Chromosome Biorientation Research Articles

Bod1, a novel kinetochore protein required for chromosome biorientation

We have combined the proteomic analysis of Xenopus laevis in vitro–assembled chromosomes with RNA interference and live cell imaging in HeLa cells to identify novel factors required for proper chromosome segregation. The first of these is Bod1, a protein conserved throughout metazoans that associates with a large macromolecular complex and localizes with kinetochores and spindle poles during mitosis. Small interfering RNA depletion of Bod1 in HeLa cells produces elongated mitotic spindles with severe biorientation defects. Bod1-depleted cells form syntelic attachments that can oscillate and generate enough force to separate sister kinetochores, suggesting that microtubule–kinetochore interactions were intact. Releasing Bod1-depleted cells from a monastrol block increases the frequency of syntelic attachments and the number of cells displaying biorientation defects. Bod1 depletion does not affect the activity or localization of Aurora B but does cause mislocalization of the microtubule depolymerase mitotic centromere- associated kinesin and prevents its efficient phosphorylation by Aurora B. Therefore, Bod1 is a novel kinetochore protein that is required for the detection or resolution of syntelic attachments in mitotic spindles.

Self-Organization of MTOCs Replaces Centrosome Function during Acentrosomal Spindle Assembly in Live Mouse Oocytes

Chromosome segregation in mammalian oocytes is driven by a microtubule spindle lacking centrosomes. Here, we analyze centrosome-independent spindle assembly by quantitative high-resolution confocal imaging in live maturing mouse oocytes. We show that spindle assembly proceeds by the self-organization of over 80 microtubule organizing centers (MTOCs) that form de novo from a cytoplasmic microtubule network in prophase and that functionally replace centrosomes. Initially distributed throughout the ooplasm, MTOCs congress at the center of the oocyte, where they contribute to a massive, Ran-dependent increase of the number of microtubules after nuclear envelope breakdown and to the individualization of clustered chromosomes. Through progressive MTOC clustering and activation of kinesin-5, the multipolar MTOC aggregate self-organizes into a bipolar intermediate, which then elongates and thereby establishes chromosome biorientation. Finally, a stable barrel-shaped acentrosomal metaphase spindle with oscillating chromosomes and astral-like microtubules forms that surprisingly exhibits key properties of a centrosomal spindle.

Bi-orientation of achiasmatic chromosomes in meiosis I oocytes contributes to aneuploidy in mice

The spindle assembly checkpoint guards against chromosomal missegregation but does not induce arrest in oocytes that contain a few achiasmatic chromosomes (univalents). We followed the fate of univalents in oocytes during the first meiotic division, and although these preserved a meiotic kinetochore structure, they were also bi-oriented in a mitotic manner. The hybrid chromosomal configuration attained by univalents allows them to evade the spindle assembly checkpoint and contribute to aneuploidy in oocytes.

Mad3 KEN Boxes Mediate both Cdc20 and Mad3 Turnover, and Are Critical for the Spindle Checkpoint

Mitotic progression is controlled by proteolytic destruction of securin and cyclin. The mitotic E3 ubiquitin ligase, known as the anaphase promoting complex or cyclosome (APC/C), in partnership with its activators Cdc20p and Cdh1p, targets these proteins for degradation. In the presence of defective kinetochore-microtubule interactions, APC/CCdc20 is inhibited by the spindle checkpoint, thereby delaying anaphase onset and providing more time for spindle assembly. Cdc20p interacts directly with Mad2p, and its levels are subject to careful regulation, but the precise mode(s) of APC/C Cdc20 inhibition remain unclear. The mitotic checkpoint complex (MCC, consisting of Mad3p, Mad2p, Bub3p and Cdc20p in budding yeast) is a potent APC/C inhibitor. Here we focus on Mad3p and how it acts, in concert with Mad2p, to efficiently inhibit Cdc20p. We identify and analyse the function of two motifs in Mad3p, KEN30 and KEN296, which are conserved from yeast Mad3p to human BubR1. These KEN amino acid sequences resemble ‘degron’ signals that confer interaction with APC/C activators and target proteins for degradation. We show that both Mad3p KEN boxes are necessary for spindle checkpoint function. Mutation of KEN30 abolished MCC formation and stabilised Cdc20p in mitosis. In addition, mutation of Mad3-KEN30, APC/C subunits, or Cdh1p, stabilised Mad3p in G1, indicating that the N-terminal KEN box could be a Mad3p degron. To determine the significance of Mad3p turnover, we analysed the consequences of MAD3 overexpression and found that four-fold overproduction of Mad3p led to chromosome bi-orientation defects and significant chromosome loss during recovery from anti-microtubule drug induced checkpoint arrest. In conclusion, Mad3p KEN30 mediates interactions that regulate the proteolytic turnover of Cdc20p and Mad3p, and the levels of both of these proteins are critical for spindle checkpoint signaling and high fidelity chromosome segregation.

The human Nup107–160 nuclear pore subcomplex contributes to proper kinetochore functions

We previously demonstrated that a fraction of the human Nup107-160 nuclear pore subcomplex is recruited to kinetochores at the onset of mitosis. However, the molecular determinants for its kinetochore targeting and the functional significance of this localization were not investigated. Here, we show that the Nup107-160 complex interacts with CENP-F, but that CENP-F only moderately contributes to its targeting to kinetochores. In addition, we show that the recruitment of the Nup107-160 complex to kinetochores mainly depends on the Ndc80 complex. We further demonstrate that efficient depletion of the Nup107-160 complex from kinetochores, achieved either by combining siRNAs targeting several of its subunits excluding Seh1, or by depleting Seh1 alone, induces a mitotic delay. Further analysis of Seh1-depleted cells revealed impaired chromosome congression, reduced kinetochore tension and kinetochore-microtubule attachment defects. Finally, we show that the presence of the Nup107-160 complex at kinetochores is required for the recruitment of Crm1 and RanGAP1-RanBP2 to these structures. Together, our data thus provide the first molecular clues underlying the function of the human Nup107-160 complex at kinetochores.

Mitotic Chromosome Biorientation in Fission Yeast Is Enhanced by Dynein and a Minus-end–directed, Kinesin-like Protein

Chromosome biorientation, the attachment of sister kinetochores to sister spindle poles, is vitally important for accurate chromosome segregation. We have studied this process by following the congression of pole-proximal kinetochores and their subsequent anaphase segregation in fission yeast cells that carry deletions in any or all of this organism's minus end-directed, microtubule-dependent motors: two related kinesin 14s (Pkl1p and Klp2p) and dynein. None of these deletions abolished biorientation, but fewer chromosomes segregated normally without Pkl1p, and to a lesser degree without dynein, than in wild-type cells. In the absence of Pkl1p, which normally localizes to the spindle and its poles, the checkpoint that monitors chromosome biorientation was defective, leading to frequent precocious anaphase. Ultrastructural analysis of mutant mitotic spindles suggests that Pkl1p contributes to error-free biorientation by promoting normal spindle pole organization, whereas dynein helps to anchor a focused bundle of spindle microtubules at the pole.

Shugoshin 2 Regulates Localization of the Chromosomal Passenger Proteins in Fission Yeast Mitosis

Fission yeast has two members of the Shugoshin family, Sgo1 and Sgo2. Although Sgo1 has clearly been established as a protector of centromere cohesion in meiosis I, the roles of Sgo2 remain elusive. Here we show that Sgo2 is required to ensure proper chromosome biorientation upon recovery from a prolonged spindle checkpoint arrest. Consistent with this, Sgo2 is essential for maintaining the Passenger proteins on centromeres upon checkpoint activation. Interestingly, lack of Sgo2 has a more penetrant effect on the localization of Survivin than on the two other Passenger proteins INCENP and Aurora B, and the Survivin-INCENP complex but not the INCENP-Aurora B complex is destabilized in the absence of Sgo2. Finally we show that the conserved C-terminus of Sgo2 is crucial to maintain Sgo2 and Passenger proteins localization on centromeres upon prolonged checkpoint activation. Taken together, our results demonstrate that Sgo2 is important for chromosome biorientation and that it controls docking of the Passenger proteins on chromosomes in early mitotic cells.

Kinetochore Orientation during Meiosis Is Controlled by Aurora B and the Monopolin Complex

Kinetochores of sister chromatids attach to microtubules emanating from the same pole (coorientation) during meiosis I and microtubules emanating from opposite poles (biorientation) during meiosis II. We find that the Aurora B kinase Ipl1 regulates kinetochore-microtubule attachment during both meiotic divisions and that a complex known as the monopolin complex ensures that the protein kinase coorients sister chromatids during meiosis I. Furthermore, the defining of conditions sufficient to induce sister kinetochore coorientation during mitosis provides insight into monopolin complex function. The monopolin complex joins sister kinetochores independently of cohesins, the proteins that hold sister chromatids together. We propose that this function of the monopolin complex helps Aurora B coorient sister chromatids during meiosis I.

The enhancement of pericentromeric cohesin association by conserved kinetochore components promotes high-fidelity chromosome segregation and is sensitive to microtubule-based tension.

Sister chromatid cohesion, conferred by the evolutionarily conserved cohesin complex, is essential for proper chromosome segregation. Cohesin binds to discrete sites along chromosome arms, and is especially enriched surrounding centromeres, but past studies have not clearly defined the roles of arm and pericentromeric cohesion in chromosome segregation. To address this issue, we developed a technique that specifically reduced pericentromeric cohesin association on a single chromosome without affecting arm cohesin binding. Under these conditions, we observed more extensive stretching of centromeric chromatin and elevated frequencies of chromosome loss, suggesting that pericentromeric cohesin enrichment is essential for high-fidelity chromosome transmission. The magnitude of pericentromeric cohesin association was negatively correlated with tension between sister kinetochores, with the highest levels of association in cells lacking kinetochore-microtubule attachments. Pericentromeric cohesin recruitment required evolutionarily conserved components of the inner and central kinetochore. Together, these observations suggest that pericentromeric cohesin levels reflect the balance of opposing forces: the kinetochore-mediated enhancement of cohesin binding and the disruption of binding by mechanical tension at kinetochores. The involvement of conserved kinetochore components suggests that this pathway for pericentromeric cohesin enrichment may have been retained in higher eukaryotes to promote chromosome biorientation and accurate sister chromatid segregation.

Review of "The Cell Cycle: Principles of Control" by David O. Morgan

Review of "The Cell Cycle: Principles of Control" by David O. Morgan

A Bir1-Sli15 Complex Connects Centromeres to Microtubules and Is Required to Sense Kinetochore Tension

Proper connections between centromeres and spindle microtubules are of critical importance in ensuring accurate segregation of the genome during cell division. Using an in vitro approach based on the sequence-specific budding yeast centromere, we identified a complex of the chromosomal passenger proteins Bir1 and Sli15 (Survivin and INCENP) that links centromeres to microtubules. This linkage does not require Ipl1/Aurora B kinase, whose targeting and activation are controlled by Bir1 and Sli15. Ipl1 is the tension-dependent regulator of centromere-microtubule interactions that ensures chromosome biorientation on the spindle. Elimination of the linkage between centromeres and microtubules mediated by Bir1-Sli15 phenocopies mutations that selectively cripple Ipl1 kinase activation. These findings lead us to propose that the Bir1-Sli15-mediated linkage, which bridges centromeres and microtubules and includes the Aurora kinase-activating domain of INCENP family proteins, is the tension sensor that relays the mechanical state of centromere-microtubule attachments into local control of Ipl1 kinase activity.

Microtubule depolymerization can drive poleward chromosome motion in fission yeast

Prometaphase kinetochores interact with spindle microtubules (MTs) to establish chromosome bi-orientation. Before becoming bi-oriented, chromosomes frequently exhibit poleward movements (P-movements), which are commonly attributed to minus end-directed, MT-dependent motors. In fission yeast there are three such motors: dynein and two kinesin-14s, Pkl1p and Klp2p. None of these enzymes is essential for viability, and even the triple deletion grows well. This might be due to the fact that yeasts kinetochores are normally juxtapolar at mitosis onset, removing the need for poleward chromosome movement during prometaphase. Anaphase P-movement might also be dispensable in a spindle that elongates significantly. To test this supposition, we have analyzed kinetochore dynamics in cells whose kinetochore-pole connections have been dispersed. In cells recovering from this condition, the maximum rate of poleward kinetochore movement was unaffected by the deletion of any or all of these motors, strongly suggesting that other factors, like MT depolymerization, can cause such movements in vivo. However, Klp2p, which localizes to kinetochores, contributed to the effectiveness of P-movement by promoting the shortening of kinetochore fibers.

Inhibition of Aurora kinases perturbs chromosome alignment and spindle checkpoint signaling in rat spermatocytes

In somatic cells, integrity of cell division is safeguarded by the spindle checkpoint, a signaling cascade that delays the separation of sister chromatids in the presence of misaligned chromosomes. Aurora kinases play important roles in this process by promoting centrosome maturation, chromosome bi-orientation, spindle checkpoint signaling, and cytokinesis. To investigate the functions of Aurora kinases in male meiosis, we applied a small molecule Aurora inhibitor, ZM447439, to seminiferous tubules in vitro. Primary and secondary spermatocytes exposed to ZM447439 exhibit defects in the spindle morphology and fail to align their chromosomes at the metaphase plate. Moreover, the treated spermatocytes undergo a forced exit from the meiotic M-phase without cytokinesis. These results suggest that the activities of Aurora kinases are required for normal spindle assembly as well as for establishment and maintenance of proper microtubule–kinetochore attachments and spindle checkpoint signaling in male mammalian meiosis.

Mps1 Phosphorylation of Dam1 Couples Kinetochores to Microtubule Plus Ends at Metaphase

Duplicated chromosomes are equally segregated to daughter cells by a bipolar mitotic spindle during cell division. By metaphase, sister chromatids are coupled to microtubule (MT) plus ends from opposite poles of the bipolar spindle via kinetochores. Here we describe a phosphorylation event that promotes the coupling of kinetochores to microtubule plus ends. Dam1 is a kinetochore component that directly binds to microtubules. We identified DAM1-765, a dominant allele of DAM1, in a genetic screen for mutations that increase stress on the spindle pole body (SPB) in Saccharomyces cerevisiae. DAM1-765 contains the single mutation S221F. We show that S221 is one of six Dam1 serines (S13, S49, S217, S218, S221, and S232) phosphorylated by Mps1 in vitro. In cells with single mutations S221F, S218A, or S221A, kinetochores in the metaphase spindle form tight clusters that are closer to the SPBs than in a wild-type cell. Five lines of experimental evidence, including localization of spindle components by fluorescence microscopy, measurement of microtubule dynamics by fluorescence redistribution after photobleaching, and reconstructions of three-dimensional structure by electron tomography, combined with computational modeling of microtubule behavior strongly indicate that, unlike wild-type kinetochores, Dam1-765 kinetochores do not colocalize with an equal number of plus ends. Despite the uncoupling of the kinetochores from the plus ends of MTs, the DAM1-765 cells are viable, complete the cell cycle with the same kinetics as wild-type cells, and biorient their chromosomes as efficiently as wild-type cells. We conclude that phosphorylation of Dam1 residues S218 and S221 by Mps1 is required for efficient coupling of kinetochores to MT plus ends. We find that efficient plus-end coupling is not required for (1) maintenance of chromosome biorientation, (2) maintenance of tension between sister kinetochores, or (3) chromosome segregation.

A mitotic topoisomerase II checkpoint in budding yeast is required for genome stability but acts independently of Pds1/securin

Topoisomerase II (Topo II) performs topological modifications on double-stranded DNA molecules that are essential for chromosome condensation, resolution, and segregation. In mammals, G2 and metaphase cell cycle delays induced by Topo II poisons have been proposed to be the result of checkpoint activation in response to the catenation state of DNA. However, the apparent lack of such controls in model organisms has excluded genetic proof that Topo II checkpoints exist and are separable from the conventional DNA damage checkpoint controls. But here, we define a Topo II-dependent G2/M checkpoint in a genetically amenable eukaryote, budding yeast, and demonstrate that this checkpoint enhances cell survival. Conversely, a lack of the checkpoint results in aneuploidy. Neither DNA damage-responsive pathways nor Pds1/securin are needed for this checkpoint. Unusually, spindle assembly checkpoint components are required for the Topo II checkpoint, but checkpoint activation is not the result of failed chromosome biorientation or a lack of spindle tension. Thus, compromised Topo II function activates a yeast checkpoint system that operates by a novel mechanism.

Human Scc4 Is Required for Cohesin Binding to Chromatin, Sister-Chromatid Cohesion, and Mitotic Progression

Sister-chromatid cohesion depends on the cohesin complex whose association with chromatin is mediated by Scc2 and Scc4 in budding yeast. Both cohesin and Scc2 have been conserved from yeast to humans, but no Scc4 orthologs have been identified. Mutation of Scc2 orthologs causes defects in cohesion, transcription, and development, resulting in Cornelia de Lange syndrome in humans. We have identified a family of tetratricopeptide repeat proteins that share weak sequence similarities with yeast Scc4. This family includes MAU-2, which is required for development of the nervous system in Caenorhabditis elegans. We show that the human member of this family is associated with Scc2, is bound to chromatin from telophase until prophase, and is required for association of cohesin with chromatin during interphase. Cells lacking Scc4 lose sister-chromatid cohesion precociously and arrest in prometaphase. Mitotic chromosomes in Scc4-depleted cells lack cohesin, even though the cohesin-protecting proteins Sgo1 and Bub1 are normally enriched at centromeres and separase does not seem to be active. Our data indicate that human Scc4 is required for the association of cohesin with chromatin, which is a prerequisite for the establishment of sister-chromatid cohesion and for chromosome biorientation in mitosis. The proteinaceous machinery that is required for loading of cohesin onto chromatin is therefore conserved from yeast to humans. The finding that Caenorhabditis elegans MAU-2 is an ortholog of Scc4 further supports the notion that the Scc2-Scc4 complex is required for developmental processes in metazoans.

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.

DrosophilaIncenp is required for cytokinesis and asymmetric cell division during development of the nervous system

The chromosomal passenger protein complex has emerged as a key player in mitosis, with important roles in chromatin modifications, kinetochore-microtubule interactions, chromosome bi-orientation and stability of the bipolar spindle, mitotic checkpoint function, assembly of the central spindle and cytokinesis. The inner centromere protein (Incenp; a subunit of this complex) is thought to regulate the Aurora B kinase and target it to its substrates. To explore the roles of the passenger complex in a developing multicellular organism, we have performed a genetic screen looking for new alleles and interactors of Drosophila Incenp. We have isolated a new null allele of Incenp that has allowed us for the first time to study the functions of the chromosomal passengers during development. Homozygous incenp(EC3747) embryos show absence of phosphorylation of histone H3 in mitosis, failure of cytokinesis and polyploidy, and defects in peripheral nervous system development. These defects are consistent with depletion of Aurora B kinase activity. In addition, the segregation of the cell-fate determinant Prospero in asymmetric neuroblast division is abnormal, suggesting a role for the chromosomal passenger complex in the regulation of this process.

Chromosomes Can Congress to the Metaphase Plate Before Biorientation

The stable propagation of genetic material during cell division depends on the congression of chromosomes to the spindle equator before the cell initiates anaphase. It is generally assumed that congression requires that chromosomes are connected to the opposite poles of the bipolar spindle ("bioriented"). In mammalian cells, we found that chromosomes can congress before becoming bioriented. By combining the use of reversible chemical inhibitors, live-cell light microscopy, and correlative electron microscopy, we found that monooriented chromosomes could glide toward the spindle equator alongside kinetochore fibers attached to other already bioriented chromosomes. This congression mechanism depended on the kinetochore-associated, plus end-directed microtubule motor CENP-E (kinesin-7).

Chromosome Alignment and Segregation Regulated by Ubiquitination of Survivin

Proper chromosome segregation requires the attachment of sister kinetochores to microtubules from opposite spindle poles to form bi-oriented chromosomes on the metaphase spindle. The chromosome passenger complex containing Survivin and the kinase Aurora B regulates this process from the centromeres. We report that a de-ubiquitinating enzyme, hFAM, regulates chromosome alignment and segregation by controlling both the dynamic association of Survivin with centromeres and the proper targeting of Survivin and Aurora B to centromeres. Survivin is ubiquitinated in mitosis through both Lys(48) and Lys(63) ubiquitin linkages. Lys(63) de-ubiquitination mediated by hFAM is required for the dissociation of Survivin from centromeres, whereas Lys(63) ubiquitination mediated by the ubiquitin binding protein Ufd1 is required for the association of Survivin with centromeres. Thus, ubiquitinaton regulates dynamic protein-protein interactions and chromosome segregation independently of protein degradation.