Journal of Science 112, 2453-2461 (1999) 2453 Printed in Great Britain © The Company of Biologists Limited 1999 JCS0524

Ran-GTP stabilises asters and inhibits nuclear assembly in Xenopus egg extracts

Chuanmao Zhang1,2, Mike Hughes1 and Paul R. Clarke1,* 1Biomedical Research Centre, University of Dundee, Level 5, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland, UK 2Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing 100871, China *Author for correspondence (e-mail: [email protected])

Accepted 25 May; published on WWW 24 June 1999

SUMMARY

Ran is an abundant GTPase of the Ras superfamily that is nucleus and blocks chromatin decondensation. In contrast, highly conserved in eukaryotes. In interphase cells, Ran is Ran GDP does not stabilise or inhibit nuclear mainly nuclear and thought to be predominantly GTP- assembly. RanT24N and RanBP1, which oppose the bound, but it is also present in the cytoplasm, probably generation of Ran-GTP by RCC1, arrest nuclear growth GDP-bound. This asymmetric distribution plays an after disappearance of the aster. Ran associates with important role in directing nucleocytoplasmic transport. microtubule asters in egg extracts and with mitotic spindles Ran has also been implicated in control, including in somatic Xenopus cells, suggesting that it may affect the transition from to interphase when the microtubule stability directly. These results show that Ran compartmentalisation of the nucleus is established. Here, has a novel function in the control of microtubule stability we have examined the role of Ran in this transition using that is clearly distinct from nucleocytoplasmic transport. a cell-free system of Xenopus egg extracts supplemented The Ran GDP/GTP switch may play a role in co-ordinating with sperm heads that provides a model for microtubule changes in the structure of microtubules and the assembly aster formation and post-M phase nuclear assembly. Ran- of the nucleus associated with the transition from mitosis GTP, added as wild-type protein, a mutant defective in to interphase. GTPase activity (Q69L), or generated by addition of the specific nucleotide exchange factor RCC1, stabilises large microtubule asters nucleated at the sperm , Key words: Ran, Microtubule, Centrosome, NuMA, Cell cycle prevents the redistribution of NuMA from the aster to the control

INTRODUCTION of Ran-GTP in the cytoplasm of interphase cells (Görlich and Mattaj, 1996; Görlich et al., 1996). This asymmetric Ran, a member of the Ras small GTPase superfamily, is highly distribution is thought to be crucial for the directionality of conserved in eukaryotes from yeast to humans (Rush et al., transport through the nuclear pore (Görlich et al., 1996; 1996). Like other GTPases, Ran exists in GTP and GDP bound Izaurralde et al., 1997), with RanGTP promoting the assembly states that interact differently with regulators and effectors. The of nuclear export complexes in the nucleus which are major nucleotide exchange factor for Ran, RCC1, is localised disassembled by hydrolysis of GTP on Ran in the cytoplasm. in the nucleus where it is thought to generate Ran-GTP Conversely, the assembly of nuclear import complexes in the (Bischoff and Ponstingl, 1991a; Klebe et al., 1995; Ohtsubo et cytoplasm is promoted by RanGDP and their disassembly after al., 1989). The intrinsic GTPase activity of Ran is very low, but transport into the nucleus is caused by the high concentration is stimulated by the interaction of a GTPase-activating protein of RanGTP there. (RanGAP1) located at the cytoplasmic face of the nuclear pore The Ran system may also play roles during the cell division (Bischoff et al., 1994; Mahajan et al., 1997; Matunis et al., cycle, being implicated in changes in nuclear architecture, 1996). Ran binding protein 1 (RanBP1) is also predominantly DNA replication and mitosis (reviewed by Sazer, 1996). cytoplasmic (Richards et al., 1996) and interacts specifically Expression of dominant Ran mutants in cultured cells arrests with the GTP-bound form of Ran (Coutavas et al., 1993), cell cycle progression (Ren et al., 1993, 1994), inhibiting entry stimulating GTPase activation by RanGAP1 (Bischoff et al., into mitosis or progression into S-phase. In a hamster fibroblast 1995). The compartmentalised localisation of these regulators cell line that has a temperature sensitive mutation in the RCC1 has been proposed to generate a high concentration of Ran gene (tsBN2), cells in S-phase undergo GTP in the nucleus, and a low or very localised concentration condensation and premature activation of mitotic Cdc2 protein 2454 C. Zhang and others kinase activity at the restrictive temperature. Loss of RCC1 Nuclear assembly and immunofluorescence microscopy also causes the formation of multiple micronuclei and cells fail Xenopus egg extracts (LSS) were prepared as described previously to pass the G1/S transition (Nishitani et al., 1991). In the fission (Hutchison, 1994), frozen and stored in aliquots in liquid nitrogen. yeast, Schizosaccharomyces pombe, a temperature sensitive After thawing, extracts were pre-incubated with added proteins for 60 mutation of the RCC1 homologue Dcd1/Pim1 produces a minutes on ice prior to addition of sperm heads and an ATP defect in exit from mitosis, resulting in condensed chromatin regenerating system (Hutchison, 1994). Nuclear assembly and microtubule aster formation was initiated by the addition of and cell cycle arrest, although Cdc2/cyclin B activity declines µ (Sazer and Nurse, 1994). Intriguingly, there is fragmentation demembranated Xenopus sperm heads (1,000/ l) followed by incubation at 23¡C. To examine nuclear morphology, samples of the nuclear envelope at the restrictive temperature, even removed at various times during the incubation were fixed and stained though the nuclear envelope normally remains intact during on a slide with DAPI (Hutchison, 1994). For immunofluorescence mitosis in S. pombe. This suggests that Dcd1/Pim1 is required labelling, samples fixed with the EGS (Hutchison, 1994) for 30 for the re-establishment of nuclear organisation following minutes at 37°C. The fixed nuclei were then recovered by centrifuging mitosis (Demeter et al., 1995). Nevertheless, it has been onto coverslips through a cushion of 30% glycerol. To monitor DNA difficult to distinguish putative distinct functions for the Ran replication, 4 µM biotin-16-dUTP was added to the extracts at the start system from possible secondary effects due to disruption of of the incubation and incorporated biotin was detected with nucleocytoplasmic transport. strepavidin conjugated to Texas Red. α-tubulin and NuMA were A useful cell-free system to dissect the roles of Ran and its detected using anti-tubulin antibody (Sigma) and anti-NuMA interacting proteins is provided by concentrated cell-free antibody (Merdes et al., 1996) followed by FITC- and Texas Red- conjugated secondary antibodies (DAKO, Jackson ImmunoResearch extracts derived from Xenopus laevis eggs. Demembranated Laboratories), respectively (Zhang et al., 1996). Ran was detected Xenopus sperm heads added to interphase extracts assemble with a mouse monoclonal antibody (Transduction Laboratories) raised into functional nuclei that undergo DNA replication against residues 7-171 of human Ran, followed by anti-mouse IgG (Hutchison, 1994). This system provides an in vitro model for secondary antibody coupled to FITC (DAKO). Images were captured the formation of the male pronucleus following fertilisation of on a Zeiss Axioskop microscope using a cooled CCD camera and the egg and for post-M phase nuclear assembly in general. processed using Improvision Openlab and Adobe Photoshop software. Previously, we have shown that Ran-GDP is required in this system for the assembly of nuclei competant for DNA Western blotting replication, whereas Ran-GTP is unable to substitute, possibly Proteins were separated on 10% polyacrylamide gels and transferred indicating a dominant inhibitory effect of Ran-GTP (Hughes to nitrocellulose. Blots were blocked with 5% (w/v) milk powder in Tween-Tris buffered saline (TTBS). Ran proteins were detected using et al., 1998). Perturbing the Ran GDP/GTP cycle by adding a the same monoclonal antibody used for immunofluorescence (1:500 mutant of Ran that is defective in GTPase activity (RanQ69L), dilution in TTBS, overnight at 4¡C) followed by HRP-coupled anti- another mutant does not bind GTP and inhibits RCC1 mouse IgG (Bio-Rad) (1:2000 dilution in TTBS, 1 hour at room (RanT24N), or excess Ran BP1, all inhibit nuclear lamina temperature) and development by ECL (Amersham). formation, chromatin decondensation and DNA replication, although there are some differences in the gross structure of Xenopus cell culture and indirect fluorescence the chromatin produced (Dasso et al., 1994; Hughes et al., microscopy 1998; Kornbluth et al., 1994; Nicolás et al., 1997; Pu and XTC cells were cultured on coverslips in L-15 medium Leibovitz Dasso, 1997). (Sigma) plus 10% FCS and 25% H2O. The cells were fixed with 4% Here, we show that Ran-GTP, in contrast to RanGDP, paraformaldehyde in TBS for 20 minutes on ice and permeablised with 0.5% Triton X-100 also in TBS for 5 minutes. Indirect arrests pronuclear assembly from sperm chromatin with a fluorescent labelling was carried out by incubating with the first large, stable microtubule aster nucleated at the sperm antibody overnight at 4¡C followed by incubation with the FITC- centrosome and blocks chromatin decondensation. Addition conjugated second antibody for 60 minutes at room temperature. of RCC1 produces a similar effect, whereas RanT24N and Images were taken and processed as described above. RanBP1 do not stabilise the asters but arrest nuclear assembly at a later stage. Ran associates with microtubule asters in egg extracts and with the mitotic spindle in cultured cells. This RESULTS work demonstrates that Ran has a role in the control of microtubule stability that is clearly distinct from Ran-GTP but not Ran-GDP stabilises microtubule nucleocytoplasmic transport and suggests that the Ran- asters and inhibits chromatin decondensation GDP/GTP switch may play an important role in co-ordinating To investigate the role of the Ran GTPase system in nuclear the transition from mitosis to interphase. assembly, we utilised Xenopus egg extracts supplemented with demembranated Xenopus sperm heads as a model cell-free system. We used interphase extracts containing cyclohexamide MATERIALS AND METHODS which inhibits synthesis of M-phase cyclins and other proteins, permitting passage through the first interphase but preventing Recombinant proteins subsequent re-entry into M-phase. As shown in Fig. 1, when sperm heads were added to these extracts, microtubules were Recominant human Ran proteins were produced in E. coli, purified and loaded with nucleotides as described previously (Hughes et al., nucleated at a centrosome formed around the sperm 1998). RanT24N was loaded with GDP, RanQ69L with GTP and wild- located at end of each sperm head where the flagellum was type Ran with the nucleotide indicated. Xenopus RanBP1 was present (Félix et al., 1994; Stearns and Kirschner, 1994). By produced as described by Nicolás et al. (1997). RCC1 (Klebe et al., 20 minutes dense asters of elongated microtubules had formed. 1993) was a kind gift from A. Wittinghofer (Dortmund). By 40 minutes, the asters diminished and the condensed Ran controls microtubule asters and nuclear assembly 2455 chromatin rounded up (Fig. 1A). Subsequently, the chromatin When the concentration of Ran-GDP in the extracts decondensed further, a nuclear envelope and lamina were (estimated at 1-2 µM; Clarke et al., 1995) was increased by formed, and DNA replication was initiated at 40-60 minutes addition of 10 µM recombinant wild-type Ran preloaded with (data not shown). In this assay, only microtubules specifically GDP (Fig. 1B), the time course of microtubule aster assembly nucleated by and recovered together with sperm and disassembly was similar to incubations lacking exogenous heads by centrifuging onto coverslips were observed, not the Ran (Fig. 1A). RanT24N, which fails to bind GTP, only had a randomly nucleated microtubules that form in interphase egg small effect on microtubule stability, slightly delaying aster extracts. disassembly (Fig. 1C, 40 minutes). Addition of RanQ69L,

Fig. 1. Time course of microtubule aster formation and sperm chromatin decondensation in Xenopus egg extracts. Extracts were pre-incubated on ice for 60 minutes with (a) buffer, (b) wild-type Ran-GDP, (c) RanT24N- GDP or (d) RanQ69L-GTP. Ran proteins were added at 10 µM. Demembranated sperm were then added and the extracts incubated for the times shown (in minutes) before fixation, recovery of nuclei by centrifugation onto coverslips, staining for DNA with DAPI and detection of α-tubulin by indirect immunofluorescence. 2456 C. Zhang and others

Fig. 2. Ran-GTP but not Ran-GDP blocks chromatin decondensation, stabilises microtubule asters and inhibits DNA replication. Egg extracts, supplemented with biotin-dUTP and 10 µM Ran proteins preloaded with GTP or GDP, were incubated for 100 minutes after addition of sperm heads. Samples were fixed, spun onto coverslips, processed for indirect immunofluorescence to detect tubulin, streptavidin to detect incorporation of biotin and assess DNA replication, and stained with DAPI to detect DNA. which is deficient in GTPase activity and is therefore locked microtubule asters and irregularly condensed chromatin (Fig. in a GTP-bound state, also had no clear effect on microtubule 3B), consistent with the effects of adding Ran-GTP. nucleation or the formation of asters. However, this mutant dramatically stabilised the asters during prolonged incubation. In the presence of RanQ69L, the chromatin remained rather elongated and irregularly condensed (Fig. 1D), whereas in extracts supplemented with RanT24N, chromatin become rounded and evenly condensed (Fig. 1C). Although we have previously shown that both of these mutants prevent lamina assembly and DNA replication in this system (Hughes et al., 1998; see also Fig. 5), these results demonstrate that RanQ69L and RanT24N disrupt nuclear assembly at different stages. Addition of 10 µM wild-type RanGTP also produced large microtubule asters that were stabilised to incubation, very similar to the effect of adding RanQ69L. The chromatin remained elongated and irregularly condensed, indicating that nuclear assembly was inhibited prior to rounding up of the chromatin. The effect of RanQ69L is therefore likely to be due to stabilisation of the GTP-bound state of Ran rather than any other effect of the mutation. No DNA replication occurred in extracts supplemented with Ran-GTP, shown by the lack of incorporation of biotin-dUTP. In contrast, addition of 10 µM exogenous Ran-GDP permitted nuclear assembly and subsequent DNA replication to occur (Fig. 2). We also perturbed the GDP/GTP state of endogenous Ran without changing the overall concentration by supplementing extracts with regulatory proteins. RCC1, a specific nucleotide exchange factor, generates Ran-GTP from Ran-GDP (Bischoff and Ponstingl, 1991b; Klebe et al., 1995). In contrast, Ran BP1 binds Ran-GTP, opposes the activity of RCC1 and, together with RanGAP1, stimulates the hydrolysis of GTP bound to Ran (Bischoff et al., 1995). Increased concentrations of either RCC1 or RanBP1 inhibit the establishment of DNA replication in this system (Nicolás et al., 1997; Pu and Dasso, 1997). Nevertheless, we found clear differences in their effects on microtubule structure. RanBP1 did not prevent the formation Fig. 3. Effect of RanBP1 and RCC1 on microtubule stability. Egg of microtubule asters, or their subsequent disappearance, extracts were supplemented with (a) 10 µM RanBP1 or (b) 10 µM but produced small nuclei, similar to the effect of adding RCC1 and incubated for the times shown before processing for DNA RanT24N (Fig. 3A). In contrast, RCC1 produced large, stable staining and indirect immunofluorescence of tubulin as in Fig. 2. Ran controls microtubule asters and nuclear assembly 2457

RanQ69L affects NuMA localisation assembly by indirect immunofluorescence using a specific The molecular mechanisms controlling changes in microtubule antibody that only detects Ran on western blots of Xenopus stability and nuclear assembly at the exit from mitosis are egg extracts and somatic Xenopus XTC cells (Fig. 5). Ran is poorly characterised. One protein that has been implicated in absent from demembranated sperm, but rapidly binds to the post-mitotic nuclear formation is NuMA, a nuclear protein that condensed chromatin after addition to egg extract (Fig. 6A, associates with the mitotic apparatus (Compton and Cleveland, 5 minutes). After 20 minutes incubation and the assembly of 1993; Gaglio et al., 1995; Merdes and Cleveland, 1998; Merdes an aster, Ran was found associated with the microtubule et al., 1996). Detection of NuMA with specific antibodies array. At 100 minutes, following loss of the aster and nuclear showed a diffuse staining of the microtubule arrays, envelope formation, Ran was present in the nucleus (Fig. 6A). concentrated around the centrosomal area, as described When asters were stabilised by RanQ69L (Fig. 6B), Ran was previously (Merdes et al., 1996). Subsequently, NuMA became dispersed on the microtubule array and was also associated concentrated in the forming interphase nuclei (Fig. 4). Small with the chromatin. With RanT24N, when a small aster was rounded nuclei asembled in the presence of RanT24N also still apparent, Ran was present on the microtubules, but with contained NuMA, but in extracts containing RanQ69L, NuMA a more punctate staining than with RanQ69L. Ran also remained dispersed on the microtubule arrays. associated with the nucleus in the presence of RanT24N, particularly around the periphery, which probably Ran associates with microtubule arrays in Xenopus corresponds to the nuclear envelope (Fig. 6B). With further egg extracts incubation of extracts containing RanT24N, the aster We determined the localisation of Ran during nuclear disappeared, as did the extranuclear Ran staining, whereas

Fig. 4. Effect of Ran mutants on microtubule asters and NuMA localisation. RanQ69L arrests extracts with elongated and irregularly condensed chromatin, large microtubule asters and NuMA dispersed on the asters. RanT24N does not stabilise microtubules, but forms nuclei which are small and rounded, with highly condensed chromatin, which contain NuMA. Sperm heads were incubated in egg extracts supplemented with buffer, wild-type Ran-GDP, RanT24N-GDP or RanQ69L-GTP (each at 10 µM) for 100 minutes before processing as in Fig. 2. 2458 C. Zhang and others

Fig. 5. A monoclonal antibody raised against human Ran (residues 7- 171) was used to probe a western blot of total proteins from human HeLa cells (lane 1), Xenopus XTC cells (lane 2), nuclei assembled in Xenopus egg extracts (lane 3) and demembranated Xenopus sperm heads (lane 4). The amino acid sequence of Xenopus Ran is almost identical to human Ran in this core region (J. Avis and P. R. Clarke, unpublished). The antibody recognises a single polypeptide in XTC cells and assembled nuclei that co-migrates with human Ran at approximately 25 kDa, the predicted molecular mass of Ran. This polypeptide is absent from sperm heads prior to their addition to extracts. On the right are shown the migration positions of molecular mass markers (Bio-Rad): from top to bottom, 103,76, 49, 33, 28 and 19.9 kDa.

Fig. 6. Ran localises to microtubule asters in egg extracts. (a) Time course of nuclear assembly, showing Ran staining on the condensed chromatin shortly after addition to egg extracts (5 minutes), on microtubule asters (20 minutes) and in the assembled interphase nucleus (100 minutes). (b) Effect of recombinant Ran proteins on Ran localisation. Incubations were performed as in Fig. 3, except that they were carried out for 50 minutes before processing for indirect immunofluorescence of Ran and detection of DNA replication by biotin-dUTP incorporation. Note that the antibody used to detect Ran does not distinguish between the endogenous protein and the added recombinant proteins. Ran controls microtubule asters and nuclear assembly 2459

Ran remained on the asters formed in the presence of became rather more diffuse throughout the cell, with some RanQ69L (Fig. 1C, data not shown). concentration at the nuclear envelope. In , Ran was present throughout the cell, but a sub-population was localised The localisation of Ran during the cell cycle in specifically to the mitotic apparatus, particularly the Xenopus somatic cells polar/ microtubules and the spindle poles, but not We also examined the localisation of Ran during the cell cycle to the condensed . In , Ran was more in cultured somatic cells (Fig. 7). Previous experiments using concentrated towards the poles of the elongating spindle, but mammalian cultured cells have shown that Ran is dispersed was excluded by the chromosomes. In early , throughout the cell during mitosis and is not specifically probably before the nuclear envelope is completed, Ran localised to the mitotic chromosomes (Ren et al., 1993). Using showed a dispersed cytoplasmic staining, and was still clearly Xenopus laevis XTC cells, we characterised the localisation of excluded from the condensed chromatin. By late telophase, Ran in more detail during each stage of mitosis. These cells there was a dramatic relocalisation of most of the Ran to the are particularly useful for this purpose, since they do not round reassembled daughter nuclei (Fig. 7). up greatly during mitosis and they allow localisation to specific structures to be seen. In interphase XTC cells, we found that Ran was mainly nuclear, as expected, although there was some Microtubule nucleation,nucleation weak, dispersed cytoplasmic staining. In , the staining atvesicle centrosome binding

Microtubule elongation,elongation further vesicle binding

Ran-GTP

Microtubule shrinking, formationvesicle fusion of nuclear to form envelopecomplete envelope

Disappearance of aster, rounding up of chromatin

RanT24N RanBP1

Nucleocytoplasmic transport, chromatin decondensation, assembly of lamina and matrix, initiation of DNA replication

Fig. 8. Schematic diagram showing the effect of disrupting the Ran system on nuclear assembly from Xenopus sperm heads. A centrosome at one end of the condensed chromatin is formed around the sperm centrioles at one end of the sperm head following addition to Xenopus egg extract. The centrosome then nucleates a dense aster of microtubules which elongate. The microtubules nucleated by the centrosome subsequently shrink, a nuclear envelope is formed and chromatin rounds up. The nuclear lamina and nuclear matrix are then assembled and DNA replication is initiated. Ran-GTP, either exogenous wild-type or Q69L mutant, or generated by RCC1, stabilises long microtubules and blocks the rounding up and Fig. 7. Localisation of Ran during the cell cycle in Xenopus XTC decondensation of the chromatin. In contrast, RanT24N or RanBP1, cells. Cells were grown on coverslips, fixed and processed for which disrupt RCC1 activity, inhibit nuclear assembly after indirect immunofluorescence to detect Ran as in Materials and completion of the envelope and rounding up of the chromatin, but Methods. DNA was stained with DAPI. Selected cells in interphase prior to restarting nucleocytoplasmic transport, nuclear lamina and at specific stages of mitosis are shown. assembly and growth of the nucleus. 2460 C. Zhang and others

DISCUSSION egg extracts (Merdes and Cleveland, 1998), blocking its relocalisation or its function might affect microtubule stability. The Ran GTPase and its interacting proteins have been However, the precise role of NuMA in this process and implicated in a wide variety of cellular processes (Rush et al., its functional interaction with Ran will require further 1996; Sazer, 1996). Of these, only the role of Ran in directing investigation. nucleocytoplasmic transport during interphase has been well The localisation of Ran to the mitotic apparatus in established. In this study, we have used a cell-free system of metaphase cells suggests that Ran, like NuMA (Merdes et al., Xenopus egg extracts to examine the function of Ran during 1996), may also have a role in formation of the mitotic spindle, the assembly of pronuclei from demembranated Xenopus although we have not addressed that question here directly. sperm heads. By characterising changes in microtubule Although the majority of Ran molecules dispersed in the structure and the formation of the nuclear envelope during cytoplasm during mitosis are probably GDP-bound, it remains nuclear assembly, we have shown that disrupting the possible that the sub-population of Ran localised to the spindle RanGTPase cycle results in defects at two distinct points (Fig. is GTP-bound. Since RCC1 is localised mainly to the 8). Increased concentrations of Ran-GTP, produced by addition condensed chromosomes during metaphase (C. Zhang and P. of recombinant Ran proteins or generated in the extracts by R. Clarke, unpublished), a localised concentration of RanGTP RCC1, produce a large, stable microtubule aster nucleated could be generated near to the chromosomes and play a role in from the sperm centrosome and arrest nuclear assembly, stabilising the mitotic spindle. At present, we are unable to blocking chromatin decondensation. These effects are clearly distinguish the localisation of the GTP- and GDP-bound forms distinct from the well characterised role of Ran in of Ran and test this possibility directly. Recently, a novel, nucleocytoplasmic transport. centrosomal protein named RanBPM has been found to interact In contrast to Ran-GTP, excess RanGDP has no inhibitory preferentially with Ran-GTP in a two-hybrid system of co- effects on aster formation or nuclear assembly. This is consistent expression in yeast (Nakamura et al., 1998). Ran could with the endogenous Ran present in the extracts being therefore play a role in microtubule nucleation at the spindle predominantly GDP bound, as indicated by interactions with poles during mitosis, although our experiments did not reveal specific partner proteins (Hughes et al., 1998; Nicolás et al., any inhibitory effect of Ran-GTP (or Ran-GDP) on 1997). Disruption of the Ran GTPase cycle with RanT24N or centrosomal nucleation in egg extracts, unlike the effect on increased concentrations of RanBP1 does not stabilise microtubule nucleation in tubulin solution reported by microtubules but arrests nuclear growth at a later stage after Nakamura et al. (1998). rounding up of the chromatin (Fig. 8). Together with previous At present, the molecular mechanism by which Ran results (Dasso et al., 1992, 1994; Hughes et al., 1998; Nicolás influences microtubule stability is unknown. Ran could et al., 1997; Pu and Dasso, 1997), this indicates that the mediate this process through specific interacting proteins (Avis generation of RanGTP by RCC1 specifically inside the nucleus and Clarke, 1996). The functions of such putative effectors is required for the re-establishment of nucleocytoplasmic could be restricted to mitosis or might also be involved in transport, the assembly of the nuclear lamina and the initiation nucleocytoplasmic transport during interphase. An alternative of DNA replication. Thus, there may be a switch in the model is that Ran co-ordinates changes in microtubule nucleotide state of Ran from GDP to GTP specifically inside the structure and the assembly of the interphase nucleus through a nucleus at exit from mitosis. In somatic cells, where we show single mechanism that is controlled by the GDP/GTP state of that there is a dramatic redistribution of most Ran molecules Ran. For instance, both microtubule dynamics and nuclear to daughter nuclei at late telophase when nuclear assembly are known to be regulated by protein phosphorylation compartmentalisation is re-established, this would correspond to (Pfaller et al., 1991; Verde et al., 1990). However, in the case a change in the predominant form of Ran, although some Ran- of control of these processes by Ran, it is unlikely that GDP would remain in the cytoplasm. In Xenopus egg extracts, stabilisation of mitotic cyclin-dependent protein kinase activity a large pool of Ran-GDP would remain outside the nuclei. is responsible, since the interphase extracts that we have used Since Ran associates with microtubule asters in Xenopus egg are released from the M-phase (CSF) arrested state of the extracts, it may play a direct role in controlling microtubule unfertilized eggs during preparation, resulting in degradation dynamics. The localisation of Ran to microtubule asters shows of the essential cyclin subunits (Murray, 1991). Furthermore, similarities with NuMA, a protein which associates with extracts containing Ran-GTP do not have high histone H1 spindle pole and microtubules during mitosis and relocalises to kinase activity, a measure of mitotic cyclin A- and cyclin B- the nucleus in interphase (Compton and Cleveland, 1993; dependent protein kinases (data not shown). Rather, Ran-GTP Gaglio et al., 1995; Merdes et al., 1996). Interestingly, may arrest the extracts in a state resembling the transition truncated NuMA mutants produce multiple micronuclei in at the end of mitosis approximately corresponding to the hamster cells. This phenotype is strikingly similar to that anaphase/telophase boundary when microtubules return to produced by the loss of RCC1 at the restrictive temperature in interphase dynamics and the nuclear envelope reforms. hamster tsBN2 cells, conditions where the localisation of In summary, we have shown that disrupting the RanGTPase NuMA to the micronuclei is defective. Remarkably, cycle results in defects in nuclear assembly at two distinct overexpression of NuMA can rescue the micronucleation points. Whereas Ran-GDP is required for nuclear assembly, phenotype produced by loss of RCC1 (Compton and excess RanGTP stabilises large microtubule asters nucleated Cleveland, 1993). Our results would support the involvement by centrosomes and arrests nuclear assembly at an early stage. of Ran in the relocalisation of NuMA from the microtubule Inhibition of the generation of RanGTP blocks nuclear growth aster to the reassembled nucleus. Although NuMA is not and initiation of DNA replication probably after completion of required for assembly of interphase nuclei in Xenopus the nuclear envelope. These results demonstrate novel effects Ran controls microtubule asters and nuclear assembly 2461 of modulating the GDP/GTP state of Ran that are distinct from is essential for transport into and out of the nucleus. EMBO J. 16, 6535- its interphase function in nucleocytoplasmic transport. They 6547. also indicate that the Ran GDP/GTP switch plays a role in Klebe, C., Nishimoto, T. and Wittinghofer, A. (1993). Functional expression in Escherichia coli of the mitotic regulator proteins p24ran and p45rcc1 and coordinating changes in microtubule structure and nuclear fluorescence measurements of their interaction. Biochemistry 32, 11923- assembly at the transition from M-phase to interphase. 11928. Klebe, C., Prinz, H., Wittinghofer, A. and Goody, R. S. (1995). The kinetic We are grateful to V. Allan, A. Merdes and A. Wittinghofer for gifts mechanism of Ran-nucleotide exchange catalyzed by RCC1. Biochemistry of anti-tubulin antibody, anti-NuMA antibody and RCC1 protein, 34, 12543-12552. respectively. RanBP1 was prepared by F. J. Nicolás in our laboratory. Kornbluth, S., Dasso, M. and Newport, J. (1994). Evidence for a dual role This work was supported by the Biotechnology and Biological Sciences for TC4 protein in regulating nuclear structure and cell cycle progression. J. Cell Biol. 125, 705-719. Research Council and The Cancer Research Campaign (CRC). Mahajan, R., Delphin, C., Guan, T., Gerace, L. and Melchior, F. (1997). A small ubiquitin-related polypeptide involved in targetting RanGAP1 to REFEENCES nuclear pore complex protein RanBP2. Cell 88, 97-107. Matunis, M. J., Coutavas, E. and Blobel, G. (1996). A novel ubiquitin-like Avis, J. A. and Clarke, P. R. (1996). Ran, a GTPase involved in nuclear modification modulates the partitioning of the Ran-GTPase-activating processes: its regulators and effectors. J. Cell Sci. 109, 2423-2427. protein RanGAP1 between the cytosol and the nuclear pore complex. J. Cell Bischoff, F. R. and Ponstingl, H. (1991a). Catalysis of guanine nucleotide Biol. 135, 1457-1470. exchange on Ran by the mitotic regulator RCC1. Nature 354, 80-82. Merdes, A., Ramyar, K., Vechio, J. D. and Cleveland, D. W. (1996). A Bischoff, F. R. and Ponstingl, H. (1991b). Mitotic regulator protein RCC1 is complex of NuMA and cytoplasmic is essential for mitotic spindle complexed with a nuclear ras-related polypeptide. Proc. Nat. Acad. Sci. USA assembly. Cell 87, 447-458. 88, 10830-10834. Merdes, A. and Cleveland, D. W. (1998). The role of NuMA in the interphase Bischoff, F. R., Klebe, C., Kretschmer, J., Wittinghofer, A. and Ponstingl, nucleus. J. Cell Sci. 111, 71-79. H. (1994). RanGAP1 induces GTPase activity of nuclear Ras-related Ran. Murray, A. W. (1991). Cell cycle extracts. In Xenopus laevis: Practical uses Proc. Nat. Acad. Sci. USA 91, 2587-2591. in Cell and Molecular Biology, vol. 36 (ed. B. J. Kay and H. B. Peng), pp. Bischoff, F. R., Krebber, H., Smirnova, E., Dong, W. and Ponstingl, H. 581-605. San Diego: Academic Press. (1995). Co-activation of RanGTPase and inhibition of GTP dissociation by Nakamura, M., Masuda, H., Horii, J., Kuma, K., Yokoyama, N., Ohba, T., Ran-GTP binding protein RanBP1. EMBO J. 14, 705-715. Nishitani, H., Miyata, T., Tanaka, M. and Nishimoto, T. (1998). When Clarke, P. R., Klebe, C., Wittinghofer, A. and Karsenti, E. (1995). overexpressed, a novel centrosomal protein, RanBPM, causes ectopic Regulation of Cdc2/cyclin B activation by Ran, a Ras-related GTPase. J. microtubule nucleation similar to γ-tubulin. J. Cell Biol. 143, 1041-1052. Cell Sci. 108, 1217-1225. Nicolás, F. J., Zhang, C., Hughes, M., Goldberg, M. W., Watton, S. J. and Compton, D. A. and Cleveland, D. W. (1993). NuMA is required for the Clarke, P. R. (1997). Xenopus Ran-binding protein 1: molecular normal completion of mitosis. J. Cell Biol. 120, 947-957. interactions and effects on nuclear assembly in Xenopus egg extracts. J. Cell Coutavas, E., Ren, M., Oppenheim, J. D., D’Eustachio, P. and Rush, M. Sci. 110, 3019-3030. G. (1993). Characterization of proteins that interact with the cell-cycle Nishitani, H., Ohtsubo, M., Yamashita, K., Iida, H., Pines, J., Yasudo, H., regulatory protein Ran/TC4. Nature 366, 585-7. Shibata, Y., Hunter, T. and Nishimoto, T. (1991). Loss of RCC1, a nuclear Dasso, M., Nishitani, H., Kornbluth, S., Nishimoto, T. and Newport, J. W. DNA-binding protein, uncouples the completion of DNA replication from (1992). RCC1, a regulator of mitosis, is essential for DNA replication. Mol. the activation of cdc2 protein kinase and mitosis. EMBO J. 10, 1555-1564. Cell. Biol. 12, 3337-3345. Ohtsubo, M., Okazaki, H. and Nishimoto, T. (1989). The RCC1 protein, a Dasso, M., Seki, T., Azuma, Y., Ohba, T. and Nishimoto, T. (1994). A regulator for the onset of chromosome condensation locates in the nucleus mutant form of the Ran/TC4 protein disrupts nuclear function in Xenopus and binds to DNA. J. Cell Biol. 109, 1389-1397. laevis egg extracts by inhibiting the RCC1 protein, a regulator of Pu, R. T. and Dasso, M. (1997). The balance of RanBP1 and RCC1 is critical chromosome condensation. EMBO J. 13, 5732-5744. for nuclear assembly and nuclear transport. Mol. Biol. Cell. 8, 1955-1970. Demeter, J., Morphew, M. and Sazer, S. (1995). A mutation in the RCC1- Ren, M., Drivas, G., D’Eustachio, P. and Rush, M. G. (1993). Ran/TC4: a related protein pim1 results in nuclear envelope fragmentation in fission small nuclear GTP-binding protein that regulates DNA synthesis. J. Cell yeast. Proc. Nat. Acad. Sci. USA 92, 1436-1440. Biol. 120, 313-23. Félix, M. A., Antony, C., Wright, M. and Maro, B. (1994). Centrosome Ren, M., Coutavas, E., D’Eustachio, P. and Rush, M. G. (1994). Effects of assembly in vitro: role of gamma-tubulin recruitment in Xenopus sperm mutant Ran/TC4 proteins on cell cycle progression. Mol. Cell Biol. 14, aster formation. J. Cell Biol. 124, 19-31. 4216-4224. Gaglio, T., Saredi, A. and Compton, D. A. (1995). NuMA is required for the Richards, S. A., Lounsbury, K. M., Carey, K. L. and Macara, I. G. (1996). organisation of microtubules into aster-like mitotic arrays. J. Cell Biol. 131, A nuclear export signal is essential for the cytosolic localization of the Ran 693-708. binding protein, RanBP1. J. Cell Biol. 134, 1157-1168. Görlich, D. and Mattaj, I. W. (1996). Nucleocytoplasmic transport. Science Rush, M. G., Drivas, G. and D’Eustachio, P. D. (1996). The small nuclear 271, 1513-1518. GTPase Ran: how much does it run? BioEssays 18, 103-112. Görlich, D., Panté, N., Kutay, U., Aebi, U. and Bischoff, F. R. (1996). Sazer, S. and Nurse, P. (1994). A fission yeast RCC1-related protein is Identification of different roles for RanGDP and RanGTP in nuclear protein required for the mitosis to interphase transition. EMBO J. 13, 606-615. import. EMBO J. 15, 5584-5594. Sazer, S. (1996). The search for the primary function of the Ran GTPase Hughes, M., Zhang, C., Avis, J. M., Hutchison, C. J. and Clarke, P. R. continues. Trends Cell Biol. 6, 81-85. (1998). The role of Ran GTPase in nuclear assembly and DNA replication: Stearns, T. and Kirschner, M. (1994). In vitro reconstitution of centrosome characterisation of the effects of Ran mutants. J. Cell Sci. 111, 3017- assembly and function: The central role of γ-tubulin. Cell 76, 623-637. 3026. Verde, F., Labbe, J. C., Doree, M. and Karsenti, E. (1990). Regulation of Hutchison, C. J. (1994). The use of cell-free extracts of Xenopus eggs for microtubule dynamics by cdc2 protein kinase in cell-free extracts of studying DNA replication in vitro. In The Cell Cycle: A Practical Approach Xenopus eggs. Nature 343, 233-238. (ed. P. Fantes and R. Brooks), pp. 177-195. Oxford: IRL Press. Zhang, C., Jenkins, H., Goldberg, M. W., Allen, T. D. and Hutchison, C. Izaurralde, E., Kutay, U., von Kobbe, C., Mattai, I. W. and Görlich, D. J. (1996). Nuclear lamina and nuclear matrix organisation in sperm (1997). The asymmetric distribution of the constituents of the Ran system pronuclei assembled in Xenopus egg extract. J. Cell Sci. 109, 2275-2286