Structure of the human inner kinetochore bound to a centromeric CENP-A nucleosome

Accurate chromosome segregation, controlled by kinetochore-mediated chromatid attachments to the mitotic spindle, ensures the faithful inheritance of genetic information. Kinetochores assemble onto specialized CENP-A nucleosomes (CENP-ANuc) of centromeric chromatin. In humans, this is mostly organized as thousands of copies of an ~171 bp α-satellite repeat. Here, we describe the cryo-EM structure of the human inner kinetochore CCAN (Constitutive Centromere Associated Network) complex bound to CENP-ANuc reconstituted onto α-satellite DNA. CCAN forms edge-on contacts with CENP-ANuc, while a linker DNA segment of the α-satellite repeat emerges from the fully-wrapped end of the nucleosome to thread through the central CENP-LN channel which tightly grips the DNA. The CENP-TWSX histone-fold module, together with CENP-HIKHead, further augments DNA binding and partially wraps the linker DNA in a manner reminiscent of canonical nucleosomes. Our study suggests that the topological entrapment of the α-satellite repeat linker DNA by CCAN provides a robust mechanism by which the kinetochore withstands the pushing and pulling of centromeres associated with chromosome congression and segregation forces.

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A quantitative analysis of cohesin decay in mitotic fidelity

The Journal of Cell Biology ◽

10.1083/jcb.201801111 ◽

2018 ◽

Vol 217 (10) ◽

pp. 3343-3353 ◽

Cited By ~ 6

Author(s):

Sara Carvalhal ◽

Alexandra Tavares ◽

Mariana B. Santos ◽

Mihailo Mirkovic ◽

Raquel A. Oliveira

Keyword(s):

Quantitative Analysis ◽

Chromosome Segregation ◽

Sister Chromatid ◽

Mitotic Spindle ◽

Sister Chromatid Cohesion ◽

Force Balance ◽

Chromosome Organization ◽

Centromeric Chromatin ◽

Chromatid Cohesion ◽

Chromosome Alignment

Sister chromatid cohesion mediated by cohesin is essential for mitotic fidelity. It counteracts spindle forces to prevent premature chromatid individualization and random genome segregation. However, it is unclear what effects a partial decline of cohesin may have on chromosome organization. In this study, we provide a quantitative analysis of cohesin decay by inducing acute removal of defined amounts of cohesin from metaphase-arrested chromosomes. We demonstrate that sister chromatid cohesion is very resistant to cohesin loss as chromatid disjunction is only observed when chromosomes lose >80% of bound cohesin. Removal close to this threshold leads to chromosomes that are still cohered but display compromised chromosome alignment and unstable spindle attachments. Partial cohesin decay leads to increased duration of mitosis and susceptibility to errors in chromosome segregation. We propose that high cohesin density ensures centromeric chromatin rigidity necessary to maintain a force balance with the mitotic spindle. Partial cohesin loss may lead to chromosome segregation errors even when sister chromatid cohesion is fulfilled.

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Intrakinetochore stretch is associated with changes in kinetochore phosphorylation and spindle assembly checkpoint activity

The Journal of Cell Biology ◽

10.1083/jcb.200808130 ◽

2009 ◽

Vol 184 (3) ◽

pp. 373-381 ◽

Cited By ~ 181

Author(s):

Thomas J. Maresca ◽

Edward D. Salmon

Keyword(s):

Drosophila Melanogaster ◽

Green Fluorescent Protein ◽

Chromosome Segregation ◽

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Fluorescent Protein ◽

Spindle Assembly ◽

Centromeric Chromatin ◽

Cell Assays ◽

Green Fluorescent

Cells have evolved a signaling pathway called the spindle assembly checkpoint (SAC) to increase the fidelity of chromosome segregation by generating a “wait anaphase” signal until all chromosomes are properly aligned within the mitotic spindle. It has been proposed that tension generated by the stretch of the centromeric chromatin of bioriented chromosomes stabilizes kinetochore microtubule attachments and turns off SAC activity. Although biorientation clearly causes stretching of the centromeric chromatin, it is unclear whether the kinetochore is also stretched. To test whether intrakinetochore stretch occurs and is involved in SAC regulation, we developed a Drosophila melanogaster S2 cell line expressing centromere identifier–mCherry and Ndc80–green fluorescent protein to mark the inner and outer kinetochore domains, respectively. We observed stretching within kinetochores of bioriented chromosomes by monitoring both inter- and intrakinetochore distances in live cell assays. This intrakinetochore stretch is largely independent of a 30-fold variation in centromere stretch. Furthermore, loss of intrakinetochore stretch is associated with enhancement of 3F3/2 phosphorylation and SAC activation.

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Integrity of the human centromere DNA repeats is protected by CENP-A, CENP-C, and CENP-T

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1615133114 ◽

2017 ◽

Vol 114 (8) ◽

pp. 1928-1933 ◽

Cited By ~ 47

Author(s):

Simona Giunta ◽

Hironori Funabiki

Keyword(s):

Chromosome Segregation ◽

Satellite Dna ◽

Replicative Senescence ◽

Alternative Methods ◽

Dna Repeats ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Satellite Repeat ◽

Culture Cells ◽

Centromeric Repeat

Centromeres are highly specialized chromatin domains that enable chromosome segregation and orchestrate faithful cell division. Human centromeres are composed of tandem arrays of α-satellite DNA, which spans up to several megabases. Little is known about the mechanisms that maintain integrity of the long arrays of α-satellite DNA repeats. Here, we monitored centromeric repeat stability in human cells using chromosome-orientation fluorescent in situ hybridization (CO-FISH). This assay detected aberrant centromeric CO-FISH patterns consistent with sister chromatid exchange at the frequency of 5% in primary tissue culture cells, whereas higher levels were seen in several cancer cell lines and during replicative senescence. To understand the mechanism(s) that maintains centromere integrity, we examined the contribution of the centromere-specific histone variant CENP-A and members of the constitutive centromere-associated network (CCAN), CENP-C, CENP-T, and CENP-W. Depletion of CENP-A and CCAN proteins led to an increase in centromere aberrations, whereas enhancing chromosome missegregation by alternative methods did not, suggesting that CENP-A and CCAN proteins help maintain centromere integrity independently of their role in chromosome segregation. Furthermore, superresolution imaging of centromeric CO-FISH using structured illumination microscopy implied that CENP-A protects α-satellite repeats from extensive rearrangements. Our study points toward the presence of a centromere-specific mechanism that actively maintains α-satellite repeat integrity during human cell proliferation.

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The fission yeast nucleoporin Alm1 is required for proteasomal degradation of kinetochore components

The Journal of Cell Biology ◽

10.1083/jcb.201612194 ◽

2017 ◽

Vol 216 (11) ◽

pp. 3591-3608 ◽

Cited By ~ 6

Author(s):

Silvia Salas-Pino ◽

Paola Gallardo ◽

Ramón R. Barrales ◽

Sigurd Braun ◽

Rafael R. Daga

Keyword(s):

Nuclear Envelope ◽

Fission Yeast ◽

Chromosome Segregation ◽

Mitotic Spindle ◽

Essential Role ◽

Spindle Assembly Checkpoint ◽

Proteasomal Degradation ◽

Nuclear Pore ◽

Checkpoint Activation ◽

Centromeric Chromatin

Kinetochores (KTs) are large multiprotein complexes that constitute the interface between centromeric chromatin and the mitotic spindle during chromosome segregation. In spite of their essential role, little is known about how centromeres and KTs are assembled and how their precise stoichiometry is regulated. In this study, we show that the nuclear pore basket component Alm1 is required to maintain both the proteasome and its anchor, Cut8, at the nuclear envelope, which in turn regulates proteostasis of certain inner KT components. Consistently, alm1-deleted cells show increased levels of KT proteins, including CENP-CCnp3, spindle assembly checkpoint activation, and chromosome segregation defects. Our data demonstrate a novel function of the nucleoporin Alm1 in proteasome localization required for KT homeostasis.

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De Novo Kinetochore Assembly Requires the Centromeric Histone H3 Variant

Molecular Biology of the Cell ◽

10.1091/mbc.e05-08-0771 ◽

2005 ◽

Vol 16 (12) ◽

pp. 5649-5660 ◽

Cited By ~ 54

Author(s):

Kimberly A. Collins ◽

Andrea R. Castillo ◽

Sean Y. Tatsutani ◽

Sue Biggins

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

De Novo ◽

Histone H3 ◽

Centromeric Chromatin ◽

Centromeric Dna ◽

Kinetochore Assembly ◽

Histone H3 Variant ◽

Chromosome Attachment ◽

Dna Specificity

Kinetochores mediate chromosome attachment to the mitotic spindle to ensure accurate chromosome segregation. Budding yeast is an excellent organism for kinetochore assembly studies because it has a simple defined centromere sequence responsible for the localization of >65 proteins. In addition, yeast is the only organism where a conditional centromere is available to allow studies of de novo kinetochore assembly. Using a conditional centromere, we found that yeast kinetochore assembly is not temporally restricted and can occur in both G1 phase and prometaphase. We performed the first investigation of kinetochore assembly in the absence of the centromeric histone H3 variant Cse4 and found that all proteins tested depend on Cse4 to localize. Consistent with this observation, Cse4-depleted cells had severe chromosome segregation defects. We therefore propose that yeast kinetochore assembly requires both centromeric DNA specificity and centromeric chromatin.

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Genes Involved in Sister Chromatid Separation and Segregation in the Budding Yeast Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/159.2.453 ◽

2001 ◽

Vol 159 (2) ◽

pp. 453-470

Author(s):

Sue Biggins ◽

Needhi Bhalla ◽

Amy Chang ◽

Dana L Smith ◽

Andrew W Murray

Keyword(s):

Chromosome Segregation ◽

Sister Chromatid ◽

Mitotic Spindle ◽

Budding Yeast ◽

Temperature Sensitive ◽

Chromosome Behavior ◽

Yeast Saccharomyces Cerevisiae ◽

Sister Chromatids ◽

Marked Chromosome ◽

Sister Chromatid Separation

Abstract Accurate chromosome segregation requires the precise coordination of events during the cell cycle. Replicated sister chromatids are held together while they are properly attached to and aligned by the mitotic spindle at metaphase. At anaphase, the links between sisters must be promptly dissolved to allow the mitotic spindle to rapidly separate them to opposite poles. To isolate genes involved in chromosome behavior during mitosis, we microscopically screened a temperature-sensitive collection of budding yeast mutants that contain a GFP-marked chromosome. Nine LOC (loss of cohesion) complementation groups that do not segregate sister chromatids at anaphase were identified. We cloned the corresponding genes and performed secondary tests to determine their function in chromosome behavior. We determined that three LOC genes, PDS1, ESP1, and YCS4, are required for sister chromatid separation and three other LOC genes, CSE4, IPL1, and SMT3, are required for chromosome segregation. We isolated alleles of two genes involved in splicing, PRP16 and PRP19, which impair α-tubulin synthesis thus preventing spindle assembly, as well as an allele of CDC7 that is defective in DNA replication. We also report an initial characterization of phenotypes associated with the SMT3/SUMO gene and the isolation of WSS1, a high-copy smt3 suppressor.

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Disruption of Centrosome Structure, Chromosome Segregation, and Cytokinesis by Misexpression of Human Cdc14A Phosphatase

Molecular Biology of the Cell ◽

10.1091/mbc.01-11-0535 ◽

2002 ◽

Vol 13 (7) ◽

pp. 2289-2300 ◽

Cited By ~ 92

Author(s):

Brett K. Kaiser ◽

Zachary A. Zimmerman ◽

Harry Charbonneau ◽

Peter K. Jackson

Keyword(s):

Cell Cycle ◽

Phosphatase Activity ◽

Chromosome Segregation ◽

Mitotic Spindle ◽

Genomic Stability ◽

Mitotic Exit ◽

Protein Abundance ◽

Cyclin Dependent Kinase ◽

Chromosome Partitioning

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and γ-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.

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Phosphorylation of Drosophila CENP-A on serine 20 regulates protein turn-over and centromere-specific loading

Nucleic Acids Research ◽

10.1093/nar/gkz809 ◽

2019 ◽

Vol 47 (20) ◽

pp. 10754-10770 ◽

Cited By ~ 2

Author(s):

Anming Huang ◽

Leopold Kremser ◽

Fabian Schuler ◽

Doris Wilflingseder ◽

Herbert Lindner ◽

...

Keyword(s):

Chromosome Segregation ◽

Posttranslational Modifications ◽

Centromeric Chromatin ◽

Phosphorylated Form ◽

Specific Loading ◽

Histone H3 Variant ◽

Proteasome Pathway ◽

Turn Over ◽

Fine Tune

Abstract Centromeres are specialized chromosomal regions epigenetically defined by the presence of the histone H3 variant CENP-A. CENP-A is required for kinetochore formation which is essential for chromosome segregation during mitosis. Spatial restriction of CENP-A to the centromere is tightly controlled. Its overexpression results in ectopic incorporation and the formation of potentially deleterious neocentromeres in yeast, flies and in various human cancers. While the contribution of posttranslational modifications of CENP-A to these processes has been studied in yeast and mammals to some extent, very little is known about Drosophila melanogaster. Here, we show that CENP-A is phosphorylated at serine 20 (S20) by casein kinase II and that in mitotic cells, the phosphorylated form is enriched on chromatin. Importantly, our results reveal that S20 phosphorylation regulates the turn-over of prenucleosomal CENP-A by the SCFPpa-proteasome pathway and that phosphorylation promotes removal of CENP-A from ectopic but not from centromeric sites in chromatin. We provide multiple lines of evidence for a crucial role of S20 phosphorylation in controlling restricted incorporation of CENP-A into centromeric chromatin in flies. Modulation of the phosphorylation state of S20 may provide the cells with a means to fine-tune CENP-A levels in order to prevent deleterious loading to extra-centromeric sites.

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Regulation of Microtubule Assembly: The View From The End

Microscopy and Microanalysis ◽

10.1017/s143192760003289x ◽

2000 ◽

Vol 6 (S2) ◽

pp. 80-81

Author(s):

L. Cassimeris ◽

C. Spittle ◽

M. Kratzer

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

Dynamic Instability ◽

Intrinsic Property ◽

Microtubule Dynamics ◽

Microtubule Assembly ◽

Chromosome Movement ◽

Spindle Formation ◽

Associated Proteins

The mitotic spindle is responsible for chromosome movement during mitosis. It is composed of a dynamic array of microtubules and associated proteins whose assembly and constant turnover are required for both spindle formation and chromosome movement. Because microtubule assembly and turnover are necessary for chromosome segregation, we are studying how cells regulate microtubule dynamics. Microtubules are polarized polymers composed of tubulin subunits; they assemble by a process of dynamic instability where individual microtubules exist in persistent phases of elongation or rapid shortening with abrupt transitions between these two states. The switch from elongation to shortening is termed catastrophe, and the switch from shortening to elongation, rescue. Although dynamic instability is an intrinsic property of the tubulin subunits, cells use associated proteins to both speed elongation (∼ 10 fold) and regulate transitions.The only protein isolated to date capable of promoting fast polymerization consistent with rates in vivo is XMAP215, a 215 kD protein from Xenopus eggs.

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