RANdezvous with the Spindle: Role of the RanGTP Cycle in Mitotic Spindle Assembly

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1 Einstein Quart. J. Biol. Med. (2001) 18(3): RANdezvous with the Spindle: Role of the RanGTP Cycle in Mitotic Spindle Assembly Rajarshi Ghosh Department of Developmental and Molecular Biology Albert Einstein College of Medicine. Bronx, New York Abstract The small GTPase Ran has long been recognized for its role in nucleocytoplasmic transport. Recent evidence using Xenopus egg extracts points to an altogether novel role of Ran, which is completely independent of its function in trafficking. Ran is now implicated in mitotic spindle assembly. The mechanism of action of RanGTP in microtubule formation is not clear at this time, but the results so far indicate that there may be an important role for RanGTP in the cell division cycle. This review discusses these reports and attempts to explain this new role of RanGTP in the context of the accepted microtubule assembly mechanisms. Introduction G-proteins act as molecular switches and clocks that regulate various important intracellular events. They can adopt different conformational states depending on the bound nucleotide and thereby interact with effector molecules to modulate cellular events or signaling cascades. Generally, the GTP-bound state is the on or active state, and the GDP-bound state is the off or inactive state. The off to on conversion is mediated by a Guanine Nucleotide Exchange Factor, or GEF, that exchanges the bound GDP for a GTP molecule (Bourne et al., 1990). Most of the G-proteins have very little intrinsic GTPase activity. This activity is stimulated by a GTPase Activating Protein (GAP) (Gideon et al., 1992). For the Ras family of GTPases, GAPs stabilize the negatively charged transition state formed during GTP hydrolysis by contributing a positively charged amino acid (usually arginine) that is conserved throughout the family (Scheffzek et al., 1998). The main focus of this review is Ran, Ras-like nuclear G- protein (Bischoff and Ponstingl, 1991), which is highly conserved from yeast to humans, and its associated GTPase cycle. This cycle has all of the features and players involved with a typical G-protein including a GEF, RCC1 (Regulator of Chromosome Condensation), a GAP, RanGAP1 (Bischoff and Ponstingl, 1995), and the GTPase Ran in its on and off forms. There are properties that make the RanGTP cycle unique. First, Ran, unlike Ras, does not have any lipid modification motif and is, consequently, not membrane-bound. Second, the RanGTP cycle is compartmentalized; the two phases of the GTPase reaction occur in two different cellular compartments. RCC1, which carries out the conversion of RanGDP to RanGTP, remains bound to chromatin in the nucleus (Ohtsubo et al., 1989). RanGAP is located on the cytoplasmic face of the nuclear pore (Matunis et al., 1996). It stimulates the very low intrinsic GTPase activity of Ran. Thus, GTP hydrolysis occurs in the cytoplasm, and the exchange reaction occurs in the nucleus. So, Ran must shuttle between the nucleus and the cytoplasm, which is consistent with its function in the nucleocytoplasmic transport of a broad range of cargo (Gorlich and Mattaj, 1996). The polarity of movement and the specificity of binding are determined by the nucleotide status of Ran. For example, the importins (nuclear import receptors) firmly bind RanGDP in the cytoplasm but dissociate upon entry into the nucleus. This is a direct result of the higher concentration of RanGDP in the cytoplasm. Likewise, the exportins (nuclear export receptors) bind RanGTP, and a high concentration of the RanGTP form in the nucleus stimulates the export of cargo molecules. Thus, both polarity of movement and binding specificity to either of the karyopherins (importins and exportins) is determined by whether Ran is GTP- or GDP-bound (Mattaj and Englmeier, 1998) (Figure 1). The Masking Problem Since the RanGTP cycle is involved in the transport of a very broad range of RNA and proteins, it is not unexpected that searching for one primary role of the Ran cycle would be very difficult. Indeed both in vivo and in vitro mutational and depletion analyses of the key players in the RanGTP cycle give rise to a large number of defects, from RNA processing to cell cycle regulation. In other words, the primary role of RanGTP cycle is likely to be shrouded by the secondary effects (Sazer, 1996). New roles for Ran have been suggested, but it is difficult to dissect them from their secondary effects, except for one case. The RanGTP cycle is operative in interphase cells. Higher eukaryotes exhibit an open mitosis, which is a disassembly of the nuclear membrane at the onset of mitosis. The regulation by Ran due to compartmentalization is therefore lost. What role, then, does the Ran cycle play during the M phase of the cell cycle? It is very likely that this open mitosis offers a mechanism to investigate the function of Ran in cell cycle-related events that may be masked by Ran's function in the import and export of RNA and proteins.

2 RANdevous with the Spindle 107 A New Player, Ran-BPM One of the initial observations that associated the RanGTP cycle with microtubule assembly was the suppression of certain tubulin mutants in Saccharomyces cerevisiae by Alpha-Tubulin Suppressor (ATS1), a structural homologue of the mammalian RCC1. These mutants undergo a growth arrest due to excess microtubule formation at the nonpermissive temperature (Kirkpatrick and Solomon, 1994). This is surprising, because there is no evidence of open mitosis in S. cerevisiae. Hence, this result may not be attributed to the direct involvement of Ran in spindle assembly. Instead, it could arise from a defect in nucleocytoplasmic transport. But, the discovery of a novel RanGTP binding protein, Ran Binding Protein in Microtubule organizing center (RanBPM) (Nakamura et al., 1998), and studies with Xenopus egg extracts (see below) have provided more convincing evidence that RanGTP may be regulating microtubule assembly independent of its function in nucleocytoplasmic transport. Direct binding of a protein to Ran depends on the presence of a highly conserved Ran binding domain. This domain stabilizes the interaction between the GTPbound state of Ran and the protein containing this domain. Best documented among these proteins are RanBP1 (Bischoff et al., 1995), located in the cytosol, and RanBP2, located in the cytoplasmic filament of the nuclear pore complex (Mahajan et al., 1997). Both of these proteins possess one or more Ran binding domains that stabilize their interaction with RanGTP or RanGDP. This domain is critical for their downstream function, which involves nucleocytoplasmic trafficking. However, Nakamura et al. (1998) found RanBPM using human Ran as bait with the yeast two-hybrid technique. Interestingly, this protein was localized at the centrosomes, the major microtubule organizing center. The typical animal cell centrosome is comprised of two centrioles surrounded by a relatively ill-defined cloud of pericentriolar material (PCM). Electron microscopy studies indicated that the microtubules originate from the PCM (Gould and Borisy, 1977) in a process involving γ-tubulin. In fact, recent evidence indicates that γ-tubulin possesses microtubule nucleation potential (Zheng et al., 1995). This job is not done by γ-tubulin per se, but involves a complex array of proteins collectively known as the γ-tubulin ring complex (γ-turc) (Zheng et al., 1995). This complex is located in the cytoplasm and is recruited to the centrosome scaffolds to initiate microtubule nucleation. Salt extraction of Spisula centrosomes, which removes γ-turc, leaves behind a centrosome scaffold (Schnackenberg et al., 1998) that does not possess any microtubule nucleating potential (MNP). These salt-stripped centrosomes (SSCs) can regain the MNP when treated with Spisula oocyte extracts (Schnackenberg et al., 1998). Moritz et al. (1998) report that γ-turc is necessary but not sufficient for complementing the nucleating potential of SSCs, and that an additional factor is required. RanBPM is a likely candidate for that additional nucleation factor. The localization of RanBPM in the PCM was confirmed by staining MRC5 cells separately with anti- RanBPM and the anti-γ-tubulin antibodies, a marker for centrosomes. Then, the cells were superimposed (Nakamura et al., 1998). Localization alone does not qualify RanBPM as an important member of the microtubule nucleation party. Overexpression of RanBPM cdna results in both ectopic nucleation and reorganization of the microtubule network in transfected COS cells (Nakamura et al., 1998). This ectopic aster formation is similar to the one due to γ-tubulin overexpression both structurally and kinetically. Also, the in vitro microtubule nucleation is inhibited by Ran bound to the nonhydrolyzable GTP analogue, GTP-γS, as well as by antibodies to RanBPM (Nakamura et al., 1998). These reports allude to the involvement of the Ran cycle in the microtuble assembly process. Bridging the Gap The implication that RanBPM and hence Ran could be involved in spindle assembly during mitosis was further supported by the recent reports of five groups. Xenopus egg extract arrested at meiotic metaphase II by the cytostatic factor CSF (Sagata et al., 1989) was the system of choice. First, the unfertilized eggs lack centrosomes, but contain all of the materials required for the assembly of greater than 1000 centrosomes, requiring no additional protein synthesis besides the maternal stores (Stearns and Kirschner, 1994). Second, addition of a proper template such as basal bodies, demembranated sperm nuclei, or artificial chromosomes tethered to beads can initiate centrosome and spindle assembly (Stearns and Kirschner, 1994). Third, concentrated cytoplasmic extracts from Xenopus can carry out many in vivo reactions (Stearns and Kirschner, 1994). And fourth, the results obtained with these extracts cannot be attributed to a nuclear transport defect, because spindle assembly occurs independently of any events in interphase (Kalab et al., 1999). Thus, the Xenopus egg extracts were a powerful system that could bridge the gap between Ran and microtubule assembly. Taking advantage of this system, the effects of several mutants of Ran and its binding partners, which alter the normal levels of either RanGDP or RanGTP, were reported. It was previously shown that RanBP1 functions by stimulating the activity of RanGAP, which increases the rate of hydrolysis of GTP bound to Ran (Bischoff et al., 1995). Kalab et al. (1999) reported that addition of exogenous RanBP1 to Xenopus egg extracts in M phase dramatically disrupts spindle assembly. The inhibitory effect of RanBP1 was quantitatively reversed by the addition of

3 108 The Einstein Quarterly Journal of Biology and Medicine Figure 1: The nuclear import and export pathways. Nuclear Pore Complex (NPC). The karyopherins (nuclear receptors) that carry the cargo into and out of the nucleus are shown. The receptor for import is called importing, and the receptor for export is called exportin (Nakielny and Dreyfuss, 1999). RCC1. Consistent with this observation, examination of the effects of Ran mutants RanG19V and RanT24N showed defects resembling that of RanBP1- and RCC1- treated egg extracts, respectively. RanG19V has higher RanGDP concentration while RanT24N has higher RanGTP concentration. In other words, this study shows that an increase in RanGDP concentration (via addition of RanBP1 or a RanG19V mutant) contributes to the destabilization of spindle, whereas an increase in concentration of the RanGTP (via addition of RCC1 or a RanT24N mutant) favors spindle assembly. That report was corroborated by other studies which strengthen the notion that it is the GTP-bound form of Ran that is involved in microtubule aster formation. Ohba et al. (1999) reported that immunodepletion of the GEF, RCC1, from meiotically arrested Xenopus egg extracts, with or without demembranated sperm nucleus, results in severe inhibition of aster formation. This effect was not restored by a mutant of RCC1, RCC1D182, that had very little GEF activity and produced very little RanGTP, but was reversed by wild-type RCC1. They also showed that the self-organization of the microtubule aster induced by RCC1 s GEF activity was dependent on the hydrolysis of the GTP associated with Ran. Experiments with RanGTPS resulted in the formation of a large number of very small asters, even after prolonged incubation. Thus, the hydrolysis of RanGTP, generated by the GEF activity of RCC1, is a key factor for proper microtubule aster formation. To find the nuclear signals involved in the formation of microtubules after nuclear envelope breakdown during mitosis, Wilde and Zheng (1999) resorted to the CSF-arrested Xenopus egg extracts. Their assay was based on the ability of the demembranated sperm nuclei to nucleate microtubule asters, which are subsequently organized into spindles. They found that a previously described GTP-locked mutant of Ran, RanL43E (Lounsbury et al. 1996), could activate cellular factors in the egg extract to stimulate microtubule polymerization in the absence of sperm nuclei. This particular mutant alone had the ability to induce the formation of a bipolar spindle in egg extracts similar to those produced by sperm nuclei, except that the spindles are smaller and less numerous. By immunodepletion of γ-turc and XMAP215, a microtubule binding protein that increases the assembly rate at the plus ends, from egg extracts, they showed that both γ-turc and XMAP215 are required for microtubule polymerization. Both of these studies reported the dependence of Ran-induced microtubule formation on the minus enddirected motor protein dynein. This was consistent with the finding that treatment of egg extracts with antibodies against dynein resulted in the inhibition of selforganization of microtubule aster (Ohba et al., 1999) and prevented the formation of microtubule asters normally induced by RanL43E (Wilde and Zheng, 1999). Zhang et al. (1999) showed that increased concentrations of RanGTP, generated either by RCC1 overexpression or by use of Ran mutants defective in GTP hydrolysis, resulted in microtubule stabilization and a subsequent block in the mitosis to interphase transition. As a corollary to this, generation of RanGDP either by RanT24N, a mutant of Ran that is basically a constitutive RanGDP source, or by RanBP1, did not stabilize the asters. The study by Carazo-Salas et al. (1999) was based on an in vitro system in which magnetic beads coupled with plasmid DNA acted as the template for spindle assembly in CSF-arrested Xenopus egg extracts (Heald et al., 1996).

4 RANdevous with the Spindle 109 Figure 2: Model for the mechanism of Ran cycle-mediated spindle assembly. Merging the localized gradient hypothesis with the mechanism of Ran action. RCC1 is bound to the chromatin through a protein complex (not shown). The putative factor that could be a part of this complex and may hold the key to link Ran to the cell cycle is shown. The two conformational states of RanGTP are shown as RanGTP(1) and RanGTP(2). The RanGTP(2) form binds to RCC1. It is released from RCC1 in the RanGTP(1) conformation that is competent for hydrolysis by RanBP1 and RanGAP. The yet undiscovered effector protein through which RanGTP may exert its effect in spindle assembly is also shown RCC1 generates a high concentration of RanGTP in the vicinity of chromatin that, with the help of RanBPM and γ-turc (recruited by RanBPM), nucleates spindle assembly. High RanGDP concentration away from chromatin is generated by the coupled action of RanGAP1 and RanBP1. The horizontal arrow indicates the gradient, with higher RanGTP concentration near the tail. The decreasing vertical block arrows depict the distance effect. Increase in distance from the chromatin results in a decrease in RanGTP concentration due to its hydrolysis by RanBP1 and RanGAP1. This corresponds to decreasing spindle assembly away from chromatin in plant mitosis and mammalian meiosis (see text). Exploiting this chromosome bead assay along with different Ran mutants, they reiterated the results of previous studies and reported that RanGTP generated by RCC1 induced microtubule assembly around DNA beads in M phase extracts. In addition, they stated that RCC1 and RanGAP/RanBP1 specifically regulated the microtubule nucleation by modulating the relative levels of RanGTP and RanGDP. They also found that RCC1 was among the proteins that firmly bind chromatin beads. They suggested that the high local concentration of chromatin-associated RCC1 caused an increase in RanGTP levels in the vicinity of chromatin, which in turn resulted in spindle assembly. Thus, extensive use of Ran mutants and the meiotically arrested Xenopus egg extracts implicated a novel physiological role of RanGTP in microtubule assembly. But, what do these studies indicate about the role of Ran in mitotic spindle assembly? Function of Ran Cycle in Mitotic Assembly In contrast to the conventional mitotic spindle assembly pathways, where microtubules arising from centrosomes associated with chromosomes to generate a bipolar spindle, spindle assembly around chromatin in Xenopus extracts are centrosome-independent. From the studies described, it appears that Ran has two important functions; one in microtubule nucleation and the other in centrosome-independent microtubule assembly. To understand how Ran influences these processes, first consider the events in the cell during the transition from interphase to the mitotic state. As the cell cycle approaches the G2/M boundary, the nuclear envelope

5 110 The Einstein Quarterly Journal of Biology and Medicine breaks down, and there is a dramatic change in the stability of microtubules. The half-life of the microtubules is reduced from 10 minutes in interphase to about 1 minute following nuclear envelope breakdown (Desai and Mitchison, 1997). So, it is quite reasonable to assume that some regulation due to compartmentalization by the nucleus is lost during mitosis. The recent reports using Xenopus egg extracts indicate Ran involvement in the process of altering microtubule stability. During interphase, RCC1 remains bound to the chromatin, and the RanGAP and RanBP1 are localized in the cytoplasm. This results in a higher concentration of RanGTP inside the nucleus and a higher concentration of RanGDP in the cytoplasm (Gorlich, 1998). Upon entry into mitosis and dissolution of the nuclear envelope, this gradient is dispersed into localized gradients. These local gradients are a consequence of the high local concentration of chromatin-associated RCC1 giving rise to high RanGTP (Carazo-Salas et al., 1999) near the chromatin. As one moves away from the chromosome, due to the presence of RanBP1 and RanGAP, cooperative hydrolysis of RanGTP takes place that ensures a high RanGDP concentration outside the immediate vicinity of the chromatin (Carazo-Salas et al., 1999). The role of gradients in microtubule assembly is supported by the reports that other forms of RanGTP such as RanGTP-S or RanL43E, which are unable to hydrolyze the GTP, can induce bipolar aster formation in Xenopus extracts (Ohba et al., 1999; Wilde and Zheng, 1999). This function is, however, dependent on γ-turc and/or XMAP215, a plus end-directed motor protein (Wilde and Zheng, 1999). RanBPM could be the bridge between the gradient of RanGTP and the generation of bipolar spindles. RanBPM, which interacts with RanGTP, is normally localized to centrosomes. In systems such as dividing plant cells and meiotic animal cells where there are no centrosomes, it is free to nucleate the microtubule assembly at the chromatin cooperatively with RanGTP (Nakamura et al., 1998). After this centrosome-independent nucleation by RanBPM, RanGTP and other proteins, chromatin-associated kinesin-like proteins capture the microtubule plus ends. These proteins bind the nascent microtubules at the plus end and attach them to the chromosomes. The organization of microtubules at the minus end is mediated by the minus end-directed motor dynein. Finally, NuMA, a protein associated with microtubule minus ends, crosslinks the minus ends of the microtubule and forms a bipolar spindle. But how do high levels of RanGTP bring about the spindle assembly? Potential Mechanism of RanGTP Action. Several hypotheses exist for the mechanism of RanGTP action. The gradient hypothesis relies on the fact that high levels of RanGTP generated by RCC1 can induce microtubule formation (Zhang et al., 1999). Taxol or DMSO can induce similar effects by stabilizing the microtubule directly (Heald et al., 1997). None of the Ran mutants (RanG19V, RanQ69L, or RanL43E) can induce microtubule polymerization from purified α-β-tubulins, which indicates the involvement of another cellular factor (Wilde and Zheng, 1999). Indeed, recent reports showed that addition of RanG19V and RCC1 results in stabilization and formation of non-centrosomal microtubules in egg extracts (Kalab et al., 1999). This has been attributed to a distance effect, which states that the frequency of catastrophe, or depolymerization, of a microtubule is reduced in the vicinity of chromatin due to a gradient of enzyme products and is inversely proportional to the distance from the chromosome (Kalab et al., 1999). The enzyme may be RCC1-generating RanGTP, while RanGAP and RanBP1 create the gradient by RanGTP hydrolysis (Kalab et al., 1999). Finally, high local concentration of RanGTP can result in microtubule assembly. The mechanism of Ran action may be analogous to the way small GTPases act. Ran can follow the general mode of signal transduction pathways through the usual receptor, adaptor, GEF, GTPase, and effector. The signal here is the chromatin status, and the GEF is RCC1 (Nishimoto, 1999). The RCC1 may bind DNA through a protein-protein complex. This is consistent with the report that the yeast homologue of RCC1, Prp20, forms a complex with at least two other proteins inside the nucleus, one of which has a Ran-binding domain. This implies that a similar protein may serve as an adapter (Taura et al., 1997). Consistent with this idea, the deletion of the DNA binding domain of RCC1 has no effect on its localization at the chromatin (Seino et al., 1992). Structural analysis of RCC1 showed that it has a sevenbladed-propeller, one side of which binds Ran (Renault et al., 1998). This structure is similar to the β-subunit of the heterotrimeric G-protein. The interaction of RCC1 with Ran is like the α-β interaction of the heterotrimeric G proteins, where RCC1 is similar to the β-subunit (Renault et al., 1998). In analogy with the heterotrimeric G-proteins, Nishimoto (1999) proposes the presence of an unidentified subunit (Figure 2), which holds the critical link between the Ran-cycle and Ran GTPase action in spindle assembly. Despite this structural similarity, there is little evidence that it functions like the typical heterotrimeric G protein. Also, RanGTP exists in two conformations; one binds RCC1, and the other binds RanBP1 (Figure 2) (Geyer et al., 1999). Therefore, it can be hypothesized that the RanGTP(2) conformation binds to RCC1 and, upon release from RCC1, it is converted to RanGTP(1), which is competent for binding RanBP1 and eventual hydrolysis. This could contribute to a high RanGDP concentration away from the chromatin, which is critical for the gradient formation that drives spindle assembly (see Figure 2).

6 RANdevous with the Spindle 111 Another important finding would be a Ran binding protein that is directly involved in spindle assembly. The working model adapted and modified from Nishimoto (1999) connects the signaling and localized gradient hypothesis (Figure 2). In vivo studies should be one of the future priorities to establish this role of RanGTP. Lower eukaryotes such as yeast do not undergo nuclear envelope breakdown so it may not be a system of choice. One probable strategy is the microinjection of antibodies against different putative Ran effectors involved in microtubule assembly into blastomeres of Xenopus embryos at the proper stage (Cha et al., 1999). Another important multicellular system that is amenable to in vivo manipulations is a plant cell. In fact, in plant cell mitosis and in mammalian meiosis, chromatin can carry out centrosome-independent microtubule assembly. Moreover, Ran is highly conserved in plants, animals, and fungi (Ach and Gruissem, 1994). In tomato, Ran mrna is expressed in all of the tissues examined. The tomato Ran protein can suppress the S. pombe pim1 mutation (Ach and Gruissem, 1994), which results in premature chromosome condensation and entry into mitosis. The role of Ran and RCC1 could be investigated using transgenic plants that express inducible mutant Ran proteins. All these studies should be able to elucidate the role of Ran in microtubule assembly in the absence of centrosomes. Conclusion There is little doubt that RanGTP is involved in centrosome-independent microtubule assembly, but how it carries out this function is largely unknown. Several key questions need to be answered for the gradient hypothesis of RanGTP involvement in the spindle assembly to be tenable. First, is there any RanGTP binding protein that is directly involved in spindle assembly? Second, how justifiable is the gradient hypothesis? As Desai and Hyman (1999) suggest, it is imperative to show the lack of spindle formation in the absence of RCC1. In an RCC1 negative background, spindle formation at any concentration of RanGTP rules out the localized gradient hypothesis. And third, is there a putative factor that along with RanGTP and RCC1 forms a heterotrimeric complex that may be important for the Ran GTPase signaling pathway? It is important to clarify these doubts to show that the function of RanGTP in microtubule assembly is indeed real. Note Three recent reports (Wiese et al., 2001; Nachury et al., 2001; Gruss et al., 2001) suggest that the effector protein in the figure may be NuMA (a nuclear assembly protein) which is generally inactive during interphase due to its binding to the importins. Absence of a nuclear membrane and a high RanGTP concentration near the chromatin causes local release of NuMA and other related factors, thereby promoting spindle assembly. 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