Proc. Nati. Acad. Sci. USA
Vol. 88, pp. 9929-9933, November 1991
Cell Biology
Centrosomes competent for parthenogenesis in Xenopus eggs
support procentriole budding in cell-free extracts
(cell cycle/protein synthesis)
FRE9DERIC TOURNIER*, MAREK CYRKLAFFt, ERIC KARSENTIt, AND MICHEL BORNENS*
*Centre de Gdn6tique Mol6culaire, Centre National de la Recherche Scientifique, 2 Avenue de la terrasse, 91198 Gif/Yvette, France; and tEuropean
Molecular Biology Laboratory, Postfach 102209, Meyerhofstrasse 1, D-6900 Heidelberg, Federal Republic of Germany
Communicated by Andre Lwoff, August 1, 1991 (received for review April 22, 1991)
cell cycle from which they have been isolated: centrosomes
isolated from G1 or G2 human lymphoid cells (KE37 cell line)
or from quiescent cells (peripheral human lymphocytes in Go)
were shown to possess a similar parthenogenetic activity (8).
We have isolated (10) centrosomes from calf thymocytes
(CTs) and shown that the two centrioles were linearly associated by their proximal ends through a mass of dense
material. Furthermore, they were unable to induce egg cleavage, although centrosomes isolated from other bovine cells
and from thymocytes of other species could induce egg
cleavage (6). These results suggest that the centrosome cycle
can be blocked when the centrioles are prevented from
separating into a nonlinear configuration, a step that might be
critical for the initiation of procentriole budding.
Therefore, we have prepared cell-free extracts from Xenopus eggs in the mitotic or interphasic stage, in which we
could directly compare the duplicative capacity of centrosomes that are competent and incompetent (such as CT
centrosomes) to induce parthenogenesis. We show that competent centrosomes from human lymphoblasts (KE37 cell
line), synchronized in G1 phase, initiate procentriole budding
in interphasic extracts from Xenopus eggs in the absence of
protein synthesis, whereas incompetent (CT) centrosomes do
not. Since CT centrosomes do not support parthenogenesis,
the present results strongly suggest that duplication of the
foreign centrosome is required for centrosome-induced parthenogenesis.
Heterologous centrosomes from diversed speABSTRACT
cies including humans promote egg cleavage when i iected into
metaphase-arrested Xenopus eggs. We have recently isolated
centrosomes from calf thymocytes and shown that they were
unable to induce egg cleavage, an inability that was apparently
correlated with the peculiar structure of these centrosomes
rather than with a lack of microtubule-nucleating activity: the
two centrioles were associated in a colinear orientation by their
proximal ends. To promote cleavage, a heterologous centrosome probably is required to duplicate, although this has not
yet been demonstrated. Therefore, we designed an in vitro assay
that would enable us to directly observe the duplication process. We show that competent centrosomes from KE37 cells
synchronized in G1 phase initiate procentriole budding in
interphasic extracts from Xenopus eggs in the absence of
protein synthesis, whereas calf thymocyte centrosomes do not.
Since calf thymocyte centrosomes do not support parthenogenesis, the present results suggest that duplication of the foreign
centrosome is required for centrosome-induced parthenogenesis. Furthermore, procentriole budding takes place in the
absence of protein synthesis in egg extracts arrested in S phase.
This in vitro assay should contribute to the identification of
molecular mechanisms involved in the initiation of centrosome
duplication.
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In somatic cells, centrosome duplication encompasses the
whole cell cycle and progression in the centrosome cycle
appears to be a marker of progression in the cell cycle. The
importance of centrosomes in establishing the spatial organization of the mitotic apparatus mandates that the cell tightly
controls the reproduction of its centrosome, which must
occur only once in each cell cycle, prior to mitosis. The
morphological events of the centrosome duplication cycle in
somatic cells have been described and involve budding of
daughter centrioles off the wall of the parent structures in late
G1 phase, elongation of the daughter centrioles during S and
G2 phases, redistribution of pericentriolar material, and separation of duplicated centrosomes in prophase (1-3). However, the molecular mechanisms that govern centrosome
duplication are not known.
As amphibian eggs apparently lack a functional centrosome, which can be replaced by the injection of foreign
centrosomes (parthenogenetic development), they represent
a favorable system to study the duplication of centrosomes.
In this system, no species specificity is apparently required
for the centrosomes: heterologous centrosomes isolated from
sea urchin (4), rodent (5, 6), or human (7-9) cells can induce
cleavage of Xenopus eggs. In this parthenogenetic test, the
heterologous centrosomes are believed to duplicate, but
direct experimental evidence is lacking. The parthenogenetic
activity of the centrosome is independent of the stage of the
MATERIALS AND METHODS
Animals. Xenopus laevis females were obtained from the
Service d'Elevage d'Amphibiens du Centre National de la
Recherche Scientifique (France). Eggs were obtained from
females injected 3-8 days before use with 100 international
units of pregnant-mare-serum gonadotropin (Intervet, Angers, France) and 1 day before with 1000 international units
of human chorionic gonadotropin (Sigma). The eggs were
collected in 100 mM NaCl to prevent activation.
Preparation of Extracts. Egg extracts were prepared according to Felix et al. (11). Egg jelly was removed in 2%
(wt/vol) cysteine hydrochloride (pH 7.8), and eggs were
washed four times in 0.25 x MMR (lx MMR = 0.1 M NaCl/2
mM KCl/1 mM MgSO4/2 mM CaCl2/5 mM Hepes/0.1 mM
EDTA, pH 7.4) containing cycloheximide (150 pug/ml; Sigma), and then activated by an electric shock (11) in the same
buffer for 90 min at 20'C. Activated eggs were transferred to
Beckman SW 50.1 rotor tubes filled with ice-cold acetate
buffer [AB = 100 mM potassium acetate/2.5 mM magnesium
acetate/cytochalasin D (10 pug/ml; Sigma)/1 mM dithiothreitol/250 mM sucrose/leupeptin (10 pug/ml)/pepstatin (10 kkg/
ml)/aprotinin (10 ,g/ml), pH 7.2]. Excess acetate buffer was
removed from the tubes containing packed eggs prior to
centrifugation. The eggs were crushed by centrifugation at
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviation: CT, calf thymocyte.
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9930
Cell Biology: Toumier et al.
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10,000 x g (with maximum acceleration rate) for 10 min at 40C
in a L5-65 Beckman centrifuge. The cytoplasmic material
between the upper lipid layer and the yolk pellet was collected (10,000 x g supernatant) and an ATP regenerating
system (10 mM creatine phosphate, creatine phosphokinase
(80 jig/ml), and 1 mM ATP (Boehringer Mannheim)] and
protease inhibitors as above were added. A sample was kept
in ice during further steps. The 100,000 x g extracts were
obtained by further centrifugation of the 10,000 x g supernatant at 100,000 X g for 60 min at 40C in the SW 50.1 rotor
with adaptors for 0.6-ml tubes. The yellow cytoplasmic layer
(100,000 x g supernatant) was collected and kept at 4TC,
because freezing the extracts gave inconsistent results in the
centrosome duplication assay.
Cell Synchronization and Isolation of Centrosomes. KE37 is
a human T-lymphoblastic cell line. KE37 cells, cultured in
RPMI 1640 medium (Eurobio, Les Ulis, France) containing
7% (vol/vol) calf serum, were first synchronized at the
G1/S-phase border with 2.5 mM thymidine (Sigma) for 20 hr
(12) and then separated in a cell-cycle-dependent manner
using centrifugal elutriation according to Tournier et al. (8).
Centrifugal elutriation led to an enriched fraction containing
>80% G1 phase cells. Centrosomes were prepared from
KE37 cells in G1 phase according to Tournier et al. (8) and
from CTs according to Komesli et al. (10).
Duplication Assay. Typically, 10 ,ul of KE37 G1 centrosomes
(1 x 104 centrosomes per ul) was mixed with 100,ul of extract
(either 10,000 x g or 100,000 X g supernatant) at 4°C. The
mixture was equally distributed into four tubes and incubated at
Proc. Natl. Acad. Sci. USA 88 (1991)
room temperature for 0-3 hr. After incubation, nocodazole was
added to 30 ,&g/ml and the tubes were kept at 4°C for 30 min.
Then 0.8 ml of lysis buffer [1 mM Tris'HCl, pH 7.5/0.1%
2-mercaptoethanol/0.5% Nonidet P-40/0.5 mM MgClJ1 mM
phenylmethylsulfonyl fluoride/aprotinin (100 pg/ml)/leupeptin
(1 ,ug/ml)/pepstatin (1 ,ug/ml)] at 4°C was added to each tube
and the material in each tube was layered onto 25% (vol/vol)
glycerol in RG2 buffer (80 mM Pipes.KOH/1 mM MgCl2/1 mM
EGTA, pH 6.8) in a 15-ml modified Corex tube with a special
adaptor containing a 12-mm round coverslip. The material was
centrifuged at 12,000 rpm (20,000 x g) in a JS-13.1 rotor
(Beckman) at 4°C for 15 min to apply the centrosomes to the
coverslips. The coverslips were removed, fixed in methanol at
-20°C for 5 min, and processed for immunofluorescence staining.
Immunofluorescence Study. Centrosome duplication was
monitored by measuring the numbers of centrioles and centrosomes by double immunofluorescence (7). A monoclonal
antibody against a-tubulin (Amersham) stained centrioles
and a human autoimmune serum stained the pericentriolar
material of human centrosomes (J. E. Dominguez, E.K., and
J. Avila, unpublished data).
Negative Staining. For negative staining of electron microscopy preparations, 15 ,ul of G0 centrosomes (1 x 104 centrosomes per ,l) was mixed at 4°C with 300 ,l of extract.
Further steps were identical to the immunofluorescence
protocol until the lysis step. For each point (at 0, 2, and 3 hr
of incubation), 100 ,l of extract was mixed with 200 Al of lysis
buffer and incubated 2 min at 4°C. The material was loaded
FIG. 1. Double immunofluorescence staining of G1 centrosomes (KE37) in Xenopus extracts arrested in S phase at 0 time (a and a') and
after 3 hr of incubation (b and b'). Centrosomes stained with the human autoimmune serum (a) appear to be doublets of tubulin-containing dots
corresponding to centrioles (a'). In this experiment, the two centrioles in a centrosome do not split (see Table 1) and after 3 hr of incubation
in the extract, the centrosomes (b) contain three or four tubulin-containing dots as judged by the anti-tubulin staining (b'). In the lower parts
of each panel, other examples at 0 time (a and a') and after 3 hr of incubation (b and b') are shown. The variable intensities of the centrioles
probably depend on their orientation on the coverslip. (x 16,900.)
Cell
~ ~1
Biology: Toumier et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
onto 200 Al of 25% glycerol in RG2 buffer in a 0.6-ml tube
containing a Teflon adaptator supporting a carbon grid. The
material was centrifuged at 14,000 rpm (25,000 x g) in a SW
50.1 rotor (Beckman) for 20 min (40C) to apply centrosomes
to the grid. Grids were then removed, rinsed in RG2 buffer,
stained with 2% (wt/vol) phosphotungstate (pH 7 with
KOH), and dried.
RESULTS
Duplication of G, Centrosomes in Cell-Free Extracts: Immunofluorescence Study. Competent centrosomes were isolated from KE37 cells synchronized in G1 phase. We have
shown (8) that these centrosomes were capable of inducing
parthenogenesis when injected into unfertilized Xenopus
eggs. The use of cell-free extracts and immunofluorescence
to study microtubule polymerization nucleated by isolated
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TIME
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(min)
FIG. 2. Duplication of KE37 centrosomes in Xenopus extracts
arrested in S phase. (a) Numbers of centrosomes (u) and centrioles
(m) were recorded at 0 time and after 1, 2, and 3 hr of incubation. After
3 hr of incubation, the number of centrosomes is constant (as the two
centrioles in a given centrosome did not split in the extract) whereas
the number of centrioles doubles. Approximately 1 x 105 centrosomes were added at 0 time in 100 1.l of extract. Ten fields (>100
centrosomes) were recorded for each point after incubation. The
number of centrioles at 0 time was standardized (1 on the Y axis). (b)
The number of duplicated forms (three or four centrioles per centrosome) increases from 1% to 48%. (c) The number of KE37
centrioles remains constant with the 10,000 x g supernatant (e)
incubated for 0-3 hr. In the corresponding 100,000 x g supernatant
(o), the number of centrioles increases after 2 hr of incubation. This
suggests that the high-speed (100,000 x g) pellet contains an inhibitory activity for centrosome duplication.
9931
centrosomes has been described (13-15). In the present
study, visualization of the centrosomes by immunofluorescent staining was done after nocodazole-induced disassembly
of the microtubules, which had been nucleated in the extract
by the added centrosomes. Double staining was used to
identify both the pericentriolar domain and the centrioles.
This allowed us to identify the centrosomes unambiguously
by distinguishing them from tubulin aggregates. More reproducible results were obtained from cytoplasmic extracts of
Xenopus eggs prepared from activated oocytes in the presence of cycloheximide (interphasic extracts).
G1 centrosomes from KE37 cells were incubated in 100,000
x g Xenopus egg extracts for 0-3 hr. At 0 time, centrosomes
were observed as pairs of centrioles (Fig. 1 a and a'), and
after 3 hr of incubation, centrosomes contained three or four
tubulin dots (Fig. 1 b and b'). We interpreted these dots as
duplicative forms of centrosomes in which the centrioles did
not separate. The numbers of centrosomes and centrioles
were recorded at various incubation times by double immunofluorescence. After 3 hr of incubation, the number of
centrosomes remained constant but the number of centrioles
increased from 1 (reference value at incubation time) to 1.95
(Fig. 2a) and the number of centrosomes containing 3 or 4
identifiable centrioles increased from 1% to 48% (Fig. 2b).
Typically, the number of KE37 centrioles doubled after 2-3
hr of incubation. We noted, however, that the efficiency of
centrosome duplication depended largely on the batch of
extract used.
In other experiments, the two centrioles in a given centrosome often separated from each other over large distances
(Table 1) during the first hour of incubation as judged by the
increase in the proportion of single centrioles counted at this
time. Duplicated forms observed after 3 hr of incubation were
identified as a pair of tubulin-containing dots associated with
centrosome labeling.
When the same kinetic experiments were done using
low-speed extracts (10,000 x g), no increase in centriole
number was detected after 2 or 3 hr of incubation (Fig. 2c) or
up to 6 hr of incubation (data not shown), suggesting that the
10,000 x g supernatant contained an activity that inhibited
centrosome duplication.
Duplicative Forms of Centrosomes Observed by Negative
Staining. To validate our scoring method by an independent
approach (electron microscopy), we examined negatively
stained centrosomes that had been incubated in the extract.
Table 1. Centriole splitting in interphasic extracts during the first
hour of incubation
Centriole form, no.
Double
Single
n
Time, hr
Cells
Exp.
488 (66)
I
5
0
388 (44)
KE37
189 (16)
1
952 (84)
148 (75)
II
49 (25)
2
0
180 (73)
67 (27)
1
183 (81)
42 (19)
2
0
CT
45 (23)
152 (77)
1
n represents the number of experiments. Centrosomes were recorded as pairs of tubulin-containing dots at 0 time and after 1 hr of
incubation. For KE37 centrioles (experiments I), a splitting of the
two centrioles in a given centrosome was observed (44% of single
centrioles at 0 time and 84% after 1 hr of incubation). In five
experiments (I), duplicated forms observed after 3 hr of incubation
in the extract were identified as pairs of tubulin-containing dots
associated with centrosome labeling. In two experiments (II), a
splitting of the two centrioles was not observed (25% of single
centrioles at 0 time and 27% after 1 hr of incubation). The duplicated
forms observed after 3 hr in the latter case (II) were recorded as three
or four tubulin-containing dots. For CT centrosomes, in all cases, we
did not observe any significant splitting of the centrioles after 1 hr of
incubation. Numbers in parentheses are percent of total centrioles.
9932
Cell Biology: Tournier et al.
Proc. Natl. Acad Sci. USA 88 (1991)
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z
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TIME (min)
FIG. 4. Centrosomes isolated from CTs do not duplicate in
100,000 x g supernatant, whereas KE37 centrosomes initiate procentriole budding, as shown by the increase of the number of
centrioles. o, CT centrioles; *, KE37 centrioles.
c
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:,
.8
'1
.
FIG. 3. Duplicating centrosomes observed by negative staining.
Centrioles were in pairs (a and c) or were single (b and d), without
(a and b) or with procentriole buds (arrows) (c and d). (Bar = 0.1
AM.)
Centrioles were scored either as single centrioles or as pairs
and with or without an associated orthogonal procentriole
(Fig. 3). A representative experiment is shown in Table 2. At
0 time, 46% of the centrioles were pairs and 52% were single.
After 2 hr of incubation, 12% of centrioles were pairs and 82%
of centrioles were single. These results were in agreement
with immunofluorescence observations and indicated extensive splitting of the centrioles during incubation in the extracts. Duplicated forms were only 4% of the total centrioles
at 0 time (Table 2), which is consistent with the fact that
centrosomes were prepared from G1 cells. After 2 hr and 3 hr
Table 2. Centriole splitting and duplicated forms by electron
microscopy after negative staining
Centriole form, no.
Duplicated
Double
Triple
forms, no.
Single
1 (2)
97
4 (4)
0
30(46)
34(52)
2
21
14 (82)
2 (12)
1 (6)
13 (62)
5 (19)
2 (7)
3
36
20 (74)
21 (58)
n represents the number of centrioles. Note that, at 0 time, 52% of
centrioles were observed as single centrioles and 46% were observed
as centriole pairs. After 2 hr of incubation, single centrioles represented 82% of the total centrioles observed. Centrioles were sometimes observed (2-7%) as triplets. Duplicated forms correspond to
centrioles observed with a centriole bud. For duplicated forms, 4%
were observed at 0 time, 62% were observed after 2 hr of incubation,
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Time,
hr
n
and 58% were observed after 3 hr of incubation. Numbers in
parentheses are percent of total centrioles.
of incubation, an orthogonal bud (procentriole) was detected
on 60% of the scored centrioles.
CT Centrosomes Do Not Support Procentriole Budding. CT
centrosomes, which display linearly associated centrioles
(10), do not support cleavage when injected into unfertilized
Xenopus eggs (6). This suggested that these centrosomes
were incompetent to initiate bud formation in an adequate
cytoplasmic environment. As shown in Fig. 4, we did not
observe an increase in centriole number in an extract incubated with CT centrosomes. Since, in the same extract, KE37
centrosomes do form buds, this result shows that CT centrosomes are incompetent for duplication and interestingly,
CT centrioles do not separate (Table 1).
DISCUSSION
We have compared the capacity of centrosomes to initiate a
centrosome duplication cycle in cell-free extracts by using an
immunofluorescence-based assay. Centrosomes competent
to induce parthenogenesis (G1 centrosomes from KE37 cells)
initiate procentriole budding whereas incompetent centrosomes (CT centrosomes) do not. The electron microscopy
study supported the conclusion that the increase in tubulincontaining dots observed by immunofluorescence during
incubation of the centrosomes in extracts corresponds to an
increase in centriole or procentriole number. But it also
indicated that only procentrioles were formed. Indeed, we
never observed pairs of centrioles tightly associated in an
orthogonal configuration in which the two centrioles had the
same length. Further incubation (up to 6 hr) did not increase
the number of centrioles (data not shown). In particular, we
never obtained evidence for successive rounds of centriolar
duplication. This suggests that the duplication process is not
completed under our in vitro conditions.
This may have several causes. First, the high-speed supernatant (100,000 x g) may lack components necessary for
bud elongation. Such components may or may not be in the
high-speed pellet, because the pellet apparently also contains
an activity from the 10,000 x g supernatant that was shown
to inhibit the initiation of centriole duplication (Fig. 2c). The
most likely explanation is that the components necessary for
assembling full-length centrioles are limiting in comparison to
the number of centrosomes incubated in the extract. We
added between 500 and 1500 centrosomes per ,ul of extract,
which is the minimum number required to make the immunofluorescence study feasible. This figure is close to the
number of centrosomes produced per embryo at the midblastula transition (16) (one embryo has a volume of about 1 "I).
From such a figure, we may conclude that a single centrosome duplication cycle at most could take place under our
Cell
Biology: Toumier et al.
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conditions. If this were the case, adding centrosomes at a
lower concentration should allow multiple rounds of duplication in the extract, but we have not succeeded in designing
an experiment to test this hypothesis.
Another implication of these results is that the first steps of
centrosome duplication are independent of the cytoplasmic
oscillator that drives the cell cycle, since procentriole budding takes place in cycloheximide-treated interphasic extracts. However, the incompleteness of the duplication cycle
could suggest that a signal from the "mitotic clock" is
necessary for the late steps of the process. This is unlikely
since multiple cycles of centrosome duplication occurred in
cycloheximide-treated Xenopus blastomeres (16) and a similar result was also reported for sea urchin eggs (17).
Centrosome duplication can be experimentally uncoupled
from the synthesis of mitotic cyclins (refs. 16 and 17 and this
report). Our experimental system should, allow investigation
ofthe coupling between the centrosome duplication cycle and
the cytoplasmic oscillator. The asynchrony of centrosome
reproduction in the absence of protein synthesis (16, 17)
suggests indeed that phasing of the cytoplasmic oscillator and
the centrosome cycle occurs in normal development. p34cdc2,
a suci homologue (18), and cyclin B (E. Bailly, J. Pines, T.
Hunter, and M.B., unpublished data), the major components
of the cytoplasmic oscillator, are associated with the centrosome in G2 and M phases. It will be of interest to study
centrosome duplication in cycling extracts and the role of
protein phosphorylation controlled by various cyclins in
synchronization of centrosome duplication with the mitotic
cycle. Preliminary experiments in mitotic extracts suggest
that procentriole budding cannot occur during mitosis (data
not shown).
The inability of CT centrosomes to initiate a centriole bud
in vitro strongly suggests that, in parthenogenetic Xenopus
eggs, cleavage requires the duplication of the injected centrosome. This also indicates that the structural configuration
of the centrosome is essential to initiate budding in a permissive cytoplasmic environment. Specific egg proteins are
able to assemble on the two centrioles of the heterologous
centrosome to form the two poles of the first mitotic spindle,
in which each pole should contain a chimeric centrosome.
The incompetence of CT centrosomes is not simply due to the
species or the tissue origin, because the linear arrangement of
the CT centrosomes apparently represents a locked configuration.
Proc. Natl. Acad. Sci. USA 88 (1991)
9933
In conclusion, the present data demonstrate the potential
of our in vitro assay to describe in molecular terms the
orthogonal budding of procentrioles from the proximal end of
parental centrioles.
We thank Dr. Spencer Brown, Dr. Pedro Santamaria, and Eric
Bailly for their helpful comments and English corrections on the
manuscript. We also thank J. E. Dominguez for the gift of the human
autoimmune serum and Daniel Meur for the art work. This work has
been supported by Centre National de la Recherche Scientifique and
by Grant 87.C.0555 from Ministere de la Recherche et de la Technologie to M.B. and by the Association pour la Recherche sur le
Cancer. F.T. is a recipient of a fellowship from the Ministere de la
Recherche et de la Technologie.
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