- Email: [email protected]
Ran GTPase: a master regulator of nuclear structure and function during the eukaryotic cell division cycle?
366
Opinion
TRENDS in Cell Biology Vol.11 No.9 September 2001
Ran GTPase: a master regulator of nuclear structure and function during the eukaryotic cell division cycle?
case, when the mitotic apparatus is exposed to the cytoplasm, how are the spindle microtubules stabilized while those in the cytoplasm are dissolved? It seems that centrosomes seed microtubule nucleation, whereas chromatin provides a stabilizing effect on microtubule growth1. At the end of mitosis, the spindle is broken down and the NE is reassembled to establish the compartmentalization of each daughter nucleus. Recent advances in understanding the functions of Ran, a small GTPase of the Ras superfamily, have shown that it has a central role in the control of both mitotic spindle assembly2,3 and NE dynamics4,5 in addition to its role in directing nucleocytoplasmic transport during interphase6. Here, we discuss these findings and their implication that Ran plays a general role in nuclear functions throughout the cell division cycle. Molecular marker of the nucleus?
Paul R. Clarke and Chuanmao Zhang Ran is an abundant GTPase that is highly conserved in eukaryotic cells and has been implicated in many aspects of nuclear structure and function, especially determining the directionality of nucleocytoplasmic transport during interphase. However, cell-free systems have recently shown that Ran plays distinct roles in mitotic spindle assembly and nuclear envelope (NE) formation in vitro. During spindle assembly, Ran controls the formation of complexes with importins, the same effectors that control nucleocytoplasmic transport. Here, we review these advances and discuss a general model for Ran in the coordination of nuclear processes throughout the cell division cycle via common biochemical mechanisms.
Paul R. Clarke Biomedical Research Centre, Level 5, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK DD1 9SY. e-mail: [email protected], Chuanmao Zhang Biomedical Research Centre, Level 5, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK DD1 9SY, and the Dept Cell Biology and Genetics, Peking University, Beijing 100871, China.
Compartmentalization of the genetic material into a distinct nucleus bounded by the nuclear envelope (NE) is the hallmark of a eukaryotic cell. Communication between the cytoplasm and nucleus is established via nuclear pore complexes (NPCs), large multiprotein structures that perforate the double membrane, forming channels that permit the diffusion of small molecules but restrict the passage of larger molecules to those carrying specific targeting signals. The NE allows a distinctive biochemical environment to be established within the nucleus and controls access to chromatin, which has many advantages for the regulation of gene expression and DNA replication. However, during cell division, enormous changes in nuclear structure are required to allow segregation of duplicated genomes to the daughter cells. In organisms such as yeast, which have a closed mitosis, segregation of sister chromatids is accomplished on a microtubule apparatus contained within the NE. Nuclear division is then required to produce one nucleus in each daughter cell. By contrast, most animal cells have an open mitosis, during which the NE breaks down and the nuclear architecture is completely reorganized. Duplicated, condensed chromosomes are arranged in the middle of a large, bipolar spindle assembled from microtubules that are focused at each pole around a centrosome. In this http://tcb.trends.com
Like other GTPases, Ran exists in GTP- and GDPbound conformations that interact differently with effectors. Conversion between these forms and the assembly or disassembly of effector complexes requires the interaction of regulator proteins. The intrinsic GTPase activity of Ran is very low, but it is greatly stimulated by a GTPase-activating protein (RanGAP1) located in the cytoplasm. By contrast, RCC1, a guanine nucleotide exchange factor that generates Ran–GTP, is bound to chromatin and confined to the nucleus. Ran itself is mobile and is actively imported into the nucleus by a mechanism involving NTF-2. Together with the compartmentalization of its regulators, this is thought to produce a relatively high concentration of Ran–GTP in the nucleus. Indeed, Ran–GTP might act as a molecular marker that distinguishes the nucleoplasm from the cytoplasm of interphase cells. However, it is difficult to demonstrate this model directly because the local concentration of Ran in the GTP or GDP form cannot yet be measured in vivo or in cell-free systems. Most of our evidence for the functions of Ran comes from the perturbation of the system by changing the levels of regulators or by introducing mutant Ran proteins that are defective in the guanine-nucleotide cycle. … Ran directs nucleocytoplasmic transport…
A gradient in Ran–GTP concentrations across the NE has been proposed to be crucial for the directionality of transport of many macromolecules through the nuclear pores7–9 (Fig. 1). In nucleocytoplasmic transport, Ran functions by controlling the assembly and disassembly of complexes formed between transported cargoes and a family of Ran-binding proteins that act as receptors for targeting sequences on the cargo6. In the nucleus, association of Ran–GTP with receptors that are required for export (exportins), such as the leptomycin-B-sensitive factor Crm1, enhances their affinity for proteins containing leucine-rich nuclear export signals. Although the
0962-8924/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(01)02071-2
Opinion
TRENDS in Cell Biology Vol.11 No.9 September 2001
Nuclear import Importin-α/β
Importin-α/β NLS-protein
NLS-protein
Ran–GDP Nucleus
Ran–GDP
GTP RCC1 GDP
RanGAP RanBP1
Pi
Cytoplasm
Ran–GTP
Ran–GTP
NES-protein
NES-protein
Exportin/Crm1
Exportin/Crm1
Leptomycin B Nuclear export TRENDS in Cell Biology
Fig. 1. Ran directs nucleocytoplasmic transport. Ran shuttles across the nuclear envelope via the nuclear pores but is concentrated in the nucleus by active import. In the nucleus, a high concentration of Ran–GTP is generated by nucleotide exchange catalysed by RCC1 bound to chromatin. Ran–GTP causes the dissociation of imported complexes containing proteins with nuclear localization signals (NLS) by binding to importin β and ejecting the cargo. Conversely, binding of Ran–GTP to exportin/Crm1 promotes the assembly of export complexes containing proteins with nuclear export signals (NES). In the cytoplasm, Ran–GTP is converted into Ran–GDP and phosphate (Pi) by Ran GTPase, which is activated by RanGAP and RanBP1, and export complexes are dissociated. The importins and exportins are recycled by transport back across the pore (not shown). In addition to this basic mechanism, other members of the importin family mediate the transport of specific cargoes6.
mechanism of translocation across the nuclear pore is still not fully understood, the export complex is dissociated by the interaction of the cytoplasmic Ran–GTP-binding proteins RanBP1 and RanBP2, followed by GTP hydrolysis by Ran stimulated by RanGAP1 (Ref. 10). In the cytoplasm, cargoes for import form complexes with a heterodimeric receptor consisting of importins α (an adaptor that recognizes lysine-rich nuclear localization signals) and β. After translocation through the nuclear pore, import complexes are dissociated in the nucleoplasm by Ran–GTP, which binds importin β and ejects importin α and the cargo. For some imported proteins, importin β interacts directly with the cargo, whereas other importin family molecules play more specialized roles in the transport of specific proteins that have distinct signal sequences6. During translocation through the pore, transport cargoes may interact with nuclear pore complex proteins (nucleoporins) in transient interactions controlled by Ran11. …mitotic spindle assembly… …
Ran or its interacting proteins have been implicated in aspects of cell cycle progression for many years, but it was difficult to distinguish direct effects from secondary effects that undoubtedly occur when http://tcb.trends.com
367
nucleocytoplasmic transport is disrupted. In 1999, the breakthrough demonstration of a role in mitosis came from a number of groups using Xenopus egg extracts as a model cell-free system that is amenable to biochemical dissection12–16. Kalab et al.13 provided the first evidence that changing the balance of Ran–GTP and Ran–GDP within a mitotic egg extract disrupted spindle assembly, a process that occurs in the absence of nuclear compartmentalization and is therefore distinct from nucleocytoplasmic transport. Ran is present in Xenopus egg extracts at a high concentration (1–2 µM) and, judging by its interaction with specific binding proteins, is predominantly GDP bound17. When the concentration of Ran–GTP is increased by adding the exchange factor RCC1, or mutants (G19V, Q69L or L43E) that are deficient in GTPase activity and are thereby stabilized in the GTP-bound state, microtubule assembly is promoted throughout the extract, resulting in ectopic asters containing typical centrosome-associated proteins such as γ tubulin, NuMA and Xgrip109 (Refs 12–15). With incubation, these asters might form spindle-like structures in the absence of chromatin or centrioles, albeit smaller than proper spindles formed following the addition of sperm heads. More recently, three groups have shown that the effects of adding Ran–GTP to mitotic Xenopus egg extracts is mediated by the same factors that are involved in nuclear protein import, namely the importins (Fig. 2). Nachury et al.18 showed that, when importin β is removed from extracts by depletion with RanQ69L, widespread microtubule polymerization is induced that is suppressed by exogenous importin β. This procedure allowed them to establish an assay for aster-promoting activities, one of which was identified as NuMA, a microtubuleassociated protein that is essential for spindle assembly. In a related study, Wiese et al.19 showed that the induction of asters by RanL43E or a fragment of NuMa (NuMA tail II) could be suppressed by importin β. Gruss et al.20 showed that importin α also inhibits aster formation in mitotic extract and that this effect can be overcome by exogenous TPX2, a microtubule-associated protein that targets the motor protein Xklp2 to microtubules. Ran–GTP displaces TPX2 from importins α and β, releasing this aster-promoting activity from inhibition. Together, these results suggest that importins have roles in controlling spindle assembly in mitotic Xenopus egg extracts by binding to and suppressing the activities of factors such as NuMA and TPX2, which are present throughout the extracts3. It is perhaps surprising that proper spindle assembly can be restored by adding single components back to importin-suppressed extracts, but such components might displace additional factors involved in spindle assembly from inhibitory complexes; in fact, crucial factors might remain to be identified. One important implication of these findings is that importins act not
368
Opinion
TRENDS in Cell Biology Vol.11 No.9 September 2001
Ran– GDP
Pi
RanGAP RCC1
Ran– GTP
Importin Importin
APA
APA
TRENDS in Cell Biology
Fig. 2. Ran directs mitotic spindle assembly. Ran–GTP releases asterpromoting activities (APA) from inhibited complexes with importins to promote mitotic spindle assembly. The chromosomal localization of RCC1 might generate Ran–GTP locally, stabilizing microtubules in the vicinity of chromatin.
only as targeting molecules but also as regulators of the proteins to which they bind. Many spindle assembly factors, such as NuMA, are sequestered mainly in the nucleus of somatic cells during interphase, but inactivation by importins would ensure that their activity is suppressed in the cytoplasm, even in Xenopus early embryos, which contain large cytoplasmic stores of such factors. It is unclear at present whether importins play a role in targeting spindle assembly factors to the correct location. It will also be interesting to see whether importins have a general role in suppressing the activity of karyophilic proteins during interphase. These experiments show how artificially increasing Ran–GTP levels in mitotic Xenopus egg extracts causes widespread aster formation. How might Ran normally function in spindle assembly? Addition of RanT24N, a mutant defective in nucleotide binding that forms a stable inhibitory complex with RCC1, or of RanGAP and RanBP1, which reduce the free concentration of Ran–GTP, all prevent the proper alignment of chromosomes on the metaphase spindle (including spindles formed around DNA-coated beads in the absence of centrosomes)12–16,21. Importantly, high concentrations of Ran–GTP throughout the extract also disrupt proper spindle assembly, resulting in dissociation of microtubules from chromatin12,13. These results indicate that Ran–GTP must be generated for spindle formation, but also that it must be localized to permit proper alignment of the chromosomes on the spindle. Chromatin has a positional effect on spindle formation by decreasing the catastrophe rate and increasing the rescue frequency of dynamic microtubules, thereby promoting the elongation of spindle microtubules specifically towards the chromatin21,22. The effect of chromatin could be due to the generation of a microtubule-stabilizing factor by http://tcb.trends.com
an enzyme located on mitotic chromosomes23. If RCC1 is localized to chromatin during mitosis, as it is in interphase, then the local generation of Ran–GTP could account for the influence of chromatin12,13. If high concentrations of Ran–GTP are generated ectopically throughout the extract then a gradient of Ran–GTP away from the chromatin would be disrupted and spindle assembly would be disorganized, as is indeed the case12,13. However, it is not yet possible to demonstrate the existence of a gradient of Ran–GTP concentrations directly and so this model remains to be verified experimentally. A requirement for RCC1 to be localized to chromatin for the positional effect also needs to be tested. Indeed, the localization of RCC1 to mitotic chromatin is uncertain because of the apparently contradictory results of immunofluorescence with different cell fixation techniques24; this question might be resolved by localizing fusions of green fluorescent protein with RCC1 in live, unfixed cells. Ran-GTP not only stabilizes microtubule elongation, through release of importin-bound factors, but also regulates motor proteins involved in spindle dynamics22. How are these functions related to microtubule nucleation at centrosomes? Factors such as NuMA and TPX2 appear to be involved in both processes18–20, but is microtubule nucleation driven by local generation of Ran–GTP at the centrosome? It has been reported that depletion of RCC1 from mitotic Xenopus egg extracts blocks microtubule aster formation14, but others12,21 have found that RanT24N (which efficiently inhibits RCC1) does not suppress centrosomal microtubule nucleation, and there is no indication that RCC1 is localized to centrosomes. One possibility is that a high concentration of Ran–GTP generated at chromatin by RCC1 extends in a cloud to envelop the whole spindle, permitting the chromatin to influence the nucleating capacity of the centrosomes and promote microtubule growth21. In this case, microtubule nucleation might be promoted specifically at centrosomes by other mechanisms such as protein phosphorylation directed by localized kinases, but Ran–GTP would be permissive for this activity. Nevertheless, high concentrations of importin-β strongly suppress centrosomal aster formation19, whereas Ran mutants stabilized in the GTP-bound form not only increase the nucleation capacity of centrosomes21,22 but also induce the formation of centrosome-like structures that nucleate microtubules in the absence of centrioles or chromatin12–15. Perhaps the assembly of centrosomes and microtubule nucleation involve the concentration and stabilization of Ran–GTP by localized binding proteins – rather than de novo generation of Ran–GTP by localized nucleotide exchange factor activity. …and NE assembly!
At the end of mitosis, the mitotic spindle is disassembled and microtubules return to interphase
Opinion
TRENDS in Cell Biology Vol.11 No.9 September 2001
dynamics. The NE is re-assembled around chromatin, nuclear pore complexes are reformed, nucleocytoplasmic transport is restarted and the distinct environment of the nucleus is re-established. Interphase Xenopus egg extracts provide a model system for studying the assembly of the nucleus from sperm chromatin. First, the chromatin undergoes decondensation, a process that involves the exchange of basic proteins for histones mediated by nucleoplasmin. Subsequently, membrane vesicles bind to chromatin and fuse to form a double membrane, nuclear pore complexes are assembled and nuclear growth occurs. Ran had been shown to be required for proper nuclear assembly in Xenopus egg extracts, but it was unclear whether or not this role could be attributed to a requirement for Ran in the establishment of nucleocytoplasmic transport and nuclear growth17,25. This question was addressed by Hetzer et al. using a novel assay4 in which chromatin was first decondensed by treatment with a heat-stable fraction of extract containing nucleoplasm. Vesicles labeled with two differently coloured lipophilic dyes were incubated with the decondensed chromatin with the addition of a soluble (200 000 g supernatant) extract fraction. Membrane formation was assayed by the mixing of the two colours as fusion between the two vesicle populations occurred. This assay showed that the addition of RanQ69L or RanT24N inhibited vesicle fusion. When extracts were depleted of RCC1 using RanT24N, exogenous RCC1 or Ran–GTP, but not Ran–GDP, were able to overcome the inhibition of vesicle fusion around chromatin assembled on bacteriophage λ DNA, indicating that the generation of Ran–GTP by RCC1 is required. However, Ran bound with the non-hydrolysable GTP analogue GTPγS failed to rescue RCC1 depletion, indicating that GTP hydrolysis by Ran is also required. Immunodepletion experiments also showed that Ran itself is essential for membrane fusion and nucleoporin incorporation. In similar experiments, we found that Ran–GDP, but not the mutants RanQ69L or RanT24N, promoted NE assembly around sperm chromatin in Xenopus egg extracts. Because Ran might associate with chromatin early in nuclear assembly16, we reasoned that concentration of Ran might play a role in NE formation. To test whether Ran could induce NE assembly directly rather than through effects on chromatin, we coupled Ran–GDP to Sepharose beads and incubated the beads in Xenopus egg extracts. Remarkably, the beads rapidly accumulated membrane vesicles that fused to form a continuous lipid layer that incorporated nucleoporins5. Using electron microscopy, nuclear pore complexes were apparent crossing a double membrane, indicating that a complete NE was assembled. The envelopes were functional, because a fluorescent dextran that is too large to diffuse across the nuclear pores was excluded, whereas a karyophilic protein containing http://tcb.trends.com
369
a nuclear localization signal was concentrated within the beads. In other words, simply concentrating Ran on the surface of beads is sufficient to induce membrane–vesicle binding and fusion, as well as the assembly of NPCs and the initiation of nucleocytoplasmic transport. In contrast to Ran, beads coated with the mutants RanT24N or RanQ69L failed to make intact NEs, indicating that both loading of Ran with GTP and GTP hydrolysis are required for envelope assembly. The ability of Ran concentrated on the surface of beads to induce the formation of NEs is not restricted to Xenopus egg extracts, because it also occurs in extracts prepared from mitotic human cultured cells26. Using human cell extracts, we were able to show that RCC1 is required to generate Ran–GTP if beads coated with Ran–GDP are used, whereas beads coated with Ran–GTP do not require RCC1. Conversely, if RanGAP is inhibited with an antibody then vesicle binding and fusion are reduced, showing that GTP hydrolysis on Ran is required. Previous work had shown that addition of Ran–GDP to extracts promotes nuclear assembly around sperm chromatin, whereas high concentrations (10 µM) of Ran–GTP are inhibitory16,17. This effect probably highlights the importance of localization and the establishment of a difference in concentration and nucleotide-bound state in determining the activity of Ran. When a high concentration of Ran–GTP is established throughout the extracts, the gradient from high Ran–GTP concentration within the nucleus or around the mitotic chromatin is disrupted and reactions that should occur only in the vicinity of the chromatin occur throughout the extract. Thus, high cytoplasmic Ran–GTP concentrations inhibit nuclear import, disorganize mitotic spindle formation and disrupt NE assembly. Conversely, increasing the concentration of Ran–GDP in the cytoplasm might in fact increase Ran–GTP produced specifically at the chromatin by RCC1 activity and thus promote NE assembly. How does Ran control vesicle binding and fusion? Does Ran–GTP dissociate importin-bound complexes to release initiating factors close to the surface of the beads, similar to the mechanism of nuclear protein import and spindle assembly, or does Ran–GTP rather stabilize interactions with specific proteins, perhaps similar to its role in nuclear export? Identification of the effectors of Ran in NE assembly will be crucial to understanding how the process is initiated. The mechanism of NE assembly is not well understood, but vesicle fusion is inhibited by GTPγS (which might be accounted for, at least in part, by Ran) or N-ethylmaleimide (NEM), a reagent that reacts with thiol groups on proteins. Vesicle fusion might be a prerequisite for NPC insertion because GTPγS or NEM prevent NPC formation. NPC insertion can be uncoupled from membrane formation, because the metal cation chelator BAPTA results in the formation of a NE without NPCs27,28.
Opinion
370
TRENDS in Cell Biology Vol.11 No.9 September 2001
Interphase
Ran–GDP
RCC1
Cell division
RanBP1 RanGAP
Ran–GTP
Ran–GDP
Ran–GDP
RCC1 RCC1
RanBP1 RanGAP Ran–GTP
RCC1 Ran–GTP Telophase
Metaphase TRENDS in Cell Biology
Fig. 3. Ran controls nuclear processes throughout the cell cycle. During interphase, Ran directs nucleocytoplasmic transport. During mitosis, local generation of Ran–GTP promotes spindle assembly. At the end of mitosis, the cycling of Ran–GTP and Ran–GDP induces nuclear envelope reassembly and the restarting of nucleocytoplasmic transport.
The initial recruitment of vesicles to chromatin might involve binding between chromatin or lamins and integral membrane proteins that become constituents of the inner membrane29. Presumably, this event is regulated by Ran, although the biochemical mechanism remains unclear. Once vesicles are bound then NPC assembly takes place, a process that might also involve Ran, because Ran mutants alter NPC configuration30. Does Ran coordinate the cell division cycle?
Does Ran play a merely passive role during the reorganization of nuclear morphology and function during the cell division cycle, or does it control the timing or otherwise coordinate these events? Ran seems to act through common biochemical mechanisms, so how are its effects directed towards specific substrates during interphase and mitosis? And how does it switch from organizing the spindle during mitosis to inducing NE assembly after mitosis? One possibility is that Ran–GTP is relatively stable in the perichromatin or spindle regions during mitosis but, at the end of mitosis, Ran GTPase is activated to promote spindle disassembly and NE formation (Fig. 3). Ran is mainly excluded from chromatin during mitosis and might be concentrated on the spindle, but it is relocated to the reassembling nuclei at telophase, suggesting that translocation of Ran might play a role in this transition16. However, evidence for changes in the activity of the components of the Ran system that would indicate an active role in controlling the timing of cell cycle transitions is currently lacking, with the possible exception of the levels of expression of RanBP1 (Ref. 31). http://tcb.trends.com
It is likely that there are interactions between the Ran system and cell cycle regulators such the cyclindependent kinases and other protein kinases. These regulators might be dominant over Ran, perhaps directing its functions. Conversely, Ran is certainly involved in the nucleocytoplasmic transport of many cell cycle regulators such as cyclins, which must be directed to the correct compartment for their function32. In addition, Ran might control their activities: in Xenopus egg extracts, RanT24N inhibits Cdc2–cyclin-B-kinase activation even in the absence of nucleocytoplasmic transport, suggesting that the generation of Ran–GTP by RCC1 is required for kinase activation33,34. Conversely, in Xenopus egg extracts that are not arrested in mitosis, high concentrations of Ran–GTP inhibit the progression from centrosome-nucleated asters to nuclear assembly around sperm chromatin, as occurs in the egg following fertilization, suggesting a cell cycle arrest16. The mechanisms of these effects remain to be characterized. Are Ran’s functions common to all eukaryotic cells?
Are the roles of Ran identified in Xenopus egg extracts, namely nucleocytoplasmic transport, mitotic spindle assembly and NE assembly, also valid in vivo and are they conserved among all eukaryotes? There is good evidence that disruption of the Ran system perturbs nucleocytoplasmic transport in intact cells6, but its roles in mitosis are less well characterized. Ran itself is a highly conserved protein and is present in all eukaryotic cells that have been examined, including the primitive Giardia lamblia35. In Caenorhabditis elegans, ablation of the expression of Ran, RanGAP or RCC1 using RNA-mediated interference results in defective mitosis and failure to reassemble post-mitotic nuclei36, and mutation of the gene for the importin-β homolog Ketel causes defects in the formation of cleavage nuclei after mitosis in Drosophila early embryos37. In cultured mammalian cells, disruption of RanBP1 (Ref. 31) or microinjection of importin-β mutants18 causes defects in mitotic spindle structure. However, although the loss of RCC1 in the temperature-sensitive hamster cell line tsBN2 causes a micronucleus phenotype consistent with a defective post-mitotic nuclear assembly, spindle assembly is permitted38. Thus, it will be important to test further the role of the Ran system in mammalian cells to confirm the significance of its activity during mitosis. In yeast, the role of Ran in nucleocytoplasmic transport is clearly conserved and there is some evidence that components of the Ran system have separable effects on microtubule integrity and chromatin condensation in Schizosaccharomyces pombe39,40. There is no evidence to indicate a subnuclear redistribution of Ran–GTP during mitosis in yeast, but closed mitosis in a microorganism might not require chromatin-directed stabilization of microtubules, just a permissive
Opinion
Acknowledgements We thank our colleagues W. Moore, J. Hutchins and F. Nicolás for helpful discussion. Work in our laboratory is supported by the Biotechnology and Biological Sciences Research Council, the Medical Research Council and The Cancer Research Campaign.
TRENDS in Cell Biology Vol.11 No.9 September 2001
environment contained within the NE. Defects in the RCC1 homolog Pim1/Dcd1 result in fragmentation of NEs at the end of mitosis in S. pombe41, indicating that the generation of Ran–GTP is required for maintenance of NE structure. It thus appears that Ran plays fundamental, conserved roles in the control of nuclear structure and function. Is one of these functions ancestral, such the role of Ran in NE assembly that creates the nuclear compartment or the organization of the mitotic spindle that allows segregation of duplicated chromosomes? This question can probably never be answered with any certainty because an ancestral eukaryotic cell is not available for study, but the evolutionary significance of Ran might be its ability to allow chromatin to direct responses from the rest of the
References 1 Hyman, A.A. and Karsenti, E. (1996) Morphogenetic properties of microtubules and mitotic spindle assembly. Cell 84, 401–410 2 Heald, R. and Weis, K. (2000) Spindles get the Ran around. Trends Cell Biol. 10, 1–4 3 Dasso, M. (2001) Running on Ran: nuclear transport and the mitotic spindle. Cell 104, 321–324 4 Hetzer, M. et al. (2000) GTP hydrolysis by Ran is required for nuclear envelope assembly. Mol. Cell 5, 1013–1024 5 Zhang, C. and Clarke, P.R. (2000) Chromatinindependent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts. Science 288, 1429–1432 6 Gorlich, D. and Kutay, U. (1999) Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15, 607–660 7 Moore, M.S. and Blobel, G. (1993) The GTPbinding protein Ran/TC4 is required for protein import into the nucleus. Nature 365, 661–663 8 Izaurralde, E. et al. (1997) The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus. EMBO J. 16, 6535–6547 9 Nachury, M.V. and Weis, K. (1999) The direction of transport through the nuclear pore can be inverted. Proc. Natl. Acad. Sci. U. S. A. 96, 9622–9627 10 Bischoff, F.R. and Görlich, D. (1997) RanBP1 is crucial for the release of RanGTP from importin βrelated nuclear transport factors. FEBS Lett. 419, 249–254 11 Rexach, R. and Blobel, G. (1995) Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors and nucleoporins. Cell 83, 683–692 12 Carazo-Salas, R.E. et al. (1999) Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation. Nature 400, 178–181 13 Kalab, P. et al. (1999) The Ran GTPase regulates mitotic spindle assembly. Curr. Biol. 9, 481–484 14 Ohba, T. et al. (1999) Self-organization of microtubule asters induced in Xenopus egg extracts by GTP-bound Ran. Science 284, 1356–1359 15 Wilde, A. and Zheng, Y. (1999) Stimulation of microtubule aster formation and spindle
http://tcb.trends.com
16
17
18
19
20
21
22
23
24
25
26
27
28
371
cell to ensure the maintenance and successful transmission of the genome. Conclusions
It is now apparent that nucleocytoplasmic transport is only one of Ran’s functions and that it plays a wider role in coordinating nuclear functions throughout the cell cycle. In nucleocytoplasmic transport and mitotic spindle assembly, Ran acts through common effectors to direct distinct processes. The challenge now is to determine how the system is regulated during the cell division cycle. Because the processes in which Ran is involved, particularly correct segregation of the genome on the mitotic spindle, might be defective in diseases such as cancer, there is at least the potential for the Ran system to play a role in human disease.
assembly by the small GTPase Ran. Science 284, 1362–1365 Zhang, C. et al. (1999) Ran–GTP stabilises microtubule asters and inhibits nuclear assembly in Xenopus egg extracts. J. Cell Sci. 112, 2453–2461 Hughes, M. et al. (1998) The role of Ran GTPase in nuclear assembly and DNA replication: characterization of the effects of Ran mutants. J. Cell Sci. 111, 3017–3026 Nachury, M.V. et al. (2001) Importin β is a mitotic target of the small GTPase Ran in spindle assembly. Cell 104, 95–106 Wiese, C. et al. (2001) Role of importin-β in coupling Ran to downstream targets in microtubule assembly. Science 291, 653–656 Gruss, O.J. et al. (2001) Ran induces spindle assembly by reversing the inhibitory effect of importin α on TPX2 activity. Cell 104, 83–93 Carazo-Salas, R.E. et al. (2001) Ran–GTP coordinates regulation of microtubule nucleation and dynamics during mitotic-spindle assembly. Nat. Cell Biol. 3, 228–234 Wilde, A. et al. (2001) Ran stimulates spindle assembly by altering microtubule dynamics and the balance of motor activities. Nat. Cell Biol. 3, 221–227 Dogterom, M. et al. (1996) Influence of M-phase chromatin on the anisotropy of microtubule asters. J. Cell Biol. 133, 125–140 Azuma, Y. et al. (1997) Inhibition by anti-RCC1 monoclonal antibodies of RCC1-stimulated guanine nucleotide exchange on Ran GTPase. J. Biochem. 122, 1133–1138 Dasso, M. et al. (1994) A mutant form of the Ran/TC4 protein disrupts nuclear function in Xenopus laevis egg extracts by inhibiting the RCC1 protein, a regulator of chromosome condensation. EMBO J. 13, 5732–5744 Zhang, C. and Clarke, P.R. (2001) The roles of Ran–GTP and Ran–GDP in precursor vesicle recruitment and fusion during nuclear envelope assembly in a human cell-free system. Curr. Biol. 11, 208–212 Macaulay, C. and Forbes, D.J. (1996) Assembly of the nuclear pore: biochemically distinct steps revealed with NEM, GTP gamma S, and BAPTA. J. Cell Biol. 132, 5–20 Goldberg, M.W. et al. (1997) Dimples, pores, starrings, and thin rings on growing nuclear
29
30
31
32 33
34
35
36
37
38
39
40
41
envelopes: evidence for structural intermediates in nuclear pore complex assembly. J. Cell Sci. 110, 409–420 Vasu, S.K. and Forbes, D.J. (2001) Nuclear pores and nuclear assembly. Curr. Opin. Cell Biol. 13, 363–375 Goldberg, M.W. et al. (2000) Ran alters nuclear pore complex conformation. J. Mol. Biol. 300, 519–529 Guarguaglini, G. et al. (2000) Regulated Ranbinding protein 1 activity is required for organization and function of the mitotic spindle in mammalian cells in vivo. Cell Growth Differ. 11, 455–465 Pines, J. (1999) Cell cycle. Checkpoint on the nuclear frontier. Nature 397, 104–105 Clarke, P.R. et al. (1995) Regulation of cdc2/cyclin B activation by Ran, a Ras-related GTPase. J. Cell Sci. 108, 1217–1225 Kornbluth, S. et al. (1994) Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression. J. Cell Biol. 125, 705–719 Chen, L.M. et al. (1994) Molecular cloning and characterization of a ras-related gene of ran/tc4/spi1 subfamily in Giardia lamblia. J. Biol. Chem. 269, 17297–17304 Gönczy, P. et al. (2000) Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 408, 331–336 Tirian, L. et al. (2000) The Ketel(D) dominantnegative mutations identify maternal function of the Drosophila importin-beta gene required for cleavage nuclei formation. Genetics 156, 1901–1912 Nishitani, H. et al. (1991) Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of Cdc2 protein kinase and mitosis. EMBO J. 10, 1555–1564 Matsusaka, T. et al. (1998) Mutations in fission yeast Cut15, an importin α homolog, lead to mitotic progression without chromosome condensation. Curr. Biol. 8, 1031–1034 Fleig, U. et al. (2000) The fission yeast Ran GTPase is required for microtubule integrity. J. Cell Biol. 151, 1101–1112 Demeter, J. et al. (1995) A mutation in the RCC1related protein Pim1 results in nuclear envelope fragmentation in fission yeast. Proc. Natl. Acad. Sci. U. S. A. 92, 1436–1440
Opinion
TRENDS in Cell Biology Vol.11 No.9 September 2001
Ran GTPase: a master regulator of nuclear structure and function during the eukaryotic cell division cycle?
case, when the mitotic apparatus is exposed to the cytoplasm, how are the spindle microtubules stabilized while those in the cytoplasm are dissolved? It seems that centrosomes seed microtubule nucleation, whereas chromatin provides a stabilizing effect on microtubule growth1. At the end of mitosis, the spindle is broken down and the NE is reassembled to establish the compartmentalization of each daughter nucleus. Recent advances in understanding the functions of Ran, a small GTPase of the Ras superfamily, have shown that it has a central role in the control of both mitotic spindle assembly2,3 and NE dynamics4,5 in addition to its role in directing nucleocytoplasmic transport during interphase6. Here, we discuss these findings and their implication that Ran plays a general role in nuclear functions throughout the cell division cycle. Molecular marker of the nucleus?
Paul R. Clarke and Chuanmao Zhang Ran is an abundant GTPase that is highly conserved in eukaryotic cells and has been implicated in many aspects of nuclear structure and function, especially determining the directionality of nucleocytoplasmic transport during interphase. However, cell-free systems have recently shown that Ran plays distinct roles in mitotic spindle assembly and nuclear envelope (NE) formation in vitro. During spindle assembly, Ran controls the formation of complexes with importins, the same effectors that control nucleocytoplasmic transport. Here, we review these advances and discuss a general model for Ran in the coordination of nuclear processes throughout the cell division cycle via common biochemical mechanisms.
Paul R. Clarke Biomedical Research Centre, Level 5, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK DD1 9SY. e-mail: [email protected], Chuanmao Zhang Biomedical Research Centre, Level 5, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK DD1 9SY, and the Dept Cell Biology and Genetics, Peking University, Beijing 100871, China.
Compartmentalization of the genetic material into a distinct nucleus bounded by the nuclear envelope (NE) is the hallmark of a eukaryotic cell. Communication between the cytoplasm and nucleus is established via nuclear pore complexes (NPCs), large multiprotein structures that perforate the double membrane, forming channels that permit the diffusion of small molecules but restrict the passage of larger molecules to those carrying specific targeting signals. The NE allows a distinctive biochemical environment to be established within the nucleus and controls access to chromatin, which has many advantages for the regulation of gene expression and DNA replication. However, during cell division, enormous changes in nuclear structure are required to allow segregation of duplicated genomes to the daughter cells. In organisms such as yeast, which have a closed mitosis, segregation of sister chromatids is accomplished on a microtubule apparatus contained within the NE. Nuclear division is then required to produce one nucleus in each daughter cell. By contrast, most animal cells have an open mitosis, during which the NE breaks down and the nuclear architecture is completely reorganized. Duplicated, condensed chromosomes are arranged in the middle of a large, bipolar spindle assembled from microtubules that are focused at each pole around a centrosome. In this http://tcb.trends.com
Like other GTPases, Ran exists in GTP- and GDPbound conformations that interact differently with effectors. Conversion between these forms and the assembly or disassembly of effector complexes requires the interaction of regulator proteins. The intrinsic GTPase activity of Ran is very low, but it is greatly stimulated by a GTPase-activating protein (RanGAP1) located in the cytoplasm. By contrast, RCC1, a guanine nucleotide exchange factor that generates Ran–GTP, is bound to chromatin and confined to the nucleus. Ran itself is mobile and is actively imported into the nucleus by a mechanism involving NTF-2. Together with the compartmentalization of its regulators, this is thought to produce a relatively high concentration of Ran–GTP in the nucleus. Indeed, Ran–GTP might act as a molecular marker that distinguishes the nucleoplasm from the cytoplasm of interphase cells. However, it is difficult to demonstrate this model directly because the local concentration of Ran in the GTP or GDP form cannot yet be measured in vivo or in cell-free systems. Most of our evidence for the functions of Ran comes from the perturbation of the system by changing the levels of regulators or by introducing mutant Ran proteins that are defective in the guanine-nucleotide cycle. … Ran directs nucleocytoplasmic transport…
A gradient in Ran–GTP concentrations across the NE has been proposed to be crucial for the directionality of transport of many macromolecules through the nuclear pores7–9 (Fig. 1). In nucleocytoplasmic transport, Ran functions by controlling the assembly and disassembly of complexes formed between transported cargoes and a family of Ran-binding proteins that act as receptors for targeting sequences on the cargo6. In the nucleus, association of Ran–GTP with receptors that are required for export (exportins), such as the leptomycin-B-sensitive factor Crm1, enhances their affinity for proteins containing leucine-rich nuclear export signals. Although the
0962-8924/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(01)02071-2
Opinion
TRENDS in Cell Biology Vol.11 No.9 September 2001
Nuclear import Importin-α/β
Importin-α/β NLS-protein
NLS-protein
Ran–GDP Nucleus
Ran–GDP
GTP RCC1 GDP
RanGAP RanBP1
Pi
Cytoplasm
Ran–GTP
Ran–GTP
NES-protein
NES-protein
Exportin/Crm1
Exportin/Crm1
Leptomycin B Nuclear export TRENDS in Cell Biology
Fig. 1. Ran directs nucleocytoplasmic transport. Ran shuttles across the nuclear envelope via the nuclear pores but is concentrated in the nucleus by active import. In the nucleus, a high concentration of Ran–GTP is generated by nucleotide exchange catalysed by RCC1 bound to chromatin. Ran–GTP causes the dissociation of imported complexes containing proteins with nuclear localization signals (NLS) by binding to importin β and ejecting the cargo. Conversely, binding of Ran–GTP to exportin/Crm1 promotes the assembly of export complexes containing proteins with nuclear export signals (NES). In the cytoplasm, Ran–GTP is converted into Ran–GDP and phosphate (Pi) by Ran GTPase, which is activated by RanGAP and RanBP1, and export complexes are dissociated. The importins and exportins are recycled by transport back across the pore (not shown). In addition to this basic mechanism, other members of the importin family mediate the transport of specific cargoes6.
mechanism of translocation across the nuclear pore is still not fully understood, the export complex is dissociated by the interaction of the cytoplasmic Ran–GTP-binding proteins RanBP1 and RanBP2, followed by GTP hydrolysis by Ran stimulated by RanGAP1 (Ref. 10). In the cytoplasm, cargoes for import form complexes with a heterodimeric receptor consisting of importins α (an adaptor that recognizes lysine-rich nuclear localization signals) and β. After translocation through the nuclear pore, import complexes are dissociated in the nucleoplasm by Ran–GTP, which binds importin β and ejects importin α and the cargo. For some imported proteins, importin β interacts directly with the cargo, whereas other importin family molecules play more specialized roles in the transport of specific proteins that have distinct signal sequences6. During translocation through the pore, transport cargoes may interact with nuclear pore complex proteins (nucleoporins) in transient interactions controlled by Ran11. …mitotic spindle assembly… …
Ran or its interacting proteins have been implicated in aspects of cell cycle progression for many years, but it was difficult to distinguish direct effects from secondary effects that undoubtedly occur when http://tcb.trends.com
367
nucleocytoplasmic transport is disrupted. In 1999, the breakthrough demonstration of a role in mitosis came from a number of groups using Xenopus egg extracts as a model cell-free system that is amenable to biochemical dissection12–16. Kalab et al.13 provided the first evidence that changing the balance of Ran–GTP and Ran–GDP within a mitotic egg extract disrupted spindle assembly, a process that occurs in the absence of nuclear compartmentalization and is therefore distinct from nucleocytoplasmic transport. Ran is present in Xenopus egg extracts at a high concentration (1–2 µM) and, judging by its interaction with specific binding proteins, is predominantly GDP bound17. When the concentration of Ran–GTP is increased by adding the exchange factor RCC1, or mutants (G19V, Q69L or L43E) that are deficient in GTPase activity and are thereby stabilized in the GTP-bound state, microtubule assembly is promoted throughout the extract, resulting in ectopic asters containing typical centrosome-associated proteins such as γ tubulin, NuMA and Xgrip109 (Refs 12–15). With incubation, these asters might form spindle-like structures in the absence of chromatin or centrioles, albeit smaller than proper spindles formed following the addition of sperm heads. More recently, three groups have shown that the effects of adding Ran–GTP to mitotic Xenopus egg extracts is mediated by the same factors that are involved in nuclear protein import, namely the importins (Fig. 2). Nachury et al.18 showed that, when importin β is removed from extracts by depletion with RanQ69L, widespread microtubule polymerization is induced that is suppressed by exogenous importin β. This procedure allowed them to establish an assay for aster-promoting activities, one of which was identified as NuMA, a microtubuleassociated protein that is essential for spindle assembly. In a related study, Wiese et al.19 showed that the induction of asters by RanL43E or a fragment of NuMa (NuMA tail II) could be suppressed by importin β. Gruss et al.20 showed that importin α also inhibits aster formation in mitotic extract and that this effect can be overcome by exogenous TPX2, a microtubule-associated protein that targets the motor protein Xklp2 to microtubules. Ran–GTP displaces TPX2 from importins α and β, releasing this aster-promoting activity from inhibition. Together, these results suggest that importins have roles in controlling spindle assembly in mitotic Xenopus egg extracts by binding to and suppressing the activities of factors such as NuMA and TPX2, which are present throughout the extracts3. It is perhaps surprising that proper spindle assembly can be restored by adding single components back to importin-suppressed extracts, but such components might displace additional factors involved in spindle assembly from inhibitory complexes; in fact, crucial factors might remain to be identified. One important implication of these findings is that importins act not
368
Opinion
TRENDS in Cell Biology Vol.11 No.9 September 2001
Ran– GDP
Pi
RanGAP RCC1
Ran– GTP
Importin Importin
APA
APA
TRENDS in Cell Biology
Fig. 2. Ran directs mitotic spindle assembly. Ran–GTP releases asterpromoting activities (APA) from inhibited complexes with importins to promote mitotic spindle assembly. The chromosomal localization of RCC1 might generate Ran–GTP locally, stabilizing microtubules in the vicinity of chromatin.
only as targeting molecules but also as regulators of the proteins to which they bind. Many spindle assembly factors, such as NuMA, are sequestered mainly in the nucleus of somatic cells during interphase, but inactivation by importins would ensure that their activity is suppressed in the cytoplasm, even in Xenopus early embryos, which contain large cytoplasmic stores of such factors. It is unclear at present whether importins play a role in targeting spindle assembly factors to the correct location. It will also be interesting to see whether importins have a general role in suppressing the activity of karyophilic proteins during interphase. These experiments show how artificially increasing Ran–GTP levels in mitotic Xenopus egg extracts causes widespread aster formation. How might Ran normally function in spindle assembly? Addition of RanT24N, a mutant defective in nucleotide binding that forms a stable inhibitory complex with RCC1, or of RanGAP and RanBP1, which reduce the free concentration of Ran–GTP, all prevent the proper alignment of chromosomes on the metaphase spindle (including spindles formed around DNA-coated beads in the absence of centrosomes)12–16,21. Importantly, high concentrations of Ran–GTP throughout the extract also disrupt proper spindle assembly, resulting in dissociation of microtubules from chromatin12,13. These results indicate that Ran–GTP must be generated for spindle formation, but also that it must be localized to permit proper alignment of the chromosomes on the spindle. Chromatin has a positional effect on spindle formation by decreasing the catastrophe rate and increasing the rescue frequency of dynamic microtubules, thereby promoting the elongation of spindle microtubules specifically towards the chromatin21,22. The effect of chromatin could be due to the generation of a microtubule-stabilizing factor by http://tcb.trends.com
an enzyme located on mitotic chromosomes23. If RCC1 is localized to chromatin during mitosis, as it is in interphase, then the local generation of Ran–GTP could account for the influence of chromatin12,13. If high concentrations of Ran–GTP are generated ectopically throughout the extract then a gradient of Ran–GTP away from the chromatin would be disrupted and spindle assembly would be disorganized, as is indeed the case12,13. However, it is not yet possible to demonstrate the existence of a gradient of Ran–GTP concentrations directly and so this model remains to be verified experimentally. A requirement for RCC1 to be localized to chromatin for the positional effect also needs to be tested. Indeed, the localization of RCC1 to mitotic chromatin is uncertain because of the apparently contradictory results of immunofluorescence with different cell fixation techniques24; this question might be resolved by localizing fusions of green fluorescent protein with RCC1 in live, unfixed cells. Ran-GTP not only stabilizes microtubule elongation, through release of importin-bound factors, but also regulates motor proteins involved in spindle dynamics22. How are these functions related to microtubule nucleation at centrosomes? Factors such as NuMA and TPX2 appear to be involved in both processes18–20, but is microtubule nucleation driven by local generation of Ran–GTP at the centrosome? It has been reported that depletion of RCC1 from mitotic Xenopus egg extracts blocks microtubule aster formation14, but others12,21 have found that RanT24N (which efficiently inhibits RCC1) does not suppress centrosomal microtubule nucleation, and there is no indication that RCC1 is localized to centrosomes. One possibility is that a high concentration of Ran–GTP generated at chromatin by RCC1 extends in a cloud to envelop the whole spindle, permitting the chromatin to influence the nucleating capacity of the centrosomes and promote microtubule growth21. In this case, microtubule nucleation might be promoted specifically at centrosomes by other mechanisms such as protein phosphorylation directed by localized kinases, but Ran–GTP would be permissive for this activity. Nevertheless, high concentrations of importin-β strongly suppress centrosomal aster formation19, whereas Ran mutants stabilized in the GTP-bound form not only increase the nucleation capacity of centrosomes21,22 but also induce the formation of centrosome-like structures that nucleate microtubules in the absence of centrioles or chromatin12–15. Perhaps the assembly of centrosomes and microtubule nucleation involve the concentration and stabilization of Ran–GTP by localized binding proteins – rather than de novo generation of Ran–GTP by localized nucleotide exchange factor activity. …and NE assembly!
At the end of mitosis, the mitotic spindle is disassembled and microtubules return to interphase
Opinion
TRENDS in Cell Biology Vol.11 No.9 September 2001
dynamics. The NE is re-assembled around chromatin, nuclear pore complexes are reformed, nucleocytoplasmic transport is restarted and the distinct environment of the nucleus is re-established. Interphase Xenopus egg extracts provide a model system for studying the assembly of the nucleus from sperm chromatin. First, the chromatin undergoes decondensation, a process that involves the exchange of basic proteins for histones mediated by nucleoplasmin. Subsequently, membrane vesicles bind to chromatin and fuse to form a double membrane, nuclear pore complexes are assembled and nuclear growth occurs. Ran had been shown to be required for proper nuclear assembly in Xenopus egg extracts, but it was unclear whether or not this role could be attributed to a requirement for Ran in the establishment of nucleocytoplasmic transport and nuclear growth17,25. This question was addressed by Hetzer et al. using a novel assay4 in which chromatin was first decondensed by treatment with a heat-stable fraction of extract containing nucleoplasm. Vesicles labeled with two differently coloured lipophilic dyes were incubated with the decondensed chromatin with the addition of a soluble (200 000 g supernatant) extract fraction. Membrane formation was assayed by the mixing of the two colours as fusion between the two vesicle populations occurred. This assay showed that the addition of RanQ69L or RanT24N inhibited vesicle fusion. When extracts were depleted of RCC1 using RanT24N, exogenous RCC1 or Ran–GTP, but not Ran–GDP, were able to overcome the inhibition of vesicle fusion around chromatin assembled on bacteriophage λ DNA, indicating that the generation of Ran–GTP by RCC1 is required. However, Ran bound with the non-hydrolysable GTP analogue GTPγS failed to rescue RCC1 depletion, indicating that GTP hydrolysis by Ran is also required. Immunodepletion experiments also showed that Ran itself is essential for membrane fusion and nucleoporin incorporation. In similar experiments, we found that Ran–GDP, but not the mutants RanQ69L or RanT24N, promoted NE assembly around sperm chromatin in Xenopus egg extracts. Because Ran might associate with chromatin early in nuclear assembly16, we reasoned that concentration of Ran might play a role in NE formation. To test whether Ran could induce NE assembly directly rather than through effects on chromatin, we coupled Ran–GDP to Sepharose beads and incubated the beads in Xenopus egg extracts. Remarkably, the beads rapidly accumulated membrane vesicles that fused to form a continuous lipid layer that incorporated nucleoporins5. Using electron microscopy, nuclear pore complexes were apparent crossing a double membrane, indicating that a complete NE was assembled. The envelopes were functional, because a fluorescent dextran that is too large to diffuse across the nuclear pores was excluded, whereas a karyophilic protein containing http://tcb.trends.com
369
a nuclear localization signal was concentrated within the beads. In other words, simply concentrating Ran on the surface of beads is sufficient to induce membrane–vesicle binding and fusion, as well as the assembly of NPCs and the initiation of nucleocytoplasmic transport. In contrast to Ran, beads coated with the mutants RanT24N or RanQ69L failed to make intact NEs, indicating that both loading of Ran with GTP and GTP hydrolysis are required for envelope assembly. The ability of Ran concentrated on the surface of beads to induce the formation of NEs is not restricted to Xenopus egg extracts, because it also occurs in extracts prepared from mitotic human cultured cells26. Using human cell extracts, we were able to show that RCC1 is required to generate Ran–GTP if beads coated with Ran–GDP are used, whereas beads coated with Ran–GTP do not require RCC1. Conversely, if RanGAP is inhibited with an antibody then vesicle binding and fusion are reduced, showing that GTP hydrolysis on Ran is required. Previous work had shown that addition of Ran–GDP to extracts promotes nuclear assembly around sperm chromatin, whereas high concentrations (10 µM) of Ran–GTP are inhibitory16,17. This effect probably highlights the importance of localization and the establishment of a difference in concentration and nucleotide-bound state in determining the activity of Ran. When a high concentration of Ran–GTP is established throughout the extracts, the gradient from high Ran–GTP concentration within the nucleus or around the mitotic chromatin is disrupted and reactions that should occur only in the vicinity of the chromatin occur throughout the extract. Thus, high cytoplasmic Ran–GTP concentrations inhibit nuclear import, disorganize mitotic spindle formation and disrupt NE assembly. Conversely, increasing the concentration of Ran–GDP in the cytoplasm might in fact increase Ran–GTP produced specifically at the chromatin by RCC1 activity and thus promote NE assembly. How does Ran control vesicle binding and fusion? Does Ran–GTP dissociate importin-bound complexes to release initiating factors close to the surface of the beads, similar to the mechanism of nuclear protein import and spindle assembly, or does Ran–GTP rather stabilize interactions with specific proteins, perhaps similar to its role in nuclear export? Identification of the effectors of Ran in NE assembly will be crucial to understanding how the process is initiated. The mechanism of NE assembly is not well understood, but vesicle fusion is inhibited by GTPγS (which might be accounted for, at least in part, by Ran) or N-ethylmaleimide (NEM), a reagent that reacts with thiol groups on proteins. Vesicle fusion might be a prerequisite for NPC insertion because GTPγS or NEM prevent NPC formation. NPC insertion can be uncoupled from membrane formation, because the metal cation chelator BAPTA results in the formation of a NE without NPCs27,28.
Opinion
370
TRENDS in Cell Biology Vol.11 No.9 September 2001
Interphase
Ran–GDP
RCC1
Cell division
RanBP1 RanGAP
Ran–GTP
Ran–GDP
Ran–GDP
RCC1 RCC1
RanBP1 RanGAP Ran–GTP
RCC1 Ran–GTP Telophase
Metaphase TRENDS in Cell Biology
Fig. 3. Ran controls nuclear processes throughout the cell cycle. During interphase, Ran directs nucleocytoplasmic transport. During mitosis, local generation of Ran–GTP promotes spindle assembly. At the end of mitosis, the cycling of Ran–GTP and Ran–GDP induces nuclear envelope reassembly and the restarting of nucleocytoplasmic transport.
The initial recruitment of vesicles to chromatin might involve binding between chromatin or lamins and integral membrane proteins that become constituents of the inner membrane29. Presumably, this event is regulated by Ran, although the biochemical mechanism remains unclear. Once vesicles are bound then NPC assembly takes place, a process that might also involve Ran, because Ran mutants alter NPC configuration30. Does Ran coordinate the cell division cycle?
Does Ran play a merely passive role during the reorganization of nuclear morphology and function during the cell division cycle, or does it control the timing or otherwise coordinate these events? Ran seems to act through common biochemical mechanisms, so how are its effects directed towards specific substrates during interphase and mitosis? And how does it switch from organizing the spindle during mitosis to inducing NE assembly after mitosis? One possibility is that Ran–GTP is relatively stable in the perichromatin or spindle regions during mitosis but, at the end of mitosis, Ran GTPase is activated to promote spindle disassembly and NE formation (Fig. 3). Ran is mainly excluded from chromatin during mitosis and might be concentrated on the spindle, but it is relocated to the reassembling nuclei at telophase, suggesting that translocation of Ran might play a role in this transition16. However, evidence for changes in the activity of the components of the Ran system that would indicate an active role in controlling the timing of cell cycle transitions is currently lacking, with the possible exception of the levels of expression of RanBP1 (Ref. 31). http://tcb.trends.com
It is likely that there are interactions between the Ran system and cell cycle regulators such the cyclindependent kinases and other protein kinases. These regulators might be dominant over Ran, perhaps directing its functions. Conversely, Ran is certainly involved in the nucleocytoplasmic transport of many cell cycle regulators such as cyclins, which must be directed to the correct compartment for their function32. In addition, Ran might control their activities: in Xenopus egg extracts, RanT24N inhibits Cdc2–cyclin-B-kinase activation even in the absence of nucleocytoplasmic transport, suggesting that the generation of Ran–GTP by RCC1 is required for kinase activation33,34. Conversely, in Xenopus egg extracts that are not arrested in mitosis, high concentrations of Ran–GTP inhibit the progression from centrosome-nucleated asters to nuclear assembly around sperm chromatin, as occurs in the egg following fertilization, suggesting a cell cycle arrest16. The mechanisms of these effects remain to be characterized. Are Ran’s functions common to all eukaryotic cells?
Are the roles of Ran identified in Xenopus egg extracts, namely nucleocytoplasmic transport, mitotic spindle assembly and NE assembly, also valid in vivo and are they conserved among all eukaryotes? There is good evidence that disruption of the Ran system perturbs nucleocytoplasmic transport in intact cells6, but its roles in mitosis are less well characterized. Ran itself is a highly conserved protein and is present in all eukaryotic cells that have been examined, including the primitive Giardia lamblia35. In Caenorhabditis elegans, ablation of the expression of Ran, RanGAP or RCC1 using RNA-mediated interference results in defective mitosis and failure to reassemble post-mitotic nuclei36, and mutation of the gene for the importin-β homolog Ketel causes defects in the formation of cleavage nuclei after mitosis in Drosophila early embryos37. In cultured mammalian cells, disruption of RanBP1 (Ref. 31) or microinjection of importin-β mutants18 causes defects in mitotic spindle structure. However, although the loss of RCC1 in the temperature-sensitive hamster cell line tsBN2 causes a micronucleus phenotype consistent with a defective post-mitotic nuclear assembly, spindle assembly is permitted38. Thus, it will be important to test further the role of the Ran system in mammalian cells to confirm the significance of its activity during mitosis. In yeast, the role of Ran in nucleocytoplasmic transport is clearly conserved and there is some evidence that components of the Ran system have separable effects on microtubule integrity and chromatin condensation in Schizosaccharomyces pombe39,40. There is no evidence to indicate a subnuclear redistribution of Ran–GTP during mitosis in yeast, but closed mitosis in a microorganism might not require chromatin-directed stabilization of microtubules, just a permissive
Opinion
Acknowledgements We thank our colleagues W. Moore, J. Hutchins and F. Nicolás for helpful discussion. Work in our laboratory is supported by the Biotechnology and Biological Sciences Research Council, the Medical Research Council and The Cancer Research Campaign.
TRENDS in Cell Biology Vol.11 No.9 September 2001
environment contained within the NE. Defects in the RCC1 homolog Pim1/Dcd1 result in fragmentation of NEs at the end of mitosis in S. pombe41, indicating that the generation of Ran–GTP is required for maintenance of NE structure. It thus appears that Ran plays fundamental, conserved roles in the control of nuclear structure and function. Is one of these functions ancestral, such the role of Ran in NE assembly that creates the nuclear compartment or the organization of the mitotic spindle that allows segregation of duplicated chromosomes? This question can probably never be answered with any certainty because an ancestral eukaryotic cell is not available for study, but the evolutionary significance of Ran might be its ability to allow chromatin to direct responses from the rest of the
References 1 Hyman, A.A. and Karsenti, E. (1996) Morphogenetic properties of microtubules and mitotic spindle assembly. Cell 84, 401–410 2 Heald, R. and Weis, K. (2000) Spindles get the Ran around. Trends Cell Biol. 10, 1–4 3 Dasso, M. (2001) Running on Ran: nuclear transport and the mitotic spindle. Cell 104, 321–324 4 Hetzer, M. et al. (2000) GTP hydrolysis by Ran is required for nuclear envelope assembly. Mol. Cell 5, 1013–1024 5 Zhang, C. and Clarke, P.R. (2000) Chromatinindependent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts. Science 288, 1429–1432 6 Gorlich, D. and Kutay, U. (1999) Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15, 607–660 7 Moore, M.S. and Blobel, G. (1993) The GTPbinding protein Ran/TC4 is required for protein import into the nucleus. Nature 365, 661–663 8 Izaurralde, E. et al. (1997) The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus. EMBO J. 16, 6535–6547 9 Nachury, M.V. and Weis, K. (1999) The direction of transport through the nuclear pore can be inverted. Proc. Natl. Acad. Sci. U. S. A. 96, 9622–9627 10 Bischoff, F.R. and Görlich, D. (1997) RanBP1 is crucial for the release of RanGTP from importin βrelated nuclear transport factors. FEBS Lett. 419, 249–254 11 Rexach, R. and Blobel, G. (1995) Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors and nucleoporins. Cell 83, 683–692 12 Carazo-Salas, R.E. et al. (1999) Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation. Nature 400, 178–181 13 Kalab, P. et al. (1999) The Ran GTPase regulates mitotic spindle assembly. Curr. Biol. 9, 481–484 14 Ohba, T. et al. (1999) Self-organization of microtubule asters induced in Xenopus egg extracts by GTP-bound Ran. Science 284, 1356–1359 15 Wilde, A. and Zheng, Y. (1999) Stimulation of microtubule aster formation and spindle
http://tcb.trends.com
16
17
18
19
20
21
22
23
24
25
26
27
28
371
cell to ensure the maintenance and successful transmission of the genome. Conclusions
It is now apparent that nucleocytoplasmic transport is only one of Ran’s functions and that it plays a wider role in coordinating nuclear functions throughout the cell cycle. In nucleocytoplasmic transport and mitotic spindle assembly, Ran acts through common effectors to direct distinct processes. The challenge now is to determine how the system is regulated during the cell division cycle. Because the processes in which Ran is involved, particularly correct segregation of the genome on the mitotic spindle, might be defective in diseases such as cancer, there is at least the potential for the Ran system to play a role in human disease.
assembly by the small GTPase Ran. Science 284, 1362–1365 Zhang, C. et al. (1999) Ran–GTP stabilises microtubule asters and inhibits nuclear assembly in Xenopus egg extracts. J. Cell Sci. 112, 2453–2461 Hughes, M. et al. (1998) The role of Ran GTPase in nuclear assembly and DNA replication: characterization of the effects of Ran mutants. J. Cell Sci. 111, 3017–3026 Nachury, M.V. et al. (2001) Importin β is a mitotic target of the small GTPase Ran in spindle assembly. Cell 104, 95–106 Wiese, C. et al. (2001) Role of importin-β in coupling Ran to downstream targets in microtubule assembly. Science 291, 653–656 Gruss, O.J. et al. (2001) Ran induces spindle assembly by reversing the inhibitory effect of importin α on TPX2 activity. Cell 104, 83–93 Carazo-Salas, R.E. et al. (2001) Ran–GTP coordinates regulation of microtubule nucleation and dynamics during mitotic-spindle assembly. Nat. Cell Biol. 3, 228–234 Wilde, A. et al. (2001) Ran stimulates spindle assembly by altering microtubule dynamics and the balance of motor activities. Nat. Cell Biol. 3, 221–227 Dogterom, M. et al. (1996) Influence of M-phase chromatin on the anisotropy of microtubule asters. J. Cell Biol. 133, 125–140 Azuma, Y. et al. (1997) Inhibition by anti-RCC1 monoclonal antibodies of RCC1-stimulated guanine nucleotide exchange on Ran GTPase. J. Biochem. 122, 1133–1138 Dasso, M. et al. (1994) A mutant form of the Ran/TC4 protein disrupts nuclear function in Xenopus laevis egg extracts by inhibiting the RCC1 protein, a regulator of chromosome condensation. EMBO J. 13, 5732–5744 Zhang, C. and Clarke, P.R. (2001) The roles of Ran–GTP and Ran–GDP in precursor vesicle recruitment and fusion during nuclear envelope assembly in a human cell-free system. Curr. Biol. 11, 208–212 Macaulay, C. and Forbes, D.J. (1996) Assembly of the nuclear pore: biochemically distinct steps revealed with NEM, GTP gamma S, and BAPTA. J. Cell Biol. 132, 5–20 Goldberg, M.W. et al. (1997) Dimples, pores, starrings, and thin rings on growing nuclear
29
30
31
32 33
34
35
36
37
38
39
40
41
envelopes: evidence for structural intermediates in nuclear pore complex assembly. J. Cell Sci. 110, 409–420 Vasu, S.K. and Forbes, D.J. (2001) Nuclear pores and nuclear assembly. Curr. Opin. Cell Biol. 13, 363–375 Goldberg, M.W. et al. (2000) Ran alters nuclear pore complex conformation. J. Mol. Biol. 300, 519–529 Guarguaglini, G. et al. (2000) Regulated Ranbinding protein 1 activity is required for organization and function of the mitotic spindle in mammalian cells in vivo. Cell Growth Differ. 11, 455–465 Pines, J. (1999) Cell cycle. Checkpoint on the nuclear frontier. Nature 397, 104–105 Clarke, P.R. et al. (1995) Regulation of cdc2/cyclin B activation by Ran, a Ras-related GTPase. J. Cell Sci. 108, 1217–1225 Kornbluth, S. et al. (1994) Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression. J. Cell Biol. 125, 705–719 Chen, L.M. et al. (1994) Molecular cloning and characterization of a ras-related gene of ran/tc4/spi1 subfamily in Giardia lamblia. J. Biol. Chem. 269, 17297–17304 Gönczy, P. et al. (2000) Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 408, 331–336 Tirian, L. et al. (2000) The Ketel(D) dominantnegative mutations identify maternal function of the Drosophila importin-beta gene required for cleavage nuclei formation. Genetics 156, 1901–1912 Nishitani, H. et al. (1991) Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of Cdc2 protein kinase and mitosis. EMBO J. 10, 1555–1564 Matsusaka, T. et al. (1998) Mutations in fission yeast Cut15, an importin α homolog, lead to mitotic progression without chromosome condensation. Curr. Biol. 8, 1031–1034 Fleig, U. et al. (2000) The fission yeast Ran GTPase is required for microtubule integrity. J. Cell Biol. 151, 1101–1112 Demeter, J. et al. (1995) A mutation in the RCC1related protein Pim1 results in nuclear envelope fragmentation in fission yeast. Proc. Natl. Acad. Sci. U. S. A. 92, 1436–1440