1 CNR Institute of Molecular Biology and Pathology, Section of Genetics, c/o
University `La Sapienza', 00185 Rome, Italy
2 Institut Curie, Section Recherche, UMR144-CNRS, 75248 Paris Cedex,
France
* Author for correspondence (e-mail: patrizia.lavia{at}uniroma1.it)
Accepted 16 April 2003
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Summary |
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Key words: RanBP1, Ran GTPase, Mitosis, Spindle pole, Centriole, Centrosome
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Introduction |
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The Ran GTPase network has attracted increasing interest during the past 10
years as the major regulator of nucleo-cytoplasmic transport in interphase
cells. The directionality of transport in and out of the nucleus has been
shown to rely on the different distributions of nucleotide-bound forms of Ran
in specific subcellular compartments: Ran-GTP is generated essentially in the
nucleus, where the RCC1 nucleotide exchange factor resides, whereas factors
activating GTP hydrolysis (RanGAP1 and RanBP1) are largely cytoplasmic
(Clarke and Zhang, 2001;
Hetzer et al., 2002
;
Dasso, 2002
). Nuclear RanGTP
promotes the dissociation of import complexes and hence the release of
nuclear proteins in the nucleoplasm as well as the assembly of export
complexes, which, conversely, mediate transport of cytoplasmic proteins and
RNAs to the cytoplasm.
More recent evidence also indicate that the Ran system carries out mitotic
regulatory functions after nuclear envelope breakdown (NEB). In
Xenopus-oocyte-extract-based in vitro systems, RanGTP and RCC1 are
required for the assembly of mitotic microtubule (MT) arrays in spindle-like
structures (Kalab et al.,
1999; Ohba et al.,
1999
; Wilde and Zheng,
1999
; Carazo-Salas et al.,
1999
). This is largely due to the ability of GTP-bound Ran to
regulate the release of active `aster-promoting activities' (APAs), including
NuMA and TPX2 (Gruss et al.,
2001
; Nachury et al.,
2001
; Wiese et al.,
2001
). In the presence of low concentrations of RanGTP, APAs are
sequestered in inactivating complexes with importin
and ß; APAs
need be released in the free form in the presence of RanGTP to promote spindle
assembly. Thus, the functional role of Ran in nucleo-cytoplasmic transport and
in spindle formation relies essentially on one same mechanism the
ability of RanGTP locally to dissociate macromolecular complexes formed by
import vectors and their partners
(Melchior, 2001
;
Dasso, 2002
). In this
framework, the ability of released NuMA and TPX2 to orchestrate spindle
assembly is essentially determined by the redistribution of nuclear and
cytoplasmic components after NEB. The underlying biochemical basis of RanGTP
activity in transport and in mitosis is otherwise identical except for the
different localization of molecules that act as downstream targets of the Ran
system before and after NEB. Because RCC1 remains largely chromatin-bound
throughout mitosis in Xenopus extract
(Carazo-Salas et al., 1999
) and
in somatic cells (Guarguaglini et al.,
2000
; Moore et al.,
2002
), GTP exchange on Ran during mitosis is expected to take
place near chromosomes. Indeed, visual evidence for the bulk of RanGTP being
concentrated near mitotic chromosomes has recently been provided
(Kalab et al., 2002
).
Different mechanisms underlie spindle assembly in mammalian somatic cells
and in meiotic Xenopus extracts, despite of the high conservation of
molecular components (Merdes and
Cleveland, 1997). One obvious difference in spindle organization
lies in the role played by centrosomes in somatic cells but not in meiotic
extract. Centrosomes act as the major organizing centers for MT nucleation in
somatic cells, and hence their function is intimately connected with the
organization of spindle poles. Thus, specific aspects of Ran-controlled
processes during mitosis might differ in these systems.
Evidence from living cells, albeit still fragmentary, clearly implicate the
Ran network in control of spindle organization and function. Injection of
anti-RanBP1 antibody in mitosis perturbs MT dynamics to the point of impairing
complete chromosome segregation
(Guarguaglini et al., 2000).
Microinjection of a deleted importin ß protein, lacking the Ran-binding
domain, causes misassembly of the spindle and chromosome misalignment
(Nachury et al., 2001
).
Aberrant chromosome alignment is also seen in cells overexpressing RanBP1,
associated with the formation of multipolar spindles
(Guarguaglini et al., 2000
).
Consistent with the absence of a specific checkpoint that would detect
multipolar spindles (Sluder et al.,
1997
), these cells do not arrest at metaphase but progress to
ana-telophase and segregate uneven groups of chromosomes. Similar defects have
been reported following expression of a RCC1 mutant that mislocalizes to the
mitotic cytoplasm (Moore et al.,
2002
). Thus, the Ran network, as well as regulating spindle
assembly in the proximity of chromatin in the Xenopus system, also
controls aspects of spindle function in mammalian cells, in which spindle pole
formation and mitotic MT nucleation are directed from centrosomes. To achieve
these functions, components of the Ran network might locally act at crucial
mitotic locations in animal cells.
Here, we focus on mitotic functions of the RanBP1 protein in mammalian
cells. Expression of the mammalian RanBP1 gene varies during the cell
cycle (Di Matteo et al., 1995;
Di Fiore et al., 1999
), with
highest protein levels in G2 and M phases, and an abrupt decline in late
telophase (Guarguaglini et al.,
2000
). As recalled above, RanBP1 overexpression yields abnormal
mitotic spindles with multiple poles
(Guarguaglini et al., 2000
).
To date, this is one of the clearest phenotypes visualized during the
mammalian mitosis under alteration of Ran network components. We have now
sought to identify the underlying defects of multipolar spindle formation.
Correct reproduction and structural organization of centrosomes are crucial
for the establishment of the spindle bipolarity. Multipolar spindles that
direct chromosome missegregation often form in consequence of centrosome
overduplication during cell transformation
(Lingle and Salisbury, 2000;
Brinkley, 2001
;
Doxsey, 2001
). Here, we report
that RanBP1 does not influence the centrosome duplication cycle but instead
induces a specific and distinct aberration (unscheduled splitting between
mother and daughter centrioles at spindle poles). This process is specifically
induced after NEB in a MT- and Eg5-dependent manner. Split centrioles retain
the ability to anchor functional MT arrays and give rise to multipolar
spindles that direct abnormal chromosome segregation. We also show that a
RanBP1 fraction localizes to centrosomes. These results uncover a novel aspect
of mitotic centrosome cohesion, the maintenance of which is important to
ensure proper chromosome segregation, and indicate that this function is
sensitive to RanBP1 levels.
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Materials and Methods |
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Cell culture and synchronization
Murine NIH/3T3 embryo fibroblasts (ATCC CRL-1658), murine L929 lung
epithelial cells (ATCC CCL-1) and derived cell lines stably transfected with
centrin 1-GFP (Piel et al.,
2000) (kindly given by M. Bornens, Institut Curie, Paris), human
HeLa epithelial cells (ATCC CCL-2), were all grown in DMEM (Dulbecco's
Modified Eagle Medium, Euroclone) supplemented with 10% fetal calf serum (FCS;
Gibco BRL) at 37°C in the presence of 5% CO2. Centrin 1-GFP
L929 cell lines were cultured with G418 (500 µg ml1,
Gibco BRL). For cell cycle synchronization experiments, cell cultures were
maintained in low FCS (0.5%) for at least 48 hours to induce quiescence, and
subsequently stimulated to synchronously re-enter the cell cycle by raising
the FCS concentration to 15%. Cells were collected 9 hours, 15 hours and 22
hours after stimulation. To analyse G1-S progression to mitosis, NIH/3T3 and
L929 cell cultures were blocked in the presence of thymidine (Sigma Aldrich, 2
mM for NIH/3T3 and 5 mM for L929 cells) for 24 hours, then released in
complete DMEM supplemented with 30 µM deoxycytidine (Sigma Aldrich) and
harvested 6-8 hours after release from thymidine arrest, when the cell
population was mostly in G2-M by fluorescence-activated cell sorting (FACS)
analysis and the mitotic index was highest by microscope scoring. Where
indicated, cell cultures were released from thymidine arrest for 4-6 hours and
subsequently exposed to 0.1 µg ml1 nocodazole (NOC; Sigma
Aldrich) or 100 µM monastrol (MA; Tocris) for 4 hours before harvesting.
Cells were then fixed, or released in drug-free medium for 45 minutes (NOC) or
30 minutes (MA). For localization experiments, thymidine-arrested and released
cultures were exposed to 1 µM Taxol (Sigma Aldrich) for 4 hours. In all
cases cell cycle phase synchronization was analysed by FACS (Beckton
Dickinson) as described (Battistoni et al.,
1997
).
Transfection experiments
NIH/3T3 cells were seeded in 60 mm Petri dishes onto sterile glass
coverslips and transfected using Fugene (Roche Diagnostic, 3 µl
µg1 DNA). L929 cells were transfected by electroporation
(950 µF, 310 V) and reseeded onto sterile glass coverslips. Six hours after
transfection, the medium was replaced with fresh medium. Cells were routinely
collected 36-48 hours after transfection (asynchronous cell cultures). Where
indicated, transfected cell cultures were submitted to synchronization
protocols starting 6-10 hours after transfection; the overall duration of
thymidine arrest and release, with or without mitosis-arresting drugs, covered
36-42 hours of culture after transfection (see above). Cells were then
harvested and processed for parallel FACS and indirect immunofluorescence (IF)
assays.
Antibodies
Goat polyclonal anti-RanBP1 (M-19 for murine cells and C-19 for human
cells) antibodies were from Santa Cruz Biotechnology and were used 0.5 µg
ml1 in western blotting and 2 µg ml1 in
IF experiments. Anti-HA (Y-11; Santa Cruz Biotechnology) antibody was used at
1:100 dilution. Monoclonal Ran antibody (clone 20; Transduction Laboratories)
was used at 0.25 µg ml1. Goat polyclonal anti-RCC1 (C-20)
and anti-RanGAP1 (N-19) antibodies (Santa Cruz Biotechnology) were used at 1
µg ml1 and 2 µg ml1, respectively.
Monoclonal -tubulin (clone B-5-1-2; Sigma Aldrich) antibody was used at
1:1000 dilution. Monoclonal (GTU-88) and rabbit polyclonal
anti-
-tubulin antibodies (both from Sigma Aldrich) were used at 1:5000
dilution for western blotting and 1:1000 for IF assays. Monoclonal anti-GT335
antibody (used 1:3000) was kindly provided by P. Denoulet (Université
Pierre et Marie Curie, Paris); rabbit polyclonal anti-centrin 2 antibody (used
at 1:2000 dilution) was from M. Bornens (Institut Curie, Paris). The
monoclonal antibody CTR453 (IgG2b) was generated in M. Bornens's
laboratory and has previously been characterized as specific for the
centrosome (Bailly et al.,
1989
). Horseradish peroxidase (HRP)-conjugated secondary
antibodies were from Santa Cruz Biotechnology. Secondary antibodies conjugated
to fluorescein-, AMCA-(Jackson ImmunoResearch Laboratories), rhodamine (Santa
Cruz Biotechnology), Texas Red (Vector) and Cy-3 (Amersham) were chosen
depending on the basis of species specificity and used as recommended by the
suppliers.
Immunofluorescence microscopy
Cells were grown on sterile glass coverslips, washed in PBS and fixed in
methanol for 6 minutes at 20°C or in 3% PFA, 30 mM sucrose for 10
minutes at room temperature. Where indicated, cells were permeabilized for 30
seconds in 0.5% Triton X-100 in PHEM (45 mM PIPES pH 6.9, 45 mM HEPES pH 6.9,
10 mM EGTA, 5 mM MgCl2, 1 mM PMSF) before fixation. Incubation with
primary antibodies was carried out for 1 hour at 37°C. Secondary
antibodies were incubated for 45 minutes. DNA was counterstained with DAPI
(0.1 µg ml1). Coverslips were then mounted in Vectashield
(Vector). IF was also performed as above using purified centrosomes from the
KE37 cell line (see below), after sedimentation onto coverslips (at 20,000
g, 15 minutes, 4°C) and fixation in methanol for 6 minutes
at 20°C.
Fixed cell preparations were examined under an upright Olympus AX70 microscope equipped for epifluorescence and images were taken (100x objective) using either a CoolSnap FX, or a Photometrics CCD camera. Where indicated, fluorescence intensity was quantified in arbitrary units using Adobe Photoshop software on CCD images of single cells acquired under identical exposure and gain setting within each experiment. Video recording of living mitotic cells was carried out on an inverted fluorescence microscope (Leica DMIRBE) controlled by Metamorph software; cells transfected with pRanBP1-RFP were identified on the red channel and images were taken every 10 minutes (10x objective). Confocal images were taken (60x objective) using a TCS-SP2 confocal microscope (Leica) with a 488 nm laser excitation line.
Statistical analysis
To assess the statistical significance of the results, each experiment was
repeated at least three times; means and standard deviations were calculated
to compare the same category in different experiments. This procedure
consistently gave extremely low, statistically insignificant deviations within
each experimental condition. Data from different experiments were therefore
pooled and P values were calculated on pooled data using the
2 test.
Protein extraction from the centrosomal fraction and immunoblotting
analysis
Centrosomes were isolated from the KE37 cell line as described by Moudjou
and Bornens (Moudjou and Bornens,
1994). Pelleted centrosomes were incubated for 1 hour at 4°C
in extraction buffer (20 mM Tris-HCl pH 7.4, 2 mM EDTA) alone or in the
presence of: (i) 0.5% NP40 (1D buffer); (ii) 0.5% NP40 and 0.5% deoxycholate
(DOC, 2D buffer); (iii) 0.5% NP40, 0.5% DOC and 0.1% SDS (3D buffer); (iv) 8 M
urea. Centrosome-associated and non-associated proteins were recovered in the
pellet and supernatant fractions, respectively, by centrifugation at 10,000
g for 15 minutes. Proteins were separated through SDS-PAGE and
transferred onto nitrocellulose filters (Schleicher & Schuell). Filters
were saturated in 5% milk in TBS (10 mM Tris-HCl pH 7.4, 150 mM NaCl)
containing 0.1% Tween 20, for 1 hour at 37°C. Primary and secondary
antibodies were incubated for 1 hour or 45 minutes, respectively, at room
temperature. HRP-conjugated secondary antibodies were revealed with ECL plus
(Amersham-Pharmacia).
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Results |
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Centrosomal abnormalities in RanBP1-overexpressing cells are induced
during mitosis
Normal cells undergo only one round of centrosome duplication, during which
each of the two centrioles composing the centrosome duplicates in a
semiconservative manner. Each centrosome eventually segregates to a daughter
cell at cytokinesis and becomes `licensed' to undergo a novel round of
duplication in the next cell cycle. Loss of the spindle bipolarity is often
related to abnormal centrosome duplication
(Lingle and Salisbury, 2000;
Brinkley, 2001
;
Doxsey, 2001
). The influence
of specific factors on centrosome duplication can be assessed after prolonged
treatment of CHO cells with hydroxyurea, which blocks DNA synthesis but not
centrosome duplication (Balczon et al.,
1995
). Ectopic expression of the cyclinA and
cdk2 genes in this system induces centrosome overduplication, whereas
pRb and p16 inhibit it, and E2F-1 overexpression
rescues the inhibition (Meraldi et al.,
1999
). Instead, overexpression of RanBP1 showed no additional
effect on centrosome duplication compared with hydroxyurea alone, nor did it
overcome the block of centrosome duplication imposed by pRb (P. Meraldi,
personal communication). Thus, RanBP1 has no direct effect on centrosome
duplication.
We next sought to restrict the cell cycle window in which centrosomal
abnormalities are generated. RanBP1-transfected cell populations were
synchronized and centrosomal components were analysed during synchronous
progression through the cell cycle phases. We first brought NIH/3T3 cell
cultures to G0/G1 arrest by serum starvation, then stimulated cell cycle
re-entry with high serum. Cell samples were fixed 9 hours, 15 hours and 22
hours after cell cycle re-stimulation to obtain G1, S and G2/M phase
enrichment, respectively, as indicated by FACS analysis (data not shown).
Centrosomal abnormalities, revealed by staining centrioles with anti-centrin-2
antibody, were quantified as for Fig.
1B: cells with one or two paired dots (corresponding to one or two
centrosomes, respectively) were taken as normal, whereas cells with more than
two pairs of dots or with scattered dots were assumed to reflect
overduplication and abnormal splitting of centrosomes, respectively, and
considered to be abnormal. Serum restimulation of quiescent cells induces per
se a high frequency of centrosome splitting in vector-transfected cells
(Table 1), in line with
previous reports (Sherline and Mascardo,
1982; Schliwa et al.,
1982
; Schliwa et al.,
1983
). RanBP1 overexpression had no additional effect on
serum-induced centrosomal abnormalities in interphase; however a significant
increase was recorded in RanBP1-overexpressing mitotic cells compared to
control cultures (Table 1). To
analyse S-to-M progression more accurately, cells were arrested at the G1/S
transition with thymidine, then released in thymidine-free medium and
centrosomes were analysed in cells that were allowed to progress towards
mitosis. Again, no difference between vector- and RanBP1-transfected cells
were observed in S or G2 interphase cells, whereas a high proportion of
centrosomal abnormalities was recorded in mitoses from RanBP1-transfected
compared to vector-transfected cultures
(Table 1). Thus, centrosomal
abnormalities induced by RanBP1 overexpression are specifically generated in
mitosis.
|
Quantification of RanBP1-associated fluorescence in CCD images of single
cells transfected with expression construct, or with vector alone, indicated
that the RanBP1 signal increased by over fourfold, on average, in
overexpressing cells: most transfected cells (55%) displayed a three- to
fivefold increase, and
30% showed a five- to sevenfold increase in RanBP1
signal intensity compared to control cells. To assess whether the induction of
centrosomal abnormalities did correlate with the level of exogenous RanBP1, we
examined 100 mitotic cells from cultures transfected with pRanBP1-HA, then
processed with anti-HA/FITC to visualize transfected cells, and
GT335/rhodamine to visualize centrioles. Cells were analysed for the presence
or absence of centrosomal abnormalities on the red channel, and the intensity
of the FITC signal, quantified on the green channel. Among RanBP1-transfected
mitoses that displayed a normal phenotype (n=61), the mean
fluorescence scored 1.9 (±0.5), taking the faintest signals in the
lowest-expressing cells as 1;
60% of them displayed relative intensities
below 2, and the remaining 40% fell between 2 and 3. Among RanBP1-transfected
mitoses that developed centrosomal abnormalities (n=39), the mean
relative fluorescence rose to 2.9 (±1.2); a minority (
23%) of
these abnormal mitoses displayed a fluorescence intensity below 2, comparable
to normal mitoses; all other cells had relative intensities above 2, with a
discrete cell population (
15%) showing more than a fourfold increase in
RanBP1 signal intensity (Table
2). Thus, RanBP1-transfected cells that develop mitotic
centrosomal abnormalities tend to express the highest levels of exogenous
RanBP1.
|
RanBP1 overexpression disrupts cohesion of sister centrioles in
mitotic diplosomes
To resolve accurately the type of centrosomal abnormality induced by RanBP1
overexpression, we made use of L929-derived cell cultures stably transfected
with centrin-1/GFP chimera (Piel et al.,
2000). The incorporation of GFP-chimerized centrin in individual
centrioles allows a higher resolution of centrosomes than indirect
immunofluorescence techniques. This cell model therefore provides a
particularly useful tool to analyse the effects of RanBP1.
We initially characterized centrin-1/GFP L929 cells from non-synchronized cultures and noticed that they spontaneously develop a somewhat higher level of centrosomal abnormalities (26.5% in 170 scored mitoses) compared with NIH/3T3 fibroblasts (10.7% in 400 mitoses). Of all centrosomal abnormalities detected among L929 mitotic cells, nearly half (12.3% of all mitoses) were represented by supernumerary, structurally integral centrosomes (arrangement II in Fig. 2B). The remaining abnormal mitoses showed diplosome splitting, either associated with a normal number of centrioles (i.e. four centrin dots, arrangement III in Fig. 2B) or concomitant with supernumerary centrosomes (i.e. more than four centrin dots, arrangement IV in Fig. 2B). For comparison, the corresponding phenotypes among NIH/3T3 mitoses scored 3.8% (centrosomes overduplication) and 6.9% (diplosome splitting).
|
We next assessed the effect of transfected RanBP1-RFP chimeras in
L929-derived cell cultures stably expressing centrin-1/GFP. Cells that reached
mitosis after thymidine synchronization and release were collected by the
`shake off' method, then immediately re-seeded on microscope slides, and
mitotic cells with supernumerary integral centrosomes or with split diplosomes
were examined by analysing the arrangement of centrin-1/GFP centrioles (see
scheme in Fig. 2B). In normal
mitoses, chromosomes were correctly aligned and centrioles were arranged in
typical diplosomes at each pole (Fig.
2A, left corner in upper row, see magnification in a). RanBP1
overexpression did not significantly affect centrosome duplication
(Fig. 2B), consistent with
results obtained in NIH/3T3 cell lines (see above), but specifically induced
sister centrioles from single diplosomes to move apart from one another
(Fig. 2A, magnification in b
and c). As shown in Fig. 2B,
45% of RanBP1-overexpressing mitotic cells showed split diplosome,
compared with 20% in vector-transfected cells (P<0.001). We also
analysed cultures that remained adherent during shaking off and were enriched
in G2-phase cells: RanBP1 overexpression in these cultures failed to increase
the frequency of abnormal centrosome numbers or splitting (data not shown), as
previously observed in NIH/3T3 cultures, thereby confirming that RanBP1
specifically induces diplosome splitting during mitosis.
Splitting of centrioles during mitosis was previously reported to occur
under induction of mitotic arrest (Sluder
and Rieder, 1985; Gallant and
Nigg, 1992
). RanBP1 overexpression actually causes some increase
in the mitotic index, as previously observed
(Guarguaglini et al., 2000
).
However, the extent of the induced delay in our experiments was in the upper
limit of the physiological range or just above it
(Table 3), different from that
induced by MT drugs or failure of cyclin-B degradation. Video recordings of
cells transfected with pRFP vector or pRanBP1-RFP depicted no dramatic delay
in the timing from prophase/prometaphase indicated by rounding-up of
the cells to anaphase in vivo: all video-recorded control cells
reached anaphase within 40 minutes from mitosis onset, and most of them took
20-30 minutes. RanBP1-transfected cells underwent some delay, with most of
them taking 30-40 minutes to execute the same stages. Thus, RanBP1-dependent
delay in early mitosis is well below that induced by MT drugs or
non-degradable cyclin B, which is in the order of hours. Furthermore,
progression through mitotic substages was analysed in transfected cultures
after IF to
-tubulin: this revealed a higher proportion of
ana/telophases among RanBP1-overexpressing mitoses compared with controls.
Thus, the induction of mitotic delay by RanBP1 overexpression is essentially
caused by prolonged duration of ana/telophase stages, possibly reflecting
hindrance in M exit (Battistoni et al.,
1997
; Guarguaglini et al.,
2000
), whereas earlier mitotic stages are not significantly
affected. RanBP1 induction of centriole splitting is instead already visible
in prometaphase. Thus, RanBP1-dependent centriole splitting is a specific
phenomenon, not attributable to prolonged duration of mitosis.
|
We next wished to ascertain whether single split centrioles were able to
assemble functional spindle poles. Centrin-1/GFP expressing L929 cultures were
transfected with the RanBP1-RFP chimera and spindle MTs were labeled with
anti--tubulin antibody, revealed with an AMCA-conjugated secondary
antibody. As shown in Fig. 3,
microtubule arrays nucleating from single centrioles are focused to form
separate poles, hence forming a multipolar spindle.
|
Diplosome splitting by RanBP1 requires integrity of mitotic
microtubules
Cohesion between parental centrioles requires MT integrity
(Jean et al., 1999) and MT
disruption by nocodazole favors the separation of parental centrosomes
(Mayor et al., 2000
). We
wondered whether MTs are implicated in RanBP1-induced splitting between
centrioles. NIH/3T3 cultures were transfected with RanBP1-HA or RanBP1-GFP and
subsequently synchronized in G2/M phases by thymidine block and release as
described above. Cells were then exposed to nocodazole (NOC) and either
collected after 4 hours, with most cells arrested in prometaphase without
spindle MTs, or allowed to resume mitosis by removing NOC and fixed 45 minutes
after release. Both FACS analysis and microscope counting of mitotic cells
(data not shown) were used to monitor synchronization. Mitotic centrosomes
were analysed using either GT335 or anti-centrin-2 antibodies. In cultures
exposed to NOC, the overall centrosomal organization was altered in interphase
cells, with centrosomes being typically distanced and displaced from the
juxtanuclear region (data not shown), consistent with the established role of
MTs in anchoring centrosomal structures to each other and to their subcellular
site (Jean et al., 1999
).
Fig. 4 shows the results
obtained in RanBP1-overexpressing cultures. In cells that underwent mitosis
after release from thymidine arrest, RanBP1 overexpression caused a highly
significant increase in diplosome splitting compared with cultures transfected
with vector. When NOC was added to G2 cultures to inhibit MT polymerization,
the effects of RanBP1 overexpression were prevented, and the frequency of
mitoses with split centrioles was comparable in RanBP1-overexpressing and in
vector-transfected cultures. Thus, NOC per se does not affect the organization
of sister centrioles within diplosomes, in contrast to its ability to induce
separation of parental centrioles, yet counteracts the disruptive effect
caused by RanBP1 excess, indicating that MT integrity is required for
induction of diplosome splitting. The specificity of this requirement was
further demonstrated by removing NOC from the culture medium and allowing the
cells to reform MTs in vivo: upon resumption of mitosis, RanBP1-overexpressing
mitoses again underwent diplosome splitting
(Fig. 4).
|
We previously reported that multipolar spindles are similarly induced by
wild-type RanBP1 and by the RanBP1L186A/V188A mutant, which carries
inactivating mutations in the nuclear export signal (NES) and hence is
retained in nuclei throughout interphase
(Richards et al., 1996;
Guarguaglini et al., 2000
). If
multipolar spindles are generated through loss of diplosome cohesion as a
truly mitotic phenomenon, then similar effects to those reported thus far are
expected in cells overexpressing the RanBP1L186A/V188A mutant,
regardless of its abnormal localization during interphase. Indeed, the
NES-defective RanBP1 mutant induced a highly significant increase in mitotic
diplosome splitting in NIH/3T3 cultures released from thymidine arrest,
similar to wild-type RanBP1 (Fig.
4). Parallel analysis of L929 centrin-1/GFP cultures enabled us to
establish that the type of mitotic diplosome splitting induced by mutant and
wild-type RanBP1 was indistinguishable (data not shown). The
RanBP1L186A/V188A mutant failed instead to induce diplosome
splitting in nocodazole-exposed cells, similar to wild-type RanBP1
(Fig. 4). Thus, the comparable
ability of export-defective and wild-type RanBP1 to disrupt centriole cohesion
in a MT-dependent manner further confirms that diplosome splitting takes place
after NEB.
Diplosome splitting by RanBP1 requires Eg5 activity
The Eg5 kinesin controls the establishment of the spindle bipolarity by
causing parental centrosome separation at the onset of mitosis
(Walczak et al., 1998) and Ran
can modulate Eg5 mobility on MTs (Wilde et
al., 2001
). Thus, we wondered whether Eg5 activity influenced
RanBP1-induced splitting within mitotic diplosomes. Inhibition of Eg5 activity
by monastrol (MA) prevents centrosome separation, yielding mitotic cells that
typically arrest with monoastral spindles
(Kapoor et al., 2000
). In our
experiments, RanBP1- or vector-transfected NIH/3T3 cell cultures were
synchronized by thymidine block and release as above, and, when in G2 as
judged by FACS analysis, MA was added for 4 hours. Cells were then fixed and
centrosomes were analysed. By
-tubulin staining of centrosomes and DAPI
staining of chromosomes, monoastral mitoses with unseparated centrosomes were
indistinguishable in RanBP1- and vector-transfected cells
(Fig. 5A). Centrosome structure
was more closely inspected using anti-centrin-2 antibody. Although all mitoses
had a monoastral spindle, different arrangements could be appreciated at the
centrosome level: monoastral mitoses in which two sets of paired centrioles
were visible at the center of the spindle were defined `normal'
(Fig. 5Ba); mitoses showing
more than two paired centrin spots (Fig.
5Bb) were assumed to reflect overduplication, whereas clearly
distanced centrioles in at least one diplosome
(Fig. 5Bc) were recorded as
abnormal splitting. By these criteria, centriole splitting occurred with
similar frequency in vector-transfected and RanBP1-overexpressing monoastral
mitoses (Fig. 5C). Eg5
inhibition by MA is reversible and so cells released in MA-free medium readily
re-establish bipolarity. Under these conditions, diplosome splitting was again
appreciated in RanBP1-overexpressing cells that progressed through mitosis 30
minutes after MA removal (Fig.
5C). Thus, Eg5 function is required for induction of diplosome
splitting by overexpressed RanBP1.
|
Centrosomal localization of RanBP1
Because RanBP1 overexpression affects centriole cohesion, we re-examined
its localization relative to centrosomes. In NIH/3T3 interphase cells fixed
with paraformaldehyde (PFA), RanBP1 is almost completely cytoplasmic; some
enrichment at the spindle can be appreciated in mitotic cells
(Guarguaglini et al., 2000).
If a fraction of RanBP1 localizes at centrosomes, such a fraction might be
masked by the abundant soluble pool and difficult to resolve. Indeed, partial
permeabilization of NIH/3T3 cells with Triton X-100 prior to methanol or PFA
fixation revealed a fraction of insoluble RanBP1 protein at the centrosome,
revealed by
-tubulin, in both interphase
(Fig. 6Aa) and mitotic cells
(Fig. 6Ab,c). A small
centrosomal fraction of RanBP1 was also visualized in mouse L929
(Fig. 6Ad) and human HeLa cells
(Fig. 6Ae) using independent
antibodies. The co-localization of RanBP1 signals with
-tubulin was
confirmed by scanning NIH/3T3 cell spreads under confocal microscopy
(Fig. 6B).
|
To extend these results, we analysed preparations of purified centrosomes
isolated from the human lymphoblastic cell line KE37. RanBP1 was retained on
isolated centrosomes analysed by IF (Fig.
6C) and showed a very similar labeling pattern to that revealed
using the CTR453 antibody, which specifically recognizes the AKAP450
centrosomal matrix protein (Bailly et al.,
1989). Western immunoblotting was then used to assess the strength
of the interaction of RanBP1 with the KE37-derived centrosomal fraction
(Fig. 6D). Purified centrosome
preparations were treated with solubilizing detergents of increasing strength,
and the soluble (supernatant) and insoluble (pellet) fractions were analysed
with anti-RanBP1 antibody. As shown in Fig.
6D, the association of a RanBP1 fraction with centrosomes was
resistant to strong solubilizing conditions: centrosomal RanBP1 was not
solubilized by NP40 alone (1D buffer), nor by NP40 combined with DOC (2D
buffer), nor with DOC and SDS simultaneously (3D buffer). Treatment of
centrosomes with 8 M urea eventually solubilized centrosomal RanBP1. Thus, a
RanBP1 fraction is actually involved in a stable interaction with
centrosomes.
To ascertain whether exogenously expressed RanBP1 also reached centrosomes,
IF experiments were performed in cultures transfected with pRanBP1-HA. After
solubilization and fixation, exogenous RanBP1 was revealed by anti-HA antibody
and centrosomes were stained for -tubulin. This showed that anti-HA
staining was concentrated in the pericentrosomal region
(Fig. 6E).
Because RanBP1 excess alters cohesion within centrosomes in the presence of intact MTs, we asked whether localization of RanBP1 at the centrosome is influenced by the status of mitotic MTs. When thymidine-released cultures were exposed to NOC, under conditions that prevent both MT polymerization and RanBP1-dependent centriole splitting, a fraction of RanBP1 was still detected at the centrosome (Fig. 7a). A comparable localization was seen in cells exposed to Taxol (Fig. 7b). Therefore, the association of a RanBP1 fraction with centrosomes is independent of MT integrity or dynamics. This result, together with the strength of the association depicted in Fig. 6D, suggests that a fraction of RanBP1 associates constitutively with centrosomes.
|
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Discussion |
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---|
Ran is an abundant GTPase (107 molecules cell1
in HeLa cells) (Bischoff and Ponstingl, 1991), and is estimated to be present
in a 25-fold excess over endogenous RCC1 and fivefold excess over endogenous
RanBP1 (Bischoff et al., 1995).
In our transfection experiments, we recorded an average fourfold increase in
RanBP1 levels; furthermore, RanBP1-transfected cells that displayed mitotic
centrosomal abnormalities typically showed higher than average levels of
overexpression. That range of increase is expected to produce a significant
shift in the balance of nucleotide hydrolysis and exchange on Ran. We
previously reported that RanBP1 overexpression induces cell cycle
abnormalities (Battistoni et al.,
1997
; Guarguaglini et al.,
2000
) comparable to those observed in the presence of Ran mutants
(Ren et al., 1993
;
Ren et al., 1994
;
Moore et al., 2002
),
supporting the idea that RanBP1 acts by altering the Ran network. In addition,
we have now sought to quantify the intracellular RanGTP levels using an
antibody (AR12, a kind gift from I. Macara) that preferentially
although not exclusively recognizes the GTP-bound conformation of Ran
(Richards et al., 1995
).
Although these experiments do not allow us to draw a precise quantitative
estimate, they do indicate that RanGTP levels are lowered in
RanBP1-overexpressing compared with normal cells (data not shown).
Induction of multipolar spindles by RanBP1 excess reflects the aberrant
splitting of single centrioles within diplosomes in mitosis. None of
duplication of centrosomes, recruitment of -tubulin or glutamylation of
centriole MTs are affected instead. Furthermore, no defects were recorded in
focusing of MT arrays to the poles. Split centrioles retain their functional
integrity and can organize polarized MT arrays, thereby giving rise to
spindles with multiple poles. This is a novel finding and begins to identify
aspects of centrosome organization and function that are influenced by members
of the Ran network.
Cohesion and dynamics of centrosomes are highly regulated processes. After
duplication, centrosomes remain tethered together throughout most of
interphase, then separate in late G2 and eventually migrate to form the
spindle poles. MTs contribute to the link between centrosomes
(Jean et al., 1999). Cohesion
in G2 and separation in mitosis are also regulated by a network of specific
factors (Meraldi and Nigg,
2001
), including the centrosomal C-Nap1 protein
(Mayor et al., 2000
), its
upstream kinase Nek2 (Meraldi and Nigg,
2001
) and the Inh2 regulator of Nek2
(Eto et al., 2002
).
Deregulated activity of these factors induces unscheduled centrosome
separation but the integrity within centrosomes is not affected and so neither
spindle assembly nor the mitotic division are necessarily perturbed
(Mayor et al., 2002
). RanBP1
overexpression influences neither the timing nor the extent of parental
centrosome separation in interphase, but selectively perturbs cohesion of
centrioles within diplosomes in mitosis.
Physiologically, centrioles undergo splitting during telophase, accompanied
by extensive motility and repositioning of the mother centriole to the
mid-body in preparation of cytokinesis
(Piel et al., 2001). In early
interphase, split centrioles act as duplication templates. Under abnormal
circumstances, however, centrioles can split during mitosis, as observed
during (for example) mitotic arrest induced by non-degradable cyclin B
(Gallant and Nigg, 1992
).
Indeed, induction of mitotic delay by mercaptoethanol or colcemid was used as
an experimental tool to study the functional relationship between centrioles
and spindle poles (Sluder and Rieder,
1985
). In RanBP1-overexpressing cultures, we recorded some
increase in the mitotic index, but this was essentially due to prolonged of
telophase. The timing of early mitotic progression was instead not
dramatically perturbed in RanBP1-overexpressing cells, whereas centriole
splitting could already be detected in prometaphase, as soon as the nuclear
envelope disappeared a stage that was not prolonged by RanBP1
overexpression. These observations support the conclusion that RanBP1-induced
centrosomal abnormalities are not a consequence of abnormally prolonged
mitosis.
The RanBP1L186A/V188A construct, which has a different
localization from wild-type RanBP1 throughout interphase, has similar
disruptive effects than wild-type on mitotic centrosomes. This is paralleled
by the ability of this mutant to induce multipolar spindles as effectively as
wild-type RanBP1 (Guarguaglini et al.,
2000). These data are consistent with the view that overexpressed
RanBP1 interferes with crucial factor(s) implicated in centrosome organization
specifically during mitosis. Such factor(s) might be activated, and/or be
capable of establishing crucial interactions at the centrosomal level,
specifically after NEB in a manner that is similarly sensitive to
NES-defective and wild-type RanBP1. The mitotic nature of the splitting
phenomenon induced by RanBP1 excess was further evidenced in cells that
resumed mitotic progression after NOC-induced arrest. NOC prevented the
disruptive effect of RanBP1 excess, yet centrosome splitting was again
appreciated after as little as 45 minutes after NOC removal and resumption of
MT reconstitution in vivo. This experiment further strengthens the conclusion
that centriole cohesion is sensitive to RanBP1 levels during mitosis and,
furthermore, implicates MTs. Induction of diplosome splitting by high RanBP1
is also dependent on Eg5 activity, suggesting that either centrosome
separation is required or that some timely regulated interaction that is
physiologically dependent upon Eg5 is required. It is noteworthy that RanBP1
was detected at centrosomes even in NOC-exposed cells. This observation and
the ability of a RanBP1 fraction to localize at centrosomes already in
interphase and to interact with centrosomes in a stable, detergent-resistant
manner, converge to suggest that a small RanBP1 fraction constitutively
associates with centrosomes. It has recently been found that a fraction of Ran
also localizes at centrosomes, in the presence or absence of NOC
(Keryer et al., 2003
). Thus,
the suppressive effect of NOC is not due to failure of RanBP1 or Ran to
localize at centrosomes. Rather, MTs themselves or motor proteins might play a
role in cohesion within diplosomes in a manner that is sensitive to high
RanBP1 levels. We previously found that inactivation of mitotic RanBP1 by
antibody microinjection impairs dynamics of the spindle MTs. RanBP1 excess
might influence MT dynamics at spindle poles, and altered dynamics might in
turn favor the aberrant separation of mother and daughter centrioles. An
alternative but not necessarily mutually exclusive possibility
is that one or more factor(s) that regulate the organization and/or the
intrinsic dynamic features of mitotic centrosomes is transported to spindle
poles in a MT-dependent manner after NEB and, once there, is sensitive to
elevated levels of RanBP1. In the presence of NOC, the hypothetical protein(s)
would not be transported to poles and so would not be in a position to
modulate the behavior of centrioles, regardless of RanBP1 levels. Based on
these observations, the mitotic role of Ran network components might be
critically dependent on their ability to associate with specific mitotic
structures. In mitosis, Ran members might reorganize in `local factories' at
specific locations and act on local downstream targets in the mitotic
apparatus. The recent observation that spindle pole defects and chromosome
misalignment are caused by a RCC1 mutant that mislocalizes to the cytoplasm
but not by wild-type RCC1 (Moore et al.,
2002
) is consistent with this view.
Interestingly, while this work was in progress, disruption of spindle pole
organization was observed in mammalian cells under interfering RNA-mediated
inactivation of an important Ran target, TPX2
(Garrett et al., 2002).
Although, in other studies, the major outcome of TPX2 inactivation was failure
of MT connections between spindle poles, probably because of differences in
the experimental conditions that yielded partial inactivation of TPX2
(Gruss et al., 2002
), in the
study by Garrett et al. (Garrett et al.,
2002
) multipolar spindles formed as a consequence of spindle pole
fragmentation. Remarkably, these abnormalities are MT and Eg5 dependent,
similar to those reported here under RanBP1 excess. The authors suggest that
multipolar spindles induced in their conditions might reflect an imbalance
between TPX2-dependent structural support and motor-driven force: when TPX2 is
inactivated, the force would be exerted freely and cause spindle pole
disruption. By analogy of reasoning, it is tempting to speculate that
defective RanGTP formation caused by RanBP1 excess causes insufficient release
of factor(s) that provide structural support to sister centrioles during
spindle assembly.
In summary, a fraction of the RanBP1 protein is present at centrosomes throughout the cell cycle, where it can interact with Ran. At this location, RanBP1 can act on factor(s) that reach the centrosomes after NEB to contribute to the organization of mitotic centrosomes. The presence of excess RanBP1 favors the aberrant separation of individual centrioles in mitosis, giving rise to multipolar spindles. Further understanding the mechanisms through which Ran network components act locally in mitosis and control downstream targets in the assembly of mitotic structures will be a major field to disentangle in the near future.
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Acknowledgments |
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