View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Dispatch R257

Ran GTPase cycle: One mechanism — two functions Frauke Melchior

The Ras-related GTPase functions in transport — the import receptor β and its adaptor nucleocytoplasmic transport by regulating interactions importin α. RanGTP functions by releasing MAPs of transport receptors with transport cargo. Three from complexes with importin α and β. From these find- recent studies suggest that Ran uses exactly the same ings, the authors propose a model in which the ability of mechanism to regulate spindle assembly during Ran to disrupt importin β-containing complexes not only . regulates nuclear import in but also spindle for- mation in mitosis. Address: Max-Planck Institute for Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany. E-mail: [email protected] The Ran GTPase cycle in interphase and mitosis Like all , Ran exists in two different states: either Current Biology 2001, 11:R257–R260 GTP-bound or GDP-bound. Depending on the nucleotide 0960-9822/01/$ – see front matter bound, Ran interacts with several different , most © 2001 Elsevier Science Ltd. All rights reserved. of which have well defined roles in nucleocytoplasmic transport ([1,2] and references therein). To change its The small GTPase Ran has a well established function in nucleotide state, Ran needs to interact with regulatory nucleocytoplasmic transport [1,2], but is also implicated proteins, the GTPase-activating protein RanGAP1 and the in mitotic spindle formation and refor- guanine nucleotide exchange factor RCC1. Vertebrate mation after mitosis [3–5]. Despite extensive work in a RanGAP1 is highly enriched at the cytoplasmic filaments number of organisms, it has remained controversial whether of complexes in interphase, and is diffusely Ran plays a direct role in mitosis, or whether perturbing distributed throughout the cell upon nuclear envelope the Ran GTPase cycle causes mitotic defects only indi- breakdown in prometaphase. There is also some indication rectly. Switching from in vivo studies to an in vitro system that RanGAP1 is enriched at the mitotic spindle but this has greatly benefited the analysis of nucleocytoplasmic awaits further analysis. RCC1 on the other hand is concen- transport in the past, and has now been a key to the analy- trated on throughout the entire cell cycle. Con- sis of Ran function during mitosis. sequently, the locations of RanGAP1 and RCC1 determine the sites for nucleotide specific interactions of Ran. Extracts prepared from Xenopus laevis oocytes arrested at metaphase of meiosis II (CSF-extracts) have high levels of In interphase, a complete GTPase cycle requires shuttling mitotic kinase activity ( B–Cdk1) and are used to of Ran into and out of the nucleus. After nuclear envelope analyse mitotic events in vitro [6]. Addition of demem- breakdown, nucleotide exchange is believed to take place branated sperm DNA to such extracts, as well as DNA- predominantly at condensed , while hydroly- coated beads, induces formation of a bipolar spindle [7]. sis takes place throughout the cell. It is at present unclear This latter finding is particularly interesting as it indicates whether the activities of RCC1 and RanGAP1 are altered that neither centrosomes nor are essential for when cells undergo mitosis, but levels of the RanGTP- bipolar spindle formation in these extracts. When added in interacting protein RanBP1 drop significantly in excess, several proteins or protein domains induce micro- [14]. As RanBP1 both stimulates GTP hydrolysis rates and tubule asters and spindle-like structures in the absence of inhibits nucleotide exchange rates in vitro, alterations in DNA. Among those are the microtubule-associated pro- RanBP1 levels may have dramatic consequences for rates teins (MAPs) NuMA and TPX2 [8,9]. Therefore, it was an of GTP hydrolysis and exchange. exciting finding that elevated levels of GTP-bound Ran (RanGTP) in CSF-extracts also result in DNA indepen- Ran in nucleocytoplasmic transport dent induction of microtubule asters (reviewed in [3–5]). Regulated transport of proteins and ribonucleoprotein Recently, it was shown that Ran also affects microtubule particles — the ‘cargo’ — through the nuclear pore complex nucleation and stability in Xenopus extracts that contain is accomplished through spatially controlled interactions reduplicated centrosomes and DNA [10]. of the cargo with their respective transport receptors [1,2]. These receptors recognize small peptide sequences in Taken together, these findings strongly implicate Ran in a the cargo, so called import (NLS) or export signals. A pro- transport-independent function in spindle formation. totype receptor is importin β, which recognizes a large Three recent publications [11–13] now reveal that the number of different NLS-containing proteins, either effect of Ran on aster formation in CSF-extracts is never- directly or in association with importin α. Vectorial move- theless mediated via proteins that are required for nuclear ment of cargo is accomplished by coupling cargo–receptor R258 Current Biology Vol 11 No 7

interactions to the Ran GTPase cycle. RanGTP binds throughout the entire cell cycle. In most with high affinity to all receptors of the importin β family, other cells, nuclear envelope breakdown takes place in irrespective of whether they are involved in import or prometaphase and disrupts the strict spatial separation of export. Association of RanGTP with import receptors dis- RCC1 and RanGAP1. Despite these striking differences, rupts their interactions with import cargo, thus releasing Ran has been implicated in both types of mitosis [3–5]. the cargo into the nucleoplasm. In contrast, association of RanGTP with export receptors enhances their affinity for Xenopus CSF-extracts have become the system of choice export cargo and is required for export. The distinct intra- for studying the mitotic function of Ran under conditions cellular localization of RCC1 and RanGAP1 to the nuclear that do not require nuclear transport. Elevated levels of and cytoplasmic compartment, respectively, ensures that RanGTP in these extracts lead to the induction of ectopic formation and disassembly of import and export com- microtubule asters and spindle-like structures even in the plexes take place only in the desired cellular compartment absence of DNA [3–5]. What is the mechanism underlying (Figure 1). this effect? The three recent studies [11–13] demonstrate that RanGTP disrupts complexes containing importin β The translocation of proteins into or out of the nucleus is and other key players in spindle formation. Interestingly, often used to regulate interactions both spatially and tem- Ran is dispensable for spindle formation once importin β porally. Temporal regulation has been well established for has been removed from the extract. This implies that Ran some cell-cycle-dependent kinases [15], and may also be has no additional role in this process. true for several MAPs that reside in the nucleus during interphase. TPX2 and NuMA are actively transported into Two proteins that are released from importin β by the nucleus during interphase which may segregate them RanGTP have been identified — NuMA and TPX2 — from microtubules until mitosis [9,16]. but there may be several others. An artificial import cargo with a classical NLS was found to induce aster formation Ran in spindle formation in extracts by competitively releasing important compo- Yeast cells progress through mitosis without breakdown nents from importin α/β complexes [11]. NuMA and of the nuclear envelope and depend on Ran-mediated TPX2 also bind to importin α/β. Consequently, their aster-promoting activity may be due to two effects in the Figure 1 extract, one directly on spindle formation, and a second one by competing with other factors for importin α/β Cytoplasmic binding. One candidate protein is the mitotic kinase microtubules cyclin B–Cdk1 which binds directly to importin β [15]. Inhibiting its function would obviously be deleterious for mitotic processes. Nuclear envelope What do these findings imply for intact cells? In higher RanGTP eukaryotes, changes in microtubule dynamics correlate β with the onset of nuclear envelope breakdown, and are RanGAP due in part to the exposure of cytoplasmic microtubules to MAPs and motor proteins that are intranuclear during NuMA interphase (for a review on spindle dynamics see [17]). RCC1 RanGDP Upon nuclear envelope breakdown, however, these intra- GTP GDP Nucleus nuclear MAPs are also exposed to nuclear import recep- tors, and could therefore be trapped in inactive complexes. Current Biology Consequently, cells must carefully control the balance of nuclear transport components even in the absence of a The RanGTPase cycle in nuclear protein import. RanGAP1-mediated GTP hydrolysis in the cytoplasm and RCC1 mediated conversion of nuclear envelope. RanGDP to RanGTP are coupled to the translocation of proteins into the nucleus. NLS-containing proteins such as NuMA bind to importin β Mitotic defects caused by interference with the Ran in the cytoplasm and are subsequently translocated into the nucleus. GTPase cycle may therefore be explained by the untimely β Binding of RanGTP to importin releases the cargo from the receptor disruption or formation of import and export complexes. inside the nucleus, and a RanGTP–importin β complex is formed. Translocation of this complex into the cytoplasm and subsequent Most importantly, the findings described above suggest RanGAP1-mediated GTP hydrolysis release importin β and allow it to that the Ran GTPase cycle may be used in mitosis, as in bind another cargo. Finally, Ran re-enters the nucleus where it is interphase, for spatial control of MAP activities. This converted back to RanGTP by RCC1. For clarity, importin α which is β could be accomplished through RanGTP-mediated disso- often needed as an adaptor between cargo and importin , and several α β other factors participating in this pathway, are omitted. ciation of complexes consisting of MAPs and importin / close to chromosomes, where RCC1 is concentrated, and Dispatch R259

re-association of these complexes in the cytoplasm, where Figure 2 RanGAP1 function is predominant (Figure 2).

Perspectives RanGTP β The model for Ran function in mitosis predicts that spindle RanGAP formation is initiated at the chromosomes through RCC1- NuMA dependent generation of RanGTP. Is this model applicable GTP GDP to all cells? In the Xenopus system, microtubules are stabi- RanGDP lized along the entire DNA, which correlates well with RCC1 localisation. On the other hand, in somatic animal RCC1 cells, microtubules are stabilized only at kinetochores, Condensed despite the fact that RCC1 is present along the entire chromosomes length of the chromatin. Moreover, there is some indication that RCC1 — and by inference RanGTP — is not needed Mitotic cytoplasm Metaphase spindle for spindle formation in vertebrate cells. This stems from the observation that tsBN2 cells, which have a tempera- Current Biology ture-sensitive RCC1 protein, enter mitosis prematurely and form a mitotic spindle at the restrictive temperature [18]. The RanGTPase cycle in spindle formation. RanGAP1-mediated GTP hydrolysis takes place throughout the mitotic cytoplasm; RCC1 mediated conversion of RanGDP to RanGTP is believed to occur How can these apparent discrepancies be reconciled? Plant preferentially at chromatin. NLS-containing proteins involved in spindle cells and some meiotic cells, including Xenopus oocytes, formation such as NuMA bind to importin β in the cytoplasm, which use a centrosome-independent mechanism for spindle for- may keep them from interacting with microtubules. RanGTP-dependent mation that requires stabilization of microtubules in the release of these proteins at chromatin is proposed to account for microtubule-stabilizing effects of DNA and the formation of a mitotic immediate vicinity of chromosomes. These microtubules spindle. As in Figure 1, importin α and several other factors are subsequently focused at their minus ends into spindle participating in this pathway are left out for clarity. poles. The centrosome-dependent mechanism, believed to be predominant in somatic animal cells, involves nucle- ation of microtubules at the two microtubule-organizing nuclear envelopes in Xenopus extracts, and that GTP hydrol- centers and subsequent stabilization of the microtubule ysis by Ran is required [20,21]. One may predict that this plus ends at kinetochores [17]. It may be that Ran only third function of the Ran GTPase cycle is also accom- plays an essential role in centrosome-independent spindle plished via Ran–transport receptor interactions that allow formation. Somatic animal cells may use additional or dif- site specific recruitment of essential factors. ferent mechanisms, for example specific phosphorylation to mask NLSs during . This would ensure that Finally, a burning question is whether the activities regu- NuMA, TPX2 and other factors are not trapped by lating the Ran GTPase cycle are altered when cells enter importin β upon nuclear envelope breakdown. Both NuMA and exit mitosis. This may provide an additional level of and TPX2 are mitotic phosphoproteins, but the conse- regulation of mitotic processes. quences of phosphorylation are not yet understood [8,9]. Alternatively, RCC1 activity may be regulated differently Acknowledgements My thanks go to Drs. Michael Knop, Sowmya Swaminathan and Ludger in the proximity of and at some distance from kinetochores. Hengst for critical reading of the manuscript.

Is the above described mitotic role for Ran conserved References in yeast? Budding yeast has nuclear and cytoplasmic 1. Mattaj IW, Englmeier L: Nucleocytoplasmic transport: the soluble phase. Annu Rev Biochem 1998, 67:265-306. microtubules even during interphase [19]. Consequently, 2. Görlich D, Kutay U: Transport between the and the changes in microtubule dynamics in these cells cannot cytoplasm. Annu Rev Cell Dev Biol 1999, 15:607-660. easily be explained by the sudden exposure of micro- 3. Desai A, Hyman A: Microtubule cytoskeleton: no longer an also Ran. Curr Biol 1999, 9:R704-R707. tubules to the stabilizing effects of RanGTP near chromo- 4. Sazer S, Dasso M: The ran decathlon: multiple roles of Ran. J Cell somes. Other regulatory mechanisms such as cell cycle- Sci 2000, 113:1111-1118. dependent transport, phosphorylation of MAPs, or alter- 5. Moore JD: The Ran-GTPase and cell-cycle control. Bioessays 2001, 23:77-85. ations in the Ran GTPase cycle may play a role here. 6. Murray AW: Cell cycle extracts. Methods Cell Biol 1991, 36:581-605. 7. Heald R, Tournebize R, Blank T, Sandaltzopoulos R, Becker P, In addition to the roles that Ran plays in nucleocytoplas- Hyman A, Karsenti E: Self-organization of microtubules into bipolar mic transport and spindle formation, it has also been spindles around artificial chromosomes in Xenopus egg extracts. implicated in nuclear envelope reformation at the end of Nature 1996, 382:420-425. 8. Merdes A, Ramyar K, Vechio JD, Cleveland DW: A complex of NuMA mitosis. Two key findings were that beads coated with and cytoplasmic is essential for mitotic spindle assembly. RanGDP are sufficient to form pseudo-nuclei with intact Cell 1996, 87:447-458. R260 Current Biology Vol 11 No 7

9. Wittmann T, Wilm M, Karsenti E, Vernos I: TPX2, A novel Xenopus MAP involved in spindle pole organization. J Cell Biol 2000, 149:1405-1418. 10. Carazo-Salas RE, Gruss OJ, Mattaj IW, Karsenti E: Ran-GTP coordinates the regulation of and dynamics during mitotic spindle assembly. Nat Cell Biol 2001, in press. 11. Gruss OJ, Carazo-Salas RE, Schatz CA, Guarguaglini G, Kast J, Wilm M, Le Bot N, Vernos I, Karsenti E, Mattaj IW: Ran induces spindle assembly by reversing the inhibitory effect of importin α on TPX2 activity. Cell 2001, 104:83-93. 12. Nachury MV, Maresca TJ, Salmon WC, Waterman-Storer CM, Heald R, Weis K: Importin β is a mitotic target of the small GTPase Ran in spindle assembly. Cell 2001, 104:95-106. 13. Wiese C, Wilde A, Moore MS, Adam SA, Merdes A, Zheng Y: Role of importin-β in coupling ran to downstream targets in microtubule assembly. Science 2001, 291:653-656. 14. Guarguaglini G, Renzi L, D’Ottavio F, Di Fiore B , Casenghi M, Cundari E, Lavia P: Regulated Ran-binding protein 1 activity is required for organization and function of the mitotic spindle in mammalian cells in vivo. Cell Growth Differ 2000, 11:455-465. 15. Moore JD, Yang J, Truant R, Kornbluth S: Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1. J Cell Biol 1999, 144:213-224. 16. Merdes A, Cleveland DW: The role of NuMA in the interphase nucleus. J Cell Sci 1998, 111:71-79. 17. Compton DA: Spindle assembly in animal cells. Annu Rev Biochem 2000, 69:95-114. 18. Nishimoto T, Eilen E, Basilico C: Premature condensation in a ts DNA- mutant of BHK cells. Cell 1978, 15:475-483. 19. Winey M, O´Toole ET: The spindle cycle in budding yeast. Nat Cell Biol 2001, 3:E23-E27. 20. Hetzer M, Bilbao-Cortes D, Walther TC, Gruss OJ, Mattaj IW: GTP hydrolysis by Ran is required for nuclear envelope assembly. Mol Cell 2000, 5:1013-1024. 21. Zhang C, Clarke PR: Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts. Science 2000, 288:1429-1432.