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.

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.

scholarly journals Cyclin A2 Regulates Nuclear-Envelope Breakdown and the Nuclear Accumulation of Cyclin B1

2007 ◽  
Vol 17 (1) ◽  
pp. 85-91 ◽  
Author(s):  
Delquin Gong ◽  
Joseph R. Pomerening ◽  
Jason W. Myers ◽  
Christer Gustavsson ◽  
Joshua T. Jones ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Cyclin B1 ◽  
Nuclear Accumulation ◽  
Nuclear Envelope Breakdown ◽  
Cyclin A2

scholarly journals An ultrastructural study of cross-fertilization (Arbacia female x Mytilus male).

1977 ◽  
Vol 73 (1) ◽  
pp. 14-26 ◽  
Author(s):  
F J Longo
Keyword(s):  
Sea Urchin ◽  
Ultrastructural Study ◽  
Cortical Granule ◽  
Male Pronucleus ◽  
Female Pronucleus ◽  
Sperm Nuclei ◽  
Cross Fertilization ◽  
Sperm Aster

Insemination of sea urchin (Arbacia) ova with mussel (Mytilus) sperm has been accomplished by treating eggs with trypsin and suspending the gametes in seawater made alkaline with NaOH. Not all inseminated eggs undergo a cortical granule reaction. Some eggs either elevate what remains of their vitelline layer or demonstrate no cortical modification whatsoever. After its incorporation into the egg, the nucleus of Mytilus sperm undergoes changes which eventually give rise to the formation of a male pronucleus. Concomitant with these transformations, a sperm aster may develop in association with the centrioles brought into the egg with the spermatozoon. Both the male pronucleus and the sperm aster may then migrate centrad to the female pronucleus. Evidence is presented which suggests that fusion of the male pronuclei from Mytilus sperm with female pronuclei from Arbacia eggs may occur, although this was not directly observed. These results demonstrate that Mytilus sperm nuclei are able to react to conditions within Arbacia eggs and differentiate into male pronuclei.

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.

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 Nonperiodic Activity of the Human Anaphase-Promoting Complex–Cdh1 Ubiquitin Ligase Results in Continuous DNA Synthesis Uncoupled from Mitosis

2000 ◽  
Vol 20 (20) ◽  
pp. 7613-7623 ◽  
Author(s):  
Claus Storgaard Sørensen ◽  
Claudia Lukas ◽  
Edgar R. Kramer ◽  
Jan-Michael Peters ◽  
Jiri Bartek ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Kinase Inhibitor ◽  
Cyclin B1 ◽  
S Phase ◽  
Anaphase Promoting Complex

ABSTRACT Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccharomyces cerevisiae andDrosophila spp., triggers exit from mitosis and during G1 prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference with the APC-Cdh1 dissociation at the G1/S transition resulted in an inability to accumulate a surprisingly broad range of critical mitotic regulators including cyclin B1, cyclin A, Plk1, Pds1, mitosin (CENP-F), Aim1, and Cdc20. Unexpectedly, although constitutively assembled APC-Cdh1 also delayed G1/S transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27Kip1 cyclin-dependent kinase inhibitor. Consequently, failure to inactivate APC-Cdh1 beyond the G1/S transition not only inhibited productive cell division but also supported slow but uninterrupted DNA replication, precluding S-phase exit and causing massive overreplication of the genome. Our data suggest that timely oscillation of the APC-Cdh1 ubiquitin ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells.

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 Nuclear-cytoplasmic compartmentalization promotes robust timing of mitotic events by cyclin B1-Cdk1

2021 ◽  
Author(s):  
Gembu Maryu ◽  
Qiong Yang
Keyword(s):  
Phase Plane ◽  
Nuclear Translocation ◽  
Spatial Models ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cyclin B1 ◽  
Nuclear Accumulation ◽  
Nuclear Envelope Breakdown ◽  
Synthetic Cells ◽  
Tunable Frequency

Studies applying well-mixed cytosolic extracts found the mitotic network centered on cyclin-dependent kinase (Cdk1) performs robust relaxation oscillations with tunable frequency. However, recent work also highlighted the importance of cyclin B1-Cdk1 nuclear translocation in mitotic timing. How nuclear compartmentalization affects the oscillator properties and the accurate ordering of mitotic events, especially in embryos lacking checkpoints, remains elusive. Here we developed a Forster resonance energy transfer (FRET) biosensor for analyzing Cdk1 spatiotemporal dynamics in synthetic cells containing nuclei compared to those without. We found cellular compartmentalization significantly impacts clock behaviors. While the amplitude-frequency dependency measured in the homogeneous cytoplasm showed highly tunable frequency for a fixed amplitude, confirming predictions by non-spatial models, the frequency remains constant against cyclin variations when nuclei are present, suggesting a possible buffering mechanism of nuclear compartments to ensure robust timing. We also found all cyclin degrades within similar mitotic durations despite variable interphase cyclin expression. This scalable degradation of cyclin may further promote the precise mitotic duration. Simultaneous measurements revealed Cdk1 and cyclin B1 cycle rigorously out of phase, producing a wide orbit on their phase plane, essential for robust oscillations. We further mapped mitotic events on the phase-plane orbits. Unlike cytoplasmic-only cells showing delayed Cdk1 activation, nucleus-containing cells exhibit steady cyclin B1-Cdk1 nuclear accumulation until nuclear envelope breakdown (NEB) followed by an abrupt cyclin-independent activation to trigger anaphase. Thus, both biphasic activation and subcellular localization of Cdk1 ensure accurate ordering of substrates.

133. SPATIAL REGULATION OF APCCdh1 INDUCED CYCLIN B1 DEGRADATION MAINTAINS GV ARREST IN MOUSE OOCYTES

2009 ◽  
Vol 21 (9) ◽  
pp. 52
Author(s):  
J. E. Holt ◽  
J. L. Weaver ◽  
K. T. Jones
Keyword(s):  
Cyclin B1 ◽  
26S Proteasome ◽  
Nuclear Accumulation ◽  
Regulatory Subunit ◽  
Anaphase Promoting Complex ◽  
Intracellular Location ◽  
Nuclear Localisation ◽  
Nuclear Envelope Breakdown ◽  
Mouse Oocytes ◽  
Nuclear Levels

Within the mammalian ovary, oocytes remain prophase I arrested until a hormonal cue triggers meiotic resumption. The E3 ubiquitin ligase, Anaphase-Promoting Complex with its co-activator Cdh1 (APCCdh1) is known to be essential for this process by promoting cyclin B1 degradation via the 26S proteasome. Cyclin B1 is the regulatory subunit of Maturation-Promoting Factor, forming a heterodimer with CDK1, which is essential for nuclear envelope breakdown (NEB). Here we describe the intracellular partitioning that would explain how Cdh1 activity is fine-tuned, such that cyclin B1 is maintained at sufficient levels to allow oocytes to resume meiosis but not so high as to cause premature meiosis re-entry. Using RT-PCR we detected only one splice variant in mouse oocytes, Cdh1α, which possessed a nuclear localisation signal. By immunofluorescence we confirmed the nuclear location of Cdh1 and degradation machinery components including essential subunits of the APC and 26S proteasome. In all systems studied, cyclin B1 shuttles between the cytoplasm and nucleus, with nuclear localisation occurring just before NEB. We reasoned therefore that the nuclear localisation of the APCCdh1 and 26S proteasome would aid in maintaining low nuclear levels of cyclin B1. Using two GFP-coupled cyclin B1 mutants, which differed in their intracellular location we found that nuclear accumulation of cyclin B1 was necessary in order for it to promote meiotic resumption because over-expression of nuclear-cyclin B1 accelerated entry into meiosis, whereas cytoplasmic-cyclin B1 did not. However, in milrinone-arrested GV oocytes rates of nuclear-cyclin B1 degradation were 5 fold higher than cytoplasmic-cyclin B1. Therefore we conclude that in oocytes, an increase in the nuclear-cytoplasmic ratio of cyclin B1 is an essential step in meiotic resumption, and that nuclear APCCdh1 activity guards against early meiotic resumption, until the degradation machinery is overwhelmed by cyclin B1 translocation.

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