Metaphase Chromosome Alignment Research Articles

Kinesin-7 CENP-E mediates chromosome alignment and spindle assembly checkpoint in meiosis I.

In eukaryotes, meiosis is the genetic basis for sexual reproduction, which is important for chromosome stability and species evolution. The defects in meiosis usually lead to chromosome aneuploidy, reduced gamete number, and genetic diseases, but the pathogenic mechanisms are not well clarified. Kinesin-7 CENP-E is a key regulator in chromosome alignment and spindle assembly checkpoint in cell division. However, the functions and mechanisms of CENP-E in male meiosis remain largely unknown. In this study, we have revealed that the CENP-E gene was highly expressed in the rat testis. CENP-E inhibition influences chromosome alignment and spindle organization in metaphase I spermatocytes. We have found that a portion of misaligned homologous chromosomes is located at the spindle poles after CENP-E inhibition, which further activates the spindle assembly checkpoint during the metaphase-to-anaphase transition in rat spermatocytes. Furthermore, CENP-E depletion leads to abnormal spermatogenesis, reduced sperm count, and abnormal sperm head structure. Our findings have elucidated that CENP-E is essential for homologous chromosome alignment and spindle assembly checkpoint in spermatocytes, which further contribute to chromosome stability and sperm cell quality during spermatogenesis.

PAK2 is essential for chromosome alignment in metaphase I oocytes

As a highly conserved and ubiquitously expressed serine/threonine kinase, p21-activated kinase 2 (PAK2) participates in diverse biologic events. However, its roles in mouse oocyte meiotic maturation remain unclear. The present study revealed that mouse oocytes depleted of Pak2 were unable to completely progress through meiosis and that a majority were arrested at metaphase I. Pak2 depletion thus prompted MI arrest and induced meiotic chromosome alignment defects in mouse oocytes, in part due to a reduction in polo-like kinase (PLK1). We demonstrated that PAK2’s interaction with PLK1 protected it from degradation by APC/CCdh1, and that it promoted meiotic progression and bipolar spindle formation. Our data collectively display critical functions for PAK2 in meiotic progression and chromosome alignment in mouse oocytes.

Kinetochore-microtubule attachment in human cells is regulated by the interaction of a conserved motif of Ska1 with EB1

The kinetochore establishes the linkage between chromosomes and the spindle microtubule plus ends during mitosis. In vertebrates, the spindle-kinetochore-associated (Ska1,2,3) complex stabilizes kinetochore attachment with the microtubule plus ends, but how Ska is recruited to and stabilized at the kinetochore-microtubule interface is not understood. Here, our results show that interaction of Ska1 with the general microtubule plus end-associated protein EB1 through a conserved motif regulates Ska recruitment to kinetochores in human cells. Ska1 forms a stable complex with EB1 via interaction with the motif in its N-terminal disordered loop region. Disruption of this interaction either by deleting or mutating the motif disrupts Ska complex recruitment to kinetochores and induces chromosome alignment defects, but it does not affect Ska complex assembly. Atomic-force microscopy imaging revealed that Ska1 is anchored to the C-terminal region of the EB1 dimer through its loop and thereby promotes formation of extended structures. Furthermore, our NMR data showed that the Ska1 motif binds to the residues in EB1 that are the binding sites of other plus end targeting proteins that are recruited to microtubules by EB1 through a similar conserved motif. Collectively, our results demonstrate that EB1-mediated Ska1 recruitment onto the microtubule serves as a general mechanism for the formation of vertebrate kinetochore-microtubule attachments and metaphase chromosome alignment.

Chromosome number, sex determination, and meiotic chromosome behavior in the praying mantid Hierodula membranacea.

Praying mantids are important models for studying a wide range of chromosome behaviors, yet few species of mantids have been characterized chromosomally. Here we show that the praying mantid Hierodula membranacea has a chromosome number of 2n = 27, and X1X1X2X2 (female): X1X2Y (male) sex determination. In male meiosis I, the X1, X2, and Y chromosomes of H. membranacea form a sex trivalent, with the Y chromosome associating with one spindle pole and the X1 and X2 chromosomes facing the opposite spindle pole. While it is possible that such a sex trivalent could experience different spindle forces on each side of the trivalent, in H. membranacea the sex trivalent aligns at the spindle equator with all of the autosomes, and then the sex chromosomes separate in anaphase I simultaneously with the autosomes. With this observation, H. membranacea can be used as a model system to study the balance of forces acting on a trivalent during meiosis I and analyze the functional importance of chromosome alignment in metaphase as a preparatory step for subsequent correct chromosome segregation.

The ciliopathy protein CCDC66 controls mitotic progression and cytokinesis by promoting microtubule nucleation and organization.

Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division and signaling via the primary cilium in quiescent cells. Microtubule-associated proteins (MAPs) govern assembly, maintenance, and remodeling of diverse microtubule arrays. While a set of conserved MAPs are only active during cell division, an emerging group of MAPs acts as dual regulators in dividing and nondividing cells. Here, we elucidated the nonciliary functions and molecular mechanism of action of the ciliopathy-linked protein CCDC66, which we previously characterized as a regulator of ciliogenesis in quiescent cells. We showed that CCDC66 dynamically localizes to the centrosomes, the bipolar spindle, the spindle midzone, the central spindle, and the midbody in dividing cells and interacts with the core machinery of centrosome maturation and MAPs involved in cell division. Loss-of-function experiments revealed its functions during mitotic progression and cytokinesis. Specifically, CCDC66 depletion resulted in defective spindle assembly and orientation, kinetochore fiber stability, chromosome alignment in metaphase as well as central spindle and midbody assembly and organization in anaphase and cytokinesis. Notably, CCDC66 regulates mitotic microtubule nucleation via noncentrosomal and centrosomal pathways via recruitment of gamma-tubulin to the centrosomes and the spindle. Additionally, CCDC66 bundles microtubules in vitro and in cells by its C-terminal microtubule-binding domain. Phenotypic rescue experiments showed that the microtubule and centrosome-associated pools of CCDC66 individually or cooperatively mediate its mitotic and cytokinetic functions. Collectively, our findings identify CCDC66 as a multifaceted regulator of the nucleation and organization of the diverse mitotic and cytokinetic microtubule arrays and provide new insight into nonciliary defects that underlie ciliopathies.

Cell division requires RNA eviction from condensing chromosomes.

During mitosis, the genome is transformed from a decondensed, transcriptionally active state to a highly condensed, transcriptionally inactive state. Mitotic chromosome reorganization is marked by the general attenuation of transcription on chromosome arms, yet how the cell regulates nuclear and chromatin-associated RNAs after chromosome condensation and nuclear envelope breakdown is unknown. SAF-A/hnRNPU is an abundant nuclear protein with RNA-to-DNA tethering activity, coordinated by two spatially distinct nucleic acid-binding domains. Here we show that RNA is evicted from prophase chromosomes through Aurora-B-dependent phosphorylation of the SAF-A DNA-binding domain; failure to execute this pathway leads to accumulation of SAF-A-RNA complexes on mitotic chromosomes, defects in metaphase chromosome alignment, and elevated rates of chromosome missegregation in anaphase. This work reveals a role for Aurora-B in removing chromatin-associated RNAs during prophase and demonstrates that Aurora-B-dependent relocalization of SAF-A during cell division contributes to the fidelity of chromosome segregation.

Transgelin-2 and phosphoregulation of the LIC2 subunit of dynein govern mitotic spindle orientation.

The molecular motor dynein is essential for mitotic spindle orientation, which defines the axis of cell division. The light intermediate chain subunits, LIC1 and LIC2, define biochemically and functionally distinct vertebrate dynein complexes, with LIC2-dynein playing a crucial role in ensuring spindle orientation. We reveal a novel, mitosis-specific interaction of LIC2-dynein with the cortical actin-bundling protein transgelin-2. Transgelin-2 is required for maintaining proper spindle length, equatorial metaphase chromosome alignment, spindle orientation and timely anaphase onset. We show that transgelin-2 stabilizes the cortical recruitment of LGN-NuMA, which together with dynein is required for spindle orientation. The opposing actions of transgelin-2 and LIC2-dynein maintain optimal cortical levels of LGN-NuMA. In addition, we show that the highly conserved serine 194 phosphorylation of LIC2 is required for proper spindle orientation, by maintaining mitotic centrosome integrity to ensure optimal astral microtubule nucleation. The work reveals two specific mechanisms through which LIC2-dynein regulates mitotic spindle orientation; namely, through a new interactor transgelin-2, which is required for engagement of LGN-NuMA with the actin cortex, and through mitotic phosphoregulation of LIC2 to control microtubule nucleation from the poles.This article has an associated First Person interview with the first author of the paper.

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations.

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, misunderstood, or misinterpreted by the fixed-cell analysis. While the fixed cell imaging and analysis is robust and sufficient to observe cellular steady-state, it can be limited in defining a temporal order of events at the cellular level and is ill-equipped to assess the transient nature of dynamic processes including mitotic progression. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular processes at the single-cell level over time and has the capacity to capture the dynamics of processes that would otherwise be poorly represented in fixed cell imaging. Here we describe an approach to generate cells carrying fluorescently labeled markers of chromatin and microtubules and their use in live cell imaging approaches to monitor metaphase chromosome alignment and mitotic exit. We describe imaging-based techniques to assess the dynamics of spindle formation and mitotic progression, including the identification of cells at various stages in mitosis, identificationand tracking of mitotic defects, and analysis of spindle dynamics and mitotic cell fate following the treatment with mitotic inhibitors.

Creation and testing of a new, local microtubule-disruption tool based on the microtubule-severing enzyme, katanin p60.

Current methods to disrupt the microtubule cytoskeleton do not easily provide rapid, local control with standard cell manipulation reagents. Here, we develop a new microtubule-disruption tool based on katanin p60 severing activity and demonstrate proof-of-principle by targeting it to kinetochores in Drosophila melanogaster S2 cells. Specifically, we show that human katanin p60 can remove microtubule polymer mass in S2 cells and an increase in misaligned chromosomes when globally overexpressed. When katanin p60 was targeted to the kinetochores via Mis12, we were able to recapitulate the misalignment only when using a phosphorylation-resistant mutant katanin p60. Our results demonstrate that targeting an active version of katanin p60 to the kinetochore can reduce the fidelity of achieving full chromosome alignment in metaphase and could serve as a microtubule disruption tool for the future.

The Hybrid Incompatibility Genes Lhr and Hmr Are Required for Sister Chromatid Detachment During Anaphase but Not for Centromere Function.

Crosses between Drosophila melanogaster females and Drosophila simulans males produce hybrid sons that die at the larval stage. This hybrid lethality is suppressed by loss-of-function mutations in the D. melanogaster Hybrid male rescue (Hmr) or in the D. simulans Lethal hybrid rescue (Lhr) genes. Previous studies have shown that Hmr and Lhr interact with heterochromatin proteins and suppress expression of transposable elements within D. melanogaster It also has been proposed that Hmr and Lhr function at the centromere. We examined mitotic divisions in larval brains from Hmr and Lhr single mutants and Hmr; Lhr double mutants in D. melanogaster In none of the mutants did we observe defects in metaphase chromosome alignment or hyperploid cells, which are hallmarks of centromere or kinetochore dysfunction. In addition, we found that Hmr-HA and Lhr-HA do not colocalize with centromeres either during interphase or mitotic division. However, all mutants displayed anaphase bridges and chromosome aberrations resulting from the breakage of these bridges, predominantly at the euchromatin-heterochromatin junction. The few dividing cells present in hybrid males showed fuzzy and irregularly condensed chromosomes with unresolved sister chromatids. Despite this defect in condensation, chromosomes in hybrids managed to align on the metaphase plate and undergo anaphase. We conclude that there is no evidence for a centromeric function of Hmr and Lhr within D. melanogaster nor for a centromere defect causing hybrid lethality. Instead, we find that Hmr and Lhr are required in D. melanogaster for detachment of sister chromatids during anaphase.

ATRX is a novel progesterone-regulated protein and biomarker of low developmental potential in mammalian oocytes.

A multi-species meta-analysis of published transcriptomic data from models of oocyte competence identified the chromatin remodelling factor ATRX as a putative biomarker of oocyte competence. The objective of the current study was to test the hypothesis that ATRX protein expression by cumulus-oocyte complexes (COCs) reflects their intrinsic quality and developmental potential. In excess of 10,000 bovine COCs were utilised to test our hypothesis. COCs were in vitro matured (IVM) under conditions associated with reduced developmental potential: IVM in the presence or absence of (1) progesterone synthesis inhibitor (Trilostane); (2) nuclear progesterone receptor inhibitor (Aglepristone) or (3) an inducer of DNA damage (Staurosporine). ATRX protein expression and localisation were determined using immunocytochemistry and Western blot analysis. A proportion of COCs matured in the presence or absence of Trilostane was in vitro fertilised and cultured, and subsequent embryo development characteristics were analysed. In addition, ATRX expression was investigated in 40 human germinal vesicle-stage COCs. Our results showed that ATRX is expressed in human and bovine germinal vesicle oocytes and cumulus cells. In bovine, expression decreases after IVM. However, this decline is not observed in COCs matured under sub-optimal conditions. Blastocyst development rate and cell number are decreased, whereas the incidence of abnormal metaphase phase spindle and chromosome alignment are increased, after IVM in the presence of Trilostane (P < 0.05). In conclusion, localisation of ATRX to the cumulus cell nuclei and oocyte chromatin, after IVM, is associated with poor oocyte quality and low developmental potential. Furthermore, ATRX is dynamically regulated in response to progesterone signalling.

Spindle Assembly and Chromosome Segregation Requires Central Spindle Proteins inDrosophilaOocytes

Oocytes segregate chromosomes in the absence of centrosomes. In this situation, the chromosomes direct spindle assembly. It is still unclear in this system which factors are required for homologous chromosome bi-orientation and spindle assembly. The Drosophila kinesin-6 protein Subito, although nonessential for mitotic spindle assembly, is required to organize a bipolar meiotic spindle and chromosome bi-orientation in oocytes. Along with the chromosomal passenger complex (CPC), Subito is an important part of the metaphase I central spindle. In this study we have conducted genetic screens to identify genes that interact with subito or the CPC component Incenp. In addition, the meiotic mutant phenotype for some of the genes identified in these screens were characterized. We show, in part through the use of a heat-shock-inducible system, that the Centralspindlin component RacGAP50C and downstream regulators of cytokinesis Rho1, Sticky, and RhoGEF2 are required for homologous chromosome bi-orientation in metaphase I oocytes. This suggests a novel function for proteins normally involved in mitotic cell division in the regulation of microtubule-chromosome interactions. We also show that the kinetochore protein, Polo kinase, is required for maintaining chromosome alignment and spindle organization in metaphase I oocytes. In combination our results support a model where the meiotic central spindle and associated proteins are essential for acentrosomal chromosome segregation.

Arginine methylation of ubiquitin-associated protein 2-like is required for the accurate distribution of chromosomes.

Proper bioriented attachment of microtubules and kinetochores is essential for the precise distribution of duplicated chromosomes to each daughter cell. An aberrant kinetochore-microtubule attachment results in chromosome instability, which leads to cellular transformation or apoptosis. In this article, we show that ubiquitin-associated protein 2-like (UBAP2L) is necessary for correct kinetochore-microtubule attachment. Depletion of UBAP2L inhibited chromosome alignment in metaphase and delayed progression to anaphase by activating spindle assembly checkpoint signaling. In addition, UBAP2L knockdown increased side-on attachment of kinetochores along the microtubules and suppressed stable kinetochore fiber formation. A proteomics analysis identified protein arginine methyltransferase (PRMT)1 as a direct interaction partner of UBAP2L. UBAP2L has an arginine- and glycine-rich motif called the RGG/RG or GAR motif in the N terminus. Biochemical analysis confirmed that arginine residues in the RGG/RG motif of UBAP2L were directly methylated by PRMT1. Finally, we demonstrated that the RGG/RG motif of UBAP2L is essential for the proper alignment of chromosomes in metaphase for the accurate distribution of chromosomes. Our results show a possible role for arginine methylation in UBAP2L for the progression of mitosis.

Expression and characterization of three Aurora kinase C splice variants found in human oocytes

Chromosome segregation is an extensively choreographed process yet errors still occur frequently in female meiosis, leading to implantation failure, miscarriage or offspring with developmental disorders. Aurora kinase C (AURKC) is a component of the chromosome passenger complex and is highly expressed in gametes. Studies in mouse oocytes indicate that AURKC is required to regulate chromosome segregation during meiosis I; however, little is known about the functional significance of AURKC in human oocytes. Three splice variants of AURKC exist in testis tissue. To determine which splice variants human oocytes express, we performed quantitative real-time PCR using single oocytes and found expression of all three variants. To evaluate the functional differences between the variants, we created green fluorescent protein-tagged constructs of each variant to express in oocytes from Aurkc−/− mice. By quantifying metaphase chromosome alignment, cell cycle progression, phosphorylation of INCENP and microtubule attachments to kinetochores, we found that AURKC_v1 was the most capable of the variants at supporting metaphase I chromosome segregation. AURKC_v3 localized to chromosomes properly and supported cell cycle progression to metaphase II, but its inability to correct erroneous microtubule attachments to kinetochores meant that chromosome segregation was not as accurate compared with the other two variants. Finally, when we expressed the three variants simultaneously, error correction was more robust than when they were expressed on their own. Therefore, oocytes express three variants of AURKC that are not functionally equivalent in supporting meiosis, but fully complement meiosis when expressed simultaneously.

Heterogeneous nuclear ribonucleoprotein K upregulates the kinetochore complex component NUF2 and promotes the tumorigenicity of colon cancer cells

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multi-functional protein involved in transcription, mRNA splicing, mRNA stabilization and translation. Although hnRNP K has been suggested to play a role in the development of many cancers, its molecular function in colorectal cancer has remained elusive. Here we show that hnRNP K plays an important role in the mitotic process in HCT116 colon cancer cells. Furthermore, we demonstrate that hnRNP K directly transactivates the NUF2 gene, the product of which is a component of the NDC80 kinetochore complex and which is known to be critical for a stable spindle microtubule-kinetochore attachment. In addition, knockdown of both hnRNP K and NUF2 caused failure in metaphase chromosome alignment and drastic decrease in the growth of colon cancer cells. These results suggest that the hnRNP K-NUF2 axis is important for the mitotic process and proliferation of colon cancer cells and that this axis could be a target for the therapy of colon cancer.

Stu2, the Budding Yeast XMAP215/Dis1 Homolog, Promotes Assembly of Yeast Microtubules by Increasing Growth Rate and Decreasing Catastrophe Frequency

Stu2 is the budding yeast member of the XMAP215/Dis1 family of microtubule polymerases. It is essential in cell division, allowing proper spindle orientation and metaphase chromosome alignment, as well as spindle elongation during anaphase. Despite Stu2 having a phenotype that suggests it promotes microtubule growth, like the other members of the XMAP215/Dis1 family, previous studies with purified Stu2 indicate only that it antagonizes microtubule growth. One potential explanation for these contradictory findings is that the assays were performed with mammalian brain tubulin, which may not be the right substrate to test the activity of Stu2 given that yeast and brain tubulins are quite divergent in sequence and that the vertebrate tubulins are subject to many post-translational modifications. To test this possibility, we reconstituted the activity of Stu2 with purified budding yeast tubulin. We found that Stu2 accelerated microtubule growth in yeast tubulin by severalfold, similar to the acceleration reported for XMAP215 in porcine brain tubulin. Furthermore, Stu2 accelerated polymerization in yeast tubulin to a much greater extent than in porcine brain tubulin, and the concentration of Stu2 required to reach 50% maximum activity in yeast tubulin was nearly 2 orders of magnitude lower than that in porcine brain tubulin. We conclude that Stu2 is a microtubule polymerase, like its relatives, and that its activity is considerably higher in yeast tubulin compared with mammalian brain tubulin. The biochemical properties of Stu2 reported here account for many of the phenotypes of Stu2 observed in cells.

Chk1 is required for the metaphase–anaphase transition via regulating the expression and localization of Cdc20 and Mad2

AimsThe checkpoint kinase 1 (Chk1) functions not only in genotoxic stresses but also in normal cell cycle progression, particularly in the initiation, progression and fidelity of unperturbed mitosis. In this study, we investigated the role of Chk1 in regulating the metaphase–anaphase transition in mammalian cells. Main methodsThe mitotic progression was monitored by flow cytometry analysis. The levels of cyclin B1, Cdc20 and Mad2 were measured by Western blotting. Metaphase chromosome alignment and the subcellular localization of Cdc20 and Mad2 were analyzed by immunofluorescence and confocal microscopy. Key findingsCyclin B1 degradation and the metaphase–anaphase transition were severely blocked by Chk1 siRNA. Depletion of Chk1 induced chromosome alignment defect in metaphase cells. The kinetochore localization of Cdc20, Mad2 was disrupted in Chk1 depleted cells. Chk1 abrogation also dramatically reduced the protein expression levels of Cdc20 and Mad2. SignificanceThese results strongly suggest that Chk1 is required for the metaphase–anaphase transition via regulating the subcellular localization and the expression of Cdc20 and Mad2.

Role and regulation of kinesin-8 motors through the cell cycle.

Members of the kinesin-8 motor family play a central role in controlling microtubule length throughout the eukaryotic cell cycle. Inactivation of kinesin-8 causes defects in cell polarity during interphase and astral and mitotic spindle length, metaphase chromosome alignment, timing of anaphase onset and accuracy of chromosome segregation. Although the biophysical mechanism by which kinesin-8 molecules influence microtubule dynamics has been studied extensively in a variety of species, a consensus view has yet to emerge. One reason for this might be that some members of the kinesin-8 family can associate to other microtubule-associated proteins, cell cycle regulatory proteins and other kinesin family members. In this review we consider how cell cycle specific modification and its association to other regulatory proteins may modulate the function of kinesin-8 to enable it to function as a master regulator of microtubule dynamics.

Katanin maintains meiotic metaphase chromosome alignment and spindle structure in vivo and has multiple effects on microtubules in vitro

Assembly of Caenorhabditis elegans female meiotic spindles requires both MEI-1 and MEI-2 subunits of the microtubule-severing ATPase katanin. Strong loss-of-function mutants assemble apolar intersecting microtubule arrays, whereas weaker mutants assemble bipolar meiotic spindles that are longer than wild type. To determine whether katanin is also required for spindle maintenance, we monitored metaphase I spindles after a fast-acting mei-1(ts) mutant was shifted to a nonpermissive temperature. Within 4 min of temperature shift, bivalents moved off the metaphase plate, and microtubule bundles within the spindle lengthened and developed a high degree of curvature. Spindles eventually lost bipolar structure. Immunofluorescence of embryos fixed at increasing temperature indicated that MEI-1 was lost from spindle microtubules before loss of ASPM-1, indicating that MEI-1 and ASPM-1 act independently at spindle poles. We quantified the microtubule-severing activity of purified MEI-1/MEI-2 complexes corresponding to six different point mutations and found a linear relationship between microtubule disassembly rate and meiotic spindle length. Previous work showed that katanin is required for severing at points where two microtubules intersect in vivo. We show that purified MEI-1/MEI-2 complexes preferentially sever at intersections between two microtubules and directly bundle microtubules in vitro. These activities could promote parallel/antiparallel microtubule organization in meiotic spindles.

The MRN-CtIP Pathway Is Required for Metaphase Chromosome Alignment

Faithful duplication of the genome in S phase followed by its accurate segregation in mitosis is essential to maintain genomic integrity. Recent studies have suggested that proteins involved in DNA transactions are also required for whole-chromosome stability. Here we demonstrate that the MRN (Mre11, Rad50, and Nbs1) complex and CtIP are required for accurate chromosome segregation. Depletion of Mre11 or CtIP, antibody-mediated inhibition of Mre11, or small-molecule inhibition of MRN using mirin results in metaphase chromosome alignment defects in Xenopus egg extracts. Similarly, loss of MRN function adversely affects spindle assembly around DNA-coated beads in egg extracts. Inhibition of MRN function in mammalian cells triggers a metaphase delay and disrupts the RCC1-dependent RanGTP gradient. Addition of the Mre11 inhibitor mirin to egg extracts and mammalian cells reduces RCC1 association with mitotic chromosomes. Thus, the MRN-CtIP pathway contributes to Ran-dependent mitotic spindle assembly by modulating RCC1 chromosome association.