Chromosome Segregation Is Biased by Kinetochore Size

Chromosome missegregation during mitosis or meiosis is a hallmark of cancer and the main cause of prenatal death in humans. The gain or loss of specific chromosomes is thought to be random, with cell viability being essentially determined by selection. Several established pathways including centrosome amplification, sister-chromatid cohesion defects, or a compromised spindle assembly checkpoint can lead to chromosome missegregation. However, how specific intrinsic features of the kinetochore—the critical chromosomal interface with spindle microtubules—impact chromosome segregation remains poorly understood. Here we used the unique cytological attributes of female Indian muntjac, the mammal with the lowest known chromosome number (2n = 6), to characterize and track individual chromosomes with distinct kinetochore size throughout mitosis. We show that centromere and kinetochore functional layers scale proportionally with centromere size. Measurement of intra-kinetochore distances, serial-section electron microscopy, and RNAi against key kinetochore proteins confirmed a standard structural and functional organization of the Indian muntjac kinetochores and revealed that microtubule binding capacity scales with kinetochore size. Surprisingly, we found that chromosome segregation in this species is not random. Chromosomes with larger kinetochores bi-oriented more efficiently and showed a 2-fold bias to congress to the equator in a motor-independent manner. Despite robust correction mechanisms during unperturbed mitosis, chromosomes with larger kinetochores were also strongly biased to establish erroneous merotelic attachments and missegregate during anaphase. This bias was impervious to the experimental attenuation of polar ejection forces on chromosome arms by RNAi against the chromokinesin Kif4a. Thus, kinetochore size is an important determinant of chromosome segregation fidelity

Memórias autobiográficas e as escolhas profissionais das meninas

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Feedback control of chromosome separation by a midzone Aurora B gradient

Accurate chromosome segregation during mitosis requires the physical separation of sister chromatids before nuclear envelope reassembly (NER). However, how these two processes are coordinated remains unknown. Here, we identified a conserved feedback control mechanism that delays chromosome decondensation and NER in response to incomplete chromosome separation during anaphase. A midzone-associated Aurora B gradient was found to monitor chromosome position along the division axis and to prevent premature chromosome decondensation by retaining Condensin I. PP1/PP2A phosphatases counteracted this gradient and promoted chromosome decondensation and NER. Thus, an Aurora B gradient appears to mediate a surveillance mechanism that prevents chromosome decondensation and NER until effective separation of sister chromatids is achieved. This allows the correction and reintegration of lagging chromosomes in the main nuclei before completion of NER.We thank all colleagues who provided reagents and P. Sampaio for technical help with FRET. H. M. is funded by FCOMP-01-0124-FEDER-015941 (PTDC/SAU-ONC/112917/2009) through COMPETE and Fundacao para a Ciencia e a Tecnologia of Portugal, the Human Frontier Science Program, and PRECISE grant from the European Research Council. Data described can be found in the main figures and supplementary materials. The authors declare no conflict of interests

An anaphase surveillance mechanism prevents micronuclei formation from frequent chromosome segregation errors

Micronuclei are a hallmark of cancer and several other human disorders. Recently, micronuclei were implicated in chromothripsis, a series of massive genomic rearrangements that may drive tumor evolution and progression. Here, we show that Aurora B kinase mediates a surveillance mechanism that integrates error correction during anaphase with spatial control of nuclear envelope reassembly to prevent micronuclei formation. Using high-resolution live-cell imaging of human cancer and non-cancer cells, we uncover that anaphase lagging chromosomes are more frequent than previously anticipated, yet they rarely form micronuclei. Micronuclei formation from anaphase lagging chromosomes is prevented by a midzone-based Aurora B phosphorylation gradient that stabilizes kinetochore-microtubule attachments and assists spindle forces required for anaphase error correction while delaying nuclear envelope reassembly on lagging chromosomes, independently of microtubule density. We propose that a midzone-based Aurora B phosphorylation gradient actively monitors and corrects frequent chromosome segregation errors to prevent micronuclei formation during human cell division.We thank António Pereira and Ana Almeida for assistance with CH-STED, Ariana Jacome for the generation of lentiviral vectors, Cristina Ferrás and Marco Novais-Cruz for drawing attention to the effects of nocodazole treatment and washout on the Aurora B phosphorylation gradient, current and former lab members for suggestions, and Jonathan Higgins and Andrew McAinsh for discussing results prior to publication. This work was funded by the European Research Council (ERC) consolidator grant CODECHECK , under the European Union’s Horizon 2020 research and innovation programme (grant agreement 681443 ), and Fundação para a Ciência e a Tecnologia of Portugal ( PTDC/MED-ONC/3479/2020 )

Microtubule detyrosination guides chromosomes during mitosis

Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination.We thank F. I. Ataullakhanov for help with the laser trap and data analysis; A. Kiyatkin, V. Mustyatsa, M. Molodtsov, A. Gautreau, G. Lakisic, and M. Barisic for technical assistance; and members of our laboratories for stimulating discussions. This work was supported by National Institutes of Health grant R01-GM098389 and RSG-14-018-01-CCG from the American Cancer Society to E.L.G.; by the Institut Curie, the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale, the L'Agence Nationale de la Recherche (ANR) award ANR-12-BSV2-0007, INCA_6517, ANR-10-LBX-0038, part of the IDEX Idex PSL, ANR-10-IDEX-0001-02 PSL to C.J.; and Fundacao Luso-Americana para o Desenvolvimento (FLAD) Life Science 2020 and PRECISE grant from the European Research Council to H.M. A.V.Z. is supported by the RAS Presidium Grants "Mechanisms of the Molecular Systems Integration," " Molecular and Cell Biology programs," and Russian Fund for Basic Research Grant 12-04-00111-a and 13-00-40188. R.S.S. is supported by a fellowship from the Programa Graduado em Areas da Biologia Basica e Aplicada (GABBA) PhD program from the University of Porto. A.L.P. is supported by fellowship SFRH/BPD/66707/2009 from Fundacao para a Ciencia e a Tecnologia of Portugal. M.B., R.S.S., S.K.T., M.M.M., C.J., E.L.G., and H.M. designed the experiments; M.B. performed all experiments in cells; M. M. M. established and performed the tubulin purification protocol from HeLa cells; R.S.S. performed single-molecule experiments; S.K.T. performed force measurements; A.L.P. provided reagents; all authors analyzed data; H.M., E.L.G., and M.B. wrote the paper, with contributions from all authors; H.M. conceived and coordinated the project. Data described can be found in the main figures and supplementary materials. The authors declare no conflict of interests

Robust gap repair in the contractile ring ensures timely completion of cytokinesis.

Cytokinesis in animal cells requires the constriction of an actomyosin contractile ring, whose architecture and mechanism remain poorly understood. We use laser microsurgery to explore the biophysical properties of constricting rings in Caenorhabditis elegans embryos. Laser cutting causes rings to snap open. However, instead of disintegrating, ring topology recovers and constriction proceeds. In response to severing, a finite gap forms and is repaired by recruitment of new material in an actin polymerization-dependent manner. An open ring is able to constrict, and rings repair from successive cuts. After gap repair, an increase in constriction velocity allows cytokinesis to complete at the same time as controls. Our analysis demonstrates that tension in the ring increases while net cortical tension at the site of ingression decreases throughout constriction and suggests that cytokinesis is accomplished by contractile modules that assemble and contract autonomously, enabling local repair of the actomyosin network. Consequently, cytokinesis is a highly robust process impervious to discontinuities in contractile ring structure.This project has received funding from the European Research Council (grants 640553, 260892, and 338410), Fundo Europeu de Desenvolvimento Regional (FED ER) funds through the Operational Competitiveness Program (COM PETE), national funds through Fundação para a Ciência e a Tecnologia (FCT) under the project FCO MP-01-0124-FED ER-028255 (PTDC/BEX-BCM/0654/2012), Fundação Luso-Americana para o Desenvolvimento Life Science 2020, and the Louis-Jeantet Young Investigator Award to H. Maiato. A.X. Carvalho, R. Gassmann, and I.A. Telley have FCT Investigator positions funded by FCT and cofunded by the European Social Fund through Programa Operacional Temático Potencial Type 4.2 promotion of scientific employment. A.M. Silva holds an FCT fellowship (SFRH/BPD/95707/2013). D.S. Osório was cofunded by the Programa Operacional Regional do Norte under the Quadro de Downloaded from jcb.rupress.org on February 27, 2018 Laser microsurgery in the contractile ring • Silva et al. 799 Referência Estratégico Nacional through FED ER and by FCT grant NOR TE-07-0124-FED ER-000003 (Cell Homeostasis Tissue Organization and Organism Biology)

The tumour suppressor DLC2 ensures mitotic fidelity by coordinating spindle positioning and cell-cell adhesion.

Dividing epithelial cells need to coordinate spindle positioning with shape changes to maintain cell-cell adhesion. Microtubule interactions with the cell cortex regulate mitotic spindle positioning within the plane of division. How the spindle crosstalks with the actin cytoskeleton to ensure faithful mitosis and spindle positioning is unclear. Here we demonstrate that the tumour suppressor DLC2, a negative regulator of Cdc42, and the interacting kinesin Kif1B coordinate cell junction maintenance and planar spindle positioning by regulating microtubule growth and crosstalk with the actin cytoskeleton. Loss of DLC2 induces the mislocalization of Kif1B, increased Cdc42 activity and cortical recruitment of the Cdc42 effector mDia3, a microtubule stabilizer and promoter of actin dynamics. Accordingly, DLC2 or Kif1B depletion promotes microtubule stabilization, defective spindle positioning, chromosome misalignment and aneuploidy. The tumour suppressor DLC2 and Kif1B are thus central components of a signalling network that guides spindle positioning, cell-cell adhesion and mitotic fidelity

Spatiotemporal control of mitotic exit during anaphase by an aurora B-Cdk1 crosstalk

According to the prevailing ‘clock’ model, chromosome decondensation and nuclear envelope reformation when cells exit mitosis are byproducts of Cdk1 inactivation at the metaphase-anaphase transition, controlled by the spindle assembly checkpoint. However, mitotic exit was recently shown to be a function of chromosome separation during anaphase, assisted by a midzone Aurora B phosphorylation gradient-the ‘ruler’ model. Here we found that Cdk1 remains active during anaphase due to ongoing APC/CCdc20- and APC/CCdh1-mediated degradation of B-type Cyclins in Drosophila and human cells. Failure to degrade B-type Cyclins during anaphase prevented mitotic exit in a Cdk1-dependent manner. Cyclin B1-Cdk1 localized at the spindle midzone in an Aurora B-dependent manner, with incompletely separated chromosomes showing the highest Cdk1 activity. Slowing down anaphase chromosome motion delayed Cyclin B1 degradation and mitotic exit in an Aurora B-dependent manner. Thus, a crosstalk between molecular ‘rulers’ and ‘clocks’ licenses mitotic exit only after proper chromosome separation.We thank Eric Griffis, Jean-René Huynh, Claudio Sunkel, Jonathon Pines, Melina Schuh and Christian Lehner for the kind gift of reagents, and Marco Gonzalez-Gaitán for supporting OA during the final stages of this work. LPC is the recipient of a Marie Skłodowska-Curie Action fellowship (grant agreement 746515). EMS holds an FCT Investigator position and his work is supported by Fundac¸ ão para a Ciência e a Tecnologia (PTDC/BEX-BCM/0432/2014). This work was supported by R01GM107026 grant to TJM and a Commonwealth Honors College grant to CMC Confocal and FLIM microscopy data collection was performed in the Light Microscopy Facility and Nikon Center of Excellence at the Institute for Applied Life Sciences, University of Massachusetts Amherst with support from the Massachusetts Life Science Center. Work in the HM lab is supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 681443) and FLAD Life Science 2020

DNA content of a functioning chicken kinetochore

© The Author(s) 2014. In order to understand the three-dimensional structure of the functional kinetochore in vertebrates, we require a complete list and stoichiometry for the protein components of the kinetochore, which can be provided by genetic and proteomic experiments. We also need to know how the chromatin-containing CENP-A, which makes up the structural foundation for the kinetochore, is folded, and how much of that DNA is involved in assembling the kinetochore. In this MS, we demonstrate that functioning metaphase kinetochores in chicken DT40 cells contain roughly 50 kb of DNA, an amount that corresponds extremely closely to the length of chromosomal DNA associated with CENP-A in ChIP-seq experiments. Thus, during kinetochore assembly, CENP-A chromatin is compacted into the inner kinetochore plate without including significant amounts of flanking pericentromeric heterochromatin. © 2014 The Author(s).Wellcome Trust [grant number 073915]; Wellcome Trust Centre for Cell Biology (core grant numbers 077707 and 092076); Darwin Trust of Edinburg