scholarly journals Entry into Mitosis in Vertebrate Somatic Cells Is Guarded by a Chromosome Damage Checkpoint That Reverses the Cell Cycle When Triggered during Early but Not Late Prophase

1998 ◽  
Vol 142 (4) ◽  
pp. 1013-1022 ◽  
Author(s):  
Conly L. Rieder ◽  
Richard W. Cole
Keyword(s):  
Cell Cycle ◽  
Somatic Cells ◽  
Early Prophase ◽  
Checkpoint Control ◽  
Late Prophase ◽  
Cytoplasmic Microtubule ◽  
Nuclear Envelope Breakdown ◽  
Prophase Nucleus ◽  
Nuclear Events ◽  
A Cell

When vertebrate somatic cells are selectively irradiated in the nucleus during late prophase (<30 min before nuclear envelope breakdown) they progress normally through mitosis even if they contain broken chromosomes. However, if early prophase nuclei are similarly irradiated, chromosome condensation is reversed and the cells return to interphase. Thus, the G2 checkpoint that prevents entry into mitosis in response to nuclear damage ceases to function in late prophase. If one nucleus in a cell containing two early prophase nuclei is selectively irradiated, both return to interphase, and prophase cells that have been induced to returned to interphase retain a normal cytoplasmic microtubule complex. Thus, damage to an early prophase nucleus is converted into a signal that not only reverses the nuclear events of prophase, but this signal also enters the cytoplasm where it inhibits e.g., centrosome maturation and the formation of asters. Immunofluorescent analyses reveal that the irradiation-induced reversion of prophase is correlated with the dephosphorylation of histone H1, histone H3, and the MPM2 epitopes. Together, these data reveal that a checkpoint control exists in early but not late prophase in vertebrate cells that, when triggered, reverses the cell cycle by apparently downregulating existing cyclin-dependent kinase (CDK1) activity.

Nucleolus behaviour during the cell cycle of a primitive dinoflagellate eukaryote, Prorocentrum micans Ehr., seen by light microscopy and electron microscopy

1992 ◽  
Vol 102 (3) ◽  
pp. 475-485 ◽  
Author(s):  
MARIE-ODILE SOYER-GOBILLARD ◽  
MARIE-LINE GERAUD
Keyword(s):  
Cell Cycle ◽  
Light Microscopy ◽  
Early Prophase ◽  
Microscopy Observation ◽  
Prorocentrum Micans ◽  
Late Prophase ◽  
Daughter Cells ◽  
Nucleolar Material ◽  
Freeze Fixation ◽  
Higher Eukaryotes

Light-microscopy observation of the dinoflagellate Prorocentrum micans after silver-staining of the argyrophilic proteins of the nucleolar organizing region (Ag-NOR staining) showed the presence of nucleolar material throughout the vegetative cell cycle, and in particular during all the mitotic stages. This contrasts with the case in most higher eukaryotes, in which nucleoli disappear at the end of prophase and are reconstituted in daughter cells during telophase. Electron-microscope (EM) observations after conventional or fast-freeze fixation revealed that during interphase several functional nucleoli with three compartments (NORs, the fibrillogranular and the preribosomal granular compartments) are present in a nucleus in which the envelope is persistent and the chromosomes are always compact. During early prophase, when chromosomes are beginning to split, the nucleoli remain functional, whereas in late prophase they contain only a NOR and the granular component, and the chromosomes are surrounded by many protein masses. In early telophase, the nucleolar material coating the chromosomes migrates along with the chromosomes. Nucleologenesis occurs through the formation of prenucleolar bodies around lateral or telomeric nucleofilaments extruding from the chromosomes. Several chromosomes can contribute to the formation of one nucleolus. The behaviour of these ‘persistent nucleoli’ in a closed-nucleus model such as that of the dinoflagellates is discussed with regard to the higher eukaryotes.

Sperm nuclear envelope: breakdown of intrinsic envelope and de novo formation in hamster oocytes or eggs

Zygote ◽  
1997 ◽  
Vol 5 (1) ◽  
pp. 35-46 ◽  
Author(s):  
Noriko Usui ◽  
Atsuo Ogura ◽  
Yasuyuki Kimura ◽  
Ryuzo Yanagimachi
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
De Novo ◽  
Germinal Vesicle ◽  
Mature Oocyte ◽  
Sperm Nucleus ◽  
Sperm Chromatin ◽  
Early Prophase ◽  
Nuclear Envelope Breakdown ◽  
M Phase

SummaryDuring fertilisation of a fully mature oocyte, the sperm intrinsic nuclear envelope (SINE) disappears soon after sperm-oocyte fusion. A new nuclear envelope appears around the decondensed sperm chromatin when the oocyte reaches telophase II. Whether the SINE persists or rapidly disappears after sperm entery into immature oocytes or fertilised eggs has been controversial. Nuclear envelopes have been demonstrated around the sperm chromatin, which cannot be decondensed within the ooplasm of these oocytes or eggs, but whether these envelopes are persisting SINEs or newly formed envelopes has been apoint of dispute. To resolve this issue, the fate of the germinal vesicle stage(GV oocytes) or fertilised eggs at the pronuclear stage(PN eggs). The SINEs disappeared quikly within these oocytes or eggs, like those within maturing or mature oocytes, suggesting that the envelops around the sperm chromatin must be newly formed after SINE breakdown. To obtain further evidence, a detergent-treated, SINE-free sperm nucleus was injected into a PN egg. A new envelope appeared around the still-condensed or partially decondensed sperm chromatin within 3h after injection. Thus, disassembly of the SINE within ooplasm, unlike that of nuclear envelopes of other cells at prophase, is independent of the cell cycle stage of the oocyte or egg, whereas the ability of the ooplasm to assemble the new envelope is restricted to certain periods of the cycle. i.e. early prophase and telophase during meiosis and interphase, periods when active M-phase Promoting factor (MPF) is absent from the ooplasm.

Repetitive sperm-induced Ca2+ transients in mouse oocytes are cell cycle dependent

Development ◽  
1995 ◽  
Vol 121 (10) ◽  
pp. 3259-3266 ◽  
Author(s):  
K.T. Jones ◽  
J. Carroll ◽  
J.A. Merriman ◽  
D.G. Whittingham ◽  
T. Kono
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Dependent Manner ◽  
Nuclear Envelope Breakdown ◽  
Mouse Oocytes ◽  
A Cell ◽  
Mature Mouse ◽  
Cycle Dependent ◽  
Stage 1

Mature mouse oocytes are arrested at metaphase of the second meiotic division. Completion of meiosis and a block to polyspermy is caused by a series of repetitive Ca2+ transients triggered by the sperm at fertilization. These Ca2+ transients have been widely reported to last for a number of hours but when, or why, they cease is not known. Here we show that Ca2+ transients cease during entry into interphase, at the time when pronuclei are forming. In fertilized oocytes arrested at metaphase using colcemid, Ca2+ transients continued for as long as measurements were made, up to 18 hours after fertilization. Therefore sperm is able to induce Ca2+ transients during metaphase but not during interphase. In addition metaphase II oocytes, but not pronuclear stage 1-cell embryos showed highly repetitive Ca2+ oscillations in response to microinjection of inositol trisphosphate. This was explored further by treating in vitro maturing oocytes at metaphase I for 4–5 hours with cycloheximide, which induced nuclear progression to interphase (nucleus formation) and subsequent re-entry to metaphase (nuclear envelope breakdown). Fertilization of cycloheximide-treated oocytes revealed that continuous Ca2+ oscillations in response to sperm were observed after nuclear envelope breakdown but not during interphase. However interphase oocytes were able to generate Ca2+ transients in response to thimerosal. This data suggests that the ability of the sperm to trigger repetitive Ca2+ transients in oocytes is modulated in a cell cycle-dependent manner.

scholarly journals Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes.

1996 ◽  
Vol 135 (6) ◽  
pp. 1701-1713 ◽  
Author(s):  
H A Lane ◽  
E A Nigg
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Regulation ◽  
Fundamental Aspect ◽  
Early Prophase ◽  
Functional Maturation ◽  
A Cell ◽  
Gamma Tubulin ◽  
Cycle Regulation ◽  
Kinetics Of

Mammalian polo-like kinase 1 (Plk1) is structurally related to the polo gene product of Drosophila melanogaster, Cdc5p of Saccharomyces cerevisiae, and plo1+ of Schizosaccharomyces pombe, a newly emerging family of serine-threonine kinases implicated in cell cycle regulation. Based on data obtained for its putative homologues in invertebrates and yeasts, human Plk1 is suspected to regulate some fundamental aspect(s) of mitosis, but no direct experimental evidence in support of this hypothesis has previously been reported. In this study, we have used a cell duplication, microinjection assay to investigate the in vivo function of Plk1 in both immortalized (HeLa) and nonimmortalized (Hs68) human cells. Injection of anti-Plk1 antibodies (Plk1+) at various stages of the cell cycle had no effect on the kinetics of DNA replication but severely impaired the ability of cells to divide. Analysis of Plk1(+)-injected, mitotically arrested HeLa cells by fluorescence microscopy revealed abnormal distributions of condensed chromatin and monoastral microtubule arrays that were nucleated from duplicated but unseparated centrosomes. Most strikingly, centrosomes in Plk1(+)-injected cells were drastically reduced in size, and the accumulation of both gamma-tubulin and MPM-2 immunoreactivity was impaired. These data indicate that Plk1 activity is necessary for the functional maturation of centrosomes in late G2/early prophase and, consequently, for the establishment of a bipolar spindle. Additional roles for Plk1 at later stages of mitosis are not excluded, although injection of Plk1+ after the completion of spindle formation did not interfere with cytokinesis. Injection of Plk1+ into nonimmortalized Hs68 cells produced qualitatively similar phenotypes, but the vast majority of the injected Hs68 cells arrested as single, mononucleated cells in G2. This latter observation hints at the existence, in nonimmortalized cells, of a centrosome-maturation checkpoint sensitive to the impairment of Plk1 function.

scholarly journals The Xenopus Chk1 Protein Kinase Mediates a Caffeine-sensitive Pathway of Checkpoint Control in Cell-free Extracts

1998 ◽  
Vol 142 (6) ◽  
pp. 1559-1569 ◽  
Author(s):  
Akiko Kumagai ◽  
Zijian Guo ◽  
Katayoon H. Emami ◽  
Sophie X. Wang ◽  
William G. Dunphy
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Checkpoint Control ◽  
Checkpoint Response ◽  
Cell Cycle Delay ◽  
Checkpoint Pathway ◽  
Egg Extracts ◽  
A Cell

We have analyzed the role of the protein kinase Chk1 in checkpoint control by using cell-free extracts from Xenopus eggs. Recombinant Xenopus Chk1 (Xchk1) phosphorylates the mitotic inducer Cdc25 in vitro on multiple sites including Ser-287. The Xchk1-catalyzed phosphorylation of Cdc25 on Ser-287 is sufficient to confer the binding of 14-3-3 proteins. Egg extracts from which Xchk1 has been removed by immunodepletion are strongly but not totally compromised in their ability to undergo a cell cycle delay in response to the presence of unreplicated DNA. Cdc25 in Xchk1-depleted extracts remains bound to 14-3-3 due to the action of a distinct Ser-287-specific kinase in addition to Xchk1. Xchk1 is highly phosphorylated in the presence of unreplicated or damaged DNA, and this phosphorylation is abolished by caffeine, an agent which attenuates checkpoint control. The checkpoint response to unreplicated DNA in this system involves both caffeine-sensitive and caffeine-insensitive steps. Our results indicate that caffeine disrupts the checkpoint pathway containing Xchk1.

scholarly journals Binding of E-MAP-115 to microtubules is regulated by cell cycle-dependent phosphorylation.

1995 ◽  
Vol 131 (4) ◽  
pp. 1015-1024 ◽  
Author(s):  
D Masson ◽  
T E Kreis
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Mitotic Spindle ◽  
Biochemical Characterization ◽  
Dynamic Properties ◽  
Cell Polarization ◽  
Early Prophase ◽  
Late Prophase ◽  
Mitotic Cells

Expression levels of E-MAP-115, a microtubule-associated protein that stabilizes microtubules, increase with epithelial cell polarization and differentiation (Masson and Kreis, 1993). Although polarizing cells contain significant amounts of this protein, they can still divide and thus all stabilized microtubules must disassemble at the onset of mitosis to allow formation of the dynamic mitotic spindle. We show here that binding of E-MAP-115 to microtubules is regulated by phosphorylation during the cell cycle. Immunolabeling of HeLa cells for E-MAP-115 indicates that the protein is absent from microtubules during early prophase and progressively reassociates with microtubules after late prophase. A fraction of E-MAP-115 from HeLa cells released from a block at the G1/S boundary runs with higher apparent molecular weight on SDS-PAGE, with a peak correlating with the maximal number of cells in early stages of mitosis. E-MAP-115 from nocodazole-arrested mitotic cells, which can be obtained in larger amounts, displays identical modifications and was used for further biochemical characterization. The level of incorporation of 32P into mitotic E-MAP-115 is about 15-fold higher than into the interphase protein. Specific threonine phosphorylation occurs in mitosis, and the amount of phosphate associated with serine also increases. Hyperphosphorylated E-MAP-115 from mitotic cells cannot bind stably to microtubules in vitro. These results suggest that phosphorylation of E-MAP-115 is a prerequisite for increasing the dynamic properties of the interphase microtubules which leads to the assembly of the mitotic spindle at the onset of mitosis. Microtubule-associated proteins are thus most likely key targets for kinases which control changes in microtubule dynamic properties at the G2- to M-phase transition.

The Spd1p S phase inhibitor can activate the DNA replication checkpoint pathway in fission yeast

2000 ◽  
Vol 113 (23) ◽  
pp. 4341-4350 ◽  
Author(s):  
A. Borgne ◽  
P. Nurse
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Checkpoint Control ◽  
Replication Checkpoint ◽  
Checkpoint Pathway ◽  
Dna Replication Checkpoint ◽  
A Cell ◽  
Checkpoint Genes

Spd1p (for S phase delayed) is a cell cycle inhibitor in Schizosaccharomyces pombe. Spd1p overexpression blocks the onset of both S phase and mitosis. In this study, we have investigated the mechanisms by which Spd1p overexpression blocks cell cycle progression, focussing on the block over mitotic onset. High levels of Spd1p lead to an increase in Y15 phosphorylation of Cdc2p and we show that the block over G(2) requires the Wee1p kinase and is dependent on the rad and chk1/cds1 checkpoint genes. We propose that high levels of Spd1p in G(2) cells activate the DNA replication checkpoint control, which leads to a Wee1p-dependent increase of Cdc2p Y15 phosphorylation blocking onset of mitosis. The Spd1p block at S phase onset may act by interfering directly with DNA replication, and also activates the G(2)rad/hus checkpoint pathway to block mitosis.

scholarly journals cdc25 is a nuclear protein expressed constitutively throughout the cell cycle in nontransformed mammalian cells.

1992 ◽  
Vol 118 (4) ◽  
pp. 785-794 ◽  
Author(s):  
F Girard ◽  
U Strausfeld ◽  
J C Cavadore ◽  
P Russell ◽  
A Fernandez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Tyrosine Phosphatase ◽  
Specific Protein ◽  
Cyclin B ◽  
Early Prophase ◽  
Cdc25 Phosphatase ◽  
Nuclear Envelope Breakdown ◽  
Cellular Compartments ◽  
Cdc25 Protein

A family of proteins homologous to the cdc25 gene product of the fission yeast bear specific protein tyrosine phosphatase activity involved in the activation of the p34cdc2-cyclin B kinase. Using affinity-purified antibodies raised against a synthetic peptide corresponding to the catalytic site of the cdc25 phosphatase, we show that cdc25 protein is constitutively expressed throughout the cell cycle of nontransformed mammalian fibroblasts and does not undergo major changes in protein level. By indirect immunofluorescence, cdc25 protein is found essentially localized in the nucleus throughout interphase and during early prophase. Just before the complete nuclear envelope breakdown at the prophase-prometaphase boundary, cdc25 proteins are redistributed throughout the cytoplasm. During metaphase and anaphase, cdc25 staining remains distributed throughout the cell and excludes the condensed chromosomes. The nuclear locale reappears during telophase. In light of the recent data describing the cytoplasmic localization of cyclin B protein (Pines, J., and T. Hunter. 1991. J. Cell Biol. 115:1-17), the data presented here suggest that separation in two distinct cellular compartments of the cdc25 phosphatase and its substrate p34cdc2-cyclin B may be of importance in the regulation of the cdc2 kinase activity.

scholarly journals Yeast Bim1p Promotes the G1-specific Dynamics of Microtubules

1999 ◽  
Vol 145 (5) ◽  
pp. 993-1007 ◽  
Author(s):  
Jennifer S. Tirnauer ◽  
Eileen O'Toole ◽  
Lisbeth Berrueta ◽  
Barbara E. Bierer ◽  
David Pellman
Keyword(s):  
Cell Cycle ◽  
Dynamic Instability ◽  
Microtubule Dynamics ◽  
Yeast Protein ◽  
Gene Encoding ◽  
Cytoplasmic Microtubule ◽  
A Cell ◽  
Cytoplasmic Microtubules

Microtubule dynamics vary during the cell cycle, and microtubules appear to be more dynamic in vivo than in vitro. Proteins that promote dynamic instability are therefore central to microtubule behavior in living cells. Here, we report that a yeast protein of the highly conserved EB1 family, Bim1p, promotes cytoplasmic microtubule dynamics specifically during G1. During G1, microtubules in cells lacking BIM1 showed reduced dynamicity due to a slower shrinkage rate, fewer rescues and catastrophes, and more time spent in an attenuated/paused state. Human EB1 was identified as an interacting partner for the adenomatous polyposis coli (APC) tumor suppressor protein. Like human EB1, Bim1p localizes to dots at the distal ends of cytoplasmic microtubules. This localization, together with data from electron microscopy and a synthetic interaction with the gene encoding the kinesin Kar3p, suggests that Bim1p acts at the microtubule plus end. Our in vivo data provide evidence of a cell cycle–specific microtubule-binding protein that promotes microtubule dynamicity.

scholarly journals Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle.

1994 ◽  
Vol 127 (5) ◽  
pp. 1301-1310 ◽  
Author(s):  
C L Rieder ◽  
A Schultz ◽  
R Cole ◽  
G Sluder
Keyword(s):  
Nuclear Envelope ◽  
Somatic Cells ◽  
Checkpoint Control ◽  
Average Interval ◽  
Nuclear Envelope Breakdown ◽  
Anaphase Onset ◽  
Sister Kinetochore ◽  
Chromosome Motion

To test the popular but unproven assumption that the metaphase-anaphase transition in vertebrate somatic cells is subject to a checkpoint that monitors chromosome (i.e., kinetochore) attachment to the spindle, we filmed mitosis in 126 PtK1 cells. We found that the time from nuclear envelope breakdown to anaphase onset is linearly related (r2 = 0.85) to the duration the cell has unattached kinetochores, and that even a single unattached kinetochore delays anaphase onset. We also found that anaphase is initiated at a relatively constant 23-min average interval after the last kinetochore attaches, regardless of how long the cell possessed unattached kinetochores. From these results we conclude that vertebrate somatic cells possess a metaphase-anaphase checkpoint control that monitors sister kinetochore attachment to the spindle. We also found that some cells treated with 0.3-0.75 nM Taxol, after the last kinetochore attached to the spindle, entered anaphase and completed normal poleward chromosome motion (anaphase A) up to 3 h after the treatment--well beyond the 9-48-min range exhibited by untreated cells. The fact that spindle bipolarity and the metaphase alignment of kinetochores are maintained in these cells, and that the chromosomes move poleward during anaphase, suggests that the checkpoint monitors more than just the attachment of microtubules at sister kinetochores or the metaphase alignment of chromosomes. Our data are most consistent with the hypothesis that the checkpoint monitors an increase in tension between kinetochores and their associated microtubules as biorientation occurs.

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