From the Centre de Recherches de Biochimie Macromoléculaire, UPR 1086 CNRS, 1919 route de Mende, 34293 Montpellier cedex 5, France
Received for publication, August 2, 2002, and in revised form, October 30, 2002
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ABSTRACT |
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The Aurora kinase family has been involved both
in vivo and in vitro in the stability of
the metaphase plate and chromosome segregation. However, to date only
one member of this family, the protein kinase Aurora B, has been
implicated in the regulation of meiotic division in
Caenorhabditis elegans. In this species, disruption of
Aurora B results in the failure of polar body extrusion. To investigate
whether Aurora A is also required in meiosis, we microinjected highly
specific From yeast to human, members of the Aurora/Ipl1 kinase
family have been implicated in many mitotic cell cycle events ranging from centrosome separation and bipolar spindle assembly to chromosome segregation (1, 2). Thus, a direct interaction of the Aurora B kinase
with the inner centromere protein (INCENP) has been reported as
required for kinetochore disjunction and chromosome segregation during
mitosis (2-4). The Aurora A kinase has also been involved in mitosis,
because either RNA-mediated interference or mutational inactivation of the Drosophila Aurora A gene perturbs the
formation of the embryonic bipolar mitotic spindle (5, 6). In
Xenopus, the disorganization of a preformed mitotic
metaphase plate in egg extracts can also be induced by adding either a
specific antibody or a catalytically inactive mutant of the Aurora A
kinase (7, 8). Additionally, the functional disruption of an Aurora A substrate, the kinesin-like protein Eg5 (9), can also induce a collapse
of a performed mitotic metaphase (10, 11). These phenotypes are
consistent with a requirement of Aurora A kinase and the kinesin-like
protein Eg5 in the dynamic events required for mitotic metaphase plate
formation (5, 9). Despite the established role of Aurora kinase family
in mitosis, little is known about its participation in the control of
meiosis. In this regard, up to this date, only Aurora B has been shown
to be involved in meiosis. Maternal depletion of Aurora B in
Caenorhabditis elegans results in a failure of polar body
extrusion during first meiotic division, because of a
cytokinesis defect (12). To gain a better understanding of the
involvement of the Aurora kinase family in meiosis, we investigated
here whether in addition to Aurora B, Aurora A, which is expressed at
low levels at the first meiotic prophase in Xenopus oocytes,
is also involved in the control of meiotic division.
Preparation and Handling of Oocytes--
Ovaries from
Xenopus laevis females were surgically removed.
Fully grown oocytes were prepared, free from follicle cells, by
collagenase treatment and transferred in MMR buffer (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 0.1 mM EGTA, 5 mM HEPES, pH 7.7) in which all experiments were performed.
The usual volume microinjected was 50 nl. Progesterone was used at a
final concentration of 2 µg/ml. To prepare homogenates from oocytes,
single oocytes were crushed in a buffer containing 50 mM
When parthenogenetically activated oocytes were used,
immature oocytes were first incubated overnight in the presence of 2 µg/ml progesterone and subsequently activated by adding ionophore A23187 (Sigma) into the buffer at a final concentration of 2 µg/ml. Oocytes were microinjected with either 100 ng of antibodies or
together with 500 ng of corresponding recombinant protein when indicated. Aliquots of Xenopus cell-free extracts were
prepared as previously described (13).
Antibodies, Proteins, and Western
Blotting--
Affinity-purified antibodies against Xenopus
cyclin B2 and the C-terminal peptide sequence of Xenopus
Cdc2 have been described elsewhere (14, 15). The polyclonal antibodies
against Xenopus Aurora A were raised in Rabbits against
recombinant full-length Aurora A-His6 and
immunopurified using the recombinant Aurora A protein cross-linked to
CNBr-activated Sepharose (Amersham Biosciences) as an affinity
matrix. Rabbit Ig used as controls were obtained from Sigma. The
secondary anti-rabbit Ig-horseradish peroxidase conjugate was diluted
according to the supplier's recommendations (Amersham Biosciences),
and Western blot was monitored using ECL (PerkinElmer Life Sciences).
Monoclonal anti-Xenopus Aurora A antibodies as well as
anti-Eg5 antibodies were kindly provided by Dr. Prigent (8), Rennes,
France, and Dr. Blangy, Montpellier, France, respectively. Recombinant
full-length Aurora A-His6 protein was affinity-purified and
then dialyzed according to the supplier's recommendations (Qiagen).
Kinase Activities of Cdc2--
H1 activities on Cdc2
immunoprecipitates were assayed as described (16).
Cytological Observation--
Oocytes were fixed for 1 h in
a HEPES buffer (100 M KCl, 3 mM
MgCl2, and 10 mM HEPES, pH 7.8) containing
3.7% formaldehyde, 0.1% glutaraldehyde and 0.1% Triton X-100 and
then incubated overnight at room temperature in the HEPES buffer
containing 20 µg/liter Hoechst. Stained oocytes were then transferred
in HEPES buffer free of Hoechst stain and viewed from the animal pole
with a UV epifluorescence immersion objective (63X/0.90W U-V-I; Leica).
Microinjection of
We microinjected prophase-arrested Xenopus oocytes with
either Hormone-stimulated Oocytes Microinjected with Microinjection of
When Xenopus metaphase II-arrested oocytes are electrically
stimulated or treated with ionophore A23187, two procedures that mimic
fertilization, they resume anaphase II concomitantly with cyclin
degradation (13). We then asked whether cyclin B2 degradation could be
impaired in A growing number of reports indicate a requirement for Aurora A
and B in mitosis. Aurora A kinase has been implicated in the maintenance of mitotic metaphase plate as well as in cytokinesis (1,
2). Moreover, the Aurora kinase family seems to be also involved in
meiotic division because maternal Aurora B depletion in C. elegans results in the failure of polar body extrusion during first meiosis (12). We investigated here whether Aurora A, which is
expressed at low levels at first meiotic prophase in Xenopus oocytes (8, 23, 24), plays a role in the meiotic cell cycle. We used
microinjection of highly specific The kinesin-like Eg5 is a well reported substrate of Aurora A in
Xenopus (9). Disruption of this protein in different cell lines leads to a disorganization of the mitotic metaphase plate (10,
11) similar to that observed when Aurora A is blocked (9). As described
previously herein for In an effort to characterize the second mechanism controlling meiosis I
exit, we investigated whether the metaphase I arrest observed in
Meiotic metaphase II-arrested oocytes of vertebrates are believed to
exit naturally this stage when cyclin degradation is triggered by
fertilization or parthenogenetic activation (13). Our results indicate
that oocytes with a metaphase I-arrested phenotype normally degrade
cyclin B2 upon parthenogenetic activation. However, despite the fact
that our data show a requirement of Aurora A during first meiosis in
Xenopus oocytes, we could not assess whether this kinase is
also involved in the second meiotic anaphase because of the early
phenotype observed. We conclude that the disruption of Aurora A
in vivo does not imply a block of the normal meiotic
cytoplasmic cell cycle events. Moreover, our data reveal, for the first
time, a requirement of the Aurora A kinase and Eg5 in the meiosis of
Xenopus oocytes. We ask now how Aurora A impinges on the
segregation of homologous chromosomes at first meiosis in
Xenopus. Thus, the Aurora kinases seem to be key factors not
only in controlling mitotic events but also in the regulation of
meiotic division.
-Aurora A antibodies in Xenopus oocytes. We
demonstrated that microinjected oocytes fail to extrude the first polar
body and are arrested with condensed chromosomes on a typical
metaphase I plate, which has not performed its normal 90° rotation.
We additionally found that, although the failure of first polar body
extrusion observed in
-Aurora A-microinjected oocytes is likely
mediated by Eg5, the impairment of the metaphase plate rotation does
not involve this kinesin-like protein. Surprisingly, although
chromosomes remain condensed at a metaphase I stage in
-Aurora
A-microinjected oocytes, the cytoplasmic cell cycle events progress
normally through meiosis until metaphase II arrest. Moreover, these
oocytes are able to undergo parthenogenetic activation. We
conclude that Aurora A and Eg5 are involved in meiosis I to meiosis II
transition in Xenopus oocytes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, 10 mM MgCl2, 7.5 mM EGTA, 1 mM dithiothreitol, pH 7.3, and then briefly centrifuged for 3 min at 13,000 rpm. Supernatants were then used for Western blots or kinase assays. Scoring experiments for
the rate of GVBD1 after
various microinjections were conducted on at least 50 oocytes that stem
from four different Xenopus females.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Aurora A Antibodies Does Not Inhibit
Progesterone-induced Activation of Cdc2 Kinase--
To asses the
putative role of Aurora A in meiosis, we produced antibodies against
recombinant Xenopus Aurora A. These antibodies specifically
recognize and immunoprecipitate Xenopus Aurora A and do not
cross-react with the other member of the Aurora A family, the Aurora B
protein (Fig. 1, A and
B, respectively; see also Ref. 4 and supplementary data in
Ref. 18). Moreover, they do not inhibit Aurora A myelin basic protein
kinase activity in vitro (data not shown). Indeed, previous
reports have already shown mitotic spindle disorganization induced by
treatment of Xenopus egg extracts with anti-Aurora A
monoclonal antibodies that neither recognize the catalytic domain of
the kinase nor inhibit its kinase activity (7). This suggests that such
antibodies may exert their effect by perturbing the functional
targeting of the kinase and/or the accessibility of its
substrate.
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Fig. 1.
Characterization of anti-Aurora A
antibodies. A, aliquots of cell-free extracts (20 µl)
prepared from metaphase II-arrested Xenopus oocytes were
immunoprecipitated (IP) with affinity-purified polyclonal
anti-Aurora A antibodies ( -Aurora, 2 µg) or the same
amount of rabbit Ig and then analyzed by Western blotting with
monoclonal anti-Aurora A antibodies. B, aliquots of 1 µl
of cell-free interphase Xenopus egg extracts
(Int) or 2 µl of Aurora A (Auro A) and
Aurora B (Auro B) programmed reticulocyte lysate were
analyzed by PAGE and Western blotting with affinity-purified antibodies
against Aurora A (left) and Aurora B
(right).
-Aurora A antibodies (final concentration 13 ng/µl) or, as a control, the same amount of rabbit Ig. Then progesterone was added
into the media to induce meiotic maturation. Both microinjected and control oocytes underwent germinal vesicle breakdown (GVBD) with
similar kinetics (Fig. 2A). To
determine the cell cycle status of the corresponding oocytes, we
monitored the H1-kinase activity of Cdc2 at GVBD as a main landmark of
the G2-M transition. No differences between control and
treated oocytes were detected in the level of H1-kinase activity at the
time of GVBD (Fig. 2B).
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Fig. 2.
-Aurora A-microinjected oocytes
readily underwent GVBD but arrested at metaphase I. A,
oocytes were microinjected with either
-Aurora A antibodies or the
same amount of rabbit Ig and then incubated in the presence of
progesterone. The time of GVBD was established by scoring white-spot
formation in the animal pole under a dissecting microscope.
B, same oocytes as in A, but homogenates were
prepared from one of these oocytes at 0 h (prophase) or
at the time of GVBD, subjected to immunoprecipitation with
anti-Xenopus Cdc2 antibodies, and analyzed by
autoradiography for H1-kinase activity. C, maturing oocytes
(left-hand panels, top and middle) and oocytes
microinjected with anti-Aurora A antibodies (right-hand panels,
top and middle), with anti-Aurora A antibodies
preincubated with the recombinant Aurora A-His6 protein (1 µg of antibody/5 µg of protein) (bottom left), and with
control immunoglobulins (bottom right) were taken at the
indicated times after GVBD. Subsequently, they were fixed,
Hoechst-stained, and examined from the animal pole under an
epifluorescence microscope. The arrows indicate the first
polar body, and the scale bar represents 10 µm.
D, 1 µl of interphase (INT) or metaphase
II-arrested (CSF) Xenopus egg extracts were
submitted to SDS-PAGE and Western blotting with either anti-Aurora A
antibody (1/500 dilution; left panel) or the same serum
previously preabsorbed using the recombinant Aurora A-His6
cross-linked to CNBr-activated Sepharose (1/500 dilution; right
panel).
-Aurora A
Antibodies Are Arrested at Metaphase I--
We then assessed
the cytological status of microinjected
-Aurora A or control oocytes
by Hoechst dye staining and direct observation under a UV
epifluorescence microscope. As shown in Fig.
3, normal meiotic maturation in
Xenopus oocytes is characterized by the formation of a first
metaphase plate (20-60 min after GVBD), which is perpendicular to the
surface of the oocyte, followed by rotation of the metaphase plate by
90° (60-80 min after GVBD). Finally, homologous chromosomes undergo
anaphase (90-120 min after GVBD) leading to the extrusion of the first
polar body and meiotic metaphase II arrest (180 min after GVBD). As
shown in Fig. 2C (top left), control oocytes
fixed 3 h after GVBD had reached second meiotic metaphase and
arrested at this step. In contrast, oocytes microinjected with
-Aurora A antibodies (Fig. 2C, top right) were
systematically (see Table I) arrested,
with condensed chromosomes aligned on a metaphase plate oriented
perpendicular to the oocyte surface. This arrested phenotype is in fact
similar to the normal early metaphase plate before rotation of the
first meiotic spindle in control oocytes at 1 h after GVBD (Fig.
2C, middle left, and Fig. 3; see Ref. 19).
Moreover, no changes in
-Aurora A-microinjected oocytes were
observed when they were fixed 10 h after GVBD (Fig. 2C, middle right), suggesting that chromosomes
moved to form the first metaphase plate and did not proceed
further in the first meiotic cell cycle. Arrest at the first meitotic
metaphase before spindle rotation was a specific consequence of
blocking some essential function of Aurora A protein, because
microinjection of control antibodies had no such effect (Fig.
2C, bottom right). Moreover, no arrest at first
meiotic metaphase was observed when oocytes were co-injected with an
excess of recombinant Aurora A protein together with the native
antibodies (Fig. 2C, bottom left). We know that
under these conditions the recombinant Aurora A protein inhibits
antigen-antibody binding because the Western blot signal corresponding
to Aurora A kinase in interphase and metaphase II-arrested egg extracts
dramatically decreased when these preabsorbed antibodies were used
(Fig. 2D). The rotation of condensed chromosomes on the
metaphase plate was never observed in
-Aurora A-microinjected oocytes arrested at the first meiotic cell cycle. However, suppression of chromosomes segregation does not appear to be associated necessarily with a failure of the metaphase plate rotation, because a very small
proportion (see Table I) of injected oocytes escaped metaphase I arrest
and progressed to first meiotic anaphase even though the metaphase
plate failed to rotate (Fig. 4,
A and B). Because these experiments were
conducted on at least four different sets of oocytes, these latter
results probably reflect a slight natural heterogeneity of the
microinjected oocytes regarding the potential effect of microinjection.
Additionally, this subset of oocytes that were less responsive to
microinjection allow us to postulate that metaphase I plate rotation is
not required for the metaphase-to-anaphase transition to take
place.
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Fig. 3.
A, schematic representation and direct
epifluorescence observation of Xenopus oocytes during a
normal course of meiotic maturation. G2/prophase-arrested
oocytes were incubated with progesterone to resume meiotic maturation
and treated for epifluorescence observation at the indicated times as
described under "Material and Methods." The double-headed
arrow indicates the space between the two sets of homologous
chromosomes undergoing anaphase I, and the single-headed
arrow indicates the first polar body. The scale bar
represents 10 µm. B, -Eg5 antibody specificity. 1 µl
of interphase (INT) or metaphase II-arrested
(CSF) Xenopus egg extracts were submitted to
SDS-PAGE and Western blot with either anti-Eg5 affinity-purified
antibodies (1/1000 dilution; left panel) or control
immunoglobulins (1/1000 dilution; right panel).
-Aurora A microinjection arrests Xenopus oocytes at meiosis I
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Fig. 4.
First meiotic anaphase in the absence of
spindle rotation and requirement of the kinesin-like Eg5 for metaphase
I exit. A and B, oocytes injected with
-Aurora A antibodies were fixed 10 h after GVBD and examined
from the animal pole as in Fig. 2. Pictures of the same oocyte were
taken at two different focal planes (A and
B) to allow detection of separate sets of chromosomes.
C-F, oocytes injected with either
-Eg5 antibodies
(C, E, F) or the same amount of rabbit Ig as a control
(D) were taken 3 (C, D) or 10 h (E,
F; two different oocytes) after GVBD and examined by
epifluorescence as described in the legend for Fig. 2. The scale
bar represents 10 µm.
-Eg5 Antibodies Induces Metaphase I Arrest in
Maturing Xenopus Oocytes--
The kinesin-like protein Eg5 is a well
reported Aurora A substrate (9) in which homologues act as regulators
of mitotic cytokinesis in different species. Disruption of Eg5 function
in cell cultures by monastrol (10, 11) as well as Aurora A-antibody treatment of mitotic Xenopus egg extracts lead to a collapse
of the mitotic metaphase plate (7). To investigate whether the role of
Aurora A in meiosis is mediated by its substrate, Eg5, we tested for
whether this kinesin-like protein is also required to develop
complete meiotic division by microinjecting oocytes with highly
specific
-Eg5 antibodies (see Fig. 3B) or with rabbit Ig
in controls. Prophase oocytes microinjected with both rabbit Ig or
-Eg5 antibodies reached GVBD with the same kinetics when treated
with progesterone (data not shown). However, only
-Eg5-microinjected oocytes failed to emit the first polar body (Table I and Fig. 4C) by the time controls had reached the second meiotic
metaphase arrest (Fig. 4D), indicating that, like Aurora A,
Eg5 is also required for the oocyte to resume meiosis. Moreover,
although the first meiotic metaphase plate rotation was not impaired
(Fig. 4C), the subsequent organization of condensed
chromosomes was greatly perturbed, as observed in fixed oocytes
10 h after GVBD (Fig. 4, E and F). This
disorganization of the condensed chromosomes resembles the previously
described phenotype on the mitotic metaphase plate observed on cultured
cells after monastrol treatment (10, 11). These results indicate that
Aurora A, likely through its substrate Eg5, controls metaphase I exit;
however, they also indicate that the failure of metaphase I plate
rotation in these oocytes is mediated mainly by Aurora A and does not
involve the kinesin-like Eg5.
-Aurora A Antibodies Do Not Impair the Kinetics of Cyclin
Degradation during Either Meiotic Maturation or Meiotic Metaphase II to
Anaphase II Transition--
During meiosis of Xenopus
oocytes, a window of cyclin degradation occurs in the course of the
metaphase I to metaphase II transition (20-22). This window of
degradation is, however, naturally hidden by a dynamic cyclin
neosynthesis. Following this window, a progressive cyclin stabilization
step occurs, which culminates with a final high and permanent level of
Cdc2 activity. This cyclin stabilization results directly from
an accumulation of cytostatic factor activity (CSF) during
meiosis and allows the installation of a permanent Cdc2 kinase activity
(21, 22). Thus, because they contain CSF activity, vertebrate oocytes
fail to degrade cyclin B and are arrested at the second meiotic
metaphase until fertilization. It is well known that protein synthesis
is not required after GVBD for completion of the first meiotic cell
cycle in Xenopus oocytes. Thus, to facilitate detection of
cyclin degradation, we used cycloheximide (CHX) to suppress protein
synthesis during progesterone-induced meiotic maturation after GVBD. We
then assessed whether the first metaphase I arrest observed in
-Aurora A-microinjected oocytes could be the result of a cyclin
stabilization, leading to a possible cell cycle arrest, by monitoring
both cyclin B2 degradation and Cdc2 activity during meiosis.
Progesterone-treated oocytes, previously microinjected or not with
-Aurora A antibodies at the prophase stage, were transferred at the
indicated times after GVBD for 1 h in a buffer containing CHX.
Then they were homogenized to allow immunoblotting detection of cyclin
B2 as well as H1-kinase assay on Cdc2 immunoprecipitates. As shown
(Fig. 5A), cyclin B2
disappeared in control and
-Aurora A-microinjected oocytes at 30-60
min after GVBD. As a result of a nonreplacement of degraded cyclin due
to the CHX, Cdc2 kinase activity remained low. Moreover, a high Cdc2
activity and no cyclin B2 degradation were detected in both
-Aurora
A- and rabbit Ig-microinjected oocytes when CHX was added 100 min or
more after GVBD, by the time that oocytes were already arrested at
metaphase II. The normal degradation of cyclin B2 observed in
-Aurora A-microinjected oocytes indicates that the metaphase I
arrest in those oocytes is not the result of a general block of the
meiotic cell cycle. Moreover, despite the fact that the time course
establishment of the CSF activity, as revealed by cyclin B2
accumulation and an increase in Cdc2 activity (Fig. 5A),
remained unaffected in
-Aurora A-treated oocytes, the cytological
condensed status of meiosis I-arrested chromosomes did not evolve
during this period (Fig. 2C, middle right).
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Fig. 5.
The -Aurora
A-microinjected oocytes show both active
anaphase-promoting complex (APC)-dependent
degradation of cyclin B2 after GVBD and metaphase II exit upon
parthenogenetic activation. A, prophase oocytes
microinjected (left panel) or not (middle and
right panels) with
-Aurora A antibodies were treated with
progesterone and transferred at the indicated times (minutes) after
GVBD for 1 h in the presence of CHX (left and
middle panels). One oocyte/point was then crushed and the
homogenate split in two aliquots for either immunoblot with anti-cyclin
B2 (upper panels) or immunoprecipitation with anti-Cdc2
antibodies and H1-kinase assay (lower panels;
autoradiographs). The prophase lane (on the
left) consists of non-progesterone-treated oocytes. Control
oocytes (upper right panel) were crushed directly at the
indicated times without CHX treatment. B, metaphase
II-arrested oocytes, previously microinjected with either
-Aurora A
(upper panel) or the same amount of rabbit Ig as control
(lower panel) were parthenogenetically activated with
ionophore A23187. Groups of three oocytes were then crushed at the
indicated times, and one aliquot of the homogenates corresponding to a
single oocyte was used for cyclin B2 detection by immunoblotting.
-Aurora A-microinjected oocytes upon ionophore
treatment. To test this possibility, prophase Xenopus oocytes were microinjected with either
-Aurora A or the same amount
of rabbit Ig. Then progesterone was added to resume meiotic maturation,
and by the time that control oocytes arrested at metaphase II, oocytes
were activated with the ionophore. Time course degradation of the
endogenous cyclin B2 was then monitored by immunoblot. As shown in Fig.
5B, both treated and control oocytes normally degraded
cyclin B2. Our results indicate that, in
-Aurora A-microinjected oocytes, the failure to resume the first meiotic anaphase does not
impair subsequent cyclin degradation upon parthenogenetic activation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Aurora A antibodies to target
Aurora A kinase in vivo. We show here that, upon
microinjection, the ability of the recipient oocytes to resume meiotic
maturation until GVBD and to activate the Cdc2 kinase is not impaired.
However, we were surprised to find that the same recipient oocytes
failed to emit the first polar body and were arrested at meiosis
I with condensed chromosomes. Moreover the condensed chromosomes remain aligned on a metaphase plate oriented perpendicular to the oocyte surface. Although spindle rotation was never observed in meiosis I-arrested oocytes, we showed, in some limited cases, however, a small
proportion of
-Aurora A-microinjected oocytes that escaped metaphase
I arrest and progressed to first meiotic anaphase. This subset of
particularly less responsive oocytes probably indicates that metaphase
I to anaphase I transition in Xenopus oocytes does not need
a previous 90° rotation of the metaphase plate. On the basis of these
results, we concluded that Aurora A kinase is involved in separation of
homologous chromosomes and spindle rotation during meiosis. Because
Aurora A is a protein kinase it makes sense to hypothesize that this
protein exerts its role by phosphorylating one or several key
substrates controlling meiosis resumption. Microinjection of
-Aurora
A antibodies into Xenopus oocytes may inhibit
phosphorylation of the Aurora A targets. However, we (data not shown)
and others (7) have demonstrated that these antibodies have no effect
on Aurora A kinase activity, indicating that their blocking activity is
probably mediated by perturbation of functional targeting and/or
substrate accessibility.
-Aurora A antibodies,
-Eg5-microinjected
oocytes fail to emit the first polar body and are arrested with
condensed chromosomes organized on a rotated metaphase plate. Thus,
because
-Eg5-microinjected oocytes always succeed in the 90°
metaphase I plate rotation, we conclude that Aurora A may be involved
in two different mechanisms, the first one regulating metaphase I exit,
which is likely mediated by Eg5, and the second one, independent of
Eg5, which may control metaphase I plate rotation. In this regard,
there is no data concerning the regulation of spindle rotation by this
kinesin-like protein either in Xenopus oocytes or
other cell types. Unlike Eg5, the role of the dynein-dynactin complex
in spindle rotation during embryogenesis and cell division has been
reported largely in several species, for example in C. elegans (23) or Saccharomyces cerevisiae (24, 25) or in
mammalian cells (26). The current model proposes that astral
microtubules interact with cortical actin and dynactin through one
dynactin subunit, the actin-capping protein. Then, tethered by
dynactin, dynein interacts with the plus-end of microtubules and pulls
toward the anterior cortex by minus-end-directed motility causing
spindle rotation (23). According to this hypothesis, dynactin is
localized to the cortical microtubules capture site, and both dynactin
and dynein are required for spindle rotation (27). Several evidences
indicate that the dynein-dynactin-dependent mechanism of
spindle orientation is regulated by phosphorylation of both
complexes. Thus, phosphorylation of p150(Glued), a subunit of the
dynactin complex, mediates dynamic binding to microtubules (28).
Moreover, dynein-dynactin interaction is also regulated by
phosphorylation of the dynein intermediate chain (29). Spindle rotation
in Xenopus oocytes at metaphase I may also be mediated by a dynactin-dynein mechanism. Accordingly, cortical F-actin is
required for spindle rotation because rotation of metaphase I spindle
is completely inhibited upon cytochalasin B treatment (30). Moreover,
two components of the dynactin-dynein complex undergo coordinated
phosphorylation during oocyte maturation (17). In this work we
found that inhibition of Aurora A kinase blocks metaphase I plate
rotation and metaphase I exit. We propose that the first phenomenon may
be mediated by phosphorylation of one or several constituents of the
dynactin-dynein complex, as for example the p150(Glued) protein or the
intermediate chain of dynein.
-Aurora A-microinjected oocytes was the result of a general block of
the cell cycle. We found that normal meiotic cytoplasmic events, such
as cyclin B2 degradation, proceed normally. Thus, we conclude that the
arrest at first meiotic metaphase observed in
-Aurora
A-microinjected oocytes is not correlated with a general block of the
cell cycle.
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ACKNOWLEDGEMENT |
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We are grateful to Pierre Travo, head of the IFR24 integrated imaging facility, for his constant interest and support.
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FOOTNOTES |
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* This work was supported by the "Ligue Nationale Contre Le Cancer" (Equipe Labellisée).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Postdoctoral fellow supported by the Ligue Nationale Contre le Cancer.
§ To whom correspondence should be addressed: CRBM, UPR 1086 CNRS, 1919 route de Mende, 34293 Montpellier cedex 5, France. Tel.: 33-467613372; Fax: 33-467521559. E-mail: galas@crbm.cnrs-mop.fr.
Published, JBC Papers in Press, November 7, 2002, DOI 10.1074/jbc.M207894200
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ABBREVIATIONS |
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The abbreviations used are: GVBD, germinal vesicle breakdown; H1-kinase, histone H1-kinase; CHX, cycloheximide; CSF, cytostatic factor.
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REFERENCES |
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