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1.
Precise timing coordinates cell proliferation with embryonic morphogenesis. As Drosophila melanogaster embryos approach cell cycle 14 and the midblastula transition, rapid embryonic cell cycles slow because S phase lengthens, which delays mitosis via the S-phase checkpoint. We probed the contributions of each of the three mitotic cyclins to this timing of interphase duration. Each pairwise RNA interference knockdown of two cyclins lengthened interphase 13 by introducing a G2 phase of a distinct duration. In contrast, pairwise cyclin knockdowns failed to introduce a G2 in embryos that lacked an S-phase checkpoint. Thus, the single remaining cyclin is sufficient to induce early mitotic entry, but reversal of the S-phase checkpoint is compromised by pairwise cyclin knockdown. Manipulating cyclin levels revealed that the diversity of cyclin types rather than cyclin level influenced checkpoint reversal. We conclude that different cyclin types have distinct abilities to reverse the checkpoint but that they collaborate to do so rapidly. 相似文献
2.
To ask what controls the periodic accumulation and destruction of the mitotic across the cell cycle, we have developed a cell-free system from clam embryos that reproduces several aspects of cyclin behavior. One or more rounds of cyclin proteolysis and resynthesis occur in vitro, and the destruction of the cyclins is highly specific. The onset, duration, and extent of cyclin destruction and the appropriately stagered disappearance of cyclin A and cyclin B are correctly regulated during the first cycle in the cell-free system. Just as in intact cells, lysates made from early interphase cells require further protein synthesis to reach the cyclin destruction point, and lysates made from later stages do not. Using the cell-free system we show that cyclin disappearance requires ATP and Mg2+. By combining lysates from different cell cycle stages, we show that (a) interphase lysates do not contain a dominant inhibitor of cyclin destruction and (b) the timing of cyclin destruction is determined by the cell cycle stage of the cytoplasm rather than the cell cycle stage of the substrate cyclins themselves. Among a large variety of agents tested, only a few affect cyclin destruction. Tosyl-lysine chlormethyl ketone (TLCK, a protease inhibitor), 6-dimethylaminopurine (6-DMAP, a kinase inhibitor), certain sulfhydryl-blocking agents, ZnCl2 and EDTA (but not EGTA) completely block cyclin destruction in vitro. Addition of 1 mM Ca2+ to the cell-free system has no effect on cyclin stability, but 5 mM Ca2+ leads to the rapid destruction of cyclins and a small number of other proteins. 相似文献
3.
R M Amikura M Ihnuma E Aikawa H Yamada H Nagano 《Molecular reproduction and development》1991,29(3):245-252
When sea urchin embryos were subjected to nucleolar organizer region (NOR)-silver staining, densely stained particles were observed in the cytoplasm. The appearance of these cytoplasmic particles (CPs) was cell-cycle dependent. During early development, the CPs were detected at interphase, but not during mitosis; they disappeared at metaphase and reappeared at telophase. The CPs appeared periodically even when embryos were treated with cytochalasin B or aphidicolin, which inhibits the progression of cytokinesis and nuclear division, respectively. By contrast, CPs were not detected in the colchicine-treated embryos in which both cytokinesis and nuclear divisions were prevented. The CPs were observed only in the embryos whose stage was early blastula (about 6th to 7th cleavage) or earlier; no CPs were detected even at interphase in the embryos at late blastula (about 8th to 9th cleavage) or later. Electron microscopic evaluation showed CPs to be granular structures, similar to heavy bodies. Also, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) showed that 95-kDa and 38-kDa proteins were the NOR-silver-staining proteins in sea urchin embryos. These proteins existed during the course of the cell cycles. These results suggest that (1) the cyclic appearance of the CPs or heavy bodies is closely related to the cell cycle as well as the programming of the embryogenesis, but independent of the cycle of cytokinesis and nuclear division; (2) 95-kDa and 38-kDa proteins are the major NOR-silver-staining proteins in sea urchin embryos. 相似文献
4.
MELK (maternal embryonic leucine zipper kinase) is a cell cycle dependent protein kinase involved in diverse cell processes including cell proliferation, apoptosis, cell cycle and mRNA processing. Noticeably, MELK expression is increased in cancerous tissues, upon cell transformation and in mitotically-blocked cells. The question of how MELK protein level is controlled is therefore important. Here, we show that MELK protein is restricted to proliferating cells derived from either cancer or normal tissues and that MELK protein level is severely decreased concomitantly with other cell cycle proteins in cells which exit the cell cycle. Moreover, we demonstrate in human HeLa cells and Xenopus embryos that approximately half of MELK protein is degraded upon mitotic exit whereas another half remains stable during interphase. We show that the stability of MELK protein in M-phase is dependent on its phosphorylation state. 相似文献
5.
In the present study, the sequential expression and cellular localization of cyclin B1 was examined in two-cell mouse embryos to elucidate the mechanism of the two-cell block. One-cell embryos derived from in vitro fertilization were cultured with oviductal tissue (nonblocking condition) or without oviductal tissue (blocking condition) to establish the experimental conditions in which the embryos either overcome the two-cell block or do not. The amount of cyclin B1 gradually increased through the second cell cycle (through S to G2 phase). However, the difference was not observed between culture conditions. This showed that even embryos exhibiting the two-cell block normally synthesize cyclin B1 through the cell cycle. Cyclin B1 in embryos cultured under nonblocking condition accumulates in the nucleus during the transition from the G2 to the M phase, whereas that in embryos cultured in blocking condition localizes in the cytoplasm throughout the cell cycle. These data indicate that two-cell embryos cultured in blocking condition are able to normally synthesize cyclin B1 but have defects in nuclear accumulation of the protein. However, when two-cell blocked embryos were treated with okadaic acid, an activator of Cdc2 kinase, part of cyclin B1 in the embryos translocated into the nucleus. Moreover, treatment with butyrolactone I, a specific inhibitor of Cdc2 kinase, inhibits nuclear translocation of cyclin B1 in those embryos. These results suggest that Cdc2 kinase regulates the nuclear accumulation of cyclin B1 in mouse two-cell embryos. 相似文献
6.
The importin alpha family of nuclear-cytoplasmic transport factors mediates the nuclear localization of proteins containing classical nuclear localization signals. Metazoan animals express multiple importin alpha proteins, suggesting their possible roles in cell differentiation and development. Adult Caenorhabditis elegans hermaphrodites express three importin alpha proteins, IMA-1, IMA-2, and IMA-3, each with a distinct expression and localization pattern. IMA-2 was expressed exclusively in germ line cells from the early embryonic through adult stages. The protein has a dynamic pattern of localization dependent on the stage of the cell cycle. In interphase germ cells and embryonic cells, IMA-2 is cytoplasmic and nuclear envelope associated, whereas in developing oocytes, the protein is cytoplasmic and intranuclear. During mitosis in germ line cells and embryos, IMA-2 surrounded the condensed chromosomes but was not directly associated with the mitotic spindle. The timing of IMA-2 nuclear localization suggested that the protein surrounded the chromosomes after fenestration of the nuclear envelope in prometaphase. Depletion of IMA-2 by RNA-mediated gene interference (RNAi) resulted in embryonic lethality and a terminal aneuploid phenotype. ima-2(RNAi) embryos have severe defects in nuclear envelope formation, accumulating nucleoporins and lamin in the cytoplasm. We conclude that IMA-2 is required for proper chromosome dynamics in germ line and early embryonic mitosis and is involved in nuclear envelope assembly at the conclusion of mitosis. 相似文献
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Qing Kang Jeyaraman Srividhya Joseph Ipe Joseph R. Pomerening 《The Journal of biological chemistry》2014,289(25):17480-17496
The mitotic kinase Aurora A (AurA) is regulated by a complex network of factors that includes co-activator binding, autophosphorylation, and dephosphorylation. Dephosphorylation of AurA by PP2A (human, Ser-51; Xenopus, Ser-53) destabilizes the protein, whereas mitotic dephosphorylation of its T-loop (human, Thr-288; Xenopus, Thr-295) by PP6 represses AurA activity. However, AurA(Thr-295) phosphorylation is restricted throughout the early embryonic cell cycle, not just during M-phase, and how Thr-295 is kept dephosphorylated during interphase and whether or not this mechanism impacts the cell cycle oscillator were unknown. Titration of okadaic acid (OA) or fostriecin into Xenopus early embryonic extract revealed that phosphatase activity other than PP1 continuously suppresses AurA(Thr-295) phosphorylation during the early embryonic cell cycle. Unexpectedly, we observed that inhibiting a phosphatase activity highly sensitive to OA caused an abnormal increase in AurA(Thr-295) phosphorylation late during interphase that corresponded with delayed cyclin-dependent kinase 1 (CDK1) activation. AurA(Thr-295) phosphorylation indeed influenced this timing, because AurA isoforms retaining an intact Thr-295 residue further delayed M-phase entry. Using mathematical modeling, we determined that one phosphatase would be insufficient to restrict AurA phosphorylation and regulate CDK1 activation, whereas a dual phosphatase topology best recapitulated our experimental observations. We propose that two phosphatases target Thr-295 of AurA to prevent premature AurA activation during interphase and that phosphorylated AurA(Thr-295) acts as a competitor substrate with a CDK1-activating phosphatase in late interphase. These results suggest a novel relationship between AurA and protein phosphatases during progression throughout the early embryonic cell cycle and shed new light on potential defects caused by AurA overexpression. 相似文献
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10.
Nuclei of differentiated cells can acquire totipotency following transfer into the cytoplasm of oocytes. While the molecular basis of this nuclear reprogramming remains unknown, the developmental potential of nuclear-transfer embryos is influenced by the cell-cycle stage of both donor and recipient. As somatic H1 becomes immunologically undetectable on bovine embryonic nuclei following transfer into ooplasm and reappears during development of the reconstructed embryo, suggesting that it may act as a marker of nuclear reprogramming, we investigated the link between cell-cycle state and depletion of immunoreactive H1 following nuclear transplantation. Blastomere nuclei at M-, G1-, or G2-phase were introduced into ooplasts at metaphase II, telophase II, or interphase, and the reconstructed embryos were processed for immunofluorescent detection of somatic histone H1. Immunoreactivity was lost more quickly from donor nuclei at metaphase than at G1 or G2. Regardless of the stage of the donor nucleus, immunoreactivity was lost most rapidly when the recipient cytoplast was at metaphase and most slowly when the recipient was at interphase. When the recipient oocyte was not enucleated, however, immunoreactive H1 remained in the donor nucleus. The phosphorylation inhibitors 6-DMAP, roscovitine, and H89 inhibited the depletion of immunoreactive H1 from G2, but not G1, donor nuclei. In addition, immunoreactive H1 was depleted from mouse blastomere nuclei following transfer into bovine oocytes. Finally, expression of the developmentally regulated gene, eIF-1A, but not of Gapdh, was extinguished in metaphase recipients but not in interphase recipients. These results indicate that evolutionarily conserved cell-cycle-regulated activities, nuclear elements, and phosphorylation-linked events participate in the depletion of immunoreactive histone H1 from blastomere nuclei transferred in oocyte cytoplasm and that this is linked to changes in gene expression in the transferred nucleus. 相似文献
11.
Shinozawa T Mizutani E Tomioka I Kawahara M Sasada H Matsumoto H Sato E 《Molecular reproduction and development》2004,68(3):313-318
In the present study, we examined the developmental ability of enucleated zygotes, MII oocytes, and parthenogenetically activated oocytes at pronuclear stages (parthenogenetic PNs) as recipient cytoplasm for rat embryonic cell nuclear transfer. Enucleated zygotes as recipient cytoplasm receiving two-cell nuclei allowed development to blastocysts, whereas the development of embryos reconstituted with MII oocytes and parthenogenetic PNs was arrested at the two-cell stage. Previous observations in rat two-cell embryos suggested that the distribution of microtubules is involved in two-cell arrest. Therefore, we also examined the distribution of microtubules using immunofluorescence. At the two-cell stage after nuclear transfer into enucleated zygotes, microtubules were distributed homogeneously in the cytoplasm during interphase, and normal mitotic spindles were observed in cleaving embryos from the two- to four-cell stage. In contrast, embryos reconstituted with MII oocytes and parthenogenetic PNs showed aberrant microtubule organization. In enucleated zygotes, fibrous microtubules were distributed homogeneously in the cytoplasm. In contrast, dense microtubules were localized at the subcortical area in the cytoplasm and strong immunofluorescence intensity was observed at the plasma membrane, while very weak intensity was detected in the central part of enucleated MII oocytes. In enucleated parthenogenetic PNs, high-density and fibrous microtubules were distributed in the subcortical and central areas, respectively. Pre-enucleated parthenogenetic PNs also showed lower intensity of microtubule immunofluorescence in the central cytoplasm than zygotes. In conclusion, the results of the present study showed that zygote cytoplasm is better as recipient than MII oocyte and parthenogenetic PNs for rat two-cell embryonic cell nuclear transfer to develop beyond four-cell stage. Furthermore, microtubule organization is involved in the development of reconstituted embryos to overcome the two-cell arrest. 相似文献
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B G Gabrielli M S Lee D H Walker H Piwnica-Worms J L Maller 《The Journal of biological chemistry》1992,267(25):18040-18046
The Xenopus cdk2 gene encodes a 32-kDa protein kinase with sequence similarity to the 34-kDa product of the cdc2 gene. Previous studies have shown that the kinase activity of the protein product of the cdk2 gene oscillates in the Xenopus embryonic cell cycle with a high in M-phase and a low in interphase. In the present study cdk2 was found not to be associated with any newly synthesized proteins during the cell cycle, but the enzyme did undergo periodic changes in phosphorylation. Upon exit from metaphase, cdk2 became increasingly phosphorylated on both tyrosine and serine residues, and labeling on these residues increased progressively until entry into mitosis, when tyrosine residues were markedly dephosphorylated. Phosphopeptide mapping of cdk2 demonstrated the major sites of phosphorylation were in a phosphopeptide with a pI of 3.7 that contained both phosphoserine and phosphotyrosine. This phosphopeptide accumulated in egg extracts blocked in S-phase with aphidicolin and was not evident in cdc2 immunoprecipitated under the same conditions. Under the same conditions cdc2 was phosphorylated primarily on a phosphopeptide containing both phosphothreonine and phosphotyrosine residues, most likely threonine 14 and tyrosine 15. Affinity-purified human GST-cdc25 was able to dephosphorylate and activate cdk2 isolated from interphase cells. Phosphopeptide mapping demonstrated that the phosphate was specifically removed from the same phosphopeptide identified as the major in vivo site of phosphorylation. These results demonstrate that cdk2 is regulated in the cell cycle by phosphorylation and dephosphorylation on both serine and tyrosine residues. Moreover, the increased phosphorylation of cdk2 in aphidicolin-blocked extracts and the ability of cdc25 to mediate cdk2 dephosphorylation in vitro suggest the possibility that cdk2 is part of the mechanism ensuring mitosis is not initiated until completion of DNA replication. It also implies cdc25 may have other functions in addition to the regulation of cdc2 kinase activity. 相似文献
15.
Mouse embryos of the ddY strain fertilized in vitro undergo the first cleavage to the 2-cell stage but not the second cleavage even 45 hr after insemination (2-cell block). We examined the phosphorylation state of p34cdc2 and histone H1 kinase activity in mouse 2-cell embryos to investigate the relationship of p34cdc2 with 2-cell block. In the first mitotic cell cycle, the amount of phosphorylated forms of p34cdc2, which were detected as the bands of retarded mobility on SDS-PAGE followed by immunoblotting with anti-p34cdc2 antibody, increased during interphase and abruptly decreased at M phase. Concomitant with this dephosphorylation, histone H1 kinase activity was increased. After the embryos cleaved to the 2-cell stage, the amounts of phosphorylated forms of p34cdc2 increased up to 33 hr after insemination. However, the activation of histone H1 kinase did not occur and the states of phosphorylation of p34cdc2 did not show any significant changes until 45 hr. In contrast, 2-cell embryos of B6C3F1 mice, which do not show a 2-cell block and develop normally to blastocysts in vitro, exhibit the dephosphorylation of p34cdc2 and an increase in histone H1 kinase activity between 31 and 45 hr after insemination. When the ddY mouse embryos arrested at the 2-cell stage were treated with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, the dephosphorylation of p34cdc2 occurred and histone H1 kinase activity increased. The chromosomes of these embryos stained with 4',6'-diamidino-2-phenylindole revealed the initiation of condensation. These results suggest that 2-cell-blocked embryos contain enough p34cdc2 to induce mitotic events but the protein remains in a latent form. 相似文献
16.
Bovine oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic cell nuclei from various mammalian species. 总被引:25,自引:0,他引:25
T Dominko M Mitalipova B Haley Z Beyhan E Memili B McKusick N L First 《Biology of reproduction》1999,60(6):1496-1502
The transfer of nuclei from one cell to another provides a powerful tool for studying the interactions between the cytoplasm of one cell and the nucleus of another. This study was designed to examine the ability of the bovine metaphase oocyte cytoplasm to support mitotic cell cycles under the direction of differentiated somatic cell nuclei of various mammalian species. Skin fibroblast cells from cows, sheep, pigs, monkeys, and rats were used as sources of donor nuclei. Nuclear transfer units produced by fusion of enucleated bovine oocytes and individual fibroblasts from all species examined underwent transition to interphase accompanied by nuclear swelling, further progression through the cell cycle, and completion of the first mitosis. Regardless of the species of donor fibroblasts used, some cleaving units progressed further and developed to advanced stages, as evidenced by continuation of cell proliferation and formation of a blastocoele cavity at the time appropriate for the donor fibroblast species. Although no pregnancies have been carried to term after transfer of embryos into surrogate animals, these observations suggest that mechanisms regulating early embryonic development may be conserved among mammalian species and that bovine oocyte cytoplasm can support the introduced differentiated nucleus regardless of chromosome number, species, or age of the donor fibroblast. 相似文献
17.
In vitro regulation of granulosa cell differentiation. Involvement of cytoskeletal protein expression 总被引:9,自引:0,他引:9
The link between the biochemical and morphological differentiation of granulosa cells was studied by investigating the organization and the expression of cytoskeletal proteins which determine cell shape and contacts. In cells treated with follicle-stimulating hormone (FSH), in a serum- and growth factor-free medium, or with other compounds which elevate cellular cAMP levels, the synthesis of the adherens junction proteins, vinculin, alpha-actinin, and actin was reduced significantly when compared to unstimulated cells (7-fold for vinculin, 5-fold for alpha-actinin, and 3-fold for actin). The in vitro translatability of the mRNAs coding for these proteins and the level of actin mRNA determined by RNA blot hybridization were generally reduced in differentiating cells. The synthesis and the organization of vimentin and tubulin was unaffected during this process, whereas the organization of actin and vinculin was dramatically affected, with FSH-treated cells displaying a diffuse pattern of actin and vinculin, with very little vinculin in adhesion plaques. Gonadotropin-releasing hormone agonist and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate which are known to antagonize the cAMP-mediated biochemical differentiation of granulosa cells by reducing cAMP levels or by activating protein kinase C and phospholipid turnover, blocked to a large extent the FSH-induced effect on the adherens junction proteins. Epidermal growth factor, which blocked the FSH-induced cAMP increase, but not the FSH-induced progesterone production, failed to block the synthesis of vinculin, alpha-actinin, and actin. Cytochalasin B could induce steroidogenesis and similar changes in the synthesis of these cytoskeletal proteins, whereas fibronectin, which causes cell spreading, blocked in part the FSH-induced effect on the expression of cytoskeletal proteins. The modulation of cytoskeletal proteins may therefore be an essential feature of programmed differentiation events leading to the final phenotype of granulosa cells. 相似文献
18.
Ultrastructural changes of the nucleolus in mitotic embryonic ectodermal cells of 7 1/2-day and 7 2/3-day rat embryos were examined. It was found that the nucleolus was broken down into small fragments during late prophase and metaphase, and that some of these fragments persisted in the cytoplasm of telophase cell (persistent nucleoli). No interphase embryonic ectodermal cells contained persistent nucleoli. Persistent nucleoli were also found in telophase cells of extraembryonic ectoderm, extraembryonic visceral endoderm and parietal endoderm of the embryos, but they disappeared in interphase cells. Persistent nucleoli in telophase cells tended to decrease in size with embryonic age, and they had almost completely disappeared in neuroectodermal cells of the telencephalon in 14 1/2-day embryos. They were concluded to be remnants of disappearing nucleoli in embryonic cells that were cycling too rapidly to permit their nucleoli to disappear completely. 相似文献
19.
Onset of nucleolus organizer activity in early mouse embryogenesis and evidence for its regulation 总被引:2,自引:0,他引:2
Ovulated mouse oocytes and preimplantation embryos were examined for NOR activity by means of selective silver staining. Evidence of the first staining activity appeared in two cell embryos, which was later followed by an increase in nucleolar activity, whereas the ovulated oocytes and pronuclei showed no such activity whatsoever. The staining of chromosomes was restricted to the nucleolus organizing region. Our results agree with earlier observations that genes for ribosomal RNA (rRNA) are transcribed as early as in the 2-cell stage in mouse embryogenesis. In addition to the nuclear staining we also observed some silver staining within the cytoplasm, at least from 4-cell stages onwards. Cytoplasmic staining was resistant to incubation with cycloheximide and actinomycin D. Nuclear staining was depressed, or even totally blocked, after actinomycin D incubation but was not blocked by cycloheximide. The onset of silver staining depends not on a specific embryonic stage but on the time interval following ovulation. This appears to indicate that the initiation of ribosomal cistrons is regulated by molecules which are activated or synthesized within the oocyte soon after ovulation. 相似文献
20.
Nava R. Shochet Amira Rudi Yoel Kashman Yaacov Hod M. Raafat El-Maghrabi Ilan Spector 《Journal of cellular physiology》1993,157(3):481-492
Six novel alkaloids that contain a fused tetracyclic pyrido[2,3,4-kl]acridine ring system were purified recently from the Red Sea purple tunicate Eudistoma sp. Evaluation of the effects of these alkaloids on cultured neuroblastoma and fibroblast cells revealed that they possess potent growth regulatory properties, and affect cell shape and adhesion. In mouse neuroblastoma cells, the Eudistoma alkaloids inhibited cell proliferation and induced a process of differentiation during which the cels flattened onto the surface, increased considerably in size, and extended long neurites. In hamster fibroblasts the alkaloids slowed down cell multiplication, and caused an exceptional cell flattening or elongation. In a virustransformed derivative of the hamster fibroblasts the alkaloids restored many aspects of normal cell growth and morphology. In addition, several of the alkaloids mimicked the effects of cAMP analogs on two well-characterized cAMP-mediated processes involved in hepatic glucose metabolism–inhibition of pyruvate kinase (PK) activity and induction of mRNA for phosphoenolpyruvate carboxykinase (PEPCK). All these effects suggest that the Eudistoma alkaloids may act on the cAMP signaling system. However, a single application of these compounds was sufficient to completely block cell multiplication and to induce and sustain differentiation and “reverse transformation”. Furthermore, these effects were not readily reversible following removal of the drugs. In contrast, a single application of agents that mimic or elevate cAMP induced a transient response that waned with time in culture, and the effects induced by constant elevation of cAMP reverse rapidly following drug removal. We propose that the Eudistoma alkaloids cause growth inhibition, differentiation, and reverse transformation by modifying the activity state of proteins that are involved in the regulation of cell shape and adhesion and serve as a target for the cAMP and/or other second messenger systems. © 1993 Wiley-Liss, Inc. 相似文献