Loss of KLP-19 polar ejection force causes misorientation and missegregation of holocentric chromosomes

Holocentric chromosomes assemble kinetochores along their length instead of at a focused spot. The elongated expanse of an individual holocentric kinetochore and its potential flexibility heighten the risk of stable attachment to microtubules from both poles of the mitotic spindle (merotelic attachment), and hence aberrant segregation of chromosomes. Little is known about the mechanisms that holocentric species have evolved to avoid this type of error. Our studies of the influence of KLP-19, an essential microtubule motor, on the behavior of holocentric Caenorhabditis elegans chromosomes suggest that it has a major role in combating merotelic attachments. Depletion of KLP-19, which associates with nonkinetochore chromatin, allows aberrant poleward chromosome motion during prometaphase, misalignment of holocentric kinetochores, and multiple anaphase chromosome bridges in all mitotic divisions. Time-lapse movies of GFP-labeled mono- and bipolar spindles demonstrate that KLP-19 generates a force on relatively stiff holocentric chromosomes that pushes them away from poles. We hypothesize that this polar ejection force minimizes merotelic misattachment by maintaining a constant tension on pole-kinetochore connections throughout prometaphase, tension that compels sister kinetochores to face directly toward opposite poles.     

Differential role of CENP-A in the segregation of holocentric C. elegans chromosomes during meiosis and mitosis

Two distinct chromosome architectures are prevalent among eukaryotes: monocentric, in which localized centromeres restrict kinetochore assembly to a single chromosomal site, and holocentric, in which diffuse kinetochores form along the entire chromosome length. During mitosis, both chromosome types use specialized chromatin, containing the histone H3 variant CENP-A, to direct kinetochore assembly. For the segregation of recombined homologous chromosomes during meiosis, monocentricity is thought to be crucial for limiting spindle-based forces to one side of a crossover and to prevent recombined chromatids from being simultaneously pulled towards both spindle poles. The mechanisms that allow holocentric chromosomes to avert this fate remain uncharacterized. Here, we show that markedly different mechanisms segregate holocentric chromosomes during meiosis and mitosis in the nematode Caenorhabditis elegans. Immediately prior to oocyte meiotic segregation, outer-kinetochore proteins were recruited to cup-like structures on the chromosome surface via a mechanism that is independent of CENP-A. In striking contrast to mitosis, both oocyte meiotic divisions proceeded normally following depletion of either CENP-A or the closely associated centromeric protein CENP-C. These findings highlight a pronounced difference between the segregation of holocentric chromosomes during meiosis and mitosis and demonstrate the potential to uncouple assembly of outer-kinetochore proteins from CENP-A chromatin.     

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.     

Kinetochores and chromatin diminution in early embryos of Parascaris univalens

In Parascaris the mitotic chromosomes of gonial germline cells are holocentric and possess a continuous kinetochore along their entire length. By contrast, in meiotic cells, the centromeric activity is restricted to the heterochromatic tips where direct insertion of spindle microtubules into chromatin without any kinetochore plate is seen. In the presomatic cells of early embryos, which undergo heterochromatin elimination, only euchromatin shows kinetic activity. After developing a technique to separate the very resistant egg shell from the embryos, we studied the cell divisions during early embryogenesis by immunochemical and EM approaches. The results reported here show that in presomatic cells microtubules bind only the euchromatin where a continuous kinetochore plate is present. We also report observations suggesting that the binding of the long kinetochores to the mitotic spindle initiates to a limited number of sites and extends along the entire length, during chromosome condensation. The existence of different centromere stages in different cell types, rends Parascaris chromosomes a very good model to study centromere organization.     

Inhibition of aurora B kinase blocks chromosome segregation,overrides the spindle checkpoint,and perturbs microtubule dynamics in mitosis

How kinetochores correct improper microtubule attachments and regulate the spindle checkpoint signal is unclear. In budding yeast, kinetochores harboring mutations in the mitotic kinase Ipl1 fail to bind chromosomes in a bipolar fashion. In C. elegans and Drosophila, inhibition of the Ipl1 homolog, Aurora B kinase, induces aberrant anaphase and cytokinesis. To study Aurora B kinase in vertebrates, we microinjected mitotic XTC cells with inhibitory antibody and found several related effects. After injection of the antibody, some chromosomes failed to congress to the metaphase plate, consistent with a conserved role for Aurora B in bipolar attachment of chromosomes. Injected cells exited mitosis with no evidence of anaphase or cytokinesis. Injection of anti-Xaurora B antibody also altered the microtubule network in mitotic cells with an extension of the astral microtubules and a reduction of kinetochore microtubules. Finally, inhibition of Aurora B in cultured cells and in cycling Xenopus egg extracts caused escape from the spindle checkpoint arrest induced by microtubule drugs. Our findings implicate Aurora B as a critical coordinator relating changes in microtubule dynamics in mitosis, chromosome movement in prometaphase and anaphase, signaling of the spindle checkpoint, and cytokinesis.     

How do kinetochores CLASP dynamic microtubules?

Maintenance of genetic stability during cell division requires binding of chromosomes to the mitotic spindle, a process that involves attachment of spindle microtubules to kinetochores. This enables chromosomes to move to the metaphase plate, to satisfy the spindle checkpoint and finally to segregate during anaphase. Recent studies on the function MAST in Drosophila and its human homologue CLASP1, have revealed that these microtubule-associated proteins play an essential role for the kinetochore-microtubule interaction. CLASP1 localizes to the plus ends of growing microtubules and to the most external kinetochore domain. Depletion of CLASP1 causes abnormal chromosome congression, collapse of the mitotic spindle and attachment of kinetochores to very short microtubules that do not show dynamic behavior. These results suggest that CLASP1 is required at kinetochores to regulate the dynamic behavior of attached microtubules.     

How Do Kinetochores CLASP Dynamic Microtubules?

Maintenance of genetic stability during cell division requires binding of chromosomes to the mitotic spindle, a process that involves attachment of spindle microtubules to kinetochores. This enables chromosomes to move to the metaphase plate, to satisfy the spindle checkpoint and finally to segregate during anaphase. Recent studies on the function MAST in Drosophila and its human homologue CLASP1, have revealed that these microtubule-associated proteins play an essential role for the kinetochore-microtubule interaction. CLASP1 localizesto the plus ends of growing microtubules and to the most external kinetochore domain. Depletion of CLASP1 causes abnormal chromosome congression, collapse of the mitotic spindle and attachment of kinetochores to very short microtubules that do not show dynamic behavior. These results suggest that CLASP1 is required at kinetochores to regulate the dynamic behavior of attached microtubules.     

A Highlights from MBoC Selection: Ipl1/Aurora-B is necessary for kinetochore restructuring in meiosis I in Saccharomyces cerevisiae

In mitosis, the centromeres of sister chromosomes are pulled toward opposite poles of the spindle. In meiosis I, the opposite is true: the sister centromeres move together to the same pole, and the homologous chromosomes are pulled apart. This change in segregation patterns demands that between the final mitosis preceding meiosis and the first meiotic division, the kinetochores must be restructured. In budding yeast, unlike mammals, kinetochores are largely stable throughout the mitotic cycle. In contrast, previous work with budding and fission yeast showed that some outer kinetochore proteins are lost in early meiosis. We use quantitative mass spectrometry methods and imaging approaches to explore the kinetochore restructuring process that occurs in meiosis I in budding yeast. The Ndc80 outer kinetochore complex, but not other subcomplexes, is shed upon meiotic entry. This shedding is regulated by the conserved protein kinase Ipl1/Aurora-B and promotes the subsequent assembly of a kinetochore that will confer meiosis-specific segregation patterns on the chromosome.     

A spindle checkpoint functions during mitosis in the early Caenorhabditis elegans embryo

During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis.     

Identification of two novel components of the human NDC80 kinetochore complex

Proper kinetochore function is essential for the accurate segregation of chromosomes during mitosis. Kinetochores provide the attachment sites for spindle microtubules and are required for the alignment of chromosomes at the metaphase plate (chromosome congression). Components of the conserved NDC80 complex are required for chromosome congression, and their disruption results in mitotic arrest accompanied by multiple spindle aberrations. To better understand the function of the NDC80 complex, we have identified two novel subunits of the human NDC80 complex, termed human SPC25 (hSPC25) and human SPC24 (hSPC24), using an immunoaffinity approach. hSPC25 interacted with HEC1 (human homolog of yeast Ndc80) throughout the cell cycle and localized to kinetochores during mitosis. RNA interference-mediated depletion of hSPC25 in HeLa cells caused aberrant mitosis, followed by cell death, a phenotype similar to that of cells depleted of HEC1. Loss of hSPC25 also caused multiple spindle aberrations, including elongated, multipolar, and fractured spindles. In the absence of hSPC25, MAD1 and HEC1 failed to localize to kinetochores during mitosis, whereas the kinetochore localization of BUB1 and BUBR1 was largely unaffected. Interestingly, the kinetochore localization of MAD1 in cells with a compromised NDC80 function was restored upon microtubule depolymerization. Thus, hSPC25 is an essential kinetochore component that plays a significant role in proper execution of mitotic events.     

Anaphase spindle mechanics prevent mis-segregation of merotelically oriented chromosomes

Merotelic kinetochore orientation is a kinetochore misattachment in which a single kinetochore is attached to microtubules from both spindle poles instead of just one. It can be favored in specific circumstances, is not detected by the mitotic checkpoint, and induces lagging chromosomes in anaphase. In mammalian cells, it occurs at high frequency in early mitosis, but few anaphase cells show lagging chromosomes. We developed live-cell imaging methods to determine whether and how the mitotic spindle prevents merotelic kinetochores from producing lagging chromosomes. We found that merotelic kinetochores entering anaphase never lost attachment to the spindle poles; they remained attached to both microtubule bundles, but this did not prevent them from segregating correctly. The two microtubule bundles usually showed different fluorescence intensities, the brighter bundle connecting the merotelic kinetochore to the correct pole. During anaphase, the dimmer bundle lengthened much more than the brighter bundle as spindle elongation occurred. This resulted in correct segregation of the merotelically oriented chromosome. We propose a model based on the ratios of microtubules to the correct versus incorrect pole for how anaphase spindle dynamics and microtubule polymerization at kinetochores prevent potential segregation errors deriving from merotelic kinetochore orientation.     

Human centromere chromatin protein hMis12, essential for equal segregation,is independent of CENP-A loading pathway

Kinetochores are the chromosomal sites for spindle interaction and play a vital role for chromosome segregation. The composition of kinetochore proteins and their cellular roles are, however, poorly understood in higher eukaryotes. We identified a novel kinetochore protein family conserved from yeast to human that is essential for equal chromosome segregation. The human homologue hMis12 of yeast spMis12/scMtw1 retains conserved sequence features and locates at the kinetochore region indistinguishable from CENP-A, a centromeric histone variant. RNA interference (RNAi) analysis of HeLa cells shows that the reduced hMis12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei without mitotic delay, while CENP-A is located at kinetochores. Further, the metaphase spindle length is abnormally extended. Spindle checkpoint protein hMad2 temporally localizes at kinetochores at early mitotic stages after RNAi. The RNAi deficiency of CENP-A leads to a similar mitotic phenotype, but the kinetochore signals of other kinetochore proteins, hMis6 and CENP-C, are greatly diminished. RNAi for hMis6, like that of a kinetochore kinesin CENP-E, induces mitotic arrest. Kinetochore localization of hMis12 is unaffected by CENP-A RNAi, demonstrating an independent pathway of CENP-A in human kinetochores.     

Connecting up and clearing out: how kinetochore attachment silences the spindle assembly checkpoint

With the goal of creating two genetically identical daughter cells, cell division culminates in the equal segregation of sister chromatids. This phase of cell division is monitored by a cell cycle checkpoint known as the spindle assembly checkpoint (SAC). The SAC actively prevents chromosome segregation while one or more chromosomes, or more accurately kinetochores, remain unattached to the mitotic spindle. Such unattached kinetochores recruit SAC proteins to assemble a diffusible anaphase inhibitor. Kinetochores stop production of this inhibitor once microtubules (MTs) of the mitotic spindle are bound, but productive attachment of all kinetochores is required to satisfy the SAC, initiate anaphase, and exit from mitosis. Although mechanisms of kinetochore signaling and SAC inhibitor assembly and function have received the bulk of attention in the past two decades, recent work has focused on the principles of SAC silencing. Here, we review the mechanisms that silence SAC signaling at the kinetochore, and in particular, how attachment to spindle MTs and biorientation on the mitotic spindle may turn off inhibitor generation. Future challenges in this area are highlighted towards the goal of building a comprehensive molecular model of this process.     

Septin 7 interacts with centromere-associated protein E and is required for its kinetochore localization

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore. Septin (SEPT) belongs to a conserved family of polymerizing GTPases localized to the metaphase spindle during mitosis. Previous study showed that SEPT2 depletion results in chromosome mis-segregation correlated with a loss of centromere-associated protein E (CENP-E) from the kinetochores of congressing chromosomes (1). However, it has remained elusive as to whether CENP-E physically interacts with SEPT and how this interaction orchestrates chromosome segregation in mitosis. Here we show that SEPT7 is required for a stable kinetochore localization of CENP-E in HeLa and MDCK cells. SEPT7 stabilizes the kinetochore association of CENP-E by directly interacting with its C-terminal domain. The region of SEPT7 binding to CENP-E was mapped to its C-terminal domain by glutathione S-transferase pull-down and yeast two-hybrid assays. Immunofluorescence study shows that SEPT7 filaments distribute along the mitotic spindle and terminate at the kinetochore marked by CENP-E. Remarkably, suppression of synthesis of SEPT7 by small interfering RNA abrogated the localization of CENP-E to the kinetochore and caused aberrant chromosome segregation. These mitotic defects and kinetochore localization of CENP-E can be successfully rescued by introducing exogenous GFP-SEPT7 into the SEPT7-depleted cells. These SEPT7-suppressed cells display reduced tension at kinetochores of bi-orientated chromosomes and activated mitotic spindle checkpoint marked by Mad2 and BubR1 labelings on these misaligned chromosomes. These findings reveal a key role for the SEPT7-CENP-E interaction in the distribution of CENP-E to the kinetochore and achieving chromosome alignment. We propose that SEPT7 forms a link between kinetochore distribution of CENP-E and the mitotic spindle checkpoint.     

Efficient mitosis in human cells lacking poleward microtubule flux

Chromosome segregation relies on the dynamic properties of spindle microtubules (MTs). Poleward MT flux contributes to spindle dynamics through the disassembly of MT minus ends at spindle poles coupled to the continuous poleward transport of spindle MTs. Despite being conserved in metazoan cells, the function of flux remains controversial because flux rates differ widely in different cell types. In meiotic systems, the rate of flux nearly matches that of chromosome movement, but in mitotic systems, flux is significantly slower than chromosome movement. Here, we show that spindles in human mitotic cells depleted of the kinesin-13 proteins Kif2a and MCAK lack detectable flux and that such cells frequently fail to segregate all chromosomes appropriately at anaphase. Elimination of flux reduces poleward chromosome velocity approximately 20%, but does not hinder bipolar spindle assembly, chromosome alignment, or mitotic progression. Thus, mitosis proceeds efficiently in human cells lacking detectable poleward MT flux. These data demonstrate that in human cultured cells, kinetochores are sufficient to effectively power chromosome movement, leading us to speculate that flux is maintained in these cells to fulfill other functional roles such as error correction or kinetochore regulation.     

KLP-7 acts through the Ndc80 complex to limit pole number in C. elegans oocyte meiotic spindle assembly

During oocyte meiotic cell division in many animals, bipolar spindles assemble in the absence of centrosomes, but the mechanisms that restrict pole assembly to a bipolar state are unknown. We show that KLP-7, the single mitotic centromere–associated kinesin (MCAK)/kinesin-13 in Caenorhabditis elegans, is required for bipolar oocyte meiotic spindle assembly. In klp-7(−) mutants, extra microtubules accumulated, extra functional spindle poles assembled, and chromosomes frequently segregated as three distinct masses during meiosis I anaphase. Moreover, reducing KLP-7 function in monopolar klp-18(−) mutants often restored spindle bipolarity and chromosome segregation. MCAKs act at kinetochores to correct improper kinetochore–microtubule (k–MT) attachments, and depletion of the Ndc-80 kinetochore complex, which binds microtubules to mediate kinetochore attachment, restored bipolarity in klp-7(−) mutant oocytes. We propose a model in which KLP-7/MCAK regulates k–MT attachment and spindle tension to promote the coalescence of early spindle pole foci that produces a bipolar structure during the acentrosomal process of oocyte meiotic spindle assembly.     

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.     

Analysis of kinetochore assembly and function in Caenorhabditis elegans embryos and human cells

All eukaryotes rely on multi-protein assemblies, called kinetochores, to direct the segregation of their chromosomes in mitosis. The list of known kinetochore components has been growing rapidly in the post-genomic era: in animal cells, there are presently more than 80 proteins that show either exclusive or partial localization at kinetochores during mitosis. The future challenge is to elucidate how these proteins contribute to kinetochore structure, spindle microtubule attachment, regulation of microtubule dynamics, and the detection, signaling, and correction of microtubule attachment errors. Cultured human tumor cells, especially HeLa cells, are widely used for the study of kinetochores. Recently, the experimental advantages offered by the nematode Caenorhabditis elegans have been exploited for functional analysis of kinetochore components in the first embryonic division. Here, we discuss basic methods, largely based on fluorescence imaging, to study kinetochore structure and function in these two metazoan model systems.     

Dynein is a transient kinetochore component whose binding is regulated by microtubule attachment, not tension

Cytoplasmic dynein is the only known kinetochore protein capable of driving chromosome movement toward spindle poles. In grasshopper spermatocytes, dynein immunofluorescence staining is bright at prometaphase kinetochores and dimmer at metaphase kinetochores. We have determined that these differences in staining intensity reflect differences in amounts of dynein associated with the kinetochore. Metaphase kinetochores regain bright dynein staining if they are detached from spindle microtubules by micromanipulation and kept detached for 10 min. We show that this increase in dynein staining is not caused by the retraction or unmasking of dynein upon detachment. Thus, dynein genuinely is a transient component of spermatocyte kinetochores.We further show that microtubule attachment, not tension, regulates dynein localization at kinetochores. Dynein binding is extremely sensitive to the presence of microtubules: fewer than half the normal number of kinetochore microtubules leads to the loss of most kinetochoric dynein. As a result, the bulk of the dynein leaves the kinetochore very early in mitosis, soon after the kinetochores begin to attach to microtubules. The possible functions of this dynein fraction are therefore limited to the initial attachment and movement of chromosomes and/or to a role in the mitotic checkpoint.     

Centrosome-kinetochore interaction in multinucleate cells

The interaction between centrosomes and kinetochores was studied in multinucleate cells induced by Colcemid treatment or by random cell fusion. Except for prematurely condensed chromosomes (PCC) of the G₂-phase, PCCs do not develop their own spindle area. Perhaps the maturation promoting factor (MPF) fails to activate these centrosomes. In such PCCs, the kinetochore-centrosome interaction was found to be non-specific: sometimes only a few chromosomes of a group could establish connections with centrosomes, sometimes chromosomes from the same PCC group developed microtubule (MT) attachment with different centrosomes (not the pair), and sometimes kinetochores of PCC groups failed to interact with MTs. These findings explain the abnormal mitotic behaviour of PCCs as seen in the light microscope. These PCCs develop micronuclei or normal nuclei by nuclear re-formation in telophase. All the different PCC groups revealed kinetochores with kinetochore plates. It was shown that transformation of presumptive kinetochores to a trilaminar kinetochore does not depend on nuclear envelope breakdown or on the degree of chromosome condensation. This may be induced by the MPF which may initiate different events like chromosome condensation, nuclear envelope breakdown and kinetochore transformation by secondary factors. Other observations like establishment of connections by different chromosome groups to a common centrosome, kinetochore attachment of PCCs to different centrosomes, interaction of one kinetochore with two centrosomes, kinetochores being stretched and bent to receive microtubules and finally the failure of some kinetochores to develop MT attachment, all strongly suggest that the kinetochores serve as the point of termination rather than the nucleation sites of kinetochore MTs.