Chromosome bi-orientation on the mitotic spindle

For proper chromosome segregation, sister kinetochores must attach to microtubules extending from opposite spindle poles prior to anaphase onset. This state is called sister kinetochore bi-orientation or chromosome bi-orientation. The mechanism ensuring chromosome bi-orientation lies at the heart of chromosome segregation, but is still poorly understood. Recent evidence suggests that mal-oriented kinetochore-to-pole connections are corrected in a tension-dependent mechanism. The cohesin complex and the Ipl1/Aurora B protein kinase seem to be key regulators for this correction. In this article, I discuss how cells ensure sister kinetochore bi-orientation for all chromosomes, mainly focusing on our recent findings in budding yeast.     

Kinetochore-microtubule interactions: steps towards bi-orientation

Eukaryotic cells segregate their chromosomes accurately to opposite poles during mitosis, which is necessary for maintenance of their genetic integrity. This process mainly relies on the forces generated by kinetochore-microtubule (KT-MT) attachment. During prometaphase, the KT initially interacts with a single MT extending from a spindle pole and then moves towards a spindle pole. Subsequently, MTs from the other spindle pole also interact with the KT. Eventually, one sister KT becomes attached to MTs from one pole while the other sister to those from the other pole (sister KT bi-orientation). If sister KTs interact with MTs with aberrant orientation, this must be corrected to attain proper bi-orientation (error correction) before the anaphase is initiated. Here, I discuss how KTs initially interact with MTs and how this interaction develops into bi-orientation; both processes are fundamentally crucial for proper chromosome segregation in the subsequent anaphase.     

Mps1 kinase promotes sister-kinetochore bi-orientation by a tension-dependent mechanism

Segregation of sister chromatids to opposite spindle poles during anaphase is dependent on the prior capture of sister kinetochores by microtubules extending from opposite spindle poles (bi-orientation). If sister kinetochores attach to microtubules from the same pole (syntelic attachment), the kinetochore-spindle pole connections must be re-oriented to be converted to proper bi-orientation. This re-orientation is facilitated by Aurora B kinase (Ipl1 in budding yeast), which eliminates kinetochore-spindle pole connections that do not generate tension. Mps1 is another evolutionarily conserved protein kinase, required for spindle-assembly checkpoint and, in some organisms, for duplication of microtubule-organizing centers. Separately from these functions, however, Mps1 has an important role in chromosome segregation. Here we show that, in budding yeast, Mps1 has a crucial role in establishing sister-kinetochore bi-orientation on the mitotic spindle. Failure in bi-orientation with inactive Mps1 is not due to a lack of kinetochore-spindle pole connections by microtubules, but due to a defect in properly orienting the connections. Mps1 promotes re-orientation of kinetochore-spindle pole connections and eliminates those that do not generate tension between sister kinetochores. We did not find evidence that Ipl1 regulates Mps1 or vice versa; therefore, they play similar, but possibly independent, roles in facilitating bi-orientation.     

Bi-orienting chromosomes: acrobatics on the mitotic spindle

To maintain their genetic integrity, eukaryotic cells must segregate their chromosomes properly to opposite poles during mitosis. This process mainly depends on the forces generated by microtubules that attach to kinetochores. During prometaphase, kinetochores initially interact with a single microtubule that extends from a spindle pole and then move towards a spindle pole. Subsequently, microtubules that extend from the other spindle pole also interact with kinetochores and, eventually, each sister kinetochore attaches to microtubules that extend from opposite poles (sister kinetochore bi-orientation). If sister kinetochores interact with microtubules in wrong orientation, this must be corrected before the onset of anaphase. Here, I discuss the processes leading to bi-orientation and the mechanisms ensuring this pivotal state that is required for proper chromosome segregation.     

Aurora controls sister kinetochore mono-orientation and homolog bi-orientation in meiosis-I

Aurora-B kinases are important regulators of mitotic chromosome segregation, where they are required for the faithful bi-orientation of sister chromatids. In contrast to mitosis, sister chromatids have to be oriented toward the same spindle pole in meiosis-I, while homologous chromosomes are bi-oriented. We find that the fission yeast Aurora kinase Ark1 is required for the faithful bi-orientation of sister chromatids in mitosis and of homologous chromosomes in meiosis-I. Unexpectedly, Ark1 is also necessary for the faithful mono-orientation of sister chromatids in meiosis-I, even though the canonical mono-orientation pathway, which depends on Moa1 and Rec8, seems intact. Our data suggest that Ark1 prevents unified sister kinetochores during metaphase-I from merotelic attachment to both spindle poles and thus from being torn apart during anaphase-I, revealing a novel mechanism promoting monopolar attachment. Furthermore, our results provide an explanation for the previously enigmatic observation that fission yeast Shugoshin Sgo2, which assists in loading Aurora to centromeres, and its regulator Bub1 are required for the mono-orientation of sister chromatids in meiosis-I.     

Molecular Mechanisms of Homologous Chromosome Pairing and Segregation in Plants

In most eukaryotic species, three basic steps of pairing, recombination and synapsis occur during prophase of meiosis I. Homologous chromosomal pairing and recombination are essential for accurate segregation of chromosomes. In contrast to the well-studied processes such as recombination and synapsis, many aspects of chromosome pairing are still obscure. Recent progress in several species indicates that the telomere bouquet formation can facilitate homologous chromosome pairing by bringing chromosome ends into close proximity, but the sole presence of telomere clustering is not sufficient for recognizing homologous pairs. On the other hand, accurate segregation of the genetic material from parent to offspring during meiosis is dependent on the segregation of homologs in the reductional meiotic division （MI） with sister kinetochores exhibiting mono-orientation from the same pole, and the segregation of sister chromatids during the equational meiotic division （MII） with kinetochores showing bi-orientation from the two poles. The underlying mechanism of orientation and segregation is still unclear. Here we focus on recent studies in plants and other species that provide insight into how chromosomes find their partners and mechanisms mediating chromosomal segregation.     

Holocentric plant meiosis: first sisters,then homologues

Meiosis is a crucial process of sexual reproduction by forming haploid gametes from diploid precursor cells. It involves 2 subsequent divisions (meiosis I and meiosis II) after one initial round of DNA replication. Homologous monocentric chromosomes are separated during the first and sister chromatids during the second meiotic division. The faithful segregation of monocentric chromosomes is realized by mono-orientation of fused sister kinetochores at metaphase I and by bi-orientation of sister kinetochores at metaphase II. Conventionally this depends on a 2-step loss of cohesion, along chromosome arms during meiosis I and at sister centromeres during meiosis II.     

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.     

The spindle assembly checkpoint promotes chromosome bi-orientation: A novel Mad1 role in chromosome alignment

Faithful chromosome segregation relies on dynamic interactions between spindle microtubules and chromosomes. Especially, all chromosomes must be aligned at the equator of the spindle to establish bi-orientation before they start to segregate. The spindle assembly checkpoint (SAC) monitors this process, inhibiting chromosome segregation until all chromosomes achieve bi-orientation. The original concept of ‘checkpoints’ was proposed as an external surveillance system that does not play an active role in the process it monitors. However, accumulating evidence from recent studies suggests that SAC components do play an active role in chromosome bi-orientation. In this review, we highlight a novel Mad1 role in chromosome alignment, which is the first conserved mechanism that links the SAC and kinesin-mediated chromosome gliding.     

The aurora B kinase AIR-2 regulates kinetochores during mitosis and is required for separation of homologous Chromosomes during meiosis

BACKGROUND: Mitotic chromosome segregation depends on bi-orientation and capture of sister kinetochores by microtubules emanating from opposite spindle poles and the near synchronous loss of sister chromatid cohesion. During meiosis I, in contrast, sister kinetochores orient to the same pole, and homologous kinetochores are captured by microtubules emanating from opposite spindle poles. Additionally, mechanisms exist that prevent complete loss of cohesion during meiosis I. These features ensure that homologs separate during meiosis I and sister chromatids remain together until meiosis II. The mechanisms responsible for orienting kinetochores in mitosis and for causing asynchronous loss of cohesion during meiosis are not well understood. RESULTS: During mitosis in C. elegans, aurora B kinase, AIR-2, is not required for sister chromatid separation, but it is required for chromosome segregation. Condensin recruitment during metaphase requires AIR-2; however, condensin functions during prometaphase, independent of AIR-2. During metaphase, AIR-2 promotes chromosome congression to the metaphase plate, perhaps by inhibiting attachment of chromatids to both spindle poles. During meiosis in AIR-2-depleted oocytes, congression of bivalents appears normal, but segregation fails. Localization of AIR-2 on meiotic bivalents suggests this kinase promotes separation of homologs by promoting the loss of cohesion distal to the single chiasma. Inactivation of the phosphatase that antagonizes AIR-2 causes premature separation of chromatids during meiosis I, in a separase-dependent reaction. CONCLUSIONS: Aurora B functions to resolve chiasmata during meiosis I and to regulate kinetochore function during mitosis. Condensin mediates chromosome condensation during prophase, and condensin-independent pathways contribute to chromosome condensation during metaphase.     

CAMP (C13orf8, ZNF828) is a novel regulator of kinetochore-microtubule attachment

Proper attachment of microtubules to kinetochores is essential for accurate chromosome segregation. Here, we report a novel protein involved in kinetochore-microtubule attachment, chromosome alignment-maintaining phosphoprotein (CAMP) (C13orf8, ZNF828). CAMP is a zinc-finger protein containing three characteristic repeat motifs termed the WK, SPE, and FPE motifs. CAMP localizes to chromosomes and the spindle including kinetochores, and undergoes CDK1-dependent phosphorylation at multiple sites during mitosis. CAMP-depleted cells showed severe chromosome misalignment, which was associated with the poor resistance of K-fibres to the tension exerted upon establishment of sister kinetochore bi-orientation. We found that the FPE region, which is responsible for spindle and kinetochore localization, is essential for proper chromosome alignment. The C-terminal region containing the zinc-finger domains negatively regulates chromosome alignment, and phosphorylation in the FPE region counteracts this regulation. Kinetochore localization of CENP-E and CENP-F was affected by CAMP depletion, and by expressing CAMP mutants that cannot functionally rescue CAMP depletion, placing CENP-E and CENP-F as downstream effectors of CAMP. These data suggest that CAMP is required for maintaining kinetochore-microtubule attachment during bi-orientation.     

Kinetochore capture and bi-orientation on the mitotic spindle

Kinetochores are large protein complexes that are formed on chromosome regions known as centromeres. For high-fidelity chromosome segregation, kinetochores must be correctly captured on the mitotic spindle before anaphase onset. During prometaphase, kinetochores are initially captured by a single microtubule that extends from a spindle pole and are then transported poleward along the microtubule. Subsequently, microtubules that extend from the other spindle pole also interact with kinetochores and, eventually, each sister kinetochore attaches to microtubules that extend from opposite poles - this is known as bi-orientation. Here we discuss the molecular mechanisms of these processes, by focusing on budding yeast and drawing comparisons with other organisms.     

Global analysis of core histones reveals nucleosomal surfaces required for chromosome bi-orientation

The attachment of sister kinetochores to microtubules from opposite spindle poles is essential for faithful chromosome segregation. Kinetochore assembly requires centromere-specific nucleosomes containing the histone H3 variant CenH3. However, the functional roles of the canonical histones (H2A, H2B, H3, and H4) in chromosome segregation remain elusive. Using a library of histone point mutants in Saccharomyces cerevisiae, 24 histone residues that conferred sensitivity to the microtubule-depolymerizing drugs thiabendazole (TBZ) and benomyl were identified. Twenty-three of these mutations were clustered at three spatially separated nucleosomal regions designated TBS-I, -II, and -III (TBZ/benomyl-sensitive regions I-III). Elevation of mono-polar attachment induced by prior nocodazole treatment was observed in H2A-I112A (TBS-I), H2A-E57A (TBS-II), and H4-L97A (TBS-III) cells. Severe impairment of the centromere localization of Sgo1, a key modulator of chromosome bi-orientation, occurred in H2A-I112A and H2A-E57A cells. In addition, the pericentromeric localization of Htz1, the histone H2A variant, was impaired in H4-L97A cells. These results suggest that the spatially separated nucleosomal regions, TBS-I and -II, are necessary for Sgo1-mediated chromosome bi-orientation and that TBS-III is required for Htz1 function.     

Shugoshins function as a guardian for chromosomal stability in nuclear division

Accurate chromosome segregation during mitosis and meiosis is regulated and secured by several distinctly different yet intricately connected regulatory mechanisms. As chromosomal instability is a hallmark of a majority of tumors as well as a cause of infertility for germ cells, extensive research in the past has focused on the identification and characterization of molecular components that are crucial for faithful chromosome segregation during cell division. Shugoshins, including Sgo1 and Sgo2, are evolutionarily conserved proteins that function to protect sister chromatid cohesion, thus ensuring chromosomal stability during mitosis and meiosis in eukaryotes. Recent studies reveal that Shugoshins in higher animals play an essential role not only in protecting centromeric cohesion of sister chromatids and assisting bi-orientation attachment at the kinetochores, but also in safeguarding centriole cohesion/engagement during early mitosis. Many molecular components have been identified that play essential roles in modulating/mediating Sgo functions. This review primarily summarizes recent advances on the mechanisms of action of Shugoshins in suppressing chromosomal instability during nuclear division in eukaryotic organisms.     

Shugoshins function as a guardian for chromosomal stability in nuclear division

Accurate chromosome segregation during mitosis and meiosis is regulated and secured by several distinctly different yet intricately connected regulatory mechanisms. As chromosomal instability is a hallmark of a majority of tumors as well as a cause of infertility for germ cells, extensive research in the past has focused on the identification and characterization of molecular components that are crucial for faithful chromosome segregation during cell division. Shugoshins, including Sgo1 and Sgo2, are evolutionarily conserved proteins that function to protect sister chromatid cohesion, thus ensuring chromosomal stability during mitosis and meiosis in eukaryotes. Recent studies reveal that Shugoshins in higher animals play an essential role not only in protecting centromeric cohesion of sister chromatids and assisting bi-orientation attachment at the kinetochores, but also in safeguarding centriole cohesion/engagement during early mitosis. Many molecular components have been identified that play essential roles in modulating/mediating Sgo functions. This review primarily summarizes recent advances on the mechanisms of action of Shugoshins in suppressing chromosomal instability during nuclear division in eukaryotic organisms.     

Centromeres: old tales and new tools

The centromere is a specialised region of the eukaryotic chromosome that directs the equal segregation of sister chromatids into two daughter cells during mitosis. In mitosis, the kinetochores mediate (1) microtubule capture and chromosome alignment at a metaphase plate; (2) the correction of improper microtubule attachments; (3) the maintenance of an active checkpoint until bi-orientation is achieved by the whole complement of chromosomes; (4) the establishment of tension within the centromere which, in turn, contributes to silencing of the spindle checkpoint and triggers the onset of anaphase. In this review, we will analyse how centromeres are organised with respect to chromatin types and arrangements.     

Phosphorylation of Microtubule-binding Protein Hec1 by Mitotic Kinase Aurora B Specifies Spindle Checkpoint Kinase Mps1 Signaling at the Kinetochore

The spindle assembly checkpoint (SAC) is a quality control device to ensure accurate chromosome attachment to spindle microtubule for equal segregation of sister chromatid. Aurora B is essential for SAC function by sensing chromosome bi-orientation via spatial regulation of kinetochore substrates. However, it has remained elusive as to how Aurora B couples kinetochore-microtubule attachment to SAC signaling. Here, we show that Hec1 interacts with Mps1 and specifies its kinetochore localization via its calponin homology (CH) domain and N-terminal 80 amino acids. Interestingly, phosphorylation of the Hec1 by Aurora B weakens its interaction with microtubules but promotes Hec1 binding to Mps1. Significantly, the temporal regulation of Hec1 phosphorylation orchestrates kinetochore-microtubule attachment and Mps1 loading to the kinetochore. Persistent expression of phosphomimetic Hec1 mutant induces a hyperactivation of SAC, suggesting that phosphorylation-elicited Hec1 conformational change is used as a switch to orchestrate SAC activation to concurrent destabilization of aberrant kinetochore attachment. Taken together, these results define a novel role for Aurora B-Hec1-Mps1 signaling axis in governing accurate chromosome segregation in mitosis.     

Dynamical Scenarios for Chromosome Bi-orientation

Chromosome bi-orientation at the metaphase spindle is essential for precise segregation of the genetic material. The process is error-prone, and error-correction mechanisms exist to switch misaligned chromosomes to the correct, bi-oriented configuration. Here, we analyze several possible dynamical scenarios to explore how cells might achieve correct bi-orientation in an efficient and robust manner. We first illustrate that tension-mediated feedback between the sister kinetochores can give rise to a bistable switch, which allows robust distinction between a loose attachment with low tension and a strong attachment with high tension. However, this mechanism has difficulties in explaining how bi-orientation is initiated starting from unattached kinetochores. We propose four possible mechanisms to overcome this problem (exploiting molecular noise; allowing an efficient attachment of kinetochores already in the absence of tension; a trial-and-error oscillation; and a stochastic bistable switch), and assess their impact on the bi-orientation process. Based on our results and supported by experimental data, we put forward a trial-and-error oscillation and a stochastic bistable switch as two elegant mechanisms with the potential to promote bi-orientation both efficiently and robustly.     

Dynamical Scenarios for Chromosome Bi-orientation

Chromosome bi-orientation at the metaphase spindle is essential for precise segregation of the genetic material. The process is error-prone, and error-correction mechanisms exist to switch misaligned chromosomes to the correct, bi-oriented configuration. Here, we analyze several possible dynamical scenarios to explore how cells might achieve correct bi-orientation in an efficient and robust manner. We first illustrate that tension-mediated feedback between the sister kinetochores can give rise to a bistable switch, which allows robust distinction between a loose attachment with low tension and a strong attachment with high tension. However, this mechanism has difficulties in explaining how bi-orientation is initiated starting from unattached kinetochores. We propose four possible mechanisms to overcome this problem (exploiting molecular noise; allowing an efficient attachment of kinetochores already in the absence of tension; a trial-and-error oscillation; and a stochastic bistable switch), and assess their impact on the bi-orientation process. Based on our results and supported by experimental data, we put forward a trial-and-error oscillation and a stochastic bistable switch as two elegant mechanisms with the potential to promote bi-orientation both efficiently and robustly.     

Role of the Number of Microtubules in Chromosome Segregation during Cell Division

Faithful segregation of genetic material during cell division requires alignment of chromosomes between two spindle poles and attachment of their kinetochores to each of the poles. Failure of these complex dynamical processes leads to chromosomal instability (CIN), a characteristic feature of several diseases including cancer. While a multitude of biological factors regulating chromosome congression and bi-orientation have been identified, it is still unclear how they are integrated so that coherent chromosome motion emerges from a large collection of random and deterministic processes. Here we address this issue by a three dimensional computational model of motor-driven chromosome congression and bi-orientation during mitosis. Our model reveals that successful cell division requires control of the total number of microtubules: if this number is too small bi-orientation fails, while if it is too large not all the chromosomes are able to congress. The optimal number of microtubules predicted by our model compares well with early observations in mammalian cell spindles. Our results shed new light on the origin of several pathological conditions related to chromosomal instability.