Vol.
11, 455– 465, August 2000
Regulated Ran-binding Protein 1 Activity Is Required for
Organization and Function of the Mitotic Spindle in
Mammalian Cells in Vivo1
Giulia Guarguaglini,2 Luigina Renzi,3
Filippo D’Ottavio, Barbara Di Fiore,
Martina Casenghi,2 Enrico Cundari, and
Patrizia Lavia4
National Research Council Centre of Evolutionary Genetics, c/o
Department of Genetics and Molecular Biology, University of Rome “La
Sapienza,” Rome 00185, Italy
Abstract
Ran-binding protein (RanBP) 1 is a major regulator of
the Ran GTPase and is encoded by a regulatory target
gene of E2F factors. The Ran GTPase network controls
several cellular processes, including nucleocytoplasmic
transport and cell cycle progression, and has recently
also been shown to regulate microtubule nucleation
and spindle assembly in Xenopus oocyte extracts. Here
we report that RanBP1 protein levels are cell cycle
regulated in mammalian cells, increase from S phase
to M phase, peak in metaphase, and abruptly decline in
late telophase. Overexpression of RanBP1 throughout
the cell cycle yields abnormal mitoses characterized by
severe defects in spindle polarization. In addition,
microinjection of anti-RanBP1 antibody in mitotic cells
induces mitotic delay and abnormal nuclear division,
reflecting an abnormal stabilization of the mitotic
spindle. Thus, regulated RanBP1 activity is required for
proper execution of mitosis in somatic cells.
Introduction
The Ran GTPase network has been implicated in control of a
puzzling variety of processes because mutations in the Ran
GTPase itself or in partner molecules affect cell cycle pro-
gression, chromosome stability, nuclear organization, and
Received 1/25/00; revised 4/17/00; accepted 6/1/00.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indi-
cate this fact.
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 Tec-
nologica. 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 Anguillar-
ese 301, S. Maria di Galeria, 00060 Rome, Italy.
4
To whom requests for reprints should be addressed, at National Re-
search Council Centre of Evolutionary Genetics, c/o Department of Ge-
netics 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@uniroma1.it.
Cell Growth & Differentiation 455
nucleocytoplasmic traffic in several organisms (for reviews,
see Refs. 1– 4). The biological activity of Ran is dependent on
the turnover rate between the GTP- and GDP-bound state.
Major regulators controlling the nucleotide-bound state of
Ran include RanGAP,5 which catalyzes GTP hydrolysis yield-
ing Ran-GDP (5), and the RCC1 protein, which acts as the
guanine exchange factor for Ran and favors the formation of
Ran-GTP (6). RanBP1 interacts with GTP-bound Ran (7, 8)
and favors its conversion to Ran-GDP, at least in vitro, by
increasing the rate of GTP hydrolysis via RanGAP and by
inhibiting the exchange activity of RCC1 (9).
The role of Ran is particularly well documented in control
of nucleocytoplasmic traffic in interphase cells. The nucle-
otide-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 increas-
ingly 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 cy-
cle 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). Fur-
thermore, work with Xenopus egg extracts has depicted a
direct role of members of the Ran network in mitotic control
(reviewed in Refs. 3, 4, 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, so-
matic 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 subcel-
lular compartments. It is important to examine in vivo pro-
cesses to assess whether components of the Ran network
are actually implicated in mitotic control in somatic cells in
vivo.
5
The abbreviations used are: RanGAP, Ran GTPase-activating protein;
RanBP, Ran-binding protein; RCC1, regulator of chromosome condensa-
tion; 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.
456 Mitotic Functions of RanBP1

Fig. 1. Components of the Ran network during cell cycle progression. A, anti-RanBP1, anti-Ran, and anti-RCC1 antibodies (see “Materials and Methods”
for details) yield specific signals in murine NIH/3T3 cells. Thirty ␮g of whole cell extract were loaded in each lane. Antibodies were used alone (⫺) or after
preincubation (⫹) with immunogenic peptides. B, Western blot assays of protein extract from staged cells (40 ␮g/lane). Protein extracts from growth-
arrested (G0), G1, and S-phase cells were prepared from serum-starved and restimulated cultures harvested at the indicated times after serum addition.
To follow-up mitotic exit, extracts from M-phase cells were prepared from cultures grown in the presence of NOC and then released and harvested at the
indicated times after release of NOC arrest.

In previous work, we cloned and characterized the regu-
latory features of the murine RanBP1 gene. We found that
RanBP1 mRNA transcription is subjected to both growth
regulation in proliferating versus quiescent cells (20, 21) 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 overex-
pressed in several transformed cell lines (20). The link be-
tween 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 trans-
port during the cell cycle phases. Indeed, previous experi-
ments in which RanBP1 transcription was rendered consti-
tutive from a cell cycle-independent promoter yielded
significant cell cycle alterations with a predominant impair-
ment 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 gen-
erated 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 estab-
lish whether RanBP1 dysfunction directly affected mitotic
progression. To that end, we microinjected anti-RanBP1 an-
tibody 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.
Results
The RanBP1 Protein Is Present in a Limited Cell Cycle
Window in Mammalian Cells. Before addressing the func-
tional role of RanBP1, we sought to compare the relative
distribution of components of the Ran network during the cell
cycle phases. Western immunoblotting experiments were
carried out with murine NIH/3T3 whole cell extract using
commercial affinity-purified antibodies against Ran, RanBP1,
and RCC1. Highly specific signals were generated, which
were abolished by preincubation with the immunogenic pep-
tides (Fig. 1A). 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 promet-
aphase-arrested cells but decline within 1 h after release of
NOC arrest, i.e., when cells traverse the mitosis to G1 tran-
sition. These results are specific by comparison with both
phase-specific (i.e., E2F-1 in the case of release from serum
 Cell Growth & Differentiation 457

Fig. 2. Immunolocalization of
RanBP1 during the cell cycle. A,
NIH/3T3 cell cultures stimulated
to cycle were examined at vari-
ous times after serum addition. a,
10 h (G1). b, 18 h (S/G2). c⫺e,
mitotic cells were seen after 24 h,
when synchronization is gradu-
ally lost. Cell spreads were incu-
bated with anti-RanBP1 antibody
followed by FITC-conjugated
secondary antibody and counter-
stained with DAPI. Bar, 10 ␮m. B,
RanBP1 during mitotic exit. Cells
were exposed to NOC for 9 h and
then fixed at regular intervals
from the block release and
stained with DAPI, MPM-2, and
anti-RanBP1 antibodies. A met-
aphase is shown in column a (20
min after release from NOC ar-
rest), anaphase and telophase
are shown in columns b (40 min
after release) and c (80 min after
release), and a late telophase is
shown in c (arrows). Bar, 10 ␮m.
C, RanBP1 transits through the
nucleus. Constructs encoding
wild-type (a, pRanBP1wt) or NES-
mutagenized (b, pNES) RanBP1
were transfected in cycling cells,
and the subcellular localization
of the protein was determined.
RanBP1 IF and DAPI staining
were performed as described for
A. RanBP1 is cytoplasmic in con-
trol cultures (c, ⫺LMB) but be-
comes nuclear within 2 h of LMB
addition (d, ⫹LMB). Bar, 10 ␮m.

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 ex-
pressed 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.
We next examined the RanBP1 protein in cell cultures
synchronized by serum starvation/restimulation using indi-
rect IF. Consistent with the immunoblotting results, the
RanBP1 signal was barely detectable in early G1 cells (Fig.
2A, a) 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 promet-
aphase accumulation and then released in NOC-free me-
dium as described above. Cells were fixed during the follow-
ing 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 gen-
erated in late G2 and mitosis (29). These experiments re-
vealed high levels of RanBP1 until early telophase (Fig. 2B, a
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 reactiv-
ity in the midbody (arrowed in the middle row). Thus, these
458 Mitotic Functions of RanBP1
experiments enabled us to visualize the stage at which
RanBP1 underwent down-regulation as revealed in Western
blot assays (see Fig. 1B).
In S and G2 phases, in which RanBP1 is abundantly ex-
pressed, a predominant cytoplasmic distribution was ob-
served, yet nuclear signals were also apparent (see Fig. 2A,
b). RanBP1 was previously reported to contain a NES (30,
31). Indeed, by mutagenizing two crucial amino acids iden-
tified 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 ob-
served 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 (32, 33). 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 compo-
nents has actually been carried out in somatic cells progress-
ing 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
Fig. 3. Ran is redistributed to the cy-
toplasm, whereas RCC1 colocalizes
with chromatin during mitosis. Cells
were synchronized by serum starva-
tion/restimulation as described in the
Fig. 2A legend. a and e were taken
from S/G2-enriched cultures 18 h after
cell cycle entry; a similar distribution
was detected throughout interphase.
Prophase cells in b and f, metaphase
cells in c and g, and anaphase cells in
d and h were seen among cultures har-
vested 24 h after cell cycle entry. Cell
spreads were fixed in paraformalde-
hyde and incubated with anti-Ran an-
tibody (a⫺d) or fixed with methanol
and incubated with anti-RCC1 anti-
body (e⫺h). FITC-conjugated second-
ary antibody was used, and DNA was
counterstained with DAPI. Bar, 10 ␮m.
Western blot analysis, with a predominantly nuclear location,
as seen in other cell types (34, 35). 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 to-
ward the nuclear periphery (Fig. 3b). Ran was massively
redistributed to the cytoplasm during mitotic progression
(Fig. 3, c 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). Chromo-
somes 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 through-
out mitosis (data not shown), consistent with previous results
in human cells (36). Examination of isolated mitotic chromo-
somes from human AHH1 cells confirmed that RCC1 is in-
deed retained on metaphase chromosomes (data not
shown).
In summary, only Ran and RCC1 are steadily expressed
throughout the cell cycle, whereas RanBP1 levels are low in
G1 and increase throughout S phase and G2 phase. In these

Table 1 Cellular abnormalities in cultures overexpressing RanBP1
Cellular phenotypes were scored among GFP-expressing cells in cul-
tures transfected with pX vector, pRanBP1 or pNES (6 ␮g of each con-
struct). Ps were calculated from the single comparison of either pRanBP1-
or pNES-transfected cultures, with control cultures using the ␹2 test.
Cell types (%) pX pRanBP1 pNES
Normal interphases 81.63 66.5 47.64a
Normal mitoses 6.08 5.4 6.8
Abnormal mitoses 0.36 5.2a 8.63a
Pyknotic nuclei 7.5 13.75 21.99a
Aberrant nuclei 4.38 9.12 14.92a
Counted cells 822 670 764
P ⬍⬍0.001 ⬍⬍0.001
a
Classes whose partial ␹2 values contribute most significantly to the
total ␹2.
phases, Ran and RCC1 are largely or almost exclusively
nuclear, whereas RanBP1 is largely cytoplasmic but appears
to be able to shuttle between the nucleus and the cytoplasm.
In mitotic cells, all components are abundantly expressed
and asymetrically redistributed: mitotic chromosomes only
retain RCC1 or at least a significant fraction(s) of the RCC1
pool; whereas Ran and RanBP1 massively localize to the
mitotic cytoplasm. RanBP1 is present at high levels until late
telophase and is dramatically down-regulated during mitotic
exit, so that cells reentering G1 again express low levels of
RanBP1.
Overexpression of RanBP1 in Asynchronously Cycling
Cells Yields Aberrant Mitotic Spindles and Nuclear Ab-
normalities. In previous work (28), we designed experi-
ments to override cell cycle regulation of endogenous
RanBP1 and forced constitutive transcription from a trans-
fected 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 under-
took 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 trans-
fected 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 ap-
pearance; about 6% were normal mitoses, and around 12%
showed an abnormal nuclear morphology and/or condensed
chromatin. The proportion of normal interphase cells de-
creased 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 de-
oxynucleotidyl transferase-mediated nick end labeling anal-
yses revealed that aberrant nuclei do not represent apoptotic
Cell Growth & Differentiation 459
products (Ref. 28; data not shown). The frequency of cells
with chromatin abnormalities was statistically higher in cul-
tures 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 gener-
ated during mitosis. Actually, several pyknotic nuclei ap-
peared in paired figures (see Fig. 4A for example), suggesting
that they derived from telophase cells that failed to decon-
dense chromatin.
Examination of DAPI staining in GFP-positive cells further
revealed that abnormal mitotic figures also accumulated in
cultures transfected with either export-defective or wild-type
RanBP1 construct (Table 1). To characterize mitotic abnor-
malities in more detail, transfection experiments were re-
peated, 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. 5, b and c) spindles
were observed. In certain cases, no spindle structure could
actually be recognized, and mitotic chromosomes were scat-
tered 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 an-
aphase/telophase, with figures often showing abnormal cen-
tral spindles in both multipolar and bipolar cells, with shorter
and thicker microtubules compared with control cells trans-
fected with vector alone. Groups of chromatids segregated
abnormally in multipolar anaphase cells (Fig. 5e). The fre-
quency of abnormal mitoses was highly significant (Table 1),
and their phenotype was indistinguishable in cultures over-
expressing either wild-type or export-defective RanBP1.
Microinjection of Anti-RanBP1 Antibody in Living Mi-
totic Cells. Results thus far indicate that the mitotic spindle
organization is sensitive to intracellular levels of RanBP1. To
directly assess the role of RanBP1 in mitosis, we injected
anti-RanBP1 antibody in living cells that were progressing
through mitotic substages, when RanBP1 is maximally ex-
pressed. To further ascertain that anti-RanBP1 antibody did
not cross-react with conserved RBDs present in other cel-
lular proteins, we carried out preliminary Western blot exper-
iments with purified GST-fusion proteins expressing single
RBDs (RBD1– 4) from the RanBP2 protein: no cross-reactiv-
ity was detected (data not shown). Microinjections were car-
ried out during mitotic substages as indicated in Table 2.
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 an-
aphase 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 con-
460 Mitotic Functions of RanBP1
sidered 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 seg-
regation. 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 be-
tween 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. 6A, b) or
ultimately failed (Fig. 6A, c). Abnormal mitotic products were
particularly frequent among cells that had been injected with
anti-RanBP1 during metaphase and anaphase (Table 3). Ex-
amples of defects of increasing severity are shown in Fig. 6B:
micronuclei generated by chromosome loss were often pres-
ent in late telophase/early G1 cells (Fig. 6B, a); most anti-
RanBP1-injected cells showed chromatin bridges (Fig. 6B, b
and c); and, in extreme cases, daughter nuclei remained
partly or largely connected, and their separation was pre-
vented (Fig. 6B, d and e).
The defects shown in Fig. 6 suggest that microinjection of
anti-RanBP1 antibody during or after metaphase impairs the
spindle dynamics and hence impairs chromatid migration to
Fig. 4. Examples of nuclear defects induced
by overexpression of wild-type or export-
defective RanBP1. Asynchronously cycling
cell cultures were transfected with vector (pX),
wild-type pRanBP1, or pNES (4 and 6 ␮g of
each). Recorded defects include (A) pyknotic
nuclei with homogeneously hypercondensed
chromatin and (B) abnormal nuclei with irreg-
ular edges and blobs of chromatin. Right, de-
fects were quantified among 400 GFP-positive
cells cotransfected with pX vector, wild-type
pRanBP1, and pNES and stained with MPM-2
antibody. Histograms represent the percent-
age of observed defects using 4 or 6 ␮g of
each construct.
the poles. As assessed by IF staining of ␣-tubulin, the anti-
RanBP1 antibody does not appear to alter the spindle mor-
phology 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 microtu-
bules. 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 coun-
teracts the effect of NOC on tubulin depolymerization and
prevents disassembly of the mitotic spindle.
Discussion
The present study reports several novel aspects of RanBP1
function in mitotic control in mammalian cells in vivo. We
have found that regulated RanBP1 activity is required in at
least three important processes associated with mitotic di-
vision, including the structural organization of the spindle,
control of microtubule dynamics in metaphase and an-

Fig. 5. Defects in spindle polarization after overexpression of wild-type
or export-defective RanBP1. Asynchronous cell cultures were transfected
with vector, wild-type pRanBP1 (a and b), or pNES (c⫺e). In all cases, the
pGFP expression plasmid was cotransfected. Cells were fixed with
paraformaldehyde to visualize GFP, processed for IF using anti-␣-tubulin
antibody (left column), and counterstained with DAPI (middle column).
Merged figures in the right column show chromosome misalignment with
respect to aberrant spindles. Examples of spindle abnormalities are
shown: a, monopolar spindle; b, tripolar spindle; c, tetrapolar spindle, d,
tubulin aggregates with no clear spindle structure; and e, tripolar
anaphase.
aphase, and nuclear chromatin reorganization after mitotic
exit.
In mammalian cells, the RanBP1 protein is abundant in the
cell cycle window included between S phase and late telo-
phase, unlike Ran and RCC1, which are both steadily ex-
pressed 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 fluctua-
tions in RanBP1 levels in nontransformed cells are expected
to determine programmed changes in the Ran-GTP:Ran-
Cell Growth & Differentiation 461
GDP ratio at the G1-S and M-G1 transitions. Between S
phase and the nuclear envelope breakdown, RanBP1 tran-
siently 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 (9, 37). Such inter-
actions may be important to convey or modulate regulatory
signal(s) between the nuclear and cytoplasmic compart-
ments in S phase and G2.
To begin to pinpoint cell cycle events sensitive to alter-
ations 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 daugh-
ter 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 mamma-
lian cell cultures overexpressing RanBP1 during serum star-
vation, during which cells ought to have completed the mi-
totic division to enter G0 (28), and in Schizosaccharomyces
pombe strains carrying an imbalance among components of
the Ran network (38, 39) and defective in the mitosis-to-
interphase transition (40). In nuclear reconstitution experi-
ments with Xenopus laevis extracts, excess RanBP1 inter-
feres with nuclear reassembly and nuclear envelope
reformation after mitosis (35, 41). 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 endog-
enous protein normally disappears, and chromatin is due to
decondense. Thus, continuous RanBP1 expression during
mitotic exit, particularly when accompanied by nuclear re-
tention, 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 ab-
normal number of poles were observed in cells that reached
mitosis after continuous expression of RanBP1. Unlike chro-
matin defects, which are preferentially generated by export-
defective RanBP1, abnormal spindles were assembled in
cells overexpressing either form of the protein. Thus, main-
taining regulated RanBP1 levels appears to represent a more
critical requirement than correctly localizing the protein, as
far as the spindle structure is concerned. Overexpressed
462 Mitotic Functions of RanBP1

Table 2 Anti-RanBP1 microinjection in mitosis induces metaphase and anaphase delay

Microinjected cells Metaphase length (min) Anaphase length (min)

Injection
Stage No. of cells Mean ⫾ SD Range Mean ⫾ SD Range

PBS Prophase a
10 6.2 ⫾ 2.0 4–10 9.5 ⫾ 1.6 9–13
Prometaphase 10 6.2 ⫾ 2.3 3–10 10.3 ⫾ 13 9–12
Metaphase 10 5.2 ⫾ 2.9 2–10 9.0 ⫾ 2.7 7–15
Anaphase 5 13.8 ⫾ 4.7 7–21
IgG Prophasea 10 6.4 ⫾ 1.6 4–10 9.5 ⫾ 2.4 7–13
Prometaphase 10 5.6 ⫾ 1.7 3–9 10.7 ⫾ 3.1 8–15
Metaphase 10 5.6 ⫾ 1.6 2–8 12.4 ⫾ 2.8 8–16
Anaphase 5 12.7 ⫾ 2.6 9–16
RanBP1 antibody Prophasea 10 7.5 ⫾ 5.4 3–16 8.6 ⫾ 1.1 7–10
Prometaphase 10 7.0 ⫾ 3.5 3–15 13.6 ⫾ 9.1 7–34
Metaphase 10 10.9 ⫾ 5.1b,c 5–20 18.2 ⫾ 7.6d 11–30
Anaphase 5 24.0 ⫾ 4.6c 18–31
a
No difference between cytoplasmic and nuclear injection.
b
Because one cell was arrested for 94 min, mean ⫾ SD values were calculated from nine cells only.
c
P ⬍ 0.01 (differences in mitotic stage lengths were evaluated using the Student’s t test).
d
P ⬍ 0.05 (differences in mitotic stage lengths were evaluated using the Student’s t test).

Fig. 6. Examples of defects induced

by microinjecting RanBP1 antibody
into mitotic cells. Mitotic progression
was timed by live image recording for
at least 90 min. A, examples of mitotic
progression in microinjected cells in
vivo (phase-contrast microscopy).
DAPI-stained nuclei after fixation are
shown in the right column. a, PBS-
injected cell progressing through
complete cytokinesis and nuclear di-
vision. Anti-RanBP1-microinjected
cells, despite forming a normal cleav-
age furrow, show partly (b) or largely
(c) incomplete nuclear division. Bar,
10 ␮m. B, abnormal mitotic products
in anti-RanBP1-microinjected cells
fixed after mitotic completion and
stained with DAPI. a, micronucleus
(arrow); b and c, DNA bridges be-
tween daughter cells, with increasing
amounts of trapped chromatin in the
actin contractile ring (arrows); d, chro-
matid sets did not migrate at an-
aphase; e, chromosomes did not go
through metaphase or anaphase, and
chromatin blobs were visualized at
telophase. Bar, 10 ␮m.

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 forma-
tion. 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. 3, 4, and
15). RanBP1 may affect the organization of the mitotic spin-
dle 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 per-
turbed in retinoblastoma-deficient cells and tumors; see

Table 3 Defects in mitotic cells microinjected with anti-RanBP1
antibody
No. of
Stage of injected cells Observed defects
injection
Total Aberrant
Prophase 10 2 Chromosomes decondensed in the
absence of proper metaphase-to-
anaphase transition; chromatin
blobs formed; no cytokinesis
(Fig. 7B, p d)
1 Chromatin bridge between daughter
nuclei; no cytokinesis
1 Tripolar cell
Prometaphase 10 1 Chromatin bridge
2 Micronuclei
1 Chromosomes decondensed in the
absence of proper metaphase-to-
anaphase transition; chromatin
blobs formed; no cytokinesis
Metaphase 10 2 Lagging chromosomes and
micronuclei (Fig. 7B, p a)
2 Chromatin bridges (Fig. 7B, p b)
2 Chromatin bridges; no cytokinesis
(p b in Fig. 7A and c in Fig. 7B)
1 Incomplete separation of
chromosome sets
1 Metaphase arrest for 94 min;
eventually, daughter nuclei
separated, forming one
micronucleus
Anaphase 5 2 Delay in chromosome migration,
incomplete anaphase, chromosome
arms entangled with the contractile
ring, no cytokinesis (p c in Fig. 6A
and e in Fig. 6B)
2 Chromatin bridges, no cytokinesis
1 Progressed from anaphase onset to
anaphase B in 10 min; aberrant
telophase after 31 min
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 re-
quired 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 spin-
dle 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 chro-
mosome 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 met-
Cell Growth & Differentiation 463
Fig. 7. Anti-RanBP1 microinjection stabilizes the mitotic spindle. Met-
aphase cells were injected with PBS or with anti-RanBP1 antibody and
exposed to NOC. After 10 min, cells were fixed and stained with anti-␣-
tubulin antibody to reveal the spindle morphology and with secondary
antibody to visualize anti-RanBP1 antibody; chromosomes were stained
with DAPI. a, normal spindle in a metaphase cell injected anti-RanBP1
antibody; b, disassembled spindle in a PBS-injected metaphase cell ex-
posed to NOC; c, polymerized spindle in an anti-RanBP1-injected met-
aphase cell exposed to NOC. Bar, 10 ␮m.
aphase alignment. Anti-RanBP1 microinjection prevented
the tubulin-depolymerizing effect of NOC on spindle micro-
tubules. 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 chromo-
somes. 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 possi-
bility that Ran and RanBP1 act in control of the spindle
dynamics through a direct association with the spindle mi-
crotubules. However, no component of the Ran network was
evidenced in coimmunoprecipitation assays with mitotic tu-
bulin; furthermore, only a minor fraction of Ran and RanBP1
cosedimented with in vitro polymerized microtubules pre-
pared 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-direct-
ed) 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 chro-
mosomes may be important to maintain local regions of high
Ran-GTP near the sites of microtubule attachment to chro-
mosomes.
In conclusion, the present results provide the first in vivo
evidence that RanBP1 is directly implicated in mitotic control
464 Mitotic Functions of RanBP1
in mammalian cells. Because the RanBP1 gene is a regula-
tory 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 asso-
ciated with RanBP1 deregulation or dysfunction may begin
to identify aberrant processes that may occur in tumors.
Materials and Methods
Cell Cultures and Cell Cycle Analysis. Murine NIH/3T3 embryo fibro-
blasts (ATCC CRL 1658) were routinely grown as described previously
(22). Cell cycle synchronization was obtained by maintaining the cultures
in low FCS (0.5%) for 48 h to induce growth arrest and subsequently
stimulating synchronous cell cycle reentry by raising the serum to 15%.
Cells were collected at regular intervals after stimulation. Cultures were
also synchronized in prometaphase by exposure to 0.2– 0.5 ␮g/ml NOC
for 9 h and then released in NOC-free medium and harvested during the
following 2 h at 20-min intervals. Cell cycle progression was analyzed by
fluorescence-activated cell sorting using a FACStar Plus flow cytometer
(Becton Dickinson) as described previously (22). To inhibit nuclear export,
cells were exposed to 20 nM LMB [a kind gift from Minuro Yoshida
(Department of Biotechnology, University of Tokyo, Tokyo, Japan)] for 2 h.
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 ␮g) from
NIH/3T3 cells by preincubating the antibodies with the corresponding
immunogenic peptides (2.5 ␮g/ml). All antibodies and immunogenic pep-
tides 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 mu-
rine 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 re-
spective 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 electro-
blotted as described previously (21). Filters were incubated with 0.5 ␮g/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 re-
vealed using the enhanced chemiluminescence detection system (Amer-
sham/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 Depart-
ment, 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 con-
ditions described in Refs. 36 and 45. In cotransfection experiments with
the pGFP construct, cells were fixed in 4% paraformaldehyde. Cell prep-
arations were incubated in 20% (v/v) FCS in PBST for 45 min. Antibodies
against Ran network components were used at 10 ␮g/ml, MPM-2 anti-
body was used at 1.3 ␮g/ml, and anti-␣-tubulin was used at 3 ␮g/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 incu-
bated for 30 min with FITC-conjugated antigoat antibody (4 ␮g/ml; Santa
Cruz Biotechnology) to detect Ran, RCC1, and RanBP1 or with Texas
Red-conjugated antimouse antibody (15 ␮g/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 ␮g/ml in distilled water) and mounted in Vectashield (Vector Labora-
tories). Images were taken using a Zeiss Axioplan microscope configured
for epifluorescence with a ⫻100 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
␮g of DNA from pX empty vector, wild-type pRanBP1, or pNES. pGFP
expression vector (2 ␮g/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 micro-
scope with a heated stage equipped with ⫻40 (0.6) and ⫻60 (0.7) objec-
tives 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 sec-
ondary antibody to reveal anti-RanBP1.
Acknowledgments
We are grateful to Rosamaria Mangiacasale for help with flow cytom-
etry and to Antonella Palena for many contributions to this work. We also
thank Minuro Yoshida for the gift of LMB, Paulo Ferreira for the gift of
RanBP2-derived Ran-binding domains and antibodies, and Takeharu
Nishimoto for anti-RCC1 antibodies.
References
1. Rush, M. G., Drivas, G., and D’Eustachio, P. The small nuclear GTPase
Ran: how much does it run? Bioessays, 18: 103–112, 1996.
2. Sazer, S. The search for the primary function of the Ran GTPase
continues. Trends Cell Biol., 6: 81– 85, 1996.
3. Nishimoto, T. A new role of Ran GTPase. Biochem. Biophys. Res.
Commun., 262: 571–574, 1999.
4. Sazer, S., and Dasso, M. The Ran decathlon: multiple roles of Ran.
J. Cell Sci., 113: 1111–1118, 2000.
5. Bischoff, R. F., Klebe, C., Kretschmer, J., Wittinghofer, A., and
Ponstingl, H. RanGAP induces GTPase activity of nuclear Ras-related
Ran. Proc. Natl. Acad. Sci. USA, 91: 2787–2791, 1994.
6. Bischoff, F. R., and Ponstingl, H. Catalysis of guanine exchange on Ran
by the mitotic regulator RCC1. Nature (Lond.), 354: 80 – 82, 1991.

7. Coutavas, E., Ren, M., Oppenheim, J., D’Eustachio, P., and Rush, M.
Characterization of proteins that interact with the cell-cycle regulatory
protein Ran/TC4. Nature (Lond.), 366: 585–587, 1993.
8. Richards, S. A., Lounsbury, K., and Macara, I. The C-terminus of the
nuclear Ran/TC4 GTPase stabilizes the GDP-bound state and mediates
interaction with RCC1, Ran-GAP and Htf9-a/RanBP1. J. Biol. Chem., 270:
14405–14411, 1995.
9. Bischoff, R. F., Krebber, H., Smirnova, E., Dong, W., and Ponstingl, H.
Co-activation of RanGTPase and inhibition of GTP dissociation by Ran-
GTP binding protein RanBP1. EMBO J., 14: 705–715, 1995.
10. Dasso, M., and Pu, R. T. Nuclear transport: run by Ran? Am. J. Hum.
Genet., 63: 311–316, 1998.
11. Adam, S. A. Transport pathways of macromolecules between the
nucleus and the cytoplasm. Curr. Opin. Cell Biol., 11: 402– 406, 1999.
12. Stochaj, U., and Rother, K. L. Nucleocytoplasmic trafficking of pro-
teins: with or without Ran? Bioessays, 21: 579 –589, 1999.
13. Ouspenski, I. I. A RanBP1 mutation which does not visibly affect
nuclear import may reveal additional functions of the Ran GTPase system.
Exp. Cell Res., 244: 171–183, 1998.
14. Nakamura, M., Masuda, H., Horii, J., Kuma, K., Yokoyama, N., Ohba,
T., Nishitani, H., Miyata, T., Tanaka, M., and Nishimoto, T. When overex-
pressed, a novel centrosomal protein, RanBPM, causes ectopic microtu-
bule nucleation similar to ␥-tubulin. J. Cell Biol., 143: 1041–1052, 1998.
15. Kahana, J. A., and Cleveland, D. W. Beyond nuclear transport. Ran-
GTP as a determinant of spindle assembly. J. Cell Biol., 146: 1205–1210,
1999.
16. Carazo-Salas, R. E., Guarguaglini, G., Gruss, O. J., Segref, A.,
Karsenti, E., and Mattaj, I. W. Generation of GTP-bound Ran by RCC1 is
required for chromatin-induced mitotic spindle formation. Nature (Lond.),
400: 178 –181, 1999.
17. Kalab, P., Pu, R. T., and Dasso, M. The Ran GTPase regulates mitotic
spindle assembly. Curr. Biol., 9: 481– 484, 1999.
18. Ohba, T., Nakamura, M., Nishitani, H., and Nishimoto, T. Self-
organization of microtubule asters induced in Xenopus egg extracts by
GTP-bound Ran. Science (Washington DC), 284: 1356 –1358, 1999.
19. Wilde, A., and Zheng, Y. Stimulation of microtubule aster formation
and spindle assembly by the small GTPase Ran. Science (Washington
DC), 284: 1359 –1362, 1999.
20. Di Matteo, G., Fuschi, P., Zerfass, K., Moretti, S., Ricordy, R., Cen-
ciarelli, C., Tripodi, M., Jansen-Dürr, P., and Lavia, P. Transcriptional
control of the Htf9-a/RanBP1 gene during the cell cycle. Cell Growth
Differ., 6: 1213–1224, 1995.
21. Di Matteo, G., Salerno, M., Guarguaglini, G., Di Fiore, B., Palitti, F.,
and Lavia, P. Interactions with single-stranded and double-stranded DNA-
binding factors and alternative promoter conformation upon transcrip-
tional activation of the Htf9-a/RanBP1 and Htf9-c genes. J. Biol. Chem.,
273: 495–505, 1998.
22. Guarguaglini, G., Battistoni, A., Pittoggi, C., Di Matteo, G., Di Fiore, B.,
and Lavia, P. Expression of the murine RanBP1 and Htf9-c genes is
regulated from a shared bidirectional promoter during cell cycle progres-
sion. Biochem. J., 325: 277–286, 1997.
23. Di Fiore, B., Guarguaglini, G., Palena, A., Kerkhoven, R. M., Bernards,
R., and Lavia, P. Two E2F-binding sites independently control growth-
regulated and cell-cycle regulated transcription of the Htf9-a/RanBP1
gene. J. Biol. Chem., 274: 10339 –10348, 1999.
24. Muller, H., and Helin, K. The E2F transcription factors: key regulators
of cell proliferation. Biochim. Biophys. Acta, 1470: M1–M12, 2000.
25. Dyson, N. The regulation of E2F by pRB-family proteins. Genes Dev.,
12: 2245–2262, 1998.
26. Nevins, J. R. Toward an understanding of the functional complexity of
the E2F and retinoblastoma families. Cell Growth Differ., 9: 585–593,
1998.
27. Lavia, P., and Jansen-Dürr, P. E2F target genes and cell cycle check-
point control. Bioessays, 21: 221–230, 1999.
Cell Growth & Differentiation 465
28. Battistoni, A., Guarguaglini, G., Degrassi, F., Pittoggi, C., Palena, A.,
Di Matteo, G., Pisano, C., Cundari, E., and Lavia, P. Deregulated expres-
sion of the RanBP1 gene alters cell cycle progression in murine fibro-
blasts. J. Cell Sci., 110: 2345–2357, 1997.
29. Davis, F. M., Tsao, T. Y., Fowler, S. K., and Rao, P. N. Monoclonal
antibodies to mitotic cells. Proc. Natl. Acad. Sci. USA, 80: 2926 –2930,
1983.
30. Richards, S. A., Lounsbury, K. M., Carey, K. L., and Macara, I. A
nuclear export signal is essential for the cytosolic localization of the Ran
binding protein, RanBP1. J. Cell Biol., 134: 1157–1168, 1996.
31. Pasquinelli, A. E., Powers, M. A., Lund, E., Forbes, D., and Dahlberg,
J. E. Inhibition of mRNA export in vertebrate cells by nuclear export signal
conjugates. Proc. Natl. Acad. Sci. USA, 94: 14394 –14399, 1997.
32. Fukuda, M., Asano, S., Nakamura, T., Adachi, M., Yoshida, M.,
Yanagida, M., and Nishida, E. CRM1 is responsible for intracellular trans-
port mediated by the nuclear export signal. Nature (Lond.), 390: 308 –311,
1997.
33. Kudo, N., Khochbin, S., Nishi, K., Kitano, K., Yanagida, M., Yoshida,
M., and Horinouchi, S. Molecular cloning and cell cycle-dependent ex-
pression of mammalian CRM1, a protein involved in nuclear export of
proteins. J. Biol. Chem., 272: 29742–29751, 1997.
34. Ren, M., Drivas, G., D’Eustachio, P., and Rush, M. G. Ran/TC4: a
small nuclear GTP-binding protein that regulates DNA synthesis. J. Cell
Biol., 120: 313–323, 1993.
35. Zhang, C., Hughes, M., and Clarke, P. R. Ran-GTP stabilizes micro-
tubule asters and inhibits nuclear assembly in Xenopus egg extracts.
J. Cell Sci., 112: 2453–2461, 1999.
36. Azuma, Y., Hachiya, T., and Nishimoto, T. Inhibition by anti-RCC1
monoclonal antibodies of RCC1-stimulated guanine nucleotide exchange
on Ran GTPase. J. Biochem. (Tokyo), 122: 1133–1138, 1997.
37. Pu, R. T., and Dasso, M. The balance of RanBP1 and RCC1 is critical
for nuclear assembly and nuclear transport. Mol. Biol. Cell, 10: 1955–
1970, 1997.
38. Demeter, J., Morphew, M., and Sazer, S. A mutation in the RCC1-
related protein pim1 results in nuclear envelope fragmentation in fission
yeast. Proc. Natl. Acad. Sci. USA, 92: 1436 –1440, 1995.
39. He, X., Hayashi, N., Walcott, N. G., Azuma, Y., Patterson, T. E.,
Bischoff, F. R., Nishimoto, T., and Sazer, S. The identification of cDNAs
that affect the mitosis-to-interphase transition in Schizosaccharomyces
pombe, including sbp1, which encodes a spi1p-GTP-binding protein.
Genetics, 148: 645– 656, 1998.
40. Matynia, A., Dimitrov, K., Mueller, U., He, X., and Sazer, S. Perturba-
tions in the spi1p GTPase cycle of Schizosaccharomyces pombe through
its GTPase-activating protein and guanine nucleotide exchange factor
components result in similar phenotypic consequences. Mol. Cell. Biol.,
16: 6352– 6362, 1996.
41. Nicolas, F., Zhang, C., Hughes, M., Goldberg, M., Watton, S., and
Clarke, P. Xenopus Ran-binding protein 1: molecular interactions and
effects on nuclear assembly in Xenopus egg extracts. J. Cell Sci., 110:
3019 –3330, 1997.
42. Meraldi, P., Lukas, J., Fry, A. M., Bartek, J., and Nigg, E. A. Centro-
some duplication in mammalian somatic cells requires E2F and Cdk2-
cyclin A. Nat. Cell Biol., 1: 88 –93, 1999.
43. Matunis, M. J., Coutavas, E., and Blobel, G. A novel ubiquitin-like
modification modulates the partitioning of the Ran-GTPase-activating
protein RanGAP between the cytosol and the nuclear pore complex.
J. Cell Biol., 135: 1457–1470, 1996.
44. Balczon, R., Varden, C. E., and Schroer, T. A. Role for microtubules in
centrosome doubling in Chinese hamster ovary cells. Cell Motil. Cytoskel-
eton, 42: 60 –72, 1999.
45. Ohtsubo, M., Yoshida, T., Seino, H., Nishitani, H., Clark, K. L.,
Sprague, G. F., Jr., Frasch, M., and Nishimoto, T. Mutation of the hamster
cell cycle gene RCC1 is complemented by the homologous genes of
Drosophila and S. cerevisiae. EMBO J., 10: 1265–1273, 1991.