From the Institut Curie, Section Recherche, UMR 144 CNRS, 26 rue d'Ulm, 75248 Paris Cedex 05, France and the ¶ INSERM
U440, Institut du Fer à Moulin, 17 rue du Fer à Moulin,
F-75005 Paris, France
Received for publication, February 15, 2001, and in revised form, April 4, 2001
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ABSTRACT |
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Stathmin/Op18 destabilizes microtubules in
vitro and regulates microtubule polymerization in
vivo. Both a microtubule catastrophe-promoting activity and a
tubulin sequestering activity were demonstrated for stathmin in
vitro, and both could contribute to microtubule depolymerization
in vivo. Stathmin activity can be turned down by extensive
phosphorylation on its four phosphorylatable serines, and
down-regulation of stathmin activity by phosphorylation is necessary
for cells to proceed through mitosis. We show here that microinjection
of a nonphosphorylatable Ser to Ala (4A) quadruple mutant in
Xenopus two-cell stage embryos results in cell cleavage arrest in the injected blastomeres and aborted development, whereas injection of a pseudo-phosphorylated Ser to Glu quadruple mutant (4E)
does not prevent normal development. Addition of these mutants to
mitotic cytostatic factor-arrested extracts in which spindle assembly was induced led to a dramatic reduction of spindle size with
4A stathmin, and to a moderate increase with 4E stathmin, but both
localized to spindle poles. Interestingly, the microtubule assembly-dependent phosphorylation of endogenous stathmin
was abolished in the presence of 4A stathmin, but not of 4E
stathmin. Altogether, this shows that the phosphorylation-mediated
regulation of stathmin activity during the cell cycle is essential for
early Xenopus embryonic development.
Stathmin/Op18 is a soluble, ubiquitous phosphoprotein originally
proposed to act as a relay for diverse intracellular signaling pathway
(1). Stathmin was subsequently isolated as a microtubule (MT)1 catastrophe-promoting
factor that was shown to interact directly with free tubulin (2).
Further work proved that stathmin causes MT destabilization in
vitro and in vivo and that its MT-destabilizing activity can be turned off by phosphorylation on its four
phosphorylatable sites (3-7).
Stathmin becomes extensively phosphorylated at mitosis (6, 8-10) on
its four phosphorylatable serines. These sites are targets both
in vitro and in vivo of multiple kinases (for a
review, see Ref. 11): Ca2+/calmodulin-dependent
kinases II and IV/Gr phosphorylate Ser16, mitogen-activated
protein kinases phosphorylate Ser25 and Ser38,
cyclin-dependent kinases phosphorylate Ser25
and Ser38, and cAMP-dependent protein kinase
phosphorylates Ser16 and Ser63. Overexpression
of phosphorylation target site-deficient mutants in mammalian somatic
cells revealed that whereas wild-type phosphorylatable stathmin allows
spindle formation at mitosis, a stathmin mutant in which the four
phosphorylatable serines have been replaced by alanines (4A mutant)
prevents formation of a normal spindle and results in a mitotic block
of the cells (6, 12, 13). As the 4A mutant mimics the constitutively
active form of stathmin, this shows that down-regulation of stathmin
MT-destabilizing activity is likely required to allow formation of the
mitotic spindle and progression through mitosis. However, it has been
recently reported that a 4E pseudo-phosphorylated mutant, in which the
four phosphorylation serines have been replaced by glutamic acid to
mimic the constitutively down-regulated form, shows only a limited
decrease in tubulin complex formation in vitro and in
MT-destabilizing activity in transfected somatic cells (14, 15).
To investigate the in vivo importance of the phosphorylation
state of stathmin in an embryonic cell system, we used wild-type human
stathmin and the two 4A and 4E mutants. We introduced these recombinants stathmin proteins in two-cell stage embryos and in mitotic
egg extracts from Xenopus laevis to examine their effects on
embryo development and on the MT network. We then performed immunofluorescence experiments to detect the specific localization of
the added forms in the mitotic egg extracts and extended these observations to somatic tissue culture cells.
Preparation of Extracts and Human Stathmin Expression--
Low
speed (15,000 × g) Xenopus egg extracts
blocked in metaphase II of meiosis (CSF extracts) and permeabilized
sperm heads were prepared as described previously (16). Spindles
were assembled at 22 °C in the interphase to mitosis pathway as
described previously (17) by adding 0.2 mM
CaCl2 for 1 h, then adding an equal volume of fresh
CSF extract kept on ice to drive the extract back into mitosis. High
speed extracts were prepared from CSF extracts spun at 245,000 × g for 20 min at 4 °C in a TLS 55 rotor (Beckman) and were
complemented with 0.05 volume of Energy Mix (150 mM
creatine phosphate, 20 mM ATP, 20 mM
MgCl2). Recombinant human wild-type 4A (serine changed to
alanine at positions 16, 25, 38, and 63) and 4E (serines substituted
with glutamic acid) stathmin were in PBS at 10 mg/ml (18). Briefly,
stathmin-expressing bacteria were sonicated, and the extracts were
centrifuged at 4 °C for 5 min at 3,000 × g followed
by 6 min at 400,000 × g. Stathmin was purified in a
two-step procedure: anion exchange chromatography on a Q-Sepharose
column (Amersham Pharmacia Biotech) and fast protein liquid
chromatography gel filtration on a superose 12 HR 10/30 column
(Amersham Pharmacia Biotech). Recombinant stathmin was used in
experiments to an amount equivalent to between three and five times the
endogenous quantity estimated to be 110 µg/ml (2).
Antibodies--
Stathmin antibodies (both sera and
affinity-purified) were polyclonal anti-COOH-terminal (C) and
anti-internal (I) peptides of human stathmin. Antibody C was thus
specific of the human form of stathmin, whereas antibody I recognized
both human and Xenopus forms of stathmin (19). Monoclonal GT
335 recognizes polyglutamylated isoforms of tubulin, which makes it a
marker of both centrioles and spindle MTs (20). Monoclonal CTR453 was
raised against centrosomes isolated from KE37 cells and specifically
recognizes a 350-kDa antigen in the pericentriolar material (21).
Monoclonal anti- Frog Embryos Microinjection and Immunofluorescence
Analysis--
Two-cell stage embryos were injected as in Ref. 22.
Embryos were left to develop at 22 °C and fixed with methanol 9 h after fertilization and processed for immunofluorescence with
anti- Microtubule Polymerization--
High speed extracts were
incubated for 45 min at 22 °C with Me2SO (5%,
ICN) and then were layered on top of a BRB80 (80 mM K-Pipes, pH 6.8, 1 mM MgCl2, 1 mM
EGTA)/40% glycerol cushion containing 10 µg/ml protease inhibitors:
leupeptin, pepstatin, and chymostatin (Boehringer). Centrifugation was
at 22 °C for 20 min at 140,000 × g in a TLS 55 rotor (Beckman). A sample from the supernatant was taken and the
remaining volume discarded; the pellet washed once with BRB80 and left
to dry before an equal volume of Buffer A (24) was added to both
supernatant and pellet fractions. An equal fraction in volume of both
samples was loaded for 12% SDS-polyacrylamide gel electrophoresis.
Western blotting was done as in Ref. 25, except that proteins were
further fixed on nitrocellulose with 0.25% glutaraldehyde at room
temperature for 20 min. The following primary antibody dilutions used
were: serum I, 1:10,000 and anti- Immunofluorescence Analysis--
Spindles assembled in
Xenopus egg extracts were fixed with cold methanol after
dilution in BRB80, 30% glycerol, 1% Triton X-100 and spun onto
coverslips. Coverslips were blocked with PBS-5% bovine serum albumin
and incubated for 1 h with primary antibodies (affinity-purified I
and C, 1:40; GT 335, 1:5,000; CTR453, 1:500; autoimmune anti-NuMA,
1:200; anti- We microinjected the stathmin 4A and 4E mutants in one blastomere
of two-cell stage X. laevis embryos, the uninjected
blastomere representing an inner control, to investigate any
interference with embryo development. The quantities injected
correspond to the double of the endogenous amount of stathmin,
estimated to be 110 µg/ml (2). Embryos were photographed (Fig.
1A) and scored for normal
development (Fig. 1B) around mid-blastula transition (MBT)
9 h after fertilization, and during late gastrulation 22 h
after fertilization. Injection of 4A stathmin resulted in cell cleavage
arrest in 95% of the injected blastomeres as observed at MBT, and in
exogastrulation or lysis at 22 h (Fig. 1, A and B). All 4A-injected embryos showed aborted development (data
not shown). On the contrary injection of the 4E stathmin mutant
resulted in no visible phenotype at all observed times and led to
normal tadpoles, as was the case for PBS-injected embryos (Fig. 1,
A and B). Immunofluorescence analysis using
anti-
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-tubulin was from Amersham Pharmacia Biotech. The
autoimmune human serum against NuMA was from Dr C. André.
-tubulin antibody according to Ref. 23. Embryos were observed
with a fluorescence stereomicroscope (Leica MZFLIII) coupled to a
Hamamatsu C5985 CCD camera.
-tubulin, 1:200. Bound antibodies
were detected by anti-rabbit coupled to alkaline phosphatase
(Promega).
-tubulin, 1:500). After three washes with PBS-Tween 20 (0.1%), coverslips were incubated for 1 h with the appropriate
rhodamine-, fluorescein-, or 7-amino-4-methylcoumarin-3-acetic acid-conjugated antibody (Jackson ImmunoResearch). DNA was stained by a
5-min incubation with DAPI. After three washes, coverslips were mounted
with AF1 solution (Citifluor) and observed with a Leica DMR
fluorescence photomicroscope equipped with a Hamamatsu C5985 CCD
camera, coupled to a Macintosh computer with Adobe Photoshop software.
HeLa cells were fixed with methanol at
20 °C for 6 min,
immediately or after a few minutes at room temperature in PBS. A Leica
DMRXA fluorescence microscope was used for observation: image stacks
(0.2 µm) were recorded using a piezoelectric objective (100 × 1.4 NA) positioning device and a MicroMAx CCD camera (Princeton Instruments). All images are maximal intensity projections.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-tubulin antibody on 4A-injected embryos at the blastula stage
revealed the absence of any polymerized tubulin in the injected half,
and in particular the absence of mitotic spindles observed in the
noninjected area where division and cleavage seemed normal (Fig.
1C). We thus show that injection of heterologous
constitutively nonphosphorylated 4A stathmin affects MT dynamics in
such a way that cell cleavage and normal embryo development is
compromised, whereas the same amount of the constitutively
pseudo-phosphorylated 4E form has no effect.
View larger version (82K):
[in a new window]
Fig. 1.
Microinjection in Xenopus
two-cell stage embryos. A, two-cell stage embryos were
injected in one of the two blastomeres with PBS, pseudo-phosphorylated
4E (250 µg/ml) or nonphosphorylatable 4A (250 µg/ml) human
recombinant stathmin mutants. Embryos were photographed 9 h
(blastula stage) and 22 h (late gastrula stage) after
fertilization. Note in 4A-injected embryos the absence of cleavage or
lysis in the area corresponding to the injected blastomere
(arrows). Bar: 500 µm. B, table
summarizing the stathmin mutants injection experiments. Embryos were
assessed for normal, retarded (showing undercleavage compared with the
uninjected blastomere), or blocked (no cleavage) development 9 h
after fertilization. Development is aborted with 4A but is not affected
by 4E, leading to apparently normal tadpoles. C, a
4A-injected embryo was fixed 9 h after fertilization and processed
for in toto immunofluorescence using anti- -tubulin
antibody. No organized microtubules are visible in the injected part.
Bar: 200 µm.
It is known that overexpression in mammalian cells of stathmin with
mutated nonphosphorylatable p34cdc2 sites prevents spindle
formation in mitosis (12). We therefore wondered if 4A stathmin, but
not 4E stathmin, could block embryo development by specifically acting
on MT dynamics at mitosis, thus preventing the formation of a
functional spindle. It has already been shown that addition of 4A
stathmin to in vitro assembled spindles results in a
decrease of spindle size (26). We reproduced this result but extended
our observations to the 4E mutant. We added the wild-type or the
different stathmin mutants (in 3-fold quantity compared with the
endogenous amount) to mitotic (CSF) egg extracts in which spindles were
then assembled from sperm heads by the "interphase to mitosis
pathway" (17). Spindles were processed for immunofluorescence and
their sizes measured (Fig. 2). Presence
of 4A stathmin resulted in an important decrease in spindle size as
compared with control conditions, whereas the spindle size was slightly
increased by 4E stathmin (Fig. 2). The size of spindles assembled in
the presence of heterologous wild-type stathmin was only moderately
reduced, since this phosphorylatable form is known to be inactivated by
extensive phosphorylation at mitosis (12, 26). Thus presence of
nonphosphorylated 4A stathmin results in considerably smaller spindles,
whereas the pseudo-phosphorylated 4E mutant allows formation of
spindles with close to normal size. These results could explain the
different effects on embryo development of the 4A and 4E mutants, as
perturbed MT dynamics and small size would make the spindles obtained
with 4A stathmin incompatible with correct cell division.
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We have recently identified both in Xenopus egg extracts and
in somatic cells a new pathway in which it is the assembly of MTs that
leads to stathmin hyperphosphorylation. Specifically, we have shown
that MT assembly results in the additional phosphorylation, on stathmin
Ser16, leading to the appearance of one or two bands with
reduced electrophoretic mobilities (27). This phosphorylation of
Ser16, which corresponds to an hyperphosphorylation, has
been shown to be critical for the down-regulation of stathmin activity
(5, 6, 28). This MT assembly-dependent phosphorylation is
likely involved in MT stabilization during spindle formation and could contribute to global control of the MT network (see Fig.
3A and Ref. 27). We wondered
if MT assembly-dependent phosphorylation of endogenous
stathmin in Xenopus egg extracts would be affected by the
addition of exogenous recombinant mutant forms of human stathmin.
The prediction was that adding 4A human stathmin would limit MT
assembly and the resulting MT assembly-dependent
phosphorylation on Xenopus stathmin. On the contrary,
addition of 4E human stathmin would not significantly affect MT
assembly and Xenopus stathmin hyperphosphorylation.
Therefore, Me2SO was added to induce MT assembly together
with mutant human proteins (in a 3-fold amount compared with the
endogenous) to high speed CSF extract for 45 min at 22 °C before MTs
were pelleted by ultracentrifugation. Stathmin was then analyzed in
supernatants by Western blot with antiserum I, which recognizes both
human and Xenopus stathmin (19) (Fig. 3B).
Tubulin was also followed by Western blot in pellets to control for its
polymerization. MT assembly by Me2SO resulted in the
appearance of two additional Xenopus stathmin bands with
reduced electrophoretic mobilities (Fig. 3B, lane
C, arrows). As expected, the addition of the
nonphosphorylatable 4A and 4E human stathmin mutants had distinct
effects: 4A prevented the Me2SO-induced
hyperphosphorylation of endogenous Xenopus stathmin, whereas
4E did not. As a control, none of the mutants did induce any
hyperphosphorylation of endogenous stathmin in the absence of
Me2SO. This shows that opposing MT assembly with the 4A
mutant prevents hyperphosphorylation of Xenopus stathmin,
whereas the pseudo-phosphorylated form does not. Therefore the effect
of the 4A stathmin mutant on embryo development could result from both a direct perturbation of MT dynamics in mitosis and from preventing the
MT assembly-dependent phosphorylation of endogenous
stathmin, which contributes to spindle MTs stabilization (27).
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We wondered next whether the significant amounts of human exogenous
stathmin added would present a specific localization in the in
vitro spindle assembly experiments. We performed therefore immunofluorescence on in vitro spindles formed in
Xenopus egg extracts (17). Spindles were assembled from
permeabilized Xenopus sperm heads in CSF extracts
supplemented with either wild-type, 4A, or 4E recombinant human
stathmin. Antibody C specific to human stathmin that does not recognize
the endogenous Xenopus form (19) was then used (Figs.
4A, panel b)
together with mAb GT335 (Fig. 4A, panel a), an
antibody that recognizes polyglutamylated forms of tubulin that are
particularly abundant on MTs in mitosis and at centrioles (20). In all
spindles, the wild-type form of human stathmin accumulated at spindle
poles (see Fig. 4A) with a peripheral localization
reminiscent of MT minus end markers such as NuMA (29).
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To extend these observations to somatic cells, HeLa cells were
processed for immunofluorescence either after saponin extraction followed by paraformaldehyde/glutaraldehyde fixation (data not shown)
or after methanol treatment (Fig. 4B). Cells were then double-stained with antiserum C, together with various anti-centrosome antibodies. Fig. 4B shows in most cells, as expected from a
soluble protein, a diffuse cytoplasmic staining for stathmin. However a
specific accumulation at mitotic spindle poles in mitosis (Fig. 4B, panel a) and at aster centers in
G2 (Fig. 4B, panel a') could be
detected. The specific accumulation at spindle poles (Fig. 4B, panels c and f) was always
peripheral to all markers used, as for NuMA (Fig. 4B,
panel b), antipericentriolar antigen antibodies (CTR453;
Fig. 4B, panel e), or anticentriolar antibodies
(GT335; not shown). There was however a partial overlap of the stathmin staining with NuMA (compare Fig. 4B, panel d with
panel g). The staining was sometimes found to be more
abundant toward the exterior of the spindle pole and having the
appearance of a horseshoe (see Fig. 4B, panels f
and g). The stathmin accumulation at spindle poles was no
longer observed if antiserum C was preincubated with the peptide C-O
(19) against which the antiserum was raised (data not shown).
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DISCUSSION |
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The phosphorylation state of stathmin, an important physiological regulator of MT dynamics, appears to play an important role in Xenopus egg embryonic development. Our results show dramatic differences in the effects of stathmin mutants with opposed phosphorylation profiles. The uncharged 4A mutant, which mimics constitutively nonphosphorylated stathmin, interferes with several important aspects of normal egg development, including cell cleavage, spindle size, MT assembly, and hence MT-dependent stathmin hyperphosphorylation. In contrast, the 4E mutant, which mimics constitutively phosphorylated stathmin by permanently harboring four negative charges, has no visible effects in this context.
A number of studies performed both in vitro and in vivo have shown that nonphosphorylated stathmin promotes MT disassembly and that this activity is turned off by phosphorylation (see Ref. 11 for a review). Thus, overexpression of 4A stathmin in mammalian somatic cells destabilizes MTs, prevents formation of a normal spindle at mitosis, and blocks the cell cycle (6, 12, 13), whereas a pseudo-phosphorylated mutant has no effect (3, 6). Overexpression of wild-type phosphorylatable stathmin also destabilizes interphase MTs but allows spindle formation and normal progression through the cell cycle, because the kinase levels at mitosis are most likely sufficient for the phosphorylation of wild-type stathmin (6, 12). This shows that stathmin inactivation by phosphorylation is required to allow mitotic spindle formation and progression through mitosis. Therefore, the effect of 4A stathmin on Xenopus egg development reported here can be explained by its constitutive MT-depolymerizing activity, resulting in interference with spindle formation and preventing normal division and cleavage.
Furthermore, we have shown in Xenopus egg extracts that MT assembly leads to hyperphosphorylation of stathmin specifically on Ser16 (27), the phosphorylation of which is critical for turning down stathmin MT-depolymerizing activity (5, 6). The MT assembly-dependent hyperphosphorylation of stathmin allows MT stabilization around chromatin in mitotic egg extracts (27), a mechanism required in mitotic spindle assembly (26). We show here that the addition of an excess of nonphosphorylatable 4A stathmin prevents Me2SO-induced MT assembly and hence phosphorylation of endogenous stathmin on Ser16 (see Fig. 3). This could contribute, at least partially, to the impairment of normal spindle assembly and egg development. In contrast, the constitutively pseudo-phosphorylated 4E mutant, which does not prevent MT-dependent hyperphosphorylation of endogenous stathmin, does not interfere with normal Xenopus egg development. Altogether, our results indicate that the MT-dependent phosphorylation of stathmin in Xenopus eggs likely contributes to the regulation of mitotic spindle assembly.
Our observation that 4E stathmin has no apparent effect on egg development apparently contradicts recent overexpression reports in somatic cells showing that the 4E mutant retains significant MT-depolymerizing activity (14, 15). However these experiments have been performed with a 20-fold overexpression level, whereas we performed 2-5-fold addition experiments, which proved to be sufficient with the 4A mutant to elicit effects on egg development. As 4E stathmin retains significant tubulin binding activity in vitro (13, 30-32), and as tubulin sequestering represents a probable mechanism to explain stathmin MT-depolymerizing activity in cells, a large excess of 4E stathmin could result in an effect on MT stability that is not seen at close to physiological levels.
Whereas the majority of stathmin is cytosolic, we observed a distinct accumulation of stathmin at spindle poles, both in HeLa cells and in spindles assembled in Xenopus egg extracts. Several factors, such as the MT-severing protein katanin (33-35), the MT-destabilizing kinesin Kar3p in yeast (36), and the MT-stabilizing microtubule-associated protein XMAP215 in Xenopus (37) have been shown to concentrate at the centrosome. Two contrasting interpretations can be proposed for the centrosomal accumulation of a minor part of stathmin. It could correspond to a phosphorylated fraction contributing to the local stabilization of short, growing microtubules nucleated at the centrosomes during spindle assembly. Alternatively, once the spindle is assembled, an active pool of stathmin at the centrosome could contribute locally to the turnover of MTs at their minus ends in assembled spindles. It has been actually established in vertebrate cells and in Xenopus eggs extracts that spindle MTs were constantly depolymerizing at their minus ends as a result of poleward flux (38, 39). However, polar accumulation was observed in Xenopus egg extracts supplemented with wild-type, 4A, or 4E human stathmin, suggesting that it is independent of the stathmin phosphorylation state. The asymmetrical accumulation of stathmin at the spindle poles of somatic cells, toward the cell cortex rather than toward the chromosomes, is also striking. It could reflect stathmin interaction with complexes associated to the MT plus ends, that contain proteins such as dynein and dynactin (40, 41), CLIP-170 (42), or EB1 (43).
Altogether, this report demonstrates for the first time in an embryonic
development system that the phosphorylation level of stathmin and its
variations during the cell cycle have dramatic effects on cell
development. We specifically show that the pseudo-phosphorylated form
of stathmin does not apparently interfere with normal progression through the cell cycle in early developmental stages, as opposed to the
nonphosphorylatable mutant. Furthermore, the fact that the effects of
the 4A and 4E stathmin mutants are similar in a control extract, but
totally different when tubulin is forced to assemble by
Me2SO, shows that the stathmin phosphorylation level
becomes critical only in conditions where the free tubulin concentration is very low (when tubulin is assembled into MTs). These
observations could suggest that the pool of tubulin dimers ready to
assemble in vivo is very low.
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ACKNOWLEDGEMENTS |
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We thank V. Doye for suggesting experiments and for stimulating discussions. We are grateful to P. Curmi for the preparation of wild-type stathmin, to D. Morineau and D. Meur for artwork, and to G. Keryer and V. Doye for critical reading of the manuscript. GT335 monoclonal antibody was a kind gift of B. Eddé and P. Denoulet, and the autoimmune serum against NuMA was kindly provided by Dr. C. André.
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FOOTNOTES |
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* This work was supported by Centre National de la Recherche Scientifique, Institut Curie, Institut National de la Santé et de la Recherche Médicale, Association pour la Recherche sur le Cancer, Association Française contre les Myopathies, and by an European Economic Community Grant HCP CHRX CT 94-0642 (to M. B.).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.
§ Received fellowships from the Ministère de l'Enseignement Supérieur et de la Recherche, from Association pour la Recherche sur le Cancer, from Société de Secours des Amis des Sciences, and from the Luxembourg Ministère de l'Education Nationale et de la Formation Professionnelle during this work.
Received a fellowship from the Ligue Nationale contre le
Cancer. Present address: Institut Curie, Section Recherche, UMR 144 CNRS, 26 rue d'Ulm, 75248 Paris Cedex 05, France.
** To whom correspondence should be addressed: Institut Curie, Section Recherche, UMR 144 CNRS, 26 rue d'Ulm, 75248 Paris Cedex 05, France. Tel.: 33-1-42-34-64-20; Fax: 33-1-42-34-64-21; E-mail: mbornens@curie.fr.
Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M101466200
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ABBREVIATIONS |
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The abbreviations used are: MT, microtubule; CSF, cytostatic factor; PBS, phosphate-buffered saline; Pipes, 1,4-piperazinediethanesulfonic acid; DAPI, 4,6-diamidino-2-phenylindole.
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