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PubMed2006

The case for Survivin as mitotic regulator.

Survivin has been proposed to inhibit apoptosis and to regulate cell division. However, controversy still exists as to whether Survivin can indeed execute these distinct functions and if Survivin somehow coordinates apoptosis and (abnormal) cell division. Recent evidence has demonstrated that Survivin acts as a subunit of the chromosomal passenger complex, which is essential for proper chromosome segregation and cytokinesis. Within this complex, the mitotic kinase Aurora B acts as the enzymatic core, whereas Survivin dictates chromosomal passenger complex localization. This function of Survivin appears to be conserved throughout evolution. Although these findings do not exclude a role for Survivin as apoptosis inhibitor, they make a very strong case for Survivin as mitotic regulator.

Animals↗
PubMed2006

Sperm-FISH analysis in a pericentric chromosome 1 inversion, 46,XY,inv(1)(p22q42), associated with infertility.

No phenotypic effect is observed in most inversion heterozygotes. However, reproductive risks may occur in the form of infertility, spontaneous abortions or chromosomally unbalanced children as a consequence of meiotic recombination between inverted and non-inverted chromosomes. An odd number of crossovers within the inverted segment results in gametes bearing recombinant chromosomes with a duplication of the region outside of the inversion segment of one arm and a deletion of the terminal segment of the other arm [dup(p)/del(q) and del(p)/dup(q)]. Using fluorescence in-situ hybridization (FISH), the chromosome segregation of a pericentric inversion of chromosome 1 was studied in spermatozoa of a inv(1)(p22q42) heterozygous carrier. Three-colour FISH was performed on sperm samples using a probe mixture consisting of chromosome 1p telomere-specific probe, chromosome 1q telomere-specific probe and chromosome 18 centromere-specific alpha satellite DNA probe. The frequency of the non-recombinant product was 80.1%. The frequencies of the two types of recombinants carrying a duplication of the short arm and a deletion of the long arm, and vice versa, were respectively 7.6 and 7.2%, and these frequencies were not statistically significant from the expected ratio of 1:1. Sperm-FISH allows the further understanding of segregation patterns and their effect on reproductive failure and allows an accurate genetic counselling.

Adult↗
PubMed2002

Ubc9 is essential for viability of higher eukaryotic cells.

Ubc9 is an enzyme involved in the conjugation of SUMO-1 (small ubiquitin related modifier 1) to target proteins. The SUMO-1 conjugation system is well conserved from yeasts to higher eukaryotes, but many SUMO-1 target proteins reported recently in higher eukaryotic cells, including IkappaBalpha, MDM2, p53, and PML, are not present in yeasts. To determine the physiological roles of SUMO-1 conjugation in higher eukaryotic cells, we constructed a conditional UBC9 mutant of chicken DT40 cells containing the UBC9 transgene under control of a tetracycline-repressible promoter and characterized their loss of function phenotypes. Ubc9 disappeared 3 days after the addition of tetracycline and the increase in viable cell number stopped 4 days after the addition of drug. In contrast to the cases of ubc9 mutants of budding and fission yeasts, which show defects in progression of G2 or early M phase and in chromosome segregation, respectively, we did not observe accumulation of cells in G2/M phase or a considerable increase in the frequency of chromosome missegregation upon depletion of Ubc9 but we did observe an increase in the number of cells containing multiple nuclei, indicating defects in cytokinesis. A considerable portion of the Ubc9-depleted cell population was committed to apoptosis without accumulating in a specific phase of the cell cycle, suggesting that chromosome damages are accumulated in Ubc9-depleted cells, and apoptosis is triggered without activating checkpoint mechanisms under conditions of SUMO-1 conjugation system impairment.

Animals↗
PubMed2005

Three-dimensional ultrastructure of Saccharomyces cerevisiae meiotic spindles.

Meiotic chromosome segregation leads to the production of haploid germ cells. During meiosis I (MI), the paired homologous chromosomes are separated. Meiosis II (MII) segregation leads to the separation of paired sister chromatids. In the budding yeast Saccharomyces cerevisiae, both of these divisions take place in a single nucleus, giving rise to the four-spored ascus. We have modeled the microtubules in 20 MI and 15 MII spindles by using reconstruction from electron micrographs of serially sectioned meiotic cells. Meiotic spindles contain more microtubules than their mitotic counterparts, with the highest number in MI spindles. It is possible to differentiate between MI versus MII spindles based on microtubule numbers and organization. Similar to mitotic spindles, kinetochores in either MI or MII are attached by a single microtubule. The models indicate that the kinetochores of paired homologous chromosomes in MI or sister chromatids in MII are separated at metaphase, similar to mitotic cells. Examination of both MI and MII spindles reveals that anaphase A likely occurs in addition to anaphase B and that these movements are concurrent. This analysis offers a structural basis for considering meiotic segregation in yeast and for the analysis of mutants defective in this process.

Anaphase↗
PubMed2005

The yeast DASH complex forms closed rings on microtubules.

The Saccharomyces cerevisiae DASH complex is an essential microtubule-binding component of the kinetochore. We coexpressed all ten subunits of this assembly in Escherichia coli and purified a single complex, a approximately 210-kDa heterodecamer with an apparent stoichiometry of one copy of each subunit. The hydrodynamic properties of the recombinant assembly are indistinguishable from those of the native complex in yeast extracts. The structure of DASH alone and bound to microtubules was visualized by EM. The free heterodecamer is relatively globular. In the presence of microtubules, DASH oligomerizes to form rings and paired helices that encircle the microtubules. We discuss potential roles for such collar-like structures in maintaining microtubule attachment and spindle integrity during chromosome segregation.

Chromosome Segregation↗
PubMed2001

Yeast Dam1p has a role at the kinetochore in assembly of the mitotic spindle.

During mitosis, replicated chromosomes are separated to daughter cells by the microtubule-based mitotic spindle. Chromosomes attach to the mitotic spindle through specialized DNA/protein structures called kinetochores, but the mechanism of attachment is not well understood. We show here that the yeast microtubule-binding protein, Dam1p, associates physically and functionally with kinetochores, suggesting a role in kinetochore attachment to the spindle. An epitope-tagged version of Dam1p colocalizes with the integral kinetochore component Ndc10p/Cbf2p in immunofluorescence analysis of chromosome spreads. In addition, Dam1p is associated preferentially with centromeric DNA as shown by chromatin immunoprecipitation experiments, and this association depends on Ndc10p/Cbf2p. We also demonstrate genetic interactions between DAM1 and CTF19 or SLK19 genes encoding kinetochore proteins. Although the defect caused by the dam1-1 mutation leads to activation of the spindle checkpoint surveillance system and consequent persistence of sister chromatid cohesion, the metaphase arrest spindle abnormally elongates, resulting in virtually complete chromosome missegregation. Execution point experiments indicate that Dam1p has a role in formation of a metaphase spindle and in anaphase spindle elongation. Finally, we have observed that the protein encoded by the dam1-1 allele becomes delocalized at the nonpermissive temperature, correlating with the subsequent onset of the mutant phenotype. Our studies are consistent with a role for Dam1p in attachment of sister chromatids through the kinetochore to the mitotic spindle before chromosome segregation.

Cell Cycle Proteins↗
PubMed2005

HTP-1 coordinates synaptonemal complex assembly with homolog alignment during meiosis in C. elegans.

During meiosis, the mechanisms responsible for homolog alignment, synapsis, and recombination are precisely coordinated to culminate in the formation of crossovers capable of directing accurate chromosome segregation. An outstanding question is how the cell ensures that the structural hallmark of meiosis, the synaptonemal complex (SC), forms only between aligned pairs of homologous chromosomes. In the present study, we find that two closely related members of the him-3 gene family in Caenorhabditis elegans function as regulators of synapsis. HTP-1 functionally couples homolog alignment to its stabilization by synapsis by preventing the association of SC components with unaligned and immature chromosome axes; in the absence of the protein, nonhomologous contacts between chromosomes are inappropriately stabilized, resulting in extensive nonhomologous synapsis and a drastic decline in chiasma formation. In the absence of both HTP-1 and HTP-2, synapsis is abrogated per se and the early association of SC components with chromosomes observed in htp-1 mutants does not occur, suggesting a function for the proteins in licensing SC assembly. Furthermore, our results suggest that early steps of recombination occur in a narrow window of opportunity in early prophase that ends with SC assembly, resulting in a mechanistic coupling of the two processes to promote crossing over.

Animals↗
PubMed2003

Identification of a subunit of a novel Kleisin-beta/SMC complex as a potential substrate of protein phosphatase 2A.

Protein phosphatase 2A (PP2A) holoenzymes consist of a catalytic C subunit, a scaffolding A subunit, and one of several regulatory B subunits that recruit the AC dimer to substrates. PP2A is required for chromosome segregation, but PP2A's substrates in this process remain unknown. To identify PP2A substrates, we carried out a two-hybrid screen with the regulatory B/PR55 subunit. We isolated a human homolog of C. elegans HCP6, a protein distantly related to the condensin subunit hCAP-D2, and we named this homolog hHCP-6. Both C. elegans HCP-6 and condensin are required for chromosome organization and segregation. HCP-6 binding partners are unknown, whereas condensin is composed of the structural maintenance of chromosomes proteins SMC2 and SMC4 and of three non-SMC subunits. Here we show that hHCP-6 becomes phosphorylated during mitosis and that its dephosphorylation by PP2A in vitro depends on B/PR55, suggesting that hHCP-6 is a B/PR55-specific substrate of PP2A. Unlike condensin, hHCP-6 is localized in the nucleus in interphase, but similar to condensin, hHCP-6 associates with chromosomes during mitosis. hHCP-6 is part of a complex that contains SMC2, SMC4, kleisin-beta, and the previously uncharacterized HEAT repeat protein FLJ20311. hHCP-6 is therefore part of a condensin-related complex that associates with chromosomes in mitosis and may be regulated by PP2A.

Adenosine Triphosphatases↗
PubMed2003

Cytokinesis: relative alignment of the cell division apparatus and the mitotic spindle.

The cell division apparatus is assembled at different stages of the cell cycle in different eukaryotic organisms. Mechanisms exist in all organisms, however, to ensure that the cell division apparatus and the mitotic spindle are aligned perpendicular to each other. Such an alignment ensures that each daughter cell receives a nucleus and that the cell division apparatus does not cleave and destroy the genetic material. The interaction(s) of astral microtubules with the cell cortex appears to play an important role in establishing perpendicularity between chromosome segregation and cell division machinery.

Animals↗
PubMed2004

The genetics and molecular biology of the synaptonemal complex.

The synaptonemal complex (SC) is a protein lattice that resembles railroad tracks and connects paired homologous chromosomes in most meiotic systems. The two side rails of the SC, known as lateral elements (LEs), are connected by proteins known as transverse filaments. The LEs are derived from the axial elements of the chromosomes and play important roles in chromosome condensation, pairing, transverse filament assembly, and prohibiting double-strand breaks (DSBs) from entering into recombination pathways that involve sister chromatids. The proteins that make up the transverse filaments of the SC also play a much earlier role in committing a subset of DSBs into a recombination pathway, which results in the production of reciprocal meiotic crossovers. Sites of crossover commitment can be observed as locations where the SC initiates and as immunostaining foci for a set of proteins required for the processing of DSBs to mature crossovers. In most (but not all) organisms it is the establishment of sites marking such crossover-committed DSBs that facilitates completion of synapsis (full-length extension of the SC). The function of the mature full-length SC may involve both the completion of meiotic recombination at the DNA level and the exchange of the axial elements of the two chromatids involved in the crossover. However, the demonstration that the sites of crossover formation are designated prior to SC formation, and the finding that these sites display interference, argues against a role of the mature SC in mediating the process of interference. Finally, in at least some organisms, modifications of the SC alone are sufficient to ensure meiotic chromosome segregation in the complete absence of meiotic recombination.

Animals↗
PubMed1997

Role of chromosomes in assembly of meiotic and mitotic spindles.

The assembly of a mitotic spindle requires the interaction of microtubules with chromosomes. As a cell enters mitosis, long microtubules are converted to short ones, as microtubules become unstable. Dynamic microtubules are then stabilised by chromosomes, forming a bipolar spindle. In this review, we discuss the different roles of kinetochores and chromosome arms during spindle assembly. Kinetochores, required for proper chromosomes segregation, capture microtubules and maintain attachment. Chromosome arms greatly enhance microtubule stability, and alone can be sufficient for spindle assembly.

Animals↗
PubMed1992

Isolation of anonymous DNA markers for human chromosome 22q11 from a flow-sorted library, and mapping using hybrids from patients with DiGeorge syndrome.

DiGeorge syndrome (DGS) is a human developmental defect of the structures derived from the third and fourth pharyngeal pouches. It apparently arises due to deletion of 22q11. We describe a strategy for the isolation of DNA probes for this region. A deleted chromosome 22, which includes 22q11, was flow-sorted from a lymphoblastoid cell line of a patient with cat eye syndrome and used as the source of DNA. A DNA library was constructed from this chromosome by cloning into the EcoR1 site of the vector Lambda gt10. Inserts were amplified by PCR and mapped using a somatic cell hybrid panel of this region. Out of 32 probes, 14 were mapped to 22q11. These probes were further sublocalised within the region by dosage analysis of DGS patients, and by the use of two new hybrid cell lines which we have produced from DGS patients. One of these lines (7939B662) contains the altered human chromosome segregated from its normal homologue. This chromosome 22 contains an interstitial deletion in 22q11, and will be useful for localising further probes to the DGS region.

Base Sequence↗
PubMed2005

Meiotic transverse filament proteins: essential for crossing over.

Meiosis is a specialized set of two nuclear divisions, meiosis I and II, by which a diploid cell produces four haploid daughters. After premeiotic DNA replication, homologous chromosomes pair and recombine, and then disjoin at meiosis I. Subsequently, at meiosis II, the sister chromatids of each chromosome segregate. In nearly all eukaryotes, meiotic chromosome pairing culminates in the formation of a ladder-like supramolecular protein structure, the synaptonemal complex (SC) (Page and Hawley, 2004). The rungs of the ladder are known as transverse filaments (TFs). Genes encoding TF proteins have been identified in a limited number of organisms, and their function has been studied by mutational analysis. Although TF proteins show little amino acid sequence conservation, their structure and function are largely conserved. In all analyzed species, TF proteins are required for meiotic reciprocal recombination (crossing over).

Animals↗
PubMed1996

The Saccharomyces cerevisiae kinetochore.

Accurate chromosome segregation is dependent on a specialized chromosomal structure, the kinetochore/centromere. The only essential constituent of the S. cerevisiae kinetochore established today is CBF3, a multisubunit complex that binds to S. cerevisiae centromere DNA. Therefore CBF3 and its four components, Cbf3a, Cbf3b, Cbf3c and Cbf3d, will form the centerpiece of this review. In addition, we will describe proteins that are putatively involved in kinetochore function specifically in the context with CBF3 interaction. Furthermore, we discuss the role of the S. cerevisiae kinetochores in a putative cell cycle checkpoint control and in microtubule attachment.

Base Sequence↗
PubMed2004

Proteolysis contributes to the exclusive centromere localization of the yeast Cse4/CENP-A histone H3 variant.

Kinetochores are the specialized protein structures that form on centromeric DNA and direct chromosome segregation. It is critical that all chromosomes assemble a single kinetochore every cell cycle. One hallmark of all eukaryotic kinetochores is CENP-A, an essential centromeric histone H3 (CenH3) variant. Overexpression of CENP-A causes mislocalization to euchromatin, which could lead to deleterious consequences because CENP-A overexpression is associated with colorectal cancer . Although CENP-A protein levels are important for genomic stability, little is known about the mechanisms of CenH3 regulation. Here, we show that the levels of the budding yeast CenH3, Cse4, are regulated by ubiquitin-proteasome-mediated proteolysis. Because mutation of all Cse4 lysine residues did not completely stabilize the protein, we isolated a dominant lethal mutant, CSE4-351, that was stable. The Cse4-351 protein localized to euchromatin, suggesting that proteolysis prevents CenH3 euchromatic localization. When wild-type Cse4 was fused to a degron signal, the soluble Cse4 protein was rapidly degraded, but the centromere bound Cse4 was stable, indicating that centromere localization protects Cse4 from degradation. Taken together, these data identify proteolysis as one mechanism that contributes to the restricted centromere localization of the yeast CenH3.

Autoantigens↗
PubMed2002

Characterization of regions in hsMAD1 needed for binding hsMAD2. A polymorphic change in an hsMAD1 leucine zipper affects MAD1-MAD2 interaction and spindle checkpoint function.

In eukaryotes, the mitotic spindle assembly checkpoint provides a monitor for the fidelity of chromosomal segregation. In this context, the mitotic arrest deficiency protein 2 (MAD2) censors chromosomal mis-segregation by monitoring microtubule attachment/tension, a role that requires its attachment to kinetochores. Studies in yeast have shown that binding of MAD1 to MAD2 is important for the checkpoint function of the latter. The interactions between human MAD1 (hsMAD1) and human MAD2 (hsMAD2) have, however, remained poorly characterized. Here we report that two leucine zipper domains (amino acids 501-522 and 557-571) in hsMAD1 are required for its contact with hsMAD2. Interestingly, in several cancer cell lines, we noted the frequent presence of a coding single nucleotide Arg to His polymorphism at codon 558 located within the second leucine zipper of hsMAD1. We found that hsMAD1H558 is less proficient than hsMAD1R558 in binding hsMAD2 and in enforcing mitotic arrest. We also document a first example of loss-of-heterozygosity for a spindle checkpoint gene (at the hsMAD1 558 locus) in a human breast cancer. Based on our findings, it is possible that hsMAD1H558 could be an at-risk polymorphism that contributes to attenuated spindle checkpoint function in human cells.

Amino Acid Sequence↗
PubMed1995

Microinjection of mitotic cells with the 3F3/2 anti-phosphoepitope antibody delays the onset of anaphase.

The transition from metaphase to anaphase is regulated by a checkpoint system that prevents chromosome segregation in anaphase until all the chromosomes have aligned at the metaphase plate. We provide evidence indicating that a kinetochore phosphoepitope plays a role in this checkpoint pathway. The 3F3/2 monoclonal antibody recognizes a kinetochore phosphoepitope in mammalian cells that is expressed on chromosomes before their congression to the metaphase plate. Once chromosomes are aligned, expression is lost and cells enter anaphase shortly thereafter. When microinjected into prophase cells, the 3F3/2 antibody caused a concentration-dependent delay in the onset of anaphase. Injected antibody inhibited the normal dephosphorylation of the 3F3/2 phosphoepitope at kinetochores. Microinjection of the antibody eliminated the asymmetric expression of the phosphoepitope normally seen on sister kinetochores of chromosomes during their movement to the metaphase plate. Chromosome movement to the metaphase plate appeared unaffected in cells injected with the antibody suggesting that asymmetric expression of the phosphoepitope on sister kinetochores is not required for chromosome congression to the metaphase plate. In antibody-injected cells, the epitope remained expressed at kinetochores throughout the prolonged metaphase, but had disappeared by the onset of anaphase. When normal cells in metaphase, lacking the epitope at kinetochores, were treated with agents that perturb microtubules, the 3F3/2 phosphoepitope quickly reappeared at kinetochores. Immunoelectron microscopy revealed that the 3F3/2 epitope is concentrated in the middle electronlucent layer of the trilaminar kinetochore structure. We propose that the 3F3/2 kinetochore phosphoepitope is involved in detecting stable kinetochore-microtubule attachment or is a signaling component of the checkpoint pathway regulating the metaphase to anaphase transition.

Anaphase↗
PubMed1998

Fission yeast bub1 is a mitotic centromere protein essential for the spindle checkpoint and the preservation of correct ploidy through mitosis.

The spindle checkpoint ensures proper chromosome segregation by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. We investigated the role of the fission yeast bub1 gene in spindle checkpoint function and in unperturbed mitoses. We find that bub1(+) is essential for the fission yeast spindle checkpoint response to spindle damage and to defects in centromere function. Activation of the checkpoint results in the recruitment of Bub1 to centromeres and a delay in the completion of mitosis. We show that Bub1 also has a crucial role in normal, unperturbed mitoses. Loss of bub1 function causes chromosomes to lag on the anaphase spindle and an increased frequency of chromosome loss. Such genomic instability is even more dramatic in Deltabub1 diploids, leading to massive chromosome missegregation events and loss of the diploid state, demonstrating that bub1(+ )function is essential to maintain correct ploidy through mitosis. As in larger eukaryotes, Bub1 is recruited to kinetochores during the early stages of mitosis. However, unlike its vertebrate counterpart, a pool of Bub1 remains centromere-associated at metaphase and even until telophase. We discuss the possibility of a role for the Bub1 kinase after the metaphase-anaphase transition.

Amino Acid Sequence↗