Molecular Cancer Research CTRC-AACR San Antonio Breast Cancer Symposium Bridging the Lab and the Clinic in Cancer Medicine
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yoshitome, S.
Right arrow Articles by Sagata, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yoshitome, S.
Right arrow Articles by Sagata, N.
Molecular Cancer Research 1:589-597 (2003)
© 2003 American Association for Cancer Research

Cell Death, Cell Cycle and Senescence

The C-Terminal Seven Amino Acids in the Cytoplasmic Retention Signal Region of Cyclin B2 are Required for Normal Bipolar Spindle Formation in Xenopus Oocytes and Embryos1

Satoshi Yoshitome1, Nobuaki Furuno2, Eikichi Hashimoto1 and Noriyuki Sagata3

1 Department of Biomedical Sciences, School of Life Sciences, Tottori University, Yonago, Japan;
2 Laboratory for Amphibian Biology, Graduate School of Science, Hiroshima University, Higashihiroshima, Japan; and
3 Department of Biology, Graduate School of Sciences, Kyushu University, Fukuoka, Japan

Requests for reprints: Satoshi Yoshitome, Department of Biomedical Sciences, School of Life Sciences, Tottori University, 86 Nishi-machi, Yonago 683-8503, Japan. Phone: 81-859-34-8044; Fax: 81-859-34-8208. E-mail: yoshitom{at}grape.med.tottori-u.ac.jp; or Nobuaki Furuno, Laboratory for Amphibian Biology, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima 739-8526, Japan. Phone & Fax: 81-824-24-7483. E-mail: nfuruno{at}hiroshima-u.ac.jp


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
In many vertebrates, cyclin B has several subtypes, but the functional differences among them are largely unclear. Previously, we have shown that Xenopus cyclin B2, not cyclin B1, is involved in bipolar spindle formation through its cytoplasmic retention signal (CRS) region. However, identification of a nuclear export signal (NES) in the CRS region of cyclin B1 raised the possibility that an NES-like sequence (NELS) present in the CRS region of cyclin B2 might be involved in bipolar spindle formation. We show here that cyclin B2 is actually exported from the nucleus via its NELS, but that overexpression of the cyclin B2 CRS region, having a mutated NELS, still inhibits bipolar spindle formation in oocytes. In contrast, overexpression of the cyclin B2 CRS region lacking its C-terminal seven amino acids no longer inhibits bipolar spindle formation in oocytes or embryos. These results suggest strongly that the CRS region, especially its C-terminal seven acidic residues, of cyclin B2 is required for bipolar spindle formation in both the meiotic and mitotic cell divisions.

Key Words: spindle apparatus • cyclin B • nuclear export • Xenopus oocytes


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
In higher eukaryotes, the mitotic cell cycle is divided into four phases, G1, S, G2, and M phases. Especially, M phase is the most prominent phase, in which nuclear envelope breakdown, chromosome condensation, and spindle formation occur. The progression through M phase in eukaryotic cells is mainly regulated by the activity of maturation/M phase-promoting factor (MPF) (1, 2). The activation of MPF is indispensable for initiating profound changes in the cellular architecture in M phase (3–6). MPF consists of two components: one is a catalytic subunit of cyclin-dependent kinase 1 (cdc2 kinase) and the other is a regulatory subunit, cyclin B (2). MPF is activated by phosphorylation of Thr161 and dephosphorylation of Thr14 and Tyr15 in cdc2 kinase and is inactivated by degradation of cyclin B at the metaphase/anaphase transition (2, 7).

The cyclin B protein consists of at least three functional domains: an N-terminal "destruction box," which is a prerequisite for the degradation of cyclin B at the metaphase/anaphase transition; a "cytoplasmic retention signal" (CRS), which is thought to be responsible for the cytoplasmic localization of cyclin B in interphase; and a "cyclin box," which is required for the binding of cyclin B to cdc2 kinase (8, 9). It is known that cyclin B has several subtypes in many vertebrate species (10–15), and that human cyclins B1 and B2 localize differently in the cytoplasm throughout interphase (9, 16). Particularly, cyclin B2 localizes to the Golgi apparatus, while cyclin B1 localizes to microtubules in interphase (14) and relocates to the nucleus at the beginning of M phase (12, 16). However, it was unclear whether the different subtypes of cyclin B have different functions or not, although they are known to be associated with the same cdc2 kinase subunit (17). Previously, we have suggested that cyclin B2, not cyclin B1, is specifically involved in bipolar spindle formation in Xenopus oocytes through its CRS region (18).

It was reported that cyclin B1 has a nuclear export signal (NES), which is located in the CRS region and contains four conserved hydrophobic residues, and shuttles between the nucleus and the cytoplasm (19–21). Thus, in interphase, the cytoplasmic localization of cyclin B1 is ensured by the NES-mediated nuclear export system, and, at the beginning of M phase, cyclin B1 is translocated to the nucleus by a nuclear localization signal (NLS) created by phosphorylation of the CRS (21–23). Cyclin B2 also has an NES-like sequence (NELS) in its CRS region (9), and, indeed, chicken cyclin B2 relocates to the nucleus at mitosis (12). These results raise the possibility that cyclin B2 might be involved in bipolar spindle formation via its nuclear export.

Here, in this study, we show that Xenopus cyclin B2 is actually exported from the nucleus by an NELS in the CRS region, but that overexpression of the cyclin B2 N-terminal fragment containing the CRS region with a mutated NELS still inhibited bipolar spindle formation in oocytes. These results suggest that the nuclear export of endogenous cyclin B2 is not involved in bipolar spindle formation. Interestingly, overexpression of the cyclin B2 CRS region lacking its C-terminal seven amino acids fails to inhibit bipolar spindle formation in oocytes. However, overexpression of these seven amino acids tagged with GST or microinjection of peptides of these amino acids cannot induce spindle defects. Moreover, similar results were obtained in embryos. These results show that C-terminal seven amino acids of the cyclin B2 CRS region is required, but not sufficient for spindle formation in meiosis and mitosis. Probably, cyclin B2 may be important for bipolar spindle formation via correct subcellular localization of cyclin B2 directed by cyclin B2 CRS region, not NELS, especially C-terminal seven amino acids.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Xenopus Cyclin B2 Is Exported From the Nucleus in an Exportin 1-Dependent Manner
The sequence of the cyclin B1 CRS region is fairly well conserved among all cyclin B subtypes (9), and the NES sequence has been shown to be located in this region (19–21). Xenopus cyclin B2 has no typical NES sequence in its CRS region but has an NELS (Fig. 1A). In fact, chicken cyclin B2 relocates to the nucleus at mitosis (12). Therefore, we asked whether Xenopus cyclin B2 would also be exported in an exportin 1-dependent manner. To do this, we used leptomycin B, a specific inhibitor of the NES-dependent intracellular transport system (24, 25). The nucleus and the cytoplasm in oocytes were manually isolated at 0, 3, 12, and 24 h after addition of 200 nM leptomycin B to the culture medium. [Because Xenopus oocytes are very large cells, effects of leptomycin B on these cells would be much slower than those on cultured cells and, indeed, it takes a long time for cyclin B1 to shuttle between the nucleus and cytoplasm (21).] The amount of endogenous cyclin B1 or B2 accumulated in the nucleus was then examined by Western blotting. In agreement with a previous report (21), we observed that cyclin B1 was progressively accumulated in the nucleus in the presence of leptomycin B (Fig. 1B, upper panel). Similar to cyclin B1, the accumulation of cyclin B2 was also observed appreciably in the nucleus, although the amount of cyclin B2 in the cytoplasm did not decrease greatly (Fig. 1B, lower panel). These results suggest strongly that Xenopus cyclin B2 is exported from the nucleus by an exportin 1-dependent manner and shuttles between the nucleus and the cytoplasm during interphase of the cell cycle as cyclin B1 does.



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 1. Inhibition of nuclear export of cyclin B by leptomycin B in oocytes. A. Comparison of the amino acid sequences of the CRS region between Xenopus cyclins B1 and B2. Conserved hydrophobic residues are shown in bold type. Dots indicate the well-conserved hydrophobic amino acids of the NES sequence of cyclin B1. Diamonds indicate hydrophobic amino acids of the NELS of cyclin B2. B. Western blot analysis with either anti-Xenopus cyclin B1 antibody (upper panel) or anti-Xenopus cyclin B2 antibody (lower panel). Oocytes were incubated with 200 nM leptomycin B, collected at the indicated times after addition of leptomycin B, and manually separated into the nucleus and the cytoplasm. Extracts from whole oocytes (Wh) and the cytoplasmic fraction (Cyt), each equivalent to one oocyte, and extracts from the nuclear fraction (germinal vesicle; GV) equivalent to two oocytes were subjected to Western blot analysis using anti-cyclin B1 antibody. The arrowhead indicates either endogenous cyclin B1 (upper panel) or endogenous cyclin B2 (lower panel).

 
The NELS in Xenopus Cyclin B2 Functions as an NES
Our results described above suggest that the NELS present in the cyclin B2 CRS region may act as an NES. Interestingly, this NELS is well conserved among cyclin B2 proteins in vertebrates including human (Ref. 9; see Fig. 2A). To address whether this NELS indeed mediates the nuclear export of cyclin B2, we microinjected immature oocytes with mRNA encoding an N-terminal fragment of cyclin B2 or B2DC (the N-terminal 127-amino acid sequence; Ref. 18). This fragment containing an NELS can be expected to compete for the nuclear export of endogenous cyclin B2 when overexpressed (cf. Ref. 21). Ten hours after injection with B2DC mRNA (at a dose of 30 ng per oocyte; Ref. 18), we enucleated immature oocytes and examined the presence of endogenous cyclin B2 in the nucleus by Western blot analysis. Only a small amount of endogenous cyclin B2 was detected in the nucleus of uninjected control oocytes, but was detected in significant amounts in the nucleus of oocytes injected with B2DC mRNA (Fig. 2B). These results indicate that overexpression of B2DC can inhibit the shuttling of endogenous cyclin B2 between the nucleus and the cytoplasm, suggesting strongly that the NELS in Xenopus cyclin B2 acts as an NES.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 2. Inhibition of nuclear export of endogenous cyclin B2 by overexpression of an NELS in oocytes. A. Alignment of the putative NELSs of various vertebrate cyclin B2 proteins. The NELS from Xenopus cyclin B2 (XlB2), chicken cyclin B2 (GgB2), mouse cyclins B2 (MmB2), hamster cyclin B2 (MaB2), and human cyclin B2 (HsB2) were taken from the DDBJ/EMBL/GenBank databases and aligned. Conserved hydrophobic residues are shown in bold type. Dots indicate the hydrophobic amino acids conserved in the cyclin B1 NES. Diamonds indicate the hydrophobic amino acids of the NELS in Xenopus cyclin B2. B. Accumulation of endogenous cyclin B2 in the nucleus after injection of B2DC mRNA. Oocytes injected with mRNA were incubated overnight, manually separated into the nucleus and the cytoplasm, and subjected to Western blot analysis using anti-cyclin B2 antibody. Wh, whole; Cyt, cytoplasm; GV, germinal vesicle (nucleus). The arrowhead indicates endogenous cyclin B2.

 
Overexpression of the Cyclin B2 CRS Region With a Mutated NELS Can Still Inhibit Bipolar Spindle Formation
Previously, we have shown that overexpression of the cyclin B2 CRS region (B2DC) is able to induce spindle defects, most likely by perturbing the subcellular localization of endogenous cyclin B2 (18). However, the above result that cyclin B2 is exported from the nucleus to the cytoplasm via the NELS in its CRS region raises the possibility that the spindle defects observed with overexpression of the CRS region might have been caused by a specific inhibition of the shuttling of endogenous cyclin B2 between the nucleus and the cytoplasm. Thus, to examine whether overexpression of the NELS of B2DC could affect bipolar spindle formation, we made two NELS mutants of B2DC (hereafter referred to as L103A and F107A, respectively), in which either of two hydrophobic amino acids in the NELS was replaced by alanine to disrupt the NELS (cf. Refs. 19–21; Fig. 3A). First, we injected immature oocytes with mRNA encoding L103A or F107A B2DC and examined the presence of endogenous cyclin B2 in the nucleus by Western blotting. As shown in Fig. 3B, endogenous cyclin B2 was very scarce in the nucleus of oocytes injected with either L103A or F107A B2DC mRNA, but was significantly present in the nucleus of oocytes injected with normal B2DC mRNA. These results indicate that L103A and F107A B2DC cannot inhibit the shuttling of endogenous cyclin B2 between the nucleus and the cytoplasm, suggesting that they have no NES function. In fact, both L103A and F107A B2DC fragments accumulated in the nucleus in greater amounts than the intact B2DC (Fig. 3C). Together, these results indicate that both L103A and F107A mutations abrogated the NES function of the NELS in the B2DC.



View larger version (52K):
[in this window]
[in a new window]
  		FIGURE 3. Bipolar spindle formation is independent of the nuclear export of cyclin B2. A. Schematic diagrams of N-terminal fragments of cyclin B2 used in this study. Regions of the cytoplasmic retention signal (CRS), NES-like sequence (NELS), and the destruction box (DB) are indicated. L103A and F107A are mutants in which Leu103 or Phe107 was replaced by Ala in the N-terminal fragment of cyclin B2 (B2DC) to disrupt the NELS. B. Accumulation of endogenous cyclin B2 into the nucleus in oocytes injected with mRNA encoding B2DC, L103A, or F107A. Oocytes injected with mRNAs were incubated overnight and then manually separated into the nucleus and the cytoplasm. Extracts from two germinal vesicles were subjected to Western blot with anti-cyclin B2 antibody. The arrowhead indicates endogenous cyclin B2. C. Accumulation of the cyclin B2 N-terminal fragments into the nucleus in oocytes injected with mRNA encoding B2DC, L103A, or F107A. Oocytes injected with mRNAs were incubated overnight and dissected into the nuclear and cytoplasmic fractions. Extracts from whole oocytes (Wh) and the cytoplasmic fraction (Cyt), each equivalent to one-fifth oocyte, and extracts from the nuclear fraction (germinal vesicle; GV) equivalent to two-fifth oocytes were subjected to Western blot analysis using anti-cyclin B2 antibody. D. Spindle morphology in oocytes injected with B2DC, L103A, or F107A mRNA. Oocytes injected with mRNAs were treated with progesterone, harvested 4 h after GVBD, and subjected to cytological examinations. Scale bar, 10 µm.

 
Next, we asked whether the two NELS mutants (L103A and F107A) were still able to inhibit the bipolar spindle formation. (If the NELS of cyclin B2 were involved in bipolar spindle formation, then overexpression of the NELS mutants would fail to inhibit bipolar spindle formation.) For this, we examined the spindle morphology in oocytes overexpressing the NELS mutants, L103A or F107A B2DC, at 240 min after germinal vesicle breakdown (GVBD). The majority (>60%) of the oocytes overexpressing either L103A or F107A B2DC was found to form a monopolar spindle, and this efficiency was nearly the same as that with overexpression of the intact B2DC (Fig. 3D). These results suggest strongly that bipolar spindle formation required the function of some other CRS region than the NES of cyclin B2.

The C-Terminal Seven Amino Acids in the CRS Region of Cyclin B2 Are Important for Bipolar Spindle Formation
As described above, we have suggested that the CRS region except for its NELS is important for cyclin B2 to be involved in bipolar spindle formation. Then, we wished to identify which region of the cyclin B2 CRS is important for bipolar spindle formation. Comparison of the amino acid sequences of cyclin B proteins revealed that the C-terminal seven amino acids of the CRS region, which are rich in acidic residues, are highly conserved among all cyclin B proteins in vertebrates (cf. Ref. 9; Fig. 4A). Hence, we made two truncation mutants of the C-terminal of B2DC, termed N114 and N121: N121 has the complete CRS region, but N114 lacks the highly conserved C-terminal seven amino acids of the CRS region (Fig. 4B). These truncation mutants have an NELS, and would be expected to compete for the nuclear export of endogenous cyclin B2 when overexpressed. To address this, we injected immature oocytes with mRNA encoding either N121 or N114 and examined the presence of endogenous cyclin B2 in the nucleus by Western blot analysis. Significant amounts of endogenous cyclin B2 were detected in the nucleus of oocytes injected with either N-terminal fragment mRNAs (Fig. 4C). These results suggest that both of the two truncation mutants can inhibit the shuttling of endogenous cyclin B2 between the nucleus and the cytoplasm.



View larger version (56K):
[in this window]
[in a new window]
  		FIGURE 4. Overexpression of the cyclin B2 CRS lacking the conserved C-terminal seven amino acids fails to induce spindle defects. A. Alignment of the conserved C-terminal seven amino acids in the CRS region of various vertebrate cyclin B proteins. The sequences of Xenopus cyclin B2 (XlB2), chicken cyclin B2 (GgB2), mouse cyclins B2 (MmB2), hamster cyclin B2 (MaB2), human cyclin B2 (HsB2), Xenopus cyclin B1 (XlB1), mouse cyclins B1 (MmB1), hamster cyclin B1 (MaB1), and human cyclin B1 (HsB1) were taken from the DDBJ/EMBL/GenBank databases and aligned. Acidic residues are shown in bold type. B. Schematic diagrams of the N-terminal fragments of cyclin B2 used in this study. For B2DC, see Fig. 3A. N114 and N121 contain N-terminal 114 and 121 amino acids of cyclin B2, respectively. N121 contains intact CRS region, but N114 lacks the conserved C-terminal seven amino acids of CRS region. C. Accumulation of endogenous cyclin B2 into the nucleus in oocytes injected with mRNA encoding either B2DC, N114, or N121. Oocytes injected with mRNAs were incubated overnight and then manually separated into the nucleus and the cytoplasm. Extracts from two germinal vesicles were subjected to Western blot with anti-cyclin B2 antibody. The arrowhead indicates endogenous cyclin B2. D. Spindle morphology in oocytes injected with either N114 or N121 mRNA. Oocytes injected with either mRNA were treated with progesterone, harvested 4 h after GVBD, and subjected to cytological examinations. Scale bar, 10 µm.

 
We next examined mitotic apparatus at 240 min after GVBD in oocytes injected with mRNA encoding either N121 or N114. In the majority (>60%) of oocytes injected with N121 mRNA, we observed a monopolar spindle, just as in oocytes injected with B2DC mRNA (Fig. 4D). In contrast, a normal bipolar spindle arrested at metaphase I was observed in most (>90%) of the oocytes injected with N114 mRNA. [This arrest was due to the competitive inhibition of the cyclin degradation machinery (18).] In these experiments, N114 and N121 proteins were found to be synthesized in comparable amounts (data not shown). Thus, these results indicate that the C-terminal seven-amino acid sequence of the cyclin B2 CRS region is important for normal bipolar spindle formation. Furthermore, the normal bipolar spindle formation in the oocytes overexpressing N114, which contains a complete NELS, indicates again that the NELS is not involved in bipolar spindle formation.

The C-Terminal Seven Amino Acids of the Cyclin B2 CRS Region Are Not Sufficient to Induce the Spindle Defects
The above result obtained suggested that the C-terminal seven amino acids of the cyclin B2 CRS region are necessary to induce the spindle defects in oocytes. Next, we asked whether the C-terminal seven amino acids of the cyclin B2 CRS region are sufficient to induce the spindle defects in oocytes. (As this region is rich in acidic residues, it is possible that these seven amino acids may be important for bipolar spindle formation.) To address this possibility, we designed the construct that triple these seven amino acids is tagged with GST, because the stretch of these is a very short one. We prepared mRNA encoding the GST-tagged triple the C-terminal seven amino acids of the cyclin B2 CRS region (GST-triple 7 a.a.). We injected mRNA into these oocytes and examined mitotic apparatus on the meiotic maturation. Most (>90%) of the oocytes injected with mRNA showed normal changes in spindle morphology during maturation (Fig. 5 left panel; Meta-II arrested spindle at 240 min after GVBD). As revealed by Western blot analysis, the overexpressed GST-triple 7 a.a protein was fairly stable in oocytes (data not shown). These results revealed that oocytes injected with GST-triple 7a.a. mRNA underwent normal changes in spindle morphology during maturation. These results suggest that the C-terminal seven amino acids may not be sufficient for bipolar spindle formation. However, the quantity of expressed GST-tagged seven amino acids may be low—not enough to disturb normal spindle formation. To circumvent this problem, we synthesized peptides corresponding to the C-terminal seven-amino acid sequence (7 a.a. peptides). We injected immature oocytes with 7 a.a. peptides (300 pmol/oocyte). This amount is about sixty thousand times as much as endogenous cyclin B2 (5 fmol/oocyte; Ref. 26). At 240 min after GVBD, we examined cytologically t-heir morphologies. In most (>90%) of the oocytes injected with 7 a.a. peptides, we observed a normal bipolar spindle arrested at metaphase II (Meta-II) showing the normal progression of the meiotic cell cycle (Fig. 5, right panel). In spite of the injection of 7 a.a. peptides 600 pmol, we observed essentially the same bipolar spindle even in these oocytes (data not shown). These results indicate that the seven amino acids alone had no appreciable effect on spindle formation throughout maturation. Therefore, these acidic rich residues are not sufficient but necessary to induce spindle defect and would be a part of region to be actually required for bipolar spindle formation.



View larger version (99K):
[in this window]
[in a new window]
  		FIGURE 5. Spindle morphology in oocytes injected with either mRNA encoding GST-tagged triple the C-terminal seven amino acids (left panel) or the synthetic peptides of the C-terminal seven-amino acid sequence (right panel). Oocytes injected with either the synthetic peptides or mRNA were treated with progesterone, harvested 4 h after GVBD, and subjected to cytological examinations. Arrowhead, first polar body; scale bar, 10 µm.

 
Overexpression of the CRS Region of Cyclin B2 Inhibits Bipolar Spindle Formation in Mitosis as Well
Our results described above and previously (18) were obtained by using oocytes, the pathway for bipolar spindle formation of which is quite different from that in mitosis, because of the absence of centrosomes in the meiotic spindle apparatus (27, 28). Thus, it is possible that our proposal that the cyclin B2 CRS region (except for the NELS) is involved in bipolar spindle formation might be confined only to oocytes. To investigate whether the cyclin B2 CRS region was also involved in the mitotic spindle formation or not, we injected one blastomere of two-cell embryos with mRNA encoding either B1mDC or B2mDC, in which two amino acids in the destruction box were mutated not to compete for the cyclin degradation machinery (18), and examined their spindle morphologies when uninjected control blastomeres reached the blastula stage. All of the embryos injected with B1mDC mRNA (14 ng/blastomere) developed normally and showed an apparently normal bipolar mitotic spindle (Fig. 6; Table 1), just as the oocytes injected with B1mDC mRNA (30 ng/oocyte; Fig. 6). In contrast, when injected with B2mDC mRNA (14 ng/blastomere), although some embryos cleaved normally, others cleaved irregularly and arrested at the 16/32-cell stages. [B2mDC protein synthesized in embryos and oocytes was found to be in comparable amounts (data not shown).] Cytological examination of these abnormal embryos revealed that cleavage furrows did not invade deeply with several abnormal multipolar spindles being formed (Fig. 6; Table 1). These results indicate that bipolar spindle formation was inhibited by overexpression of the CRS region of cyclin B2, but not of cyclin B1, even in mitotic cell divisions, although the morphology of the mitotic spindle was different from that of the meiotic spindle in oocytes injected with B2mDC mRNA (30 ng/oocyte; Fig. 6) (see "Discussion").



View larger version (182K):
[in this window]
[in a new window]
  		FIGURE 6. Spindle morphology in oocytes or embryos injected with mRNA encoding either B1mDC or B2mDC. Oocytes injected with either B1mDC or B2mDC mRNA were treated with progesterone, harvested 4 h after GVBD, and subjected to cytological examinations. Embryos injected with either B1mDC or B2mDC mRNA were collected at the early blastula stage and subjected to cytological examinations. Scale bar, 10 µm.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Spindle Defects in Embryos Injected With Various Cyclin B2 N-Terminal Fragments

 
We also examined whether the C-terminal seven amino acids of the cyclin B2 CRS region were important for bipolar spindle formation in mitosis. To do this, we made non-degradable mutants of both N114 and N121, termed N114mD and N121mD, respectively. (Mutation in the destruction box is essential for this type of experiment; see above.) We injected one blastomere of two-cell embryos with either N121mD or N114mD mRNA and examined their spindle morphologies. We observed that abnormal multipolar spindles were formed in the embryos injected with N121mD mRNA, just as in those embryos injected with B2mDC mRNA (Table 1). In contrast, all of the spindles observed in the embryos injected with N114mD mRNA showed normal bipolar mitotic spindles (Table 1). These results suggest strongly that the C-terminal seven amino acids of the cyclin B2 CRS region are implicated in bipolar spindle formation in both meiosis and mitosis. The difference of spindle morphology between oocytes and embryos injected with B2mDC mRNA might be due to the different pathways of spindle formation between them (see "Discussion").


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
In this study, by using leptomycin B and cyclin B2 CRS mutants, we have shown that like cyclin B1, cyclin B2 has an NES in the CRS, but that the NES is not essential for the overexpression of cyclin B2 CRS to disrupt bipolar spindles in oocytes and embryos. Instead, we have shown that the C-terminal seven amino acids of the CRS are required for spindle defects by overexpression of B2 CRS.

The consensus sequence of the NES contains four hydrophobic residues with characteristic spacings (19–21, 24, 25, 29). However, our results on the NELS of Xenopus cyclin B2 indicate that three of the four well-conserved hydrophobic residues are sufficient for the NELS to act as an NES. In fact, since cyclin B1 has a classical NES, we observed that B2DC is able to inhibit the nuclear export of endogenous cyclin B1. When B2DC was overexpressed, cyclin B1 accumulated in the nucleus (data not shown). These results indicate that B2DC inhibits the nuclear export of endogenous cyclin B1. The NELS is well conserved in cyclin B2 proteins in vertebrates (cf. Ref. 9; Fig. 2A), suggesting that all cyclin B2 proteins may be exported from the nucleus during interphase of the cell cycle.

Cyclin B1 is localized to the cytoplasm during interphase of the cell cycle by dynamic equilibrium of the nuclear export and import, and translocates to the nucleus just before nuclear envelope breakdown (30). This translocation is mediated by an NLS created by phosphorylation of serines in the CRS region (22, 23) and hence by the direct interaction of cyclin B1 with importin-{alpha} (31). Similar to cyclin B1, a fraction of cyclin B2 is translocated from the cytoplasm to the nucleus at late prophase in Xenopus oocytes (32), although the timing of its nuclear translocation seems to be slightly slower than that of cyclin B1 (our unpublished data). Moreover, the CRS region of cyclin B2 is phosphorylated in Xenopus oocytes (our unpublished data). Thus, it seems likely that cyclin B2 is translocated to the nucleus by an NLS created by serine phosphorylation in the CRS region.

Spindle defects we reported before by overexpression of the cyclin B2 CRS seemed to be caused by the perturbation of the subcellular localization of endogenous cyclin B2 (18). However, we have shown here that the cyclin B2 CRS region contains an NELS, and that cyclin B2 is exported from the nucleus via the NELS. These observations raised the possibility that nuclear export of endogenous cyclin B2 might have been perturbed by overexpression of the NELS that was present in the CRS region. However, the NELS mutants, L103A and F107A, still cause spindle defects when overexpressed in oocytes (Fig. 3C). In contrast, the N114 construct, which has a normal NELS but not the C-terminal seven amino acids of the CRS, had no effect on spindle formation (Fig. 4, C and D). Together, these results suggest strongly that the nuclear export of endogenous cyclin B2 is not involved in bipolar spindle formation in Xenopus oocytes. Rather, the present results support our previous proposal (18) that the subcellular localization of endogenous cyclin B2 that is directed by the CRS region itself is important for bipolar spindle formation in oocytes, although we cannot observe the change of subcellular localization of endogenous cyclin B2 when cyclin B2 CRS region was overexpressed in oocytes. Interestingly, Kotani et al. revealed that cyclin B2 localizes on the spindle by immunocytochemist. Moreover they showed that antisense RNA-mediated inhibition of translation of cyclin B2, but not cyclin B1, and in Rana japonica oocytes induced the monopolar spindle similar to it by overexpression of the B2DC (33). Coupling these observations with our findings, cyclin B2 would be involved in the formation of bipolar spindle by correct subcellular localization of cyclin B2 in Xenopus oocytes and embryos.

We showed that the C-terminal seven amino acids of the cyclin B2 CRS region are a part of the region for bipolar spindle formation, although this acidic rich residues are not sufficient to induce the spindle defects in oocytes (probably, in embryos, too). This C-terminal sequence is well conserved among cyclin B proteins in many vertebrate species (Fig. 4A), although the subcellular localization (9) and the role in bipolar spindle formation of cyclins B1 and B2 are apparently different between them. However, careful comparison of the acidic rich residues among cyclin B proteins reveals that the first amino acid of the seven amino acids (Glu 115; Fig. 4A) is conserved only in cyclin B2 proteins. Thus, the first glutamic acid of the C-terminal seven amino acids may be involved in bipolar spindle formation, suggesting that other domain in cyclin B2 CRS region, in addition to this acidic rich residues, would determine the difference of cyclin B2 from cyclin B1 on bipolar spindle formation. Although we did not address whether the seven amino acids are actually required for correct subcellular localization of cyclin B proteins, our findings would provide a good clue to dissolve the functional differences of cyclin B subtypes. However, recent work by Hochegger et al. (34) has shown that three additional subtypes of cyclin B are present in Xenopus (cyclin B3, B4, and B5). Comparison among them has revealed that cyclin B5 strongly resembles cyclin B2, especially the CRS region including seven amino acids. Thus, it is possible that overexpression of cyclin B2 CRS could inhibit the subcellular localization of not only cyclin B2 but also cyclin B5, consequently leading to spindle defects.

We also examined the role of the cyclin B2 CRS region in bipolar spindle formation in Xenopus embryos. By overexpression of the cyclin B2 CRS region, we could observe spindle defects in cleaving embryos as in oocytes. These results suggest that correct subcellular localization of cyclin B2 via its CRS region would be important for bipolar spindle formation not only in meiosis but also in mitosis in Xenopus. However, the phenotype of spindle defects induced by overexpression of the cyclin B2 CRS was very different between oocytes and embryos: monopolar spindles in oocytes versus multipolar spindles in embryos. This difference might be due to the different pathways of bipolar spindle formation between meiosis and mitosis (27, 28), as discussed below.

The microtubule organization centers (MTOCs) in the mitotic spindle are centrosomes, and bipolar spindle formation is initiated by separation of the centrosomes (35). A growing body of evidence indicates that many kinds of MT-based motor proteins are commonly involved in mitotic and meiotic bipolar spindle formation (36). For example, cytoplasmic dynein (minus end-directed motor protein) is involved in the initial separation of the spindle poles and organizes microtubules, irrespective of the presence or absence of centrosomes, to form the spindle poles (37). After nuclear envelope breakdown, bipolar plus end-directed motor proteins such as the BimC kinesin family members (Eg5, KRP130, and Xklp2) further push spindle poles apart. These bipolar kinesins are also involved in spindle bipolarity (27, 38–41). In Xenopus oocytes, however, centrosomes are absent; instead, MTOCs with a compact aggregate of microtubules (MTs) (and condensed chromosomes) are formed just beneath the germinal vesicle during oocyte maturation, and bipolar spindle formation starts from the MTs (42, 43). Moreover, in the course of meiotic spindle formation, a monaster spindle is transiently observed (43). Our observations that overexpression of the cyclin B2 N-terminal fragments containing the CRS region can induce monopolar spindles in Xenopus oocytes could be best understood if we assume that the overexpression perturbed subcellular localization of endogenous cyclin B2 by inhibiting bipolar plus end-directed motor proteins such as klp2 and Eg5. This idea is consistent with the monopolar spindle formation previously observed with inhibition of klp as well as with the co-localization of cyclin B2 and klp2 or Eg5 (see Discussion of Ref. 18). Moreover, as the monopolar spindle is transiently formed during oocyte maturation (43), it might be maintained if Eg5 or klp2 were inhibited.

In embryos injected with either B2mDC or N121mD mRNA, multipolar spindles were observed; typically, four spindle poles were clearly observed in several blastomeres, with spindle bipolarity being disrupted (Fig. 6). Apparently, this phenotype was different from that in oocytes injected with B2DC or N121mD mRNA, suggesting, formally, that the "target(s)" of cyclin B2/cdc2 in the course of mitotic spindle formation may be different from that in meiotic spindle formation. Normally, in mitosis, the centrosome acts as an MTOC and cannot be duplicated in M phase (44). Apparently, however, the centrosome seemed to be duplicated at least once when the B2mDC or N121mD construct was overexpressed, and the blastomere seemed to be arrested in M phase. In fact, overexpression of non-degradable cyclin B has been shown to result in multipolar spindle formation in HeLa cells and Xenopus embryos (Ref. 12; our unpublished results). It seems likely, therefore, that ectopic expression of the non-degradable cyclin B2 N-terminal fragment containing the CRS region did not inhibit the destruction of endogenous cyclin B2, allowing the centrosome to be duplicated at least once. (When the wild type, degradable cyclin B2 N-terminal fragments were overexpressed, we could not observe any multipolar spindle formation in embryos.) Thus, in embryos overexpressing the non-degradable fragments containing the CRS region, cytoplasmic dynein might have normally functioned to separate the spindle poles, and multipolar spindles might have been maintained by inhibition of some bipolar kinesin(s) such as klp2 or Eg5. It has been reported that inactivation of Eg5 can lead to the formation of monopolar spindles in mitosis in intact cells (38) or in vitro (45, 46). If bipolar kinesins were only partially inhibited by overexpression of cyclin B2mDC or N121mD, however, multipolar spindle could be formed. Thus, the most plausible possibility is that cyclin B2/cdc2 targets bipolar kinesins such as the BimC kinesin family members in both mitotic and meiotic spindle formation.

In conclusion, our results suggest that correct subcellular localization of cyclin B2 directed by the CRS region would be essential for (the initiation of) bipolar spindle formation in Xenopus oocytes and embryos. Given that both cyclin B1/and B2/Cdc2 complexes have very similar substrate specificities (17), the specific functions of the respective cyclin B subtypes would be dependent on their different subcellular localizations that are directed by the CRS. In this study, we did not determine to which compartment(s) in oocytes and embryos the endogenous cyclin B2 localizes. This important issue remains to be resolved.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Preparation, Culture, Microinjection, Enucleation, and Treatment of Oocytes and Embryos
Oocytes were prepared, cultured, and microinjected as described (47, 48). Staging of the oocytes was done according to Dumont (49). Nuclear and cytoplasmic fractions were prepared as described by Paine et al. (50). To induce maturation, stage VI oocytes were treated with progesterone (5 µg/ml); to inhibit nuclear export, oocytes were treated with leptomycin B (200 nM). For cytological analysis of oocytes, maturing (and mature) oocytes were collected routinely at 240 min after GVBD, that is, at a time that corresponded to Meta-II in normally matured oocytes (18). Ovulated eggs were in vitro fertilized, and embryos were staged according to Nieuwkoop and Faber (51).

Construction of Recombinant Plasmids
All cyclin B2 N-terminal fragments were constructed by PCR, as described previously (18). The 5' primer used for both N114 and N121 was 5'-GCGAGATCTCGGCTAGATTTTATCGGTT-3', while the 3' primer for N114 was 5'-CGGGGATCCTCAAACACTGGTCAGTGCATC-3' and that for N121 was 5'-CGGGGATCCTCAATCATCTGCATCAATGTCTTC-3'. The 5' primer contained an artificial BglII site, and the 3' primers contained an artificial BamHI. After digestion with BglII and BamHI, the PCR products were subcloned into a BglII and BamHI-digested pT7-G (UK+) vector (52).

NELS mutants (L103A and F107A) were made by using a mutagenesis kit (Stratagene, La Jolla, CA). The primers for L103A were 5'-GAAAGAGGAAGAGGCGTGCCAGGCATTC-3' and 5'-GAATGCCTGGCACGCCTCTTCCTCTTTC-3'; the primers for F107A were 5'-GTGCCAGGCAGCCTCCGATGCACTG-3' and 5'-CAGTGCATCGGAGGCTGCCTGGCAC-3'. The primers for GST-triple 7 a.a. were 5'-CATGGTTGAAGACATTGATGCAGATGA-3' and 5'-CATGTCATCTGCATCAATGTCTTCAAC-3'. These primers were annealed and subcloned into a NcoI- digested pT7-G (UK-) vector fused with GST.

In Vitro Transcription
All the constructs subcloned into the pT7-G vector or pT7-G (UK+) vector were cut singly with an appropriate restriction enzyme (EcoRI or NotI) and then in vitro transcribed into 5'-capped mRNA, as described (48).

Antibodies and Western Blot Analysis
Anti-Xenopus cyclin B1 antibody was raised in rabbits against bacterially produced Xenopus cyclin B1 protein by standard methods. Routinely, protein equivalents to one oocyte were subjected to Western blot analysis with anti-Xe cyclin B1 antibody (1:500) or anti-Xe cyclin B2 antibody (1:5000; Ref. 53; a gift from James Maller). The secondary antibody, either donkey anti-rabbit IgG antibody (1:500; Amersham, Buckinghamshire, UK) or donkey anti-sheep antibody (1:5000; Jackson ImmunoResearch Laboratories, West Grove, PA), was detected by using the ECL system (Amersham).

Cytological Examination
Either oocytes or embryos were fixed in either Smith's or Bouin's solution, dehydrated and embedded, sectioned, stained with Feulgen's stain, and counterstained with fast green, as described previously (18, 54).


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Dr. T. Hunt for the kind gift of Xenopus cyclin B1 and B2 cDNAs, Dr. J. Maller for anti-Xenopus cyclin B1 and B2 antibodies, Dr. M. Yoshida for leptomycin B, and K. Gotou for editing the manuscript.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 Grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan. Back

Received June 25, 2002; revised April 14, 2003; accepted May 5, 2003.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

  1. Masui, Y. and Markert, C. L. Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. Zool., 177: 129–145, 1971.[Medline]
  2. Nurse, P. Universal control mechanism regulating onset of M-phase. Nature (Lond.), 344: 503–508, 1990.[Medline]
  3. Chou, Y. H., Bischoff, J. R., Beach, D., and Goldman, R. D. Intermediate filament reorganization during mitosis is mediated by p34cdc2 phosphorylation of vimentin. Cell, 62: 1063–1071, 1990.[Medline]
  4. Peter, M., Heitlinger, E., Haner, M., Aebi, U., and Nigg, E. A. Disassembly of in vitro formed lamin head-to-tail polymers by CDC2 kinase. EMBO J., 10: 1535–1544, 1991.[Medline]
  5. Yamashiro, S., Yamakita, Y., Hosoya, H., and Matsumura, F. Phosphorylation of non-muscle caldesmon by p34cdc2 kinase during mitosis. Nature (Lond.), 349: 169–172, 1991.[Medline]
  6. Verde, F., Dogterom, M., Stelzer, E., Karsenti, E., and Leibler, S. Control of microtubule dynamics and length by cyclin A- and cyclin B-dependent kinases in Xenopus egg extracts. J. Cell Biol., 118: 1097–1108, 1992.[Abstract/Free Full Text]
  7. Coleman, T. R. and Dunphy, W. G. Cdc2 regulatory factors. Curr. Opin. Cell Biol., 6: 877–882, 1994.[Medline]
  8. Hunt, T. Cyclins and their partners: from a simple idea to complicated reality. Semin. Cell Biol., 2: 213–222, 1991.[Medline]
  9. Pines, J. and Hunter, T. The differential localization of human cyclins A and B is due to a cytoplasmic retention signal in cyclin B. EMBO J., 13: 3772–3781, 1994.[Medline]
  10. Pines, J. and Hunter, T. Isolation of a human cyclin cDNA: evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2. Cell, 58: 833–846, 1989.[Medline]
  11. Minshull, J., Blow, J. J., and Hunt, T. Translation of cyclin mRNA is necessary for extracts of activated Xenopus eggs to enter mitosis. Cell, 56: 947–956, 1989.[Medline]
  12. Gallant, P. and Nigg, E. A. Cyclin B2 undergoes cell cycle-dependent nuclear translocation and, when expressed as a non-destructible mutant, causes mitotic arrest in HeLa cells. J. Cell Biol., 117: 213–224, 1992.[Abstract/Free Full Text]
  13. Gallant, P. and Nigg, E. A. Identification of a novel vertebrate cyclin: cyclin B3 shares properties with both A- and B-type cyclins. EMBO J., 13: 595–605, 1994.[Medline]
  14. Jackman, M., Firth, M., and Pines, J. Human cyclins B1 and B2 are localized to strikingly different structures: B1 to microtubules, B2 primarily to the Golgi apparatus. EMBO J., 14: 1646–1654, 1995.[Medline]
  15. Brandeis, M., Rosewell, I., Carrington, M., Crompton, T., Jacobs, M. A., Kirk, J., Gannon, J., and Hunt, T. Cyclin B2-null mice develop normally and are fertile whereas cyclin B1-null mice die in utero. Proc. Natl. Acad. Sci. USA, 95: 4344–4349, 1998.[Abstract/Free Full Text]
  16. Pines, J. and Hunter, T. Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport. J. Cell Biol., 115: 1–17, 1991.[Abstract/Free Full Text]
  17. Nigg, E. A. Cellular substrates of p34cdc2 and its companion cyclin-dependent kinases. Trends Cell Biol., 3: 296–301, 1993.[Medline]
  18. Yoshitome, S., Furuno, N., and Sagata, N. Overexpression of the cytoplasmic retention signal region of cyclin B2, but not of cyclin B1, inhibits bipolar spindle formation in Xenopus oocytes. Biol. Cell, 90: 509–518, 1998.[Medline]
  19. Toyoshima, F., Moriguchi, T., Wada, A., Fukuda, M., and Nishida, E. Nuclear export of cyclin B1 and its possible role in the DNA damage G2 checkpoint. EMBO J., 17: 2728–2735, 1998.[Medline]
  20. Hagting, A., Karlsson, C., Clute, P., Jackman, M., and Pines, J. MPF localization is controlled by nuclear export. EMBO J., 17: 4127–4138, 1998.[Medline]
  21. Yang, J., Bardes, E. S., Moore, J. D., Brennan, J., Powers, M. A., and Kornbluth, S. Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1. Genes Dev., 12: 2131–2143, 1998.[Abstract/Free Full Text]
  22. Hagting, A., Jackman, M., Simpson, K., and Pines, J. Translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation-dependent nuclear import signal. Curr. Biol., 9: 680–689, 1999.[Medline]
  23. Li, J., Meyer, A. N., and Donoghue, D. J. Nuclear localization of cyclin B1 mediates its biological activity and is regulated by phosphorylation. Proc. Natl. Acad. Sci. USA, 94: 502–507, 1997.[Abstract/Free Full Text]
  24. Fornerod, M., Ohno, M., Yoshida, M., and Mattaj, I. W. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell, 90: 1051–1060, 1997.[Medline]
  25. Fukuda, M., Asano, S., Nakamura, T., Adachi, M., Yoshida, M., Yanagida, M., and Nishida, E. CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature (Lond.), 390: 308–311, 1997.[Medline]
  26. Kobayashi, H., Minshull, J., Ford, C., Golsteyn, R., Poon, R., and Hunt, T. On the synthesis and destruction of A- and B-type cyclins during oogenesis and meiotic maturation in Xenopus laevis. J. Cell Biol., 114: 755–765, 1991.[Abstract/Free Full Text]
  27. Vernos, I. and Karsenti, E. Chromosomes take the lead in spindle assembly. Trends Cell Biol., 5: 297–301, 1995.[Medline]
  28. Merdes, A. and Cleveland, D. W. Pathways of spindle pole formation: different mechanisms; conserved components. J. Cell Biol., 138: 953–956, 1997.[Free Full Text]
  29. Fischer, U., Huber, J., Boelens, W. C., Mattaj, I. W., and Luhrmann, R. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell, 82: 475–483, 1995.[Medline]
  30. Furuno, N., den Elzen, N., and Pines, J. Human cyclin A is required for mitosis until mid prophase. J. Cell Biol., 147: 295–306, 1999.[Abstract/Free Full Text]
  31. Moore, J. D., Yang, J., Truant, R., and Kornbluth, S. Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1. J. Cell Biol., 144: 213–224, 1999.[Abstract/Free Full Text]
  32. Oe, T., Nakajo, N., Katsuragi, Y., Okazaki, K., and Sagata, N. Cytoplasmic occurrence of the Chk1/Cdc25 pathway and regulation of Chk1 in Xenopus oocytes. Dev. Biol., 229: 250–261, 2001.[Medline]
  33. Kotani, T., Yoshida, N., Mita, K., and Yamashita, M. Requirement of cyclin B2, but not cyclin B1, for bipolar spindle formation in frog (Rana japonica) oocytes. Mol. Reprod. Dev., 59: 199–208, 2001.[Medline]
  34. Hochegger, H., Klotzbucher, A., Kirk, J., Howell, M., le Guellec, K., Fletcher, K., Duncan, T., Sohail, M., and Hunt, T. New B-type cyclin synthesis is required between meiosis I and II during Xenopus oocyte maturation. Development, 128: 3795–807, 2001.[Abstract/Free Full Text]
  35. Kirschner, M. and Mitchison, T. Beyond self-assembly: from microtubules to morphogenesis. Cell, 45: 329–342, 1986.[Medline]
  36. Sharp, D. J., Roger, G. C., and Scholey, J. M. Microtubule motors in mitosis. Nature (Lond.), 407: 41–47, 2000.[Medline]
  37. Heald, R., Tournebize, R., Blank, T., Sandaltzopoulos, R., Becker, P., Hyman, A., and Karsenti, E. Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature (Lond.), 382: 420–425, 1996.[Medline]
  38. Blangy, A., Lane, H. A., d'Herin, P., Harper, M., Kress, M., and Nigg, E. A. Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. Cell, 83: 1159–1169, 1995.[Medline]
  39. Sawin, K. E. and Mitchison, T. J. Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle. Proc. Natl. Acad. Sci. USA, 92: 4289–4293, 1995.[Abstract/Free Full Text]
  40. Kashina, A. S., Scholey, J. M., Leszyk, J. D., and Saxton, W. M. An essential bipolar mitotic motor. Nature (Lond.), 384: 225, 1996.[Medline]
  41. Boleti, H., Karsenti, E., and Vernos, I. Xklp2, a novel Xenopus centrosomal kinesin-like protein required for centrosome separation during mitosis. Cell, 84: 49–59, 1996.[Medline]
  42. Gard, D. L. Organization, nucleation, and acetylation of microtubules in Xenopus laevis oocytes: a study by confocal immunofluorescence microscopy. Dev. Biol., 143: 346–362, 1991.[Medline]
  43. Gard, D. L. Microtubule organization during maturation of Xenopus oocytes: assembly and rotation of the meiotic spindles. Dev. Biol., 151: 516–530, 1992.[Medline]
  44. Lacey, K. R., Jackson, P. K., and Stearns, T. Cyclin-dependent kinase control of centrosome duplication. Proc. Natl. Acad. Sci. USA, 96: 2817–2822, 1999.[Abstract/Free Full Text]
  45. Sawin, K. E., LeGuellec, K., Philippe, M., and Mitchison, T. J. Mitotic spindle organization by a plus-end-directed microtubule motor. Nature (Lond.), 359: 540–543, 1992.[Medline]
  46. Walczak, C. E., Vernos, I., Mitchison, T. J., Karsenti, E., and Heald, R. A. model for the proposed roles of different microtubule-based motor proteins in establishing spindle bipolarity. Curr. Biol., 8: 903–913, 1998.[Medline]
  47. Sagata, N., Oskarsson, M., Copeland, T., Brumbaugh, J., and Vande Woude, G. F. Function of c-mos proto-oncogene product in meiotic maturation in Xenopus oocytes. Nature (Lond.), 335: 519–525, 1988.[Medline]
  48. Furuno, N., Nishizawa, M., Okazaki, K., Tanaka, H., Iwashita, J., Nakajo, N., Ogawa, Y., and Sagata, N. Suppression of DNA replication via Mos function during meiotic divisions in Xenopus oocytes. EMBO J., 13: 2399–2410, 1994.[Medline]
  49. Dumont, J. N. Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J. Morphol., 136: 153–179, 1972.[Medline]
  50. Paine, P. L., Johnson, M. E., Lau, Y. T., Tluczek, L. J. M., and Miller, D. S. The oocyte nucleus isolated in oil retains in vivo structure and functions. Biotechniques, 13: 238–246, 1992.[Medline]
  51. Nieuwkoop, P. D. and Faber, J. Normal Table of Xenopus laevis (Daudin). Amsterdam: North-Holland Publ., 1956.
  52. Nakajo, N., Yoshitome, S., Iwashita, J., Iida, M., Uto, K., Ueno, S., Okamoto, K., and Sagata, N. Absence of Wee1 ensure the meiotic cell cycle in Xenopus oocytes. Genes Dev., 14: 328–338, 2000.[Abstract/Free Full Text]
  53. Gautier, J., Minshull, J., Lohka, M., Glotzer, M., Hunt, T., and Maller, J. L. Cyclin is a component of maturation-promoting factor from Xenopus. Cell, 60: 487–494, 1990.[Medline]
  54. Nishizawa, M., Okazaki, K., Furuno, N., Watanabe, N., and Sagata, N. The "second-codon rule" and autophosphorylation govern the stability and activity of Mos during the meiotic cell cycle in Xenopus oocytes. EMBO J., 11: 2433–2446, 1992.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yoshitome, S.
Right arrow Articles by Sagata, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yoshitome, S.
Right arrow Articles by Sagata, N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online