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SWITCH1 (SWI1): a novel protein required for the establishment of sister chromatid cohesion and for bivalent formation at meiosis.

We have characterized a new gene, SWI1, involved in sister chromatid cohesion during both male and female meiosis in Arabidopsis thaliana. A first allele, swi1.1, was obtained as a T-DNA tagged mutant and was described previously as abnormal exclusively in female meiosis. We have isolated a new allele, swi1.2, which is defective for both male and female meiosis. In swi1.2 male meiosis, the classical steps of prophase were not observed, especially because homologs do not synapse. Chromatid arms and centromeres lost their cohesion in a stepwise manner before metaphase I, and 20 chromatids instead of five bivalents were seen at the metaphase plate, which was followed by an aberrant segregation. In contrast, swi1.2 female meiocytes performed a mitotic-like division instead of meiosis, indicating a distinct role for SWI1 or a different effect of the loss of SWI1 function in both processes. The SWI1 gene was cloned; the putative SWI1 protein did not show strong similarity to any known protein. Plants transformed with a SWI1-GFP fusion indicated that SWI1 protein is present in meiocyte nuclei, before meiosis and at a very early stage of prophase. Thus, SWI1 appears to be a novel protein involved in chromatid cohesion establishment and in chromosome structure during meiosis, but with clear differences between male and female meiosis.

Alleles↗

Cohesin release is required for sister chromatid resolution, but not for condensin-mediated compaction, at the onset of mitosis.

The establishment of metaphase chromosomes is an essential prerequisite of sister chromatid separation in anaphase. It involves the coordinated action of cohesin and condensin, protein complexes that mediate cohesion and condensation, respectively. In metazoans, most cohesin dissociates from chromatin at prophase, coincident with association of condensin. Whether loosening of cohesion at the onset of mitosis facilitates the compaction process, resolution of the sister chromatids, or both, remains unknown. We have found that the prophase release of cohesin is completely blocked when two mitotic kinases, aurora B and polo-like kinase (Plx1), are simultaneously depleted from Xenopus egg extracts. Condensin loading onto chromatin is not affected under this condition, and rod-shaped chromosomes are produced that show an apparently normal level of compaction. However, the resolution of sister chromatids within these chromosomes is severely compromised. This is not because of inhibition of topoisomerase II activity that is also required for the resolution process. We propose that aurora B and Plx1 cooperate to destabilize the sister chromatid linkage through distinct mechanisms that may involve phosphorylation of histone H3 and cohesin, respectively. More importantly, our results strongly suggest that cohesin release at the onset of mitosis is essential for sister chromatid resolution but not for condensin-mediated compaction.

Adenosine Triphosphatases↗

The cytokinin requirement for cell division in cultured Nicotiana plumbaginifolia cells can be satisfied by yeast Cdc25 protein tyrosine phosphatase: implications for mechanisms of cytokinin response and plant development.

Cultured cells of Nicotiana plumbaginifolia, when deprived of exogenous cytokinin, arrest in G2 phase prior to mitosis and then contain cyclin-dependent protein kinase (CDK) that is inactive because phosphorylated on tyrosine (Tyr). The action of cytokinin in stimulating the activation of CDK by removal of inhibitory phosphorylation from Tyr is not a secondary downstream consequence of other hormone actions but is the key primary effect of the hormone in its stimulation of cell proliferation, since cytokinin could be replaced by expression of cdc25, which encodes the main Cdc2 (CDK)-Tyr dephosphorylating enzyme of yeast (Saccharomyces cerevisiae). The cdc25 gene, under control of a steroid-inducible promoter, induced a rise in cdc25 mRNA, accumulation of p67(Cdc25) protein, and increase in Cdc25 phosphatase activity that was measured in vitro with Tyr-phosphorylated Cdc2 as substrate. Cdc25 phosphatase activity peaked during mitotic prophase at the time CDK activation was most rapid. Mitosis that was induced by cytokinin also involved increase in endogenous plant CDK Tyr phosphatase activity during prophase, therefore indicating that this is a normal part of plant mitosis. These results suggest a biochemical mechanism for several previously described transgene phenotypes in whole plants and suggest that a primary signal from cytokinin leading to progression through mitosis is the activation of CDK by dephosphorylation of Tyr.

Cell Cycle Proteins↗

Gamma-tubulin in basal land plants: characterization, localization, and implication in the evolution of acentriolar microtubule organizing centers.

Although seed plants have gamma-tubulin, a ubiquitous component of centrosomes associated with microtubule nucleation in algal and animal cells, they do not have discrete microtubule organizing centers (MTOCs) comparable to animal centrosomes, and the organization of microtubule arrays in plants has remained enigmatic. Spindle development in basal land plants has revealed a surprising variety of MTOCs that may represent milestones in the evolution of the typical diffuse acentrosomal plant spindle. We have isolated and characterized the gamma-tubulin gene from a liverwort, one of the extant basal land plants. Sequence similarity to the gamma-tubulin gene of higher plants suggests that the gamma-tubulin gene is highly conserved in land plants. The G9 antibody to fission yeast gamma-tubulin recognized a single band of 55 kD in immunoblots from bryophytes. Immunohistochemistry with the G9 antibody clearly documented the association of gamma-tubulin with various MTOC sites in basal land plants (e.g., discrete centrosomes with and without centrioles and the plastid surface in monoplastidic meiosis of bryophytes). Changes in the distribution of gamma-tubulin occur in a cell cycle-specific manner during monoplastidic meiosis in the liverwort Dumortiera hirsuta. gamma-Tubulin changes its localization from the plastid surface in prophase I to the spindle, from the spindle to phragmoplasts and the nuclear envelope in telophase I, and back to the plastid surfaces in prophase II. In vitro experiments show that gamma-tubulin is detectable on the surface of isolated plastids and nuclei of D. hirsuta, and microtubules can be repolymerized from the isolated plastids. gamma-Tubulin localization patterns on plastid and nuclear surfaces are not affected by the destruction of microtubules by oryzalin. We conclude that gamma-tubulin is a highly conserved protein associated with microtubule nucleation in basal land plants and that it has a cell cycle-dependent distribution essential for the orderly succession of microtubule arrays.

Amino Acid Sequence↗

The Tangled1 gene is required for spatial control of cytoskeletal arrays associated with cell division during maize leaf development.

The cytoskeleton plays a major role in the spatial regulation of plant cell division and morphogenesis. Arrays of microtubules and actin filaments present in the cell cortex during prophase mark sites to which phragmoplasts and associated cell plates are guided during cytokinesis. During interphase, cortical microtubules are believed to influence the orientation of cell expansion by guiding the pattern in which cell wall material is laid down. Little is known about the mechanisms that regulate these cytoskeleton-dependent processes critical for plant development. Previous work showed that the Tangled1 (Tan1) gene of maize is required for spatial regulation of cytokinesis during maize leaf development but not for leaf morphogenesis. Here, we examine the cytoskeletal arrays associated with cell division and morphogenesis during the development of tan1 and wild-type leaves. Our analysis leads to the conclusion that Tan1 is required both for the positioning of cytoskeletal arrays that establish planes of cell division during prophase and for spatial guidance of expanding phragmoplasts toward preestablished cortical division sites during cytokinesis. Observations on the organization of interphase cortical microtubules suggest that regional influences may play a role in coordinating cell expansion patterns among groups of cells during leaf morphogenesis.

Actins↗

The kinesin ATK5 functions in early spindle assembly in Arabidopsis.

During cell division, the mitotic spindle partitions chromosomes into daughter nuclei. In higher plants, the molecular mechanisms governing spindle assembly and function remain largely unexplored. Here, live cell imaging of mitosis in Arabidopsis thaliana plants lacking a kinesin-14 (ATK5) reveals defects during early spindle formation. Beginning during prophase and lasting until late prometaphase, spindles of atk5-1 plants become abnormally elongated, are frequently bent, and have splayed poles by prometaphase. The period of spindle elongation during prophase and prometaphase is prolonged in atk5-1 cells. Time-lapse imaging of yellow fluorescent protein:ATK5 reveals colocalization with perinuclear microtubules before nuclear envelope breakdown, after which it congresses inward from the poles to the midzone, where it becomes progressively enriched at regions of overlap between antiparallel microtubules. In vitro microtubule motility assays demonstrate that in the presence of ATK5, two microtubules encountering one another at an angle can interact and coalign, forming a linear bundle. These data indicate that ATK5 participates in the search and capture of antiparallel interpolar microtubules, where it aids in generating force to coalign microtubules, thereby affecting spindle length, width, and integrity.

Arabidopsis↗

Localization of the Functional p34cdc2 Homolog of Maize in Root Tip and Stomatal Complex Cells: Association with Predicted Division Sites.

We have used an antibody against the functional homolog of the cdc2 kinase of maize to localize the p34cdc2 protein within dividing cells of the root apex and the stomatal complex of leaf epidermis. The microtubule cytoskeletal structure of plant cells was visualized concomitantly with a monoclonal antibody specific for [alpha]-tubulin. We found that the cdc2 protein is localized mainly to the nucleus in plant cells at interphase and early prophase. This finding contrasts markedly with the predominantly cytoplasmic staining obtained using antibody to the PSTAIRE motif, which is common to cdc2 and numerous cdc2-like proteins. In a subpopulation of root cells at early prophase, the p34cdc2 protein is also distributed in a band bisecting the nucleus. Double labeling with the maize p34cdc2Zm antibody and tubulin antibody revealed that this band colocalizes with the preprophase band (PPB) of microtubules, which predicts the future division site. Root cells in which microtubules had been disrupted with oryzalin did not contain this band of p34cdc2 protein, suggesting that formation of the microtubule PPB is necessary for localization of the p34cdc2 kinase to the plane of the PPB. The p34cdc2 protein is also localized to the nucleus and PPB in cells that give rise to the stomatal complex, including those cells preparing for the highly asymmetrical divisions that produce subsidiary cells. Association of the p34cdc2 protein with the PPB suggests that the cdc2 kinase has a role in establishing the division site of plant cells and, therefore, a role in plant morphogenesis.

Journal Article↗

The effect of environmental stress stimuli on cell division in rat palatal epithelium.

The effect on cell proliferation of procedures commonly used in studies in laboratory animals was examined in the palatal epithelium of rats. Groups of rats were exposed to ether anaesthesia, immobilization in the supine position or to cage change. Animals left undisturbed in the cages during the experiment were used as controls. At intervals of 30 min, four animals of each group were killed and histological sections of the palatal mucosa were prepared. The number of prophases, metaphases and ana/telophases in the epithelium was counted and related to 1 mm2 of the surface. Thirty minutes after the various procedures the number of metaphases and ana/telophases were extremely low. After a further 30 min a high number of prophases was observed. The number of metaphases rose within the following 30 min, during which time the number of ana/telophases also reached a maximum. These observations indicate that the environmental stimuli have blocked the entrance of cells into mitosis and have resulted in an accumulation of cells, which, following the raising of the blockade, pass through the various stages of the mitotic phase. In studies of cell population kinetics the blockade of cell division as a result of environmental stress stimuli may lead to misleading results.

Animals↗

A comparison between the morphology and cell kinetics of gonocytes and adult type undifferentiated spermatogonia in the mouse.

A comparison has been made between gonocytes and adult type undifferentiated spermatogonia in the mouse. The following morphological resemblances were noted: 1. Proliferating gonocytes, at days 13 and 14 p.c., resemble proliferating undifferentiated spermatogonia between stages IX and IV in the adult. 2. Gonocytes in G1 arrest, from day 15 p.c. until birth, resemble undifferentiated spermatogonia in G1 arrest from stage IV until stage VIII. Both populations of cells undergo the same morphological changes during this period, known to accompany transformation of most Aal spermatogonia into A1 spermatogonia in the adult. 3. Gonocytes in late G2 or prophase, 24 h p.p., resemble A1 spermatogonia in late G2 and prophase in stage IX. 4. Most of the daughter cells of the gonocytes resemble A2 spermatogonia, the others resembling adult undifferentiated spermatogonia. The hypothesis is put forward that gonocytes are identical to adult type undifferentiated spermatogonia. The primordial germ cells then give rise to adult type spermatogonial stem cells (As).

Animals↗

Expression of Mina53, a product of a Myc target gene in mouse testis.

Recently we have identified a novel gene mina53 (mina), which is a direct transcriptional target of oncoprotein Myc. Mina53 protein was shown to be highly expressed in tumour cells and to play a role in cell proliferation. Here we report the expression of Mina53 in mouse testis, which contains proliferating cells and expresses many cancer-related genes. Immunohistochemical studies by using newly produced monoclonal antibody to Mina53 showed that Mina53 was expressed in the nuclei of spermatogonia. Mina53 was also expressed in meiotic prophase cells such as preleptotene, leptotene and zygotene, and weakly in early pachytene spermatocytes, but was absent in late pachytene spermatocytes, spermatids and mature sperm. The expression pattern of Mina53 was quite similar to that of proliferation cell nuclear antigen (PCNA). Using experimental cryptorchid testis, it was found that Mina53 was highly expressed in undifferentiated spermatogonia, which were PCNA-positive. These results suggest that Mina53 is prominently expressed in proliferating, undifferentiated spermatogonia, and plays a role in cell proliferation from the spermatogonial stage to the meiotic prophase in spermatogenesis, but not in meiotic divisions per se.

Animals↗

Histone deacetylation is required for progression through mitosis in tobacco cells.

Post-translational modifications of core histone proteins play a key role in chromatin structure and function. Here, we study histone post-translational modifications during reentry of protoplasts derived from tobacco mesophyll cells into the cell cycle and evaluate their significance for progression through mitosis. Methylation of histone H3 at lysine residues 4 and 9 persisted in chromosomes during all phases of the cell cycle. However, acetylation of H4 and H3 was dramatically reduced during mitosis in a stage-specific manner; while deacetylation of histone H4 commenced at prophase and persisted up to telophase, histone H3 remained acetylated up to metaphase but was deacetylated at anaphase and telophase. Phosphorylation of histone H3 at serine 10 was initiated at prophase, concomitantly with deacetylation of histone H4, and persisted up to telophase. Preventing histone deacetylation by the histone deacetylase inhibitor trichostatin A (TSA) led to accumulation of protoplasts at metaphase-anaphase, and reduced S10 phosphorylation during anaphase and telophase; in cultured tobacco cells, TSA significantly reduced the frequency of mitotic figures. Our results indicate that deacetylation of histone H4 and H3 in tobacco protoplasts occurs during mitosis in a phase-specific manner, and is important for progression through mitosis.

Acetylation↗

Cytoplasmic events in human meiotic arrest as revealed by immunolabelling of spermatocyte proacrosin.

Proacrosin appears in the Golgi complex as early as the mid-pachytene stage and immediately thereafter initiates partition to be equally distributed in spermatids. The anti-proacrosin monoclonal antibody 4D4 (mAb 4D4) was used as a marker of these cytoplasmic events in ten men exhibiting spermatogenesis arrest in three specific stages: (i) leptotene/zygotene spermatocyte I with impaired chromosome pairing (six cases), (ii) early pachytene I (one case) and (iii) metaphase/anaphase I (three cases). Prophase arrest stages were identified on testis sections stained by silver nitrate. MAb 4D4 labelling revealed two types of leptotene/zygotene arrest depending on whether proacrosin was expressed or not. The data obtained enabled us to distinguish between: (i) nuclear blockages due to chromosome and/or nuclear matrix anomalies, when cytoplasmic events were either inhibited or not inhibited, and (ii) nuclear anomalies due to microtubular disturbances. In this latter case, cytokinesis was impaired as early as the prophase I, thus indicating a relationship between the Golgi partitioning and the microtubule network. Data show that meiotic arrest investigations, by means of an appropriate marker of the cytoplasmic events, provide valuable information on spermatogenic developmental processes.

Acrosin↗

Association of calmodulin with nuclear structures in starfish oocytes and its role in the resumption of meiosis.

The resumption of meiosis in prophase-arrested starfish oocytes is induced by the hormone 1-methyladenine, which has been shown previously to induce a calcium transient in the nucleus which at this stage is called the germinal vesicle. This transient precedes the breakdown of the germinal vesicle (GVBD). Experiments were performed to establish whether nuclear calmodulin (CaM) was involved in the progression of the meiotic cycle. CaM antagonists, antibodies, and an inhibitory peptide corresponding to the CaM-binding domain of myosin-light-chain kinase have been injected into the nucleus of prophase-arrested starfish oocytes. The antagonists failed to affect the final response to 1-methyladenine, i.e. GVBD, although two antagonists delayed it, whereas the peptide inhibitor and the antibodies completely inhibited it. The antibodies suppressed the nuclear Ca2+ spikes that were shown by previous work to be induced by the photoreleasing of caged adenosine 3',5'-(cyclic)diphosphate ribose in the germinal vesicle. Immunofluorescence staining of isolated starfish oocyte nuclei with CaM antibodies showed CaM in the envelope and in the nucleolus. Immunogold labelling of oocytes revealed aggregates of CaM and of a 36-kDa protein, of the heterogeneous ribonucleoprotein particles (hnRNP), in electron-dense hnRNP in the nuclear matrix. 1-Methyladenine induced the disappearance of these hnRNP from the nucleoplasm and the translocation of CaM and the 36-kDa protein previously associated with them to the cytoplasm, prior to the breakdown of the nuclear envelope.

Adenine↗

Sperm binding capacity of human zona pellucida derived from oocytes obtained from different sources.

The important contributions of sperm-oocyte interaction to infertility diagnostics is well established. Scientists are urged to search for methods to improve the assessment of gamete interaction. Sperm binding and penetration assays have frequented the literature, reporting on various aspects of sperm-oocyte interaction using either microbisected or whole human oocytes during the assay procedure. The objective of the study was to evaluate additional zona pellucida sources which can be used during zona binding studies. Hemizonae were obtained from the following oocytes: 1) experiment 1, prophase I oocytes from post-mortem ovarian tissue from different age groups namely, 7 months, 5 years, 7 years, 12 years and 30 years; 2) experiment 2 used donated immature Prophase I oocytes from the IVF treatment program and 3) experiment 3 evaluated zona binding for hemizonae which were previously used in hemizona assays. Results indicated that, in experiment 1, ovarian age does not have any influence on the zona pellucida's capacity to bind spermatozoa. The mean number of bound sperm among the different age groups did not differ significantly, namely 38.9 +/- 17 (7 months), 31.0 +/- 27 (5 years), 49.3 +/- 21 (7 years), 32.8 +/- 18 (12 years) and 39.5 +/- 17 (30 years). The pooled mean +/- SD binding for all the age groups in experiment 1 was 37.7 +/- 7. Likewise, the mean number of sperm bound (experiment 2) to zonae collected from oocytes using different ovulation induction regimes were 31.1 +/- 20 (unstimulated), 54.4 +/- 12 (HMG/HCG) and 15.3 +/- 9 (HMG alone).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Sumoylation of a meiosis-specific RecA homolog, Lim15/Dmc1, via interaction with the small ubiquitin-related modifier (SUMO)-conjugating enzyme Ubc9.

Sumoylation is a post-translational modification system that covalently attaches the small ubiquitin-related modifier (SUMO) to target proteins. Ubc9 is required as the E2-type enzyme for SUMO-1 conjugation to targets. Here, we show that Ubc9 interacts with the meiosis-specific RecA homolog, Lim15/Dmc1 in the basidiomycete Coprinus cinereus (CcLim15), and mediates sumoylation of CcLim15 during meiosis. In vitro protein-protein interaction assays revealed that CcUbc9 interacts with CcLim15 and binds to the C-terminus (amino acids 105-347) of CcLim15, which includes the ATPase domain. Immunocytochemistry demonstrates that CcUbc9 and CcLim15 colocalize in the nuclei from the leptotene stage to the early pachytene stage during meiotic prophase I. Coimmunoprecipitation experiments indicate that CcUbc9 interacts with CcLim15 in vivo during meiotic prophase I. Furthermore, we show that CcLim15 is a target protein of sumoylation both in vivo and in vitro, and identify the C-terminus (amino acids 105-347) of CcLim15 as the site of sumoylation in vitro. These results suggest that sumoylation is a candidate modulator of meiotic recombination via interaction between Ubc9 and Lim15/Dmc1.

Adenosine Triphosphatases↗

Regulation of the meiotic cycle in oocytes of domestic mammals.

The progression of meiosis from prophase to metaphase II involves modifications to somatic signals, activation of a master regulator system in the cytoplasm and the manipulation of independent components in the nucleus. Studies on amphibian oocytes suggest that the cytoplasmic component, maturation promoting factor (MPF), is a key component of the master regulator system. A similar and ubiquitous role for MPF in mammalian oocytes is indicated by cell fusion studies. However, the nature of the somatic signals responsible for prophase arrest and the sequence of intracellular events necessary for the synthesis and activation of MPF differ markedly between species. The most fundamental of these differences is in the G2- to M-phase transition where, in rodents, phosphorylation changes alone activate the MPF cascade while in domestic mammals both transcription and new protein synthesis is required for MPF production. One of the proteins (47,000 daltons) involved in the MPF cascade has been tentatively identified in ovine oocytes and has been shown to be dependent upon transcription at the initiation of maturation. The synthesis of additional protein, probably with anti-MPF activity, is thereafter required to drive the cycle from metaphase I to anaphase. The final phase of cell cycle-related protein synthesis occurs immediately before the formation of the metaphase II plate. The Ca2+-sensitive cytostatic proteins synthesized at this stage maintain the MII chromatin in a condensed state and prevent the completion of the meiotic cycle until fertilization when the cytostatic proteins disappear and meiosis is completed.

Animals↗

NDT80, a meiosis-specific gene required for exit from pachytene in Saccharomyces cerevisiae.

We describe the identification of a new meiosis-specific gene of Saccharomyces cerevisiae, NDT80. The ndt80 null and point mutants arrest at the pachytene stage of meiosis, with homologs connected by full-length synaptonemal complexes and spindle pole bodies duplicated but unseparated. Meiotic recombination in an ndt80 delta mutant is relatively normal, although commitment to heteroallelic recombination is elevated two- to threefold and crossing over is decreased twofold compared with those of the wild type. ndt80 arrest is not alleviated by mutations in early recombination genes, e.g., SPO11 or RAD50, and thus cannot be attributed to an intermediate block in prophase chromosome metabolism like that observed in several other mutants. The ndt80 mutant phenotype during meiosis most closely resembles that of a cdc28 mutant, which contains a thermolabile p34, the catalytic subunit of maturation-promoting factor. Cloning and molecular analysis reveal that the NDT80 gene maps on the right arm of chromosome VIII between EPT1 and a Phe-tRNA gene, encodes a 627-amino-acid protein which exhibits no significant homology to other known proteins, and is transcribed specifically during middle meiotic prophase. The NDT80 gene product could be a component of the cell cycle regulatory machinery involved in the transition out of pachytene, a participant in an unknown aspect of meiosis sensed by a pachytene checkpoint, or a SPO11- and RAD50-independent component of meiotic chromosomes that is the target of cell cycle signaling.

Amino Acid Sequence↗

An alternative splicing event which occurs in mouse pachytene spermatocytes generates a form of DNA ligase III with distinct biochemical properties that may function in meiotic recombination.

Three mammalian genes encoding DNA ligases have been identified. However, the role of each of these enzymes in mammalian DNA metabolism has not been established. In this study, we show that two forms of mammalian DNA ligase III, alpha and beta, are produced by a conserved tissue-specific alternative splicing mechanism involving exons encoding the C termini of the polypeptides. DNA ligase III-alpha cDNA, which encodes a 103-kDa polypeptide, is expressed in all tissues and cells, whereas DNA ligase III-beta cDNA, which encodes a 96-kDa polypeptide, is expressed only in the testis. During male germ cell differentiation, elevated expression of DNA ligase III-beta mRNA is restricted, beginning only in the latter stages of meiotic prophase and ending in the round spermatid stage. In 96-kDa DNA ligase III-beta, the C-terminal 77 amino acids of DNA ligase III-alpha are replaced by a different 17- to 18-amino acid sequence. As reported previously, the 103-kDa DNA ligase III-alpha interacts with the DNA strand break repair protein encoded by the human XRCC1 gene. In contrast, the 96-kDa DNA ligase III-beta does not interact with XRCC1, indicating that DNA ligase III-beta may play a role in cellular functions distinct from the DNA repair pathways involving the DNA ligase III-alpha x XRCC1 complex. The distinct biochemical properties of DNA ligase III-beta, in combination with the tissue- and cell-type-specific expression of DNA ligase III-beta mRNA, suggest that this form of DNA ligase III is specifically involved in the completion of homologous recombination events that occur during meiotic prophase.

Alternative Splicing↗