Human centromere chromatin protein hMis12, essential for equal segregation, is independent of CENP-A loading pathway

Kinetochores are the chromosomal sites for spindle interaction and play a vital role for chromosome segregation. The composition of kinetochore proteins and their cellular roles are, however, poorly understood in higher eukaryotes. We identified a novel kinetochore protein family conserved from yeast to human that is essential for equal chromosome segregation. The human homologue hMis12 of yeast spMis12/scMtw1 retains conserved sequence features and locates at the kinetochore region indistinguishable from CENP-A, a centromeric histone variant. RNA interference (RNAi) analysis of HeLa cells shows that the reduced hMis12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei without mitotic delay, while CENP-A is located at kinetochores. Further, the metaphase spindle length is abnormally extended. Spindle checkpoint protein hMad2 temporally localizes at kinetochores at early mitotic stages after RNAi. The RNAi deficiency of CENP-A leads to a similar mitotic phenotype, but the kinetochore signals of other kinetochore proteins, hMis6 and CENP-C, are greatly diminished. RNAi for hMis6, like that of a kinetochore kinesin CENP-E, induces mitotic arrest. Kinetochore localization of hMis12 is unaffected by CENP-A RNAi, demonstrating an independent pathway of CENP-A in human kinetochores.

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Kinetochore–microtubule interactions in chromosome segregation: lessons from yeast and mammalian cells

Biochemical Journal ◽

10.1042/bcj20170518 ◽

2017 ◽

Vol 474 (21) ◽

pp. 3559-3577 ◽

Cited By ~ 4

Author(s):

Geethu Emily Thomas ◽

Marira R. Renjith ◽

Tapas K. Manna

Keyword(s):

Chromosome Segregation ◽

Mammalian Cells ◽

Structural Organization ◽

Vital Role ◽

Present Review ◽

The Past ◽

Chromosome Congression ◽

Spindle Microtubules ◽

Higher Eukaryotes ◽

Structural Aspects

Chromosome congression and segregation require robust yet dynamic attachment of the kinetochore with the spindle microtubules. Force generated at the kinetochore–microtubule interface plays a vital role to drive the attachment, as it is required to move chromosomes and to provide signal to sense correct attachments. To understand the mechanisms underlying these processes, it is critical to describe how the force is generated and how the molecules at the kinetochore–microtubule interface are organized and assembled to withstand the force and respond to it. Research in the past few years or so has revealed interesting insights into the structural organization and architecture of kinetochore proteins that couple kinetochore attachment to the spindle microtubules. Interestingly, despite diversities in the molecular players and their modes of action, there appears to be architectural similarity of the kinetochore-coupling machines in lower to higher eukaryotes. The present review focuses on the most recent advances in understanding of the molecular and structural aspects of kinetochore–microtubule interaction based on the studies in yeast and vertebrate cells.

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Aneuploid abortion correlates positively with MAD1 overexpression and miR-125b down-regulation

Molecular Cytogenetics ◽

10.1186/s13039-021-00538-1 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Juan Zhao ◽

Hui Li ◽

Guangxin Chen ◽

Lijun Du ◽

Peiyan Xu ◽

...

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Mitotic Checkpoint ◽

Early Embryo ◽

Vital Role ◽

Control Group ◽

Embryo Abortion ◽

Protein Levels ◽

Mitotic Checkpoint Complex

Abstract Background Aneuploidy is the most frequent cause of early-embryo abortion. Any defect in chromosome segregation would fail to satisfy the spindle assembly checkpoint (SAC) during mitosis, halting metaphase and causing aneuploidy. The mitotic checkpoint complex (MCC), comprising MAD1, MAD2, Cdc20, BUBR1 and BUB3, plays a vital role in SAC activation. Studies have confirmed that overexpression of MAD2 and BUBR1 can facilitate correct chromosome segregation and embryo stability. Research also proves that miR-125b negatively regulates MAD1 expression by binding to its 3′UTR. However, miR-125b, Mad1 and Bub3 gene expression in aneuploid embryos of spontaneous abortion has not been reported to date. Methods In this study, embryonic villi from miscarried pregnancies were collected and divided into two groups (aneuploidy and euploidy) based on High-throughput ligation-dependent probe amplification (HLPA) and Fluorescence in situ hybridization (FISH) analyses. RNA levels of miR-125b, MAD1 and BUB3 were detected by Quantitative real-time PCR (qRT-PCR); protein levels of MAD1 and BUB3 were analysed by Western blotting. Results statistical analysis (p < 0.05) showed that miR-125b and BUB3 were significantly down-regulated in the aneuploidy group compared to the control group and that MAD1 was significantly up-regulated. Additionally, the MAD1 protein level was significantly higher in aneuploidy abortion villus, but BUB3 protein was only mildly increased. Correlation analysis revealed that expression of MAD1 correlated negatively with miR-125b. Conclusion These results suggest that aneuploid abortion correlates positively with MAD1 overexpression, which might be caused by insufficient levels of miR-125b. Taken together, our findings first confirmed the negative regulatory mode between MAD1 and miR-125b, providing a basis for further mechanism researches in aneuploid abortion.

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Determinants of Drosophila zw10 protein localization and function

Journal of Cell Science ◽

10.1242/jcs.107.4.785 ◽

1994 ◽

Vol 107 (4) ◽

pp. 785-798 ◽

Cited By ~ 13

Author(s):

B.C. Williams ◽

M.L. Goldberg

Keyword(s):

Chromosome Segregation ◽

Sister Chromatid ◽

Protein Localization ◽

Metaphase Plate ◽

Obvious Effect ◽

Anaphase Onset ◽

Protein Functions ◽

Kinetochore Region ◽

And Function ◽

Feedback Pathway

We have examined several issues concerning how the Drosophila l(1)zw10 gene product functions to ensure proper chromosome segregation. (a) We have found that in zw10 mutant embryos and larval neuroblasts, absence of the zw10 protein has no obvious effect on either the congression of chromosomes to the metaphase plate or the morphology of the metaphase spindle, although many aberrations are observed subsequently in anaphase. This suggests that activity of the zw10 protein becomes essential at anaphase onset, a time at which the zw10 protein is redistributed to the kinetochore region of the chromosomes. (b) The zw10 protein appears to bind to kinetochores in mitotically arrested cells, eventually accumulating to high levels within the chromosome mass. Our results imply that zw10 may act as part of a novel feedback pathway that normally renders sister chromatid separation dependent upon spindle integrity. (c) The localization of zw10 protein is altered by two mitotic mutations, rough deal and abnormal anaphase resolution, that specifically disrupt anaphase. These findings indicate that the zw10 protein functions as part of a multicomponent mechanism ensuring proper chromosome segregation at the beginning of anaphase.

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Antisense Inhibition of CAS, the Human Homologue of the Yeast Chromosome Segregation Gene CSE1, Interferes with Mitosis in HeLa Cells

Biochemistry ◽

10.1021/bi970236o ◽

1997 ◽

Vol 36 (31) ◽

pp. 9493-9500 ◽

Cited By ~ 21

Author(s):

Vasia V. Ogryzko ◽

Elisabeth Brinkmann ◽

Bruce H. Howard ◽

Ira Pastan ◽

Ulrich Brinkmann

Keyword(s):

Chromosome Segregation ◽

Hela Cells ◽

Antisense Inhibition ◽

Human Homologue ◽

Yeast Chromosome

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Regional centromeres in the yeastCandida lusitaniaelack pericentromeric heterochromatin

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1508749112 ◽

2015 ◽

Vol 112 (39) ◽

pp. 12139-12144 ◽

Cited By ~ 35

Author(s):

Shivali Kapoor ◽

Lisha Zhu ◽

Cara Froyd ◽

Tao Liu ◽

Laura N. Rusche

Keyword(s):

Chromosome Segregation ◽

Consensus Sequence ◽

Distinct Type ◽

Distinct Pattern ◽

Pericentromeric Heterochromatin ◽

Centromeric Chromatin ◽

Chip Sequencing ◽

Conserved Sequence ◽

Centromere Proteins ◽

Candida Lusitaniae

Point centromeres are specified by a short consensus sequence that seeds kinetochore formation, whereas regional centromeres lack a conserved sequence and instead are epigenetically inherited. Regional centromeres are generally flanked by heterochromatin that ensures high levels of cohesin and promotes faithful chromosome segregation. However, it is not known whether regional centromeres require pericentromeric heterochromatin. In the yeastCandida lusitaniae, we identified a distinct type of regional centromere that lacks pericentromeric heterochromatin. Centromere locations were determined by ChIP-sequencing of two key centromere proteins, Cse4 and Mif2, and are consistent with bioinformatic predictions. The centromeric DNA sequence was unique for each chromosome and spanned 4–4.5 kbp, consistent with regional epigenetically inherited centromeres. However, unlike other regional centromeres, there was no evidence of pericentromeric heterochromatin inC. lusitaniae. In particular, flanking genes were expressed at a similar level to the rest of the genome, and aURA3reporter inserted adjacent to a centromere was not repressed. In addition, regions flanking the centromeric core were not associated with hypoacetylated histones or a sirtuin deacetylase that generates heterochromatin in other yeast. Interestingly, the centromeric chromatin had a distinct pattern of histone modifications, being enriched for methylated H3K79 and H3R2 but lacking methylation of H3K4, which is found at other regional centromeres. Thus, not all regional centromeres require flanking heterochromatin.

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Fractionation and initial characterization of the kinetochore from mammalian metaphase chromosomes.

The Journal of Cell Biology ◽

10.1083/jcb.101.3.1124 ◽

1985 ◽

Vol 101 (3) ◽

pp. 1124-1134 ◽

Cited By ~ 53

Author(s):

M M Valdivia ◽

B R Brinkley

Keyword(s):

Micrococcal Nuclease ◽

Esophageal Dysmotility ◽

Chromosomal Dna ◽

Metaphase Chromosomes ◽

Centromeric Chromatin ◽

Chromosomal Proteins ◽

Initial Characterization ◽

Total Dna ◽

Kinetochore Region

We have partially isolated the kinetochore and associated centromeric structures from mammalian metaphase chromosomes. Human autoantibodies from scleroderma CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) patients were used as immunofluorescent probes to monitor fractionation. The procedure includes digestion of total chromosomal DNA with micrococcal nuclease, dehistonization with heparin, and dissociation of the remaining material with detergent and urea. We used a density gradient (metrizamide) to obtain an enriched fraction of stained material (kinetochore). When examined by electron microscopy, the kinetochore fraction is seen to contain numerous small immunoperoxidase-positive masses which are morphologically similar to the centromere/kinetochore region of intact metaphase chromosomes. The particulate fraction that contains kinetochore components represents less than 5% of total chromosomal proteins and contains less than 1% of total DNA. Two polypeptides of 18 and 80 kD were identified as kinetochore antigens by immunoblotting with CREST antiserum. In this paper we discuss the distribution of these kinetochore polypeptides with the associated centromeric chromatin.

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Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore–microtubule dynamics

The Journal of Cell Biology ◽

10.1083/jcb.201707160 ◽

2017 ◽

Vol 217 (1) ◽

pp. 163-177 ◽

Cited By ~ 41

Author(s):

Keith F. DeLuca ◽

Amanda Meppelink ◽

Amanda J. Broad ◽

Jeanne E. Mick ◽

Olve B. Peersen ◽

...

Keyword(s):

Chromosome Segregation ◽

Microtubule Dynamics ◽

Aurora B ◽

Aurora A Kinase ◽

Aurora A ◽

Tail Domain ◽

Mitotic Chromosomes ◽

Metaphase Chromosomes ◽

Mitotic Kinases ◽

Microtubule Attachment

Precise regulation of kinetochore–microtubule attachments is essential for successful chromosome segregation. Central to this regulation is Aurora B kinase, which phosphorylates kinetochore substrates to promote microtubule turnover. A critical target of Aurora B is the N-terminal “tail” domain of Hec1, which is a component of the NDC80 complex, a force-transducing link between kinetochores and microtubules. Although Aurora B is regarded as the “master regulator” of kinetochore–microtubule attachment, other mitotic kinases likely contribute to Hec1 phosphorylation. In this study, we demonstrate that Aurora A kinase regulates kinetochore–microtubule dynamics of metaphase chromosomes, and we identify Hec1 S69, a previously uncharacterized phosphorylation target site in the Hec1 tail, as a critical Aurora A substrate for this regulation. Additionally, we demonstrate that Aurora A kinase associates with inner centromere protein (INCENP) during mitosis and that INCENP is competent to drive accumulation of the kinase to the centromere region of mitotic chromosomes. These findings reveal that both Aurora A and B contribute to kinetochore–microtubule attachment dynamics, and they uncover an unexpected role for Aurora A in late mitosis.

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Co-ordination of cytokinesis with chromosome segregation

Biochemical Society Transactions ◽

10.1042/bst0360387 ◽

2008 ◽

Vol 36 (3) ◽

pp. 387-390 ◽

Cited By ~ 10

Author(s):

Manuel Mendoza ◽

Yves Barral

Keyword(s):

Schizosaccharomyces Pombe ◽

Chromosome Segregation ◽

Cleavage Plane ◽

Budding Yeast ◽

Sister Chromatids ◽

Higher Eukaryotes ◽

Spindle Elongation

During anaphase, the spindle pulls the sister kinetochores apart until the sister chromatids are fully separated from each other. Subsequently, cytokinesis cleaves between the two separated chromosome masses to form two nucleated cells. Results from Schizosaccharomyces pombe suggested that cytokinesis and chromosome segregation are not co-ordinated with each other. However, recent studies indicate that, at least in budding yeast, a checkpoint called NoCut prevents abscission when spindle elongation is impaired, and might delay cytokinesis until all chromosomes are pulled out of the cleavage plane. Here, we discuss this possibility and summarize evidence suggesting that such a checkpoint is likely to be conserved in higher eukaryotes.

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AIR-2: An Aurora/Ipl1-related Protein Kinase Associated with Chromosomes and Midbody Microtubules Is Required for Polar Body Extrusion and Cytokinesis in Caenorhabditis elegans Embryos

The Journal of Cell Biology ◽

10.1083/jcb.143.6.1635 ◽

1998 ◽

Vol 143 (6) ◽

pp. 1635-1646 ◽

Cited By ~ 221

Author(s):

Jill M. Schumacher ◽

Andy Golden ◽

Peter J. Donovan

Keyword(s):

Caenorhabditis Elegans ◽

Chromosome Segregation ◽

Polar Body ◽

Mitotic Division ◽

Mitotic Chromosomes ◽

Metaphase Chromosomes ◽

Cell Cycles ◽

Polar Bodies ◽

Multiple Cell ◽

Polar Body Extrusion

An emerging family of kinases related to the Drosophila Aurora and budding yeast Ipl1 proteins has been implicated in chromosome segregation and mitotic spindle formation in a number of organisms. Unlike other Aurora/Ipl1-related kinases, the Caenorhabditis elegans orthologue, AIR-2, is associated with meiotic and mitotic chromosomes. AIR-2 is initially localized to the chromosomes of the most mature prophase I–arrested oocyte residing next to the spermatheca. This localization is dependent on the presence of sperm in the spermatheca. After fertilization, AIR-2 remains associated with chromosomes during each meiotic division. However, during both meiotic anaphases, AIR-2 is present between the separating chromosomes. AIR-2 also remains associated with both extruded polar bodies. In the embryo, AIR-2 is found on metaphase chromosomes, moves to midbody microtubules at anaphase, and then persists at the cytokinesis remnant. Disruption of AIR-2 expression by RNA- mediated interference produces entire broods of one-cell embryos that have executed multiple cell cycles in the complete absence of cytokinesis. The embryos accumulate large amounts of DNA and microtubule asters. Polar bodies are not extruded, but remain in the embryo where they continue to replicate. The cytokinesis defect appears to be late in the cell cycle because transient cleavage furrows initiate at the proper location, but regress before the division is complete. Additionally, staining with a marker of midbody microtubules revealed that at least some of the components of the midbody are not well localized in the absence of AIR-2 activity. Our results suggest that during each meiotic and mitotic division, AIR-2 may coordinate the congression of metaphase chromosomes with the subsequent events of polar body extrusion and cytokinesis.

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SPDL-1 functions as a kinetochore receptor for MDF-1 in Caenorhabditis elegans

The Journal of Cell Biology ◽

10.1083/jcb.200805185 ◽

2008 ◽

Vol 183 (2) ◽

pp. 187-194 ◽

Cited By ~ 42

Author(s):

Takaharu G. Yamamoto ◽

Sonoko Watanabe ◽

Anthony Essex ◽

Risa Kitagawa

Keyword(s):

Rna Interference ◽

Caenorhabditis Elegans ◽

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Spindle Pole ◽

Cell Stage ◽

Anaphase Onset ◽

Mitotic Delay ◽

Bipolar Spindles

The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation by delaying anaphase onset until all sister kinetochores are attached to bipolar spindles. An RNA interference screen for synthetic genetic interactors with a conserved SAC gene, san-1/MAD3, identified spdl-1, a Caenorhabditis elegans homologue of Spindly. SPDL-1 protein localizes to the kinetochore from prometaphase to metaphase, and this depends on KNL-1, a highly conserved kinetochore protein, and CZW-1/ZW10, a component of the ROD–ZW10–ZWILCH complex. In two-cell–stage embryos harboring abnormal monopolar spindles, SPDL-1 is required to induce the SAC-dependent mitotic delay and localizes the SAC protein MDF-1/MAD1 to the kinetochore facing away from the spindle pole. In addition, SPDL-1 coimmunoprecipitates with MDF-1/MAD1 in vivo. These results suggest that SPDL-1 functions in a kinetochore receptor of MDF-1/MAD1 to induce SAC function.

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