Assembly and disassembly of the mitotic spindle are essential for both chromosome segregation and cell division. The small G-protein Ran has emerged as an important regulator of spindle assembly. In this review, we look at the role of Ran in different aspects of spindle assembly, including its effects on microtubule assembly dynamics and microtubule organization. In addition, we examine the possibility of a spindle matrix and the role Ran might play in such a structure.
Mitotic spindle assembly involves many factors that model the microtubule cytoskeleton into a bipolar structure consisting of three main types of microtubules. The astral microtubules emanate from two centrosomes at the spindle poles and interact with the cell cortex. The ‘pole-to-pole’ microtubules form anti-parallel interactions in the spindle. Finally, the K-fibres (kinetochore microtubule fibres) capture chromosomes and assist their alignment and segregation (Figure 1). The challenge in understanding spindle assembly is to decipher how the many factors involved co-ordinate with one another to build this complex and dynamic machine during mitosis, a relatively brief period of the cell cycle in somatic cells.
A schematic diagram of a metazoan mitotic spindle
Extensive studies have shown that mitotic entry driven by CDK1 (cyclin-dependent kinase 1) initiates and regulates spindle assembly by direct or indirect phosphorylation of a large number of SAFs (spindle assembly factors). These SAFs in turn regulate changes in both microtubule dynamic instability and microtubule organization that are essential for spindle assembly. Although the cell-cycle paradigm has proven critical in studying spindle morphogenesis, several early observations implicated the involvement of a nuclear signal in spindle assembly. For example, rupturing the prophase nuclear envelope of the grasshopper spermatocytes using a micro-needle was shown to induce spindle assembly next to condensed chromosomes . Similarly, it was found that chromosomes and nuclei could stimulate microtubule nucleation from the nearby centrosomes in Xenopus eggs . Studies in the past 7 years have now firmly established the role of the G-protein Ran as a nuclear signal that regulates spindle assembly in mitosis.
In this review, we will first provide an overview of the effect of the Ran system on the microtubule cytoskeleton and how this system may communicate with the cell-cycle machinery to regulate mitosis. Then, we will describe the current understanding of how Ran regulates spindle assembly through a number of SAFs. After a discussion of the studies that have begun to shed light on how Ran could co-ordinate both microtubule assembly and organization through the mitotic kinase Aurora A, we will end with a section describing some more recent studies on Ran and the mitotic spindle matrix.
Ran-GTP as the nuclear signal in spindle assembly
Ran is an abundant nuclear G-protein with a well-established role in regulating nucleocytoplasmic transport in interphase  that predates knowledge of its role in mitosis. Ran is a member of the Ras family of monomeric G-proteins and has one known GAP (GTPase-activating protein) called RanGAP1 and one known GEF (guanine nucleotide-exchange factor) called RCC1. RCC1 is associated with chromatin, where it catalyses Ran to exchange its GDP for GTP, whereas RanGAP1 is located in the cytoplasm where it stimulates the hydrolysis of Ran-GTP into Ran-GDP. With this unique distribution of Ran regulators in interphase cells, a much higher concentration of Ran-GTP is maintained in the nucleus as compared with the cytoplasm. This large Ran-GTP concentration difference across the nuclear envelope, often referred to as the Ran-GTP concentration gradient, is essential for nucleocytoplasmic transport. Extensive studies have demonstrated that Ran uses its GTPase cycle to regulate both nuclear import and export with the help of transport receptors (Figure 2).
The Ran system controls nuclear import and export through the use of specialized receptors
Although most of the early research on Ran had focused on its role in nuclear trafficking, studies of a number of mutations in various components of the Ran system in yeast suggested that it might have a function in mitosis [4–6]. To define the mitosis-specific function of Ran, it is necessary to assay the mitotic function of Ran directly without the complication of its interphase role. A number of groups used extracts made from mature Xenopus eggs arrested in the second meiosis to study the effect of Ran on the microtubule cytoskeleton in mitosis because this egg extract system is known to support spindle assembly from the demembranated Xenopus sperm in vitro . Amazingly, these groups found that mutant Ran proteins that are constitutively bound to GTP (Ran-GTP) could induce assembly of both microtubule asters and spindle-like structures in the egg extracts in the absence of chromatin and centrosomes . Further studies showed that Ran-GTP regulates several aspects of microtubule behaviours in mitosis, from promoting microtubule stabilization and nucleation to stimulating microtubule organization into antiparallel arrays found in the spindle [9,10].
At least in animal cells, Ran-GTP appears to form a gradient in mitosis with the highest concentration on the condensed chromosomes that tapers off towards the periphery of the cell [11,12]. Experiments in Xenopus egg extracts, in conjunction with computer simulations, further suggested that a high Ran-GTP concentration near the chromosomes stimulates microtubule nucleation, whereas microtubule stabilization is favoured by the lower concentration of Ran-GTP found farther away from the chromosomes . These differential effects of Ran-GTP on microtubules could be critical for spindle assembly. Together, these studies indicate that the mitotic Ran-GTP concentration gradient is important to navigate spindle assembly towards the condensed, RCC1-rich chromosomes.
Studies using mammalian cultured cells and Xenopus egg extracts have shown that production of Ran-GTP by RCC1 in mitosis is regulated directly by the cell-cycle machinery (Figure 3A). RCC1 exhibits dynamic interactions with chromatin in both interphase and mitosis with short mean residence times (interphase, 50±12 s; mitosis, 20±5 s) . Thus efficient production of a Ran-GTP gradient in mitosis requires RCC1 to couple its chromosome binding with its GEF activity towards Ran . This coupling is dependent on CDK1 phosphorylation of the N-terminus of RCC1 that contains an NLS (nuclear localization signal) [11,15,16]. The phosphorylation event prevents binding of importins α and β to the NLS of RCC1, allowing RCC1 to produce Ran-GTP only during the period when RCC1 is bound to mitotic chromosomes.
The mitotic functions of the Ran system
Interestingly, the Ran system may also influence cell-cycle progression in mitosis by regulating the spindle checkpoint (Figure 3A). Artificially increasing the concentration of Ran-GTP by adding excess RCC1 overrides the spindle checkpoint and activates the anaphase-promoting complex in Xenopus egg extracts . Conversely, increasing the concentration of RanGAP1 or RanBP1, which facilitates Ran-GTP hydrolysis, can restore checkpoint activity to the egg extracts. Therefore a high Ran-GTP concentration can override the spindle checkpoint induced in egg extracts. In addition, a number of proteins in the Ran system such as Crm1, RanGAP1 and RanBP2 (also called Nup358) are found at the kinetochores in mitosis and their perturbation leads to defects in kinetochore–microtubule interactions . Although exactly how the Ran system regulates the spindle checkpoint remains to be elucidated, further studies of the cross-regulation between this system and the cell-cycle machinery should provide additional insights into the control of mitotic progression.
Ran-GTP activates SAFs in mitosis
The knowledge of nucleocytoplasmic transport (Figure 2) has helped the effort to decipher how Ran-GTP regulates spindle assembly in mitosis in metazoans. A number of SAFs were known to be shuttled into the nucleus during interphase because they contain NLSs [19,20]. Studies have shown that some of these SAFs are inactivated upon binding to nuclear import receptors such as importin-β and importin-7 through their NLS after nuclear envelope breakdown in mitosis. Ran-GTP can bind to the importins, releasing associated SAFs and allowing them to mediate spindle formation (Figure 3B). Therefore Ran uses a similar mechanism to regulate nuclear import in interphase and spindle assembly in mitosis.
Two SAFs, NuMA (nuclear mitotic apparatus protein) and TPX2 (targeting protein for Xklp2), were among the first shown as downstream targets of Ran-GTP in mitosis. Both SAFs contain NLSs and are found in the nucleus in interphase. They both interact with importin-β through the adaptor importin-α in mitosis and Ran-GTP has been shown to inhibit such interactions in mitosis [21–23]. Studies have demonstrated that Ran-GTP-mediated release of TPX2 from importin α/β activates this SAF to participate in both microtubule nucleation and activation of a mitotic kinase called Aurora A (see below). However, it remains unclear which function(s) of NuMA is stimulated upon its release from importin α/β by Ran-GTP. NuMA is localized to the spindle poles where it functions along with the minus end-directed motor dynein and the dynein activator dynactin to focus the minus ends of microtubules . More recent studies suggest that NuMA can also regulate spindle assembly by interacting with another protein called LGN (lysine/glycine/asparagine-repeat-containing protein) . The release of importins might be necessary for NuMA to function more efficiently with dynein/dynactin, with LGN, or with both during spindle assembly.
Additional SAFs that are regulated by Ran-GTP and importin-β include XCTK2 (Xenopus C-terminal kinesin 2), Kid, and Rae1. The kinesin-C motor XCTK2 cross-links parallel microtubules through its C-terminally located motor domain and the non-motor microtubule-binding domain located at the N-terminus of the protein . XCKT2 can facilitate the congressing of microtubule minus-ends at spindle poles similar to that of dynein. Importin-β was shown to bind to the NLS located in the non-motor domain of XCTK2 through importin-α, which blocks association of this domain with microtubules. Ran-GTP removes importins to activate the cross-linking activity of XCTK2 on microtubules. Kid (Xkid in Xenopus), another motor, is a kinesin-like protein that associates with both condensed mitotic chromosomes (via the C-terminally located DNA-binding domain) and spindle microtubules (via the N-terminally located motor domain). These interactions are important for Kid to help push the chromosome arms away from spindle poles and assist in chromosome alignment in mitosis [27,28]. Ran-GTP appears to activate the microtubule-binding function of Kid by releasing the importin α/β bound to the NLS in N-terminal portion of the protein .
Unlike the SAFs mentioned above, Rae-1, a component of a ribonucleoprotein complex involved in mRNA export, binds importin-β independently of importin-α in a Ran-GTP-sensitive manner . It was identified through a series of biochemical fractionations aimed at isolating factors of spindle assembly that are inhibited directly by importin-β binding in mitosis. Rae-1 is required for proper spindle assembly in both culture cells and Xenopus egg extracts. The mitotic function of Rae-1 may involve RNA, but precisely what type(s) of RNA and how it might function in spindle assembly remains unclear. So far, the only SAF found to be negatively regulated by more than one import receptor is NuSAP (nucleolar and spindle-associated protein). NuSAP is imported into the nucleus by either importin-β or importin-7 and, as indicated by the name, it is localized to the nucleolus in interphase . In mitosis, NuSAP regulates spindle assembly by cross-linking microtubules. Its microtubule cross-linking role can be disrupted by either importin-β or importin-7 and Ran-GTP can at least partially block this disruption .
Ran-GTP and the Aurora A kinase in spindle assembly
As an increasing number of the Ran-regulated SAFs are uncovered, a logical next question is whether Ran plays a role in co-ordinating the functions of these SAFs to nucleate and organize microtubules into the mitotic spindle. Studies revealed that part of the co-ordination of spindle assembly by Ran-GTP evident in mitosis may be mediated by Aurora A, a mitotic kinase highly concentrated at the centrosomes and spindle poles . Aurora A (also called Eg2 in Xenopus) was first found in a genetic screen in Drosophila as a gene whose mutant alleles caused mitotic defects . In Xenopus eggs, Aurora A is essential for the maintenance of spindle bipolarity . Additional studies carried out in Caenorhabditis elegans and Drosophila using RNA interference and Aurora A mutants respectively not only verified the function of Aurora A in spindle assembly but also revealed an additional requirement of this kinase for microtubule nucleation from the mitotic centrosomes . Indeed, this kinase is required for the recruitment of a number of proteins to the mitotic centrosomes including known microtubule nucleators such as the γTuRC (γ-tubulin ring complex)  and XMAP215 (Xenopus microtubule-associated protein 215; also called Zyg9 in C. elegans and Msps in Drosophila) [34–36]. In addition, Aurora A was shown to interact with and possibly phosphorylate the plus-end-directed kinesin Eg5 required for maintaining spindle bipolarity . Since Ran-GTP was also shown to regulate microtubule assembly and microtubule-based motor activities [9,10], these studies of Aurora A imply a connection between the kinase and the G-protein. Consistent with this idea, biochemical studies revealed that Ran-GTP activates Aurora A by releasing TPX2 from importin α/β. The free TPX2 in turn binds and activates Aurora A with the help of microtubules [29,38]. Several more recent reports have begun to shed light on how Aurora A activation mediated by Ran-GTP could function to co-ordinate multiple SAFs including those that regulate microtubule assembly dynamics and those that organize microtubules.
As mentioned above, Aurora A regulates Eg5, XMAP215 and γTuRC during spindle assembly. Several studies have independently shown that Aurora A recruits and phosphorylates Maskin [also called D-TACC (Drosophila transforming acidic coiled-coil) in Drosophila] at centrosomes, which allows Maskin to recruit XMAP215 to facilitate microtubule nucleation and assembly at the mitotic centrosomes [34–36]. Interestingly, Aurora A is present in a complex containing XMAP215, Eg5, TPX2 and a newly characterized SAF called HURP (hepatoma up-regulated protein) in Xenopus egg extracts and this HURP complex can convert astral microtubule arrays induced by Ran-GTP into antiparallel arrays found in the spindle . Since Aurora A appears to be essential for the assembly of the HURP complex, these findings provide a biochemical explanation for how Ran-GTP could activate both microtubule assembly and microtubule organization via Aurora A kinase. It also helps to explain why mitotic centrosomes (which have high concentrations of Aurora A) or magnetic beads coated with Aurora A protein  can function as potent microtubule-organizing centres for spindle assembly. It is tempting to speculate that perhaps by bringing XMAP215 and Eg5 into the same complex via Aurora A, any polymerized microtubules (stimulated by XMAP215 for example) would have the proper assembly dynamics for Eg5 to organize them into antiparallel configurations found in the spindle. Clearly, additional studies are necessary to further elucidate mechanistically how Ran-GTP and Aurora A could regulate the activities of different components of the HURP complex during spindle assembly. It is also important to further explore whether and how other SAFs, including γTuRC, are regulated by the Ran-GTP–Aurora A pathway to achieve co-ordinated assembly and organization of mitotic microtubules.
Besides participating in bipolar spindle assembly as part of the HURP complex, the HURP protein itself is found on K-fibres near kinetochores and is phosphorylated by both CDK1  and Aurora A . HURP is also controlled by Ran-GTP, which dissociates importin-β from HURP and allows it to associate with K-fibres. This may allow HURP to regulate proper chromosome alignment and segregation by binding and stabilizing microtubules in general and K-fibres in particular [43,44]. Since HURP is regulated by both Ran-GTP and Aurora A, it is possible that the functions ascribed to HURP are accomplished with the help of other proteins in the HURP complex described above. If this was the case, the Ran-GTP–Aurora A pathway may regulate both the establishment of bipolarity of the spindle and chromosome movements in mitosis.
Ran-GTP and the mitotic spindle matrix
Assembly of multiple SAFs into one complex, as described above, represents one way to co-ordinate several activities during spindle formation. Another potential way to accomplish the same task might be through the mitotic spindle matrix that was proposed decades ago and revisited by several studies more recently [45,46]. Such a matrix was presumed to allow molecular motors to gain traction when pushing or pulling, provide anchor points for SAFs, and help organize and stabilize the spindle structure. However, a suitable building block for such a matrix has remained elusive. Such a structural candidate would be expected to be capable of polymerization, branching/cross-linking, absorbing spindle forces and tethering SAFs. In addition, it would remain static relative to the spindle microtubules and its existence would be independent of microtubules.
A number of recent studies have uncovered proteins with certain features ascribed to the spindle matrix. Four Drosophila nuclear proteins, called Skeletor, Chromator, Megator and EAST (Enhanced Adult Sensory Threshold locus product), have been shown to decorate spindles in mitosis and remain in spindle-like structures after microtubule depolymerization [47–50]. These proteins interact with one another and disruption of each by various means leads to spindle assembly defects. It is currently unclear whether the putative spindle matrix defined by these proteins in Drosophila can tether SAFs and regulate their activities in mitosis.
Another candidate for spindle matrix is PAR [poly(ADP-ribose)] attached to proteins on the spindle. PAR represents a protein modification capable of polymerization and branching . Spindle-associated PAR is static and perturbing PAR modification causes defects in spindle structures assembled in Xenopus egg extracts . One PAR polymerase, Tankyrase-1, is found to be required for spindle assembly in HeLa cells . Since Tankyrase-1 can poly(ADP-ribosyl)ate NuMA [53,54], PAR modification of certain SAFs such as NuMA could be important for spindle assembly. It remains unclear whether it is PAR itself or SAFs that PAR modifies that function as the spindle matrix.
A third protein implicated as a component of spindle matrix is called Fin1p in Saccharomyces cerevisiae . Fin1p is a nuclear protein in interphase and it decorates yeast spindles in mitosis. Purified Fin1p can assemble into filaments with a similar dimension to the intermediate filaments . However, it is not yet known whether Fin1p is required for spindle assembly or function in yeast.
A more recent study implicated the intermediate filament protein lamin B as a structural component of the spindle matrix . Lamin B is found in the nucleus during interphase where it serves a structural role as a component of the nuclear lamina . Reduction of lamin B in either HeLa cells or in Xenopus egg extracts led to spindle defects. Further studies in Xenopus egg extracts revealed that Ran-GTP stimulates assembly of a lamin B-based membranous matrix that tethers a number of SAFs . This matrix associates with spindles and remains in an assembled state after microtubule depolymerization. Also, lamin B mutants known to disrupt interphase nuclear lamina disrupted the mitotic lamin matrix and caused spindle defects in Xenopus egg extracts, indicating that this intermediate filament protein could mediate assembly of the spindle matrix. The presence of lipids within this lamin B matrix and its detergent sensitivity suggest that the spindle matrix might contain certain mitotic membrane compartment(s). In this context, it is worth noting that a number of proteins with interphase membrane-related functions have been recently shown to regulate spindle morphogenesis and/or chromosome segregation in mitosis [58–60]. We speculate that these proteins might carry out their mitotic functions either through their ability to tether to the membranous spindle matrix or by regulating the assembly of the membrane component of this matrix. Clearly, studies of the spindle matrix are in their infancy. However, the identification of candidate components of the spindle matrix and the finding that Ran could regulate the assembly of the matrix promise to stimulate further interests in studying spindle morphogenesis and chromosome segregation in a broader context that goes beyond the microtubule cytoskeleton.
Co-ordination of Cellular Processes: A Focus Topic at BioScience2006, held at SECC Glasgow, U.K., 23–27 July 2006. Edited by P. Clarke (Dundee, U.K.), P. Coffer (Utrecht, The Netherlands), M. Cousin (Edinburgh, U.K.), I. Dransfield (Edinburgh, U.K.), S. High (Manchester, U.K.) and G. Rutter (Imperial College London, U.K.).
guanine nucleotide-exchange factor
hepatoma up-regulated protein
kinetochore microtubule fibres
nuclear localization signal
nuclear mitotic apparatus protein
nucleolar and spindle-associated protein
spindle assembly factor
targeting protein for Xklp2
γ-tubulin ring complex
Xenopus C-terminal kinesin 2
Xenopus microtubule-associated protein 215
We thank Dan Ducat, Queenie Vong and Zhonghua Liu for critical reading of this paper. We apologize for omitted citations owing to space limitations. This work was supported by grants from the Howard Hughes Medical Institution and the National Institute of General Medical Sciences (GM56312).