Anaphase progression and furrow establishment in nocodazole-arrested PtK1 cells

The relationship between progression through anaphase and furrow establishment was investigated in PtK₁ cells using the anti-mitotic agent Nocodazole to arrest cells at different points in anaphase. The capacity of cells to furrow was compared to the kinetochore-kinetochore separation attained at the time of arrest. For the stages of anaphase examined, furrowing capacity increased directly with kinetochore-kinetochore separation until complete furrows were formed after kinetochore-kinetochore separations of 14 m or more were reached. Furrow establishment thus occurs during a definite interval during anaphase in PtK₁ cells. Results from electron microscopy of both Nocodazole-treated and control cells suggest that a population of astral microtubules may be important for furrow establishment.     

Cdk1 Inactivation Terminates Mitotic Checkpoint Surveillance and Stabilizes Kinetochore Attachments in Anaphase

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Intact Cohesion,Anaphase, and Chromosome Segregation in Human Cells Harboring Tumor-Derived Mutations in STAG2

Somatic mutations of the cohesin complex subunit STAG2 are present in diverse tumor types. We and others have shown that STAG2 inactivation can lead to loss of sister chromatid cohesion and alterations in chromosome copy number in experimental systems. However, studies of naturally occurring human tumors have demonstrated little, if any, correlation between STAG2 mutational status and aneuploidy, and have further shown that STAG2-deficient tumors are often euploid. In an effort to provide insight into these discrepancies, here we analyze the effect of tumor-derived STAG2 mutations on the protein composition of cohesin and the expected mitotic phenotypes of STAG2 mutation. We find that many mutant STAG2 proteins retain their ability to interact with cohesin; however, the presence of mutant STAG2 resulted in a reduction in the ability of regulatory subunits WAPL, PDS5A, and PDS5B to interact with the core cohesin ring. Using AAV-mediated gene targeting, we then introduced nine tumor-derived mutations into the endogenous allele of STAG2 in cultured human cells. While all nonsense mutations led to defects in sister chromatid cohesion and a subset induced anaphase defects, missense mutations behaved like wild-type in these assays. Furthermore, only one of nine tumor-derived mutations tested induced overt alterations in chromosome counts. These data indicate that not all tumor-derived STAG2 mutations confer defects in cohesion, chromosome segregation, and ploidy, suggesting that there are likely to be other functional effects of STAG2 inactivation in human cancer cells that are relevant to cancer pathogenesis.     

BuGZ Is Required for Bub3 Stability,Bub1 Kinetochore Function,and Chromosome Alignment

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Shugoshin 1 Plays a Central Role in Kinetochore Assembly and is Required for Kinetochore Targeting of Plk1

Physical connection between the sister chromatids is mediated by the cohesin protein complex. During prophase, cohesin is removed from the chromosome arms while the centromeres remain united. Shugoshin1 (Sgo1) is required for maintenance of centromeric cohesion from prophase to the metaphase-anaphase transition. Furthermore, Sgo1 has been proposed to regulate kinetochore microtubule stability and sense interkinetochore tension, two tasks which are tightly coupled with the function of the Chromosomal Passenger Complex (CPC) and Polo-like kinase 1 (Plk1). Here we show that depletion or chemical inhibition of Aurora B kinase (AurB), the catalytic subunit of the CPC, disrupts accumulation of Sgo1 on the kinetochores in HeLa cells and causes Sgo1 to localize on the chromosome arms. RNAi assays show that depletion of Sgo1 did not affect AurB localization but diminished Plk1 kinetochore binding. Furthermore, we demonstrate that vertebrate Sgo1 is phosphorylated by both AurB and Plk1 in vitro. The data presented here includes an extensive analysis of kinetochore targeting interdependencies of mitotic proteins that propose a novel branch in kinetochore assembly where Sgo1 and Plk1 have central roles. Furthermore our studies implicate Sgo1 in the tension sensing mechanism of the spindle checkpoint by regulating Plk1 kinetochore affinity.     

Histone Deacetylase 2 (HDAC2) Regulates Chromosome Segregation and Kinetochore Function via H4K16 Deacetylation during Oocyte Maturation in Mouse

Changes in histone acetylation occur during oocyte development and maturation, but the role of specific histone deacetylases in these processes is poorly defined. We report here that mice harboring Hdac1 ^−/+/Hdac2 ^−/− or Hdac2 ^−/− oocytes are infertile or sub-fertile, respectively. Depleting maternal HDAC2 results in hyperacetylation of H4K16 as determined by immunocytochemistry—normal deacetylation of other lysine residues of histone H3 or H4 is observed—and defective chromosome condensation and segregation during oocyte maturation occurs in a sub-population of oocytes. The resulting increased incidence of aneuploidy likely accounts for the observed sub-fertility of mice harboring Hdac2 ^−/− oocytes. The infertility of mice harboring Hdac1 ^−/+/Hdac2 ^−/−oocytes is attributed to failure of those few eggs that properly mature to metaphase II to initiate DNA replication following fertilization. The increased amount of acetylated H4K16 likely impairs kinetochore function in oocytes lacking HDAC2 because kinetochores in mutant oocytes are less able to form cold-stable microtubule attachments and less CENP-A is located at the centromere. These results implicate HDAC2 as the major HDAC that regulates global histone acetylation during oocyte development and, furthermore, suggest HDAC2 is largely responsible for the deacetylation of H4K16 during maturation. In addition, the results provide additional support that histone deacetylation that occurs during oocyte maturation is critical for proper chromosome segregation.     

Ionic Mechanisms for Electrical Heterogeneity between Rabbit Purkinje Fiber and Ventricular Cells

The intrinsic heterogeneity of electrical action potential (AP) properties between Purkinje fibers (PFs) and the ventricular wall, as well as within the wall, plays an important role in ensuring successful excitation of the ventricles. It can also be proarrhythmic due to nonuniform repolarization across the Purkinje-ventricular junction. However, the ionic mechanisms that underlie the marked AP differences between PFs and ventricular cells are not fully characterized. We studied such mechanisms by developing a new family of biophysically detailed AP models for rabbit PF cells and three transmural ventricular cell types. The models were based on and validated against experimental data recorded from rabbit at ionic channel, single cell, and tissue levels. They were then used to determine the functional roles of each individual ionic channel current in modulating the AP heterogeneity at the rabbit Purkinje-ventricular junction, and to identify specific currents responsible for the differential response of PFs and ventricular cells to pharmacological interventions.     

秋枫属的染色体数目及其进化意义

对秋枫属两个种Bischofia javanica和B·polycarpa的体细胞进行了染色体计数研究。结果表明这两个种在形态学上虽然存在差异,如秋枫是圆椎花序,重阳木是总状花序,但染色体数目均为2n=196。同时,结合细胞学、形态学和生态学特点探讨了秋枫属的染色体基数,多倍化的起源及其演化意义。     

卵母细胞成熟调控的分子机制及进展

卵母细胞的成熟是人类配子发育成熟,进而形成胚胎的必然阶段。目前已知有多种因素调控卵母细胞的成熟。成熟促进因子(MPF)是卵母细胞成熟调控的最重要分子,它通过CDK1亚基的磷酸化及cyclin B累积合成调节卵母细胞的成熟。MAPK/Mos及cAMP均可通过影响MPF的活性从而间接调控卵母细胞成熟。这三者之间又存在相互影响相互作用,形成一个复杂的调控网络。阐明卵母细胞成熟的分子机制有利于为治疗女性不孕症及卵母细胞体外培养成熟提供可靠的理论依据。     

The Role of Chromosome Architecture in Spindle Assembly and Anaphase: The Condensed Version

No abstract available.     

Problems of Oocyte Maturation and the Control of Chromosome Cycles*

During oocyte maturation and zygote development chromosomes undergo cyclic changes, alternaing the condensed and decondensed states. In oocytes, zygotes and perhaps in other cells, the chromosome cycle appears to be controlled in same way by common cytoplasmic factors. Among them, maturation-promoting factor (MPF) plays a particularly important role, although the germinal vesicle substances and cytoplasmic membrane vesicles are indispensable for the chromosomal changes. MPF precursor is stored in fully grown oocytes of most species, but replenishing MPF after its fall during cell cycles requires protein synthesis. During oocyte maturation protein synthesis increases following the activation of MPF, and the synthesized proteins bind with chromosomes that have condensed to a metaphase state. The temporal correlation between the appearance of MPF with chromosome condensation activity and spindle formation observed in various cells suggest a major role played by MPF in the control of chromosome and microtubule assembly cycles. Thus, MPF is a regulator that coordinates the functions of various cell components to advance the chromosome cycle from interphase to metaphase. Therefore, a key to understanding the control of the chromosome cycle lies in knowing factors on which MPF activity is dependent. Although some physiological parameters of the cell are known to affect MPF activity, including Ca ion levels, intracellular pH, protein synthesis activity, cAMP levels, and protein phosphorylation, it seems difficult to assign the control of MPF cycles to any of these parameters. On the contrary, MPF cycles appear to regulate changes in these parameters. Rather, since MPF has the ability to amplify itself by activating its precursor, thus being involved in the MPF-generating system in the cell, the MPF cycle may be an autonomous process. This notion may be supported by the recent observation of the oscillatory activity of MPF in cytosols extracted from frog eggs. We propose theoretical models to explain the MPF oscillator in the cell.     

Lack of Underdominance in a Naturally Occurring Pericentric Inversion in Drosophila Melanogaster and Its Implications for Chromosome Evolution

In(2LR)PL is a large pericentric inversion polymorphic in populations of Drosophila melanogaster on two Indian Ocean islands. This polymorphism is puzzling: because crossing over in female heterokaryotypes produces inviable zygotes, such inversions are thought to be underdominant and should be quickly eliminated from populations. The observed fixation for such inversions among related species has led to the idea that genetic drift can cause chromosome evolution in opposition to natural selection. We found, however, that In(2LR)PL is not underdominant for fertility, as heterokaryotypic females produce perfectly viable eggs. Genetic analysis shows that the lack of underdominance results from the nearly complete absence of crossing over in the inverted region. This phenomenon is probably caused by mechanical and not genetic factors, because crossing over is not suppressed in In(2LR)PL homokaryotypes. Our observations do not support the idea that the fixation of pericentric inversions among closely related species implies the action of genetic drift overcoming strong natural selection in very small populations. If chromosome arrangements vary in their underdominance, it is those with the least disadvantage as heterozygotes, like In(2LR)PL, that will be polymorphic or fixed in natural populations.     

Acidic Nucleoplasmic DNA-binding Protein (And-1) Controls Chromosome Congression by Regulating the Assembly of Centromere Protein A (CENP-A) at Centromeres

The centromere is an epigenetically designated chromatin domain that is essential for the accurate segregation of chromosomes during mitosis. The incorporation of centromere protein A (CENP-A) into chromatin is fundamental in defining the centromeric loci. Newly synthesized CENP-A is loaded at centromeres in early G₁ phase by the CENP-A-specific histone chaperone Holliday junction recognition protein (HJURP) coupled with other chromatin assembly factors. However, it is unknown whether there are additional HJURP-interacting factor(s) involving in this process. Here we identify acidic nucleoplasmic DNA-binding protein 1 (And-1) as a new factor that is required for the assembly of CENP-A nucleosomes. And-1 interacts with both CENP-A and HJURP in a prenucleosomal complex, and the association of And-1 with CENP-A is increased during the cell cycle transition from mitosis to G₁ phase. And-1 down-regulation significantly compromises chromosome congression and the deposition of HJURP-CENP-A complexes at centromeres. Consistently, overexpression of And-1 enhances the assembly of CENP-A at centromeres. We conclude that And-1 is an important factor that functions together with HJURP to facilitate the cell cycle-specific recruitment of CENP-A to centromeres.     

Distribution of Replicating Simian Virus 40 DNA in Intact Cells and Its Maturation in Isolated Nuclei

The maturation of replicating simian virus 40 (SV40) chromosomes into superhelical viral DNA monomers [SV40(I) DNA] was analyzed in both intact cells and isolated nuclei to investigate further the role of soluble cytosol factors in subcellular systems. Replicating intermediates [SV40(RI) DNA] were purified to avoid contamination by molecules broken at their replication forks, and the distribution of SV40(RI) DNA as a function of its extent of replication was analyzed by gel electrophoresis and electron microscopy. With virus-infected CV-1 cells, SV40(RI) DNA accumulated only when replication was 85 to 95% completed. These molecules [SV40(RI^*) DNA] were two to three times more prevalent than an equivalent sample of early replicating DNA, consistent with a rate-limiting step in the separation of sibling chromosomes. Nuclei isolated from infected cells permitted normal maturation of SV40(RI) DNA into SV40(I) DNA when the preparation was supplemented with cytosol. However, in the absence of cytosol, the extent of DNA synthesis was diminished three- to fivefold (regardless of the addition of ribonucleotide triphosphates), with little change in the rate of synthesis during the first minute; also, the joining of Okazaki fragments to long nascent DNA was inhibited, and SV40(I) DNA was not formed. The fraction of short-nascent DNA chains that may have resulted from dUTP incorporation was insignificant in nuclei with or without cytosol. Pulse-chase experiments revealed that joining, but not initiation, of Okazaki fragments required cytosol. Cessation of DNA synthesis in nuclei without cytosol could be explained by an increased probability for cleavage of replication forks. These broken molecules masqueraded during gel electrophoresis of replicating DNA as a peak of 80% completed SV40(RI) DNA. Failure to convert SV40(RI^*) DNA into SV40(I) DNA under these conditions could be explained by the requirement for cytosol to complete the gap-filling step in Okazaki fragment metabolism: circular monomers with their nascent DNA strands interrupted in the termination region [SV40(II^*) DNA] accumulated with unjoined Okazaki fragments. Thus, separation of sibling chromosomes still occurred, but gaps remained in the terminal portions of their daughter DNA strands. These and other data support a central role for SV40(RI^*) and SV40(II^*) DNAs in the completion of viral DNA replication.     

The structure of the cold-stable kinetochore fiber in metaphase PtK1 cells

When metaphase PtK₁ cells are cooled to 6–8 ° C for 4–6 h the free, polar, and astral spindle microtubules (MTs) disassemble while the MTs of each kinetochore fiber cluster together and persist as bundles of cold-stable MTs. These cold-stable kinetochore fibers are similar to untreated kinetochore fibers in both their length (i.e., 5–6 m) and in the number of kinetochore-associated MTs (i.e., 20–45) of which they are comprised. Quantitative information concerning the lengths of MTs within these fibers was obtained by tracking individual MTs between serial transverse sections. Approximately 1/2 of the kinetochore MTs in each fiber were found to run uninterrupted into the polar region of the spindle. It can be inferred from this and other data that a substantial number of MTs run uninterrupted between the kinetochore and the polar region in untreated metaphase PtK₁ cells.     

辐照大麦M~1花粉母细胞染色体畸变及其与M~2 性状变异的关系

大麦种子经5种不同剂量γ射线辐照后, M1植株的花粉母细胞出现多种染色体畸变类型,例如后Ⅰ、后Ⅱ中出现桥、落后,后Ⅰ中出现不对称分裂,后Ⅱ中出现不同步分裂,中期Ⅰ出现环状四价体,末期Ⅰ中有三极分裂和微核,四分体中出现微核且有的微核独立于四分体,从而形成多分体。花粉母细胞的染色体畸变率随剂量的增高而升高,二者呈极显著直线相关关系。 M1花粉母细胞的染色体畸变率与M2的叶绿素突变率及农艺性状变异率呈显著或极显著指数函数关系。 M2花粉母细胞的染色体畸变率低于M1。 Abstract:Barley seeds were irradiated with five different doses of 60 Co-γ ray.There were many kinds of chromosome aberration in pollen mother cells,such as bridge,laggards in anaphase I and anaphase II,asymmetrical division in anaphase I and asynchronous division in anaphase II,ring quadrivalent in metaphase I,three pole division and micro-nuclei in telophase I,micro-nuclei in tetrad and some micro-nucleus separates from the tetrad to form polyad.The frequency of chromosome aberration in pollen mother cells of M1 increased with the increase of dose.There was a significant linear relationship between them.The frequencies of agronomic character mutation and chlorophyll mutation in M2 significantly correlated with the frequencies of chromosome aberration in pollen mother cells of M1 and showed an exponential function.The chromosome aberration frequency in pollen mother cells of M2 was lower than that of M1.