Ran genes encode a family of well-conserve small nuclear GTPases (Ras-related nuclear proteins), whose function is implicated in both normal cell cycle progression and the transport of RNA and proteins between the nucleus and the cytoplasm. Previous studies of Ran proteins have utilized cell-free systems, yeasts, and cultured mammalian cells. The patterns of Ran gene expression have been characterized in the mouse. Serum starvation suppressed Ran gene transcription in mouse 3T3 cells. Ran mRNA reappeared in cells within 3 h after refeeding. A single Ran mRNA species was detected at low levels in most somatic tissues of the adult mouse. In testis, this Ran mRNA was abundant, as were other larger transcripts. Analysis of testis-derived Ran cDNA clones revealed the presence of two transcripts, one specifying an amino acid sequence identical to that of human Ran/TC4 and one specifying an amino acid sequence 94% identical. Northern blotting and reverse transcriptase-PCR assays with oligonucleotide probes and primers specific for each transcript demonstrated that the isoform identical to Ran/TC4 was expressed in both somatic tissues and testis, while the variant form was transcribed only in testis. The existence of tissue-specific Ran isoforms may help to rationalize the diverse roles suggested for Ran by previous biochemical studies (Coutavas, 1994).
The Ran gene family encodes small GTP binding proteins that are associated with a variety of nuclear processes. A Xenopus Ran cDNA has been isolated and the pattern of expression of this gene was analyzed during embryogenesis. Ran is expressed maternally and later in the CNS, neural crest, mesenchyme, eyes, and otic vesicles. However, expression is not detected in the somites or the notochord (Onuma, 2000).
Human Ran is the prototype of a well conserved family of GTPases that can regulate both cell-cycle progression and messenger RNA transport. Ran has been proposed to undergo tightly controlled cycles of GTP binding and hydrolysis, to operate as a GTPase switch whose GTP- and GDP-bound forms interact differentially with regulators and effectors. One known regulator, the protein RCC1, interacts with Ran to catalyse guanine nucleotide exchange, and both RCC1 and Ran are components of an intrinsic checkpoint control that prevents the premature initiation of mitosis. To test and extend the GTPase-switch model, a Ran-specific GTPase-activating protein (GAP) was sought, as well as putative effectors (proteins that interact specifically with Ran/TC4-GTP). This study reports the identification of a Ran GAP and its use to characterize the GTP-hydrolysing properties of mutant Ran proteins, and the identification and cloning of a binding protein specific for Ran-GTP (Coutavas, 1993).
RCC1 (the regulator of chromosome condensation) stimulates guanine nucleotide dissociation on the Ras-related nuclear protein Ran. Both polypeptides are components of a regulatory pathway that has been implicated in regulating DNA replication, onset of and exit from mitosis, mRNA processing and transport, and import of proteins into the nucleus. In a search for further members of the RCC1-Ran signal pathway, proteins of 23, 45 and 300 kDa were identified that tightly bind to Ran-GTP but not Ran-GDP. The purified soluble 23 kDa Ran binding protein RanBP1 does not activate RanGTPase, but increases GTP hydrolysis induced by the RanGTPase-activating protein RanGAP1 by an order of magnitude. In the absence of RanGAP, it strongly inhibits RCC1-induced exchange of Ran-bound GTP. In addition, it forms a stable complex with nucleotide-free RCC1-Ran. With these properties, it differs markedly from guanine diphosphate dissociation inhibitors which preferentially prevent the exchange of protein-bound GDP and in some cases were shown to inhibit GAP-induced GTP hydrolysis. RanBP1 is the first member of a new class of proteins regulating the binding and hydrolysis of GTP by Ras-related proteins (Bischoff, 1995).
Ran is a nuclear GTPase implicated in nucleocytoplasmic transport, the maintenance of nuclear structure, mRNA processing, and cell cycle regulation. By two-hybrid interaction in yeast, a Xenopus homologue of Ran-binding protein 1 (RanBP1) has been identified. Xenopus RanBP1 interacts specifically with the GTP-bound form of Ran and forms complexes in Xenopus egg extracts with Ran, importin-beta/karyopherin-beta and importin-alpha/karyopherin-alpha, but not p10, p120/RanBP7, RanBP2 or other nucleoporins. These complexes may play roles in the recycling of Ran and importins/karyopherins during nucleocytoplasmic transport. Increased concentrations of RanBP1 stabilise an interaction between Ran and RCC1 in egg extracts, inhibiting the exchange activity of RCC1 towards Ran. Under these conditions, the assembly of nuclei from chromatin is dramatically affected: the nuclei do not assemble a lamina and become very small with homogeneously condensed chromatin. They fail to actively import proteins and do not undergo DNA replication. By field emission in-lens scanning electron microscopy, it was shown that these nuclei have an intact nuclear envelope containing pore complexes, but the envelope is highly convoluted. However, RanBP1 does not directly inhibit nuclear protein import in assembled nuclei. These results suggest that RCC1 and/or Ran have a function early in nuclear assembly that is disrupted by RanBP1 (Nicolas, 1997).
CRM1/Exportin1 mediates the nuclear export of proteins bearing a leucine-rich nuclear export signal (NES) by forming a cooperative ternary complex with the NES-bearing substrate and the small GTPase Ran. A structural model of human CRM1 is presented based on a combination of X-ray crystallography, homology modeling, and electron microscopy. The architecture of CRM1 resembles that of the import receptor transportin1, with 19 HEAT repeats and a large loop implicated in Ran binding. Residues critical for NES recognition are identified adjacent to the cysteine residue targeted by leptomycin B (LMB), a specific CRM1 inhibitor. Evidence is presented that a conformational change of the Ran binding loop accounts for the cooperativity of Ran- and substrate binding and for the selective enhancement of CRM1-mediated export by the cofactor RanBP3. These findings indicate that a single architectural and mechanistic framework can explain the divergent effects of RanGTP on substrate binding by many import and export receptors (Petosa, 2004).
The small GTPase Ran functions in several critical processes in eukaryotic cells including nuclear transport, nuclear envelope formation, and spindle formation. A RanGDP-binding protein, NTF2, facilitates translocation of RanGDP through the nuclear pore complex and also acts to stabilize RanGDP against nucleotide exchange. This study idenifies a novel activity that stimulates release of GDP from Ran in the presence of NTF2. Hydrolyzable ATP enhances the GDP dissociation activity, and this enhancement is inhibited by nonhydrolyzable ATP analogues. In contrast, neither hydrolyzable ATP nor nonhydrolyzable ATP analogues affect GDP dissociation from Ran catalyzed by recombinant RCC1 or inhibition of GDP dissociation from Ran by recombinant NTF2. The ATP-dependent RanGDP dissociation activity therefore has the properties of a RanGDP dissociation inhibitor (GDI) displacement factor (RanGDF) where the GDI is NTF2. A protein phosphatase inhibitor mixture stimulates the RanGDF activity, suggesting the activity is regulated by phosphorylation. It is proposed that the ATP-dependent NTF2 releasing factor may have a role in the RanGDP/GTP cycle (Yamada, 2004).
The Ran protein is a small GTPase that has been implicated in a large number of nuclear processes including transport. RNA processing and cell cycle checkpoint control. A similar spectrum of nuclear activities has been shown to require RCC1, the guanine nucleotide exchange factor (GEF) for Ran. The Xenopus laevis egg extract system and in vitro assays of purified proteins were used to examine how Ran or RCC1 could be involved in these numerous processes. In these studies, mutant Ran proteins were employed to perturb nuclear assembly and function. The addition of a bacterially expressed mutant form of Ran (T24N-Ran), which is predicted to be primarily in the GDP-bound state, profoundly disrupts nuclear assembly and DNA replication in extracts. The molecular mechanism by which T24N-Ran disrupts normal nuclear activity was examined, and T24N-Ran was found to bind tightly to the RCC1 protein within the extract, resulting in its inactivation as a GEF. The capacity of T24N-Ran-blocked interphase extracts to assemble nuclei from de-membranated sperm chromatin and to replicate their DNA can be restored by supplementing the extract with excess RCC1 and thereby providing excess GEF activity. Conversely, nuclear assembly and DNA replication are both rescued in extracts lacking RCC1 by the addition of high levels of wild-type GTP-bound Ran protein, indicating that RCC1 does not have an essential function beyond its role as a GEF in interphase Xenopus extracts (Dasso, 1994).
Ran, a member of the Ras superfamily of GTPases, is predominantly localized in the nucleus and is a necessary component in the active transport of proteins through nuclear pores. Disruption of Ran function affects the regulation of mitosis, DNA synthesis, and RNA processing and export. To explore the mechanisms of Ran function, mutants of the Ran GTPase were characterized, several of which are capable of dominantly interfering with nuclear protein import. Unlike wild-type Ran, the putative gain-of-function mutant (G19V Ran) is not sensitive to the exchange factor, RCC1. In addition the G19V Ran and effector domain mutants (L43E and E46G Ran) are not sensitive to the GTPase-activating protein, Fug1. Epitope-tagged G19V Ran and L43E Ran isolated from transfected BHK21 cells are each about 50% GTP-bound, whereas the wild-type and a C-terminal deletion mutant (Delta-DE Ran) are primarily bound to GDP. While G19V Ran interacts with known Ran-binding proteins and with an isolated Ran-binding domain, the T24N Ran does not, and binding by L43E Ran is substantially reduced. Wild-type HA1-tagged Ran expressed in BHK21 cells is nuclear, whereas the G19V, T24N, L43E, and E46G forms of Ran are predominantly localized at the nuclear envelope, and Delta-DE Ran is primarily cytosolic. Similar results are observed when permeabilized BHK21 cells are incubated with extracts of COS cells expressing the mutants. Thus mutations that affect the interaction of Ran with regulatory proteins and effectors can disrupt the normal subcellular localization of Ran, lending support for the current model of Ran-mediated nuclear import (Lounsbury, 1996).
The Ran GTPase plays a critical role in nucleocytoplasmic transport and has been implicated in the maintenance of nuclear structure and cell cycle control. This study has investigated its role in nuclear assembly and DNA replication using recombinant wild-type and mutant Ran proteins added to a cell-free system of Xenopus egg extracts. RanQ69L and RanT24N prevent lamina assembly, PCNA accumulation and DNA replication. These effects may be due to the disruption of nucleocytoplasmic transport, since both mutants inhibit nuclear import of a protein carrying a nuclear localisation signal (NLS). RanQ69L, which is deficient in GTPase activity, sequesters importins in stable complexes that are unable to support the docking of NLS-proteins at the nuclear pore complex (NPC). RanT24N, in contrast to wild-type Ran-GDP, interacts only weakly with importin alpha and nucleoporins, and not at all with the import factor p10, consistent with its poor activity in nuclear import. However, RanT24N does interact stably with importin beta, Ran binding protein 1 and RCC1, an exchange factor for Ran. Ran-GDP is essential for proper nuclear assembly and DNA replication, the requirement being primarily before the initiation of DNA replication. Ran-GDP therefore mediates the active transport of necessary factors or otherwise controls the onset of S-phase in this system (Hughes, 1998).
The importin-alpha/beta heterodimer and the GTPase Ran play key roles in nuclear protein import. Importin binds the nuclear localization signal (NLS). Translocation of the resulting import ligand complex through the nuclear pore complex (NPC) requires Ran and is terminated at the nucleoplasmic side by its disassembly. The principal GTP exchange factor for Ran is the nuclear protein RCC1, whereas the major RanGAP is cytoplasmic, predicting that nuclear Ran is mainly in the GTP form and cytoplasmic Ran is in the GDP-bound form. This study shows that nuclear import depends on cytoplasmic RanGDP and free GTP, and that RanGDP binds to the NPC. Therefore, import might involve nucleotide exchange and GTP hydrolysis on NPC-bound Ran. RanGDP binding to the NPC is not mediated by the Ran binding sites of importin-beta, suggesting that translocation is not driven from these sites. Consistently, a mutant importin-beta deficient in Ran binding can deliver its cargo up to the nucleoplasmic side of the NPC. However, the mutant is unable to release the import substrate into the nucleoplasm. Thus, binding of nucleoplasmic RanGTP to importin-beta probably triggers termination, i.e. the dissociation of importin-alpha from importin-beta and the subsequent release of the import substrate into the nucleoplasm (Gorlich, 1996).
Nucleocytoplasmic transport appears mediated by shuttling transport receptors that bind RanGTP as a means to regulate interactions with their cargoes. The receptor-RanGTP complexes are kinetically very stable with nucleotide exchange and GTP hydrolysis being blocked, predicting that a specific disassembly mechanism exists. This study shows in three cases receptor RanGTP x RanBP1 complexes to be the key disassembly intermediates, where RanBP1 stimulates the off-rate at the receptor/RanGTP interface by more than two orders of magnitude. The transiently released RanGTP x RanBP1 complex is then induced by RanGAP to hydrolyse GTP, preventing the receptor to rebind RanGTP. The efficient release of importin beta from RanGTP requires importin alpha, in addition to RanBP1 (Bischoff, 1997).
The inhibitor of kappa B alpha (IkappaBalpha) protein is able to shuttle between the cytoplasm and the nucleus. A combination of in vivo and in vitro approaches were used to provide mechanistic insight into nucleocytoplasmic shuttling by IkappaBalpha. IkappaBalpha contains multiple functional domains that contribute to shuttling of IkappaBalpha between the cytoplasm and the nucleus. Nuclear import of IkappaBalpha is mediated by the central ankyrin repeat domain. Similar to previously described nuclear import pathways, nuclear import of IkappaBalpha is temperature and ATP dependent and is blocked by a dominant-negative mutant of importin beta. However, in contrast to classical nuclear import pathways, nuclear import of IkappaBalpha is independent of soluble cytosolic factors and is not blocked by the dominant-negative RanQ69L protein. Nuclear export of IkappaBalpha is mediated by an N-terminal nuclear export sequence. Nuclear export of IkappaBalpha requires the CRM1 nuclear export receptor and is blocked by the dominant-negative RanQ69L protein. The results are consistent with a model in which nuclear import of IkappaBalpha is mediated through direct interactions with components of the nuclear pore complex, while nuclear export of IkappaBalpha is mediated via a CRM1-dependent pathway (Sachdev, 2000).
An assay in which nuclear export of the shuttling transcription factor NFAT (nuclear factor of activated T cells) can be reconstituted in permeabilized cells with the GTPase Ran and the nuclear export receptor CRM1. This assay has been used to identify another export factor. After preincubation of permeabilized cells with a Ran mutant that cannot hydrolyze GTP (RanQ69L), cytosol supports NFAT export, but CRM1 and Ran alone do not. The RanQ69L preincubation leads to accumulation of CRM1 at the cytoplasmic periphery of the nuclear pore complex (NPC) in association with the p62 complex and Can/Nup214. RanGTP-dependent association of CRM1 with these nucleoporins was reconstituted in vitro. By biochemical fractionation and reconstitution, it was shown that RanBP1 restores nuclear export after the RanQ69L preincubation. It also stimulates nuclear export in cells that have not been preincubated with RanQ69L. RanBP1 as well as Ran-binding domains of the cytoplasmic nucleoporin RanBP2 promote the release of CRM1 from the NPC. Taken together, these results indicate that RanGTP is important for the targeting of export complexes to the cytoplasmic side of the NPC and that RanBP1 and probably RanBP2 are involved in the dissociation of nuclear export complexes from the NPC in a terminal step of transport (Kehlenback, 2001).
Receptor-mediated nucleocytoplasmic transport is dependent on the GTPase Ran and Ran-binding protein 1 (RanBP1). The acidic C terminus of Ran is required for high affinity interaction between Ran and RanBP1. A novel Ran mutant with four of its five acidic C-terminal amino acids modified to alanine (RanC4A) has an approximately 20-fold reduced affinity for RanBP1. The effects of RanC4A on nuclear import was investigated, along with export in permeabilized HeLa cells. Although RanC4A promotes accumulation of the nuclear export receptor CRM1 at the cytoplasmic nucleoporin Nup214, it strongly stimulates nuclear export of GFP-NFAT. Since RanC4A exhibits an elevated affinity for CRM1 and other nuclear transport receptors, this suggests that formation of the export complex containing CRM1, Ran-GTP, and substrate is a rate-limiting step in export, not release from Nup214. Conversely, importin alpha/beta-dependent nuclear import of bovine serum albumin, coupled to a classical nuclear localization sequence is strongly inhibited by RanC4A. Inhibition can be reversed by additional importin alpha, which promotes the formation of an importin alpha/beta complex. These results provide physiological evidence that release of Ran-GTP from importin beta by RanBP1 and importin alpha is critical for the recycling of importin beta to a transport-competent state (Kehlenback, 2001).
PU.1 is a transcription factor of the Ets family with important functions in hematopoietic cell differentiation. Using green fluorescent protein-PU.1 fusions, it has been shown that the Ets DNA binding domain of PU.1 is necessary and sufficient for its nuclear localization. Fluorescence and ultrastructural nuclear import assays showed that PU.1 nuclear import requires energy but not soluble carriers. PU.1 interacts directly with two nucleoporins, Nup62 and Nup153. The binding of PU.1 to Nup153, but not to Nup62, increases dramatically in the presence of RanGMPPNP, indicating the formation of a PU.1.RanGTP.Nup153 complex. The Ets domain accounts for the bulk of the interaction of PU.1 with Nup153 and RanGMPPNP. Because Nup62 is located close to the midplane of the nuclear pore complex whereas Nup153 is at its nuclear side, these findings suggest a model whereby RanGTP propels PU.1 toward the nuclear side of the nuclear pore complex by increasing its affinity for Nup153. This notion was confirmed by ultrastructural studies using gold-labeled PU.1 in permeabilized cells (Zhong, 2005).
Targeting of newly synthesized integral membrane proteins to the appropriate cellular compartment is specified by discrete sequence elements, many of which have been well characterized. An understanding of the signals required to direct integral membrane proteins to the inner nuclear membrane (INM) remains a notable exception. This study shows that integral INM proteins possess basic sequence motifs that resemble 'classical' nuclear localization signals. These sequences can mediate direct binding to karyopherin-alpha and are essential for the passage of integral membrane proteins to the INM. Furthermore, karyopherin-alpha, karyopherin-beta1 and the Ran GTPase cycle are required for INM targeting, underscoring parallels between mechanisms governing the targeting of integral INM proteins and soluble nuclear transport. Evidence is provided that specific nuclear pore complex proteins contribute to this process, suggesting a role for signal-mediated alterations in the nuclear pore complex to allow for passage of INM proteins along the pore membrane (King, 2006).
Nucleus/cytosol exchange requires a GTPase, Ran. In yeast Rna1p is the GTPase activating protein for Ran (RanGAP) and Prp20p is the Ran GDP/GTP exchange factor (GEF). RanGAP is primarily cytosolic and GEF is nuclear. Their subcellular distributions led to the prediction that Ran-GTP hydrolysis takes place solely in the cytosol and GDP/GTP exchange solely in the nucleus. Current models propose that the Ran-GTP/Ran-GDP gradient across the nuclear membrane determines the direction of exchange. Three lines of evidence are provided that Rna1p enters and leaves the nuclear interior. (1) Rna1p possesses leucine-rich nuclear export sequences (NES) that are able to relocate a passenger karyophilic protein to the cytosol; alterations of consensus residues re-establish nuclear location. (2) Rna1p possesses other sequences that function as a novel nuclear localization sequence able to deliver a passenger cytosolic protein to the nucleus. (3) Endogenous Rna1p location is dependent upon Xpo1p/Crm1p, the yeast exportin for leucine-rich NES-containing proteins. The data support the hypothesis that Rna1p exists on both sides of the nuclear membrane, perhaps regulating the Ran-GTP/Ran-GDP gradient, participating in a complete RanGTPase nuclear cycle or serving a novel function (Feng, 1999).
In yeast and mammalian cells, the spindle assembly checkpoint proteins Mad1p and Mad2p localize to the nuclear pore complex (NPC) during interphase. Deletion of MAD1 or MAD2 does not affect steady-state nucleocytoplasmic distribution of a classical nuclear localization signal-containing reporter, a nuclear export signal-containing reporter, or Ran localization. Cells with conditional mutations in the yeast Ran GTPase pathway were used to examine the relationship between Ran and targeting of checkpoint regulators to the NPC. Mutations that disrupt the concentration of Ran in the nucleus displace Mad2p but not Mad1p from the NPC. The displacement of Mad2p in M-phase cells is correlated with activation of the spindle checkpoint. These observations demonstrate that Mad2p localization at NPCs is sensitive to nuclear levels of Ran and suggest that release of Mad2p from NPCs is closely linked with spindle assembly checkpoint activation in yeast. This is the first evidence indicating that Ran affects the localization of Mad2p to the NPC (Quimby, 2005).
Although the Ran GTPase-activating protein RanGAP mainly functions in the cytoplasm, several lines of evidence indicate a nuclear function of RanGAP. Schizosaccharomyces pombe RanGAP, SpRna1, binds the core of histone H3 (H3) and enhances Clr4-mediated H3-lysine 9 (K9) methylation. This enhancement is not observed for methylation of the H3-tail containing K9 and is independent of SpRna1-RanGAP activity, suggesting that SpRna1 itself enhances Clr4-mediated H3-K9 methylation via H3. Although most SpRna1 is in the cytoplasm, some cofractionates with H3. Sprna1(ts) mutations causes decreases in Swi6 localization and H3-K9 methylation at all three heterochromatic regions of S. pombe. Thus, nuclear SpRna1 seems to be involved in heterochromatin assembly. All core histones bind SpRna1 and inhibit SpRna1-RanGAP activity. In contrast, Clr4 abolishes the inhibitory effect of H3 on the RanGAP activity of SpRna1 but partially affects the other histones. SpRna1 forms a trimeric complex with H3 and Clr4, suggesting that nuclear SpRna1 is reciprocally regulated by histones, especially H3, and Clr4 on the chromatin to function for higher order chromatin assembly. It was also found that SpRna1 forms a stable complex with Xpo1/Crm1 plus Ran-GTP, in the presence of H3 (Nishijima, 2006).
The small guanosine triphosphatase Ran is loaded with guanosine triphosphate (GTP) by the chromatin-bound guanine nucleotide exchange factor RCC1 and releases import cargoes in the nucleus during interphase. In mitosis, Ran-GTP promotes spindle assembly around chromosomes by locally discharging cargoes that regulate microtubule dynamics and organization. Fluorescence resonance energy transfer-based biosensors were used to visualize gradients of Ran-GTP and liberated cargoes around chromosomes in mitotic Xenopus egg extracts. Both gradients were required to assemble and maintain spindle structure. During interphase, Ran-GTP was highly enriched in the nucleoplasm, and a steep concentration difference between nuclear and cytoplasmic Ran-GTP is established, providing evidence for a Ran-GTP gradient surrounding chromosomes throughout the cell cycle (Kalab, 2002).
The RanGTP gradient across the interphase nuclear envelope and on the condensed mitotic chromosomes is essential for many cellular processes, including nucleocytoplasmic transport and spindle assembly. Although the chromosome-associated enzyme RCC1 is responsible for RanGTP production, the mechanism of generating and maintaining the RanGTP gradient in vivo remains unknown. This study reports that regulator of chromosome condensation (RCC1) rapidly associates and dissociates with both interphase and mitotic chromosomes in living cells, and that this mobility is regulated during the cell cycle. Kinetic modeling suggests that RCC1 couples its catalytic activity to chromosome binding to generate a RanGTP gradient. Indeed, it has been demonstrated experimentally that the interaction of RCC1 with the chromatin is coupled to the nucleotide exchange on Ran in vivo. The coupling is due to the stable binding of the binary complex of RCC1-Ran to chromatin. Successful nucleotide exchange dissociates the binary complex, permitting the release of RCC1 and RanGTP from the chromatin and the production of RanGTP on the chromatin surface (Li, 2003).
The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactions between cargoes and nuclear transport receptors of the importin-beta family. The Ran-importin-beta system also functions in mitotic spindle assembly and nuclear pore and nuclear envelope formation. The common principle underlying these diverse functions throughout the cell cycle is thought to be anisotropy of the distribution of RanGTP (the RanGTP gradient), driven by the chromatin-associated guanine nucleotide exchange factor RCC1. However, the existence and function of a RanGTP gradient during mitosis in cells is unclear. This study examined the Ran-importin-beta system in cells by conventional and fluorescence lifetime microscopy using a biosensor, termed Rango, that increases its fluorescence resonance energy transfer signal when released from importin-beta by RanGTP. Rango is predominantly free in mitotic cells, but is further liberated around mitotic chromatin. In vitro experiments and modelling show that this localized increase of free cargoes corresponds to changes in RanGTP concentration sufficient to stabilize microtubules in extracts. In cells, the Ran-importin-beta-cargo gradient kinetically promotes spindle formation but is largely dispensable once the spindle has been established. Consistent with previous reports, the Ran system also affects spindle pole formation and chromosome congression in vivo. These results demonstrate that conserved Ran-regulated pathways are involved in multiple, parallel processes required for spindle function, but that their relative contribution differs in chromatin- versus centrosome/kinetochore-driven spindle assembly systems (Kalab, 2006).
The small GTPase Ran has been found to play pivotal roles in several aspects of cell function. This study investigated the role of the Ran GTPase cycle in spindle formation and nuclear envelope assembly in dividing Caenorhabditis elegans embryos in real time. Ran and its cofactors RanBP2, RanGAP, and RCC1 are all essential for reformation of the nuclear envelope after cell division. Reducing the expression of any of these components of the Ran GTPase cycle by RNAi leads to strong extranuclear clustering of integral nuclear envelope proteins and nucleoporins. Ran, RanBP2, and RanGAP are also required for building a mitotic spindle, whereas astral microtubules are normal in the absence of these proteins. RCC1(RNAi) embryos have similar abnormalities in the initial phase of spindle formation but eventually recover to form a bipolar spindle. Irregular chromatin structures and chromatin bridges due to spindle failure were frequently observed in embryos where the Ran cycle was perturbed. In addition, connection between the centrosomes and the male pronucleus, and thus centrosome positioning, depends upon the Ran cycle components. Finally, both IMA-2 and IMB-1, the homologues of vertebrate importin alpha and beta, are essential for both spindle assembly and nuclear formation in early embryos (Askjaer, 2002).
The GTPase Ran is known to regulate transport of proteins across the nuclear envelope. Recently, Ran has been shown to promote microtubule polymerization and spindle assembly around chromatin in Xenopus mitotic extracts and to stimulate nuclear envelope assembly in Xenopus or HeLa cell extracts. However, these in vitro findings have not been tested in living cells and do not necessarily describe the generalized model of Ran functions. This study present several lines of evidence that Ran is indispensable for correct chromosome positioning and nuclear envelope assembly in C. elegans. Embryos deprived of Ran by RNAi showed metaphase chromosome misalignment and aberrant chromosome segregation, while astral microtubules seemed unaffected. Depletion of RCC1 or RanGAP by RNAi resulted in essentially the same defects. The immunofluorescent staining showed that Ran localizes to kinetochore regions of metaphase and anaphase chromosomes, suggesting the role of Ran in linking chromosomes to kinetochore microtubules. Ran was shown to localize to the nuclear envelope at telophase and during interphase in early embryos, and the depletion of Ran resulted in failure of nuclear envelope assembly. Thus, Ran is crucially involved in chromosome positioning and nuclear envelope assembly in C. elegans (Bamba, 2002).
Ran is an abundant nuclear GTPase with a clear role in nuclear transport during interphase but with roles in mitotic regulation that are less well understood. The nucleotide-binding state of Ran is regulated by a GTPase activating protein, RanGAP1, and by a guanine nucleotide exchange factor, RCC1. Ran also interacts with a guanine nucleotide dissociation inhibitor, RanBP1. RanBP1 has a high affinity for GTP-bound Ran, and it acts as a cofactor for RanGAP1, increasing the rate of GAP-mediated GTP hydrolysis on Ran approximately tenfold. RanBP1 levels oscillate during the cell cycle, and increased concentrations of RanBP1 prolong mitosis in mammalian cells and in Xenopus egg extracts. This study investigated how increased concentrations of RanBP1 disturb mitosis. Spindle assembly is dramatically disrupted when exogenous RanBP1 is added to M phase Xenopus egg extracts. Evidence is presented that the role of Ran in spindle assembly is independent of nuclear transport and is probably mediated through changes in microtubule dynamics (Kalab, 1999).
The nucleotide exchange activity of RCC1, the only known nucleotide exchange factor for Ran, a Ras-like small guanosine triphosphatase, is required for microtubule aster formation with or without demembranated sperm in Xenopus egg extracts arrested in meiosis II. Consistently, in the RCC1-depleted egg extracts, Ran guanosine triphosphate (RanGTP), but not Ran guanosine diphosphate (RanGDP), induces self-organization of microtubule asters, and the process required the activity of dynein. Thus, Ran was shown to regulate formation of the microtubule network (Ohba, 1999).
Chromosomes are segregated by two antiparallel arrays of microtubules arranged to form the spindle apparatus. During cell division, the nucleation of cytosolic microtubules is prevented and spindle microtubules nucleate from centrosomes (in mitotic animal cells) or around chromosomes (in plants and some meiotic cells). The molecular mechanism by which chromosomes induce local microtubule nucleation in the absence of centrosomes is unknown, but it can be studied by adding chromatin beads to Xenopus egg extracts. The beads nucleate microtubules that eventually reorganize into a bipolar spindle. RCC1, the guanine-nucleotide-exchange factor for the GTPase protein Ran, is a component of chromatin. Using the chromatin bead assay, this study shows that the activity of chromosome-associated RCC1 protein is required for spindle formation. Ran itself, when in the GTP-bound state (Ran-GTP), induces microtubule nucleation and spindle-like structures in M-phase extract. It is proposed that RCC1 generates a high local concentration of Ran-GTP around chromatin which in turn induces the local nucleation of microtubules (Carazo-Salas, 1999).
Ran, a small guanosine triphosphatase, is suggested to have additional functions beyond its well-characterized role in nuclear trafficking. Guanosine triphosphate-bound Ran, but not guanosine diphosphate-bound Ran, stimulates polymerization of astral microtubules from centrosomes assembled on Xenopus sperm. Moreover, a Ran allele with a mutation in the effector domain (RanL43E) induces the formation of microtubule asters and spindle assembly, in the absence of sperm nuclei, in a gammaTuRC (gamma-tubulin ring complex)- and XMAP215 (Xenopus microtubule associated protein)-dependent manner. Therefore, Ran could be a key signaling molecule regulating microtubule polymerization during mitosis (Wilde, 1999).
The guanosine tri-phosphatase Ran stimulates assembly of microtubule spindles. However, it is not known what aspects of the microtubule cytoskeleton are subject to regulation by Ran in mitosis. This study shows that Ran-GTP stimulates microtubule assembly by increasing the rescue frequency of microtubules three- to eightfold. In addition to changing microtubule dynamics, Ran-GTP also alters the balance of motor activities, partly as a result of an increase in the amount of motile Eg5, a plus-end-directed microtubule motor that is essential for spindle formation. Thus, Ran regulates multiple processes that are involved in spindle assembly (Wilde, 2001).
The small GTPase Ran, bound to GTP, is required for the induction of spindle formation by chromosomes in M phase. High concentrations of Ran.GTP are proposed to surround M phase chromatin. The action of Ran.GTP in spindle formation requires TPX2, a microtubule-associated protein previously known to target a motor protein, Xklp2, to microtubules. TPX2 is normally inactivated by binding to the nuclear import factor, importin alpha, and is displaced from importin alpha by the action of Ran.GTP. TPX2 is required for Ran.GTP and chromatin-induced microtubule assembly in M phase extracts and mediates spontaneous microtubule assembly when present in excess over free importin alpha. Thus, components of the nuclear transport machinery serve to regulate spindle formation in M phase (Gruss, 2001).
The guanosine triphosphatase Ran stimulates assembly of microtubule asters and spindles in mitotic Xenopus egg extracts. A carboxyl-terminal region of the nuclear-mitotic apparatus protein (NuMA), a nuclear protein required for organizing mitotic spindle poles, mimics Ran's ability to induce asters. This NuMA fragment also specifically interacts with the nuclear transport factor, importin-beta. Importin-beta is an inhibitor of microtubule aster assembly in Xenopus egg extracts, and Ran regulates the interaction between importin-beta and NuMA. Importin-beta therefore links NuMA to regulation by Ran. This suggests that similar mechanisms regulate nuclear import during interphase and spindle assembly during mitosis (Wiese, 2001).
GTP-bound Ran induces microtubule and pseudo-spindle assembly in mitotic egg extracts in the absence of chromosomes and centrosomes, and that chromosomes induce the assembly of spindle microtubules in these extracts through generation of Ran-GTP. This study examined the effects of Ran-GTP on microtubule nucleation and dynamics and shows that Ran-GTP has independent effects on both the nucleation activity of centrosomes and the stability of centrosomal microtubules. It was also shown that inhibition of Ran-GTP production, even in the presence of duplicated centrosomes and kinetochores, prevents assembly of a bipolar spindle in M-phase extracts (Carazo-Salas, 2001).
The GTPase Ran has recently been shown to stimulate microtubule polymerization in mitotic extracts, but its mode of action is not understood. This study shows that the mitotic role of Ran is largely mediated by the nuclear transport factor importin beta. Importin beta inhibits spindle formation in vitro and in vivo and sequesters an aster promoting activity (APA) that consists of multiple, independent factors. One component of APA is the microtubule-associated protein NuMA. NuMA and other APA components are discharged from importin beta by RanGTP and induce spindle-like structures in the absence of centrosomes, chromatin, or Ran. It is proposed that RanGTP functions in mitosis as in interphase by locally releasing cargoes from transport factors. In mitosis, this promotes spindle assembly by organizing microtubules in the vicinity of chromosomes (Nachury, 2001).
In Xenopus laevis egg extracts, TPX2 is required for the Ran-GTP-dependent assembly of microtubules around chromosomes. Interfering with the function of the human homologue of TPX2 in HeLa cells causes defects in microtubule organization during mitosis. Suppressing the expression of human TPX2 by RNA interference leads to the formation of two microtubule asters that do not interact and do not form a spindle. These results suggest that in vivo, even in the presence of duplicated centrosomes, spindle formation requires the function of TPX2 to generate a stable bipolar spindle with overlapping antiparallel microtubule arrays. This indicates that chromosome-induced microtubule production is a general requirement for the formation of functional spindles in animal cells (Gruss, 2002).
Ran GTPase is involved in several aspects of nuclear structure and function, including nucleocytoplasmic transport and nuclear envelope formation. Experiments using Xenopus egg extracts have shown that generation of Ran-GTP by the guanine nucleotide exchange factor RCC1 also plays roles in mitotic spindle assembly. This study has examined the localization and function of RCC1 in mitotic human cells. RCC1, either the endogenous protein or that expressed as a fusion with green fluorescent protein (GFP), is localized predominantly to chromosomes in mitotic cells. This localization requires an N-terminal lysine-rich region that also contains a nuclear localization signal and is enhanced by interaction with Ran. Either mislocalization of GFP-RCC1 by removal of the N-terminal region or the expression of dominant Ran mutants that perturb the GTP/GDP cycle causes defects in mitotic spindle morphology, including misalignment of chromosomes and abnormal numbers of spindle poles. These results indicate that the generation of Ran-GTP in the vicinity of chromosomes by RCC1 is important for the fidelity of mitotic spindle assembly in human cells. Defects in this system may result in abnormal chromosome segregation and genomic instability, which are characteristic of many cancer cells (Moore, 2002).
Ran, a GTPase in the Ras superfamily, is proposed to be a spatial regulator of microtubule spindle assembly by maintaining key spindle assembly factors in an active state close to chromatin. RanGTP is hypothesized to maintain the spindle assembly factors in the active state by binding to importin beta, part of the nuclear transport receptor complex, thereby preventing the inhibitory binding of the nuclear transport receptors to spindle assembly factors. To directly test this hypothesis, two putative downstream targets of the Ran spindle assembly pathway, TPX2, a protein required for correct spindle assembly and Kid, a chromokinesin involved in chromosome arm orientation on the spindle, were analyzed to determine if their direct binding to nuclear transport receptors inhibited their function. In the amino-terminal domain of TPX2 nuclear targeting information, microtubule-binding and Aurora A (see Drosophila Aurora) binding activities were identified. Nuclear transport receptor binding to TPX2 inhibited Aurora A binding activity but not the microtubule-binding activity of TPX2. Inhibition of the interaction between TPX2 and Aurora A prevents Aurora A activation and recruitment to microtubules. In addition nuclear targeting information was identified in both the amino-terminal microtubule-binding domain and the carboxy-terminal DNA binding domain of Kid. However, the binding of nuclear transport receptors to Kid only inhibited the microtubule-binding activity of Kid. Therefore, by regulating a subset of TPX2 and Kid activities, Ran modulates at least two processes involved in spindle assembly (Trieselmann, 2003).
The activated form of Ran (Ran-GTP) stimulates spindle assembly in Xenopus laevis egg extracts, presumably by releasing spindle assembly factors, such as TPX2 (target protein for Xenopus kinesin-like protein 2) and NuMA (nuclear-mitotic apparatus protein) from the inhibitory binding of importin-alpha and -beta. Ran-GTP stimulates the interaction between TPX2 and the Xenopus Aurora A kinase, Eg2. This interaction causes TPX2 to stimulate both the phosphorylation and the kinase activity of Eg2 in a microtubule-dependent manner. TPX2 and microtubules promote phosphorylation of Eg2 by preventing phosphatase I (PPI)-induced dephosphorylation. Activation of Eg2 by TPX2 and microtubules is inhibited by importin-alpha and -beta, although this inhibition is overcome by Ran-GTP both in the egg extracts and in vitro with purified proteins. Since the phosphorylation of Eg2 stimulated by the Ran-GTP-TPX2 pathway is essential for spindle assembly, it is hypothesized that the Ran-GTP gradient established by the condensed chromosomes is translated into the Aurora A kinase gradient on the microtubules to regulate spindle assembly and dynamics (Tsai, 2003).
Spindle assembly is subject to the regulatory controls of both the cell-cycle machinery and the Ran-signaling pathway. An important question is how the two regulatory pathways communicate with each other to achieve coordinated regulation in mitosis. Cdc2 kinase phosphorylates the serines located in or near the nuclear localization signal (NLS) of human RCC1, the nucleotide exchange factor for Ran. This phosphorylation is necessary for RCC1 to generate RanGTP on mitotic chromosomes in mammalian cells, which in turn is required for spindle assembly and chromosome segregation. Moreover, phosphorylation of the NLS of RCC1 is required to prevent the binding of importin alpha and beta to RCC1, thereby allowing RCC1 to couple RanGTP production to chromosome binding. These findings reveal that the cell-cycle machinery directly regulates the Ran-signaling pathway by placing a high RanGTP concentration on the mitotic chromosome in mammalian cells (Li, 2004).
The roles of the kinase Aurora A (AurA) in centrosome function and spindle assembly have been established in Drosophila, C. elegans, and Xenopus egg extracts. AurA has been shown to act downstream of the RanGTPase signaling pathway to stimulate spindle assembly in mitosis. However, it is still not clear whether AurA can stimulate the formation of microtubule organizing centers (MTOC) on its own. Moreover, whether AurA is essential for spindle assembly in the absence of centrosomes has remained unclear. This study reports the development of functional assays that facilitate the observation that activation of AurA by TPX2 is essential for Ran-stimulated spindle assembly in the presence or absence of centrosomes. Furthermore, AurA-coated magnetic beads function as MTOCs in the presence of RanGTP in Xenopus egg extracts and RanGTP stimulates AurA to recruit activities responsible for both MT nucleation and organization to the beads. The MTOC function of AurA-coated beads require both MT nucleators and motors. Compared to XMAP215-coated beads, AurA-coated beads increase the rate of bipolar spindle assembly in the presence of RanGTP, and the kinase activity of AurA is essential for the beads to function as MTOCs (Tsai, 2005).
TPX2 has multiple functions during mitosis, including microtubule nucleation around the chromosomes and the targeting of Xklp2 and Aurora A to the spindle. A detailed domain functional analysis of TPX2 was performed and a large N-terminal domain containing the Aurora A binding peptide was found to interact directly with and nucleates microtubules in pure tubulin solutions. However, it cannot substitute the endogenous TPX2 to support microtubule nucleation in response to Ran guanosine triphosphate (GTP) and spindle assembly in egg extracts. By contrast, a large C-terminal domain of TPX2 that does not bind directly to pure microtubules and does not bind Aurora A kinase rescues microtubule nucleation in response to RanGTP and spindle assembly in TPX2-depleted extract. These and previous results suggest that under physiological conditions, TPX2 is essential for microtubule nucleation around chromatin and functions in a network of other molecules, some of which also are regulated by RanGTP (Brunet, 2004).
Spatial control is a key issue in cell division. The Ran GTPase regulates several fundamental processes for cell life, largely acting through importin molecules. In mammalian cells, in which centrosomes are major spindle organizers, a link is emerging between the Ran network, centrosomes and spindle poles. This study shows that, after nuclear envelope breakdown, importin beta is transported to the spindle poles in mammalian cells. This localization is temporally regulated from prometaphase until anaphase, when importin beta dissociates from poles and is recruited back around reforming nuclei. Importin beta sediments with mitotic microtubules in vitro and its accumulation at poles requires microtubule integrity and dynamics in vivo. Furthermore, RNA interference-dependent inactivation of TPX2, the major Ran-dependent spindle organizer, abolishes importin beta accumulation at poles. Importin beta has a functional role in spindle pole organization, because overexpression yields mitotic spindles with abnormal, fragmented poles. Coexpression of TPX2 with importin beta mitigates these abnormalities. Together, these results indicate that the balance between importins and spindle regulators of the TPX2 type is crucial for spindle formation. Targeting of TPX2/importin-beta complexes to poles is a key aspect in Ran-dependent control of the mitotic apparatus in mammalian cells (Ciciarello, 2004).
During cell division, chromosomes are distributed to daughter cells by the mitotic spindle. This system requires spatial cues to reproducibly self-organize. Such cues are provided by chromosome-mediated interaction gradients between the small guanosine triphosphatase (GTPase) Ran and importin-beta. This produces activity gradients that determine the spatial distribution of microtubule nucleation and stabilization around chromosomes and that are essential for the self-organization of microtubules into a bipolar spindle (Caudron, 2005).
GTP-loaded Ran induces the assembly of microtubules into aster-like and spindle-like structures in Xenopus egg extract. The microtubule-associated protein (MAP), TPX2, can mediate Ran's role in aster formation, but factors responsible for the transition from aster-like to spindle-like structures have not been described. This study identifies a complex that is required for the conversion of aster-like to spindle-like structures. The complex consists of two characterized MAPs (TPX2, XMAP215), a plus end-directed motor (Eg5), a mitotic kinase (Aurora A), and HURP, a protein associated with hepatocellular carcinoma. Formation and function of the complex is dependent on Aurora A activity. HURP protein was further characterized and shown to bind microtubules and affect their organization both in vitro and in vivo. In egg extract, anti-HURP antibodies disrupt the formation of both Ran-dependent and chromatin and centrosome-induced spindles. HURP is also required for the proper formation and function of mitotic spindles in HeLa cells. It is concluded that HURP is a new and essential component of the mitotic apparatus. HURP acts as part of a multicomponent complex that affects the growth or stability of spindle MTs and is required for spindle MT organization (Koffa, 2006).
Nucleolar and spindle-associated protein (NuSAP) was recently identified as a microtubule- and chromatin-binding protein in vertebrates that is nuclear during interphase. Small interfering RNA-mediated depletion of NuSAP results in aberrant spindle formation, missegregation of chromosomes, and ultimately blocks cell proliferation. NuSAP is enriched on chromatin-proximal microtubules at meiotic spindles in Xenopus oocytes. When added at higher than physiological levels to Xenopus egg extract, NuSAP induces extensive bundling of spindle microtubules and causes bundled microtubules within spindle-like structures to become longer. In vitro reconstitution experiments reveal two direct effects of NuSAP on microtubules: first, it can efficiently stabilize microtubules against depolymerization, and second, it can cross-link large numbers of microtubules into aster-like structures, thick fibers, and networks. With defined components it was shown that the activity of NuSAP is differentially regulated by Importin (Imp) alpha, Impbeta, and Imp7. While Impalpha and Imp7 appear to block the microtubule-stabilizing activity of NuSAP, Impbeta specifically suppresses aspects of the cross-linking activity of NuSAP. It is proposed that to achieve full NuSAP functionality at the spindle, all three importins must be dissociated by RanGTP. Once activated, NuSAP may aid to maintain spindle integrity by stabilizing and cross-linking microtubules around chromatin (Ribbeck, 2006).
Mitotic spindle morphogenesis is a series of highly coordinated movements that lead to chromosome segregation and cytokinesis. The intermediate filament protein lamin B, a component of the interphase nuclear lamina, functions in spindle assembly. Lamin B assembles into a matrix-like network in mitosis through a process that depends on the presence of the guanosine triphosphate-bound form of the small guanosine triphosphatase Ran. Depletion of lamin B results in defects in spindle assembly. Dominant negative mutant lamin B proteins that disrupt lamin B assembly in interphase nuclei also disrupt spindle assembly in mitosis. Furthermore, lamin B is essential for the formation of the mitotic matrix that tethers a number of spindle assembly factors. It is proposed that lamin B is a structural component of the long-sought-after spindle matrix that promotes microtubule assembly and organization in mitosis (Tsai, 2006).
The Ran GTPase is required for nuclear assembly, nuclear transport, spindle assembly, and mitotic regulation. While the first three processes are relatively well understood, details of Ran's role in mitotic progression remain obscure. This study found that elevated levels of Ran's exchange factor (RCC1) abrogate the spindle assembly checkpoint in Xenopus egg extracts, restore APC/C activity, and disrupt the kinetochore localization of checkpoint regulators, including Mad2, CENP-E, Bub1, and Bub3. Depletion of Ran's GTPase activating protein (RanGAP1) and its accessory factor (RanBP1) similarly abrogates checkpoint arrest. By contrast, the addition of RanGAP1 and RanBP1 to extracts with exogenous RCC1 restores the spindle checkpoint. Together, these observations suggest that the spindle checkpoint is directly responsive to Ran-GTP levels. Finally, a clear wave of RCC1 association to mitotic chromosomes at the metaphase-anaphase transition was observed in normal cycling extracts, suggesting that this mechanism has an important role in unperturbed cell cycles (Arnaoutov, 2003).
The Ran GTPase controls multiple cellular processes, including nuclear transport, mitotic checkpoints, spindle assembly and post-mitotic nuclear envelope reassembly. This study examined the mitotic function of Crm1, the Ran-GTP-binding nuclear export receptor for leucine-rich cargo (bearing nuclear export sequence) and Snurportin-1. Crm1 localizes to kinetochores, and Crm1 ternary complex assembly is essential for Ran-GTP-dependent recruitment of Ran GTPase-activating protein 1 (Ran-GAP1) and Ran-binding protein 2 (Ran-BP2) to kinetochores. Crm1 inhibition by leptomycin B disrupts mitotic progression and chromosome segregation. Analysis of spindles within leptomycin B-treated cells shows that their centromeres are under increased tension. In leptomycin B-treated cells, centromeres frequently associate with continuous microtubule bundles that span the centromeres, indicating that their kinetochores do not maintain discrete end-on attachments to single kinetochore fibres. Similar spindle defects are observed in temperature-sensitive Ran pathway mutants (tsBN2 cells). Taken together, these findings demonstrate that Crm1 and Ran-GTP are essential for Ran-BP2/Ran-GAP1 recruitment to kinetochores, for definition of kinetochore fibres and for chromosome segregation at anaphase. Thus, Crm1 is a critical Ran-GTP effector for mitotic spindle assembly and function in somatic cells (Arnaoutov, 2005).
The Nup107-160 complex is a critical subunit of the nuclear pore. This complex localizes to kinetochores in mitotic mammalian cells, where its function is unknown. To examine Nup107-160 complex recruitment to kinetochores, human cells were stained with antisera to four complex components. Each antibody stained not only kinetochores but also prometaphase spindle poles and proximal spindle fibers, mirroring the dual prometaphase localization of the spindle checkpoint proteins Mad1, Mad2, Bub3, and Cdc20. Indeed, expanded crescents of the Nup107-160 complex encircled unattached kinetochores, similar to the hyperaccumulation observed of dynamic outer kinetochore checkpoint proteins and motors at unattached kinetochores. In mitotic Xenopus egg extracts, the Nup107-160 complex localized throughout reconstituted spindles. When the Nup107-160 complex was depleted from extracts, the spindle checkpoint remained intact, but spindle assembly was rendered strikingly defective. Microtubule nucleation around sperm centrosomes seemed normal, but the microtubules quickly disassembled, leaving largely unattached sperm chromatin. Notably, Ran-GTP caused normal assembly of microtubule asters in depleted extracts, indicating that this defect was upstream of Ran or independent of it. It is concluded that the Nup107-160 complex is dynamic in mitosis and that it promotes spindle assembly in a manner that is distinct from its functions at interphase nuclear pores (Orjalo, 2006).
The small Ran GTPase, a key regulator of nucleocytoplasmic transport, is also involved in microtubule assembly and nuclear membrane formation. This study shows by immunofluorescence, immunoelectron microscopy, and biochemical analysis that a fraction of Ran is tightly associated with the centrosome throughout the cell cycle. Ran interaction with the centrosome is mediated by the centrosomal matrix A kinase anchoring protein (AKAP450). Accordingly, when AKAP450 is delocalized from the centrosome, Ran is also delocalized, and as a consequence, microtubule regrowth or anchoring is altered, despite the persisting association of gamma-tubulin with the centrosome. Moreover, Ran is recruited to Xenopus sperm centrosome during its activation for microtubule nucleation. Centrosomal proteins such as centrin and pericentrin, but not gamma-tubulin, AKAP450, or ninein, undertake a nucleocytoplasmic exchange as they concentrate in the nucleus upon export inhibition by leptomycin B. Together, these results suggest a challenging possibility, namely, that centrosome activity could depend upon nucleocytoplasmic exchange of centrosomal proteins and local Ran-dependent concentration at the centrosome (Keryer, 2003).
The Ran GTPase plays a central function in control of nucleo-cytoplasmic transport in interphase. Mitotic roles of Ran have also been firmly established in Xenopus oocyte extracts. In this system, Ran-GTP, or the RCC1 exchange factor for Ran, drive spindle assembly by regulating the availability of 'aster-promoting activities'. In studies to assess whether the Ran network also influences mitosis in mammalian cells, it was found that overexpression of Ran-binding protein 1 (RanBP1), a major effector of Ran, induces multipolar spindles. These abnormal spindles are generated through loss of cohesion in mitotic centrosomes. Specifically, RanBP1 excess induces splitting of mother and daughter centrioles at spindle poles; the resulting split centrioles can individually organize functional microtubule arrays, giving rise to functional spindle poles. RanBP1-dependent centrosome splitting is specifically induced in mitosis and requires microtubule integrity and Eg5 activity. In addition, a fraction of RanBP1 was identified at the centrosome. These data indicate that overexpressed RanBP1 interferes with crucial factor(s) that control structural and dynamic features of centrosomes during mitosis and contribute to uncover novel mitotic functions downstream of the Ran network (Di Fiore, 2004).
In metazoa, the nuclear envelope breaks down and reforms during each cell cycle. Nuclear pore complexes (NPCs), which serve as channels for transport between the nucleus and cytoplasm, assemble into the reforming nuclear envelope in a sequential process involving association of a subset of NPC proteins, nucleoporins, with chromatin followed by the formation of a closed nuclear envelope fenestrated by NPCs. How chromatin recruitment of nucleoporins and NPC assembly are regulated is unknown. This study demonstrates that RanGTP production is required to dissociate nucleoporins Nup107, Nup153 and Nup358 from Importin beta, to target them to chromatin and to induce association between separate NPC subcomplexes. Additionally, either an excess of RanGTP or removal of Importin beta induces formation of NPC-containing membrane structures--annulate lamellae--both in vitro in the absence of chromatin and in vivo. Annulate lamellae formation is strongly and specifically inhibited by an excess of Importin beta. The data demonstrate that RanGTP triggers distinct steps of NPC assembly, and suggest a mechanism for the spatial restriction of NPC assembly to the surface of chromatin (Walther, 2003).
The small GTPase Ran is essential for spindle assembly. Ran is proposed to act through its nuclear import receptors importin alpha and/or importin beta to control the sequestration of proteins necessary for spindle assembly. To date, the molecular mechanisms by which the Ran pathway functions remain unclear. Using purified proteins, Ran-regulated microtubule binding of the C-terminal kinesin XCTK2, a kinesin important for spindle assembly, was reconstituted. The tail of XCTK2 binds to microtubules and that this binding is inhibited in the presence of importin alpha and beta (alpha/beta) and restored by addition of Ran-GTP. The bipartite nuclear localization signal (NLS) in the tail of XCTK2 is essential to this process, because mutation of the NLS abolishes importin alpha/beta-mediated regulation of XCTK2 microtubule binding. The data show that importin alpha/beta directly regulates the activity of XCTK2 and that one of the molecular mechanisms of Ran-regulated spindle assembly is identical to that used in classical NLS-driven nuclear transport (Ems-McClung, 2004).
Transport of macromolecules into and out of the nucleus is a highly regulated process. The RanGTP/RanGDP gradient controls the trafficking of molecules exceeding the diffusion limit of the nuclear pore across the nuclear envelope. Genetic interaction was found between genes establishing the Ran gradient, nuclear transport factor 2 (ntf-2), Ran GTPase activating protein (Sd), and the gene encoding Drosophila Profilin, chickadee (chic). The severe eye phenotype caused by reduction of NTF2 is suppressed by loss of function mutations in chic and gain of function mutations in Sd (RanGAP). In chic mutants, as in Sd-RanGAP, nuclear export is impaired. These data suggest that Profilin and the organization of the actin cytoskeleton play an important role in nuclear trafficking (Minakhena, 2005).
ntf-2 is an X-linked essential gene. Depending on the allele, animals die between the 2nd larval instar and the pupal stage. Some alleles have an adult survival rate of 8%-15% of expected, and all survivors show a small eye phenotype, strongly reduced numbers of ommatidia. The eye phenotype varies from 30% of normal size to a more severe phenotype displaying one or two small patches of 10-40 ommatidia (Minakhena, 2005).
The mutant eye-imaginal discs are smaller than wild-type and are often abnormally shaped. Overall, the structure of the mutant eye discs is perturbed and the organization of the actin cytoskeleton is strongly altered. Only few disorganized, irregularly spaced rabdomere-like structures are apparent in the posterior compartment of the eye disc (Minakhena, 2005).
Advantage was taken of the partial loss of function eye phenotype of ntf-2 alleles to identify genes functioning with ntf-2, and a dominant suppressor screen of the eye phenotype was performed. Males from 2nd and 3rd chromosomal deficiency stocks (deficiency/balancer) uncovering 70% to 80% of the two autosomes, or about 60% of the Drosophila genome, were crossed with ntf-2P7/FM7 females. In the next generation the number of surviving ntf-2 males also carrying a deletion was counted and the survivors monitored for their eye phenotype. For this screen, 136 individual crosses were set up, many of them repeatedly in order to obtain at least 150 adult progeny to screen for the eye phenotype. Deletions and rearrangements were identified in only four regions of the second chromosome that showed suppression. The suppression was confirmed using a second ntf-2 (P49) allele (Minakhena, 2005).
DNA rearrangements affecting regions 22A and 60B-D showed different results with the two ntf-2 alleles tested and were not pursued. Df(2l)cl-h2 (25D-F) appeared to rescue both viability and the eye phenotype, but the gene responsible for the suppression could not be identified. Df(2L)GpdhA (25D-26A) rescued the eye phenotype, but not viability. To identify the gene(s) responsible for the suppression of the eye phenotype, mutations were tested in several genes that are uncovered by Df(2L)GpdhA and are available from the Drosophila stock center (Minakhena, 2005).
Mutants in one gene, chickadee (chic), encoding Drosophila Profilin, uncovered by Df(2L)GpdhA, showed suppression of the ntf-2 eye phenotype. Several loss-of-function alleles of chic were tested, including a complete lethal null allele (chic221) and other partially viable alleles, that are either female, or male and female sterile. All chic alleles were crossed with at least 2 ntf-2 alleles, except chic221 that was tested with 4 different ntf-2 alleles. The suppression of the eye phenotype was observed in all crosses and the majority of surviving trans-heterozygous males showed suppression of the ntf-2 eye phenotype, restoration of wild-type eyes. The percent of males with wild-type eyes varied in different allele combinations. Surprisingly, the eye phenotype was usually either small or wild-type and virtually no eyes of intermediate size were observed (Minakhena, 2005).
To investigate the cause underlying the suppression of the ntf-2 phenotype and possible function of Profilin in nuclear transport, a reporter gene approach was used. Nuclear transport was assayed using UAS-NLS-NES reporter constructs C-terminally tagged with GFP in different mutant backgrounds. One construct contains a wild-type NLS and NES (UAS-NLS-NES-GFP), the other a wild-type NLS but a mutant NES that is not recognized by the nuclear export machinery (UAS-NLS-NESP12-GFP). Expression of the transgenes was driven by a heatshock-GAL4 driver, and the distribution of GFP was analyzed in salivary glands. The activity of the wild-type NES is stronger then that of the NLS. Hence, in wild-type the NLS-NES-GFP is usually localized in the cytoplasm. In contrast, NLS-NESP12-GFP has impaired nuclear export and strongly accumulates in nuclei. In homozygous chic01320 and the hetero-allelic combination chic2/chic221, the distribution of the GFP reporter is altered. In contrast to the cytoplasmic distribution of NLS-NES-GFP in wild-type, in the chic mutant salivary glands the GFP reporter is found predominantly in the nucleus. The localization of NLS-NESP12-GFP is similar in chic and wild-type, indicating that NLS-mediated import is not affected (Minakhena, 2005).
RanGAP functions in nuclear export of cargo and in Sd-RanGAP mutants the NLS-NES-GFP is found in the nucleus and NLS-NESP12-GFP is distributed the same as in wild-type. This failure of exporting NLS-NES-GFP in Sd-RanGAP mutants is reminiscent of what was observed in chic alleles (Minakhena, 2005).
Given the similarity in nuclear export phenotypes in Sd and chic mutants, tests were performed to see if Sd would also suppress the eye phenotype of ntf-2 alleles. The Sd (Sd72) chromosome was crossed with two ntf-2 alleles and it was found that the eye phenotype was suppressed in both of them. To confirm that the SD-RanGAP mutation, and not other genes on the Sd chromosome, is responsible for the suppression, a mutated Sd-RanGAP transgene (UAS-Sd-RanGAP12A-6) was expressed driven by hsp70-GAL4 or arm-GAL4 in ntf-2P7 and ntf-2P49 males and similar levels of suppression was observed as seen with Sd72 (Minakhena, 2005).
The genetic interaction between Sd-RanGAP and ntf-2 is not altogether surprising because both RanGAP and NTF2 are known to function in the formation of the RanGTP-GDP gradient. To investigate if RanGAP is affected in ntf-2 mutants the distribution of RanGAP was studied in eye discs (Minakhena, 2005).
In wild-type cells Ran-Gap is present in low levels in the cytoplasm and forms a clearly visible punctuated circle around the nucleus. The punctuate pattern of RanGAP is due to its association with nuclear pores. This distribution is different in ntf-2 discs. Patches of cells are observed in which RanGAP aggregates in small or large clumps near the nuclei, but in other cells the distribution of the protein looks relatively normal. This observation suggests, that the clumping of RanGAP is an effect of the abnormal organization of the cells within the ntf-2 disc. The cells with clumped RanGAP are usually in close proximity to cells with high levels of F-actin (Minakhena, 2005).
To investigate a connection between Profilin, RanGAP, and actin, it was next asked whether the function of Profilin or actin polymerization might have an effect on RanGAP localization. Clones were generated in eye discs of null alleles of the two genes chic (chic221) and, as a control, act up/capulet (acuE636). Acu participates in actin de-polymerization, the opposite function of Profilin (Minakhena, 2005).
In chic clones RanGAP protein is increased around the nuclear envelope and its distribution is uneven and patchy on the nuclear envelope surface. In wild-type even, punctuated circles are observed. This abnormal distribution was found in 100% of examined clones. In chic clones the level of F-actin was reduced. In the acu control clones high levels of F-actin are detected as expected, but the distribution of RanGAP is not significantly changed (Minakhena, 2005).
To test whether this patchy protein distribution of RanGAP on nuclear pores of chic22 cells is caused by problems in nuclear envelope assembly, the distribution of Lamin and nuclear pore proteins (Nups) was analyzed in chic221 clones. The distribution of both Lamin and Nups is affected in about 30% of clones. This is likely due to the mislocalization of RanGAP. It has been shown previously that RanGTPase functions in nuclear pore and envelope formation (Minakhena, 2005).
The staining experiments show higher levels of RanGAP around nuclei in chic eye disc clones. Whether this is due to overall higher levels of RanGAP in mutant cells was examined. The chic alleles used in the clonal analysis are homozygous lethal; therefore extracts were prepared from wild-type and mutant 1st instar larvae. In Western blots from extracts of chic221 (lethal at first and early second larval instar) and chic01320 (viable and female sterile) larvae, the amount of RanGAP present in mutants is not dramatically changed compared to wild-type. This may be because RanGAP and Profilin are maternally contributed and therefore at these early stages a difference in levels is not detected. Eye-antennal discs were dissected from normal larvae and larvae with chic clones. The dissected tissues also contained some brain material because eye-antennal discs are next to the brain hemispheres and are difficult to separate. In two separate experiments an increase of 30%-50% was seen in the intensity of the RanGAP band in extracts from discs carrying chic221 somatic clones compared to normal eye discs from chic221/+ larvae. The intensity of the RanGAP bands were normalized to that of the control Bic-D band and equals 2.6 for discs with clones and 1.8 for wild-type discs (Minakhena, 2005).
Why lowering the level of Profilin, which functions in actin polymerization, suppresses the ntf-2 phenotype is not immediately apparent, but there are several possible explanations. Lower levels of Profilin may result in reduction of the abnormal actin polymerization in ntf-2 mutant eye discs. But the finding that the ntf-2 eye phenotype is suppressed by the over-expression of RanGAP suggests that the disorganized appearance of F-actin is an indirect result of abnormal nuclear trafficking. Therefore lowering Profilin seems to also affect the abnormal nuclear trafficking inherent to ntf-2 eye discs. This supposition is bolstered by the finding that Profilin is essential for normal nuclear export. The results are consistent with F-actin being regulated by nuclear transport, and in turn, Profilin and Actin controlling aspects of nuclear trafficking (Minakhena, 2005).
Unpolymerized actin is found on NPC-attached nucleoplasmic filaments. It has been shown to function in the nuclear export of proteins and RNA. Unpolymerized actin also associates with Profilin and is exported from the nuclei in a Ran-dependant manner. It is not thought that these processes have a primary role in the mutant phenotypes because staining of ntf-2 eye discs and chic clones with anti-actin antibody display no obvious difference in the distribution of non-polymerized actin. Nevertheless, these processes have to be considered as part of the crosstalk between the actin cytockeleton and Ran-mediated nuclear trafficking (Minakhena, 2005).
That Profilin controls the localization of RanGAP is evident from the abnormal distribution of the protein in chic clones. The uneven distribution of RanGAP at the nuclear envelope is not due simply to higher levels of protein. In Sd transgenic lines that express wild-type or mutant RanGAP, higher levels of protein are found uniformly distributed in the cytoplasm and nucleus. In chic mutant cells, the RanGAP level is about doubled, but the protein distribution is different than that observed in the over-expressing lines (Minakhena, 2005).
The molecular basis for asymmetric meiotic divisions in mammalian oocytes that give rise to mature eggs and polar bodies remains poorly understood. Asymmetrically positioned meiotic chromosomes provide the cue for cortical polarity in mouse oocytes. This study shows that the chromatin-induced cortical response can be fully reconstituted by injecting DNA-coated beads into metaphase II-arrested eggs. The injected DNA beads induce a cortical actin cap, surrounded by a myosin II ring, in a manner that depends on the number of beads and their distance from the cortex. The Ran GTPase plays a critical role in this process, because dominant-negative and constitutively active Ran mutants disrupt DNA-induced cortical polarization. The Ran-mediated signaling to the cortex is independent of the spindle but requires cortical myosin II assembly. It is hypothesized that a RanGTP gradient serves as a molecular ruler to interpret the asymmetric position of the meiotic chromatin (Deng, 2007).
It appears that a unique characteristic of the mouse female meiotic system is that cortical polarity is cued by an internal asymmetry coming from the position of the DNA. Although it remains unclear whether any in vivo predetermined cortical cues exist to bias the movement of the meiotic chromatin, these experiments demonstrate that the egg is capable of establishing cortical polarity in any orientation in response to the DNA cue. It is interesting to note that DNA beads placed near the center of the oocyte failed to induce any cortical actomyosin assembly but were only effective within 20 µm of the plasma membrane. This distance-dependent signal propagation explains why oocytes with a defect in chromosome migration fail to undergo polar body extrusion. An intrinsic dependence of cortical actomyosin assembly on asymmetrically positioned chromosomes helps to ensure that polar body extrusion occurs in a highly restricted cortex overlying the chromosomes, therefore minimizing the loss of oocyte cytoplasm (Deng, 2007).
Because neither actin nor microtubules are required for chromatin-induced myoII cortical assembly, propagation of the signal through the cytoplasm is unlikely to be mediated through cytoskeleton-based transport. The distance dependence in the DNA bead-induced cortical response suggests that the signal decays rapidly as the distance from the chromatin increases, with a signaling range of up to 20 µm. This is consistent with the spatial range of the RanGTP gradient measured in Xenopus oocytes and somatic cells. Signal decay through Ran GTP hydrolysis could provide a convenient molecular ruler that ensures the assembly of actin and myosin occurs only when the chromosomes are within a certain distance of the cortex (Deng, 2007).
Involvement of a RanGTP gradient in mediating DNA signal to the cortex is consistent with the quantitative observation that the actin caps became narrower as bead distance to the nearest cortex increased. Similarly, a smaller gradient, for example, that generated by a single DNA bead, would be expected to result in a narrower actin cap, which in fact was observed. It is interesting to note that injection of the constitutively active RanQ69L at a high concentration, which could flatten the endogenous RanGTP gradient, inhibited DNA-induced cortical polarity as opposed to inducing multiple caps. This may suggest that some other factors critical for cortical cytoskeleton assembly exist in limited quantities and may become dispersed due to the global increase in active Ran concentration. Additionally, it was found that neither RanGTP- nor RCC1-coated beads were sufficient to induce cortical polarity or spindle assembly in mouse oocytes, suggesting that whereas these proteins are essential for chromatin signaling, chromatin may play additional roles during these processes (Deng, 2007).
Surprisingly, activation of myoII, which is regulated by MLCK, is required for the cortical accumulation of both actin and the PAR-3 polarity protein in response to the chromatin signal, suggesting that myoII activation may be a critical step downstream of the Ran signal. Although RanT24N did not inhibit global activation of MAP kinase, it appears that RanGTP is required for concentrating MAPK kinase activity to the vicinity of the chromosomes, which could result in local activation of MLCK and stimulation of myoII assembly. The function of myoII during this process may be distinct from the role of myoII in asymmetrically dividing C. elegans zygotes. In this mitotic system, myoII is proposed to concentrate polarity determinants to the anterior cortical domain through its actin-based motor activity, whereas the polarity function of myoII in mouse oocytes may not require its motor activity. MyoII may instead play a scaffolding role in tethering actin filaments and the PAR-3/aPKC polarity complex (Deng, 2007).
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