Prophase Nuclei Research Articles

Liganded ERα Stimulates the E3 Ubiquitin Ligase Activity of UBE3C to Facilitate Cell Proliferation.

Estrogen receptor (ER)α is a well-characterized ligand-dependent transcription factor. However, the global picture of its nongenomic functions remains to be illustrated. Here, we demonstrate a novel function of ERα during mitosis that facilitates estrogen-dependent cell proliferation. An E3 ubiquitin ligase, UBE3C, was identified in an ERα complex from estrogen-treated MCF-7 breast cancer cells arrested at mitosis. UBE3C interacts with ERα during mitosis in an estrogen-dependent manner. In vitro, estrogen dramatically stimulates the E3 activity of UBE3C in the presence of ERα. This effect was inhibited by the estrogen antagonist tamoxifen. Importantly, estrogen enhances the ubiquitination of cyclin B1 (CCNB1) and destabilizes CCNB1 during mitosis in a manner dependent on endogenous UBE3C. ERα, UBE3C, and CCNB1 colocalize in prophase nuclei and at metaphase spindles before CCNB1 is degraded in anaphase. Depletion of UBE3C attenuates estrogen-dependent cell proliferation without affecting the transactivation function of ERα. Collectively, these results demonstrate a novel ligand-dependent action of ERα that stimulates the activity of an E3 ligase. The mitotic role of estrogen may contribute to its effects on proliferation in addition to its roles in target gene expression.

Phenolic acid allelochemicals induced morphological, ultrastructural, and cytological modification on Cassia sophera L. and Allium cepa L.

The allelopathic potential of leaf aqueous extract (LAE) of Calotropis procera on growth behavior, ultrastructural changes on Cassia sophera L., and cytological changes on Allium cepa L. was investigated. LAE at different concentrations (0.5, 1, 2, and 4%) significantly reduced the root length, shoot length, and dry biomass of C. sophera. Besides, the ultrastructural changes (through scanning electron microscopy, SEM) induced in epidermal cells of 15-day-old seedlings of Cassia leaf were also noticed. The changes induced were shrinking and contraction of epidermal cells along with the formation of major grooves, canals, and cyst-like structures. The treated samples of epidermal cells no longer seem to be smooth as compared to control. LAE at different concentrations induces chromosomal aberrations and variation in shape of the interphase and prophase nucleus in A. cepa root tip cells when compared with control groups. The mitotic index in treated onion root tips decreased with increasing concentrations of the extracts. The most frequent aberrations were despiralization at prophase with the formation of micronuclei, sticky anaphase with bridges, sticky telophase, C-metaphase, etc. The results also show the induction of ghost cells, cells with membrane damage, and cells with heterochromatic nuclei by extract treatment. Upon HPLC analysis, nine phenolic acids (caffeic acid, gentisic acid, catechol, gallic acid, syringic acid, ellagic acid, resorcinol, p-coumaric acid, and p-hydroxy benzoic acid) were identified. Thus, the phenolic acids are mainly responsible for the allelopathic behavior of C. procera.

Nucleosome Assembly Proteins Get SET to Defeat the Guardian of Chromosome Cohesion

Nucleosome Assembly Proteins Get SET to Defeat the Guardian of Chromosome Cohesion

Evolutionary comparisons reveal a positional switch for spindle pole oscillations in Caenorhabditis embryos

During the first embryonic division in Caenorhabditis elegans, the mitotic spindle is pulled toward the posterior pole of the cell and undergoes vigorous transverse oscillations. We identified variations in spindle trajectories by analyzing the outwardly similar one-cell stage embryo of its close relative Caenorhabditis briggsae. Compared with C. elegans, C. briggsae embryos exhibit an anterior shifting of nuclei in prophase and reduced anaphase spindle oscillations. By combining physical perturbations and mutant analysis in both species, we show that differences can be explained by interspecies changes in the regulation of the cortical Gα-GPR-LIN-5 complex. However, we found that in both species (1) a conserved positional switch controls the onset of spindle oscillations, (2) GPR posterior localization may set this positional switch, and (3) the maximum amplitude of spindle oscillations is determined by the time spent in the oscillating phase. By investigating microevolution of a subcellular process, we identify new mechanisms that are instrumental to decipher spindle positioning.

Female gametogenesis and female gamete germination in the anisogamous green alga Codium fragile subsp. novae‐zelandiae (Bryopsidophyceae, Chlorophyta)

SUMMARYFemale gametogenesis was studied in the dioecious siphonous green alga Codium fragile subsp. novae‐zelandiae (J. Agardh) P. C. Silva using light and electron microscopy. Early during gametogenesis the protoplasm was uniform; then it separated in portions, while fusiform chloroplasts and nuclei increased in numbers. Some features of the nuclear divisions were similar to those of other Bryopsidophyceae. They were acentric and semi‐open. Pairs of parallel electron‐dense lines resembling synaptonemic complexes were observed in several prophase nuclei indicating meioses. In metaphase the nuclear envelope showed polar fenestrae from which the spindle emerged. No spindle microtubule nucleating material was visible and chromosome kinetochores were evident. Mature female gametes were pyriform with a hyaline anterior end from which the two flagella emerged. Mature gametes had a spherical nucleus surrounded by a mitochondrion and numerous discoid chloroplasts. Female gametes germinated parthenogenetically in culture and also inside gametangia, involving loss of flagella, rounding and lengthening of cells, multiplication of chloroplasts with well developed thylakoid systems, vacuolization and synthesis of a fibrillar cell wall.

The Tetrahymena meiotic chromosome bouquet is organized by centromeres and promotes interhomolog recombination

In order to form crossovers and to undergo reductional segregation during meiosis, homologous chromosomes must pair. In Tetrahymena, meiotic prophase nuclei elongate immensely, and, within the elongated nucleus, chromosomes are arranged with telomeres assembled at one pole and centromeres at the opposite pole. This organisation is an exaggerated form of the bouquet, a meiotic chromosome arrangement that is widely conserved among eukaryotes. We show that centromere function is crucial for the formation of Tetrahymena's stretched bouquet and, thereby, for homologue pairing. This finding adds to previous reports of the importance of centromeres in chromosome pairing in budding yeast and in Drosophila. Tetrahymena's bouquet is an ataxia telangiectasia- and RAD3-related (ATR)-dependent meiotic DNA damage response that is triggered by meiotic DNA double-strand breaks (DSBs), suggesting that the bouquet is needed for DSB repair. However, in the present study we show that although homologous pairing is impeded in the absence of the bouquet, DSB repair takes place nevertheless. Moreover, recombinational DSB repair, as monitored by bromodeoxyuridine incorporation, takes place only after exit from the bouquet stage. Therefore, we conclude that the bouquet is not required for DSB repair per se, but may be necessary for the alignment of homologous loci in order to promote homologous crossovers over alternative repair pathways.

Genetic polymorphism of flax Linum usitatissimum based on the use of molecular cytogenetic markers

Using a set of approaches based on the use of molecular cytogenetic markers (DAPI/C-banding, estimation of the total area of DAPI-positive regions in prophase nuclei, FISH with 26S and 5S rDNA probes) and the microsatellite (SSR-PCR) assay, we studied genomic polymorphism in 15 flax (Linum usitatissimum L.) varieties from different geographic regions belonging to three directions of selection (oil, fiber, and intermediate flaxes) and in the k-37 x Viking hybrid. All individual chromosomes have been identified in the karyotypes of these varieties on the basis of the patterns of differential DAPI/C-banding and the distribution of 26S and 5S rDNA, and idiograms of the chromosomes have been generated. Unlike the oil flax varieties, the chromosomes in the karyotypes of the fiber flax varieties have, as a rule, pericentromeric and telomeric DAPI-positive bands of smaller size, but contain larger intercalary regions. Two chromosomal rearrangements (chromosome 3 inversions) were discovered in the variety Luna and in the k-37 x Viking hybrid. In both these forms, no colocalization of 26S rDNA and 5S rDNA on the satellite chromosome was detected. The SSR assay with the use of 20 polymorphic pairs of primers revealed 22 polymorphic loci. Based on the SSR data, we analyzed genetic similarity of the flax forms studied and constructed a genetic similarity dendrogram. The genotypes studied here form three clusters. The oil varieties comprise an independent cluster. The genetically related fiber flax varieties Vita and Luna, as well as the landrace Lipinska XIII belonging to the intermediate type, proved to be closer to the oil varieties than the remaining fiber flax varieties. The results of the molecular chromosomal analysis in the fiber and oil flaxes confirm their very close genetic similarity. In spite of this, the combined use of the chromosomal and molecular markers has opened up unique possibilities for describing the genotypes of flax varieties and creating their genetic passports.

Sgo1 establishes the centromeric cohesion protection mechanism in G2 before subsequent Bub1-dependent recruitment in mitosis

Bub1 was one of the first protein kinases identified as a component of the spindle-assembly checkpoint, a surveillance mechanism that delays anaphase onset until all chromosomes are stably attached to spindle microtubules. Whereas the kinase activity of Bub1 is not required for checkpoint function in yeast, its requirement in mammalian cells is still unclear. Using a complementation assay with bona fide BUB1-null mouse embryonic fibroblasts, we show that the kinase activity of Bub1 is not required for checkpoint function or chromosome alignment. Its activity is, however, required for centromeric localisation of Sgo1, a known protector of centromeric cohesion. Despite the absence of Sgo1 from mitotic centromeres in cells devoid of Bub1 activity, centromeric cohesion is still maintained until anaphase. An explanation for this comes from observations showing that Sgo1 is first recruited to centromeric heterochromatin in G2, but then becomes diffusely localised throughout the nucleus in early prophase, before returning to centromeres later in prophase. Importantly, whereas centromeric localisation of Sgo1 in prophase is dependent on the kinase activity of Bub1, its recruitment to centromeric heterochromatin in G2 is not. Rather, the localisation of Sgo1 in G2 is abolished when heterochromatin protein 1 is not bound to centromeric heterochromatin. Thus, it seems that Sgo1 sets up the centromeric protection mechanism in G2, but that its Bub1-dependent localisation to centromeres during mitosis is not required to maintain cohesion.

Nuclear-localized subtype of end-binding 1 protein regulates spindle organization in Arabidopsis

End-binding 1 (EB1) proteins are evolutionarily conserved plus-end-tracking proteins that localize to growing microtubule plus ends where they regulate microtubule dynamics and interactions with intracellular targets. Animal EB1 proteins have acidic C-terminal tails that might induce an autoinhibitory conformation. Although EB1 proteins with the same structural features occur in plants (EB1a and EB1b in Arabidopsis thaliana), a variant form (EB1c) is present that lacks the characteristic tail. We show that in Arabidopsis the tail region of EB1b, but not of EB1c, inhibits microtubule assembly in vitro. EB1a and EB1b form heterodimers with each other, but not with EB1c. Furthermore, the EB1 genes are expressed in various cell types of Arabidopsis, but the expression of EB1c is particularly strong in the meristematic cells where it is targeted to the nucleus by a nuclear localization signal in the C-terminal tail. Reduced expression of EB1c compromised the alignment of spindle and phragmoplast microtubules and caused frequent lagging of separating chromosomes at anaphase. Roots of the eb1c mutant were hypersensitive to a microtubule-disrupting drug and complete rescue of the mutant phenotype required the tail region of EB1c. These results suggest that a plant-specific EB1 subtype has evolved to function preferentially on the spindle microtubules by accumulating in the prophase nucleus.

Phosphorylation of histone H3 at Thr3 is part of a combinatorial pattern that marks and configures mitotic chromatin

We have previously shown that histone H3 is transiently phosphorylated at Thr3 during mitosis. Extending these studies, we now report that phosphorylated Thr3 is always in cis to trimethylated Lys4 and dimethylated Arg8, forming a new type of combinatorial modification, which we have termed PMM. PMM-marked chromatin emerges at multiple, peripheral sites of the prophase nucleus, then forms distinct clusters at the centric regions of metaphase chromosomes, and finally spreads (as it wanes) to the distal areas of segregating chromatids. The characteristic prophase pattern can be reproduced by expressing ectopically the kinase haspin at interphase, suggesting that the formation of the PMM signature does not require a pre-existing mitotic environment. On the other hand, the ;dissolution' and displacement of PMM clusters from a centric to distal position can be induced by partial dephosphorylation or chromosome unravelling, indicating that these changes reflect the regulated grouping and scrambling of PMM subdomains during cell division. Formation of PMM is prevented by haspin knockdown and leads to delayed exit from mitosis. However, PMM-negative cells do not exhibit major chromosomal defects, suggesting that the local structures formed by PMM chromatin may serve as a ;licensing system' that allows quick clearance through the metaphase-anaphase checkpoint.

Electron microscopy of mitotic cells in a measles-carrier human cell line.

The occurrence and localization of measles virus nucleocapsids during mitosis of a measles-carrier human cell line (Lu106) were studied in the transmission electron microscope. During interphase, the virus nucleocapsids were present mainly in the cytoplasm and less frequently in the nucleus. Nucleocapsids were also observed at the different stages of mitosis, always in the cytoplasm outside prophase and telophase nuclei and outside the mitotic apparatus at metaphase and anaphase. The presence of viral materials during mitosis and at the two daughter cell stage indicates a vertical transmission of infection through cell division. The frequency of infected cells in the cell population was generally high and sometimes relatively low inferring a fluctuation state in the virus/cell equilibrium.

Condensation of chromosomes during mitotic prophase. Whole mount electron microscopy of cerebral ganglion cells of Drosophila.

Condensation of chromosomes during mitotic prophase was studied by means of whole mount electron microscopy on cerebral ganglion cells of Drosophila melanogaster after short treatment with alkaliurea. In interphase and early mitotic prophase nuclei some core-like structures, i.e. interchromomeric axial fibers, were observed. During later prophase the chromatid axis seems to be polynemic in most of the cells. Later on the primary axes of chromatids disappear more and more due to supercoiling and folding of chromatin fibrils, and through the shortening of the whole linear organization of chromosomes. Some traces of subsequent coiling orders can be locally observed in the chromosome arms. The largest gyres of metaphase chromosomes are especially clear in whole mount preparations.

Plant TPX2 and related proteins

At the onset of mitosis, microtubules form a bipolar spindle around the prophase nucleus. TPX2 is phosphorylated during mitosis and acts as a spindle assembly factor that nucleates microtubules in the close vicinity of chromosomes, independent of the centrosomes. Furthermore, it activates the kinase Aurora A and targets the Xenopus kinesin-like protein 2 to spindle poles. We have characterized the plant orthologue of TPX2 that possesses all identified functional domains of its animal counterpart. Moreover, we have demonstrated that it is exported before nuclear envelope breakdown and that its activity around the nuclear envelope is essential for prospindle assembly. Here, we compare the sequences of several characterized TPX2 domains, allowing us to define TPX2. We propose that true TPX2 orthologues share simultaneously all these conserved domains and that other proteins possessing only some of these functional blocks may be considered as TPX2-related proteins.

Mitotic Spindle Organization by the Preprophase Band

In higher plants, the preprophase band (PPB) of microtubules (MTs) forecasts the cell division site prior to mitosis and specifies the organization of MTs into a bipolar prophase spindle surrounding the nucleus. However, the mechanisms governing this PPB-dependent establishment of bipolarity are unclear. Here, we present evidence from live cell imaging studies that suggest a role for the MTs bridging the PPB and the prophase nucleus in mediating this function. Results from drug treatments, along with genetic evidence from null kinesin plants, suggest that these MTs contribute to the bipolarity, orientation, and position of the prophase spindle. Specifically, the absence of these bridge MTs is associated with lack of bipolarity, while non-uniform distributions of bridge MTs correlate with prophase spindle migration, deformation, and enhanced bipolarity toward the region of highest bridge MT density. This behavior does not require actomyosin-based forces, and is enhanced by suppressing MT dynamics with taxol. These observations occur during late prophase, and are coincident with the gradual closing of annular spindle poles. Based on these data, we describe a hypothetical mechanism for bridge MT-dependent organization of prophase spindles.

The Drosophila Cytosine-5 Methyltransferase Dnmt2 Is Associated with the Nuclear Matrix and Can Access DNA during Mitosis

Cytosine-5 methyltransferases of the Dnmt2 family are highly conserved in evolution and their biological function is being studied in several organisms. Although all structural DNA methyltransferase motifs are present in Dnmt2, these enzymes show a strong tRNA methyltransferase activity. In line with an enzymatic activity towards substrates other than DNA, Dnmt2 has been described to localize to the cytoplasm. Using molecular and biochemical approaches we show here that Dnmt2 is both a cytoplasmic and a nuclear protein. Sub-cellular fractionation shows that a significant amount of Dnmt2 is bound to the nuclear matrix. Sub-cellular localization analysis reveals that Dnmt2 proteins are enriched in actively dividing cells. Dnmt2 localization is highly dynamic during the cell cycle. Using live imaging we observed that Dnmt2-EGFP enters prophase nuclei and shows a spindle-like localization pattern during mitotic divisions. Additional experiments suggest that this localization is microtubule dependent and that Dnmt2 can access DNA during mitotic cell divisions. Our results represent the first comprehensive characterization of Dnmt2 proteins on the cellular level and have important implications for our understanding of the molecular activities of Dnmt2.

Total area of C-heterochromatin in nuclei and chromosomes as a measure of cultivated flax genome variability

Comparative statistical analysis of total C-heterochromatin content (total area of darkly stained C-bands) in prophase nuclei and metaphase chromosomes of eight flax (Linum usitatissimum L.) varieties, viz., three oil flax varieties (Shafir, Oliver, Linotte), three modern (Orshansky-2, Belinka, Baltuchai) and two ancient fiber flax varieties (Zaretsky Kryazh, K-37) has been carried out. Karyotypes of flax varieties selected for different agronomical traits differed from one another in C-heterochromatin content. The total C-band area of oil flax varieties exceeded that of fiber flax varieties. Genomic polymorphism in C-heterochromatin markers is a testimony of different breeding directions. A simple test for determining total C-heterochromatin content in prophase nuclei is recommended as a useful tool for screening specimens with predetermined characteristics during selection of new varieties.

Two X family DNA polymerases, λ and μ, in meiotic tissues of the basidiomycete, Coprinus cinereus

The X family DNA polymerases lambda (CcPollambda) and mu (CcPolmu) were shown to be expressed during meiotic prophase in the basidiomycete, Coprinus cinereus. These two polymerases are the only members of the X family in the C. cinereus genome. The open reading frame of CcPollambda encoded a predicted product of 800 amino acid residues and that of CcPolmicro of 621 amino acid residues. Both CcPollambda and CcPolmicro required Mn(2+) ions for activity, and both were strongly inhibited by dideoxythymidine triphosphate. Unlike their mammalian counterparts, CcPollambda and CcPolmicro had no terminal deoxynucleotidyl transferase activity. Immunostaining analysis revealed that CcPollambda was present at meiotic prophase nuclei in zygotene and pachytene cells, which is the period when homologous chromosomes pair and recombine. CcPolmicro was present in a slightly wider range of cell stages, zygotene to diplotene. In analyses using D-loop recombination intermediate substrates, we found that both CcPollambda and CcPolmicro could promote primer extension of an invading strand in a D-loop structure. Moreover, both polymerases could fully extend the primer in the D-loop substrate, suggesting that D-loop extension is an activity intrinsic to CcPollambda and CcPolmicro. Based on these data, we discuss the possible roles of these polymerases in meiosis.

Cyclin B1–Cdk1 Activation Continues after Centrosome Separation to Control Mitotic Progression

Activation of cyclin B1–cyclin-dependent kinase 1 (Cdk1), triggered by a positive feedback loop at the end of G2, is the key event that initiates mitotic entry. In metaphase, anaphase-promoting complex/cyclosome–dependent destruction of cyclin B1 inactivates Cdk1 again, allowing mitotic exit and cell division. Several models describe Cdk1 activation kinetics in mitosis, but experimental data on how the activation proceeds in mitotic cells have largely been lacking. We use a novel approach to determine the temporal development of cyclin B1–Cdk1 activity in single cells. By quantifying both dephosphorylation of Cdk1 and phosphorylation of the Cdk1 target anaphase-promoting complex/cyclosome 3, we disclose how cyclin B1–Cdk1 continues to be activated after centrosome separation. Importantly, we discovered that cytoplasmic cyclin B1–Cdk1 activity can be maintained even when cyclin B1 translocates to the nucleus in prophase. These experimental data are fitted into a model describing cyclin B1–Cdk1 activation in human cells, revealing a striking resemblance to a bistable circuit. In line with the observed kinetics, cyclin B1–Cdk1 levels required to enter mitosis are lower than the amount of cyclin B1–Cdk1 needed for mitotic progression. We propose that gradually increasing cyclin B1–Cdk1 activity after centrosome separation is critical to coordinate mitotic progression.

Cytoskeletal asymmetry in Zea mays subsidiary cell mother cells: A monopolar prophase microtubule half‐spindle anchors the nucleus to its polar position

Double labeling of microtubules and actin filaments revealed that in prophase subsidiary mother cells of Zea mays a monopolar prophase microtubule "half-spindle" is formed, which lines the nuclear hemisphere distal to the inducing guard mother cell. The nuclear hemisphere proximal to the guard mother cell is lined by an F-actin cap, consisting of a cortical F-actin patch and actin filaments originating from it. The microtubules of the "half-spindle" decline from the nuclear surface and terminate to the preprophase microtubule band. After disintegration of the latter, a bipolar metaphase spindle is organized. The polar F-actin cap persists during mitosis and early cytokinesis, extending to the chromosomes and the subsidiary cell daughter nucleus. In oryzalin treated subsidiary mother cells the prophase nuclei move away from the polar site. Cytochalasin B and latrunculin-B block the polar migration of subsidiary mother cell nuclei, but do not affect those already settled to the polar position. The prophase nuclei of latrunculin-B treated subsidiary mother cells are globally surrounded by microtubules, while the division plane of latrunculin-B treated subsidiary mother cells is misaligned. The prophase nuclei of brick 1 mutant Zea mays subsidiary mother cells without F-actin patch are also globally surrounded by microtubules. The presented data show that the prophase microtubule "half-spindle"-preprophase band complex anchors the subsidiary mother cell nucleus to the polar cell site, while the polar F-actin cap stabilizes the one metaphase spindle pole proximal to the inducing guard mother cell.

Polar organizers and girdling bands of microtubules are associated with γ-tubulin and act in establishment of meiotic quadripolarity in the hepatic Aneura pinguis (Bryophyta)

Meiosis in Aneura pinguis is preceded by extensive cytoplasmic preparation for quadripartitioning of the diploid sporocyte into a tetrad of haploid spores. In early prophase the four future spore domains are defined by lobing of the cytoplasm and development of a quadripolar prophase spindle focused at polar organizers (POs) centered in the lobes. Cells entering the reproductive phase become isolated and, instead of hooplike cortical microtubules, have endoplasmic microtubule systems centered on POs. These archesporial cells proliferate by mitosis before entering meiosis. In prophase of each mitosis, POs containing a distinct concentration of gamma-tubulin appear de novo at tips of nuclei and initiate the bipolar spindle. Cells entering meiosis become transformed into quadrilobed sporocytes with four POs, one in each lobe. This transition is a complex process encompassing assembly of two opposite POs which subsequently disperse into intersecting bands of microtubules that form around the central nucleus. The girdling bands define the future planes of cytokinesis and the cytoplasm protrudes through the restrictive bands becoming quadrilobed. Two large POs reappear in opposite cleavage furrows. Each divides and the resulting POs migrate into the tetrahedral lobes of cytoplasm. Cones of microtubules emanating from the four POs interact to form a quadripolar microtubule system (QMS) that surrounds the nucleus in meiotic prophase. The QMS is subsequently transformed into a functionally bipolar metaphase spindle by migration of poles in pairs to opposite cleavage furrows. These findings contribute to knowledge of microtubule organization and the role of microtubules in spatial regulation of cytokinesis in plants.