Number Of Kinetochores Research Articles

Centromere-proximal suppression of meiotic crossovers in Drosophila is robust to changes in centromere number, repetitive DNA content, and centromere-clustering.

Accurate segregation of homologous chromosomes during meiosis depends on both the presence and the regulated placement of crossovers (COs). The centromere effect, or CO exclusion in pericentromeric regions of the chromosome, is a meiotic CO patterning phenomenon that helps prevent nondisjunction, thereby protecting against chromosomal disorders and other meiotic defects. Despite being identified nearly a century ago, the mechanisms behind this fundamental cellular process remain unknown, with most studies of the Drosophila centromere effect focusing on local influences of the centromere and pericentric heterochromatin. In this study, we sought to investigate whether dosage changes in centromere number and repetitive DNA content affect the strength of the centromere effect, using phenotypic recombination mapping. Additionally, we studied the effects of repetitive DNA function on centromere effect strength using satellite DNA-binding protein mutants displaying defective centromere-clustering in meiotic nuclei. Despite what previous studies suggest, our results show that the Drosophila centromere effect is robust to changes in centromere number, repetitive DNA content, as well as repetitive DNA function. Our study suggests that the centromere effect is unlikely to be spatially controlled, providing novel insight into the mechanisms behind the Drosophila centromere effect.

Robertsonian Fusion and Centromere Repositioning Contributed to the Formation of Satellite-free Centromeres During the Evolution of Zebras.

Centromeres are epigenetically specified by the histone H3 variant CENP-A and typically associated with highly repetitive satellite DNA. We previously discovered natural satellite-free neocentromeres in Equus caballus and Equus asinus. Here, through ChIP-seq with an anti-CENP-A antibody, we found an extraordinarily high number of centromeres lacking satellite DNA in the zebras Equus burchelli (15 of 22) and Equus grevyi (13 of 23), demonstrating that the absence of satellite DNA at the majority of centromeres is compatible with genome stability and species survival and challenging the role of satellite DNA in centromere function. Nine satellite-free centromeres are shared between the two species in agreement with their recent separation. We assembled all centromeric regions and improved the reference genome of E. burchelli. Sequence analysis of the CENP-A binding domains revealed that they are LINE-1 and AT-rich with four of them showing DNA amplification. In the two zebras, satellite-free centromeres emerged from centromere repositioning or following Robertsonian fusion. In five chromosomes, the centromeric function arose near the fusion points, which are located within regions marked by traces of ancestral pericentromeric sequences. Therefore, besides centromere repositioning, Robertsonian fusions are an important source of satellite-free centromeres during evolution. Finally, in one case, a satellite-free centromere was seeded on an inversion breakpoint. At 11 chromosomes, whose primary constrictions seemed to be associated with satellite repeats by cytogenetic analysis, satellite-free neocentromeres were instead located near the ancestral inactivated satellite-based centromeres; therefore, the centromeric function has shifted away from a satellite repeat containing locus to a satellite-free new position.

Kinetochore tracking in 3D from lattice light-sheet imaging data with KiT.

MotivationLattice light-sheet microscopy (LLSM) is revolutionizing cell biology since it enables fast, high-resolution extended imaging in three dimensions combined with a drastic reduction in photo-toxicity and bleaching. However, analysis of such datasets still remains a major challenge.ResultsAutomated tracking of kinetochores, the protein complex facilitating and controlling microtubule attachment of the chromosomes within the mitotic spindle, provides quantitative assessment of chromosome dynamics in mitosis. Here, we extend existing open-source kinetochore tracking software (KiT) to track (and pair) kinetochores throughout prometaphase to anaphase in LLSM data. One of the key improvements is a regularization term in the objective function to enforce biological information about the number of kinetochores in a human mitotic cell, as well as improved diagnostic tools. This software provides quantitative insights into how kinetochores robustly ensure congression and segregation of chromosomes during mitosis.Availability and implementationKiT is free, open-source software implemented in MATLAB and can be downloaded as a package from https://github.com/cmcb-warwick/KiT. The source repository is available at https://bitbucket.org/jarmond/kit (tag v2.4.0) and under continuing development.Supplementary information Supplementary data are available at Bioinformatics online.

Oocyte cytoplasmic diameter of ≥130 μm can be used to determine human giant oocytes

To determine if a cytoplasmic diameter of ≥130 μm can help identify human giant oocytes (GOs) in clinical practice and confirm the presence of genetic abnormalities in GOs by assessing the spindle length and centromere numbers. Case-control study. Private invitro fertilization clinic. The subjects were women aged 20-49 years who underwent oocyte retrieval after ovarian stimulation from January 2014 to December2020. None. The oocyte diameter was measured; immunofluorescent staining was performed to assess the spindle diameter and centromere numbers. Among the 254,337 oocytes examined, 561 (0.22%) had a diameter of ≥130 μm. The mean diameter ranges in the normal-sized metaphase II (MII) oocytes (MII group) and GO group were 103.0-119.0 and 132.3-175.9 μm. Spindle size could be measured in 6 GOs with 1 spindle (GO1), 10 GOs with 2 spindles (GO2), and 16 MII groups. The equatorial plane and pole-to-pole distance in the GO1 were significantly longer than in the GO2 and MII groups. The median numbers of centromeres were 86 in GOs with 1 spindle and 42 in each spindle for GOs with 2 spindles among 11 GO1s and 5 GO2s. This study is the first to define GOs as oocytes with a diameter of ≥130 μm and is a large-scale study surveying the incidence of GO. It is also the first study to analyze and elucidate the relationship between spindle numbers within the cytoplasm of GOs and spindle size and centromeres.

The differences of clinicopathologic characteristics among subgroups of reclassified HER2 fluorescence in situ hybridization (FISH) according to the ASCO/CAP 2018 breast cancer HER2 testing guidelines

AimsThe aim of this study is to analyse differences in clinicopathologic features among reclassified human epidermal growth factor receptor-2 (HER2) fluorescence in situ hybridization (FISH) results in breast cancers according...

Ectopic Activation of the Spindle Assembly Checkpoint Signaling Cascade Reveals Its Biochemical Design

Switch-like activation of the spindle assembly checkpoint (SAC) is critical for accurate chromosome segregation and for cell division in a timely manner. To determine the mechanisms that achieve this, we engineered an ectopic, kinetochore-independent SAC activator: the "eSAC." The eSAC stimulates SAC signaling by artificially dimerizing Mps1 kinase domain and a cytosolic KNL1 phosphodomain, the kinetochore signaling scaffold. By exploiting variable eSAC expression in a cell population, we defined the dependence of the eSAC-induced mitotic delay on eSAC concentration in a cell to reveal the dose-response behavior of the core signaling cascade of the SAC. These quantitative analyses and subsequent mathematical modeling of the dose-response data uncover two crucial properties of the core SAC signaling cascade: (1) a cellular limit on the maximum anaphase-inhibitory signal that the cascade can generate due to the limited supply of SAC proteins and (2) the ability of the KNL1 phosphodomain to produce the anaphase-inhibitory signal synergistically, when it recruits multiple SAC proteins simultaneously. We propose that these properties together achieve inverse, non-linear scaling between the signal output per kinetochore and the number of signaling kinetochores. When the number of kinetochores is low, synergistic signaling by KNL1 enables each kinetochore to produce a disproportionately strong signal output. However, when many kinetochores signal concurrently, they compete for a limited supply of SAC proteins. This frustrates synergistic signaling and lowers their signal output. Thus, the signaling activity of unattached kinetochores will adapt to the changing number of signaling kinetochores to enable the SAC to approximate switch-like behavior.

Birth, evolution, and transmission of satellite-free mammalian centromeric domains.

Mammalian centromeres are associated with highly repetitive DNA (satellite DNA), which has so far hindered molecular analysis of this chromatin domain. Centromeres are epigenetically specified, and binding of the CENPA protein is their main determinant. In previous work, we described the first example of a natural satellite-free centromere on Equus caballus Chromosome 11. Here, we investigated the satellite-free centromeres of Equus asinus by using ChIP-seq with anti-CENPA antibodies. We identified an extraordinarily high number of centromeres lacking satellite DNA (16 of 31). All of them lay in LINE- and AT-rich regions. A subset of these centromeres is associated with DNA amplification. The location of CENPA binding domains can vary in different individuals, giving rise to epialleles. The analysis of epiallele transmission in hybrids (three mules and one hinny) showed that centromeric domains are inherited as Mendelian traits, but their position can slide in one generation. Conversely, centromere location is stable during mitotic propagation of cultured cells. Our results demonstrate that the presence of more than half of centromeres void of satellite DNA is compatible with genome stability and species survival. The presence of amplified DNA at some centromeres suggests that these arrays may represent an intermediate stage toward satellite DNA formation during evolution. The fact that CENPA binding domains can move within relatively restricted regions (a few hundred kilobases) suggests that the centromeric function is physically limited by epigenetic boundaries.

Spatial organization of fibroblast and spermatocyte nuclei with different B-chromosome content in Korean field mouse, Apodemus peninsulae (Rodentia, Muridae).

Korean field mouse (Apodemus peninsulae) shows a wide variation in the number of B chromosomes composed of constitutive heterochromatin. For this reason, it provides a good model to study the influence of the number of centromeres and amount of heterochromatin on spatial organization of interphase nuclei. We analyzed the three-dimensional organization of fibroblast and spermatocyte nuclei of the field mice carrying a different number of B chromosomes using laser scanning microscopy and 3D fluorescence in situ hybridization. We detected a co-localization of the B chromosomes with constitutive heterochromatin of the chromosomes of the basic set. We showed a non-random distribution of B chromosomes in the spermatocyte nuclei. Unpaired B chromosomes showed a tendency to occur in the compartment formed by the unpaired part of the XY bivalent.

Developing an Automated Cytogenetic Imaging System for Detection of Dicentric Chromosomes in Biological Dosimetry

Background: High-energy ionizing radiation is harmful and changes the genetic makeup of DNA, which can lead to increased risk of cancer. Thus, the exposure of radiation dose should be under control and limited. Ionizing radiation might lead to some chromosome aberrations like dicentric. There is a strong relation between the frequency of dicentric chromosome in metaphase, and the received dose. Objective: Identifying the frequency of dicentric chromosomes is an important task especially in large-scale radiation accident for making rapid clinical decision. Given this, a large number of metaphases should be investigated for an accurate estimating of dose. It is well known that non-automated (visual) scoring of chromosome aberrations such as dicentrics requires highly trained experts. On the other hand, as thousands of cells should be scored, it is time-consuming and results in fatigue that can lead to poor concentration. Methods: Biological dosimetry technique for assessing radiation dose is based on analyzing chromosome in peripheral blood lymphocyte. Our study describes a technique to speed up this procedure by automatically distinguishing abnormal chromosomes from normal ones. The most important feature of dicentric chromosomes is their two centromeres. Therefore, the main approach in our study is to design an automated system to identify the number and position of centromeres. We presented a method for classifying chromosomes into four groups in accordance with the number of centromeres in each chromosome. Results: The image dataset of 311 chromosomes of normal and dicentric chromosomes is used to test the scheme. The sensitivity about 90% and specificity more than 95% for classification of chromosomes into dicentric and non-dicentric as well as accuracy more than 90% for centromere identification, are obtained. Conclusion: If an automated system with the capability of straightening bent chromosome was applied prior to the proposed algorithm, even more accurate results would be achieved.

A Balance between Nuclear and Cytoplasmic Volumes Controls Spindle Length.

Proper assembly of the spindle apparatus is crucially important for faithful chromosome segregation during anaphase. Thanks to the effort over the last decades, we have very detailed information about many events leading to spindle assembly and chromosome segregation, however we still do not understand certain aspects, including, for example, spindle length control. When tight regulation of spindle size is lost, chromosome segregation errors emerge. Currently, there are several hypotheses trying to explain the molecular mechanism of spindle length control. The number of kinetochores, activity of molecular rulers, intracellular gradients, cell size, limiting spindle components, and the balance of the spindle forces seem to contribute to spindle size regulation, however some of these mechanisms are likely specific to a particular cell type. In search for a general regulatory mechanism, in our study we focused on the role of cell size and nuclear to cytoplasmic ratio in this process. To this end, we used relatively large cells isolated from 2-cell mouse embryos. Our results showed that the spindle size upper limit is not reached in these cells and suggest that accurate control of spindle length requires balanced ratio between nuclear and cytoplasmic volumes.

Cell Size Determines the Strength of the Spindle Assembly Checkpoint during Embryonic Development

The spindle assembly checkpoint (SAC) delays mitotic progression when chromosomes are not properly attached to microtubules of the mitotic spindle. Cells vary widely in the extent to which they delay mitotic progression upon SAC activation. To explore the mechanisms that determine checkpoint strength in different cells, we systematically measured the mitotic delay induced by microtubule disruption at different stages of embryogenesis in Caenorhabditis elegans. Strikingly, we observed a gradual increase in SAC strength after each round of division. Analysis of mutants that alter cell size or ploidy revealed that SAC strength is determined primarily by cell size and the number of kinetochores. These findings provide clear evidence in vivo that the kinetochore-to-cytoplasm ratio determines the strength of the SAC, providing new insights into why cells exhibit such large variations in their SAC responses.

Assessment and significance of human epidermal growth factor receptor 2 gene status in breast cancer with polysomy of chromosome 17

To explore the impact of polysomy of chromosome 17 on the interpretation of human epidermal growth factor receptor 2 (HER2) status and its clinical significance in breast cancers. Clincopathological data of 338 breast cancer patients were collected, and the association of clinicopathological characteristics with polysomy of chromosome 17 was analyzed. Based on the American Society of Clinical Oncology /College of American Pathologists updated recommendations of HER2 testing in breast cancer in 2013, the relationships between the copy number of centromeres on chromosome 17 (CEP17) and HER2 status, other important biological markers and clincopathological features of breast cancer were analyzed statistically. Immunohistochemical examination revealed that among the 338 breast cancer cases, there were 9 (2.7%) HER2-negative cases, 309 (91.4%) HER2-equivocal cases, and 20 (5.9%) HER2-positive cases. The FISH analysis showed that there were 226 (66.9%) HER2-negative cases, 13 (3.8%) HER2-equivocal cases, and 99 (29.3%) HER2-positive cases. Among the 338 breast cancer patients, 42 (12.4%) cases were identified as chromosome 17 polysomy (polysomy 17), 291 (86.1%) cases were chromosome 17 disomy, and 5 (1.3%) cases were chromosome 17 monosomy. Polysomy 17 showed significant correlation with the intensity of HER2 protein expression (P=0.046). The HER2 FISH results and HER2 gene copy number of polysomy 17 cases showed significant differences from the non-polysomy 17 cases (P<0.001). There were no significant differences of HER2/CEP17 ratio between the polysomy 17 and non-polysomy 17 groups (P=0.930). Polysomy 17 cases showed a significant relationship with tumor grading and Ki-67 index (P<0.05), but not with the age of patient, tumor size, lymph node metastasis, ER and PR expressions, and effect evaluation of neoadjuvant chemotherapy (P>0.05). In the breast cancer with polysomy of chromosome 17, assessment of the absolute copy number of HER2 signals is even more important, and the HER2/CEP17 ratio and HER2 protein expression levels should be comprehensively evaluated together.

Extreme chromosomal instability forecasts improved outcome in ER-negative breast cancer: a prospective validation cohort study from the TACT trial

Chromosomal instability (CIN) has been shown to be associated with drug resistance and poor clinical outcome in several cancer types. However, in oestrogen receptor (ER)-negative breast cancer we have previously demonstrated that extreme CIN is associated with improved clinical outcome, consistent with a negative impact of CIN on tumour fitness and growth. The aim of this current study was to validate this finding using previously defined CIN thresholds in a much larger prospective cohort from a randomised, controlled, clinical trial. As a surrogate measurement of CIN, dual centromeric fluorescence in situ hybridisation was performed for both chromosomes 2 and 15 on 1173 tumours from the breast cancer TACT trial (CRUK01/001). Each tumour was scored manually and the mean percentage of cells deviating from the modal centromere number was used to define four CIN groups (MCD1-4), where tumours in the MCD4 group were defined as having extreme CIN. In a multivariate analysis of disease-free survival, with a median follow-up of 91 months, increasing CIN was associated with improved outcome in patients with ER-negative cancer (P trend = 0.03). A similar pattern was seen in ER-negative/HER2-negative cancers (Ptrend = 0.007). This prospective validation cohort study further substantiated the association between extreme CIN and improved outcome in ER-negative breast cancers. Identifying such patients with extreme CIN may help distinguish good from poor prognostic groups, and therefore support treatment and risk stratification in this aggressive breast cancer subtype.

Determination of Centromere Number of Chromosomes Involved in Robertsonian Translocation by using Fluorescence in Situ Hybridization (FISH)

Determination of Centromere Number of Chromosomes Involved in Robertsonian Translocation by using Fluorescence in Situ Hybridization (FISH)

Increased MYC gene copy number correlates with increased mRNA levels in diffuse large B-cell lymphoma

Translocations involving the MYC gene and increased MYC mRNA levels are associated with poor outcome in diffuse large B-cell lymphoma. However, the presence of increased MYC gene copy number and/or polysomy of chromosome 8 have not been previously described. Utilizing dual color chromogenic in situ hybridization, we investigated MYC gene copy and chromosome 8 centromere numbers in 52 cases of diffuse large B-cell lymphoma. Cases were divided into those with "increased" or "not increased" MYC gene copy number for comparison with MYC mRNA levels, Ki-67 values, and survival. Increased MYC gene copy number was present in 38% of cases. Overall, the average MYC mRNA level was 2398 (range, 342 - 9783) and the percentage of nuclei positive for Ki-67 was 57.5% (range, 20-87%). Within the group with increased MYC copy number, the MYC mRNA values ranged from 816 to 5912 (average, 2843) and the Ki-67 values ranged from 23% to 83% (average, 57%). Within the group with not increased MYC copy number, MYC mRNA values ranged from 342 to 9783 (average, 2118) and the Ki-67 values ranged from 20% to 87% (average, 58%). There was a statistically significant relationship between increased MYC gene copy number and increased MYC mRNA (P=0.034) and a trend toward a relationship between increased mRNA and higher Ki-67 values. This is the first report that low level copy number increases are common in diffuse large B-cell lymphoma and that these changes correlate with MYC mRNA in a statistically significant manner. MYC copy number changes are an additional possible molecular mechanism that may result in increased mRNA and, likely, high proliferation and poor outcome.

Human Sgo1 downregulation leads to chromosomal instability in colorectal cancer

Background and aims:Chromosomal instability (CIN) is recognised as a hallmark of cancer and is caused by a spindle assembly checkpoint disorder or chromosome mis-segregation during mitosis. Although the recent identification...

Genes in conflict: the biology of selfish genetic elements

Genes in conflict: the biology of selfish genetic elements

Unstable microtubule capture at kinetochores depleted of the centromere-associated protein CENP-F

Centromere protein F (CENP-F) (or mitosin) accumulates to become an abundant nuclear protein in G2, assembles at kinetochores in late G2, remains kinetochore-bound until anaphase, and is degraded at the end of mitosis. Here we show that the absence of nuclear CENP-F does not affect cell cycle progression in S and G2. In a subset of CENP-F depleted cells, kinetochore assembly fails completely, thereby provoking massive chromosome mis-segregation. In contrast, the majority of CENP-F depleted cells exhibit a strong mitotic delay with reduced tension between kinetochores of aligned, bi-oriented sister chromatids and decreased stability of kinetochore microtubules. These latter kinetochores generate mitotic checkpoint signaling when unattached, recruiting maximum levels of Mad2. Use of YFP-marked Mad1 reveals that throughout the mitotic delay some aligned, CENP-F depleted kinetochores continuously recruit Mad1. Others rebind YFP-Mad1 intermittently so as to produce 'twinkling', demonstrating cycles of mitotic checkpoint reactivation and silencing and a crucial role for CENP-F in efficient assembly of a stable microtubule-kinetochore interface.

Polysomy 17 in HER-2/neu Status Elaboration in Breast Cancer: Effect on Daily Practice

To assess the effect of chromosome 17 copy number on HER-2/neu status determination in breast cancers. HER-2/neu gene copy and chromosome 17 centromere numbers were evaluated on 893 breast carcinomas using double color fluorescence in situ hybridization (FISH). The net and chromosome 17 corrected (ratio) HER-2/neu copy numbers were compared and related to immunohistochemistry done according to the Food and Drug Administration (FDA)-approved scoring system (0, 1+, 2+, and 3+) as a first screening step in 584 cases. When a ratio > or = 2 was considered as criterion for FISH positivity, 49.3% (440 of 893) of cases showed amplification versus 56.2% (502 of 893) by using a net HER-2/neu gene copy number >4 as a alternative criterion; 14.8% (67 of 453) of cases having a ratio <2 had a net HER-2/neu gene copy number >4 and 1.1% (5 of 440) with a ratio > or = 2 had a net HER-2/neu gene copy number <4. Among discordant cases, 88.8% (64 of 72) were polysomic (>2.25 chromosomes 17/cell) and among polysomic cases, 12.8% (40 of 312) of the low polysomic (2.26-3.75 chromosomes 17/cell) and 36.9% (24 of 65) of the highly polysomic (>3.75 chromosomes 17/cell) cases showed discordance. In cases with a ratio <2, polysomy 17 incidences were 85.7% (6 of 7) in IHC 3+, 42.4% (79 of 186) in IHC 2+, 33.3% (15 of 45) in IHC 1+, and 29.1% (16 of 55) in IHC 0. A net increase in HER-2/neu gene copy number consecutive to polysomy 17 in the absence of specific gene amplification might lead to a strong protein overexpression in a small subset of breast carcinomas. HER-2/neu status determination by FISH is dependent on the criterion considered for positivity in clinical practice.

Chromosome organization in wheat endosperm and embryo

We have analysed the chromosome organization in endosperm and embryo of bread wheat (Triticum aestivum L.), in order to compare these tissues with developing anthers, in which the centromeres associate, and the developing root xylem vessel cells, in which the chromosomes endoreduplicate to become polytene and associate via their centromeres. Both endosperm and embryo showed a typical Rabl configuration and a degree of non-homologous centromere association and the endosperm also showed extensive telomere association. Wheat endosperm is initially triploid and during its development a percentage of the nuclei increase their DNA content to 6C and 12C. 6C nuclei showed twice as many centromeres as 3C nuclei and the centromere number increased further in 12C nuclei. The higher the C-content of a nucleus the more the telomeres associated in endosperm. The vast majority of 12C nuclei showed six rye chromosome arms, although a few showed three associated groups of rye chromosome arms. This means that during endosperm development wheat nuclei show both polyploidization and polytenization.