Making an effective switch at the kinetochore by phosphorylation and dephosphorylation

The kinetochore, the proteinaceous structure on the mitotic centromere, functions as a mechanical latch that hooks onto microtubules to support directional movement of chromosomes. The structure also brings in a number of signaling molecules, such as kinases and phosphatases, which regulate microtubule dynamics and cell cycle progression. Erroneous microtubule attachment is destabilized by Aurora B-mediated phosphorylation of multiple microtubule-binding protein complexes at the kinetochore, such as the KMN network proteins and the Ska/Dam1 complex, while Plk-dependent phosphorylation of BubR1 stabilizes kinetochore–microtubule attachment by recruiting PP2A-B56. Spindle assembly checkpoint (SAC) signaling, which is activated by unattached kinetochores and inhibits the metaphase-to-anaphase transition, depends on kinetochore recruitment of the kinase Bub1 through Mps1-mediated phosphorylation of the kinetochore protein KNL1 (also known as Blinkin in mammals, Spc105 in budding yeast, and Spc7 in fission yeast). Recruitment of protein phosphatase 1 to KNL1 is necessary to silence the SAC upon bioriented microtubule attachment. One of the key unsolved questions in the mitosis field is how a mechanical change at the kinetochore upon microtubule attachment is converted to these and other chemical signals that control microtubule attachment and the SAC. Rapid progress in the field is revealing the existence of an intricate signaling network created right on the kinetochore. Here we review the current understanding of phosphorylation-mediated regulation of kinetochore functions and discuss how this signaling network generates an accurate switch that turns on and off the signaling output in response to kinetochore–microtubule attachment.     

Mechanisms of kinetochore‐microtubule attachment errors in mammalian oocytes

Proper kinetochore‐microtubule attachment is essential for correct chromosome segregation. Therefore, cells normally possess multiple mechanisms for the prevention of errors in kinetochore‐microtubule attachments and for selective stabilization of correct attachments. However, the oocyte, a cell that produces an egg through meiosis, exhibits a high frequency of errors in kinetochore‐microtubule attachments. These attachment errors predispose oocytes to chromosome segregation errors, resulting in aneuploidy in eggs. This review aims to provide possible explanations for the error‐prone nature of oocytes by examining key differences among other cell types in the mechanisms for the establishment of kinetochore‐microtubule attachments.     

Mechanisms of kinesin‐7 CENP‐E in kinetochore–microtubule capture and chromosome alignment during cell division

Chromosome congression is essential for faithful chromosome segregation and genomic stability in cell division. Centromere‐associated protein E (CENP‐E), a plus‐end‐directed kinesin motor, is required for congression of pole‐proximal chromosomes in metaphase. CENP‐E accumulates at the outer plate of kinetochores and mediates the kinetochore‐microtubule capture. CENP‐E also transports the chromosomes along spindle microtubules towards the equatorial plate. CENP‐E interacts with Bub1‐related kinase, Aurora B and core kinetochore components during kinetochore–microtubule attachment. In this review, we introduce the structures and mechanochemistry of kinesin‐7 CENP‐E. We highlight the complicated interactions between CENP‐E and partner proteins during chromosome congression. We summarise the detailed roles and mechanisms of CENP‐E in mitosis and meiosis, including the kinetochore–microtubule capture, chromosome congression/alignment in metaphase and the regulation of spindle assembly checkpoint. We also shed a light on the roles of CENP‐E in tumourigenesis and CENP‐E's specific inhibitors.     

Three wise centromere functions: see no error,hear no break,speak no delay

The main function of the centromere is to promote kinetochore assembly for spindle microtubule attachment. Two additional functions of the centromere, however, are becoming increasingly clear: facilitation of robust sister‐chromatid cohesion at pericentromeres and advancement of replication of centromeric regions. The combination of these three centromere functions ensures correct chromosome segregation during mitosis. Here, we review the mechanisms of the kinetochore–microtubule interaction, focusing on sister‐kinetochore bi‐orientation (or chromosome bi‐orientation). We also discuss the biological importance of robust pericentromeric cohesion and early centromere replication, as well as the mechanisms orchestrating these two functions at the microtubule attachment site.     

Cyclin B1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis

Cyclin B1 is a key regulatory protein controlling cell cycle progression in vertebrates. Cyclin B1 binds CDK1, a cy-clin-dependent kinase catalytic subunit, forming a complex that orchestrates mitosis through phosphorylation of key proteins. Cyclin B1 regulates both the activation of CDK1 and its subcellular localization, which may be critical for substrate selection. Here, we demonstrate that cyclin B1 is concentrated on the outer plate of the kinetochore during prometaphase. This localization requires the cyclin box region of the protein. Cyclin B1 is displaced from individual kinetochores to the spindle poles by microtubule attachment to the kinetochores, and this displacement is dependent on the dynein/dynactin complex. Depletion of cyclin B1 by vector-based siRNA causes inefficient attachment between kinetochores and microtubules, and chromosome alignment defects, and delays the onset of anaphase. We conclude that cyclin B1 accumulates at kinetochores during prometaphase, where it contributes to the correct attachment of mi- crotubules to kinetochores and efficient alignment of the chromosomes, most likely through localized phosphorylation of specific substrates by cyclin B1-CDK1. Cyclin B1 is then transported from each kinetochore as microtubule attachment is completed, and this relocalization may redirect the activity of cyclin B1-CDK1 and contribute to inactivation of the spindle assembly checkpoint.     

Kinetochore function is controlled by a phospho-dependent coexpansion of inner and outer components

It is widely accepted that the kinetochore is built on CENP-A–marked centromeric chromatin in a hierarchical order from inner to outer kinetochore. Recruitment of many kinetochore proteins depends on microtubule attachment status, but it remains unclear how their assembly/disassembly is orchestrated. Applying 3D structured illumination microscopy to Xenopus laevis egg extracts, here we reveal that in the absence of microtubule attachment, proteins responsible for lateral attachment and spindle checkpoint signaling expand to form micrometer-scale fibrous structures over CENP-A–free chromatin, whereas a core module responsible for end-on attachment (CENP-A, CENP-T, and Ndc80) does not. Both outer kinetochore proteins (Bub1, BubR1, Mad1, and CENP-E) and the inner kinetochore component CENP-C are integral components of the expandable module, whose assembly depends on multiple mitotic kinases (Aurora B, Mps1, and Plx1) and is suppressed by protein phosphatase 1. We propose that phospho-dependent coexpansion of CENP-C and outer kinetochore proteins promotes checkpoint signal amplification and lateral attachment, whereas their selective disassembly enables the transition to end-on attachment.     

Driving chromosome segregation: lessons from the human and Drosophila centromere-kinetochore machinery

The kinetochore is a complex molecular machine that serves as the interface between sister chromatids and the mitotic spindle. The kinetochore assembles at a particular chromosomal locus, the centromere, which is essential to maintain genomic stability during cell division. The kinetochore is a macromolecular puzzle of subcomplexes assembled in a hierarchical manner and fulfils three main functions: microtubule attachment, chromosome and sister chromatid movement, and regulation of mitotic progression though the spindle assembly checkpoint. In the present paper we compare recent results on the assembly, organization and function of the kinetochore in human and Drosophila cells and conclude that, although essential functions are highly conserved, there are important differences that might help define what is a minimal chromosome segregation machinery.     

The Toxoplasma gondii kinetochore is required for centrosome association with the centrocone (spindle pole)

The kinetochore is a multi‐protein structure assembled on eukaryotic centromeres mediating chromosome attachment to spindle microtubules. Here we identified the kinetochore proteins Nuf2 and Ndc80 in the apicomplexan parasite Toxoplasma gondii. Localization revealed that kinetochores remain clustered throughout the cell cycle and colocalize with clustered centromeres at the centrocone, a structure containing the spindle pole embedded in the nuclear envelope. Pharmacological disruption of microtubules resulted in partial loss of some kinetochore and centromere clustering, indicating microtubules are necessary but not strictly required for kinetochore clustering. Generation of a TgNuf2 conditional knock‐down strain revealed it is essential for chromosome segregation, but dispensable for centromere clustering. The centromeres actually remained associated with the centrocone suggesting microtubule binding is not required for their interaction with the spindle pole. The most striking observation upon TgNuf2 depletion was that the centrosome behaved normally, but that it lost its association with the centrocone. This suggests that microtubules are essential to maintain contact between the centrosome and chromosomes, and this interaction is critical for the partitioning of the nuclei into the two daughter parasites. Finally, genetic complementation experiments with mutated TgNuf2 constructs highlighted an apicomplexan‐specific motif with a putative role in nuclear localization.     

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.     

The behaviour of kinetochore microtubules during meiosis in the fungusSaprolegnia

Summary InSaprolegnia, kinetochore microtubules persist throughout the mitotic nuclear cycle but, whilst present at leptotene, they disappear coincidently with the formation of synaptonemal complexes at pachytene and reform at metaphase I. In some other fungi chromosomal segregation is random in meiosis and non-random in mitosis. The attachment of chromosomes to persistent kinetochore microtubules in mitosis, but not meiosis, inSaprolegnia provides a plausible explanation for such behaviour. At metaphase I each bivalent is connected to the spindle by 2 laterally paired kinetochore microtubules whereas at metaphase II (as in mitosis) each univalent bears only one kinetochore microtubule, thus showing that all kinetochores are fully active at all stages of meiosis.     

Aurora B switches relative strength of kinetochore–microtubule attachment modes for error correction

To establish chromosome biorientation, aberrant kinetochore–microtubule interaction must be resolved (error correction) by Aurora B kinase. Aurora B differentially regulates kinetochore attachment to the microtubule plus end and its lateral side (end-on and lateral attachment, respectively). However, it is still unclear how kinetochore–microtubule interactions are exchanged during error correction. Here, we reconstituted the budding yeast kinetochore–microtubule interface in vitro by attaching the Ndc80 complexes to nanobeads. These Ndc80C nanobeads recapitulated in vitro the lateral and end-on attachments of authentic kinetochores on dynamic microtubules loaded with the Dam1 complex. This in vitro assay enabled the direct comparison of lateral and end-on attachment strength and showed that Dam1 phosphorylation by Aurora B makes the end-on attachment weaker than the lateral attachment. Similar reconstitutions with purified kinetochore particles were used for comparison. We suggest the Dam1 phosphorylation weakens interaction with the Ndc80 complex, disrupts the end-on attachment, and promotes the exchange to a new lateral attachment, leading to error correction.     

Propagation of centromeric chromatin requires exit from mitosis

Centromeres direct chromosomal inheritance by nucleating assembly of the kinetochore, a large multiprotein complex required for microtubule attachment during mitosis. Centromere identity in humans is epigenetically determined, with no DNA sequence either necessary or sufficient. A prime candidate for the epigenetic mark is assembly into centromeric chromatin of centromere protein A (CENP-A), a histone H3 variant found only at functional centromeres. A new covalent fluorescent pulse-chase labeling approach using SNAP tagging has now been developed and is used to demonstrate that CENP-A bound to a mature centromere is quantitatively and equally partitioned to sister centromeres generated during S phase, thereby remaining stably associated through multiple cell divisions. Loading of nascent CENP-A on the megabase domains of replicated centromere DNA is shown to require passage through mitosis but not microtubule attachment. Very surprisingly, assembly and stabilization of new CENP-A-containing nucleosomes is restricted exclusively to the subsequent G1 phase, demonstrating direct coupling between progression through mitosis and assembly/maturation of the next generation of centromeres.     

Stable kinetochore–microtubule interactions depend on the Ska complex and its new component Ska3/C13Orf3

Ska1 and Ska2 form a complex at the kinetochore–microtubule (KT–MT) interface and are required for timely progression from metaphase to anaphase. Here, we use mass spectrometry to search for additional components of the Ska complex. We identify C13Orf3 (now termed Ska3) as a novel member of this complex and map the interaction domains among the three known components. Ska3 displays similar characteristics as Ska1 and Ska2: it localizes to the spindle and KT throughout mitosis and its depletion markedly delays anaphase transition. Interestingly, a more complete removal of the Ska complex by concomitant depletion of Ska1 and Ska3 results in a chromosome congression failure followed by cell death. This severe phenotype reflects a destabilization of KT–MT interactions, as demonstrated by reduced cold stability of KT fibres. Yet, the depletion of the Ska complex only marginally impairs KT localization of the KMN network responsible for MT attachment. We propose that the Ska complex functionally complements the KMN, providing an additional layer of stability to KT–MT attachment and possibly signalling completion of attachment to the spindle checkpoint.     

The Kinetochore Protein Kis1/Eic1/Mis19 Ensures the Integrity of Mitotic Spindles through Maintenance of Kinetochore Factors Mis6/CENP-I and CENP-A

Microtubules play multiple roles in a wide range of cellular phenomena, including cell polarity establishment and chromosome segregation. A number of microtubule regulators have been identified, including microtubule-associated proteins and kinases, and knowledge of these factors has contributed to our molecular understanding of microtubule regulation of each relevant cellular process. The known regulators, however, are insufficient to explain how those processes are linked to one another, underscoring the need to identify additional regulators. To find such novel mechanisms and microtubule regulators, we performed a screen that combined genetics and microscopy for fission yeast mutants defective in microtubule organization. We isolated approximately 900 mutants showing defects in either microtubule organization or the nuclear envelope, and these mutants were classified into 12 categories. We particularly focused on one mutant, kis1, which displayed spindle defects in early mitosis. The kis1 mutant frequently failed to assemble a normal bipolar spindle. The responsible gene encoded a kinetochore protein, Mis19 (also known as Eic1), which localized to the interface of kinetochores and spindle poles. We also found that the inner kinetochore proteins Mis6/CENP-I and Cnp1/CENP-A were delocalized from kinetochores in the kis1 cells and that kinetochore-microtubule attachment was defective. Another mutant, mis6, also displayed similar spindle defects. We conclude that Kis1 is required for inner kinetochore organization, through which Kis1 ensures kinetochore-microtubule attachment and spindle integrity. Thus, we propose an unexpected relationship between inner kinetochore organization and spindle integrity.     

The spindle assembly checkpoint in Caenorhabditis elegans: One who lacks Mad1 becomes mad one

The spindle assembly checkpoint (SAC) monitors the microtubule attachment status of the kinetochore and arrests cells before anaphase until all pairs of sister kinetochores achieve bipolar attachment of microtubules, thereby ensuring faithful chromosome transmission. The evolutionarily conserved coiled-coil protein MAD1 has been implicated in the SAC signaling pathway. MAD1 forms a complex with another SAC component MAD2 and specifically localizes to unattached kinetochores to facilitate efficient binding of MAD2 to its target, CDC20, the mitotic substrate-specific activator of the anaphase promoting complex or cyclosome (APC/C). Thus, MAD1 connects 2 sequential events in the SAC signaling pathway – recognition of unattached kinetochores and inhibition of APC/C activity. However, the molecular mechanisms by which it specifically localizes to unattached kinetochores are largely unknown. Studies in multicellular organisms have revealed the role of MAD1 in development and tumor suppression, but the precise time at which MAD1 activity is required is unknown. Investigation of cellular and organismic functions of MAD1 in the simple multicellular organism C. elegans identified functional interactors of MAD1 in both kinetochore-oriented SAC signaling and kinetochore-independent cell cycle regulation. Studying the function of SAC components in C. elegans provides a new molecular insight into the SAC-regulated cell cycle progression in a context of a multicellular organism.     

N-Terminal Polyubiquitination of the ARF Tumor Suppressor,A Natural Lysine-Less Protein

The Spindle Assembly Checkpoint ensures the fidelity of chromosome segregation at each cell division cycle. Previous reports have indicated that in higher eukaryotes checkpoint proteins, such as BubR1, are also implicated in chromosome congression, more specifically that BubR1 regulates chromosome-spindle attachments. Also, several studies have shown that BubR1 interacts with the microtubule motor protein CENP-E. Whether this association contributes to the regulation of chromosome-spindle attachments is not yet known. Accordingly, we performed a detailed analysis of microtubule-kinetochore interactions after depletion of BubR1 and the Drosophila CENP-E homologue, CENP-meta by RNAi. We find that depletion of BubR1 affects mitosis very differently from depletion of CENP-meta. While BubR1-depleted cells exit mitosis prematurely due to loss of SAC activity, CENP-meta-depleted cells accumulate in prometaphase and do not exit mitosis after spindle damage. Also, in contrast to cells depleted for CENP-meta, cells depleted for BubR1 very rarely reach full metaphase alignment even if arrested in mitosis with the proteasome inhibitor MG132. More importantly, we show for the first time that BubR1-depleted cells contain a high frequency of either monoriented or fully unattached chromosomes while most CENP-meta dsRNAi-treated cells have chromosomes attached to spindle microtubules. Moreover, simultaneous depletion of both proteins reveals that absence of CENP-meta is able to partially rescue the unattached chromosome phenotype observed after BubR1 depletion. These results strongly suggest that while BubR1 is required to promote stable microtubule kinetochore attachment, CENP-E appears to be required to destabilize kinetochore attachment. Overall our results suggest that activation of the mechanism that corrects inappropriate kinetochore attachment requires the antagonistic effects of BubR1 and CENP-E.     

Cooperation of the Dam1 and Ndc80 kinetochore complexes enhances microtubule coupling and is regulated by aurora B

The coupling of kinetochores to dynamic spindle microtubules is crucial for chromosome positioning and segregation, error correction, and cell cycle progression. How these fundamental attachments are made and persist under tensile forces from the spindle remain important questions. As microtubule-binding elements, the budding yeast Ndc80 and Dam1 kinetochore complexes are essential and not redundant, but their distinct contributions are unknown. In this study, we show that the Dam1 complex is a processivity factor for the Ndc80 complex, enhancing the ability of the Ndc80 complex to form load-bearing attachments to and track with dynamic microtubule tips in vitro. Moreover, the interaction between the Ndc80 and Dam1 complexes is abolished when the Dam1 complex is phosphorylated by the yeast aurora B kinase Ipl1. This provides evidence for a mechanism by which aurora B resets aberrant kinetochore–microtubule attachments. We propose that the action of the Dam1 complex as a processivity factor in kinetochore–microtubule attachment is regulated by conserved signals for error correction.