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Small supernumerary marker chromosomes (sSMCs) are characterized as additional centric chromosome fragments which are too small to be classified by cytogenetic banding alone and smaller than or equal to the size of chromosome 20 of the same metaphase spread. Here, we report a patient who presented with slight neutropenia and oral aphthous ulcers. A mosaic de novo sSMC, which originated from 5 discontinuous regions of chromosome 8, was detected in the patient. Formation of the sSMC(8) can probably be explained by a multi-step process beginning with maternal meiotic nondisjunction, followed by post-zygotic anaphase lag, and resulting in chromothripsis. Chromothripsis is a chromosomal rearrangement which occurs by breakage of one or more chromosomes leading to a fusion of surviving chromosome pieces. This case is a good example for emphasizing the importance of conventional karyotyping from PHA-induced peripheral blood lymphocytes and examining tissues other than bone marrow in patients with inconsistent genotype and phenotype.

Inhibitory “wait anaphase” signals derived from unbound kinetochores in a mitotic spindle diffuse into the cytoplasm. These diffusible signals can synchronize anaphase onset of neighboring spindles in multinucleate cells. The extent and activity of these signals are subject to diffusion barriers and cytoplasmic dilution., The spindle assembly checkpoint ensures the faithful inheritance of chromosomes by arresting mitotic progression in the presence of kinetochores that are not attached to spindle microtubules. This is achieved through inhibition of the anaphase-promoting complex/cyclosome by a kinetochore-derived “wait anaphase” signal known as the mitotic checkpoint complex. It remains unclear whether the localization and activity of these inhibitory complexes are restricted to the mitotic spindle compartment or are diffusible throughout the cytoplasm. Here we report that “wait anaphase” signals are indeed able to diffuse outside the confines of the mitotic spindle compartment. Using a cell fusion approach to generate multinucleate cells, we investigate the effects of checkpoint signals derived from one spindle compartment on a neighboring spindle compartment. We find that spindle compartments in close proximity wait for one another to align all chromosomes before entering anaphase synchronously. Synchrony is disrupted in cells with increased interspindle distances and cellular constrictions between spindle compartments. In addition, when mitotic cells are fused with interphase cells, “wait anaphase” signals are diluted, resulting in premature mitotic exit. Overall our studies reveal that anaphase inhibitors are diffusible and active outside the confines of the mitotic spindle from which they are derived.

To ensure accurate chromosome segregation, the spindle assembly checkpoint (SAC) delays anaphase onset by preventing the premature activation of anaphase-promoting complex/cyclosome (APC/C) until all kinetochores are attached to the spindle. Although an escape from mitosis in the presence of unsatisfied SAC has been shown in several cancer cells, it has not been reported in oocyte meiosis. Here, we show that CDK7 activity is required to prevent a bypass of SAC during meiosis I in mouse oocytes. Inhibition of CDK7 using THZ1 accelerated the first meiosis, leading to chromosome misalignment, lag of chromosomes during chromosome segregation, and a high incidence of aneuploidy. Notably, this acceleration occurred in the presence of SAC proteins including Mad2 and Bub3 at the kinetochores. However, inhibition of APC/C-mediated cyclin B degradation blocked the THZ1-induced premature polar body extrusion. Moreover, chromosomal defects mediated by THZ1 were rescued when anaphase onset was delayed. Collectively, our results show that CDK7 activity is required to prevent premature anaphase onset by suppressing the bypass of SAC, thus ensuring chromosome alignment and proper segregation. These findings reveal new roles of CDK7 in the regulation of meiosis in mammalian oocytes.

Genomic instability is widespread during early embryo development. Aneuploidy, mosaicism, and copy number variants (CNVs) commonly appear in human preimplantation embryos. Both age-dependent meiotic aneuploidy and age-independent mitotic aneuploidy and CNVs occur In human embryos. Telomere attrition, which contributes to genomic instability in somatic cells, also may promote genomic instability in preimplantation embryos. Telomere dynamics during gametogenesis are strikingly dimorphic between females and males. Sperm telomeres lengthen with advancing paternal age, while oocyte telomeres are among the shortest in the body. Spermatogonia express telomerase activity throughout the life of the male, while oocytes and cleavage stage embryos express low or un-measureable levels of telomerase activity. Telomere attrition in oocytes contributes to meiotic dysfunction, including spindle dysmorphologies, reduced synapsis and chiasmata, as well as delayed, arrested and fragmented embryos. Cleavage stage embryos, with such inefficient telomere reconstitution, likely undergo NHEJ, which produces anaphase lag, chromosome bridges, micronuclei, and genomic instability, including mosaicism and CNVs. Cleavage stage embryos reconstitute the short telomeres inherited from their mothers by Alternative Lengthening of Telomeres (ALT), a DNA recombination based method involving RAD 50, MRE 11, Werner and Bloom proteins, as well as telomere sister chromatid exchange. ALT robustly reconstitutes telomeres, but also predisposes to genomic instability.

Mixed gonadal dysgenesis is characterised by unilateral chromosomal abnormality, which is probably the result of anaphase lag during mitosis. The 45, XO/46, XY karyotype is the most common form of mosaicism involving the Y chromosome. It is a rare clinical entity with a worldwide incidence of 1.5/10,000 live births. Its epidemiology in Sub-Saharan Africa is not known. This study reports experience in the management of 3 cases at the Yaounde Gynecologic-Obstetric and Paediatric Hospital. From November 2009 to November 2014, 3 cases were successfully managed at our institution. Results: All patients presented with asymmetrical gonadal differentiation. On one side of the body, a poorly-developed testicular gonad and on the other side a gonadal streak. A persistent Mόllerian remnant was equally found in the 3 cases. Management of mixed gonadal dysgenesis should be done in tertiary health care centres. A multidisciplinary team approach is recommended.

Summary Condensin is a conserved chromosomal complex necessary to promote mitotic chromosome condensation and sister chromatid resolution during anaphase. Here, we report that yeast condensin binds to replicated centromere regions. We show that centromeric condensin relocalizes to chromosome arms as cells undergo anaphase segregation. We find that condensin relocalization is initiated immediately after the bipolar attachment of sister kinetochores to spindles and requires Polo kinase activity. Moreover, condensin localization during anaphase involves a higher binding rate on DNA and temporally overlaps with condensin’s DNA overwinding activity. Finally, we demonstrate that topoisomerase 2 (Top2) is also recruited to chromosome arms during anaphase in a condensin-dependent manner. Our results uncover a functional relation between condensin and Top2 during anaphase to mediate chromosome segregation., Graphical Abstract, Highlights • Condensin recruitment to centromeric regions requires DNA replication • Centromeric condensin spreads to chromosome arms during anaphase • Condensin promotes recruitment of Top2 during anaphase • Condensin localization requires Polo kinase and correlates with DNA overwinding, Chromosome condensation requires condensin and topoisomerase 2 (Top2) and is necessary for segregation. Leonard et al. show that condensin localizes to yeast centromeres following DNA replication and spreads to chromosome arms during anaphase when Top2 is also recruited. Condensin and Top2 activities are coordinated to shape mitotic chromosomes.

During mitosis, Aurora B is necessary for telomere dispersion through effects on Swi6/HP1 and cohesin and for subsequent telomere disjunction and chromosome arm separation through effects on condensin., The segregation of centromeres and telomeres at mitosis is coordinated at multiple levels to prevent the formation of aneuploid cells, a phenotype frequently observed in cancer. Mitotic instability arises from chromosome segregation defects, giving rise to chromatin bridges at anaphase. Most of these defects are corrected before anaphase onset by a mechanism involving Aurora B kinase, a key regulator of mitosis in a wide range of organisms. Here, we describe a new role for Aurora B in telomere dispersion and disjunction during fission yeast mitosis. Telomere dispersion initiates in metaphase, whereas disjunction takes place in anaphase. Dispersion is promoted by the dissociation of Swi6/HP1 and cohesin Rad21 from telomeres, whereas disjunction occurs at anaphase after the phosphorylation of condensin subunit Cnd2. Strikingly, we demonstrate that deletion of Ccq1, a telomeric shelterin component, rescued cell death after Aurora inhibition by promoting the loading of condensin on chromosome arms. Our findings reveal an essential role for telomeres in chromosome arm segregation.

Trisomy and triploidy, defined as the presence of a third copy of one or all chromosomes, respectively, are deleterious in many species including humans. Previous studies have demonstrated that Caenorhabditis elegans with a third copy of the X chromosome are viable and fertile. However, the extra X chromosome was shown to preferentially segregate into the first polar body during oocyte meiosis to produce a higher frequency of euploid offspring than would be generated by random segregation. Here, we demonstrate that extra autosomes are preferentially eliminated by triploid C. elegans and trisomy IV C. elegans. Live imaging of anaphase-lagging chromosomes and analysis of REC-8 staining of metaphase II spindles revealed that, in triploids, some univalent chromosomes do not lose cohesion and preferentially segregate intact into the first polar body during anaphase I, whereas other autosomes segregate chromatids equationally at anaphase I and eliminate some of the resulting single chromatids during anaphase II. We also demonstrate asymmetry in the anaphase spindle, which may contribute to the asymmetric segregation. This study reveals a pathway that allows aneuploid parents to produce euploid offspring at higher than random frequency.

Purpose of review Embryonic mosaicism represents an ongoing challenge for contemporary comprehensive chromosome screening platforms due to the unknown reproductive potential of mosaic embryos and technical difficulties of its detection from a single embryo biopsy. Recent findings Mosaicism in preimplantation embryos is a product of mitotic errors arising primarily from anaphase lag and chromosome nondisjunction. To date, there is high variability among estimations of prevalence of mosaicism in blastocysts, the most recent ranging from 3.3 to 83%. It has been reported that alleged mosaic embryos can develop into healthy babies, although the proper study evaluating this question remains to be completed. Technical artefacts from comprehensive chromosome screening platforms may also hinder correct classification of embryos as genuine mosaics. Summary Although complex, embryonic mosaicism is a phenomenon that deserves further investigation. Many embryos classified as mosaic may have actual reproductive potential. The predictive value of intermediate chromosome copy number assignments for the remaining embryo and for ongoing reproductive potential needs more careful consideration. In addition, recent advancements in extended embryo culture raise the possibility of investigating whether preferential segregation, selective advantage of normal cells or surveillance of abnormal chromosome numbers occur at postimplantation stages.

Post-translational protein modification such as phosphorylation and ubiquitination are critical during mitosis to ensure proper timing and progression of chromosome segregation. It has been recently recognized that another type of protein modification – neddylation – may also regulate mitosis and chromosome segregation. The conserved protein DCN1 (defective cullin neddylation 1) has been shown, when knocked-down by RNAi, to result in multinucleated cells and/or blockage of cell proliferation. However, how DCN1 functions in mitosis and chromosome segregation is not known. We report here the fission yeast dcn1+ and its role in mitosis and chromosome segregation. Dcn1-GFP localizes to the nucleus throughout the cell cycle. dcn1-deletion (dcn1Δ) leads to chromosome and kinetochore lagging at anaphase, resulting from delayed and attenuated cohesin cleavage and sister chromatids separation. These results put Dcn1 upstream of the anaphase promoting complex/cyclosome (APC/C) pathway. We propose a mechanism for Dcn1 function at mitosis., Summary: The fission yeast neddylation ligase Dcn1 plays a role in chromosome segregation. dcn1-deletion results in activation of the Mad2-dependent spindle assembly checkpoint, and lagging chromosomes at anaphase.

To prevent unbalanced chromosome segregation, meiotic metaphase I – anaphase I transition is carefully regulated by delaying anaphase until all kinetochores are well oriented (anaphase checkpoint) in mammals and insects. In plants this has not yet been established. In heterozygotes of two reciprocal translocations of Secale cereale, with one chromosome replaced by its two telocentric arms, anaphase delay was correlated with the orientation of the kinetochores of the complex of five chromosomes. The terminal kinetochores of the half chromosomes were readily elongated and pole oriented. Chains of five chromosomes with all five kinetochores orienting on alternate poles where the first to start anaphase. Kinetochores of two adjacent chromosomes when oriented on the same pole were partly shielded and less well pole directed. Anaphase was delayed. Cells with this configuration accumulated during anther development. Kinetochores in metacentric chromosomes lacking chiasmata in one arm (in trivalents and bivalents) were slightly better pole oriented and delayed anaphase less. Release of chromatid cohesion as triggered by kinetochore stretch is apparently delayed by inadequate exposition and pole orientation of the kinetochores. It is a mild form of an anaphase checkpoint, in normal material synchronizing bivalent segregation.

Study question: What is the incidence of aneuploidy and mosaicism in all cells of top-quality Day-4 embryos analysed by array-based comparative genomic hybridization (array CGH)? Summary answer: Our data show extensive abnormalities in Day-4 embryos. What is known already: Numerous studies on human embryos at Day 3 and Day 5 of development show that they frequently contain aneuploid cells and are mosaic, although Day-5 embryos contain proportionally more normal cells than at Day 3. In contrast, only limited data exist on Day 4 of preimplantation development, despite the fact that it is the suggested stage for the initiation of the process of self-correction. Study design, size, duration: Thirteen embryos were analysed: four fresh good-quality preimplantation genetic diagnosis (PGD) embryos and nine good-quality surplus embryos cryopreserved on Day 3 and donated for research. On Day 4, following removal of the zona pellucida, all blastomeres were disaggregated and collected. Participants/materials,setting, methods: The genomic DNA of 283 single blastomeres from disaggregated embryos was amplified. Array CGH was carried out using 24SureTM Cytochip microarrays. After scanning of the microarray slides, the images were analysed using BlueFuse Software (BlueGnome). Combined with selective microsatellite analysis, hypothetical reconstructions of embryo chromosome complements were made following each of the first four cleavage divisions. Main results and the role of chance: No chromosome imbalance was detected for one PGD embryo, the other three were mosaic containing between 16 and 75% abnormal cells. All nine frozen - thawed embryos were abnormal. Six were mosaic with between 30 and 100% abnormal cells; three had abnormalities of meiotic origin, two of which displayed mitotic abnormalities. Evidence was also found of mitotic unbalanced structural chromosome rearrangements. The higher rate of abnormality of frozen - thawed embryos is based on a small number of embryos and cannot be tested statistically. The aneuploidy can mostly be explained by anaphase lag and non-disjunction. In some cases, we hypothesize endoreduplication followed by a cellular division with multipolar spindles to explain the results. Limitations, reasons for caution: Array CGH technology determines relative quanti?cation of chromosomal domains but does not allow for the visualization of chromosomal rearrangements, assessment of ploidy or detection of uniparental isodisomy. Conclusions drawn on segmental abnormalities should be treated with caution. The division trees presented are hypothetical models projecting back in time that try to explain observations in single blastomeres of Day 4 embryos. The limited number of embryos analysed does not allow drawing ?rm conclusions, but nevertheless provides valuable data on the origin of aneuploidy in human embryos. Wider implications of the findings: Our data show extensive abnormalities in Day-4 embryos. We found no evidence of self-correction at this stage of development, suggesting that this process may start at a later stage of development. Study funding/competing interest (s): This research was supported by the Instituut voor de aanmoediging van innovatie door Wetenschap en Technologie in Vlaanderen (IWT-Vlaanderen). C.S. is a postdoctoral fellow at the Flemish Fund for Scienti?c Research [Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen]. There are no competing interests.

Despite the clinical importance of aneuploidy, surprisingly little is known concerning its impact during the earliest stages of human development. This study aimed to shed light on the genesis, progression, and survival of different types of chromosome anomaly from the fertilized oocyte through the final stage of preimplantation development (blastocyst). 2,204 oocytes and embryos were examined using comprehensive cytogenetic methodology. A diverse array of chromosome abnormalities was detected, including many forms never recorded later in development. Advancing female age was associated with dramatic increase in aneuploidy rate and complex chromosomal abnormalities. Anaphase lag and congression failure were found to be important malsegregation causing mechanisms in oogenesis and during the first few mitotic divisions. All abnormalities appeared to be tolerated until activation of the embryonic genome, after which some forms started to decline in frequency. However, many aneuploidies continued to have little impact, with affected embryos successfully reaching the blastocyst stage. Results from the direct analyses of female meiotic divisions and early embryonic stages suggest that chromosome errors present during preimplantation development have origins that are more varied than those seen in later pregnancy, raising the intriguing possibility that the source of aneuploidy might modulate impact on embryo viability. The results of this study also narrow the window of time for selection against aneuploid embryos, indicating that most survive until the blastocyst stage and, since they are not detected in clinical pregnancies, must be lost around the time of implantation or shortly thereafter. © 2013 Springer-Verlag Berlin Heidelberg.

During conventional anaphase A, chromosomes move outward toward spindle poles. Caenorhabditis elegans meiotic spindle poles move inward toward chromosomes to achieve the same end., In a wide range of eukaryotes, chromosome segregation occurs through anaphase A, in which chromosomes move toward stationary spindle poles, anaphase B, in which chromosomes move at the same velocity as outwardly moving spindle poles, or both. In contrast, Caenorhabditis elegans female meiotic spindles initially shorten in the pole-to-pole axis such that spindle poles contact the outer kinetochore before the start of anaphase chromosome separation. Once the spindle pole-to-kinetochore contact has been made, the homologues of a 4-μm-long bivalent begin to separate. The spindle shortens an additional 0.5 μm until the chromosomes are embedded in the spindle poles. Chromosomes then separate at the same velocity as the spindle poles in an anaphase B–like movement. We conclude that the majority of meiotic chromosome movement is caused by shortening of the spindle to bring poles in contact with the chromosomes, followed by separation of chromosome-bound poles by outward sliding.

Equal partitioning of the duplicated chromosomes into two daughter cells during cell division is a coordinated process and is initiated only after completion of DNA synthesis. However, this strict order of execution breaks down in CDC6-deficient cells. Cdc6, an evolutionarily conserved protein, is required for the assembly of pre-replicative complexes (pre-RCs) and is essential for the initiation of DNA replication. Yeast cells lacking Cdc6 function, though unable to initiate DNA replication, proceed to undergo "reductional anaphase" by partitioning the unreplicated chromosomes and lose viability rapidly. This extreme form of genomic instability in cdc6 cells is thought to be due to inactivation of a pre-RC based, Cdc6-dependent checkpoint mechanism that, during normal cell cycle, inhibits premature onset of mitosis until pre-RC is assembled. Here, we show that chromosome segregation in cdc6 mutant is caused not by precocious initiation of mitosis in the absence of a checkpoint, but by the deregulation of spindle dynamics induced via a regulatory network involving the ubiquitin-conjugating enzyme Cdc34, microtubule-associated proteins (MAPs) and the anaphase-promoting complex (APC) activator Cdh1. This regulatory circuit governs spindle behavior in the early part of the division cycle and precipitates catastrophic chromosome segregation in the absence of DNA replication.

Repo-Man and Sds22 counteract Aurora B phosphorylation of Dsn1 and thus regulate the kinetochore–microtubule interface during anaphase., During mitotic spindle assembly, Aurora B kinase is part of an error correction mechanism that detaches microtubules from kinetochores that are under low mechanical tension. During anaphase, however, kinetochore–microtubule attachments must be maintained despite a drop of tension after removal of sister chromatid cohesion. Consistent with this requirement, Aurora B relocates away from chromosomes to the central spindle at the metaphase–anaphase transition. By ribonucleic acid interference screening using a phosphorylation biosensor, we identified two PP1-targeting subunits, Sds22 and Repo-Man, which counteracted Aurora B–dependent phosphorylation of the outer kinetochore component Dsn1 during anaphase. Sds22 or Repo-Man depletion induced transient pauses during poleward chromosome movement and a high incidence of chromosome missegregation. Thus, our study identifies PP1-targeting subunits that regulate the microtubule–kinetochore interface during anaphase for faithful chromosome segregation.

SummarySegregation of chromosomes at the time of cell division is achieved by the microtubules and associated molecules of the spindle. Chromosomes attach to kinetochore microtubules (kMTs), which extend from the spindle pole region to kinetochores assembled upon centromeric DNA. In most animal cells studied, chromosome segregation occurs as a result of kMT shortening, which causes chromosomes to move toward the spindle poles (anaphase A). Anaphase A is typically followed by a spindle elongation that further separates the chromosomes (anaphase B) [1–5]. The experiments presented here provide the first detailed analysis of anaphase in a live vertebrate oocyte and show that chromosome segregation is initially driven by a significant spindle elongation (anaphase B), which is followed by a shortening of kMTs to fully segregate the chromosomes (anaphase A). Loss of tension across kMTs at anaphase onset produces a force imbalance, allowing the bipolar motor kinesin-5 to drive early anaphase B spindle elongation and chromosome segregation. Early anaphase B spindle elongation determines the extent of chromosome segregation and the size of the resulting cells. The vertebrate egg therefore employs a novel mode of anaphase wherein spindle elongation caused by loss of k-fiber tension is harnessed to kick-start chromosome segregation prior to anaphase A.

Nsk1 is a novel fission yeast protein that binds the nucleolus during interphase and the nucleoplasm during early mitosis. After anaphase and following dephosphorylation by Clp1, Nsk1 binds the kinetochore–spindle pole junction and maintains accurate chromosome segregation by promoting the association of kinetochores to spindle poles during anaphase B., Type 1 phosphatase (PP1) antagonizes Aurora B kinase to stabilize kinetochore–microtubule attachments and to silence the spindle checkpoint. We screened for factors that exacerbate the growth defect of Δdis2 cells, which lack one of two catalytic subunits of PP1 in fission yeast, and identified Nsk1, a novel protein required for accurate chromosome segregation. During interphase, Nsk1 resides in the nucleolus but spreads throughout the nucleoplasm as cells enter mitosis. Following dephosphorylation by Clp1 (Cdc14-like) phosphatase and at least one other phosphatase, Nsk1 localizes to the interface between kinetochores and the inner face of the spindle pole body during anaphase. In the absence of Nsk1, some kinetochores become detached from spindle poles during anaphase B. If this occurs late in anaphase B, then the sister chromatids of unclustered kinetochores segregate to the correct daughter cell. These unclustered kinetochores are efficiently captured, retrieved, bioriented, and segregated during the following mitosis, as long as Dis2 is present. However, if kinetochores are detached from a spindle pole early in anaphase B, then these sister chromatids become missegregated. These data suggest Nsk1 ensures accurate chromosome segregation by promoting the tethering of kinetochores to spindle poles during anaphase B.

Drosophila melanogaster RecQ5, a member of the RecQ family, is expressed in early embryos. The loss of maternally-derived RecQ5 leads to spontaneous mitotic defects in syncytial embryos. We demonstrate that the mitotic defects are derived from anaphase DNA bridges. Pairs of daughter nuclei that had been linked by the bridges concurrently exited from the cycle and were eliminated by Chk2-dependent centrosome inactivation. These results suggest that the lack of RecQ5 leads to spontaneous double-stranded DNA breaks (DSBs). RecQ5 may function in the resolution of anaphase DNA bridges during mitosis or in DSB repair during interphase in syncytial Drosophila embryos.

The dynactin complex is required for activation of the dynein motor complex, which plays a critical role in various cell functions including mitosis. During metaphase, the dynein-dynactin complex removes spindle checkpoint proteins from kinetochores to facilitate the transition to anaphase. Three components (p150(Glued), dynamitin, and p24) compose a key portion of the dynactin complex, termed the projecting arm. To investigate the roles of the dynactin complex in mitosis, we used RNA interference to down-regulate p24 and p150(Glued) in human cells. In response to p24 down-regulation, we observed cells with delayed metaphase in which chromosomes frequently align abnormally to resemble a "figure eight," resulting in cell death. We attribute the figure eight chromosome alignment to impaired metaphasic centrosomes that lack spindle tension. Like p24, RNA interference of p150(Glued) also induces prometaphase and metaphase delays; however, most of these cells eventually enter anaphase and complete mitosis. Our findings suggest that although both p24 and p150(Glued) components of the dynactin complex contribute to mitotic progression, p24 also appears to play a role in metaphase centrosome integrity, helping to ensure the transition to anaphase.

The self-organized assembly of acentrosomal meiotic spindles has been extensively studied1 but little is known about how chromosomes segregate on these spindles. Here, we investigate two chromosome-microtubule interaction mechanisms—kinetochores and chromokinesins—during meiosis in fertilized C. elegans oocytes. We show that the conserved kinetochore protein KNL-1 directs assembly of meiotic kinetochores that orient chromosomes on the acentrosomal spindles. However, in contrast to mitosis, chromosome separation during meiotic anaphase was kinetochore-independent. The chromokinesin KLP-19 did not contribute to chromosome orientation or anaphase, but stabilized late anaphase spindles. Prior to anaphase separation, meiotic kinetochores and spindle poles disassembled along with microtubules on the poleward side of the chromosomes; during anaphase, microtubules were formed between the separating chromosomes. Functional analysis implicated a set of proteins that localize to a ring-shaped domain between the kinetochores in pre-anaphase spindle assembly and anaphase separation. Ring domain proteins are localized by the chromosomal passenger complex (CPC), whose local enrichment is patterned by recombination to control step-wise loss of meiotic cohesion2–4. Thus, meiotic segregation in C. elegans is a two-stage process where kinetochores orient chromosomes but are dispensable for their separation. We suggest that separation is instead controlled by a meiosis-specific chromosomal domain to coordinate step-wise dissolution of cohesion with chromosome segregation.

Summary Sister chromatid separation is initiated at anaphase onset by the activation of separase, which removes cohesins from chromosomes. However, it remains elusive how sister chromatid separation is completed along the entire chromosome length. Here we found that, during early anaphase in Saccharomyces cerevisiae, sister chromatids separate gradually from centromeres to telomeres, accompanied by regional chromosome stretching and subsequent recoiling. The stretching results from residual cohesion between sister chromatids, which prevents their immediate separation. This residual cohesion is at least partly dependent on cohesins that have escaped removal by separase at anaphase onset. Meanwhile, recoiling of a stretched chromosome region requires condensins and generates forces to remove residual cohesion. We provide evidence that condensins promote chromosome recoiling directly in vivo, which is distinct from their known function in resolving sister chromatids. Our work identifies residual sister chromatid cohesion during early anaphase and reveals condensins' roles in chromosome recoiling, which eliminates residual cohesion to complete sister chromatid separation., Graphical Abstract Highlights ► Sister chromatid cohesion partially persists after anaphase onset in budding yeast ► Residual cohesion is partly dependent on cohesins and causes chromosome stretching ► Condensins directly promote recoiling of stretched chromosomes during early anaphase ► Chromosome recoiling generates forces to remove residual sister chromatid cohesion

The synchronous movement of chromosomes during anaphase ensures their correct inheritance in every cell division. This reflects the uniformity of spindle forces acting on chromosomes and their simultaneous entry into anaphase. Although anaphase onset is controlled by the spindle assembly checkpoint, it remains unknown how spindle forces are uniformly distributed among different chromosomes. In this paper, we show that tension uniformity at metaphase kinetochores and subsequent anaphase synchrony in Drosophila S2 cells are promoted by spindle microtubule flux. These results can be explained by a mechanical model of the spindle where microtubule poleward translocation events associated with flux reflect relaxation of the kinetochore–microtubule interface, which accounts for the redistribution and convergence of kinetochore tensions in a timescale comparable to typical metaphase duration. As predicted by the model, experimental acceleration of mitosis precludes tension equalization and anaphase synchrony. We propose that flux-dependent equalization of kinetochore tensions ensures a timely and uniform maturation of kinetochore–microtubule interfaces necessary for error-free and coordinated segregation of chromosomes in anaphase.

We examined spindle elongation in anaphase in Saccharomyces cerevisiae cells mutated for the kinesin-5 motor proteins Cin8 and Kip1. Cells were deleted for KIP1 and/or expressed one of two motor-domain Cin8 mutants (Cin8-F467A or Cin8-R196K, which differ in their ability to bind microtubules in vitro, with Cin8-F467A having the weakest ability). We found that, in kinesin-5-mutated cells, predominantly in kip1 Δ cin8-F467A cells, anaphase spindle elongation was frequently interrupted after the fast phase, resulting in a mid-anaphase pause. Expression of kinesin-5 mutants also caused an asymmetric midzone location and enlarged midzone size, suggesting that proper organization of the midzone is required for continuous spindle elongation. We also examined the effects of components of the FEAR pathway, which is involved in the early-anaphase activation of Cdc14 regulatory phosphatase, on anaphase spindle elongation in kip1 Δ cin8-F467A cells. Deletion of SLK19 , but not SPO12 , eliminated the mid-anaphase pause, caused premature anaphase onset and defects in DNA division during anaphase, and reduced viability in these cells. Finally, overriding of the pre-anaphase checkpoint by overexpression of Cdc20 also eliminated the mid-anaphase pause and caused DNA deformation during anaphase in kip1 Δ cin8 - F467A cells. We propose that transient activation of the pre-anaphase checkpoint in kinesin-5-mutated cells induces a Slk19-dependent mid-anaphase pause, which might be important for proper DNA segregation.

During mitosis, the spindle assembly checkpoint (SAC) inhibits the Cdc20-activated anaphase-promoting complex/cyclosome (APC/CCdc20), which promotes protein degradation, and delays anaphase onset to ensure accurate chromosome segregation. However, the SAC function in meiotic anaphase regulation is poorly understood. Here, we examined the SAC function in fission yeast meiosis. As in mitosis, a SAC factor, Mad2, delayed anaphase onset via Slp1 (fission yeast Cdc20) when chromosomes attach to the spindle improperly. However, when the SAC delayed anaphase I, the interval between meiosis I and II shortened. Furthermore, anaphase onset was advanced and the SAC effect was reduced at meiosis II. The advancement of anaphase onset depended on a meiosis-specific, Cdc20-related factor, Fzr1/Mfr1, which contributed to anaphase cyclin decline and anaphase onset and was inefficiently inhibited by the SAC. Our findings show that impacts of SAC activation are not confined to a single division at meiosis due to meiosis-specific APC/C regulation, which has probably been evolved for execution of two meiotic divisions.

In budding yeast, the mitotic spindle is comprised of 32 kinetochore microtubules (kMTs) and ∼8 interpolar MTs (ipMTs). Upon anaphase onset, kMTs shorten to the pole, whereas ipMTs increase in length. Overlapping MTs are responsible for the maintenance of spindle integrity during anaphase. To dissect the requirements for anaphase spindle stability, we introduced a conditionally functional dicentric chromosome into yeast. When centromeres from the same sister chromatid attach to opposite poles, anaphase spindle elongation is delayed and a DNA breakage-fusion-bridge cycle ensues that is dependent on DNA repair proteins. We find that cell survival after dicentric chromosome activation requires the MT-binding proteins Kar3p, Bim1p, and Ase1p. In their absence, anaphase spindles are prone to collapse and buckle in the presence of a dicentric chromosome. Our analysis reveals the importance of Bim1p in maintaining a stable ipMT overlap zone by promoting polymerization of ipMTs during anaphase, whereas Kar3p contributes to spindle stability by cross-linking spindle MTs.

PICH (Plk1-interacting checkpoint helicase) was recently identified as an essential component of the spindle assembly checkpoint and shown to localize to kinetochores, inner centromeres, and thin threads connecting separating chromosomes even during anaphase. In this paper, we have used immuno-fiber fluorescence in situ hybridization and chromatin-immunoprecipitation to demonstrate that PICH associates with centromeric chromatin during anaphase. Furthermore, by careful analysis of PICH-positive anaphase threads through FISH as well as bromo-deoxyurdine and CREST labeling, we strengthen the evidence that these threads comprise mainly alphoid centromere deoxyribonucleic acid. Finally, by timing the addition of ICRF-193 (a specific inhibitor of topoisomerase-II alpha) to cells synchronized in anaphase, we demonstrate that topoisomerase activity is required specifically to resolve PICH-positive threads during anaphase (as opposed to being required to prevent the formation of such threads during earlier cell cycle stages). These data indicate that PICH associates with centromeres during anaphase and that most PICH-positive threads evolve from inner centromeres as these stretch in response to tension. Moreover, they show that topoisomerase activity is required during anaphase for the resolution of PICH-positive threads, implying that the complete separation of sister chromatids occurs later than previously assumed. Electronic supplementary material The online version of this article (doi:10.1007/s00412-007-0131-7) contains supplementary material, which is available to authorized users.

Mutations in BLM cause Bloom's syndrome, a disorder associated with cancer predisposition and chromosomal instability. We investigated whether BLM plays a role in ensuring the faithful chromosome segregation in human cells. We show that BLM-defective cells display a higher frequency of anaphase bridges and lagging chromatin than do isogenic corrected derivatives that eptopically express the BLM protein. In normal cells undergoing mitosis, BLM protein localizes to anaphase bridges, where it colocalizes with its cellular partners, topoisomerase IIIalpha and hRMI1 (BLAP75). Using BLM staining as a marker, we have identified a class of ultrafine DNA bridges in anaphase that are surprisingly prevalent in the anaphase population of normal human cells. These so-called BLM-DNA bridges, which also stain for the PICH protein, frequently link centromeric loci, and are present at an elevated frequency in cells lacking BLM. On the basis of these results, we propose that sister-chromatid disjunction is often incomplete in human cells even after the onset of anaphase. We present a model for the action of BLM in ensuring complete sister chromatid decatenation in anaphase.

Maloriented chromosomes can evade the spindle assembly checkpoint and generate aneuploidy, a common feature of tumorigenesis. But chromosome missegregation in non-transformed cells triggers a p53-dependent fail-safe mechanism that blocks proliferation of normal cells that inadvertently become aneuploid. How this fail-safe is triggered is not known. Here we identify a conserved feedback mechanism that monitors missegregating chromosomes during anaphase through the differential phosphorylation of histone H3.3 at Ser31. We do this by inducing transient chromosome missegregation in diploid cells. During anaphase, H3.3 Ser31 is phosphorylated along the arms of lagging or misaligned chromosomes. Within minutes, Ser31 phosphorylation (Ser31P) spreads to all of the chromatids of both daughter cells, which persists into G1. Masking H3.3 Ser31P by antibody microinjection prevents nuclear p53 accumulation in the aneuploid daughters. Previous work demonstrated that prolonged prometaphase and DNA damage during abnormal mitosis can activate p53. We show that p53 activation in response to chromosome missegregation can occur without prolonged mitosis or DNA damage. Our study provides insight into how aneuploidy caused by chromosome missegregation is normally monitored and suppressed.

Aneuploidy is a broad term used to describe gross chromosomal imbalance in an organism. For the sake of this chapter, only aneuploidy in an embryo will be considered. Aneuploidy typically presents as either an additional chromosome (e.g., trisomy) or a missing chromosome (e.g., monosomy). Such abnormalities arise during cell division (either meiosis or mitosis) when chromosomes fail to separate equally between the two new daughter cells [1]. Aneuploidy may be present in all cells of the embryo (uniform aneuploidy) or be confined to a subpopulation of the cells (mosaicism). Aneuploidy in embryos has varied effects during reproduction, from early embryonic arrest and lack of implantation, pregnancy loss (spontaneous abortion) with trisomy 16 being the most common chromosome abnormality seen in products of conception (POC), to live born trisomic births with varying phenotypic abnormalities, the best known being Down Syndrome (trisomy 21). Aneuploidy originates during the meiotic divisions (principally in the ovary) and the early cleavage divisions (mitotic) of the preimplantation embryo. Nondisjunction, precocious separation of sister chromatids, and anaphase lag are thought to be the most common causes of aneuploidy during gamete formation and embryogenesis [1]. The impact of aneuploidy in families can be devastating, with patients being faced with the potential of pregnancy losses, stillbirths, and/or a severely affected child. In all cases, aneuploid embryos result in an unfavorable outcome for the family in question and are a major contributing factor to the relatively low fecundity of humans when compared with other species.

The reversible condensation of chromosomes during cell division remains a classic problem in cell biology. Condensation requires the condensin complex in certain experimental systems, but not in many others. Anaphase chromosome segregation almost always fails in condensin-depleted cells, leading to the formation of prominent chromatin bridges and cytokinesis failure. Here, live-cell analysis of chicken DT40 cells bearing a conditional knockout of condensin subunit SMC2 revealed that condensin-depleted chromosomes abruptly lose their compact architecture during anaphase and form massive chromatin bridges. The compact chromosome structure can be preserved and anaphase chromosome segregation rescued by preventing the targeting subunit Repo-Man from recruiting protein phosphatase 1 (PP1) to chromatin at anaphase onset. This study identifies an activity critical for mitotic chromosome structure that is inactivated by Repo-Man-PP1 during anaphase. This activity, provisionally termed 'regulator of chromosome architecture' (RCA), cooperates with condensin to preserve the characteristic chromosome architecture during mitosis.

It is well established that DNA damage induces checkpoint-mediated interphase arrest in higher eukaryotes, but recent studies demonstrate that DNA damage delays entry into anaphase as well. Damaged DNA in syncytial and gastrulating Drosophila embryos delays the metaphase/anaphase transition [1–6]. In human cultured cells, DNA damage also induces a delay in mitosis [7–9]. However, the mechanism by which DNA damage delays the anaphase onset is controversial. Some studies implicate a DNA damage checkpoint [6, 7, 10], whereas other studies invoke a spindle checkpoint [8]. To resolve this issue, we compared the effects of random DNA breaks induced by X-irradiation to site-specific I-CreI endonuclease-induced chromosome breaks on cell-cycle progression in wild-type and checkpoint-defective Drosophila neuroblasts. We found that both the BubR1 spindle checkpoint pathway and the Grp/Chk1 DNA damage checkpoint pathway are involved in delaying the metaphase/anaphase transition after extensive X-irradiation-induced DNA damage, whereas Grp/Chk1, but not BubR1, is required to delay anaphase onset in the presence of I-CreI-induced double-strand breaks. On the basis of these results, we propose that DNA damage in nonkinetochore regions produces a Grp/Chk1 DNA-damage-checkpoint-mediated delay in the metaphase/anaphase transition.

Microtubules of the mitotic spindle form the structural basis for chromosome segregation. In metaphase, microtubules show high dynamic instability, which is thought to aid the 'search and capture' of chromosomes for bipolar alignment on the spindle. Microtubules suddenly become more stable at the onset of anaphase, but how this change in microtubule behaviour is regulated and how important it is for the ensuing chromosome segregation are unknown. Here we show that in the budding yeast Saccharomyces cerevisiae, activation of the phosphatase Cdc14 at anaphase onset is both necessary and sufficient for silencing microtubule dynamics. Cdc14 is activated by separase, the protease that triggers sister chromatid separation, linking the onset of anaphase to microtubule stabilization. If sister chromatids separate in the absence of Cdc14 activity, microtubules maintain high dynamic instability; this correlates with defects in both the movement of chromosomes to the spindle poles (anaphase A) and the elongation of the anaphase spindle (anaphase B). Cdc14 promotes localization of microtubule-stabilizing proteins to the anaphase spindle, and dephosphorylation of the kinetochore component Ask1 contributes to both the silencing of microtubule turnover and successful anaphase A.

The faithful segregation of chromosomes during meiosis is vital for sexual reproduction. Currently, little is known about the molecular mechanisms regulating the initiation and completion of meiotic anaphase. We show that inactivation of CUL-2, a member of the cullin family of ubiquitin ligases,delays or abolishes meiotic anaphase II with no effect on anaphase I,indicating differential regulation during the two meiotic stages. In cul-2 mutants, the cohesin REC-8 is removed from chromosomes normally during meiosis II and sister chromatids separate, suggesting that the failure to complete anaphase results from a defect in chromosome movement rather than from a failure to sever chromosome attachments. CUL-2 is required for the degradation of cyclin B1 in meiosis and inactivation of cyclin B1 partially rescued the meiotic delay in cul-2 mutants. In cul-2mutants, the failure to degrade cyclin B1 precedes the metaphase II arrest. CUL-2 is also required for at least two aspects of embryonic polarity. The extended meiosis II in cul-2 mutants induces polarity reversals that include reversed orientation of polarity proteins, P granules, pronuclei migration and asymmetric cell division. Independently of its role in meiotic progression, CUL-2 is required to limit the initiation/spread of the polarity protein PAR-2 in regions distant from microtubule organizing centers. Finally,we show that inactivation of the leucine-rich repeat protein ZYG-11 produces meiotic and polarity reversal defects similar to those observed in cul-2 mutants, suggesting that the two proteins function in the same pathways.

In budding yeast, cells lacking separase function exit mitosis with an undivided nucleus localized to the daughter cell. Here we show that the inability to separate sister chromatids per se is not sufficient to cause the daughter preference. Rather, separase affects nuclear positioning as part of the Cdc14 early anaphase release (FEAR) pathway. The role of the FEAR pathway in nuclear positioning is exerted during anaphase and is not shared by the mitotic exit network. We find that the nuclear segregation defect in FEAR mutants does not stem from nonfunctional spindle poles or the absence of cytoplasmic microtubules. Instead, the concomitant inactivation of sister chromatid separation and the FEAR pathway uncovered a mother-directed force in anaphase that was previously masked by the elongating spindle. We propose that at anaphase onset, the FEAR pathway activates cytoplasmic microtubule-associated forces that facilitate chromosome segregation to the mother cell.

The effect of the mastv40 mutation was studied using neural ganglion cells of third-instar larvae of Drosophila melanogaster. The distributions of the cells by the interphase nucleus diameter and by the distance between the sister chromosome sets in anaphase were analyzed. Three following types of defects induced by the mutation were described: (1) Monopolar mitosis or, in the case of bipolar mitosis, an abnormally short distance between the sister chromosome sets in anaphase and early telophase. We suppose that these abnormalities are caused by damage of the start and (or) motor mechanisms of centrosome separation at the beginning and in the end of mitosis. (2) Lagging and bridging of chromosomes in anaphase and early telophase. These defects seem to be related to the disruption of functioning of mitotic spindle microtubules and (or) their defective attachment to the appropriate kinetochores. (3) Unlimited division of aneuploid and polyploid cells, which may be explained either by inactivation of the checkpoint system controlling the genome ploidy or by checkpoint adaptation. Taken collectively, our results and literature data suggest that the MAST protein is an element of the checkpoint system and that division of aneuploid and polyploid cells results from inactivation of the checkpoints.

Disjunction of maternal and paternal centromeres during meiosis I requires crossing over between homologous chromatids, which creates chiasmata that hold homologs together. It also depends on a mechanism ensuring that maternal and paternal sister kinetochore pairs attach to oppositely oriented microtubules. Proteolytic cleavage of cohesin's Rec8 subunit by separase destroys cohesion between sister chromatid arms at anaphase I and thereby resolves chiasmata. The Spo12 and Slk19 proteins have been implicated in regulating meiosis I kinetochore orientation and/or in preventing cleavage of Rec8 at centromeres. We show here that the role of these proteins is instead to promote nucleolar segregation, including release of the Cdc14 phosphatase required for Cdk1 inactivation and disassembly of the anaphase I spindle. Separase is also required but surprisingly not its protease activity. It has two mechanistically different roles during meiosis I. Loss of the protease-independent function alone results in a second meiotic division occurring on anaphase I spindles in spo12Δ and slk19Δ mutants.

The effect of mutation aarV158 on anaphase separation of chromatids was studied on fixed cells of neural ganglia of Drosophila melanogaster larvae. It was shown that mutation aarV158 causes three types of defective chromosome segregation manifested as (1) monopolar anaphase, (2) separation of chromatids to an abnormally short distance in anaphase, and (3) bridging and lagging of some chromatids or prolonged asynchronous separation of sister chromatid sets to the poles in anaphase. We believe that the former two types of defective segregation are caused by disturbed centrosome separation at the beginning of mitosis and the third type, by defects in chromatid separation during anaphase. During the two-year maintenance of the mutation in a heterozygous state, partial correction (adaptive modification) of the defects of type 1 and type 2 (but not type 3) occurred. The correction of type 1 and type 2 defects during adaptogenesis depended on the genotype: in heterozygotes and homozygotes, respectively type 1 and type 2 were preferentially corrected. The frequency of type 3 defects remained constant during the two-year period of maintenance of the mutation in a heterozygous state. However, in all variants of the experiment, their frequency decreased with increasing distance between the sister chromatid sets. In the cells that completed the previous division with abnormalities, the checkpoint system is supposed to effectively arrest the cell cycle in the subsequent division.

Objectives Constitutional aneuploidy occurs in at least 5% of recognised pregnancies, with apparent preferential involvement of the X chromosome and the smaller autosomes. Molecular cytogenetic investigations of cleavage-stage embryos have revealed anomalies affecting all sizes of chromosomes. The aim was to investigate the variety of anomalies arising during maternal meiosis I by analysis of unfertilised oocytes and polar bodies to gain insight into aneuploidy mechanisms. Methods Sequential FISH analysis was carried out with specific probes derived from eight chromosomes, representing all sizes. Only imbalance due to a gain of a whole chromosome or chromatid, represented by extra signals, was counted to avoid artefact. Results Data were obtained on 236 eggs from 124 patients of average age 32.5 years (range 22–44). Ten patients (average 32.6 years) had abnormal eggs. The abnormality rate for oocytes and for polar bodies was close to 4% for each. Fourteen hyperploidies were found, seven involving additional single chromatids. The abnormalities affected chromosomes 13,16,18, 21 and X but not chromosomes 1, 9 or 12. Conclusion The data provide evidence for several mechanisms leading to aneuploidy, including classical non-disjunction of whole univalents; pre-division of chromatids prior to anaphase I, leading to imbalance detected at metaphase II; gonadal mosaicism for a trisomic cell line and preferential involvement of the smaller chromosomes. Monosomy for the large autosomes is not uncommon in cleavage-stage embryos and may additionally arise from anaphase lag preferentially affecting such chromosomes. Copyright © 2003 John Wiley & Sons, Ltd.

Human embryonic aneuploidy can have a meiotic or a mitotic origin. The majority of meiotic chromosome errors arise during oogenesis. Two main aneuploidy-causing mechanisms have been defined: the first involves the nondisjunction of entire chromosomes and takes place during both meiotic divisions, whereas the second involves the premature division of a chromosome into its two sister chromatids during meiosis I, followed by their random segregation. Mitotic aneuploidy can arise as a consequence of problems such as nondisjunction, endoreduplication and anaphase lag and occurs most often during the first three divisions after fertilisation. The cleavage stage of development is characterised by the highest rates of aneuploidy, after which the incidence of cytogenetic abnormality decreases significantly. A large number of oocytes and embryos have been examined in order to define the spectrum of aneuploidies during the first few days of life and to shed light upon their origins. Various classical and molecular cytogenetic methods have been employed for this purpose, and valuable data of biological and clinical relevance have been obtained. Key Concepts: Aneuploidy is the most important genetic cause of human reproductive wastage (i.e. the principal reason for embryo implantation failure and miscarriage). The outcome of assisted reproductive treatments (e.g. in vitro fertilisation (IVF)) and natural reproductive cycles is negatively affected by aneuploidy. Most meiotically derived abnormalities arise during oogenesis. There is a strong relationship between advancing female age and increasing aneuploidy rates in oocytes. Two distinct mechanisms of oocyte chromosome malsegregation have been described, whole chromosome nondisjunction and unbalanced chromatid predivision. Post-zygotic aneuploidy usually arises during the first few mitotic divisions and leads to mosaicism in the embryo. There are three main mechanisms responsible for aneuploidy of mitotic origin: anaphase lag, endoreduplication and mitotic nondisjunction. The cleavage stage of preimplantation development is associated with the highest aneuploidy rates. The frequency of chromosome abnormalities and mosaicism declines as embryos progress to the blastocyst stage, presumably due to loss of abnormal cells or demise of affected embryos. Keywords: oocyte; preimplantation embryo; chromosome; aneuploidy; mosaicism

Sister chromatid separation creates a sudden loss of tension on kinetochores, which could, in principle, re-activate the spindle checkpoint in anaphase. This so-called “anaphase problem” is probably avoided by timely inactivation of cyclin B1-Cdk1, which may prevent the spindle tension sensing Aurora B kinase from destabilizing kinetochore–microtubule interactions as they lose tension in anaphase. However, exactly how spindle checkpoint re-activation is prevented remains unclear. Here, we investigated how different degrees of cyclin B1 stabilization affected the spindle checkpoint in metaphase and anaphase. Cells expressing a strongly stabilized (R42A) mutant of cyclin B1 degraded APC/CCdc20 substrates normally, showing that checkpoint release was not inhibited by high cyclin B1-Cdk1 activity. However, after this initial wave of APC/CCdc20 activity, the spindle checkpoint returned in cells with uncohesed sister chromatids. Expression of a lysine mutant of cyclin B1 that is degraded only slightly inefficiently allowed a normal metaphase-to-anaphase transition. Strikingly, however, the spindle checkpoint returned in cells that had not degraded the cyclin B1 mutant 10–15 min after anaphase onset. When cyclin B1 remained in late anaphase, cytokinesis stalled, and translocation of INCENP from separated sister chromatids to the spindle midzone was blocked. This late anaphase arrest required the activity of Aurora B and Mps1. In conclusion, our results reveal that complete removal of cyclin B1 is essential to prevent the return of the spindle checkpoint following sister chromatid disjunction. Speculatively, increasing activity of APC/CCdc20 in late anaphase helps to keep cyclin B1 levels low.

Upon establishment of proper kinetochore–microtubule attachment, the spindle assembly checkpoint (SAC) must be silenced to allow onset of anaphase, which is when sister chromatids segregate equally to two daughter cells. However, how proper kinetochore–microtubule attachment leads to timely anaphase onset remains elusive. Furthermore, the molecular mechanisms of chromosome movement during anaphase A remain unclear. In this study, we show that the fission yeast Alp7/TACC protein recruits a protein complex consisting of the kinesin-8 (Klp5–Klp6) and protein phosphatase 1 (PP1) to the kinetochore upon kinetochore–microtubule attachment. Accumulation of this complex at the kinetochore, on the one hand, facilitates SAC inactivation through PP1, and, on the other hand, accelerates polewards chromosome movement driven by the Klp5–Klp6 motor. We identified an alp7 mutant that had specific defects in binding to the Klp5–Klp6–PP1 complex but with normal localisation to the microtubule and kinetochore. Consistent with our proposition, this mutant shows delayed anaphase onset and decelerated chromosome movement during anaphase A. We propose that the recruitment of kinesin-8–PP1 to the kinetochore through Alp7/TACC interaction plays a crucial role in regulation of timely mitotic progression and chromosome movement during anaphase A.

In mitotic cells, an error in chromosome segregation occurs when a chromosome is left near the spindle equator after anaphase onset (lagging chromosome). In PtK1 cells, we found 1.16% of untreated anaphase cells exhibiting lagging chromosomes at the spindle equator, and this percentage was enhanced to 17.55% after a mitotic block with 2 μM nocodazole. A lagging chromosome seen during anaphase in control or nocodazole-treated cells was found by confocal immunofluorescence microscopy to be a single chromatid with its kinetochore attached to kinetochore microtubule bundles extending toward opposite poles. This merotelic orientation was verified by electron microscopy. The single kinetochores of lagging chromosomes in anaphase were stretched laterally (1.2–5.6-fold) in the directions of their kinetochore microtubules, indicating that they were not able to achieve anaphase poleward movement because of pulling forces toward opposite poles. They also had inactivated mitotic spindle checkpoint activities since they did not label with either Mad2 or 3F3/2 antibodies. Thus, for mammalian cultured cells, kinetochore merotelic orientation is a major mechanism of aneuploidy not detected by the mitotic spindle checkpoint. The expanded and curved crescent morphology exhibited by kinetochores during nocodazole treatment may promote the high incidence of kinetochore merotelic orientation that occurs after nocodazole washout.

The typical behavior of chromosomes in meiosis is that homologous pairs synapse, recombine, and then separate at anaphase I. At anaphase II, sister chromatids separate. However, studies of small chromosomes in maize derived from a variety of sources typically have failure of sister chromatid cohesion at anaphase I. This failure occurs whether there is pairing of two copies of a minichromosome or not. These characteristics have implications for managing the transmission of the first generation artificial chromosomes in plants. Procedures to address these issues of minichromosomes are discussed.

In yeast, anaphase depends on cohesin cleavage. How anaphase is controlled in vertebrates is unknown because their cohesins dissociate from chromosomes before anaphase. We show that residual amounts of the cohesin SCC1 remain associated with human centromeres until the onset of anaphase when a similarly small amount of SCC1 is cleaved. In Xenopus extracts, SCC1 cleavage depends on the anaphase-promoting complex and separin. Separin immunoprecipitates are sufficient to cleave SCC1, indicating that separin is associated with a protease activity. Separin activation coincides with securin destruction and partial separin cleavage, suggesting that several mechanisms regulate separin activity. We propose that in vertebrates, a cleavage-independent pathway removes cohesin from chromosome arms during prophase, whereas a separin-dependent pathway cleaves centromeric cohesin at the metaphase–anaphase transition.

Sister chromatid cohesion is established during DNA replication and depends on a multiprotein complex called cohesin. At the onset of anaphase the cohesive structures that hold sisters together must be destroyed to allow segregation of sisters. In the budding yeast Saccharomyces cerevisiae loss of sister chromatid cohesion depends on a separating protein (separin) called Esp1. At the metaphase to anaphase transition, separin is activated by proteolysis of its inhibitory subunit (securin) called Pds1. This process is mediated by the anaphase promoting complex and an accessory protein Cdc20. In meiosis a single round of DNA replication is followed by two successive rounds of segregation. Thus loss of cohesion is spun out over two divisions. By studying the mechanisms that initiate anaphase in meiotic division we show that the yeast securin Pds1p is present in meiotic nuclei and is destroyed at the onset of each meiotic division. We also show that securin destruction depends on Cdc20p which accumulates within nuclei around the time of Pds1p's disappearance.

The correct transmission of chromosomes from mother to daughter cells is fundamental for genetic inheritance. Separation and segregation of sister chromatids in growing cells occurs in the cell cycle stage called 'anaphase'. The basic process of sister chromatid separation is similar in all eukaryotes: many gene products required are conserved. In this review, the roles of two proteins essential for the onset of anaphase in fission yeast, Cut2/securin and Cut1/separin, are discussed with regard to cell cycle regulation, and compared with the postulated roles of homologous proteins in other organisms. Securin, like mitotic cyclins, is the target of the anaphase promoting complex (APC)/cyclosome and is polyubiquitinated before destruction in a manner dependent upon the destruction sequence. The anaphase never occurs properly in the absence of securin destruction. In human cells, securin is an oncogene. Separin is a large protein (MW approximately 180 kDa), the C-terminus of which is conserved, and is thought to be inhibited by association with securin at the nonconserved N-terminus. In the budding yeast, Esp1/separin is thought to be a component of proteolysis against Scc1, an essential subunit of cohesin which is thought to link duplicated sister chromatids up to the anaphase. Whether fission yeast Cut1/separin is also implicated in proteolysis of cohesin is discussed.

SummaryAt anaphase onset the mitotic spindle undergoes dramatic changes in order to segregate sister chromatids. Surprisingly, the monopolin complex, best known for its role at kinetochores in meiosis, is now shown to localize to, and stabilize, the mitotic anaphase spindle.