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Cell Growth & Differentiation Vol. 11, 455-465, August 2000
© 2000 American Association for Cancer Research

Regulated Ran-binding Protein 1 Activity Is Required for Organization and Function of the Mitotic Spindle in Mammalian Cells in Vivo1

Giulia Guarguaglini2, Luigina Renzi3, Filippo D’Ottavio, Barbara Di Fiore, Martina Casenghi2, 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
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
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
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
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 progression, chromosome stability, nuclear organization, and 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 yielding 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 (78) 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 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.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
The RanBP1 Protein Is Present in a Limited Cell Cycle Window in Mammalian Cells.
Before addressing the functional 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 peptides (Fig. 1ACitation ). 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. 1BCitation 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. 1BCitation ). 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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  		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 µg 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 µg/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.

 
We next examined the RanBP1 protein in cell cultures synchronized by serum starvation/restimulation using indirect IF. Consistent with the immunoblotting results, the RanBP1 signal was barely detectable in early G1 cells (Fig. 2AaCitation Citation ) yet was effectively visualized in cells beyond the G1-S transition (Fig. 2ACitation , b). In metaphase and anaphase cells, RanBP1 immunostaining reached the highest intensity and was confined to the mitotic cytoplasm (Fig. 2ACitation , c and d). However, the signal abruptly declined during mitotic exit and became barely detectable in late telophase (compare Fig. 2ACitation , e with Fig. 2ACitation , 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. 2BaCitation Citation and b) and a massive decrease in RanBP1 levels in late telophase, concomitant with chromatin decondensation; for example, the arrowed nuclei in Fig. 2BCitation , 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. 1BCitation ).



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  		Fig. 2. Immunolocalization of RanBP1 during the cell cycle. A, NIH/3T3 cell cultures stimulated to cycle were examined at various 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 gradually lost. Cell spreads were incubated with anti-RanBP1 antibody followed by FITC-conjugated secondary antibody and counterstained with DAPI. Bar, 10 µm. 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 metaphase is shown in column a (20 min after release from NOC arrest), 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 µm. 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 control cultures (c, -LMB) but becomes nuclear within 2 h of LMB addition (d, +LMB). Bar, 10 µm.

 
In S and G2 phases, in which RanBP1 is abundantly expressed, a predominant cytoplasmic distribution was observed, yet nuclear signals were also apparent (see Fig. 2AbCitation Citation ). 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. 2CCitation , compare b with a). Furthermore, we observed nuclear accumulation of the endogenous RanBP1 in cell cultures exposed to LMB (Fig. 2CCitation , 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. 3aCitation ), 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. 3bCitation ). Ran was massively redistributed to the cytoplasm during mitotic progression (Fig. 3c and d)Citation , with an increased intensity associated with the mitotic spindle in metaphase (Fig. 3cCitation ). In contrast, RCC1 immunostaining coincided with chromatin at all interphase stages (examples of S-phase cells are shown in Fig. 3eCitation ) and throughout mitotic progression, including the moment of chromosome alignment in metaphase (Fig. 3gCitation ). Chromosomes were selectively stained by the RCC1 antibody in methanol-fixed cells (Fig. 3Citation , 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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  		Fig. 3. Ran is redistributed to the cytoplasm, whereas RCC1 colocalizes with chromatin during mitosis. Cells were synchronized by serum starvation/restimulation as described in the Fig. 2Citation A 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 harvested 24 h after cell cycle entry. Cell spreads were fixed in paraformaldehyde and incubated with anti-Ran antibody (a-d) or fixed with methanol and incubated with anti-RCC1 antibody (e-h). FITC-conjugated secondary antibody was used, and DNA was counterstained with DAPI. Bar, 10 µm.

 
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 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 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. 2CCitation ) was used for comparison. GFP-expressing cells were examined 36–48 h after transfection. Data from eight independent experiments are summarized in Table 1Citation . 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. 4ACitation ), and nuclei with irregular edges and blobs of condensed chromatin (Fig. 4BCitation ). 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)Citation and increased with the dose of transfected construct (Fig. 4)Citation . 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. 4ACitation for example), suggesting that they derived from telophase cells that failed to decondense chromatin.


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  		Table 1 Cellular abnormalities in cultures overexpressing RanBP1

Cellular phenotypes were scored among GFP-expressing cells in cultures transfected with pX vector, pRanBP1 or pNES (6 µg of each construct). Ps were calculated from the single comparison of either pRanBP1- or pNES-transfected cultures, with control cultures using the {chi}2 test.

 


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  		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 µg of each). Recorded defects include (A) pyknotic nuclei with homogeneously hypercondensed chromatin and (B) abnormal nuclei with irregular edges and blobs of chromatin. Right, defects 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 percentage of observed defects using 4 or 6 µg of each construct.

 
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)Citation . To characterize mitotic abnormalities in more detail, transfection experiments were repeated, and cells were immunostained using an anti-{alpha}-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. 5aCitation ) and multipolar (Fig. 5b and c)Citation 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. 5dCitation ). 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. 5eCitation ). The frequency of abnormal mitoses was highly significant (Table 1)Citation , and their phenotype was indistinguishable in cultures overexpressing either wild-type or export-defective RanBP1.



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  		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-{alpha}-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.

 
Microinjection of Anti-RanBP1 Antibody in Living Mitotic 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 expressed. To further ascertain that anti-RanBP1 antibody did not cross-react with conserved RBDs present in other cellular proteins, we carried out preliminary Western blot experiments with purified GST-fusion proteins expressing single RBDs (RBD1–4) from the RanBP2 protein: no cross-reactivity was detected (data not shown). Microinjections were carried out during mitotic substages as indicated in Table 2Citation . 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)Citation : 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 2Citation . 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. 6ACitation ), 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. 6ACitation , 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. 6AbCitation Citation ) or ultimately failed (Fig. 6AcCitation Citation ). Abnormal mitotic products were particularly frequent among cells that had been injected with anti-RanBP1 during metaphase and anaphase (Table 3Citation ). Examples of defects of increasing severity are shown in Fig. 6BCitation : micronuclei generated by chromosome loss were often present in late telophase/early G1 cells (Fig. 6BaCitation Citation ); most anti-RanBP1-injected cells showed chromatin bridges (Fig. 6BbCitation Citation and c); and, in extreme cases, daughter nuclei remained partly or largely connected, and their separation was prevented (Fig. 6Citation B, d and e).


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

 


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  		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 division. Anti-RanBP1-microinjected cells, despite forming a normal cleavage furrow, show partly (b) or largely (c) incomplete nuclear division. Bar, 10 µm. 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 between daughter cells, with increasing amounts of trapped chromatin in the actin contractile ring (arrows); d, chromatid sets did not migrate at anaphase; e, chromosomes did not go through metaphase or anaphase, and chromatin blobs were visualized at telophase. Bar, 10 µm.

 

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  		Table 3 Defects in mitotic cells microinjected with anti-RanBP1 antibody

 
The defects shown in Fig. 6Citation 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 {alpha}-tubulin, the anti-RanBP1 antibody does not appear to alter the spindle morphology in microinjected cells (Fig. 7aCitation ). 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-{alpha}-tubulin antibody to visualize microtubules. Control metaphase cells injected with PBS alone showed a disassembled spindle and a diffuse {alpha}-tubulin staining throughout the mitotic cytoplasm within 10 min of NOC addition (Fig. 7bCitation ), indicating that microtubules were completely depolymerized. In contrast, when anti-RanBP1-microinjected metaphase cells were exposed to NOC (Fig. 7cCitation ), 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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  		Fig. 7. Anti-RanBP1 microinjection stabilizes the mitotic spindle. Metaphase cells were injected with PBS or with anti-RanBP1 antibody and exposed to NOC. After 10 min, cells were fixed and stained with anti-{alpha}-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 exposed to NOC; c, polymerized spindle in an anti-RanBP1-injected metaphase cell exposed to NOC. Bar, 10 µm.

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
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 division, including the structural organization of the spindle, control of microtubule dynamics in metaphase and anaphase, 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 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. 5Citation 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.


   Materials and Methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Cell Cultures and Cell Cycle Analysis.
Murine NIH/3T3 embryo fibroblasts (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 µg/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 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-{alpha}-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-{alpha}-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 {alpha}-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 {alpha}-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-{alpha}-tubulin antibody to visualize the spindle and FITC-conjugated secondary antibody to reveal anti-RanBP1.


   Acknowledgments
 
We are grateful to Rosamaria Mangiacasale for help with flow cytometry 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.


   Footnotes
 
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 indicate 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 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. Back

2 Present address: Department of Cell Biology, Max-Planck Institute of Biochemistry, Am Klopferspitz 18a, D-82152 Martinsried, Germany. Back

3 Present address: ENEA Centro Ricerche della Casaccia, Via Anguillarese 301, S. Maria di Galeria, 00060 Rome, Italy. Back

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 Back

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. Back

Received for publication 1/25/00. Revision received 4/17/00. Accepted for publication 6/ 1/00.


   References
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 Discussion
 Materials and Methods
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