Small Ubiquitin-related Modifier Ligase Activity of Mms21 Is Required for Maintenance of Chromosome Integrity during the Unperturbed Mitotic Cell Division Cycle in Saccharomyces cerevisiae

The SUMO ligase activity of Mms21/Nse2, a conserved member of the Smc5/6 complex, is required for resisting extrinsically induced genotoxic stress. We report that the Mms21 SUMO ligase activity is also required during the unchallenged mitotic cell cycle in Saccharomyces cerevisiae. SUMO ligase-defective cells were slow growing and spontaneously incurred DNA damage. These cells required caffeine-sensitive Mec1 kinase-dependent checkpoint signaling for survival even in the absence of extrinsically induced genotoxic stress. SUMO ligase-defective cells were sensitive to replication stress and displayed synthetic growth defects with DNA damage checkpoint-defective mutants such as mec1, rad9, and rad24. MMS21 SUMO ligase and mediator of replication checkpoint 1 gene (MRC1) were epistatic with respect to hydroxyurea-induced replication stress or methyl methanesulfonate-induced DNA damage sensitivity. Subjecting Mms21 SUMO ligase-deficient cells to transient replication stress resulted in enhancement of cell cycle progression defects such as mitotic delay and accumulation of hyperploid cells. Consistent with the spontaneous activation of the DNA damage checkpoint pathway observed in the Mms21-mediated sumoylation-deficient cells, enhanced frequency of chromosome breakage and loss was detected in these mutant cells. A mutation in the conserved cysteine 221 that is engaged in coordination of the zinc ion in Loop 2 of the Mms21 SPL-RING E3 ligase catalytic domain resulted in strong replication stress sensitivity and also conferred slow growth and Mec1 dependence to unchallenged mitotically dividing cells. Our findings establish Mms21-mediated sumoylation as a determinant of cell cycle progression and maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in budding yeast.

A natural triploid in Trichomycterus davisi (Siluriformes, Trichomycteridae): mitotic and meiotic characterization by chromosome banding and synaptonemal complex analyses

Within a total of 50 analyzed specimens a male individual of Trichomycterus davisi has been recorded with 81 chromosomes including 60 metacentric, 18 submetacentric and three subtelocentric chromosomes. When compared with diploid individuals (2n = 54) and the morphological standard of chromosomes, this male is a triploid with 3 = 81 chromosomes. Since staining with silver nitrate indicates three active nucleolar organizer regions (NORs), the three NOR- bearing chromosomes in this individual are genetically active. Analysis of the synaptonemal complex (SC) by electronic microscopy shows that there is an incomplete pairing of the third set of chromosomes in the triploid individual.

Mitotic mapping of a novel tumor suppressor locus on human chromosome 3q important in osteosarcoma tumorigenesis

Tumor-specific loss of constitutional heterozygosity by deletion, mitotic recombination or nondisjunction is a common mechanism for tumor suppressor allele inactivation. When loss of heterozygosity is the result of mitotic recombination, or a segmental deletion event, only a portion of the chromosome is lost. This information can be used to map the location of new tumor suppressor genes. In osteosarcoma, the highest frequencies of loss of heterozygosity have been reported for chromosomes 3q, 13q, 17p. On chromosomes 13q and 17p, allelic losses are associated with loss of function at the retinoblastoma susceptibility locus (RB1) and the p53 locus, respectively. Chromosome 3q is also of particular interest because the high percent of loss of heterozygosity (62%-75%) suggests the presence of another tumor suppressor important for osteosarcoma tumorigenesis. To localize this putative tumor suppressor gene, we used polymorphic markers on chromosome 3q to find the smallest common region of allele loss. This putative tumor suppressor was localized to a 700 kb region on chromosome 3q26.2 between the polymorphic loci D3S1282 and D3S1246. ^

The human CAS protein which is homologous to the CSE1 yeast chromosome segregation gene product is associated with microtubules and mitotic spindle.

Human CAS cDNA contains a 971-aa open reading frame that is homologous to the essential yeast gene CSE1. CSE1 is involved in chromosome segregation and is necessary for B-type cyclin degradation in mitosis. Using antibodies to CAS, it was shown that CAS levels are high in proliferating and low in nonproliferating cells. Here we describe the distribution of CAS in cells and tissues analyzed with antibodies against CAS. CAS is an approximately 100-kDa protein present in the cytoplasm of proliferating cells at levels between 2 x 10(5) and 1 x 10(6) molecules per cell. The intracellular distribution of CAS resembles that of tubulin. In interphase cells, anti-CAS antibody shows microtubule-like patterns and in mitotic cells it labels the mitotic spindle. CAS is removed from microtubules by mild detergent treatment (cytoskeleton preparations) and in vincristine- or taxol-treated cells. CAS is diffusely distributed in the cytoplasm with only traces present in tubulin paracrystals or bundles. Thus, CAS appears to be associated with but not to be an integral part of microtubules. Immunohistochemical staining of frozen tissues shows elevated amounts of CAS in proliferating cells such as testicular spermatogonia and cells in the basal layer cells of the colon. CAS was also concentrated in the respiratory epithelium of the trachea and in axons and Purkinje cells in the cerebellum. These cells contain many microtubules. The cellular location of CAS is consistent with an important role in cell division as well as in ciliary movement and vesicular transport.

Association of the NuMA region on chromosome 11q13 with breast cancer susceptibility

The development of breast cancer is a complex process that involves multiple genes at many stages, from initial cell cycle dysregulation to disease progression. To identify genetic variations that influence this process, we conducted a large-scale association study using a collection of German cases and controls and >25,000 SNPs located within 16,000 genes. One of the loci identified was located on chromosome 11q13 [odds ratio (OR)=1.85, P=0.017]. The initial association was subsequently tested in two independent breast cancer collections. In both sample sets, the frequency of the susceptibility allele was increased in the cases (OR=1.6, P=0.01). The susceptibility allele was also associated with an increase in cancer family history (P=0.1). Fine mapping showed that the region of association extends approximately 300 kb and spans several genes, including the gene encoding the nuclear mitotic apparatus protein (NuMA). A nonsynonymous SNP (A794G) in NuMA was identified that showed a stronger association with breast cancer risk than the initial marker SNP (OR=2.8, P=0.005 initial sample; OR=2.1, P=0.002 combined). NuMA is a cell cycle-related protein essential for normal mitosis that is degraded in early apoptosis. NuMA-retinoic acid receptor alpha fusion proteins have been described in acute promyelocytic leukemia. Although the potential functional relevance of the A794G variation requires further biological validation, we conclude that variations in the NuMA gene are likely responsible for the observed increased breast cancer risk.

Daxx regulates mitotic progression and prostate cancer predisposition

Mitotic progression of mammalian cells is tightly regulated by the E3 ubiquitin ligase anaphase promoting complex (APC)/C. Deregulation of APC/C is frequently observed in cancer cells and is suggested to contribute to chromosome instability and cancer predisposition. In this study, we identified Daxx as a novel APC/C inhibitor frequently overexpressed in prostate cancer. Daxx interacts with the APC/C coactivators Cdc20 and Cdh1 in vivo, with the binding of Cdc20 dependent on the consensus destruction boxes near the N-terminal of the Daxx protein. Ectopic expression of Daxx, but not the D-box deleted mutant (DaxxΔD-box), inhibited the degradation of APC/Cdc20 and APC/Cdh1 substrates, leading to a transient delay in mitotic progression. Daxx is frequently upregulated in prostate cancer tissues; the expression level positively correlated with the Gleason score and disease metastasis (P = 0.027 and 0.032, respectively). Furthermore, ectopic expression of Daxx in a non-malignant prostate epithelial cell line induced polyploidy under mitotic stress. Our data suggest that Daxx may function as a novel APC/C inhibitor, which promotes chromosome instability during prostate cancer development.

Centrobin regulates the assembly of functional mitotic spindles

The proper function of the spindle is crucial to the high fidelity of chromosome segregation and is indispensable for tumor suppression in humans. Centrobin is a recently identified centrosomal protein that has a role in stabilizing the microtubule structure. Here we functionally characterize the defects in centrosome integrity and spindle assembly in Centrobin-depleted cells. Centrobin-depleted cells show a range of spindle abnormalities including unfocused poles that are not associated with centrosomes, S-shaped spindles and mini spindles. These cells undergo mitotic arrest and subsequently often die by apoptosis, as determined by live cell imaging. Co-depletion of Mad2 relieves the mitotic arrest, indicating that cells arrest due to a failure to silence the spindle checkpoint in metaphase. Consistent with this, Centrobin-depleted metaphase cells stained positive for BubR1 and BubR1 S676. Staining with a panel of centrosome markers showed a loss of centrosome anchoring to the mitotic spindle. Furthermore, these cells show less cold-stable microtubules and a shorter distance between kinetochore pairs. These results show a requirement of Centrobin in maintaining centrosome integrity, which in turn promotes anchoring of mitotic spindle to the centrosomes. Furthermore, this anchoring is required for the stability of microtubule–kinetochore attachments and biogenesis of tension-ridden and properly functioning mitotic spindle.

WT1 interacts with MAD2 and regulates mitotic checkpoint function

Tumour suppressors safeguard the fidelity of the mitotic checkpoint by transcriptional regulation of genes that encode components of the mitotic checkpoint complex (MCC). Here we report a new role for the tumour suppressor and transcription factor, WT1, in the mitotic checkpoint. We show that WT1 regulates the MCC by directly interacting with the spindle assembly checkpoint protein, MAD2. WT1 colocalizes with MAD2 during mitosis and preferentially binds to the functionally active, closed-conformer, C-MAD2. Furthermore, WT1 associates with the MCC containing MAD2, BUBR1 and CDC20, resulting in prolonged inhibition of the anaphase-promoting complex/cyclosome (APC/C) and delayed degradation of its substrates SECURIN and CYCLIN B1. Strikingly, RNA interference-mediated depletion of WT1 leads to enhanced turnover of SECURIN, decreased lag time to anaphase and defects in chromosome segregation. Our findings identify WT1 as a regulator of the mitotic checkpoint and chromosomal stability.

Angelman Syndrome Protein UBE3A Interacts with Primary Microcephaly Protein ASPM, Localizes to Centrosomes and Regulates Chromosome Segregation

Many proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. All the seven autosomal recessive primary microcephaly (MCPH) proteins localize at the centrosome. Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome (also characterized by severe microcephaly) protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. To gain insight into the function of the most commonly mutated MCPH gene ASPM, we used the yeast two-hybrid technique to screen a human fetal brain cDNA library with an ASPM bait. The analysis identified Angelman syndrome gene product UBE3A as an ASPM interactor. Like ASPM, UBE3A also localizes to the centrosome. The identification of UBE3A as an ASPM interactor is not surprising as more than 80% of Angelman syndrome patients have microcephaly. However, unlike in MCPH, microcephaly is postnatal in Angelman syndrome patients. Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis. The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis. Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time. We suggest that a defective chromosome segregation mechanism is responsible for the development of microcephaly in Angelman syndrome.

Suppressing mitotic catastrophe: identifying mcs3 in Schizosaccharomyces pombe

In Schizosaccharomyces pombe (fission yeast), the transition from G2 phase of the cell cycle to mitosis is under strict regulation. The activation of Cdc2, a cyclin dependent serine/threonine protein kinase, is the critical control step in this process. The Cdc2/Cyclin-B (Cdc13) complex is regulated by Wee1 tyrosine kinase and Cdc25 tyrosine phosphatase, which work antagonistically to control progression into mitosis. Hyperactivation of the Cdc2/Cdc13 complex by phosphorylation results in premature mitosis, and as a consequence leads to genome instability. This is referred to as mitotic catastrophe, a lethal phenotype associated with chromosomal segregation abnormalities including chromosome breakage. Six mitotic catastrophe loci were found, five of which have been characterized and identified as various activators and repressors of the core mitotic control. The locus for mcs3 remains unknown. I used tetrad analysis in this study to determine the linkage distance between three genes suspected of flanking the region in which mcs3 is located. Linkage distances obtained in this study confirm that the SPBC428.10 and met17, as well as SPBC428.10 and wpl1 are tightly linked, suggesting this is an area of low recombination. Further linkage analysis should be conducted to determine the precise location of mcs3-12.

BubR1 is required for the mitotic block induced by combretastatin-A4 and a novel cis-restricted ß-lactam analogue in human cancer cells

BubR1 is a well-defined guardian of the mitotic spindle, initiating mitotic arrest in response to the lack of tension and/or chromosome alignment across the mitotic plate. However, the role of BubR1 in combretastatin-induced cell death remains unknown. In this study, we describe the effects of combretastatin A-4 (CA-4) and a synthetic cis-restricted 3,4-diaryl-2-azetidinone (ß-lactam) analogue (CA-432) on the modulation and phosphorylation of BubR1 in human cervical cancer-derived cells. We demonstrate that CA-4 and CA-432 depolymerise the microtubular network of human cervical carcinoma-derived cells. Both compounds induced the disassembly of the microtubules and the loss of microtubule tension led to the early phosphorylation of BubR1 and the late cleavage of BubR1. The phosphorylation of BubR1 correlated with the onset of G2M cell cycle arrest whilst the cleavage of BubR1 coincided with apoptosis induced by the combretastatins. The combretastatin-induced apoptosis and the BubR1 cleavage were caspase-dependent. In vitro enzyme digests demonstrated that combretastatin-activated BubR1 is a substrate for caspase-3. Gene silencing of BubR1 with small interfering RNA severely compromised combretastatin-induced G2M cell cycle arrest with a corresponding increase in the formation of polyploid cells in both cervical and breast cancer-derived cells. In summary, BubR1 is required to maintain the G2M arrest and limit the formation of polyploid cells in response to continued combretastatin exposure. Moreover, substitution of the ethylene bridge with 3,4-diaryl-2-azetidinone did not alter the tubulin depolymerising properties or the subsequent mitotic spindle checkpoint response to CA-4 in human cancer cells.

Chromosome structure and behaviour in Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) germ cells and early embryo

Chromosome structure and behaviour in both meiosis of the germ cells and mitosis of the embryo from fertilisation to the two-cell stage in Bursaphelenchus xylophilus were examined by DAPI staining and three-dimensional reconstruction of serial-section images from confocal laser-scanning microscopy. By this method, each chromosome’s shape and behaviour were clearly visible in early embryogenesis from fertilisation through the formation and fusion of the male and female pronuclei to the first mitotic division. The male pronucleus was bigger than that of the female, although the oocyte is larger and richer in nutrients than the sperm. From the shape of the separating chromosomes at anaphase, the mitotic chromosomes appeared to be polycentric or holocentric rather than monocentric. Each chromosome was clearly distinguishable in the male and female germ cells, pronuclei of the one-cell stage embryo, and the early embryonic nuclei. The haploid number of chromosomes (N) was six (2n = 12), and all chromosomes appeared similar. The chromosome pair containing the ribosomal RNA-coding site was visualised by fluorescence in situ hybridisation. Unlike the sex determination system in Caenorhabditis elegans (XX in hermaphrodite and XO in male), the system for B. xylophilus may consist of an XX female and an XY male.

Elucidating the crosstalk between condensin subunits and its relevance in chromosome condensation

ADN subit une série de transformations structurelles complexes au cours de la division cellulaire, ce qui entraîne dans son compactage chromosomes mitotiques par un processus appelé la condensation des chromosomes. Le complexe de condensine pentamérique est fortement impliqué comme un effecteur majeur de ce phénomène. Il s'agit d'un complexe protéine de sous-unités multiples avec deux sous-unités catalytiques [SMC- Structural Maintenance of Chromosomes] et de trois sous-unités de régulation, hautement conservés de la levure à l'homme. Le complexe de condensine dans Saccharomyces cerevisiae est constitué de deux sous-unités de SMC [Smc2 et Smc4] et trois protéines non réglementaires [Brn1, Ycs4, Ycg1]. Malgré son importance, le mécanisme d'action de condensine reste largement inconnu. Par conséquent, l'objectif de cette recherche est de comprendre le mécanisme d'action de condensine et comment elle est affectée par l'interaction entre ses sous-unités réglementaires et non-réglementaires. Cette thèse identifie quatre morphologies dépendants du cycle cellulaire distincts du locus d'ADNr. Cette transformation du phénotype ADNr de G1 à la mitose dépend condensine. Afin de déterminer le rôle de l'interaction entre les sous-unités catalytiques et réglementaires de condensine dans la régulation du complexe condensine, nous avons identifié six résidus positifs sur l'extrémité C-terminale de BRN1 qui affectent la formation du complexe condensine, l'activité de la condensation et l'interaction avec tubuline, ce qui suggère que ces résidus ont un rôle dans la régulation de condensine. Ensemble, nos résultats suggèrent un modèle de règlement du condensine par l'interaction entre les sous-unités de condensine.