Mitotic functions of the Ran GTPase network: the importance of being in the right place at the right time

The Ran GTPase has important roles in nucleocytoplasmic transport, cell cycle progression, nuclear organization and nuclear envelope (NE) assembly. In this review, we discuss emerging evidence that implicate the Ran GTPase system in mitotic control in mammalian cells. Recent work indicates that members of the Ran network control two fundamental aspects of the mammalian mitotic apparatus: (i) centrosome and spindle pole function, and (ii) kinetochore function. It is also emerging that, after NE breakdown, specific Ran network components assemble in local combinations at crucial sites of the mitotic apparatus. In the light of these findings, the original notion that nucleotide-bound forms of the Ran GTPase are distributed along a unique "gradient" in mitotic cells should be re-examined. Available data also suggest that the Ran system is deregulated in certain cellular contexts: this may represent a favoring condition for the onset and propagation of mitotic errors that can predispose cells to become genetically unstable and facilitate neoplastic growth.     

Mitotic Functions of the Ran GTPase Network: the Importance of Being in the Right Place at the Right Time

The Ran GTPase has important roles in nucleocytoplasmic transport, cell cycle progression, nuclear organization and nuclear envelope (NE) assembly. In this review, we discuss emerging evidence that implicate the Ran GTPase system in mitotic control in mammalian cells. Recent work indicates that members of the Ran network control two fundamental aspects of the mammalian mitotic apparatus: (i) centrosome and spindle pole function, and (ii) kinetochore function. It is also emerging that, after NE breakdown, specific Ran network components assemble in local combinations at crucial sites of the mitotic apparatus. In the light of these findings, the original notion that nucleotide-bound forms of the Ran GTPase are distributed along a unique “gradient” in mitotic cells should be re-examined. Available data also suggest that the Ran system is deregulated in certain cellular contexts: this may represent a favoring condition for the onset and propagation of mitotic errors that can predispose cells to become genetically unstable and facilitate neoplastic growth.     

Role of importin-beta in the control of nuclear envelope assembly by Ran

Compartmentalization of the genetic material into a nucleus bounded by a nuclear envelope (NE) is the hallmark of a eukaryotic cell. The control of NE assembly is poorly understood, but in a cell-free system made from Xenopus eggs, NE assembly involves the small GTPase Ran. In this system, Sepharose beads coated with Ran induce the formation of functional NEs in the absence of chromatin. Here, we show that importin-beta, an effector of Ran involved in nucleocytoplasmic transport and mitotic spindle assembly, is required for NE assembly induced by Ran. Concentration of importin-beta on beads is sufficient to induce NE assembly in Xenopus egg extracts. The function of importin-beta in NE assembly is disrupted by a mutation that decreases affinity for nucleoporins containing FxFG repeats. By contrast, a truncated protein that cannot interact with importin-alpha is functional. Thus, importin-beta functions in NE assembly by recruiting FxFG nucleoporins rather than by interaction through importin-alpha with karyophilic proteins carrying classical nuclear localization signals. Importin-beta links NE assembly, mitotic spindle assembly, and nucleocytoplasmic transport to regulation by Ran and may coordinate these processes during cell division.     

RanGTP and CLASP1 cooperate to position the mitotic spindle

Accurate positioning of the mitotic spindle is critical to ensure proper distribution of chromosomes during cell division. The small GTPase Ran, which regulates a variety of processes throughout the cell cycle, including interphase nucleocytoplasmic transport and mitotic spindle assembly, was recently shown to also control spindle alignment. Ran is required for the correct cortical localization of LGN and nuclear-mitotic apparatus protein (NuMA), proteins that generate pulling forces on astral microtubules (MTs) through cytoplasmic dynein. Here we use importazole, a small-molecule inhibitor of RanGTP/importin-β function, to study the role of Ran in spindle positioning in human cells. We find that importazole treatment results in defects in astral MT dynamics, as well as in mislocalization of LGN and NuMA, leading to misoriented spindles. Of interest, importazole-induced spindle-centering defects can be rescued by nocodazole treatment, which depolymerizes astral MTs, or by overexpression of CLASP1, which does not restore proper LGN and NuMA localization but stabilizes astral MT interactions with the cortex. Together our data suggest a model for mitotic spindle positioning in which RanGTP and CLASP1 cooperate to align the spindle along the long axis of the dividing cell.     

The interaction between Ran and NTF2 is required for cell cycle progression

The small GTPase Ran is required for the trafficking of macromolecules into and out of the nucleus. Ran also has been implicated in cell cycle control, specifically in mitotic spindle assembly. In interphase cells, Ran is predominately nuclear and thought to be GTP bound, but it is also present in the cytoplasm, probably in the GDP-bound state. Nuclear transport factor 2 (NTF2) has been shown to import RanGDP into the nucleus. Here, we examine the in vivo role of NTF2 in Ran import and the effect that disruption of Ran imported into the nucleus has on the cell cycle. A temperature-sensitive (ts) mutant of Saccharomyces cerevisiae NTF2 that does not bind to Ran is unable to import Ran into the nucleus at the nonpermissive temperature. Moreover, when Ran is inefficiently imported into the nucleus, cells arrest in G(2) in a MAD2 checkpoint-dependent manner. These findings demonstrate that NTF2 is required to transport Ran into the nucleus in vivo. Furthermore, we present data that suggest that depletion of nuclear Ran triggers a spindle-assembly checkpoint-dependent cell cycle arrest.     

The BRCA1/BARD1 heterodimer modulates ran-dependent mitotic spindle assembly

The heterodimeric tumor-suppressor complex BRCA1/BARD1 exhibits E3 ubiquitin ligase activity and participates in cell proliferation and chromosome stability control by incompletely defined mechanisms. Here we show that, in both mammalian cells and Xenopus egg extracts, BRCA1/BARD1 is required for mitotic spindle-pole assembly and for accumulation of TPX2, a major spindle organizer and Ran target, on spindle poles. This function is centrosome independent, operates downstream of Ran GTPase, and depends upon BRCA1/BARD1 E3 ubiquitin ligase activity. Xenopus BRCA1/BARD1 forms endogenous complexes with three spindle-pole proteins, TPX2, NuMA, and XRHAMM--a known TPX2 partner--and specifically attenuates XRHAMM function. These observations reveal a previously unrecognized function of BRCA1/BARD1 in mitotic spindle assembly that likely contributes to its role in chromosome stability control and tumor suppression.     

Coordination of Chromosome Alignment and Mitotic Progression Chromosome-Based Ran Signal

Mitotic spindle assembly and chromosome segregation are controlled by the cell cycle machinery and by the guanosine triphosphatase Ran (RanGTPase). We developed a spatial model that allows us to simulate RanGTP production with different degrees of chromosome alignment in mitosis. Aided by this model, we defined three factors that modulate mitotic RanGTP gradients and mitotic progression in somatic cells. First, the concentration of RanGTPtransport-receptor (represented by RanGTP-importin β) and its spatial distribution are very sensitive to the level of RanBP1. Reduction of RanBP1 leads to an elevated RanGTP-transport receptor concentration throughout the cell, which disrupts spindle assembly and weakens spindle checkpoint control. Second, the completion of chromosome alignment at the metaphase plategenerates highest local RanGTP concentrations on chromosomes that could lead to spindle checkpoint silencing and metaphase-anaphase transition. Finally, chromosomal RanGTP production could be dampened by a reduction of RCC1 phosphorylation in mitosis. Our spatialsimulation of RanGTP production using individual chromosomes should provide means to further understand how the Ran system and the cell cycle machinery coordinately regulate mitosis.     

Subgroup II PAK-mediated phosphorylation regulates Ran activity during mitosis

Ran is an essential GTPase that controls nucleocytoplasmic transport, mitosis, and nuclear envelope formation. These functions are regulated by interaction of Ran with different partners, and by formation of a Ran-GTP gradient emanating from chromatin. Here, we identify a novel level of Ran regulation. We show that Ran is a substrate for p21-activated kinase 4 (PAK4) and that its phosphorylation on serine-135 increases during mitosis. The endogenous phosphorylated Ran and active PAK4 dynamically associate with different components of the microtubule spindle during mitotic progression. A GDP-bound Ran phosphomimetic mutant cannot undergo RCC1-mediated GDP/GTP exchange and cannot induce microtubule asters in mitotic Xenopus egg extracts. Conversely, phosphorylation of GTP-bound Ran facilitates aster nucleation. Finally, phosphorylation of Ran on serine-135 impedes its binding to RCC1 and RanGAP1. Our study suggests that PAK4-mediated phosphorylation of GDP- or GTP-bound Ran regulates the assembly of Ran-dependent complexes on the mitotic spindle.     

Ran GTPase: a master regulator of nuclear structure and function during the eukaryotic cell division cycle?

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.     

Regulated Ran-binding protein 1 activity is required for organization and function of the mitotic spindle in mammalian cells in vivo.

Ran-binding protein (RanBP) 1 is a major regulator of the Ran GTPase and is encoded by a regulatory target gene of E2F factors. The Ran GTPase network controls several cellular processes, including nucleocytoplasmic transport and cell cycle progression, and has recently also been shown to regulate microtubule nucleation and spindle assembly in Xenopus oocyte extracts. Here we report that RanBP1 protein levels are cell cycle regulated in mammalian cells, increase from S phase to M phase, peak in metaphase, and abruptly decline in late telophase. Overexpression of RanBP1 throughout the cell cycle yields abnormal mitoses characterized by severe defects in spindle polarization. In addition, microinjection of anti-RanBP1 antibody in mitotic cells induces mitotic delay and abnormal nuclear division, reflecting an abnormal stabilization of the mitotic spindle. Thus, regulated RanBP1 activity is required for proper execution of mitosis in somatic cells.     

Subcellular localization and role of Ran1 in Tetrahymena thermophila amitotic macronucleus

Amitosis, a direct method of cell division is common in ciliated protozoan, fungi and some animal and plant cells. During amitosis, intranuclear microtubules are reorganized into specified arrays which assist in separation of nucleus, despite lack of a bipolar spindle. However, the regulation of amitosis is not understood. Here, we focused on the localization and role of mitotic spindle assembly regulator: Ran GTPase (Ran1) in macronuclear amitosis in binucleated protozoan Tetrahymena thermophila. HA-tagged Ran1 was localized in the macronucleus throughout the cell cycle of Tetrahymena during vegetative growth, and the accessory factor binding domains of Ran1 contributed to its macronuclear localization. Incomplete somatic knockout of RAN1 resulted in aberrant intramacronuclear microtubule array formation, missegregation of macronuclear chromosomes and ultimately blocked macronuclei proliferation. When the Ran1 cycle was perturbed by overexpression of Ran1T25N (GDP-bound Ran1-mimetic) or Ran1Q70L (GTP-bound Ran1-mimetic), intramacronuclear microtubule assembly was inhibited or multi-micronucleate cells formed. These results suggest that Ran GTPase pathway is involved in assembly of a specialized intramacronuclear microtubule network and coordinates amitotic progression in Tetrahymena.     

Spindles get the ran around

Despite its fundamental role in cell division, the mitotic spindle remains an enigmatic figure in cell biology. This is due to the complex dynamic behaviour of microtubules, which form the spindle fibres responsible for segregating chromosomes to opposite ends of the cell during mitosis. Recent reports indicate that the small GTPase Ran, which plays a key role in nuclear transport, also has a role in mitosis by regulating microtubule nucleation and/or growth. The race is now on to determine how Ran exerts its effects on spindle assembly.     

Overexpression of RAN1 in rice and Arabidopsis alters primordial meristem, mitotic progress, and sensitivity to auxin

Ran is an evolutionarily conserved eukaryotic GTPase. We previously identified a cDNA of TaRAN1, a novel Ran GTPase homologous gene in wheat (Triticum aestivum) and demonstrated that TaRAN1 is associated with regulation of genome integrity and cell division in yeast (Saccharomyces cerevisiae) systems. However, much less is known about the function of RAN in plant development. To analyze the possible biological roles of Ran GTPase, we overexpressed TaRAN1 in transgenic Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). TaRAN1 overexpression increased the proportion of cells in the G2 phase of the cell cycle, which resulted in an elevated mitotic index and prolonged life cycle. Furthermore, it led to increased primordial tissue, reduced number of lateral roots, and stimulated hypersensitivity to exogenous auxin. The results suggest that Ran protein was involved in the regulation of mitotic progress, either in the shoot apical meristem or the root meristem zone in plants, where auxin signaling is involved. This article determines the function of RAN in plant development mediated by the cell cycle and its novel role in meristem initiation mediated by auxin signaling.     

Phosphorylation regulates the dynamic interaction of RCC1 with chromosomes during mitosis

The small GTPase Ran has multiple roles during the cell division cycle, including nuclear transport, mitotic spindle assembly, and nuclear envelope formation. However, regulation of Ran during cell division is poorly understood. Ran-GTP is generated by the guanine nucleotide exchange factor RCC1, the localization of which to chromosomes is necessary for the fidelity of mitosis in human cells. Using photobleaching techniques, we show that the chromosomal interaction of human RCC1 fused to green fluorescent protein (GFP) changes during progression through mitosis by being highly dynamic during metaphase and more stable toward the end of mitosis. The interaction of RCC1 with chromosomes involves the interface of RCC1 with Ran and requires an N-terminal region containing a nuclear localization signal. We show that this region contains sites phosphorylated by mitotic protein kinases. One site, serine 11, is targeted by CDK1/cyclin B and is phosphorylated in mitotic human cells. Phosphorylation of the N-terminal region of RCC1 inhibits its binding to importin alpha/beta and maintains the mobility of RCC1 during metaphase. This mechanism may be important for the localized generation of Ran-GTP on chromatin after nuclear envelope breakdown and may play a role in the coordination of progression through mitosis.     

The Ran GTPase: theme and variations

The small GTPase Ran has roles in multiple cellular processes, including nuclear transport, mitotic spindle assembly, the regulation of cell cycle progression and nuclear assembly. The past year has seen a remarkable unification of these different roles with respect to the effectors and mechanisms through which they function. Our emergent understanding of Ran suggests that it plays a central role in spatial and temporal organization of the vertebrate cell.     

Ran is required before metaphase for spindle assembly and chromosome alignment and after metaphase for chromosome segregation and spindle midbody organization

The Ran pathway has been shown to have a role in spindle assembly. However, the extent of the role of the Ran pathway in mitosis in vivo is unclear. We report that perturbation of the Ran pathway disrupted multiple steps of mitosis in syncytial Drosophila embryos and uncovered new mitotic processes that are regulated by Ran. During the onset of mitosis, the Ran pathway is required for the production, organization, and targeting of centrosomally nucleated microtubules to chromosomes. However, the role of Ran is not restricted to microtubule organization, because Ran is also required for the alignment of chromosomes at the metaphase plate. In addition, the Ran pathway is required for postmetaphase events, including chromosome segregation and the assembly of the microtubule midbody. The Ran pathway mediates these mitotic events, in part, by facilitating the correct targeting of the kinase Aurora A and the kinesins KLP61F and KLP3A to spindles.     

Polo-like kinase 1-mediated phosphorylation of the GTP-binding protein Ran is important for bipolar spindle formation

Polo-like kinase functions are essential for the establishment of a normal bipolar mitotic spindle, although precisely how Plk1 regulates the spindle is uncertain. In this study, we report that the small GTP/GDP-binding protein Ran is associated with Plk1. Plk1 is capable of phosphorylating co-immunoprecipitated Ran in vitro on serine-135 and Ran is phosphorylated in vivo at the same site during mitosis when Plk1 is normally activated. Cell cultures over-expressing a Ran S135D mutant have significantly higher numbers of abnormal mitotic cells than those over-expressing either wild-type or S135A Ran. The abnormalities in S135D mutant cells are similar to cells over-expressing Plk1. Our data suggests that Ran is a physiological substrate of Plk1 and that Plk1 regulates the spindle organization partially through its phosphorylation on Ran.     

Checkpoint controls that couple mitosis to completion of DNA replication

Cells treated with inhibitors of DNA synthesis do not normally enter mitosis. Incompletely replicated DNA apparently activates a regulatory mechanism that prevents activation of the mitotic inducer M-phase kinase by controlling the dephosphorylation of a critical tyrosine residue in the active site of the kinase. The control system may also target a second mitotic inducer, possibly the NIMA protein kinase. Unreplicated DNA may be detected and signalled by a complex of RCC1, a DNA-binding protein, and Ran, a Ras-related protein. This article reviews these recent developments and discusses the possibility that the control system also operates in the normal cell cycle, to ensure that mitosis strictly follows S phase.     

Dynamic localisation of Ran GTPase during the cell cycle

Background  

Ran GTPase has multiple functions during the cell division cycle, including nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. The activity of Ran is determined by both its guanine nucleotide-bound state and its subcellular localization.     

Spatial and temporal control of nuclear envelope assembly by Ran GTPase

Using evidence derived primarily from studies using Xenopus egg extracts, a model for the role of Ran in multiple stages during NE assembly can be proposed (Figure 2). Ran is concentrated on chromatin prior to NE assembly and recruits RCC1 that generates Ran-GTP locally. Recruitment of RCC1 to chromatin may be a specialized mechanism to initiate NE assembly following fertilization of the egg, whereas in somatic cells, RCC1 may be present on chromatin throughout mitosis. Ran-GTP recruits vesicles to the surface of chromatin, and promotes vesicle fusion to form the double membrane of the NE. Ran-GTP may recruit membrane vesicles to chromatin through binding to integral membrane proteins through importin-beta. A transient complex would be formed between Ran-GTP, importin-beta and the target protein, which would be released locally to promote assembly of a precursor complex. GTP hydrolysis by Ran would release importin-beta, but may also play a role in vesicle fusion. Ran-GTP also promotes NPC assembly by releasing nucleoporins such as Nup107 from inhibitory complexes with importin-beta. In vertebrate cells undergoing mitosis, the majority of Ran molecules are excluded from the chromosomes and dispersed into the cytoplasm. Relocalization of Ran to chromatin at the end of mitosis may co-ordinate the initiation of NE assembly with disassembly of the mitotic spindle. The function of Ran in this transition is likely to be coupled to changes in the activity of cyclin-dependent protein kinases and other activities that control the progression of the cell cycle. Thus, changes in the localization of Ran and its regulators provide temporal and spatial control of NE assembly at the end of mitosis.