Chromosome Segregation Errors Research Articles

Heterochromatin: Guardian of the Genome.

Constitutive heterochromatin is a major component of the eukaryotic nucleus and is essential for the maintenance of genome stability. Highly concentrated at pericentromeric and telomeric domains, heterochromatin is riddled with repetitive sequences and has evolved specific ways to compartmentalize, silence, and repair repeats. The delicate balance between heterochromatin epigenetic maintenance and cellular processes such as mitosis and DNA repair and replication reveals a highly dynamic and plastic chromatin domain that can be perturbed by multiple mechanisms, with far-reaching consequences for genome integrity. Indeed, heterochromatin dysfunction provokes genetic turmoil by inducing aberrant repeat repair, chromosome segregation errors, transposon activation, and replication stress and is strongly implicated in aging and tumorigenesis. Here, we summarize the general principles of heterochromatin structure and function, discuss the importance of its maintenance for genome integrity, and propose that more comprehensive analyses of heterochromatin roles in tumorigenesis will be integral to future innovations in cancer treatment.

A quantitative analysis of cohesin decay in mitotic fidelity.

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.

Abstract NG03: Chromosomal instability promotes metastasis through a cytosolic DNA response

Abstract Chromosomal instability (CIN) is a hallmark of cancer and it results from ongoing errors in chromosome segregation during mitosis. While CIN is a major driver of tumor evolution, its role in metastasis has not been established. Here we show that CIN promotes metastasis by sustaining a tumor-cell autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signaling. Genetic suppression of CIN significantly delays metastasis even in highly aneuploid tumor models, whereas inducing continuous chromosome segregation errors promotes cellular invasion and metastasis in a STING-dependent manner. Using single-cell RNA sequencing, we uncover a CIN-induced transcriptional switch from a proliferative and metabolically active state to a mesenchymal phenotype associated with inflammatory pathways, offering an opportunity to target chromosome segregation errors for therapeutic benefit. Our work reveals an unexpected link between CIN, cytosolic DNA sensing pathways, and metastasis. The use of an isogenic system has enabled us to dissect the role of CIN from that of aneuploidy. Importantly, while we do not discount the role of CIN in generating karyotypic heterogeneity that can serve as the substrate for natural selection, our work demonstrates that continuous chromosome missegregation is also required to replenish cytosolic DNA pools leading to chronic upregulation of inflammatory pathways. In non-transformed settings, cytosolic DNA sensing is incompatible with viability. Unlike normal cells, chromosomally unstable cells are awash with cytosolic DNA and have adapted to coexist with a chronically active cGAS-STING pathway by suppressing downstream type I interferon signaling and instead upregulating the alternative NF-κB pathway. Persistent STING activation mediates carcinogen-induced tumor formation and we now show that tumor cells co-opt this otherwise lethal program to spread to distant organs. The evolutionary benefit of the noncanonical pathway might justify the scarcity of inactivating mutations in cGAS and STING among human cancers. The emergence, and subsequent tolerance, of CIN represents an important bottleneck during tumor evolution. Our single-cell analysis revealed that CIN induces a transcriptional switch whereby cells shift from a proliferative and highly metabolic state, ideally suited for primary tumor growth, to a chromosomally unstable and mesenchymal state associated with upregulation of inflammatory pathways. These two largely mutually exclusive states likely account for the reversibility in chromosome missegregation rates seen in primary tumors and metastases, and provide an explanation for the negative effect of aneuploidy during early tumorigenesis. Interestingly, this mutual exclusivity was recently observed in a pan-cancer genomic analysis of metastatic tumors, and it leads us to suggest that CIN underlies the subset of metastases that are characterized by EMT and inflammation. By providing a mechanistic link between CIN and metastasis, our work opens new avenues to target chromosomally unstable tumors for therapeutic benefit. Citation Format: Samuel F. Bakhoum, Bryon Ngo, Ashley L. Bakhoum, Julie-Ann Cavallo, Charles J. Murphy, Peter Ly, Pragya Shah, Roshan K. Sriram, Thomas B.k. Watkins, Neil K. Taunk, Mercedes Duran, Chantal Pauli, Christine Shaw, Kalyani Chadalavada, Vinagolu K. Rajasekhar, Giulio Genovese, Subramanian Venkatesan, Nicolai J. Birkbak, Nicholas McGranahan, Mark Lundquist, Quincy LaPlant, John H. Healey, Olivier Elemento, Christine H. Chung, Nancy Y. Lee, Marcin Imielinski, Gouri Nanjangud, Dana Pe'er, Don W. Cleveland, Simon N. Powell, Jan Lammerding, Charles Swanton, Lewis C. Cantley. Chromosomal instability promotes metastasis through a cytosolic DNA response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr NG03.

Dynamic kinetochore size regulation promotes microtubule capture and chromosome biorientation in mitosis.

Faithful chromosome segregation depends on the ability of sister kinetochores to attach to spindle microtubules. The outer layer of kinetochores transiently expands in early mitosis to form a fibrous corona, and compacts upon microtubule capture. Here we show that the dynein adaptor Spindly and the RZZ (ROD-Zwilch-ZW10) complex drive kinetochore expansion in a dynein-independent manner. C-terminal farnesylation and MPS1 kinase activity cause conformational changes of Spindly that promote oligomerization of RZZ:Spindly complexes into a filamentous meshwork in cells and in vitro. Concurrent with kinetochore expansion, Spindly potentiates kinetochore compaction by recruiting dynein via three conserved short linear motifs. Expanded kinetochores unable to compact engage in extensive, long-lived lateral microtubule interactions that persist to metaphase, and result in merotelic attachments and chromosome segregation errors in anaphase. Thus, dynamic kinetochore size regulation in mitosis is coordinated by a single, Spindly-based mechanism that promotes initial microtubule capture and subsequent correct maturation of attachments.

Protein kinase Cδ (PKCδ) involved in the regulation of pAkt1 (Ser473) on the release of mouse oocytes from diplotene arrest.

As a drug target, Akt is still the focus of research in somatic and fertilized eggs. Here, our data depicted a series of molecular events of pAkt1 (Ser473) and PKCδ via their special inhibitor and antibody in order to analyse their role and relationship on the release of mouse oocytes from diplotene arrest. Our results may offer important data for clinic research scientist to recognize the target role of pAkt1 (Ser473) via PKCδ in cell cycle regulation.

MASTL overexpression promotes chromosome instability and metastasis in breast cancer

MASTL kinase is essential for correct progression through mitosis, with loss of MASTL causing chromosome segregation errors, mitotic collapse and failure of cytokinesis. However, in cancer MASTL is most commonly amplified and overexpressed. This correlates with increased chromosome instability in breast cancer and poor patient survival in breast, ovarian and lung cancer. Global phosphoproteomic analysis of immortalised breast MCF10A cells engineered to overexpressed MASTL revealed disruption to desmosomes, actin cytoskeleton, PI3K/AKT/mTOR and p38 stress kinase signalling pathways. Notably, these pathways were also disrupted in patient samples that overexpress MASTL. In MCF10A cells, these alterations corresponded with a loss of contact inhibition and partial epithelial–mesenchymal transition, which disrupted migration and allowed cells to proliferate uncontrollably in 3D culture. Furthermore, MASTL overexpression increased aberrant mitotic divisions resulting in increased micronuclei formation. Mathematical modelling indicated that this delay was due to continued inhibition of PP2A-B55, which delayed timely mitotic exit. This corresponded with an increase in DNA damage and delayed transit through interphase. There were no significant alterations to replication kinetics upon MASTL overexpression, however, inhibition of p38 kinase rescued the interphase delay, suggesting the delay was a G2 DNA damage checkpoint response. Importantly, knockdown of MASTL, reduced cell proliferation, prevented invasion and metastasis of MDA-MB-231 breast cancer cells both in vitro and in vivo, indicating the potential of future therapies that target MASTL. Taken together, these results suggest that MASTL overexpression contributes to chromosome instability and metastasis, thereby decreasing breast cancer patient survival.

SHOC1 is a ERCC4-(HhH)2-like protein, integral to the formation of crossover recombination intermediates during mammalian meiosis.

Chromosome segregation errors during meiosis result in the formation of aneuploid gametes and are the leading cause of pregnancy loss and birth defects in humans. Proper chromosome segregation requires pairwise associations of maternal and paternal homologous chromosomes. Chiasmata, which are the cytological manifestations of crossovers (COs), provide a physical link that holds the homologs together as a pair, facilitating their orientation on the spindle at meiosis I. Although CO-promoting activities ensure a balanced number and position of COs, their identity and mechanism of action in mammals remain understudied. Previous work in yeast and Arabidopsis has shown that Zip2 and Shoc1 are ortholog proteins with an important role in promoting the formation of COs. Our work is the first study in mammals showing the in vivo and in vitro function of mouse and human SHOC1. We show that purified recombinant human SHOC1, an XPF/MUS81 family member, preferentially binds branched DNA molecules but apparently lacks in vitro endonuclease activity, despite its conserved ERCC4-(HhH)2 core structure. Cytological observations suggest that initial steps of recombination are normal in a majority of spermatocytes from SHOC1 hypomorphic mice. However, late stages of recombination appear abnormal, as chromosomal localization of MLH1 is reduced. In agreement, chiasma formation is reduced, and cells arrest at metaphase I with a few lagging chromosomes and subsequent apoptosis. This analysis of SHOC1-deficient mice and the selective localization of SHOC1 to a subset of recombination sites show that SHOC1 acts at key mid-stage steps of the CO formation process. The formation of chromosome axial elements and homologous pairing are apparently normal, but synapsis is altered with SYCP1 frequently failing to extend the full length of the chromosome axes. Finally, we describe that SHOC1 interacts with TEX11, another protein important for the formation of COs, connecting SHOC1 to chromosome axis and structure.

Mps1 inhibitors synergise with low doses of taxanes in promoting tumour cell death by enhancement of errors in cell division

BackgroundChromosomal instability (CIN) is a common trait of cancer characterised by the continuous gain and loss of chromosomes during mitosis. Excessive levels of CIN can suppress tumour growth, providing a possible therapeutic strategy. The Mps1/TTK kinase has been one of the prime targets to explore this concept, and indeed Mps1 inhibitors synergise with the spindle poison docetaxel in inhibiting the growth of tumours in mice.MethodsTo investigate how the combination of docetaxel and a Mps1 inhibitor (Cpd-5) promote tumour cell death, we treated mice transplanted with BRCA1−/−;TP53−/− mammary tumours with docetaxel and/or Cpd-5. The tumours were analysed regarding their histopathology, chromosome segregation errors, copy number variations and cell death to understand the mechanism of action of the drug combination.ResultsThe enhanced efficacy of combining an Mps1 inhibitor with clinically relevant doses of docetaxel is associated with an increase in multipolar anaphases, aberrant nuclear morphologies and cell death. Tumours treated with docetaxel and Cpd-5 displayed more genomic deviations, indicating that chromosome stability is affected mostly in the combinatorial treatment.ConclusionsOur study shows that the synergy between taxanes and Mps1 inhibitors depends on increased errors in cell division, allowing further optimisation of this treatment regimen for cancer therapy.

Embryos classified as low-grade mosaic (

Mosaic preimplantation embryos contain two or more cell lines with different chromosome content, the consequence of errors in chromosome segregation occurring during mitotic divisions. Recently the NGS was validated and implemented for PGS. There are several different platforms, with different resolutions, that can detect mosaicism to a different extent. Veriseq NGS (Illumina) that was used in our study, can detect mosaicism when aneuploidy is present in 20%-80% of TE cells biopsied from a blastocyst. The purpose of the present study was to retrospectively analyze the pregnancy outcome of replaced low rate mosaic embryos (20%-50% abnormal cells in the trophoctoderm biopsy) as diagnosed with the use of hr-NGS. The molecular analyses were provided by Nexgenomics, LLC, Pasadena, CA. The platform used was the Veryseq NGS (Illumina). Nexgenomics position is to use an artificial cutoff of 50% to classify all the mosaicism events as normal or abnormal. Mosaicism <20% is considered not differentiable from technical noise. Low rate mosaic embryos were transferred when euploid embryos were not available. This study included only patients that received a single embryo transfer of low rate mosaic embryos and for which the delivery of a newborn could be confirmed. The patients generating the control group (only single euploid embryo transferred) were matched with the patients of the study group for age, embryo quality, use of an egg donor and/or gestational carrier and period of IVF treatment. All pregnancies that went to term were confirmed, by means of chorionic villus sampling or amniocentesis, to have a normal Karyotype. The results are summarized in Table 1. A total of 33 low mosaic embryos were transferred and they resulted in an overall live birth rate of 51.5%, similar to the live birth rate obtained from the transfer of euploid embryos (48.5%). No birth defects were reported.Table 1Comparison of the pregnancy outcomes of low rate mosaic and control embryos according to the mosaicism type and the chromosomes involved.Type of mosaicismSegmental*Single-Double1-12**Single-Double13-22***X-YComplexTotalControlsPAge38.8 ± 6.440.8 ± 6.936.3 ± 5.0-34.639.1 ± 6.439.3 ± 6.3NSN of transfer cycles13127013366NSEmbryos transferred13127013366NSEmbryos Implanted875-02039NSImplantation rate (IR)61.5%58.3%71.4%-060.6%59.1%NSAbortions111--37NSAbortion rate (AR)12.5%14.3%20%--15%17.9%NSDeliveries764-01732NSLive birth rate (LBR)53.8%50.0%57.1%-051.5%48.5%NS*Segmental: regarding a piece of chromosome**Single-Double 1-12: regarding one or two entire chromosomes from number 1 to number 12***Single-Double 13-22: regarding one or two entire chromosomes from number 13 to number 12-22 Open table in a new tab *Segmental: regarding a piece of chromosome **Single-Double 1-12: regarding one or two entire chromosomes from number 1 to number 12 ***Single-Double 13-22: regarding one or two entire chromosomes from number 13 to number 12-22 Our results indicate that low rate mosaic embryos have the same IR and LBR when compared to euploid embryos. Due to the low number of embryos in our study, further studies are required to verify this observation, but for clinical purposes, these embryos could be considered euploid. As far as we know this is the second study that provides data about the LBR of the mosaic embryos (Greco et al., 2015). A recent study (Munnè et al., 2017) concluded that, overall, mosaic embryos implant less and miscarry more than euploid embryos. However, embryos with >40% abnormal cells and complex mosaic embryos had the lower ongoing implantation rate. We transferred only one complex mosaic embryo and low rate mosaic embryos, which can explain the difference in our findings.

Chromosomal instability drives metastasis through a cytosolic DNA response

Chromosomal instability (CIN) is a hallmark of cancer and it results from ongoing errors in chromosome segregation during mitosis. While CIN is a major driver of tumor evolution, its role in metastasis has not been established. Here we show that CIN promotes metastasis by sustaining a tumor-cell autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signaling. Genetic suppression of CIN significantly delays metastasis even in highly aneuploid tumor models, whereas inducing continuous chromosome segregation errors promotes cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumor cells co-opt chronic activation of innate immune pathways to spread to distant organs.

Abstract CN01-02: Chromosomal instability as a driver of tumor metastasis

Abstract Chromosomal instability (CIN) is a hallmark of cancer, and it results from ongoing errors in chromosome segregation during mitosis. While CIN is a major driver of tumor evolution, its role in metastasis has not been established. Here we show that CIN promotes metastasis by sustaining a tumor-cell autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of rupture-prone micronuclei that expose their DNA content to the cytosol. This leads to the activation of the cGAS-STING cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signaling. Genetic suppression of CIN significantly delays metastasis even in highly aneuploid tumor models, whereas inducing continuous chromosome segregation errors promotes cellular invasion and metastasis in a STING-dependent manner. Using single-cell RNA sequencing, we uncover a CIN-induced transcriptional switch from a proliferative and metabolically active state to a mesenchymal phenotype associated with inflammatory pathways, offering an opportunity to target chromosome segregation errors for therapeutic benefit. Citation Format: Samuel F. Bakhoum. Chromosomal instability as a driver of tumor metastasis [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr CN01-02.

Abstract B065: TTK inhibitors as a targeted therapy for β-catenin mutant cancers

Abstract The dual-specificity protein kinase TTK, commonly referred to as Mps1, is a component of the spindle assembly checkpoint, a surveillance mechanism that ensures the fidelity of chromosome segregation. Inhibition of TTK kinase activity with small molecule kinase inhibitors leads to chromosome segregation errors by allowing mitotic exit in the presence of unattached kinetochores (1). After several rounds of cell division, the accumulation of chromosome segregation errors results in cancer cell death by apoptosis (2). Several TTK inhibitors have been shown to reduce the growth of xenografts of human cancer cell lines from diverse tumor tissue origin in mice. In an immunocompetent mouse model of triple-negative breast cancer (TNBC), TTK inhibitors increased the efficacy of taxane chemotherapy (1). While the first TTK inhibitors have entered phase 1 dose escalating studies in combination with taxane chemotherapy, a patient stratification strategy is still missing. To enable the selection of patients most likely to respond to TTK inhibitor therapy, we profiled a set of ten preclinical and clinical inhibitors (3) on a panel of 66 genetically characterized cancer cell lines derived from different tumor tissues (Oncolines) (4). Drug sensitivity was related to the mutation status of 114 cancer genes in an unbiased way. Cell lines harboring activating mutations in the CTNNB1 gene, encoding the Wnt pathway signaling regulator β-catenin, were on average up to five times more sensitive to TTK inhibitors than cell lines wild-type for CTNNB1. The association of CTNNB1 mutant status and increased cancer cell line sensitivity to TTK inhibition was confirmed with isogenic cell line pairs harboring either mutant or wild-type CTNNB1. Treatment of a xenograft model of a CTNNB1 mutant cell line with the TTK inhibitor NTRC 0066-0 resulted in complete inhibition of tumor growth. Mutations in CTNNB1 occur at relatively high frequency in endometrial cancer and hepatocellular carcinoma, which are known to express high TTK levels (5). Interestingly, β-catenin signaling has also been implicated in intrinsic resistance against immunotherapy in melanoma by regulation of T cell infiltration (6). We propose mutant CTNNB1 as a prognostic drug response biomarker, enabling the selection of patients most likely to respond to TTK inhibitor therapy in proof-of-concept clinical trials.

Quantitative methods to measure aneuploidy and chromosomal instability.

Cell viability requires accurate chromosome segregation during meiosis and mitosis so that the daughter cells produced have the correct chromosome complement. In contrast, chromosome segregation errors lead to aneuploidy, a state of abnormal chromosome numbers. Furthermore, a persistently high rate of chromosome segregation errors causes the related phenomenon of whole chromosomal instability (w-CIN). Aneuploidy and w-CIN are common characteristics of several human conditions and diseases including birth defects and cancers. Thus, methods to measure aneuploidy and w-CIN have important research applications in many areas of cell biology. In this chapter, we describe methods to measure chromosome missegregation rates and aneuploid cell survival with a focus on cells grown in culture; however, we also highlight methods that are amenable to primary tissue samples. Together, these methods provide a comprehensive approach to determining the frequency of aneuploidy and w-CIN in cells.

Mechanisms of kinetochore-microtubule attachment errors in mammalian oocytes.

Proper kinetochore-microtubule attachment is essential for correct chromosome segregation. Therefore, cells normally possess multiple mechanisms for the prevention of errors in kinetochore-microtubule attachments and for selective stabilization of correct attachments. However, the oocyte, a cell that produces an egg through meiosis, exhibits a high frequency of errors in kinetochore-microtubule attachments. These attachment errors predispose oocytes to chromosome segregation errors, resulting in aneuploidy in eggs. This review aims to provide possible explanations for the error-prone nature of oocytes by examining key differences among other cell types in the mechanisms for the establishment of kinetochore-microtubule attachments.

Gravin regulates centrosome function through PLK1.

We propose to understand how the mitotic kinase PLK1 drives chromosome segregation errors, with a specific focus on Gravin, a PLK1 scaffold. In both three-dimensional primary prostate cancer cell cultures that are prone to Gravin depletion and Gravin short hairpin RNA (shRNA)–treated cells, an increase in cells containing micronuclei was noted in comparison with controls. To examine whether the loss of Gravin affected PLK1 distribution and activity, we utilized photokinetics and a PLK1 activity biosensor. Gravin depletion resulted in an increased PLK1 mobile fraction, causing the redistribution of active PLK1, which leads to increased defocusing and phosphorylation of the mitotic centrosome protein CEP215 at serine-613. Gravin depletion further led to defects in microtubule renucleation from mitotic centrosomes, decreased kinetochore-fiber integrity, increased incidence of chromosome misalignment, and subsequent formation of micronuclei following mitosis completion. Murine Gravin rescued chromosome misalignment and micronuclei formation, but a mutant Gravin that cannot bind PLK1 did not. These findings suggest that disruption of a Gravin–PLK1 interface leads to inappropriate PLK1 activity contributing to chromosome segregation errors, formation of micronuclei, and subsequent DNA damage.

The induction of endoreduplication and polyploidy by elevated expression of 14-3-3γ.

Several studies have demonstrated that specific 14-3-3 isoforms are frequently elevated in cancer and that these proteins play a role in human tumorigenesis. 14-3-3γ, an isoform recently demonstrated to function as an oncoprotein, is overexpressed in a variety of human cancers; however, its role in promoting tumorigenesis remains unclear. We previously reported that overexpression of 14-3-3γ caused the appearance of polyploid cells, a phenotype demonstrated to have profound tumor promoting properties. Here we examined the mechanism driving 14-3-3γ-induced polyploidization and the effect this has on genomic stability. Using FUCCI probes we showed that these polyploid cells appeared when diploid cells failed to enter mitosis and subsequently underwent endoreduplication. We then demonstrated that 14-3-3γ-induced polyploid cells experience significant chromosomal segregation errors during mitosis and observed that some of these cells stably propagate as tetraploids when isolated cells were expanded into stable cultures. These data lead us to conclude that overexpression of the 14-3-3γ promotes endoreduplication. We further investigated the role of 14-3-3γ in human NSCLC samples and found that its expression is significantly elevated in polyploid tumors. Collectively, these results suggests that 14-3-3γ may promote tumorigenesis through the production of a genetically unstable polyploid intermediate.

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes.

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Modeling the contributions of chromosome segregation errors and aneuploidy to Saccharomyces hybrid sterility.

Errors in meiosis can be important postzygotic barriers between different species. In Saccharomyces hybrids, chromosomal missegregation during meiosis I produces gametes with missing or extra chromosomes. Gametes with missing chromosomes are inviable, but we do not understand how extra chromosomes (disomies) influence hybrid gamete inviability. We designed a model predicting rates of missegregation in interspecific hybrid meioses assuming several different mechanisms of disomy tolerance, and compared predictions from the model with observations of sterility in hybrids between Saccharomyces yeast species. Sterility observations were consistent with the hypothesis that chromosomal missegregation causes hybrid sterility, and the model indicated that missegregation probabilities of 13-50% per chromosome can cause observed values of 90-99% hybrid sterility regardless of how cells tolerate disomies. Missing chromosomes in gametes are responsible for most infertility, but disomies may kill as many as 11% of the gametes produced by hybrids between Saccharomyces cerevisiae and Saccharomyces paradoxus. Copyright © 2017 John Wiley & Sons, Ltd.

Causes and consequences of chromosome segregation error in preimplantation embryos.

Errors in chromosome segregation are common during the mitotic divisions of preimplantation development in mammalian embryos, giving rise to so-called 'mosaic' embryos possessing a mixture of euploid and aneuploid cells. Mosaicism is widely considered to be detrimental to embryo quality and is frequently used as criteria to select embryos for transfer in human fertility clinics. However, despite the clear clinical importance, the underlying defects in cell division that result in mosaic aneuploidy remain elusive. In this review, we summarise recent findings from clinical and animal model studies that provide new insights into the fundamental mechanisms of chromosome segregation in the highly unusual cellular environment of early preimplantation development and consider recent clues as to why errors should commonly occur in this setting. We furthermore discuss recent evidence suggesting that mosaicism is not an irrevocable barrier to a healthy pregnancy. Understanding the causes and biological impacts of mosaic aneuploidy will be pivotal in the development and fine-tuning of clinical embryo selection methods.

Virulence protein VirD5 of Agrobacterium tumefaciens binds to kinetochores in host cells via an interaction with Spt4

The bacterium Agrobacterium tumefaciens causes crown gall tumor formation in plants. During infection the bacteria translocate an oncogenic piece of DNA (transferred DNA, T-DNA) into plant cells at the infection site. A number of virulence proteins are cotransported into host cells concomitantly with the T-DNA to effectuate transformation. Using yeast as a model host, we find that one of these proteins, VirD5, localizes to the centromeres/kinetochores in the nucleus of the host cells by its interaction with the conserved protein Spt4. VirD5 promotes chromosomal instability as seen by the high-frequency loss of a minichromosome in yeast. By using both yeast and plant cells with a chromosome that was specifically marked by a lacO repeat, chromosome segregation errors and the appearance of aneuploid cells due to the presence of VirD5 could be visualized in vivo. Thus, VirD5 is a prokaryotic virulence protein that interferes with mitosis.