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PubMed2001

The fas locus of the phytopathogen Rhodococcus fascians affects mitosis of tobacco BY-2 cells.

The effect of Rhodococcus fascians, the causal agent of leafy gall disease, on the mitotic behavior of synchronized tobacco Bright Yellow-2 (BY-2) cells was investigated. Incubation of aphidicolin-synchronized BY-2 cells with R. fascians cells specifically resulted in a broader mitotic index peak, an effect that was linked to an intact and expressed fas virulence locus. The obtained results pointed towards an effect of R. fascians on the prophase of mitosis. The relevance of these results to the virulence of the bacterium is discussed.

Cell Cycle↗
PubMed2001

Checkpoint and DNA-repair proteins are associated with the cores of mammalian meiotic chromosomes.

Meiotic checkpoints are manifested through protein complexes capable of detecting an abnormality in chromosome metabolism and signaling it to effector molecules that subsequently delay or arrest the progression of meiosis. Some checkpoints act during the first meiotic prophase to monitor the repair of chromosomal DSBs, predominantly by meiotic recombination, or to ensure the correct establishment of synapsis and its well-timed dissolution. In mammals, a number of checkpoint and repair proteins localize to the meiotic chromosomal cores, sometimes in the context of the synaptonemal complex (SC). Here we discuss possible functions of these proteins in the accomplishment of meiotic recombination and normal progression of the meiotic pathway. Also, we present arguments for a structural role of cores and SCs in the assembly of the repair and checkpoint protein complexes on the chromosomes.

Animals↗
PubMed1998

Caught in the act: deducing meiotic function from protein immunolocalization.

Meiotic division comprises a complex series of events, many of which are unique in the life cycle of the organism. The process utilizes both proteins that participate in normal mitotic cell cycle progression and DNA damage repair and proteins expressed only during meiosis. Until recently, few meiotic protein participants had been identified and characterized, but several recent developments have changed this situation. Proteins can be selected for study based on their cDNA sequence and similarity to known proteins with "suspicious" repair/recombination or cell cycle activity and antibodies against these proteins applied to meiotic nuclei to test for activity. With the development of gene sequence data bases from many organisms, similarity to a known protein need not be based on the same or even a closely related species. Potential interactions between two or more proteins can be identified and involvement in a common process inferred based on antibody colocalization. The gene sequence can be disrupted and the effect on meiotic progression directly examined. Previously identified structures, the synaptonemal complex (SC) and both early and late recombination nodules (RNs), provide structural and temporal landmarks that assist in inferring meiotic activity of the protein being studied. Mammalian meiosis is especially attractive for these kinds of studies since spermatocyte and oocyte nuclei are large with distinct nuclear organelles and since meiosis is highly protracted, occurring over a period of several days. In this chapter, an approach to the study of mammalian meiosis based on use of specific antibodies is outlined and methods of coupling this approach to other techniques, such as targeted gene disruption or chromosome aberrations, are described. Some of the proteins already identified as participants in meiotic prophase are reviewed and their presumed functions discussed.

Animals↗
PubMed1999

Spo76p is a conserved chromosome morphogenesis protein that links the mitotic and meiotic programs.

Spo76p is conserved and related to the fungal proteins Pds5p and BIMD and the human AS3 prostate proliferative shutoff-associated protein. Spo76p localizes to mitotic and meiotic chromosomes, except at metaphase(s) and anaphase(s). During meiotic prophase, Spo76p assembles into strong lines in correlation with axial element formation. As inferred from spo76-1 mutant phenotypes, Spo76p is required for sister chromatid cohesiveness, chromosome axis morphogenesis, and chromatin condensation during critical transitions at mitotic prometaphase and meiotic midprophase. Spo76p is also required for meiotic interhomolog recombination, likely at postinitiation stage(s). We propose that a disruptive force coordinately promotes chromosomal axial compaction and destabilization of sister connections and that Spo76p restrains and channels the effects of this force into appropriate morphogenetic mitotic and meiotic outcomes.

Amino Acid Sequence↗
PubMed1998

RNA-assisted nuclear transport of the meiotic regulator Mei2p in fission yeast.

Fission yeast Mei2p is an RNA-binding protein required for both premeiotic DNA synthesis and meiosis I. Mei2p binds to a polyadenylated RNA molecule, meiRNA, loss of which blocks meiosis I. Mei2p forms a dot in meiotic prophase nuclei. Here, we show that meiRNA is required for the nuclear localization of Mei2p and is detectable in the dot. However, Mei2p carrying a nuclear localization signal can produce a nuclear dot and promote meiosis I in the absence of meiRNA. Mei2p expressed in cultured mammalian cells stays in the cytoplasm, but it accumulates in the nucleolus if meiRNA is coexpressed. These results indicate that meiRNA contributes to the promotion of meiosis I exclusively as a cofactor that assists nuclear transport of Mei2p.

Animals↗
PubMed1997

Effects of 1-methyladenine on nuclear Ca2+ transients and meiosis resumption in starfish oocytes are mimicked by the nuclear injection of inositol 1,4,5-trisphosphate and cADP-ribose.

The treatment of prophase-arrested starfish oocytes with the hormone 1-methyladenine (1-MA) induces the elevation of Ca2+ in both the cytoplasm and the germinal vesicle (nucleus), and is followed by the resumption of meiosis. The injection of the modulators of the intracellular Ca2+ channels inositol 1,4,5-trisphosphate (InsP3) or cyclic adenosine diphosphate ribose (cADPr) into the germinal vesicle promoted meiosis resumption in the absence of 1-MA in about 50% of the oocytes. Caged InsP3 or caged cADPr were injected into the nuclei of oocytes together with the Ca2+ indicator calcium green dextran; their photoreleasing elicited nuclear calcium spikes which, in the case of cADPr, had repetitive behaviour. The spikes were abolished by the nuclear injection of antagonists or antibodies to the InsP3 or cADPr-sensitive Ca2+ channels. cADPr-modulated channels were localized on the membranes of the nuclear envelope using specific antibodies conjugated with IgG-gold complexes.

Adenine↗
PubMed2003

Coprinus cinereus DNA ligase I during meiotic development.

DNA ligase I is thought to be essential for DNA replication, repair and recombination, at least in the mitotic cell cycle, but whether this is also the case during the meiotic cell cycle is still obscure. To investigate the role of DNA ligase I during the meiotic cell cycle, we cloned the Coprinus cinereus DNA ligase I cDNA (CcLIG1). Northern blotting analysis indicated that CcLIG1 is expressed not only in the premeiotic S-phase but also during the meiotic cell cycle itself. Especially, intense signals were observed in the leptotene and zygotene stages. Western blotting analysis indicated that CcLIG1 is expressed through the meiotic cell cycle and immunofluorescence also showed CcLIG1 protein staining in meiotic cells. Interestingly, the patterns was similar to that for the C. cinereus proliferating cell nuclear antigen gene (CcPCNA) and immunoprecipitation analysis suggested that CcPCNA binds to CcLIG1 in crude extracts of meiotic prophase I tissues. Based on these observations, relationships and roles during the meiotic cell cycle are discussed.

Amino Acid Sequence↗
PubMed2000

Dynamic changes in microtubule organization during division of the primitive dinoflagellate Oxyrrhis marina.

The marine dinoflagellate Oxyrrhis marina has three major microtubular systems: the flagellar apparatus made of one transverse and one longitudinal flagella and their appendages, cortical microtubules, and intranuclear microtubules. We investigated the dynamic changes of these microtubular systems during cell division by transmission and scanning electron microscopy, and confocal fluorescent laser microscopy. During prophase, basal bodies, both flagella and their appendages were duplicated. In the round nucleus situated in the cell centre, intranuclear microtubules appeared radiating toward the centre of the nucleus from densities located in some nuclear pores. During metaphase, both daughter flagellar apparatus separated and moved apart along the main cell axis. Microtubules of ventral cortex were also duplicated and moved with the flagellar apparatus. The nucleus flattened in the longitudinal direction and became discoid-shaped close to the equatorial plane. Many bundles of microtubules ran parallel to the short axis of the nucleus (cell long axis), between which chromosomes were arranged in the same direction. During ana-telophase, the nucleus elongated along the longitudinal axis and took a dumbbell shape. At this stage a contractile ring containing actin was clearly observed in the equatorial cortex. The cortical microtubule network seemed to be cut into two halves at the position of the actin bundle. Shortly after, the nucleus divided into two nuclei, then the cell body was constricted at its equator and divided into one anterior and one posterior halves which were soon rebuilt to produce two cells with two full sets of cortical microtubules. From our observations, several mechanisms for the duplication of the microtubule networks during mitosis in O. marina are discussed.

Actins↗
PubMed2002

Nucleus behavior during the closed mitosis of Tritrichomonas foetus.

We present observations on the fine structure and the division process of the nucleus in the protist Tritrichomonas foetus, parasite of the urogenital tract of cattle. The nucleus was followed by immunofluorescence and electron microscopy during interphase and mitosis. Conventional karyotyping coupled to image processing and bright field Panotic staining were used to follow nucleus modifications, chromosome number and condensation pattern along the whole cell cycle. Confocal laser scanning microscopy (CLSM) using DNA fluorescent probes, followed by image processing in the SURF-Driver program, produced three-dimensional reconstruction data of the mitotic nucleus under each phase of the division process. Immunocytochemistry in thin-sections revealed the chromosome spatial arrangement after bromodeoxyuridine incorporation and immunogold labeling using anti-DNA monoclonal antibodies. Our results indicate that: (1) the nucleus assumes different size and shapes along mitosis: it appears oval in interphase, becoming lobed or concave in prophase, then undergoing torsion and constriction, displaying an 'S' shape (metaphase). Next, it becomes elongated and it is finally separated in two nuclei at the transition of anaphase to telophase; (2) T. foetus nucleus harbors five chromosomes; (3) chromosomes become condensed in a pre-mitotic phase; (4) the nucleolus persists during the mitosis.

Anaphase↗
PubMed2003

MAPK is involved in metaphase I arrest in oyster and mussel oocytes.

Oocytes of Crassostrea gigas and Mytilus galloprovincialis are arrested in metaphase I when they are spawned and ready to be fertilized. To investigate the role of MAP kinase in maintaining metaphase I arrest, oocytes were exposed to the MEK inhibitor U0126, and the effects on chromosome behavior and MAPK activity were examined by bisbenzimide staining and in immunoblots with anti-phospho MAPK antibodies. Following treatment with 50 microM U0126, active MAPK was undetectable and oocytes resumed meiosis, forming enlarged polar bodies and undergoing chromosome decondensation. Prophase stage oyster oocytes maturing spontaneously in seawater completed germinal vesicle breakdown in the presence of U0126, but failed to arrest in metaphase I, and also formed polar bodies and underwent chromosome decondensation. Treatment of oyster oocytes with the protein synthesis inhibitor, emetine (500 microM), also caused them to resume meiosis, although substantial MAPK activity remained. Levels of phospho-MEK also decreased during emetine treatment. 35 S-methionine incorporation in emetine treated oocytes was reduced to only 5% of control values. These data show that, while active MAPK is necessary to maintain metaphase I arrest, other proteins are also required.

Animals↗
PubMed2000

Control of mitosis by changes in the subcellular location of cyclin-B1-Cdk1 and Cdc25C.

Nuclear events of mitosis are initiated when the protein kinase cyclin-B1-Cdk1 is translocated into the nucleus during prophase. Recent work has unveiled many of the mechanisms that govern the localization of cyclin-B1-Cdk1 and its regulator Cdc25C. Phosphorylation-dependent changes in the rate of nuclear import and export of these proteins help to control the onset of mitosis both in normal cells and in cells delayed before mitosis by DNA damage.

Animals↗
PubMed1994

Meiosis. Telomeres lead chromosome movement.

The telomeres of fission yeast chromosomes are attached to the moving spindle pole body during karyogamy and meiotic prophase. Nuclear movement may also contribute to homologous chromosome pairing.

Cell Nucleus↗
PubMed2002

The dynamics of homologous chromosome pairing during male Drosophila meiosis.

BACKGROUND: Meiotic pairing is essential for the proper orientation of chromosomes at the metaphase plate and their subsequent disjunction during anaphase I. In male Drosophila melanogaster, meiosis occurs in the absence of recombination or a recognizable synaptonemal complex (SC). Due to limitations in available cytological techniques, the early stages of homologous chromosome pairing in male Drosophila have not been observed, and the mechanisms involved are poorly understood. RESULTS: Chromosome tagging with GFP-Lac repressor protein allowed us to track, for the first time, the behavior of meiotic chromosomes at high resolution, live, at all stages of male Drosophila meiosis. Homologous chromosomes pair throughout the euchromatic regions in spermatogonia and during the early phases of spermatocyte development. Extensive separation of homologs and sister chromatids along the chromosome arms occurs in mid-G2, several hours before the first meiotic division, and before the G2/M transition. Centromeres, on the other hand, show complex association patterns, with specific homolog pairing taking place in mid-G2. These changes in chromosome pairing parallel changes in large-scale chromosome organization. CONCLUSIONS: Our results suggest that widespread interactions along the euchromatin are required for the initiation, but not the maintenance, of meiotic pairing of autosomes in male Drosophila. We propose that heterochromatic associations, or chromatid entanglement, may be responsible for the maintenance of homolog association during late G2. Our data also suggest that the formation of chromosome territories in the spermatocyte nucleus may play an active role in ensuring the specificity of meiotic pairing in late prophase by disrupting interactions between nonhomologous chromosomes.

Anaphase↗
PubMed2000

Centrosome-independent mitotic spindle formation in vertebrates.

BACKGROUND: In cells lacking centrosomes, the microtubule-organizing activity of the centrosome is substituted for by the combined action of chromatin and molecular motors. The question of whether a centrosome-independent pathway for spindle formation exists in vertebrate somatic cells, which always contain centrosomes, remains unanswered, however. By a combination of labeling with green fluorescent protein (GFP) and laser microsurgery we have been able to selectively destroy centrosomes in living mammalian cells as they enter mitosis. RESULTS: We have established a mammalian cell line in which the boundaries of the centrosome are defined by the constitutive expression of gamma-tubulin-GFP. This feature allows us to use laser microsurgery to selectively destroy the centrosomes in living cells. Here we show that this method can be used to reproducibly ablate the centrosome as a functional entity, and that after destruction the microtubules associated with the ablated centrosome disassemble. Depolymerization-repolymerization experiments reveal that microtubules form in acentrosomal cells randomly within the cytoplasm. When both centrosomes are destroyed during prophase these cells form a functional bipolar spindle. Surprisingly, when just one centrosome is destroyed, bipolar spindles are also formed that contain one centrosomal and one acentrosomal pole. Both the polar regions in these spindles are well focused and contain the nuclear structural protein NuMA. The acentrosomal pole lacks pericentrin, gamma-tubulin, and centrioles, however. CONCLUSIONS: These results reveal, for the first time, that somatic cells can use a centrosome-independent pathway for spindle formation that is normally masked by the presence of the centrosome. Furthermore, this mechanism is strong enough to drive bipolar spindle assembly even in the presence of a single functional centrosome.

Animals↗
PubMed2003

Organization of the endoplasmic reticulum in dividing cells of the gymnosperms Pinus brutia and Pinus nigra, and of the pterophyte Asplenium nidus.

Endoplasmic reticulum (ER) organization in the dividing cells of the pterophyte Asplenium nidus and of the gymnosperms Pinus brutia and Pinus nigra has been studied by immunolocalization techniques using the monoclonal antibody 2E7, which recognizes luminar ER resident proteins containing C-terminal HDEL sequences. In the pterophyte, the ER reorganization during cell cycle is similar to that in angiosperms. Among others, prominent ER gatherings were found at the mitotic spindle poles and in the phragmoplast during cytokinesis. However, in the gymnosperms examined, the ER displays a unique pattern of reorganization not described so far. In both the Pinus species, well-defined ER patterns are successively formed during cell cycle. They are the preprophase ER-band, the prophase- metaphase- and anaphase ER-spindle, the interzonal ER-system, the ER-phragmoplast and an ER-system lining the daughter cell wall. The ER patterns are closely similar to that of the correspondent microtubule (MT) arrangements with which they are co-organized. Observations made on P. nigra root-cells affected by oryzalin, colchicine and cytochalasin D favour the conclusion that the pattern of ER organization is controlled during mitosis and cytokinesis by the MT cytoskeleton.

Actin Cytoskeleton↗
PubMed2003

A two-step scaffolding model for mitotic chromosome assembly.

Topoisomerase IIalpha (topoIIalpha) and 13S condensin are both required for mitotic chromosome assembly. Here we show that they constitute the two main components of the chromosomal scaffold on histone-depleted chromosomes. The structural stability and chromosomal shape of the scaffolding toward harsh extraction procedures are shown to be mediated by ATP or its nonhydrolyzable analogs, but not ADP. TopoIIalpha and 13S condensin components immunolocalize to a radially restricted, longitudinal scaffolding in native-like chromosomes. Double staining for topoIIalpha and condensin generates a barber pole appearance of the scaffolding, where topoIIalpha- and condensin-enriched "beads" alternate; this structure appears to be generated by two juxtaposed, or coiled, chains. Cell cycle studies establish that 13S condensin appears not to be involved in the assembly of prophase chromatids; they lack this complex but contain a topoIIalpha-defined (-mediated?) scaffolding. Condensin associates only during the pro- to metaphase transition. This two-step assembly process is proposed to generate the barber pole appearance of the native-like scaffolding.

Adenosine Diphosphate↗
PubMed1998

Pachytene chromosomes in trisomy 19 male mice with Robertsonian translocations.

Three male mice with trisomy 19 induced by a Robertsonian translocation system were used for the study of meiotic prophase cells and germ cell differentiation. Present in these males were two Robertsonian chromosomes each with a chromosome 19 arm in common, two acrocentric chromosomes corresponding to the second arms of the two Rbs and one acrocentric chromosome 19. These five chromosomes showed a wide range of meiotic pairing configurations. One particular observation was the formation of a true double synaptonemal complex (SC) with three lateral axes and two central elements, which joined the three chromosomes 19 together. Integration of the acrocentric chromosome 19 in a complex pentavalent configuration was seen in 45% of the pachytene nuclei. The proportion of spermatocytes showing association between a quadrivalent and the acrocentric no. 19 was 26%. In 29% of the nuclei, the acrocentric no. 19 was free, integrated or associated with the XY complex, paired with the X chromosome or associated with a bivalent. Finally, in 57% of pachytene cells, the meiotic multivalents or the free univalent 19 were associated with the proximal part of the X chromosome or integrated in the sex chromatin. Therefore, the question arises with regard to the fate of these spermatocytes. The testicular histology shows an arrest of germ cell development at the spermatocyte stage. Several mechanisms seem to be the cause of germ cell depletion in a sequence of different, impaired developmental processes.

Animals↗
PubMed1998

Meiosis of wheat x lymegrass hybrids.

Meiosis was examined in pollen mother cells of F1 hybrids made from crosses between wheat (Triticum aestivum) and lymegrass (Leymus arenarius and L. mollis). Fluorescence genomic in situ hybridization detected pairing between wheat and lymegrass chromosomes during prophase I and metaphase I. Such pairing, when resulting in bivalent formation, was likely to yield correct disjunction, and hence intergenomic recombination could be incorporated into the gametes. Bivalents in these hybrids, however, were more frequently formed between chromosomes of the same parental origin. Univalents were common, whereas multivalents were not clearly detected. Meiotic behaviour in some cells was not totally aberrant, and this may have accounted for the presence of normal pollen. The results are discussed in relation to intergenomic pairing, meiotic behaviour in wide-hybrids and genome relationships, including the Leymus genome origin.

Hybridization, Genetic↗