Correct spindle elongation at the metaphase/anaphase transition is an APC-dependent event in budding yeast.

At the metaphase to anaphase transition, chromosome segregation is initiated by the splitting of sister chromatids. Subsequently, spindles elongate, separating the sister chromosomes into two sets. Here, we investigate the cell cycle requirements for spindle elongation in budding yeast using mutants affecting sister chromatid cohesion or DNA replication. We show that separation of sister chromatids is not sufficient for proper spindle integrity during elongation. Rather, successful spindle elongation and stability require both sister chromatid separation and anaphase-promoting complex activation. Spindle integrity during elongation is dependent on proteolysis of the securin Pds1 but not on the activity of the separase Esp1. Our data suggest that stabilization of the elongating spindle at the metaphase to anaphase transition involves Pds1-dependent targets other than Esp1.     

Extracentromeric connections between sister chromatids demonstrated in human chromosomes induced to condense asymmetrically

Summary Extracentromeric chromatin fibers were proposed to hold sister chromatids together in mitotic chromosomes examined by electron microscopy, but their existence in living cells has not been demonstrated yet. We have performed an in vitro BrdU-H33258 treatment which induced a differential rate of condensation to each sister chromatid, thus producing asymmetrically condensing chromosomes. The fast condensing chromatid pulled the slower sister one, both bending in parallel. Bent chromatids appeared reciprocally connected by loops of chromatin fibers, suggesting they were the links which permitted the physical interplay between the differently condensing chromatids. When sister chromatid exchanges (SCE) intercalated a fast-condensing fragment in the slow-condensing chromatid or vice versa, the chromosome inverted its curvature at the SCE-point.     

Cohesin ensures bipolar attachment of microtubules to sister centromeres and resists their precocious separation

The multisubunit protein complex cohesin is required to establish cohesion between sister chromatids during S phase and to maintain it during G2 and M phases. Cohesin is essential for mitosis, and even partial defects cause very high rates of chromosome loss. In budding yeast, cohesin associates with specific sites which are distributed along the entire length of a chromosome but are more dense in the vicinity of the centromere. Real-time imaging of individual centromeres tagged with green fluorescent protein suggests that cohesin bound to centromeres is important for bipolar attachment to microtubules. This cohesin is, however, incapable of resisting the consequent force, which leads to sister centromere splitting and chromosome stretching. Meanwhile, cohesin bound to sequences flanking the centromeres prevents sister chromatids from completely unzipping and is required to pull back together sister centromeres that have already split. Cohesin therefore has a central role in generating a dynamic tension between microtubules and sister chromatid cohesion at centromeres, which lasts until chromosome segregation is finally promoted by separin-dependent cleavage of the cohesin subunit Scc1p.     

Mouse satellite DNA, centromere structure, and sister chromatid pairing

The experiments described were directed toward understanding relationships between mouse satellite DNA, sister chromatid pairing, and centromere function. Electron microscopy of a large mouse L929 marker chromosome shows that each of its multiple constrictions is coincident with a site of sister chromatid contact and the presence of mouse satellite DNA. However, only one of these sites, the central one, possesses kinetochores. This observation suggests either that satellite DNA alone is not sufficient for kinetochore formation or that when one kinetochore forms, other potential sites are suppressed. In the second set of experiments, we show that highly extended chromosomes from Hoechst 33258-treated cells (Hilwig, I., and A. Gropp, 1973, Exp. Cell Res., 81:474-477) lack kinetochores. Kinetochores are not seen in Miller spreads of these chromosomes, and at least one kinetochore antigen is not associated with these chromosomes when they were subjected to immunofluorescent analysis using anti-kinetochore scleroderma serum. These data suggest that kinetochore formation at centromeric heterochromatin may require a higher order chromatin structure which is altered by Hoechst binding. Finally, when metaphase chromosomes are subjected to digestion by restriction enzymes that degrade the bulk of mouse satellite DNA, contact between sister chromatids appears to be disrupted. Electron microscopy of digested chromosomes shows that there is a significant loss of heterochromatin between the sister chromatids at paired sites. In addition, fluorescence microscopy using anti-kinetochore serum reveals a greater inter-kinetochore distance than in controls or chromosomes digested with enzymes that spare satellite. We conclude that the presence of mouse satellite DNA in these regions is necessary for maintenance of contact between the sister chromatids of mouse mitotic chromosomes.     

Epigenetic differences between sister chromatids?

Semi-conservative replication ensures that the DNA sequence of sister chromatids is identical except for replication errors and variation in the length of telomere repeats resulting from replicative losses and variable end processing. What happens with the various epigenetic marks during DNA replication is less clear. Many chromatin marks are likely to be copied onto both sister chromatids in conjunction with DNA replication, whereas others could be distributed randomly between sister chromatids. Epigenetic differences between sister chromatids could also emerge in a more predictable manner, for example, following processes that are associated with lagging strand DNA replication. The resulting epigenetic differences between sister chromatids could result in different gene expression patterns in daughter cells. This possibility has been difficult to test because techniques to distinguish between parental sister chromatids require analysis of single cells and are not obvious. Here, we briefly review the topic of sister chromatid epigenetics and discuss how the identification of sister chromatids in cells could change the way we think about asymmetric cell divisions and stochastic variation in gene expression between cells in general and paired daughter cells in particular.     

Unusual chromosome cleavage dynamic in rodent neonatal germ cells

At the metaphase/anaphase transition in the mouse and rat male germ lines during the perinatal period, sister centromeres separate before sister chromatids. This gives the chromosomes an unusual appearance that resembles the premature centromere division described in some human pathological conditions such as Roberts syndrome. At the same period, there is also an unusual pattern of DNA methylation, with strongly demethylated heterochromatin and methylated euchromatin. This suggests that chromosome DNA methylation may modulate chromatid and centromere splitting, without altering normal chromosome segregation.     

DNA-DEPENDENT FORMATION OF THE SYNAPTINEMAL COMPLEX AT MEIOTIC PROPHASE

Evalaution of microsporocytes cultured during discrete periods of meiotic prophase in the presence of deoxyadenosine, an inhibitor of DNA synthesis, indicate that: (1) late leptonema or early zygonema DNA synthesis is required to initiate the formation of the synaptinemal complex; (2) DNA synthesized during late zygonema is necessary for the disjunction of the paired homologs at diplonema; and (3) DNA synthesis in pachynema is a requisite for normal anaphase II separation of sister chromatids.     

Differential fluorescence of sister chromatids with 4'-6-diamidino-2-phenylindole.

Differential fluorescence of sister chromatids and sister chromatid exchanges (SCE) in chromosomes from human lymphocytes grown two replication cycles in medium containing 5-bromodeoxyuridine can be detected by fluorescence microscopy after staining with 4'-6-diamidino-2-phenylindole (DAPI). The DAPI fluorescence appears to be more stable than that of the dye 33258 Hoechst and may provide a more sensitive method for the detection of SCE.     

Aurora controls sister kinetochore mono-orientation and homolog bi-orientation in meiosis-I

Aurora-B kinases are important regulators of mitotic chromosome segregation, where they are required for the faithful bi-orientation of sister chromatids. In contrast to mitosis, sister chromatids have to be oriented toward the same spindle pole in meiosis-I, while homologous chromosomes are bi-oriented. We find that the fission yeast Aurora kinase Ark1 is required for the faithful bi-orientation of sister chromatids in mitosis and of homologous chromosomes in meiosis-I. Unexpectedly, Ark1 is also necessary for the faithful mono-orientation of sister chromatids in meiosis-I, even though the canonical mono-orientation pathway, which depends on Moa1 and Rec8, seems intact. Our data suggest that Ark1 prevents unified sister kinetochores during metaphase-I from merotelic attachment to both spindle poles and thus from being torn apart during anaphase-I, revealing a novel mechanism promoting monopolar attachment. Furthermore, our results provide an explanation for the previously enigmatic observation that fission yeast Shugoshin Sgo2, which assists in loading Aurora to centromeres, and its regulator Bub1 are required for the mono-orientation of sister chromatids in meiosis-I.     

Kinetochore orientation during meiosis is controlled by Aurora B and the monopolin complex

Kinetochores of sister chromatids attach to microtubules emanating from the same pole (coorientation) during meiosis I and microtubules emanating from opposite poles (biorientation) during meiosis II. We find that the Aurora B kinase Ipl1 regulates kinetochore-microtubule attachment during both meiotic divisions and that a complex known as the monopolin complex ensures that the protein kinase coorients sister chromatids during meiosis I. Furthermore, the defining of conditions sufficient to induce sister kinetochore coorientation during mitosis provides insight into monopolin complex function. The monopolin complex joins sister kinetochores independently of cohesins, the proteins that hold sister chromatids together. We propose that this function of the monopolin complex helps Aurora B coorient sister chromatids during meiosis I.     

The STRUCTURAL MAINTENANCE OF CHROMOSOMES 5/6 Complex Promotes Sister Chromatid Alignment and Homologous Recombination after DNA Damage in Arabidopsis thaliana

Sister chromatids are often arranged as incompletely aligned entities in interphase nuclei of Arabidopsis thaliana. The STRUCTURAL MAINTENANCE OF CHROMOSOMES (SMC) 5/6 complex, together with cohesin, is involved in double-strand break (DSB) repair by sister chromatid recombination in yeasts and mammals. Here, we analyzed the function of genes in Arabidopsis. The wild-type allele of SMC5 is essential for seed development. Each of the two SMC6 homologs of Arabidopsis is required for efficient repair of DNA breakage via intermolecular homologous recombination in somatic cells. Alignment of sister chromatids is enhanced transiently after X-irradiation (and mitomycin C treatment) in wild-type nuclei. In the smc5/6 mutants, the x-ray–mediated increase in sister chromatid alignment is much lower and delayed. The reduced S phase–established cohesion caused by a knockout mutation in one of the α-kleisin genes, SYN1, also perturbed enhancement of sister chromatid alignment after irradiation, suggesting that the S phase–established cohesion is a prerequisite for correct DSB-dependent cohesion. The radiation-sensitive51 mutant, deficient in heteroduplex formation during DSB repair, showed wild-type frequencies of sister chromatid alignment after X-irradiation, implying that the irradiation-mediated increase in sister chromatid alignment is a prerequisite for, rather than a consequence of, DNA strand exchange between sister chromatids. Our results suggest that the SMC5/6 complex promotes sister chromatid cohesion after DNA breakage and facilitates homologous recombination between sister chromatids.     

GFP tagging of budding yeast chromosomes reveals that protein–protein interactions can mediate sister chromatid cohesion

Background Precise control of sister chromatid separation is essential for the accurate transmission of genetic information. Sister chromatids must remain linked to each other from the time of DNA replication until the onset of chromosome segregation, when the linkage must be promptly dissolved. Recent studies suggest that the machinery that is responsible for the destruction of mitotic cyclins also degrades proteins that play a role in maintaining sister chromatid linkage, and that this machinery is regulated by the spindle-assembly checkpoint. Studies on these problems in budding yeast are hampered by the inability to resolve its chromosomes by light or electron microscopy.Results We have developed a novel method for visualizing specific DNA sequences in fixed and living budding yeast cells. A tandem array of 256 copies of the Lac operator is integrated at the desired site in the genome and detected by the binding of a green fluorescent protein (GFP)–Lac repressor fusion expressed from the HIS3 promoter. Using this method, we show that sister chromatid segregation precedes the destruction of cyclin B. In mad or bub cells, which lack the spindle-assembly checkpoint, sister chromatid separation can occur in the absence of microtubules. The expression of a tetramerizing form of the GFP–Lac repressor, which can bind Lac operators on two different DNA molecules, can hold sister chromatids together under conditions in which they would normally separate.Conclusions We conclude that sister chromatid separation in budding yeast can occur in the absence of microtubule-dependent forces, and that protein complexes that can bind two different DNA molecules are capable of holding sister chromatids together.     

分离酶的作用及其调节

姐妹染色单体的分离是一精确时空调控事件,分离的紊乱会造成遗传物质传递的不稳定,从而可能引起严重的后果-细胞或个体的死亡或病态。在真核生物细胞中,一种比较保守的机制调控着姐妹染色单体的分离:随DNA复制过程建立由黏合素维持的姐妹染色单体的结合,在有丝分裂中期向后期转变过程中,随保全素的降解,分离酶发挥活性,裂解黏合素一个亚单位,促成黏合素蛋白质复合体的解离和姐妹染色单体的分离。     

Chromosome segregation: Samurai separation of Siamese sisters.

How do cells ensure that sister chromatids are precisely partitioned in mitosis? New studies on budding yeast have revealed that sister chromatid separation at anaphase requires endoproteolytic cleavage of a protein that maintains the association between sister chromatids.     

Shugoshin1 may play important roles in separation of homologous chromosomes and sister chromatids during mouse oocyte meiosis

Background

Homologous chromosomes separate in meiosis I and sister chromatids separate in meiosis II, generating haploid gametes. To address the question why sister chromatids do not separate in meiosis I, we explored the roles of Shogoshin1 (Sgo1) in chromosome separation during oocyte meiosis.

Methodology/Principal Findings

Sgo1 function was evaluated by exogenous overexpression to enhance its roles and RNAi to suppress its roles during two meioses of mouse oocytes. Immunocytochemistry and chromosome spread were used to evaluate phenotypes. The exogenous Sgo1 overexpression kept homologous chromosomes and sister chromatids not to separate in meiosis I and meiosis II, respectively, while the Sgo1 RNAi promoted premature separation of sister chromatids.

Conclusions

Our results reveal that prevention of premature separation of sister chromatids in meiosis I requires the retention of centromeric Sgo1, while normal separation of sister chromatids in meiosis II requires loss of centromeric Sgo1.     

Micromanipulation of chromosomes reveals that cohesion release during cell division is gradual and does not require tension

In mitosis, cohesion appears to be present along the entire length of the chromosome, between centromeres and along chromosome arms. By metaphase, sister chromatids appear as two adjacent but visibly distinct rods. Sister chromatids separate from one another in anaphase by releasing all chromosome cohesion. This is different from meiosis I, in which pairs of sister chromatids separate from one another, moving to each spindle pole by releasing cohesion only between sister chromatid arms. Then, in anaphase II, sister chromatids separate by releasing centromere cohesion. Our objective was to find where cohesion is present or absent on chromosomes in mitosis and meiosis and when and how it is released. We determined cohesion directly by pulling on chromosomes with two micromanipulation needles. Thus, we could distinguish for the first time between apparent doubleness as seen in the microscope and physical separability. We found that apparent doubleness can be deceiving: Visibly distinct sister chromatids often cannot be separated. We also demonstrated that cohesion is released gradually in anaphase, with chromosomes looking as if they were unzipped or pulled apart. This implied that tension from spindle forces was required, but we showed directly that no tension was necessary to pull chromatids apart.     

Sister chromatid exchanges in Tradescantia paludosa

A modified fluorescence-plus-Giemsa technique is described that allows differential staining of sister chromatids in root tip cells from cuttings of Tradescantia paludosa. With this staining technique, chromatids with both DNA strands unsubstituted are differentiated from chromatids containing 5-bromouracil in place of thymine in one of the strands of the DNA duplex. The baseline level of sister chromatid exchanges was shown to be dependent on the concentration of 5-bromodeoxyuridine in the treatment solution, the mean frequency being 43.5 sister chromatid exchanges per cell for the experimental protocol suggested.     

Random Segregation of Chromatids at Mitosis in Saccharomyces Cerevisiae

Previous experiments suggest that mitotic chromosome segregation in some fungi is a nonrandom process in which chromatids of the same replicative age are destined for cosegregation. We have investigated the pattern of chromatid segregation in Saccharomyces cerevisiae by labeling the DNA of a strain auxotrophic for thymidine with 5-bromodeoxyuridine. The fate of DNA strands was followed qualitatively by immunofluorescence microscopy and quantitatively by microphotometry using an anti-5-bromodeoxyuridine monoclonal antibody. Chromatids of the same replicative age were distributed randomly to daughter cells at mitosis. Quantitative measurements showed that the amount of fluorescence in the daughter nuclei derived from parents with hemilabeled chromosomes diminished in intensity by one half. The concentration of 5-bromodeoxyuridine used in the experiments had little effect on the frequency of either homologous or sister chromatid exchanges. We infer that the 5-bromodeoxyuridine was distributed randomly due to mitotic segregation of chromatids and not via sister chromatid exchanges.     

Slk19p is necessary to prevent separation of sister chromatids in meiosis I

BACKGROUND: A fundamental difference between meiotic and mitotic chromosome segregation is that in meiosis I, sister chromatids remain joined, moving as a unit to one pole of the spindle rather than separating as they do in mitosis. It has long been known that the sustained linkage of sister chromatids through meiotic anaphase I is accomplished by association of the chromatids at the centromere region. The localization of the cohesin Rec8p to the centromeres is essential for maintenance of sister chromatid cohesion through meiosis I, but the molecular basis for the regulation of Rec8p and sister kinetochores in meiosis remains a mystery. RESULTS: We show that the SLK19 gene product from Saccharomyces cerevisiae is essential for proper chromosome segregation during meiosis I. When slk19 mutants were induced to sporulate they completed events characteristic of meiotic prophase I, but at the first meiotic division they segregated their sister chromatids to opposite poles at high frequencies. The vast majority of these cells did not perform a second meiotic division and proceeded to form dyads (asci containing two spores). Slk19p was found to localize to centromere regions of chromosomes during meiotic prophase where it remained until anaphase I. In the absence of Slk19p, Rec8p was not maintained at the centromere region through anaphase I as it is in wild-type cells. Finally, we demonstrate that Slk19p appears to function downstream of the meiosis-specific protein Spo13p in control of sister chromatid behavior during meiosis I. CONCLUSIONS: Our results suggest that Slk19p is essential at the centromere of meiotic chromosomes to prevent the premature separation of sister chromatids at meiosis I.     

Holding chromatids together to ensure they go their separate ways

Association between sister chromatids is essential for their attachment and segregation to opposite poles of the spindle in mitosis and meiosis II. Sister-chromatid cohesion is also likely to be involved in linking homologous chromosomes together in meiosis I. Cytological observations provide evidence that attachment between sister chromatids is different in meiosis and mitosis and suggest that cohesion between the chromatid arms may differ mechanistically from that at the centromere. The physical nature of cohesion is addressed, and proteins that are candidates for holding sister chromatids together are discussed. Dissolution of sister-chromatid cohesion must be regulated precisely, and potential mechanisms to release cohesion are presented.