Effects of 6-dimethylaminopurine on microtubules and putative intermediate filaments in sea urchin embryos

1991 ◽  
Vol 99 (4) ◽  
pp. 721-730
Author(s):  
L. Dufresne ◽  
I. Neant ◽  
J. St-Pierre ◽  
F. Dube ◽  
P. Guerrier
Keyword(s):  
Nuclear Envelope ◽  
Protein Phosphorylation ◽  
Sea Urchin ◽  
Intermediate Filaments ◽  
Spatial Organization ◽  
Chromatin Condensation ◽  
Cortical Microtubule ◽  
The State ◽  
Strongylocentrotus Droebachiensis ◽  
Nuclear Envelope Breakdown

The effects of 6-dimethylaminopurine (6-DMAP) (a putative phosphorylation inhibitor) on the state of assembly of microtubules and intermediate filaments have been studied during the first cell cycle of the sea urchin Strongylocentrotus droebachiensis. Changes in the spatial organization of cytoskeletal structures were studied by indirect immunofluorescence with anti-tubulin and anti-IFa antibodies. The rates and patterns of protein phosphorylation in control and treated eggs were also investigated. The transfer of fertilized eggs to 600 microM 6-DMAP within 4 min following insemination inhibits pronuclear migration and syngamy. This also prevents male pronuclear decondensation, while chromatin condensation and nuclear envelope breakdown do not occur in the female pronucleus. Immunolabeling with anti-tubulin antibodies reveals the presence of cortical microtubules as early as 15 min after fertilization in both control and treated eggs. However, no sperm astral microtubules could be detected in the treated eggs. At later stages, from syngamy (40 min) up to nuclear envelope breakdown (90 min), 6-DMAP affects neither cortical microtubule organization nor the state of chromatin condensation but it precludes nuclear envelope breakdown and entry into mitosis. Treatment of the fertilized eggs after nuclear envelope breakdown induces permanent chromosome decondensation and premature disappearance of the mitotic apparatus. This last event involves disruption of the spatial organization of both microtubules and putative intermediate filaments. Quantitative measurements of protein phosphorylation show that 6-DMAP efficiently and reversibly inhibits 32P incorporation into proteins. Qualitative analysis of the autoradiograms of 32P-labeled proteins separated by SDS-PAGE reveals that a major protein band, migrating with an apparent molecular weight of 31 × 10(3)Mr, is specifically dephosphorylated in eggs treated with 6-DMAP. This study suggests that protein phosphorylation is required for sperm aster microtubule growth and migration, but not for cortical microtubule polymerization. It also strengthens the hypothesis that, in sea urchin eggs, putative intermediate filaments are tightly associated with spindle microtubules. Finally, it confirms that inhibiting protein phosphorylation before nuclear envelope breakdown reversibly prevents the entry into mitosis.

Nuclear envelope removal/maintenance determines the structural and functional remodelling of embryonic red blood cell nuclei in activated mouse oocytes

Zygote ◽  
1998 ◽  
Vol 6 (1) ◽  
pp. 65-73 ◽  
Author(s):  
Daniel Szöllösi ◽  
Renata Czołowska ◽  
Ewa Borsuk ◽  
Maria S. Szöllösi ◽  
Pascale Debey
Keyword(s):  
Nuclear Envelope ◽  
Transcriptional Activity ◽  
Chromatin Condensation ◽  
Oocyte Activation ◽  
Cell Nuclei ◽  
Nuclear Envelope Breakdown ◽  
Mouse Oocytes ◽  
Female Pronucleus ◽  
Mouse Fetuses

SummaryNuclei of embryonic red blood cells (e-RBC) from 12-day mouse fetuses are arrested in Go phase of the cell cycle and have low transcriptional activity. These nuclei were transferred with help of polyethylene glycol (PEG)-mediated fusion to parthenogenetically activated mouse oocytes and heterokaryons were analysed for nuclear structure and transcriptional activity. If fusion proceeded 25–45 min after oocyte activation, e-RBC nuclei were induced to nuclear envelope breakdown and partial chromatin condensation, followed by formation of nuclei structurally identical with pronuclei. These ‘pronuclei’, similar to egg (female) pronuclei, remained transcriptionally silent over several hours of in vitro culture. If fusion was performed 1 h or later (up to 7 h) after activation, the nuclear envelope of e-RBC nuclei remained intact and nuclear remodelling was less spectacular (slight chromatin decondensation, formation of nucleolus precursor bodies). These nuclei, however, reinforced polymerase-II-dependent transcription within a few hours of in vitro culture. Our present experiments, together with our previous work, demonstrate that nuclear envelope breakdown/maintenance are critical events for nuclear remodelling in activated mouse oocytes and that somatic dormant nuclei can be stimulated to renew transcription at a time when the female pronucleus remains transcriptionally silent.

Metaphase protein phosphorylation in Xenopus laevis eggs

1987 ◽  
Vol 7 (2) ◽  
pp. 760-768
Author(s):  
M J Lohka ◽  
J L Kyes ◽  
J L Maller
Keyword(s):  
Xenopus Laevis ◽  
Nuclear Envelope ◽  
Protein Phosphorylation ◽  
Tyrosine Kinases ◽  
Chromosome Condensation ◽  
Nuclear Envelope Breakdown ◽  
M Phase ◽  
Nuclear Events ◽  
Gamma S

Cytoplasmic extracts of metaphase (M-phase)-arrested Xenopus laevis eggs support nuclear envelope breakdown and chromosome condensation in vitro. Induction of nuclear breakdown is inhibited by AMPP(NH)P, a nonhydrolyzable ATP analog, but not by ATP or gamma-S-ATP, a hydrolyzable ATP analog, suggesting that protein phosphorylation may be required for M-phase nuclear events in vitro. By addition of [gamma-32P]ATP, we have identified in cytoplasmic extracts and in intact eggs at least six phosphoproteins that are present during M-phase but absent in G1/S-phase. These phosphoproteins also appear in response to partially purified preparations of maturation-promoting factor. A subset of these proteins are thiophosphorylated by gamma-S-ATP under conditions that promote nuclear envelope breakdown and chromosome condensation. Each of these proteins is phosphorylated on serine and threonine, and one, a 42-kilodalton protein, is also phosphorylated on tyrosine both in extracts and in intact eggs. These results indicate that activation of protein kinases accounts for at least part of the increased phosphorylation in M-phase and that both protein-serine-threonine kinases and protein-tyrosine kinases may play a role in controlling M-phase nuclear behavior.

scholarly journals Role of spindle microtubules in the control of cell cycle timing.

1979 ◽  
Vol 80 (3) ◽  
pp. 674-691 ◽  
Author(s):  
G Sluder
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Control Cell ◽  
Microtubule Assembly ◽  
Nuclear Envelope Breakdown ◽  
Anaphase Onset ◽  
Spindle Cells ◽  
Spindle Microtubules

Sea urchin eggs are used to investigate the involvement of spindle microtubules in the mechanisms that control the timing of cell cycle events. Eggs are treated for 4 min with Colcemid at prophase of the first mitosis. No microtubules are assembled for at least 3 h, and the eggs do not divide. These eggs show repeated cycles of nuclear envelope breakdown (NEB) and nuclear envelope reformation (NER). Mitosis (NEB to NER) is twice as long in Colcemid-treated eggs as in the untreated controls. Interphase (NER to NEB) is the same in both. Thus, each cycle is prolonged entirely in mitosis. The chromosomes of treated eggs condense and eventually split into separate chromatids which do not move apart. This "canaphase" splitting is substantially delayed relative to anaphase onset in the control eggs. Treated eggs are irradiated after NEB with 366-nm light to inactivate the Colcemid. This allows the eggs to assemble normal spindles and divide. Up to 14 min after NEB, delays in the start of microtubule assembly give equal delays in anaphase onset, cleavage, and the events of the following cell cycle. Regardless of the delay, anaphase follows irradiation by the normal prometaphase duration. The quantity of spindle microtubules also influences the timing of mitotic events. Short Colcemid treatments administered in prophase of second division cause eggs to assemble small spindles. One blastomere is irradiated after NEB to provide a control cell with a normal-sized spindle. Cells with diminished spindles always initiate anaphase later than their controls. Telophase events are correspondingly delayed. This work demonstrates that spindle microtubules are involved in the mechanisms that control the time when the cell will initiate anaphase, finish mitosis, and start the next cell cycle.

scholarly journals Experimental manipulation of the amount of tubulin available for assembly into the spindle of dividing sea urchin eggs.

1976 ◽  
Vol 70 (1) ◽  
pp. 75-85 ◽  
Author(s):  
G Sluder
Keyword(s):  
Nuclear Envelope ◽  
Sea Urchin ◽  
Maximum Rate ◽  
Spindle Assembly ◽  
Experimental Manipulation ◽  
Lytechinus Variegatus ◽  
Tubulin Polymerization ◽  
Treated Cell ◽  
Sea Urchin Eggs ◽  
Nuclear Envelope Breakdown

Spindle assembly is studied in the eggs of the sea urchin Lytechinus variegatus by experimentally varying the amount of polymerizable tubulin within the egg. Aliquots of fertilized eggs from the same female are individually pulsed for 1-6 min with 1 X 10(-6) M Colcemid at least 20 min before first nuclear envelope breakdown. This treatment inactivates a portion of the cellular tubulin before the spindle is formed. Upon entering mitosis, treated eggs form functional spindles that are reduced in length and birefringent retardation but not width. With increased exposure to Colcemid, the length and retardation of the metaphase spindles are progressively reduced. Similar results are obtained by pulsing the eggs with Colcemid before fertilization, which demonstrates that the tubulin found in unfertilized sea urchin eggs is later used in spindle formation. Spindles, once assembled, are responsive to increases in the amount of polymerizable tubulin within the cell. Rapid increases in the amount of polymerizable tubulin within a Colcemid-treated cell can be experimentally effected by irradiating the cells with 366-nm light. This treatment photochemically inactivates the Colcemid, thereby freeing the tubulin to polymerize. Upon irradiation, the small prometaphase spindles of Colcemid-treated cells immediately increase in length and retardation. In these irradiated cells, spindle length and retardation increase as much as four times faster than they do during prometaphase for normal spindles. This suggests that the rate of the normal prometaphase increase in retardation and spindle size may be determined by factors other than the maximum rate of tubulin polymerization in the cell.

scholarly journals Dynamics of the Endoplasmic Reticulum and Golgi Apparatus during Early Sea Urchin Development

2000 ◽  
Vol 11 (3) ◽  
pp. 897-914 ◽  
Author(s):  
Mark Terasaki
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Permeability Barrier ◽  
Cell Cycles ◽  
Nuclear Envelope Breakdown ◽  
Quantitative Measurements ◽  
Green Fluorescent

The endoplasmic reticulum (ER) and Golgi were labeled by green fluorescent protein chimeras and observed by time-lapse confocal microscopy during the rapid cell cycles of sea urchin embryos. The ER undergoes a cyclical microtubule-dependent accumulation at the mitotic poles and by photobleaching experiments remains continuous through the cell cycle. Finger-like indentations of the nuclear envelope near the mitotic poles appear 2–3 min before the permeability barrier of the nuclear envelope begins to change. This permeability change in turn is ∼30 s before nuclear envelope breakdown. During interphase, there are many scattered, disconnected Golgi stacks throughout the cytoplasm, which appear as 1- to 2-μm fluorescent spots. The number of Golgi spots begins to decline soon after nuclear envelope breakdown, reaches a minimum soon after cytokinesis, and then rapidly increases. At higher magnification, smaller spots are seen, along with increased fluorescence in the ER. Quantitative measurements, along with nocodazole and photobleaching experiments, are consistent with a redistribution of some of the Golgi to the ER during mitosis. The scattered Golgi coalesce into a single large aggregate during the interphase after the ninth embryonic cleavage; this is likely to be preparatory for secretion of the hatching enzyme during the following cleavage cycle.

scholarly journals Nucleo-cytoplasmic interactions that control nuclear envelope breakdown and entry into mitosis in the sea urchin zygote

1999 ◽  
Vol 112 (8) ◽  
pp. 1139-1148 ◽  
Author(s):  
E.H. Hinchcliffe ◽  
E.A. Thompson ◽  
F.J. Miller ◽  
J. Yang ◽  
G. Sluder
Keyword(s):  
Nuclear Envelope ◽  
Dna Synthesis ◽  
Sea Urchin ◽  
Mammalian Cells ◽  
Cyclin B1 ◽  
Nuclear Accumulation ◽  
Dna Breaks ◽  
Male Pronucleus ◽  
Nuclear Envelope Breakdown ◽  
Female Pronucleus

In sea urchin zygotes and mammalian cells nuclear envelope breakdown (NEB) is not driven simply by a rise in cytoplasmic cyclin dependent kinase 1-cyclin B (Cdk1-B) activity; the checkpoint monitoring DNA synthesis can prevent NEB in the face of mitotic levels of Cdk1-B. Using sea urchin zygotes we investigated whether this checkpoint prevents NEB by restricting import of regulatory proteins into the nucleus. We find that cyclin B1-GFP accumulates in nuclei that cannot complete DNA synthesis and do not break down. Thus, this checkpoint limits NEB downstream of both the cytoplasmic activation and nuclear accumulation of Cdk1-B1. In separate experiments we fertilize sea urchin eggs with sperm whose DNA has been covalently cross-linked to inhibit replication. When the pronuclei fuse, the resulting zygote nucleus does not break down for >180 minutes (equivalent to three cell cycles), even though Cdk1-B activity rises to greater than mitotic levels. If pronuclear fusion is prevented, then the female pronucleus breaks down at the normal time (average 68 minutes) and the male pronucleus with cross-linked DNA breaks down 16 minutes later. This male pronucleus has a functional checkpoint because it does not break down for >120 minutes if the female pronucleus is removed just prior to NEB. These results reveal the existence of an activity released by the female pronucleus upon its breakdown, that overrides the checkpoint in the male pronucleus and induces NEB. Microinjecting wheat germ agglutinin into binucleate zygotes reveals that this activity involves molecules that must be actively translocated into the male pronucleus.

Intracellular free calcium rise triggers nuclear envelope breakdown in the sea urchin embryo

Nature ◽  
1988 ◽  
Vol 332 (6162) ◽  
pp. 364-366 ◽  
Author(s):  
Richard A. Steinhardt ◽  
Janet Alderton
Keyword(s):  
Nuclear Envelope ◽  
Sea Urchin ◽  
Intracellular Free Calcium ◽  
Free Calcium ◽  
Nuclear Envelope Breakdown ◽  
Sea Urchin Embryo

scholarly journals Centripetal nuclear shape fluctuations associate with chromatin condensation towards mitosis

2021 ◽  
Author(s):  
Viola Introini ◽  
Gururaj Rao Kidiyoor ◽  
Giancarlo Porcella ◽  
Marco Foiani ◽  
Pietro Cicuta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Cell Cycle Progression ◽  
Chromatin Condensation ◽  
Nuclear Shape ◽  
Cycle Progression ◽  
Cellular Processes ◽  
Nuclear Envelope Breakdown ◽  
Nuclear Mechanics ◽  
Nuclear Processes

The cell nucleus plays a central role in several key cellular processes, including chromosome organisation, replication and transcription. Recent work intriguingly suggests an association between nuclear mechanics and cell-cycle progression, but many aspects of this connection remain unexplored. Here, by monitoring nuclear shape fluctuations at different cell cycle stages, we uncover increasing inward fluctuations in late G2 and early mitosis, which are initially transient, but develop into instabilities that culminate into nuclear-envelope breakdown in mitosis. Perturbation experiments and correlation analysis reveal an association of these processes with chromatin condensation. We propose that the contrasting forces between an extensile stress and centripetal pulling from chromatin condensation could link mechanically chromosome condensation and nuclear-envelope breakdown, the two main nuclear processes during mitosis.

scholarly journals Nuclear envelope breakdown is under nuclear not cytoplasmic control in sea urchin zygotes.

1995 ◽  
Vol 129 (6) ◽  
pp. 1447-1458 ◽  
Author(s):  
G Sluder ◽  
E A Thompson ◽  
C L Rieder ◽  
F J Miller
Keyword(s):  
Nuclear Envelope ◽  
Dna Synthesis ◽  
Sea Urchin ◽  
Control Mechanisms ◽  
Checkpoint Control ◽  
Intact Male ◽  
Male Pronucleus ◽  
Nuclear Envelope Breakdown ◽  
Sperm Nuclei ◽  
Histone Kinase

Nuclear envelope breakdown (NEB) and entry into mitosis are though to be driven by the activation of the p34cdc2-cyclin B kinase complex or mitosis promoting factor (MPF). Checkpoint control mechanisms that monitor essential preparatory events for mitosis, such as DNA replication, are thought to prevent entry into mitosis by downregulating MPF activation until these events are completed. Thus, we were surprised to find that when pronuclear fusion in sea urchin zygotes is blocked with Colcemid, the female pronucleus consistently breaks down before the male pronucleus. This is not due to regional differences in the time of MPF activation, because pronuclei touching each other break down asynchronously to the same extent. To test whether NEB is controlled at the nuclear or cytoplasmic level, we activated the checkpoint for the completion of DNA synthesis separately in female and male pronuclei by treating either eggs or sperm before fertilization with psoralen to covalently cross-link base-paired strands of DNA. When only the maternal DNA is cross-linked, the male pronucleus breaks down first. When the sperm DNA is cross-linked, male pronuclear breakdown is substantially delayed relative to female pronuclear breakdown and sometimes does not occur. Inactivation of the Colcemid after female NEB in such zygotes with touching pronuclei yields a functional spindle composed of maternal chromosomes and paternal centrosomes. The intact male pronucleus remains located at one aster throughout mitosis. In other experiments, when psoralen-treated sperm nuclei, over 90% of the zygote nuclei do not break down for at least 2 h after the controls even though H1 histone kinase activity gradually rises close to, or higher than, control mitotic levels. The same is true for normal zygotes treated with aphidicolin to block DNA synthesis. From these results, we conclude that NEB in sea urchin zygotes is controlled at the nuclear, not cytoplasmic, level, and that mitotic levels of cytoplasmic MPF activity are not sufficient to drive NEB for a nucleus that is under checkpoint control. Our results also demonstrate that the checkpoint for the completion of DNA synthesis inhibits NEB by acting primarily within the nucleus, not by downregulating the activity of cytoplasmic MPF.

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