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.

Two proteins that cycle asynchronously between centrosomes and nuclear structures: Drosophila CP60 and CP190

1997 ◽  
Vol 110 (14) ◽  
pp. 1573-1583 ◽  
Author(s):  
K. Oegema ◽  
W.F. Marshall ◽  
J.W. Sedat ◽  
B.M. Alberts
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Wide Field ◽  
Dependent Manner ◽  
Independent Manner ◽  
Nuclear Envelope Breakdown ◽  
Nuclear Structures ◽  
Dynamic Structures ◽  
Cycle Dependent

Both the nucleus and the centrosome are complex, dynamic structures whose architectures undergo cell cycle-specific rearrangements. CP190 and CP60 are two Drosophila proteins of unknown function that shuttle between centrosomes and nuclei in a cell cycle-dependent manner. These two proteins are associated in vitro, and localize to centrosomes in a microtubule independent manner. We injected fluorescently labeled, bacterially expressed CP190 and CP60 into living Drosophila embryos and followed their behavior during the rapid syncytial blastoderm divisions (nuclear cycles 10–13). Using quantitative 3-D wide-field fluorescence microscopy, we show that CP190 and CP60 cycle between nuclei and centrosomes asynchronously with the accumulation of CP190 leading that of CP60 both at centrosomes and in nuclei. During interphase, CP190 is found in nuclei. Immediately following nuclear envelope breakdown, CP190 localizes to centrosomes where it remains until telophase, thereafter accumulating in reforming nuclei. Unlike CP190, CP60 accumulates at centrosomes primarily during anaphase, where it remains into early interphase. During nuclear cycles 10 and 11, CP60 accumulates in nuclei simultaneous with nuclear envelope breakdown, suggesting that CP60 binds to an unknown nuclear structure that persists into mitosis. During nuclear cycles 12 and 13, CP60 accumulates gradually in nuclei during interphase, reaching peak levels just before nuclear envelope breakdown. Once in the nucleus, both CP190 and CP60 appear to form fibrous intranuclear networks that remain coherent even after nuclear envelope breakdown. The CP190 and CP60 networks do not co-localize extensively with each other or with DNA. This work provides direct evidence, in living cells, of a coherent protein network that may represent a nuclear skeleton.

scholarly journals Cell Cycle-Dependent Dynamics of the Golgi-Centrosome Association in Motile Cells

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1069 ◽  
Author(s):  
Keyada Frye ◽  
Fioranna Renda ◽  
Maria Fomicheva ◽  
Xiaodong Zhu ◽  
Lisa Gong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Golgi Complex ◽  
Cell Polarization ◽  
Dependent Manner ◽  
Nuclear Envelope Breakdown ◽  
Motile Cells ◽  
A Cell ◽  
Cycle Dependent ◽  
Prevailing View

Here, we characterize spatial distribution of the Golgi complex in human cells. In contrast to the prevailing view that the Golgi compactly surrounds the centrosome throughout interphase, we observe characteristic differences in the morphology of Golgi ribbons and their association with the centrosome during various periods of the cell cycle. The compact Golgi complex is typical in G1; during S-phase, Golgi ribbons lose their association with the centrosome and extend along the nuclear envelope to largely encircle the nucleus in G2. Interestingly, pre-mitotic separation of duplicated centrosomes always occurs after dissociation from the Golgi. Shortly before the nuclear envelope breakdown, scattered Golgi ribbons reassociate with the separated centrosomes restoring two compact Golgi complexes. Transitions between the compact and distributed Golgi morphologies are microtubule-dependent. However, they occur even in the absence of centrosomes, which implies that Golgi reorganization is not driven by the centrosomal microtubule asters. Cells with different Golgi morphology exhibit distinct differences in the directional persistence and velocity of migration. These data suggest that changes in the radial distribution of the Golgi around the nucleus define the extent of cell polarization and regulate cell motility in a cell cycle-dependent manner.

A direct measurement of increased divalent cation influx in fertilised mouse oocytes

Development ◽  
1996 ◽  
Vol 122 (7) ◽  
pp. 2199-2206 ◽  
Author(s):  
O.M. McGuinness ◽  
R.B. Moreton ◽  
M.H. Johnson ◽  
M.J. Berridge
Keyword(s):  
Cell Cycle ◽  
Dependent Manner ◽  
Continuous Increase ◽  
Mouse Oocytes ◽  
A Cell ◽  
Cycle Dependent ◽  
Precise Role ◽  
Entry Mechanism ◽  
Stimulation Of

On fertilisation of mouse oocytes, the fusing spermatozoon triggers a series of repetitive calcium (Ca2+) spikes. The Ca2+ spikes seem to be necessary for successful progression through the cell cycle and are regulated in a cell-cycle-dependent manner. The spikes appear to require the linkage of continuous Ca2+ influx to the periodic release of Ca2+ from intracellular stores by a process of Ca(2+)-induced Ca2+ release. The precise role of Ca2+ influx was explored using the manganese (Mn2+)-quench technique to monitor unidirectional cation influx into single mouse oocytes. There was a marked stimulation of cation influx associated closely with the upsweep of the first and subsequent fertilisation Ca2+ spikes. A smaller but significant increase in the rate of cation influx persisted in the interspike period in fertilised oocytes. Spike-associated entry was not as apparent in oocytes stimulated to spike repetitively by thimerosal or acetylcholine application. Instead, there was a continuous increase in cation influx underlying Ca2+ spiking which commenced with the onset of the first spike. Using the specific microsomal inhibitor thapsigargin and the Ca2+ ionophore ionomycin, we found evidence for a capacitative entry mechanism in mouse oocytes. We propose that the persistent influx of Ca2+ observed in response to all stimuli examined is controlled by a capacitative mechanism and sets the frequency of spiking by determining the time taken to refill the internal stores to a point where they are again sensitive enough to initiate the next spike.

scholarly journals NSun2 Promotes Cell Growth via Elevating Cyclin-Dependent Kinase 1 Translation

2015 ◽  
Vol 35 (23) ◽  
pp. 4043-4052 ◽  
Author(s):  
Junyue Xing ◽  
Jie Yi ◽  
Xiaoyu Cai ◽  
Hao Tang ◽  
Zhenyun Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Cell Division Cycle ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Cyclin Dependent Kinase ◽  
A Cell ◽  
Cycle Dependent ◽  
Insight Into

The tRNA methytransferase NSun2 promotes cell proliferation, but the molecular mechanism has not been elucidated. Here, we report that NSun2 regulates cyclin-dependent kinase 1 (CDK1) expression in a cell cycle-dependent manner. Knockdown of NSun2 decreased the CDK1 protein level, while overexpression of NSun2 elevated it without alteringCDK1mRNA levels. Further studies revealed that NSun2 methylatedCDK1mRNAin vitroand in cells and that methylation by NSun2 enhanced CDK1 translation. Importantly, NSun2-mediated regulation of CDK1 expression had an impact on the cell division cycle. These results provide new insight into the regulation of CDK1 during the cell division cycle.

scholarly journals A cell cycle-associated change in Ca2+ releasing activity leads to the generation of Ca2+ transients in mouse embryos during the first mitotic division.

1996 ◽  
Vol 132 (5) ◽  
pp. 915-923 ◽  
Author(s):  
T Kono ◽  
K T Jones ◽  
A Bos-Mikich ◽  
D G Whittingham ◽  
J Carroll
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Fluorescent Dyes ◽  
Mitotic Division ◽  
Mouse Embryos ◽  
Dependent Manner ◽  
Nuclear Envelope Breakdown ◽  
A Cell ◽  
Dose Dependent ◽  
Parthenogenetic Embryos

We have used Ca2+-sensitive fluorescent dyes to monitor intracellular Ca2+ during mitosis in one-cell mouse embryos. We find that fertilized embryos generate Ca2+ transients at nuclear envelope breakdown (NEBD) and during mitosis. In addition, fertilized embryos arrested in metaphase using colcemid continue to generate Ca2+ transients. In contrast, parthenogenetic embryos produced by a 2-h exposure to strontium containing medium do not generate detectable Ca2+ transients at NEBD or in mitosis. However, when parthenogenetic embryos are cultured continuously in strontium containing medium Ca2+ transients are detected in mitosis but not in interphase. This suggests that mitotic Ca2+ transients are detected in the presence of an appropriate stimulus such as fertilization or strontium. The Ca2+ transient detected in fertilized embryos is not necessary for inducing NEBD since parthenogenetic embryos undergo nuclear envelope breakdown (NEBD). Also the first sign that NEBD is imminent occurs several minutes before the Ca2+ transient. The Ca2+ transient at NEBD appears to be associated with the nucleus since nuclear transfer experiments show that the presence of a karyoplast from a fertilized embryo is essential. Finally, we show that the intracellular Ca2+ chelator Bapta inhibits NEBD in fertilized and parthenogenetic embryos in a dose-dependent manner. These studies show that during mitosis there is an endogenous increase in Ca2+ releasing activity that leads to the generation of Ca2+ transients specifically during mitosis. The ability of Ca2+ buffers to inhibit NEBD regardless of the presence of global Ca2+ transients suggests that the underlying cell cycle-associated Ca2+ releasing activity may take the form of localized Ca2+ transients.

scholarly journals Changes in the nuclear distribution of DNA polymerase alpha and PCNA/cyclin during the progress of the cell cycle, in a cell-free extract of Xenopus eggs

1989 ◽  
Vol 93 (4) ◽  
pp. 605-613
Author(s):  
C. Hutchison ◽  
I. Kill
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Dna Polymerase ◽  
Cell Free Extract ◽  
Dna Polymerase Alpha ◽  
Nuclear Envelope Breakdown ◽  
Nuclear Distribution ◽  
A Cell ◽  
Mitotic Figures

The nuclear distribution of DNA polymerase alpha and PCNA/cyclin in embryonic nuclei has been investigated, in a cell-free extract of Xenopus eggs that recapitulates a basic cell-cycle in vitro, by indirect immunofluorescence microscopy. Both antigens co-distribute with the chromatin in S-phase nuclei; however, as DNA replication is completed and nuclei progress into a G2 state anti-PCNA fluorescence disappears and anti-DNA polymerase alpha fluorescence becomes resolved into bright spots. These spots are initially associated with the chromatin strands and can be seen to share both anti-PCNA and anti-DNA polymerase alpha fluorescence, but as anti-PCNA fluorescence fades the spots become dissociated from the chromatin and are redistributed throughout the nucleus until they are dispersed during nuclear envelope breakdown. The loss of anti-PCNA fluorescence and displacement of anti-DNA polymerase alpha fluorescence from the chromatin can be prevented by inhibiting DNA synthesis with aphidicolin. Under these conditions both antigens remain associated with the chromatin even after nuclear envelope breakdown and lamin dispersal. The association of these antigens with mitotic figures appears to be functional, as both biotin-11-dUTP and [32P]dCTP can be incorporated efficiently into DNA during the mitotic period.

scholarly journals Cell Cycle-Dependent Regulation of Human DNA Polymerase α-Primase Activity by Phosphorylation

1999 ◽  
Vol 19 (1) ◽  
pp. 646-656 ◽  
Author(s):  
Christian Voitenleitner ◽  
Christoph Rehfuess ◽  
Melissa Hilmes ◽  
Lynda O’Rear ◽  
Pao-Chi Liao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Polymerase ◽  
Cyclin E ◽  
Cyclin A ◽  
Human Cells ◽  
Dependent Manner ◽  
Dna Polymerase Α ◽  
A Cell ◽  
Cycle Dependent

ABSTRACT DNA polymerase α-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase α-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase α-primase from human cells, synchronized and labeled in G1/S and in G2, revealed a cyclin E/cdk2-like pattern in G1/S and a cyclin A/cdk2-like pattern in G2. The slower-electrophoretic-mobility form of p68 was absent in human cells in G1/S and appeared as the cells entered G2/M. Consistent with this, the ability of DNA polymerase α-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G1/S, decreased as the cells completed S phase, and reached a minimum in G2/M. These results suggest that the replication activity of DNA polymerase α-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.

scholarly journals Regulation of Melanosome Movement in the Cell Cycle by Reversible Association with Myosin V

1999 ◽  
Vol 146 (6) ◽  
pp. 1265-1276 ◽  
Author(s):  
Stephen L. Rogers ◽  
Ryan L. Karcher ◽  
Joseph T. Roland ◽  
Alexander A. Minin ◽  
Walter Steffen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Actin Filaments ◽  
Organelle Transport ◽  
Dependent Manner ◽  
Myosin V ◽  
In Vitro Motility ◽  
Egg Extracts ◽  
A Cell ◽  
Cycle Dependent

Previously, we have shown that melanosomes of Xenopus laevis melanophores are transported along both microtubules and actin filaments in a coordinated manner, and that myosin V is bound to purified melanosomes (Rogers, S., and V.I. Gelfand. 1998. Curr. Biol. 8:161–164). In the present study, we have demonstrated that myosin V is the actin-based motor responsible for melanosome transport. To examine whether myosin V was regulated in a cell cycle-dependent manner, purified melanosomes were treated with interphase- or metaphase-arrested Xenopus egg extracts and assayed for in vitro motility along Nitella actin filaments. Motility of organelles treated with mitotic extract was found to decrease dramatically, as compared with untreated or interphase extract-treated melanosomes. This mitotic inhibition of motility correlated with the dissociation of myosin V from melanosomes, but the activity of soluble motor remained unaffected. Furthermore, we find that myosin V heavy chain is highly phosphorylated in metaphase extracts versus interphase extracts. We conclude that organelle transport by myosin V is controlled by a cell cycle-regulated association of this motor to organelles, and that this binding is likely regulated by phosphorylation of myosin V during mitosis.

scholarly journals Truncated APC regulates the transcriptional activity of β-catenin in a cell cycle dependent manner

2006 ◽  
Vol 16 (2) ◽  
pp. 199-209 ◽  
Author(s):  
Jean Schneikert ◽  
Annette Grohmann ◽  
Jürgen Behrens
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activity ◽  
Dependent Manner ◽  
A Cell ◽  
Cycle Dependent

The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly

1998 ◽  
Vol 111 (5) ◽  
pp. 557-572 ◽  
Author(s):  
C. Roghi ◽  
R. Giet ◽  
R. Uzbekov ◽  
N. Morin ◽  
I. Chartrain ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Mitotic Spindle ◽  
Cultured Cells ◽  
Dependent Manner ◽  
Egg Extracts ◽  
Pericentriolar Material ◽  
Spindle Microtubules ◽  
Cycle Dependent

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 ‘invades’ the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

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