irreversible progression of the cell cycle. On the other hand, in many fungi, chromosome segregation takes place without NEBD (designated "closed" mitosis). Here we report that in the fission yeast Schizosaccharomyces pombe, despite the nuclear envelope and the nuclear pore complex remaining intact throughout both the meiotic and mitotic cell cycles, nuclear proteins diffuse into the cytoplasm transiently for a few minutes at the onset of anaphase of meiosis II. We also found that nuclear protein diffusion into the cytoplasm occurred coincidently with nuclear localization of Rna1, an S. pombe RanGAP1 homolog that is usually localized in the cytoplasm. These results suggest that nuclear localization of RanGAP1 and depression of RanGTP activity in the nucleus may be mechanistically tied to meiosis-specific diffusion of nuclear proteins into the cytoplasm. This nucleocytoplasmic shuffling of RanGAP1 and nuclear proteins represents virtual breakdown of the nuclear envelope.     

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.     

Correcting improper chromosome-spindle attachments during cell division

For accurate segregation of chromosomes during cell division, microtubule fibres must attach sister kinetochores to opposite poles of the mitotic spindle (bi-orientation). Aurora kinases are linked to oncogenesis and have been implicated in the regulation of chromosome-microtubule attachments. Although loss of Aurora kinase activity causes an accumulation of mal-orientated chromosomes in dividing cells, it is not known how the active kinase corrects improper chromosome attachments. The use of reversible small-molecule inhibitors allows activation of protein function in living vertebrate cells with temporal control. Here we show that by removal of small-molecule inhibitors, controlled activation of Aurora kinase during mitosis can correct chromosome attachment errors by selective disassembly of kinetochore-microtubule fibres, rather than by alternative mechanisms involving initial release of microtubules from either kinetochores or spindle poles. Observation of chromosomes and microtubule dynamics with real-time high-resolution microscopy showed that mal-orientated, but not bi-orientated, chromosomes move to the spindle pole as both kinetochore-microtubule fibres shorten, followed by alignment at the metaphase plate. Our results provide direct evidence for a mechanism required for the maintenance of genome integrity during cell division.