scholarly journals The dynamics of cyclin B1 distribution during meiosis I in mouse oocytes

Reproduction ◽  
2004 ◽  
Vol 128 (2) ◽  
pp. 153-162 ◽  
Author(s):  
Petros Marangos ◽  
John Carroll
Keyword(s):  
Fluorescent Protein ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Nuclear Accumulation ◽  
Leptomycin B ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes ◽  
Living Mouse ◽  
Green Fluorescent ◽  
Meiosis I

Cdk1-cyclin B1 kinase activity drives oocytes through meiotic maturation. It is regulated by the phosphorylation status of cdk1 and by its spatial organisation. Here we used a cyclin B1-green fluorescent protein (GFP) fusion protein to examine the dynamics of cdk1-cyclin B1 distribution during meiosis I (MI) in living mouse oocytes. Microinjection of cyclin B1-GFP accelerated germinal vesicle breakdown (GVBD) and, as previously described, overrides cAMP-mediated meiotic arrest. GVBD was pre-empted by a translocation of cyclin B1-GFP from the cytoplasm to the germinal vesicle (GV). After nuclear accumulation, cyclin B1-GFP localised to the chromatin. The localisation of cyclin B1-GFP is governed by nuclear import and export. In GV intact oocytes, cyclin export was demonstrated by showing that cyclin B1-GFP injected into the GV is exported to the cytoplasm while a similar size dextran is retained. Import was revealed by the finding that cyclin B1-GFP accumulated in the GV when export was inhibited using leptomycin B. These studies show that GVBD in mouse oocytes is sensitive to cyclin B1 abundance and that the changes in distribution of cyclin B1 contribute to progression through MI.

scholarly journals Fer tyrosine kinase is required for germinal vesicle breakdown and meiosis-I in mouse oocytes

2011 ◽  
Vol 78 (1) ◽  
pp. 33-47 ◽  
Author(s):  
Lynda K. McGinnis ◽  
Xiaoman Hong ◽  
Lane K. Christenson ◽  
William H. Kinsey
Keyword(s):  
Tyrosine Kinase ◽  
Germinal Vesicle ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes ◽  
Meiosis I

scholarly journals Prophase I arrest and progression to metaphase I in mouse oocytes are controlled by Emi1-dependent regulation of APCCdh1

2006 ◽  
Vol 176 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Petros Marangos ◽  
Emmy W. Verschuren ◽  
Ruby Chen ◽  
Peter K. Jackson ◽  
John Carroll
Keyword(s):  
Meiotic Division ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Anaphase Promoting Complex ◽  
Germinal Vesicle Breakdown ◽  
Prophase I ◽  
Mouse Oocytes ◽  
Dependent Inhibition ◽  
Morpholino Oligonucleotides ◽  
Mitotic Inhibitor

Mammalian oocytes are arrested in prophase of the first meiotic division. Progression into the first meiotic division is driven by an increase in the activity of maturation-promoting factor (MPF). In mouse oocytes, we find that early mitotic inhibitor 1 (Emi1), an inhibitor of the anaphase-promoting complex (APC) that is responsible for cyclin B destruction and inactivation of MPF, is present at prophase I and undergoes Skp1–Cul1–F-box/βTrCP-mediated destruction immediately after germinal vesicle breakdown (GVBD). Exogenous Emi1 or the inhibition of Emi1 destruction in prophase-arrested oocytes leads to a stabilization of cyclin B1–GFP that is sufficient to trigger GVBD. In contrast, the depletion of Emi1 using morpholino oligonucleotides increases cyclin B1–GFP destruction, resulting in an attenuation of MPF activation and a delay of entry into the first meiotic division. Finally, we show that Emi1-dependent effects on meiosis I require the presence of Cdh1. These observations reveal a novel mechanism for the control of entry into the first meiotic division: an Emi1-dependent inhibition of APCCdh1.

scholarly journals Requirement of mosXe protein kinase for meiotic maturation of Xenopus oocytes induced by a cdc2 mutant lacking regulatory phosphorylation sites.

1992 ◽  
Vol 12 (7) ◽  
pp. 3192-3203 ◽  
Author(s):  
K M Pickham ◽  
A N Meyer ◽  
J Li ◽  
D J Donoghue
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Phosphorylation Sites ◽  
Germinal Vesicle Breakdown ◽  
Regulatory Phosphorylation

The p34cdc2 protein kinase is a component of maturation-promoting factor, the master regulator of the cell cycle in all eukaryotes. The activity of p34cdc2 is itself tightly regulated by phosphorylation and dephosphorylation. Predicted regulatory phosphorylation sites of Xenopus p34cdc2 were mutated in vitro, and in vitro-transcribed RNAs were injected into Xenopus oocytes. The cdc2 single mutants Thr-14----Ala and Tyr-15----Phe did not induce germinal vesicle breakdown (BVBD) upon microinjection into oocytes. In contrast, the cdc2 double mutant Ala-14/Phe-15 did induce GVBD. Both the Ala-14 and Ala-14/Phe-15p34cdc2 mutants were shown to coimmunoprecipitate cyclin B1 and to phosphorylate histone H1 in immune complex kinase assays. Microinjection of antisense oligonucleotides to c-mosXe was used to demonstrate the role of mos protein synthesis in the induction of GVBD by the Ala-14/Phe-15 cdc2 mutant. Thr-161 was also mutated. p34cdc2 single mutants Ala-161 and Glu-161 and triple mutants Ala-14/Phe-15/Ala-161 and Ala-14/Phe-15/Glu-161 failed to induce GVBD in oocytes and showed a decreased binding to cyclin B1 in coimmunoprecipitations. Each of the cdc2 mutants was also assayed by coinjection with cyclin B1 or c-mosXe RNA into oocytes. Several of the cdc2 mutants were found to affect the kinetics of cyclin B1 and/or mos-induced GVBD upon coinjection, although none affected the rate of progesterone-induced maturation. We demonstrate here the significance of Thr-14, Tyr-15, and Thr-161 of p34cdc2 in Xenopus oocyte maturation. In addition, these results suggest a regulatory role for mosXe in induction of oocyte maturation by the cdc2 mutant Ala-14/Phe-15.

A global survey of CRM1-dependent nuclear export sequences in the human deubiquitinase family

2011 ◽  
Vol 441 (1) ◽  
pp. 209-217 ◽  
Author(s):  
Iraia García-Santisteban ◽  
Sonia Bañuelos ◽  
Jose A. Rodríguez
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
The Novel ◽  
Sequence Motifs ◽  
Leptomycin B ◽  
Specific Amino Acid ◽  
Green Fluorescent ◽  
Nuclear Export Receptor

The mechanisms that regulate the nucleocytoplasmic localization of human deubiquitinases remain largely unknown. The nuclear export receptor CRM1 binds to specific amino acid motifs termed NESs (nuclear export sequences). By using in silico prediction and experimental validation of candidate sequences, we identified 32 active NESs and 78 inactive NES-like motifs in human deubiquitinases. These results allowed us to evaluate the performance of three programs widely used for NES prediction, and to add novel information to the recently redefined NES consensus. The novel NESs identified in the present study reveal a subset of 22 deubiquitinases bearing motifs that might mediate their binding to CRM1. We tested the effect of the CRM1 inhibitor LMB (leptomycin B) on the localization of YFP (yellow fluorescent protein)- or GFP (green fluorescent protein)-tagged versions of six NES-bearing deubiquitinases [USP (ubiquitin-specific peptidase) 1, USP3, USP7, USP21, CYLD (cylindromatosis) and OTUD7B (OTU-domain-containing 7B)]. YFP–USP21 and, to a lesser extent, GFP–OTUD7B relocated from the cytoplasm to the nucleus in the presence of LMB, revealing their nucleocytoplasmic shuttling capability. Two sequence motifs in USP21 had been identified during our survey as active NESs in the export assay. Using site-directed mutagenesis, we show that one of these motifs mediates USP21 nuclear export, whereas the second motif is not functional in the context of full-length USP21.

scholarly journals Live-Cell Fluorescence Imaging Reveals the Dynamics of Protein Kinase CK2 Individual Subunits

2003 ◽  
Vol 23 (3) ◽  
pp. 975-987 ◽  
Author(s):  
Odile Filhol ◽  
Arsenio Nueda ◽  
Véronique Martel ◽  
Delphine Gerber-Scokaert ◽  
Maria José Benitez ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Protein Kinase Ck2 ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Nucleocytoplasmic Trafficking ◽  
Green Fluorescent ◽  
Nucleocytoplasmic Distribution ◽  
Kinase Ck2

ABSTRACT Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (α or α′) and two regulatory (β) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic α and regulatory β subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2α or GFP-CK2β revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2β, CK2α can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2α is dramatically changed by its association with CK2β, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.

scholarly journals Mouse Emi2 is required to enter meiosis II by reestablishing cyclin B1 during interkinesis

2006 ◽  
Vol 174 (6) ◽  
pp. 791-801 ◽  
Author(s):  
Suzanne Madgwick ◽  
David V. Hansen ◽  
Mark Levasseur ◽  
Peter K. Jackson ◽  
Keith T. Jones
Keyword(s):  
Fluorescent Protein ◽  
Polar Body ◽  
Cyclin B1 ◽  
Anaphase Promoting Complex ◽  
Morpholino Knockdown ◽  
Short Time ◽  
Mitotic Inhibitor ◽  
Antisense Morpholino ◽  
Meiosis I

During interkinesis, a metaphase II (MetII) spindle is built immediately after the completion of meiosis I. Oocytes then remain MetII arrested until fertilization. In mouse, we find that early mitotic inhibitor 2 (Emi2), which is an anaphase-promoting complex inhibitor, is involved in both the establishment and the maintenance of MetII arrest. In MetII oocytes, Emi2 needs to be degraded for oocytes to exit meiosis, and such degradation, as visualized by fluorescent protein tagging, occurred tens of minutes ahead of cyclin B1. Emi2 antisense morpholino knockdown during oocyte maturation did not affect polar body (PB) extrusion. However, in interkinesis the central spindle microtubules from meiosis I persisted for a short time, and a MetII spindle failed to assemble. The chromatin in the oocyte quickly decondensed and a nucleus formed. All of these effects were caused by the essential role of Emi2 in stabilizing cyclin B1 after the first PB extrusion because in Emi2 knockdown oocytes a MetII spindle was recovered by Emi2 rescue or by expression of nondegradable cyclin B1 after meiosis I.

Pleiotropic effect of okadaic acid on maturing mouse oocytes

Development ◽  
1991 ◽  
Vol 112 (4) ◽  
pp. 971-980 ◽  
Author(s):  
H. Alexandre ◽  
A. Van Cauwenberge ◽  
Y. Tsukitani ◽  
J. Mulnard
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Protein Phosphatases ◽  
Chromatin Condensation ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Inhibitory Effect ◽  
Negative Control ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes

Okadaic acid (OA), a potent inhibitor of types 1 and 2A protein phosphatases, was shown recently to induce chromatin condensation and germinal vesicle breakdown (GVBD) in mouse oocytes arrested at the dictyate stage by dibutyryl cAMP (dbcAMP), isobutyl methylxanthine (IBMX) and 12,13-phorbol dibutyrate (PDBu). We confirm these results using IBMX and another phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and show that OA also bypasses the inhibitory effect of 6-dimethylaminopurine (6-DMAP). It has been concluded that protein phosphatases 1 and/or 2A (PP1, 2A), involved in the negative control of MPF activation, are thus operating downstream from both the protein kinase A and protein kinase C catalysed phosphorylation steps that prevent the breakdown of GV. Similar enzymatic activities are also able to counteract the general inhibition of protein phosphorylation. However, PP1 and/or PP2A are positively involved in the activation of pericentriolar material (PCM) into microtubule organizing centres (MTOCs). This explains the inhibitory effect of OA on spindle assembly. Finally, OA interferes with the integrity and/or function of actomyosin filaments. This results in a dramatic ruffling of the plasma membrane leading to the internalization of large vacuoles, the inhibition of chromosome centrifugal displacement and, consequently, the prevention of polar body extrusion.

The Role of Tissue Transglutaminase in the Germinal Vesicle Breakdown of Mouse Oocytes

2001 ◽  
Vol 286 (2) ◽  
pp. 229-234 ◽  
Author(s):  
Sung Woo Kim ◽  
Zee-Won Lee ◽  
ChangKyu Lee ◽  
Kyung Soon Im ◽  
Kwon-Soo Ha
Keyword(s):  
Tissue Transglutaminase ◽  
Germinal Vesicle ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes

Several protein regions contribute to determine the nuclear and cytoplasmic localization of the influenza A virus nucleoprotein

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 135-142 ◽  
Author(s):  
Rosario Bullido ◽  
Paulino Gómez-Puertas ◽  
Carmen Albo ◽  
Agustín Portela
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Phosphorylation Site ◽  
Nuclear Accumulation ◽  
Systematic Analysis ◽  
Ribonucleoprotein Complexes ◽  
Cytoplasmic Transport ◽  
Green Fluorescent

A systematic analysis was carried out to identify the amino acid signals that regulate the nucleo-cytoplasmic transport of the influenza A virus nucleoprotein (NP). The analysis involved determining the intracellular localization of eight deleted recombinant NP proteins and 14 chimeric proteins containing the green fluorescent protein fused to different NP fragments. In addition, the subcellular distribution of NP derivatives that contained specific substitutions at serine-3, which is the major phosphorylation site of the A/Victoria/3/75 NP, were analysed. From the results obtained, it is concluded that the NP contains three signals involved in nuclear accumulation and two regions that cause cytoplasmic accumulation of the fusion proteins. One of the karyophilic signals was located at the N terminus of the protein, and the data obtained suggest that the functionality of this signal can be modified by phosphorylation at serine-3. These findings are discussed in the context of the transport of influenza virus ribonucleoprotein complexes into and out of the nucleus.

Sign in / Sign up

Export Citation Format

Share Document