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National Research Council Centre of Evolutionary Genetics, c/o Department of Genetics and Molecular Biology, University of Rome "La Sapienza," Rome 00185, Italy
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Abstract |
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 TOP Abstract IntroductionResultsDiscussionMaterials and MethodsReferences |
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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The role of Ran is particularly well documented in control of nucleocytoplasmic traffic in interphase cells. The nucleotide-bound state of Ran is crucial for the assembly and disassembly of transport complexes (for recent reviews, see Refs. 10-12 ). These findings have led to the proposal that many pleiotropic effects ascribed to the RanGTPase and its regulators may in fact reflect a primary effect of Ran over nuclear transport. However, recent lines of evidence increasingly implicate the Ran network in mitotic control. In yeast, a mutant allele of the RanBP1 gene (yrb1 in Saccharomyces cerevisiae) causes mitotic spindle misalignment and cell cycle arrest in late mitosis or G1, with no apparent perturbation of nuclear import (13) . In mammalian cells, a Ran-interacting component named RanBPM localizes at centrosomes and controls microtubule nucleation from centrosomes (14) . Furthermore, work with Xenopus egg extracts has depicted a direct role of members of the Ran network in mitotic control (reviewed in Refs. 34, and 15 ): the addition of purified Ran-GTP or RCC1 (which generates Ran-GTP) promotes microtubule nucleation; whereas the addition of purified Ran-GDP, RanGAP, or RanBP1 (which favor Ran-GDP formation) inhibits microtubule nucleation and spindle assembly (16-19) . Frog egg extracts provide a useful experimental system to pinpoint biochemical requirements for the organization of microtubules into a functional mitotic spindle. However, somatic cells often show higher regulatory constraints: specific components may be present in limited amounts and are often subjected to regulated expression both in time, during cell cycle progression, and in space, in specialized subcellular compartments. It is important to examine in vivo processes to assess whether components of the Ran network are actually implicated in mitotic control in somatic cells in vivo.
In previous work, we cloned and characterized the regulatory features of the murine RanBP1 gene. We found that RanBP1 mRNA transcription is subjected to both growth regulation in proliferating versus quiescent cells (2021) and cell cycle phase-specific regulation, being specifically up-regulated at the G1-S-phase boundary (22) . This control is exerted by E2F and retinoblastoma-related proteins (23) , which act as major cell cycle regulators (see Refs. 24-26 for reviews). Thus, E2F-dependent control couples RanBP1 transcription to that of many genes required for cell cycle progression (reviewed in Ref. 27 ). This control is lost in several tumor types, and RanBP1 is consistently overexpressed in several transformed cell lines (20) . The link between RanBP1 gene expression and the cell cycle machinery suggests that RanBP1 protein plays an important role in cell cycle control. Such a role could be exerted either directly or via modulation of the efficiency of nucleocytoplasmic transport during the cell cycle phases. Indeed, previous experiments in which RanBP1 transcription was rendered constitutive from a cell cycle-independent promoter yielded significant cell cycle alterations with a predominant impairment in mitotic exit (28) . In retrospect, mitotic defects or arrest may be consistent with the regulatory role over spindle assembly ascribed to purified Ran network components in cell-free extracts.
The experiments described here were undertaken to gain further insight into the mitotic role of RanBP1 in somatic cells in vivo. Two major experimental approaches were taken. Firstly, we sought to characterize cellular phenotypes generated in cell cultures constitutively expressing RanBP1. We report that two major processes are impaired under those conditions: (a) mitotic spindles showed an abnormal number of poles; and (b) after mitotic exit, the reorganization of nuclear chromatin was aberrant. Given that both classes of defects were typically found either in mitosis or in cells that had just passed through mitosis, we further sought to establish whether RanBP1 dysfunction directly affected mitotic progression. To that end, we microinjected anti-RanBP1 antibody into living cells progressing through mitosis, when RanBP1 is maximally expressed. This induced mitotic delay, segregation defects, and incomplete separation of daughter nuclei, reflecting, at least in part, an abnormal stabilization of the spindle microtubules. Both lines of evidence therefore indicate that RanBP1 dysfunction or deregulation impairs mitotic spindle function and hence proper mitotic division in mammalian cells in vivo.
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Results |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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). Cell cultures were induced to reach growth arrest
(G0) by serum starvation and subsequently
stimulated to cycle by serum refeeding. To examine reentry into S phase
after mitotic completion and hence distinguish between cell cycle
phase-specific and growth-dependent regulation, cells were also induced
to accumulate in prometaphase in the presence of NOC and then released
from prometaphase arrest in NOC-free medium and allowed to progress
into mitosis and into the following G1 phase.
Most cells exited mitosis and resumed a normal interphase appearance
within 2 h after release of the NOC block. Fig. 1B
shows that RanBP1 protein levels, but not those of Ran or RCC1, are low
in G0-early G1 cells and
are up-regulated during progression from S phase to mitosis. RanBP1
levels are high in prometaphase-arrested cells but decline within
1 h after release of NOC arrest, i.e., when cells
traverse the mitosis to G1 transition. These
results are specific by comparison with both phase-specific
(i.e., E2F-1 in the case of release from serum starvation
and cyclin B1 after release from NOC arrest) and phase-independent
(i.e., actin) protein markers (data not shown). In contrast,
both Ran and RCC1 are steadily expressed under the same conditions
(Fig. 1B
). These findings delimit a specific cell cycle
window during which RanBP1 is present, whereas Ran and RCC1 are both
expressed in a constitutive manner.
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) yet was effectively visualized in cells beyond the
G1-S transition (Fig. 2A
,
b). In metaphase and anaphase cells, RanBP1 immunostaining
reached the highest intensity and was confined to the mitotic cytoplasm
(Fig. 2A
, c and d). However, the
signal abruptly declined during mitotic exit and became barely
detectable in late telophase (compare Fig. 2A
, e
with Fig. 2A
, c and d). To follow-up
mitotic exit more accurately, cells were cultured in NOC to induce
prometaphase accumulation and then released in NOC-free medium as
described above. Cells were fixed during the following 2 h at
regular intervals. To distinguish late telophase figures from early
interphase figures, cells were labeled using MPM-2 antibody,
which reacts with phosphoepitopes generated in late
G2 and mitosis (29)
. These
experiments revealed high levels of RanBP1 until early telophase (Fig. 2Ba
and b) and a massive decrease in RanBP1
levels in late telophase, concomitant with chromatin decondensation;
for example, the arrowed nuclei in Fig. 2B
,
c have reestablished a chromatin organization resembling
that of G1 nuclei yet represent late telophase
products based on MPM-2 reactivity in the midbody (arrowed
in the middle row). Thus, these experiments enabled us to
visualize the stage at which RanBP1 underwent down-regulation as
revealed in Western blot assays (see Fig. 1B
).
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). RanBP1
was previously reported to contain a NES (3031)
. Indeed,
by mutagenizing two crucial amino acids identified by Richards et
al. (30)
within the NES (i.e., L186A and
V188A), we were able to visualize nuclear retention of the
protein (Fig. 2C
, compare b with a).
Furthermore, we observed nuclear accumulation of the endogenous RanBP1
in cell cultures exposed to LMB (Fig. 2C
, compare
d with c), which blocks nuclear export by
specifically inhibiting the CRM1/exportin 1 factor (3233)
. These results are consistent with previous competition
experiments with functional NES sequences that had implicated CRM1 in
RanBP1 export (31)
. Together, the IF results indicate that
RanBP1 does indeed enter and exit nuclei in a regulated manner in the S
and G2 phases of the cell cycle.
Because no comparative analysis of Ran network components has actually
been carried out in somatic cells progressing through the cell cycle,
IF methods were also used to examine staged cell cultures for their Ran
and RCC1 content. Ran was detected throughout the cell cycle (examples
of S-phase cells are shown in Fig. 3a
), consistent with the Western blot analysis, with a
predominantly nuclear location, as seen in other cell types (3435)
. Cytoplasmic signals were also detected in certain cells,
consistent with the intrinsic shuttling activity of the protein. In
prophase, IF signals were excluded from condensing chromatin and
accumulated toward the nuclear periphery (Fig. 3b
). Ran was
massively redistributed to the cytoplasm during mitotic progression
(Fig. 3c and d)
, with an increased intensity
associated with the mitotic spindle in metaphase (Fig. 3c
).
In contrast, RCC1 immunostaining coincided with chromatin at all
interphase stages (examples of S-phase cells are shown in Fig. 3e
) and throughout mitotic progression, including the moment
of chromosome alignment in metaphase (Fig. 3g
). Chromosomes
were selectively stained by the RCC1 antibody in methanol-fixed cells
(Fig. 3
, f-h), whereas formaldehyde fixation
enabled us to visualize a fraction of RCC1 in the mitotic cytoplasm in
addition to a high proportion of the RCC1 pool that remained
chromosome associated throughout mitosis (data not shown), consistent
with previous results in human cells (36)
. Examination of
isolated mitotic chromosomes from human AHH1 cells confirmed that RCC1
is indeed retained on metaphase chromosomes (data not shown).
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Overexpression of RanBP1 in Asynchronously Cycling Cells Yields
Aberrant Mitotic Spindles and Nuclear Abnormalities.
In previous work (28)
, we designed experiments to override
cell cycle regulation of endogenous RanBP1 and forced constitutive
transcription from a transfected cytomegalovirus-based vector:
transfected cells failed to reach the G0 state
during serum starvation and remained arrested at various stages of
mitosis or during mitotic exit. To extend those earlier observations,
we undertook the characterization of mitotic defects in cycling cells
overexpressing RanBP1. Cells were cotransfected with mammalian pRanBP1
expression construct and with pGFP plasmid, which enabled us to
unambiguously identify transfected cells. The export-defective RanBP1
derivative (pNES construct; see Fig. 2C
) was used for
comparison. GFP-expressing cells were examined 36–48 h after
transfection. Data from eight independent experiments are summarized in
Table 1
. In cultures transfected with empty vector (pX), over 80% of
GFP-expressing cells had a normal interphase appearance; about 6% were
normal mitoses, and around 12% showed an abnormal nuclear morphology
and/or condensed chromatin. The proportion of normal interphase cells
decreased in cultures overexpressing pRanBP1 (66.5%) and
decreased more dramatically in cultures transfected with pNES (47.6%).
Concomitantly, abnormal mitotic figures and cells with aberrantly
condensed nuclei accumulated in these cultures. Nuclear abnormalities
included pyknotic nuclei, in which chromatin was homogenously condensed
(Fig. 4A
), and nuclei with irregular edges and blobs of condensed
chromatin (Fig. 4B
). Flow cytometry and terminal
deoxynucleotidyl transferase-mediated nick end labeling analyses
revealed that aberrant nuclei do not represent apoptotic products (Ref.
28
; data not shown). The frequency of cells with chromatin
abnormalities was statistically higher in cultures transfected with
pNES than in cultures transfected with pRanBP1 construct (Table 1)
and increased with the dose of transfected construct (Fig. 4)
.
Immunostaining of transfected cells using the MPM-2 antibody to
visualize mitotic proteins revealed that nuclear abnormalities were
typically associated with expression of MPM-2 antigens and hence were
generated during mitosis. Actually, several pyknotic nuclei
appeared in paired figures (see Fig. 4A
for example),
suggesting that they derived from telophase cells that failed to
decondense chromatin.
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To characterize mitotic abnormalities in more detail, transfection
experiments were repeated, and cells were immunostained using an
anti-
-tubulin antibody to visualize the spindle. Severe spindle
defects were observed in the presence of either construct. In most
cases, the number of spindle poles was abnormal; monopolar (Fig. 5a
) and multipolar (Fig. 5b and c)
spindles were observed. In certain cases, no spindle structure could
actually be recognized, and mitotic chromosomes were scattered around
tubulin aggregates that were largely unfocused (Fig. 5d
).
Polarization defects were particularly evident in metaphase cells and
were accompanied by misaligned or unattached chromosomes. These defects
persisted in anaphase/telophase, with figures often showing abnormal
central spindles in both multipolar and bipolar cells, with shorter and
thicker microtubules compared with control cells transfected with
vector alone. Groups of chromatids segregated abnormally in multipolar
anaphase cells (Fig. 5e
). The frequency of abnormal mitoses
was highly significant (Table 1)
, and their phenotype was
indistinguishable in cultures overexpressing either wild-type or
export-defective RanBP1.
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. Control cells were injected with either PBS alone or mouse IgG. After
microinjection, cells were allowed to progress through mitosis, and the
timing of each mitotic substage was recorded until completion of the
mitotic division. We found that anti-RanBP1 injection during metaphase
and early anaphase delayed further mitotic progression (Table 2)
: the
average length of metaphase and anaphase actually doubled compared with
that of control cells. One anti-RanBP1-injected cell was blocked in
metaphase for as long as 94 min; however, that particular delay was
unique and was not considered in statistical calculations of the
average mitotic delay in Table 2
. Most importantly, after recovery of
the delay and resumption of mitotic progression, most injected
metaphase and anaphase cells failed to achieve complete nuclear
segregation. The formation of the contractile ring was apparently not
affected (examples of microinjected cells progressing through
cytokinesis are shown in Fig. 6A
), hence daughter cells began to part, but the nuclear
contents remained partly or largely connected. This was best visualized
by fixing the cells after termination of the in vivo
observation and staining the DNA with DAPI. In control (PBS- or
IgG-injected) cells, daughter nuclei were fully separated, and the
distance between them clearly indicated that the spindle motion toward
the poles had proceeded normally (Fig. 6A
, a). In
contrast, variable amounts of nuclear DNA remained trapped in the
cleavage furrow in many anti-RanBP1-injected cells, so that the nuclear
division was either incomplete (Fig. 6Ab
) or ultimately
failed (Fig. 6Ac
). Abnormal mitotic products were
particularly frequent among cells that had been injected with
anti-RanBP1 during metaphase and anaphase (Table 3
). Examples of defects of increasing severity are shown in Fig. 6B
: micronuclei generated by chromosome loss were often
present in late telophase/early G1 cells (Fig. 6Ba
); most anti-RanBP1-injected cells showed chromatin
bridges (Fig. 6Bb
and c); and, in extreme
cases, daughter nuclei remained partly or largely connected, and their
separation was prevented (Fig. 6
B, d and
e).
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suggest that microinjection of anti-RanBP1
antibody during or after metaphase impairs the spindle dynamics and
hence impairs chromatid migration to the poles. As assessed by IF
staining of -tubulin, the anti-RanBP1 antibody does not appear to
alter the spindle morphology in microinjected cells (Fig. 7a
). We therefore sought to assess whether the spindle
dynamics were affected as revealed by testing the sensitivity of
mitotic microtubules to the tubulin-depolymerizing activity of NOC in
the presence or absence of the anti-RanBP1 antibody. Metaphase cells
were microinjected with anti-RanBP1 antibody and immediately exposed to
NOC. After 10 min, cells were fixed and labeled using a secondary
antibody to reveal the microinjected anti-RanBP1 and anti--tubulin
antibody to visualize microtubules. Control metaphase cells injected
with PBS alone showed a disassembled spindle and a diffuse -tubulin
staining throughout the mitotic cytoplasm within 10 min of NOC addition
(Fig. 7b
), indicating that microtubules were completely
depolymerized. In contrast, when anti-RanBP1-microinjected metaphase
cells were exposed to NOC (Fig. 7c
), microtubules remained
polymerized and effectively stained. Thus, RanBP1 inactivation during
metaphase counteracts the effect of NOC on tubulin depolymerization and
prevents disassembly of the mitotic spindle.
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Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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In mammalian cells, the RanBP1 protein is abundant in the cell cycle window included between S phase and late telophase, unlike Ran and RCC1, which are both steadily expressed during the cell cycle. Given the antagonism between RanBP1 and RCC1 in modulating Ran activity (9) , these data suggest that two major changes in the ratio of RanBP1:RCC1 take place during the cell cycle: (a) a first switch is expected to occur at the onset of S phase, when RanBP1 begins to accumulate; and (b) a second switch would take place in late telophase, when RanBP1 is down-regulated. Cyclic fluctuations in RanBP1 levels in nontransformed cells are expected to determine programmed changes in the Ran-GTP:Ran-GDP ratio at the G1-S and M-G1 transitions. Between S phase and the nuclear envelope breakdown, RanBP1 transiently enters the nucleus. RanBP1 export from the nucleus depends on NES integrity and on the availability of functional CRM1 (Refs. 30 and 31 and this study). The fact that we never saw nuclear accumulation of the endogenous RanBP1 except in the presence of LMB suggests that rapid cycles of import and export continuously take place, such that only a fraction of the RanBP1 pool localizes to the nucleus at any given time. During nuclear transit, RanBP1 may transiently interact with RCC1 in vivo, consistent with the demonstrated ability of both proteins to interact in vitro (937) . Such interactions may be important to convey or modulate regulatory signal(s) between the nuclear and cytoplasmic compartments in S phase and G2.
To begin to pinpoint cell cycle events sensitive to alterations in RanBP1 activity, overexpression experiments were carried out in asynchronous cycling cells. Recorded defects fall into two major classes: (a) mitotic spindles with abnormal poles; and (b) completely or locally hypercondensed nuclei in interphase cells. The latter were associated with MPM-2 reactivity, indicating that an aberrant chromatin organization is established during mitosis and persists through mitotic exit, preventing a normal interphase reorganization in daughter cells during the following cell cycle. Nuclear chromatin organization is differentially sensitive to overexpression of mislocalized as opposed to wild-type RanBP1.
Chromatin defects were observed previously in mammalian cell cultures overexpressing RanBP1 during serum starvation, during which cells ought to have completed the mitotic division to enter G0 (28) , and in Schizosaccharomyces pombe strains carrying an imbalance among components of the Ran network (3839) and defective in the mitosis-to-interphase transition (40) . In nuclear reconstitution experiments with Xenopus laevis extracts, excess RanBP1 interferes with nuclear reassembly and nuclear envelope reformation after mitosis (3541) . In our experiments, this phenotype is induced with significantly higher frequency by export-defective RanBP1 as compared with wild-type RanBP1. Both constructs yielded continuous synthesis of RanBP1, which persisted in late telophase, when the endogenous protein normally disappears, and chromatin is due to decondense. Thus, continuous RanBP1 expression during mitotic exit, particularly when accompanied by nuclear retention, appears to interfere with the relocalization and/or activity of components regulating cyclic condensation and decondensation of mitotic chromatin.
A central aspect of the present work is represented by the finding that
the mitotic spindle is severely affected in cells in which RanBP1
activity was deregulated during the preceding interphase or impaired
during mitosis. Spindles with an abnormal number of poles were observed
in cells that reached mitosis after continuous expression of RanBP1.
Unlike chromatin defects, which are preferentially generated by
export-defective RanBP1, abnormal spindles were assembled in cells
overexpressing either form of the protein. Thus, maintaining regulated
RanBP1 levels appears to represent a more critical requirement than
correctly localizing the protein, as far as the spindle structure is
concerned. Overexpressed wild-type RanBP1 will abnormally activate
RanGAP in the cytoplasm, yielding an excess of Ran-GDP. On the other
hand, the export-defective version is expected to yield an excess of
RanBP1/RCC1/Ran interacting complexes in the nucleus. Based on
biochemical evidence (9)
, RCC1 would become inactive in
those complexes, which would also yield an excess of Ran-GDP. Thus,
RanBP1 overexpression could interfere through either mechanism with the
timing of Ran-GTP-dependent interactions important for the spindle
formation. Experiments with cell-free Xenopus oocyte
extracts indicate that the nucleotide-bound state of Ran is crucial for
regulation of nucleation activities (reviewed in Refs. 34,
and 15
). RanBP1 may affect the organization of
the mitotic spindle in various ways: it may contribute to a regulatory
pathway directly regulating the spindle structure [for example, the
centrosome-associated RanBPM protein (14)
may be viewed as
a putative target]; or else the spindle abnormalities may result from
impaired transport of particular proteins, which would in turn perturb
spindle assembly. In either framework, the results in Fig. 5
link for
the first time the requirement for regulated levels of RanBP1 (a
situation that in vivo is perturbed in
retinoblastoma-deficient cells and tumors; see Refs.
20
and 23
) to balanced chromosome segregation
in daughter cells. RanBP1 expression is normally induced at the
G1-S transition under the control of E2F factors
(23)
, as is the expression of several genes whose products
are required for DNA replication (27)
. The centrosome
duplication cycle has also been recently shown to implicate E2F1 and
cyclin A/cyclin-dependent kinase 2 activities (42)
. It may
be speculated that coordinated control of RanBP1 and of
genes involved in the DNA replication and centrosome duplication
machineries is important for coupling formation of the spindle poles to
the cell cycle.
Direct evidence for a distinct role of RanBP1 in spindle control was shown by microinjecting anti-RanBP1 antibody into mitotic cells. In these experiments, mitotic products showed chromatin bridges and micronuclei reflecting chromosome loss or failed separation in extreme cases in which daughter nuclei remained mutually entangled. This in vivo function is clearly distinct from the nucleation regulatory activities ascribed to purified Ran network components in Xenopus oocyte extracts. In our experiments, the mitotic spindle had evidently been functional before microinjection of anti-RanBP1 antibody, to the point of orchestrating metaphase alignment. Anti-RanBP1 microinjection prevented the tubulin-depolymerizing effect of NOC on spindle microtubules. These results suggest that the role of the RanBP1 protein in vivo is exerted, at least in part, through control of microtubule dynamics after metaphase alignment. At this stage, this effect is clearly transport independent. During mitosis, Ran and RanBP1 localize to the mitotic cytoplasm, whereas RCC1 remains largely associated with chromosomes. Part of Ran colocalizes with the mitotic spindle. RanGAP, although not examined here due to the lack of a reliable antibody against the murine protein, was also shown to colocalize with the spindle in human cells (43) . Because microinjection results indicated that functional RanBP1 is required in metaphase and anaphase, we tested the possibility that Ran and RanBP1 act in control of the spindle dynamics through a direct association with the spindle microtubules. However, no component of the Ran network was evidenced in coimmunoprecipitation assays with mitotic tubulin; furthermore, only a minor fraction of Ran and RanBP1 cosedimented with in vitro polymerized microtubules prepared from mitotic extracts as described in Ref. 44 (data not shown). Thus, the largest pool of Ran and RanBP1 does not appear to be directly engaged in a structural interaction with tubulin in the spindle. These observations rather suggest that RanBP1 activity (and hence Ran-GTP hydrolysis) near the spindle may be important to either inactivate a (+end-directed) protein important for microtubule growth until metaphase or to activate a protein important for spindle motion toward the poles after metaphase. The presence of RCC1 on chromosomes may be important to maintain local regions of high Ran-GTP near the sites of microtubule attachment to chromosomes.
In conclusion, the present results provide the first in vivo evidence that RanBP1 is directly implicated in mitotic control in mammalian cells. Because the RanBP1 gene is a regulatory target of E2F- and retinoblastoma-related factors, which are often deregulated in many tumors, these results may be relevant in terms of regulatory mechanisms that may be altered during oncogenesis. In addition to an abnormal rate of cell proliferation, many solid tumors in which members of the E2F/retinoblastoma pathway are unbalanced also show high frequencies of aneuploidy. The present findings, which place RanBP1 in a crucial regulatory pathway for mitotic execution, and the characterization of mitotic defects associated with RanBP1 deregulation or dysfunction may begin to identify aberrant processes that may occur in tumors.
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Materials and Methods |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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Antibody Assays and Western Immunoblotting Experiments.
The specificity of antibodies against components of the Ran network was
assessed in Western blot assays with whole cell extract (30 µg) from
NIH/3T3 cells by preincubating the antibodies with the corresponding
immunogenic peptides (2.5 µg/ml). All antibodies and immunogenic
peptides were from Santa Cruz Biotechnology. Affinity-purified
antibodies include the N-19 series (against
NH2-terminal residues) and the C-20 series
(against COOH-terminal residues) for both murine Ran (sc-1155 and
sc-1156) and RCC1 (sc-1161 and sc-1162) and the M-19 (sc-1159) antibody
against the CKLEALSVREAREEAEEKSE unique peptide of murine RanBP1. To
further ensure that the anti-RanBP1 antibody did not react with other
RBDs, Western blot experiments were set up using GST-fusion proteins
with single RBDs (RBD1–4) from the RanBP2 protein, and no
cross-reactivity was detected. Purified GST-RBDs and their respective
antibodies were kindly provided by Paulo Ferreira (Pharmacology
Department, Medical College of Wisconsin, Milwaukee, WI). Anti-p21
antibody (sc-397) was from Santa Cruz Biotechnology. Protein extracts
were prepared from staged cell cultures, electrophoresed, and
electroblotted as described previously (21)
. Filters were
incubated with 0.5 µg/ml primary antibody in 5% low-fat milk in TBST
buffer [10 mM Tris (pH 8.0,) 150 mM NaCl, and
0.1% Tween 20] for 1 h at room temperature, washed, and further
incubated for 40 min with secondary horseradish peroxidase-conjugated
antibody (Santa Cruz Biotechnology). Immunoblots were revealed using
the enhanced chemiluminescence detection system (Amersham/Pharmacia).
Indirect IF.
Components of the Ran network were examined using various antibodies
and fixation procedures. Affinity-purified antibodies (Santa Cruz
Biotechnology) are specified above; clones directed against the
NH2-terminal and the COOH-terminal region of each
protein were used. RCC1 was also examined using antibodies against
X. laevis RCC1 (45)
kindly provided by Takeharu
Nishimoto (Molecular Biology Department, Graduate School of Medical
Science, Fukuoka, Japan). Similarly, RanBP1 IF experiments were carried
out using either affinity-purified antipeptide antibody (M-19; Santa
Cruz Biotechnology) or a polyclonal antibody against the entire murine
RanBP1 generated in our laboratory (28)
. All tested
antibodies gave similar results. MPM-2 antibody against
G2/mitotic phosphoepitopes (29)
was
from DAKO. Anti--tubulin antibody was from Amersham/Pharmacia. Cells
were fixed by two washes in cold absolute methanol or, alternatively,
3% paraformaldehyde under the conditions described in Refs.
36
and 45
. In cotransfection experiments with
the pGFP construct, cells were fixed in 4% paraformaldehyde.
Cell preparations were incubated in 20% (v/v) FCS in PBST for
45 min. Antibodies against Ran network components were used at 10
µg/ml, MPM-2 antibody was used at 1.3 µg/ml, and anti--tubulin
was used at 3 µg/ml. Primary antibodies were diluted in 5% (v/v) FCS
in PBST, and coverslips were incubated for 45 min. After rinsing in
PBST, coverslips were incubated for 30 min with FITC-conjugated
antigoat antibody (4 µg/ml; Santa Cruz Biotechnology) to detect Ran,
RCC1, and RanBP1 or with Texas Red-conjugated antimouse antibody (15
µg/ml; Vector Laboratories) to detect MPM-2 and -tubulin.
Incubations were carried out at 37°C in a humid chamber. After
washing, coverslips were counterstained with DAPI (0.5 µg/ml in
distilled water) and mounted in Vectashield (Vector Laboratories).
Images were taken using a Zeiss Axioplan microscope configured for
epifluorescence with a x100 oil immersion 1.3 objective and
equipped with a charge-coupled device camera (Photometrics).
Mammalian Expression Constructs and Transfections.
The pRanBP1 wild-type construct was generated by inserting
Htf9-a/RanBP1 cDNA (GenBank X56045) under the
cytomegalovirus promoter/enhancer region in the pBluescript-derived pX
vector (28)
. An export-defective version (termed pNES) was
synthesized using the Quick Change site-directed mutagenesis kit
(Stratagene) and the oligonucleotide
5'-GAGAAGCTGGAAGCCGCTTCAGCTAGGGAGGCCAGAGAG-3'.
pNES carries two amino acidic substitutions, i.e., L186A and
V188A, within the NES (30)
. NIH/3T3 cells were seeded in
Petri dishes (21 cm2) onto sterile coverslips and
transfected using FUGENE (Boehringer/Roche) and 4 or 6 µg of DNA from
pX empty vector, wild-type pRanBP1, or pNES. pGFP expression vector (2
µg/dish; Clontech) was also cotransfected. Cells were collected
36–48 h after transfection and processed for indirect IF to visualize
either GFP and MPM-2 or GFP and -tubulin.
Antibody Microinjection Experiments.
Cells were cultured on etched coverslips, and the medium was changed
just before injection. A concentrated stock (2 mg/ml in PBS) of
affinity-purified anti-RanBP1 antibody (Santa Cruz Biotechnology) was
used. Microinjections were performed using borosilicate glass
microneedles mounted on a manual micromanipulator and a microinjector
(Narishige, Tokyo, Japan). Cells were microinjected at different
stages of mitosis, from prophase to early anaphase. PBS or mouse IgG (2
mg/ml) was used in control experiments. Live cell imaging was recorded
using a Nikon Eclipse 300 inverted microscope with a heated stage
equipped with x40 (0.6) and x60 (0.7) objectives and a Nikon F70
camera with ISO400 films. Images were taken at 2-min intervals. The
length of mitotic substages was calculated from live cell imaging.
Mitotic progression of injected cells was followed in vivo
until completion and in all cases for at least 90 min after injection.
Cells were then fixed and stained with DAPI. In some experiments, cells
were injected with the anti-RanBP1 antibody, exposed immediately to
NOC, fixed after 10 min, and processed for immunostaining as described
above using anti--tubulin antibody to visualize the spindle and
FITC-conjugated secondary antibody to reveal anti-RanBP1.
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Acknowledgments |
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Footnotes |
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1 This work was supported by grants from the
Consiglio Nazionale delle Ricerche and the European Union. G. G. and
B. D. F. were supported by fellowships from the Ministero
dell’Università e Ricerca Scientifica e Tecnologica. L. R. was
supported by a fellowship from the European Union/Consiglio Nazionale
delle Ricerche. F. D. was supported by a grant from the Fondazione
Buzzati-Traverso. G. G. and L. R. contributed equally to this work. 
2 Present address: Department of Cell Biology,
Max-Planck Institute of Biochemistry, Am Klopferspitz 18a, D-82152
Martinsried, Germany.
3 Present address: ENEA Centro Ricerche
della Casaccia, Via Anguillarese 301, S. Maria di Galeria, 00060
Rome, Italy.
4 To whom requests for reprints should be
addressed, at National Research Council Centre of Evolutionary
Genetics, c/o Department of Genetics and Molecular Biology, University
of Rome "La Sapienza," Via degli Apuli 4, Rome 00185, Italy. Phone:
39-06-4457528; Fax: 39-06-4457529; E-mail: patrizia.lavia{at}uniroma1.it
5 The abbreviations used are: RanGAP, Ran
GTPase-activating protein; RanBP, Ran-binding protein; RCC1, regulator
of chromosome condensation; NOC, nocodazole; IF, immunofluorescence;
NES, nuclear export signal; LMB, leptomycin B; GFP, green fluorescent
protein; DAPI, 4',6-diamidino-2-phenylindole; GST, glutathione
S-transferase; RBD, Ran-binding domain; PBST, PBS
containing 0.05% Tween 20.
Received for publication 1/25/00. Revision received 4/17/00. Accepted for publication 6/ 1/00.
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-tubulin. J. Cell Biol., 143: 1041-1052, 1998.This article has been cited by other articles:
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 S. Swaminathan, F. Kiendl, R. Korner, R. Lupetti, L. Hengst, and F. Melchior RanGAP1*SUMO1 is phosphorylated at the onset of mitosis and remains associated with RanBP2 upon NPC disassembly J. Cell Biol., March 29, 2004; 164(7): 965 - 971. [Abstract] [Full Text] [PDF]
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 H.-Y. Li and Y. Zheng Phosphorylation of RCC1 in mitosis is essential for producing a high RanGTP concentration on chromosomes and for spindle assembly in mammalian cells Genes & Dev., March 1, 2004; 18(5): 512 - 527. [Abstract] [Full Text] [PDF]
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I. Cushman, D. Stenoien, and M. S. Moore The Dynamic Association of RCC1 with Chromatin Is Modulated by Ran-dependent Nuclear Transport Mol. Biol. Cell, January 1, 2004; 15(1): 245 - 255. [Abstract] [Full Text] [PDF]
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G. Keryer, B. Di Fiore, C. Celati, K. F. Lechtreck, M. Mogensen, A. Delouvee, P. Lavia, M. Bornens, and A.-M. Tassin Part of Ran Is Associated with AKAP450 at the Centrosome: Involvement in Microtubule-organizing Activity Mol. Biol. Cell, October 1, 2003; 14(10): 4260 - 4271. [Abstract] [Full Text] [PDF]
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P. Askjaer, V. Galy, E. Hannak, and I. W. Mattaj Ran GTPase Cycle and Importins alpha and beta Are Essential for Spindle Formation and Nuclear Envelope Assembly in Living Caenorhabditis elegans Embryos Mol. Biol. Cell, December 1, 2002; 13(12): 4355 - 4370. [Abstract] [Full Text] [PDF]
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M. Kunzler and E. Hurt Targeting of Ran: variation on a common theme? J. Cell Sci., March 11, 2002; 114(18): 3233 - 3241. [Abstract] [Full Text] [PDF]
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 Y. Zou, C. F. Millette, and A. O. Sperry KRP3A and KRP3B: Candidate Motors in Spermatid Maturation in the Seminiferous Epithelium Biol Reprod, March 1, 2002; 66(3): 843 - 855. [Abstract] [Full Text] [PDF]
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 S.-H. Kim, D. Arnold, A. Lloyd, and S. J. Roux Antisense Expression of an Arabidopsis Ran Binding Protein Renders Transgenic Roots Hypersensitive to Auxin and Alters Auxin-Induced Root Growth and Development by Arresting Mitotic Progress PLANT CELL, December 1, 2001; 13(12): 2619 - 2630. [Abstract] [Full Text] [PDF]
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2 test.
