InteractiveFly: GeneBrief

Ran: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Ran guanine triphosphatase (GTPase) is a central molecule required for several cellular functions including nucleo- cytoplasmic transport, cell-cycle progression, and nuclear envelope assembly (reviewed by Melchior, 1998; Nakielny, 1999; Sazer, 2000). Ran appears to fulfill at least two distinct functions in the nuclear import process: (1) the energy required to effect nucleo-cytoplasmic transport is provided in the form of GTP hydrolysis by Ran (Melchior, 1993; Moore, 1993) and (2) Ran regulates the interaction between proteins that transport substrates across the nuclear membrane (Rexach, 1995; Gorlich, 1996).

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. Perturbation of the Ran pathway disrupts multiple steps of mitosis in syncytial Drosophila embryos and new mitotic processes have been uncovered 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 (Silverman-Gavrila, 2006).

During the cell cycle, the GTPase Ran regulates multiple cellular functions, including nucleocytoplasmic transport, nuclear envelope formation, and spindle assembly (Hetzer, 2002). The nucleotide-bound state of Ran (GTP or GDP) is spatially regulated by RCC1, the chromatin-bound guanine nucleotide exchange factor for Ran, and by RanGAP, which localizes to the cytoplasm and stimulates the intrinsic GTPase activity of Ran. The localization of these proteins is essential in regulating the activity of Ran throughout the cell cycle (Silverman-Gavrila, 2006 and references therein).

In vitro, RanGTP induces spindle assembly in mitotic Xenopus egg extracts in the absence of centrosomes, kinetochores, and chromatin (Carazo-Salas, 1999; Kalab, 1999; Ohba, 1999; Wilde, 1999) by altering microtubule (MT) dynamics (Carazo-Salas, 2001; Wilde, 2001), changing the balance of motor activity (Wilde, 2001), and increasing centrosomal MT nucleation (Carazo-Salas, 2001). The Ran pathway can regulate spindle assembly by RanGTP binding to nuclear transport receptors (NTRs) and preventing them from interacting with and inhibiting spindle assembly factors (SAFs) that posses a nuclear localization signal (NLS) (Trieselmann, 2003; Tsai, 2003; Ems-McClung, 2004). For this process to drive spindle assembly, RanGTP must be generated at chromosomes where it maintains SAFs in an active form. Indeed, many in vivo and in vitro studies infer that RanGTP exists around mitotic chromatin and spindles, whereas NTRs have a much broader distribution resulting in the creation of an environment around chromosomes where NLS-containing SAFs are active (Kalab, 2002; Trieselmann, 2002; Li, 2004; Caudron, 2005; Silverman-Gavrila, 2006 and references therein).

To define the mitotic role of the Ran pathway in vivo, previous studies injected anti-RanBP1 antibodies or NTRs into mammalian cells (Guarguaglini, 2000; Nachury, 2001). However, these studies did not define the dynamic nature of the observed defects and may have missed the complete series of events that led to the observed phenotype at the time of fixation. In an alternative approach, RNA interference (RNAi) was used in Caenorhabditis elegans to reduce expression levels of different Ran pathway proteins (Askjaer, 2002; Bamba, 2002). These studies defined a role for the Ran pathway in the production of spindle MTs. However, because all spindle MT assembly was inhibited, these studies were unable to define any other mitotic processes regulated by Ran. Furthermore, because spindle assembly was not examined until at least 16-48 h after RNAi application, it is possible that the observed defects could result from the disruption of other cellular processes. Indeed, Bamba (2002) showed that RNAi of Ran pathway components did inhibit nuclear transport. In addition, embryos and pronuclei were smaller after RNAi treatment for Ran pathway components (Askjaer, 2002; Silverman-Gavrila, 2006 and references therein).

To overcome these limitations, a microinjection approach was used to disrupt the Ran pathway just before mitosis and then immediately analyze the consequences of the perturbation on mitotic cellular function by time-lapse microscopy. By manipulating the Ran pathway in multiple ways and by microinjecting different concentrations of perturbants, it was possible to 'turn down' the pathway rather than completely or severely block it. This approach revealed multiple mitotic phenotypes that were not observed when all spindle MT production was inhibited (Askjaer, 2002; Bamba, 2002). By comparing the effects with known phenotypes from different mutants and RNAi screens, an integrative view has been generated of how Ran regulates mitosis in syncytial Drosophila embryos. Indeed, the Ran pathway was found to regulate both pre- and postmetaphase events (Silverman-Gavrila, 2006).

The data suggest that centrosomal MT production is regulated by NTR-sensitive SAFs. Severe disruption of the Ran pathway by injection of high concentrations of RanT24N inhibits MT production at centrosomes. However, less severe disruption of Ran pathway leads to MT production around chromosomes. These MTs begin to organize into a spindle in a manner reminiscent of acentrosomal spindle assembly in higher plants and female meiosis. In these conditions, it is possible that a small amount of RCC1 remains active and capable of producing a small amount of RanGTP. This reduced level of RanGTP may be sufficient to maintain active SAFs close to the chromatin and not around centrosomes some distance from the chromosomes. The MTs fail to resolve into stable spindles, possibly because of the rapid onset of the next interphase (Silverman-Gavrila, 2006).

MTs nucleated from centrosomes when the Ran pathway was only partially inhibited (upon injection of lower concentrations of RanT24N, RanGAP, and NTRs) were short and failed to grow toward the chromosomes. These data suggest that the Ran pathway is required for the stabilization of centrosomal nucleated MTs in vivo, consistent with recent in vitro findings (Caudron, 2005). Thus, embryonic spindle assembly might be driven by both a dominant centrosomal-mediated pathway required for the rapid assembly of spindles and a backup chromosomal pathway as in mammalian cells. The molecular targets of this pathway are unclear because TPX2 and XMAP310, which are required in vertebrates, have no known orthologues in Drosophila (Silverman-Gavrila, 2006 and references therein).

Inhibition of the Ran pathway often causes centrosomally nucleated MTs to randomly probe their environment rather than grow rapidly toward chromosomes. Previous studies have implied that RanGTP complexed with importin ß (see Drosophila Ketel) persists around the spindle throughout mitosis (Kalab, 2002; Li, 2004; Caudron, 2005). Therefore, RanGTP generated at chromosomes could form a spatial cue to guide MTs toward chromosomes. The data support this hypothesis and suggest that MT targeting to chromosomes may not proceed by a random search and capture mechanism. Instead, a 'directed' process operates where chromosomally generated RanGTP creates a molecular environment around chromosomes that facilitates MT growth preferentially toward the chromosomes. Such a scenario has recently been predicted in a mathematical model (Silverman-Gavrila, 2006 and references therein).

This study showed that the Ran pathway has a role in spindle pole/centrosome regulation in vivo as perturbation of the Ran pathway caused defects in spindle pole and centrosome organization. When the Ran pathway is disrupted centrosomes are released, suggesting that the Ran pathway regulates factors required for centrosome attachment to spindle poles. Perturbation of the Ran pathway also leads to the formation of multipolar spindles, sometimes by the splitting of existing poles. The findings are consistent with previous studies in which overexpression of RanBP1 (Di Fiore, 2003) led to centrosome abnormalities (Silverman-Gavrila, 2006).

The mechanism by which Ran regulates the spindle pole/centrosome remains unclear. However, many of the effects have been described upon inhibition of dynein and Aurora A. Aurora A has been shown to be regulated by the Ran pathway in vitro requiring RanGTP for its targeting and activation (Trieselmann, 2003; Tsai, 2003). Whether dynein or a regulator of dynein function is regulated by Ran remains to be tested. However, Ran itself may directly affect centrosomes because it has been localized to centrosomes through an interaction with AKAP450 (Keryer, 2003, Di Fiore, 2004). Interestingly, multipolar spindles form upon injection of RCC1, suggesting that elevated levels of RanGTP also disrupt this process (Silverman-Gavrila, 2006).

In addition, the failure of KLP61F to be recruited to centrosomes correlates with shorter spindles with broad poles. Previous studies that directly inhibit KLP61F have not pointed to a role in these processes. However, because the current experiments inhibit a broad range of processes that are downstream of Ran, it suggests that either a subset of KLP61F regulators, or factors working in coordination with KLP61F, may have a combined role in pole organization (Silverman-Gavrila, 2006).

The Ran pathway is required for the organization of spindle MTs. Strikingly, the disorganization did not result in a single spindle phenotype, suggesting that in Drosophila embryos, Ran regulates multiple factors involved in spindle organization. Many defects were consistent with a disruption of the balance of forces generated by antagonistic motors that are crucial for spindle assembly. Indeed, KLP61F and KLP3A, two motors that operate in balance with other motors, require the Ran pathway for their correct localization to the spindle. The spindle phenotypes observed do not correspond to just the inhibition of KLP61F. This could be due to the motor that operates in balance with KLP61F, Ncd, also being regulated by the Ran pathway. Indeed, the Xenopus homologue of Ncd, XCTK2 has been shown in vitro to be regulated by the Ran pathway (Ems-McClung, 2004). Inhibition of both motors in Drosophila embryos or of homologues of both motors in mammalian system largely cancels out the individual phenotypes. The phenotypes that are seen upon inhibition of these two motors in Drosophila embryos were seen upon perturbing the Ran pathway. The disruption of a balance of forces is unlikely to occur uniformly throughout an embryo due to the gradual diffusion of the injected material and would therefore result in a variety of spindle organization defects (Silverman-Gavrila, 2006).

Injection of RanT24N caused the metaphase plate to be three times wider (when spindle assembly proceeded) than in controls. A widening of the metaphase plate could be due to defects in MT attachment to kinetochores or inhibition of chromokinesins, proteins that generate the 'polar ejection force' that move chromosomes to the metaphase plate. Recently, the Ran pathway was shown to affect MT attachment to kinetochores (Arnaoutov, 2005). This study shows that the Ran pathway can regulate chromosome congression by regulating the MT attachment of KLP3A (a Kinesin-4 family member) reminiscent of regulation by Ran of the chromokinesin Kid (a Kinesin-10 family member) (Trieselmann, 2003). These findings suggest that chromokinesins from different kinesin families may share a common regulatory mechanism. The role of Ran in chromosome congression may not be restricted to the direct regulation of MT kinetochore attachment and chromokinesins because Aurora A, which is also regulated by Ran, is required for chromosome alignment at the metaphase plate in vertebrates (Silverman-Gavrila, 2006).

Anaphase A chromosome movement to the poles was slower in nuclei that otherwise progressed normally through mitosis upon Ran pathway perturbation. The reduced chromosome to pole velocity is identical to chromosome velocity in KLP59C-inhibited embryos. KLP59C, a kinetochore-localized MT depolymerase of the Kinesin-13 family is not an obvious candidate to be regulated by Ran, because it is cytosolic. Therefore, either a regulator of KLP59C or a protein that works in coordination with KLP59C, such as dynein, could be a target of the Ran pathway. Indeed, dynein inhibition causes similar reductions in chromosome movement to the poles. Another potential candidate is KLP3A, which is involved in chromosome segregation and whose targeting is dependent upon the Ran pathway (Silverman-Gavrila, 2006).

Nuclear divisions were also disrupted upon RCC1 injection, resulting in chromosome decondensation without chromosome disjunction. This could be caused by a chromosome decondesation defect or an override of the spindle checkpoint. The latter would be consistent with a study in Xenopus egg extracts, where increased levels of RCC1 circumvent the spindle checkpoint (Arnaoutov, 2003) by disrupting the localization of checkpoint regulators (Silverman-Gavrila, 2006).

Disruption of the Ran pathway prevents the correct assembly of the midbody in morphologically normal spindles. A failure to bundle midbody MTs was the most common defect. Bundling of midbody MTs requires the action of multiple kinesins, including Ncd, KLP3A, KLP61F, and Pavarotti. Indeed, KLP61F fails to localize to midbodies in embryos where the Ran pathway is perturbed. In extreme cases, no midbody MTs formed, suggesting that the Ran pathway, either through MT stabilizing factors or through regulating MT bundling, is required to for midbody stability (Silverman-Gavrila, 2006).

In vitro biochemical assays have shown that Aurora A activation is a downstream event of the mitotic Ran pathway (Trieselmann, 2003; Tsai, 2003). Injection of both RanT24N and DeltaN importin ß cause similar defects in Aurora A localization; suggesting that in vivo the Ran pathway via importins regulates Aurora A by facilitating its targeting to spindle MTs. Therefore, regulating the targeting and subsequent activation of Aurora A could be a major in vivo role for the Ran pathway in coordinating spindle assembly, especially because Aurora A has been implicated in both pre- and post-metaphase mitotic processes (Silverman-Gavrila, 2006).

Together, these data suggest that the Ran pathway has to remain active postmetaphase for chromosomes to successfully segregate. Indeed, proteins expected to increase RanGTP levels (RCC1) and those expected to decrease RanGTP levels (RanT24N and RanGAP) affect mitosis, suggesting that a balance of RanGTP/GDP may be crucial for mitotic progression. The data also suggests that a major mitotic role for Ran in Drosophila embryos is to prevent NTRs inhibiting SAFs during mitosis. Furthermore, the mitotic role of the Ran pathway is not restricted to MT-dependent events; Skeletor organization, which is independent of MTs, is dependent upon Ran. Therefore, the Ran pathway has the potential to regulate mitosis to a far greater extent than previously thought (Silverman-Gavrila, 2006).

REGULATION

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

To determine the mitotic role of Ran in vivo, proteins were injected that would perturb the Ran pathway into Drosophila embryos and their effects on MT organization and karyokinesis were assessed. Early embryonic Drosophila nuclei are contained within a syncytial cytoplasm and undergo mitosis synchronously for the first 14 nuclear cycles, followed by cellularization. At nuclear cycle 10, nuclei are at the embryo cortex where spindle assembly and nuclear division can be monitored by four-dimensional confocal microscopy. To reduce effects on Ran-dependent events during interphase (e.g., nuclear transport), embryos were injected just before mitotic entry, and cellular events were followed during the first mitosis after injection. However, it is still possible that a small percentage of defects arise from inhibiting Ran just before mitosis. Initially, when material is injected into an embryo, it will form a concentration gradient within the embryo with the highest concentration around the injection site. This concentration gradient of perturbant generates more severe effects proximal to the injection site and less severe effects distal to the injection site and has been described in other studies. This facet of the system results in different phenotypes depending on the amount of injection material reaching the spindle despite all the spindles sharing the same cytosol. This offers the current study a significant advantage because weak phenotypes can be seen that may be masked by more severe phenotypes when the Ran pathway is more severely inhibited (Silverman-Gavrila, 2006).

To perturb the Ran pathway, recombinant proteins were injected that are expected to either inhibit or activate the pathway. Multiple strategies were used to inhibit the Ran pathway. One approach was to inject a dominant negative allele of Ran, RanT24N, which is locked in the GDP bound form (Kornbluth, 1994). RanT24N could inhibit the Ran pathway by binding to and inhibiting RCC1, the nuclear exchange factor that generates RanGTP (Dasso, 1994), and/or by binding to importin ß (Hughes, 1998), although this binding is indirect and its affect on importin ß function is unknown (Lounsbury, 1996). To gain insight into which inhibitory process predominates during mitosis in Drosophila embryos, the localization of rhodamine-labeled GST-RanT24N was visualized. Rhodamine-labeled RanT24N localizes to condensed mitotic chromosomes throughout mitosis, as does RCC1 (Trieselmann, 2002). This localization pattern differs markedly from mitotic importin ß, which localized throughout the embryo with some concentration at the residual nuclear envelope (Trieselmann, 2002). Rhodamine-labeled GST-RanT24N binds equally well to RCC1 as unlabeled GST-RanT24N. Thus, the predominant effect of injected RanT24N will likely be the inhibition of RCC1, thereby preventing the continual generation of RanGTP at chromosomes. In addition to RanT24N, the Ran pathway can be inhibited by injecting RanGAP, which activates the intrinsic GTPase activity of Ran, thus reducing the level of RanGTP in the embryo. This inhibition should be less severe than that obtained with RanT24N, because RanGAP does not affect the production of RanGTP (Silverman-Gavrila, 2006).

In mitosis, the Ran pathway is proposed to function through RanGTP, which prevents the inhibition of NLS-containing SAFs via its NTR binding activity. Injecting RCC1 could elevate RanGTP levels, thereby up-regulating the pathway (Silverman-Gavrila, 2006).

Ran is involved in spindle assembly:
Inhibiting the Ran pathway in embryos disrupts spindle assembly. As expected, injection of RanT24N causes the greatest frequency of severe spindle assembly defects. The most dramatic phenotype was the complete inhibition of spindle assembly, where no spindle MTs were nucleated from either centrosomes or chromatin. When MTs were observed, they emanated around condensed chromatin rather than from centrosomes as in control and failed to resolve into spindles. These MTs persisted around condensed chromatin, which stained positive for phospho-histone H3, suggesting that it remained in a mitotic state. Although these effects were observed most frequently with the highest concentrations of RanT24N, they were also observed upon injection of Ran-GAP proximal to the injection site (Silverman-Gavrila, 2006).

To observe more subtle effects of Ran, lower concentrations of RanT24N and RanGAP that allowed some MT production were injected. Under these conditions, spindles initially formed, but they soon became unstable and disorganized. Bundles of centrosomally nucleated MTs grew outward, away from the chromatin. Often, these MTs encountered MTs from neighboring spindles causing the spindles to fuse. Bipolar spindles that did form had altered dimensions (Silverman-Gavrila, 2006).

Spindle pole organization was also disrupted: poles became broader and less focused and in extreme cases, monopolar spindles formed. Where nuclei possessed multiple centrosomes, multipolar spindles formed. Sometimes, poles split or centrosomes were released. Injection of recombinant RCC1 also disrupted spindles. Although many defects were similar to those caused by RanT24N, RCC1 did stimulate the formation of a higher proportion of multipolar spindles (Silverman-Gavrila, 2006).

These data suggest that Ran is required for spindle assembly in vivo in Drosophila embryos (Silverman-Gavrila, 2006).

Nuclear transport receptors inhibit spindle assembly:
Previous studies strongly suggest that the mitotic function of Ran stems from RanGTP, generated at chromosomes, binding to NLS-containing SAFs and inhibiting them (reviewed in Di, 2004). Therefore, to determine whether this mechanism applies in vivo, NTRs were microinjected to elevate their levels within the embryo. These additional NTRs would be predicted to counteract the action of RanGTP and inhibit RanGTP-dependent events. One method was to inject recombinant nuclear import receptors either individually (importin alpha or importin ß) or in combination, because they act together in a complex. In addition, a fragment of importin ß was used that lacks the amino-terminal domain, making it insensitive to RanGTP. Another way to elevate NTRs would be to inject RanBP1. RanBP1 can bind to RanGTP and stimulate the release of NTRs from Ran, in particular nuclear export factors and their cargo from RanGTP (Bischoff, 1997; Kehlenbach, 1999). Alternatively, RanBP1 could stimulate a decrease in RanGTP levels by enhancing RanGAP activity (Coutavas, 1993; Bischoff, 1995) or by inhibiting RCC1 (Nicolas, 1997). Although no homologue of RanBP1 exists in Drosophila, the Ran binding domain in human RanBP1 is 43.8% identical to those in Drosophila RanBP2 (NUP358) and would therefore act as a diffusible source of Ran binding domains to inhibit the Ran pathway (Silverman-Gavrila, 2006).

Injection of NTRs affected spindles in a similar way as the injection of moderate concentrations of RanT24N, resulting in a comparable array and degree of phenotype severity. These data suggest that Ran regulates spindle assembly through regulating the binding of NTRs to SAFs. Interestingly, upon injection of pairs of importins, many more MTs initially formed in prometaphase as bundles around condensed chromosomes. In contrast, MTs in control embryos nucleate from centrosomes and rapidly grew toward chromosomes. In NTR-injected embryos, bundles of MTs slowly reorganized into spindle-like structures but never fully resolved into stable spindles (Silverman-Gavrila, 2006).

Injection of RanBP1 also caused similar defects to injected RanT24N and NTRs. However, uniquely some spindles exhibited consecutive defects. First centrosomes were released, then the spindle elongated. Because one function of RanBP1 is to stimulate the release of nuclear exported cargo from exportins on the cytoplasmic face of the nuclear pore complex (Kehlenbach, 1999), tests were performed to see whether a nuclear exportin has a role in spindle assembly. Injection of CAS, the nuclear export factor for importin alpha, resulted in defects similar to those caused by low doses of RanT24N. However, there were differences: CAS caused the highest frequency of multipolar spindles (Silverman-Gavrila, 2006).

Together, these data suggest that Ran, through its modulation of NTR binding to SAFs, regulates multiple aspects of spindle assembly in syncytial Drosophila embryos (Silverman-Gavrila, 2006).

MT targeting to chromosomes:
A common phenotype seen in all injections that inhibit the Ran pathway (RanT24N, RanGAP, NTRs, and RanBP1) was a failure of centrosomally nucleated MTs to grow rapidly and directly toward chromosomes. On inhibition of the Ran pathway, bundles of MTs, still focused at the poles, began to grow in random directions, probing their environment by repeated cycles of growth and shrinkage. This is in stark contrast to control embryos where centrosomally nucleated MTs grew rapidly toward chromosomes. These data suggest that Ran is required for correct MT targeting to chromosomes by preventing NTRs binding to and inhibiting SAFs (Silverman-Gavrila, 2006).

Chromosome organization:
To analyze the role of the Ran pathway in mitotic chromosome organization, a similar strategy was used to that described above, using a Drosophila line expressing GFP-histone to visualize the chromatin. In a small number of nuclei, inhibiting the Ran pathway prevented nuclear division. Chromatin masses seemed to stay in an interphaselike state (decondensed) or early prophase-like state (slightly condensed) and did not divide, suggesting that either the Ran pathway has a role in chromosome condensation or Ran has a role in mitotic entry. Some nuclei persisted at the cortex, and others exhibited nuclear fallout. However, the DNA in most nuclei condensed and chromosomes segregated. In some cases, even where chromosomes condensed normally, congression to the metaphase plate was disrupted causing a broadening (~3-fold) of the metaphase plate in the centrosome-to-centrosome axis. These data suggest that the Ran pathway is required for chromosome congression to the metaphase plate (Silverman-Gavrila, 2006).

The Ran pathway is required for postmetaphase events

Chromosome movement and segregation:
Inhibiting the Ran pathway using factors that directly affect Ran (RanT24N, RanGAP, and RCC1) or downstream factors of Ran (NTRs), disrupted chromosome segregation to different degrees compared with uninjected and control-injected embryos. Frequently, inhibition of the Ran pathway resulted in the formation of anaphase bridges. Although some bridges resolved, others did not, leading to the formation of nuclei with a 4N DNA content (judged by a doubling of the fluorescence intensity compared with adjacent nuclei that had normal division). In less severe cases, individual chromosomes lagged behind the main chromosome mass. Occasionally, disjoined chromosomes fused with their sister chromosomes resulting in nuclei with a 4N DNA content. In extreme cases, separated chromosomes from two or more nuclei fused, leading to the formation of large chromosomal masses or two or more nuclei fused (Silverman-Gavrila, 2006).

Other chromosome segregation phenotypes stemmed from apparent defects in cell cycle progression as chromosomes remained in a metaphase-condensed state. Occasionally, there seemed to be an override of the spindle checkpoint, as evidenced from the observation that metaphase chromosomes decondensed without disjunction and transited into an interphaselike state. This was also the most common effect upon injection of RCC1, which should up-regulate the Ran pathway (Silverman-Gavrila, 2006).

Distal to the injection site on morphologically normal spindles inhibition of the Ran pathway caused up to a 50% reduction in the velocity of chromosome movement to the spindle poles. In contrast, up-regulation of the pathway upon the injection of RCC1 did not alter the velocity of chromosome segregation. Together, these data demonstrate that the Ran pathway is required for multiple steps in chromosome segregation during anaphase (Silverman-Gavrila, 2006).

Midbody organization:
The effect of inhibiting the Ran pathway was not restricted to spindle assembly. Distal to the injection site spindles formed normally and progressed to anaphase but were unable to form normal midbodies. In some instances, midbody formation was completely inhibited, but when midbodies did assemble they had unbundled MTs, were bent or were narrow. These data demonstrate a role for the Ran pathway after spindle assembly and metaphase, suggesting that the Ran pathway is required throughout mitosis for correct MT organization (Silverman-Gavrila, 2006).

The Ran pathway is required for Aurora A targeting to spindle MTs

Perturbing the Ran pathway in vivo has dramatic effects on spindle assembly and function. Ran could achieve this by directly regulating the activity of a large number of SAFs, by regulating signal transduction pathways, or both. One candidate target is the Aurora A kinase, which in vitro is suggested to be downstream of Ran in the Ran spindle assembly pathway (Trieselmann, 2003; Tsai, 2003, 2005). Aurora A is recruited to centrosomes in interphase by Centrosomin where it is activated by ajuba and HEF-1. During mitosis in somatic mammalian cells Aurora A relocates to spindle MTs in a TPX2-dependent manner (Silverman-Gavrila, 2006 and references therein).

To address whether Aurora A could be affected by Ran, Aurora A localization was assessed in control and RanT24N-injected embryos. In control embryos, Aurora A localized to centrosomes in interphase and prophase, but in prometaphase it began to redistribute along spindle MTs. On injection of RanT24N, 83.3% of spindles showed mislocalization of Aurora A, which now concentrated at centrosomes and did not localize to spindle MTs (Silverman-Gavrila, 2006).

To determine whether Aurora A targeting to MTs is regulated by NTRs as demonstrated in vitro (Trieselmann, 2003), DeltaN importin ß was injected and Aurora A localization was examined. DeltaN importin ß injection also prevented Aurora A redistribution to spindle MTs in 92% of spindles. This strongly suggests that in vivo Aurora A function is regulated by a NTR sensitive SAF (Silverman-Gavrila, 2006).

The Ran pathway is required for Skeletor organization

Whether the Ran pathway is required for MT-independent mitotic events was investigated. One such process is the assembly of a matrix as defined by one of its components, Skeletor (Walker, 2000). RanT24N disrupted skeletor distribution within the embryo in 80.9% of spindles. Skeletor was asymmetrically distributed outside the area of disrupted spindles, persisting in the vicinity of condensed chromatin rather than around the residual spindle. These data suggest that Skeletor organization is dependent upon the Ran pathway (Silverman-Gavrila, 2006).

The Ran pathway is required for the localization of the motors KLP61F and KLP3A

Many of the spindle defects observed upon disruption of the Ran pathway could stem from misregulation of motor proteins. Indeed, previous in vitro studies showed that the Ran pathway affects the function of Eg5, a tetrameric kinesin of the kinesin-5 family (Lawrence, 2004) involved in spindle assembly (Wilde, 2001), of Kid (Trieselmann, 2003), a chromokinesin of the kinesin-10 family (Lawrence, 2004), and XCTK2 (Ems-McClung, 2004), a mitotic kinesin of kinesin-14 family (Lawrence, 2004). Therefore, the consequence were examined of disrupting the Ran pathway on the localization of KLP61F (the Drosophila homologue of Eg5) and KLP3A, a Drosophila chromokinesin from the kinesin-4 family (Lawrence, 2004; Silverman-Gavrila, 2006 and references therein).

After the injection of RanT24N, KLP61F distribution was disrupted in 91.8% of spindles. In control embryos, phospho-KLP61F (phosphorylated at T933 and the form of KLP61F that is recruited to spindles) (Sharp, 1999) has a cell cycle-dependent distribution: it is nuclear in interphase and localizes to spindle MTs in prometaphase and metaphase, with a small proportion at centrosomes. In anaphase and telophase, KLP61F localizes to the central spindle (Sharp, 1999). However, upon RanT24N injection KLP61F was barely detectable on spindles with disorganized MTs. Furthermore, an absence of KLP61F staining at centrosomes correlated with metaphase spindles that were shorter and had broader poles. In addition, in RanT24N injected embryos KLP61F did not localize to 93.7% of midbodies (Silverman-Gavrila, 2006).

KLP3A has a cell cycle-dependent distribution localizing to the nucleus in interphase and relocating to the central spindle region, including chromosomes, from prometaphase to anaphase. In telophase, KLP3A persists on the central midbody MTs (Kwon, 2004). After injection of RanT24N, KLP3A distribution was disrupted in 96.2% of spindles: KLP3A localized to chromosomes but not to central spindle MTs. Together, these data demonstrate that one function of the Ran pathway is to regulate the targeting of mitotic motors in vivo (Silverman-Gavrila, 2006).

Involvement of Ran in the regulation of phagocytosis against virus infection in S2 cells

Phagocytosis plays important roles in innate and adaptive immunity in animals. Some small G proteins are found to be related to phagocytosis. However, the Ran GTPase has not been intensively characterized in immunity. In this paper, the sequence analysis showed that the Ran is highly conserved in animals, suggesting that its function is preserved during animal evolution. The results showed that Ran is upregulated in S2 cells in response to DCV infection. It was further revealed that the antiviral phagocytosis is mediated by Ran in S2 cells. By comparison with the early marker and late marker of phagosomes, the results showed that the Ran protein plays an essential role at the early stage of phagocytosis or throughout the entire phagocytic process. Therefore these findings enlarged the limited knowledge about the phagocytosis regulation by small G proteins concerning to the nucleus (Ye, 2014).

Protein Interactions

Nuclear mislocalization of enzymatically active RanGAP causes segregation distortion in Drosophila

Segregation Distorter (SD) is a meiotic drive system in Drosophila that causes preferential transmission of the SD chromosome from SD/SD+ males owing to dysfunction of SD+ spermatids. The Sd locus, which is essential for distortion, encodes a truncated RanGAP (Ran GTPase activating protein), a key nuclear transport factor. This study shows that Sd-RanGAP retains normal enzyme activity but is mislocalized to nuclei. Distortion is abolished when enzymatic activity or nuclear localization of Sd-RanGAP is perturbed. Overexpression of Ran or RanGEF (Ran GTPase exchange factor) in the male germline fully suppresses distortion. It is concluded that mislocalization of Sd-RanGAP causes distortion by reducing nuclear RanGTP, thereby disrupting the Ran signaling pathway. Nuclear transport of a GFP reporter in salivary glands is impaired by SD, suggesting that a defect in nuclear transport may underlie sperm dysfunction (Kusano, 2001).

Segregation distortion induced by wild-type RanGAP in Drosophila

Segregation Distorter (SD) is a meiotic drive system in Drosophila that causes preferential transmission of the SD chromosome from SD/SD⁺ males owing to the induced dysfunction of SD⁺ spermatids. The key distorter locus, Sd, is a dominant neomorphic allele encoding a truncated, but enzymatically active, RanGAP (RanGTPase-activating protein) whose nuclear mislocalization underlies distortion by disrupting the Ran signaling pathway. This study shows that even wild-type RanGAP can cause segregation distortion when it is overexpressed in the male germ line or when the gene dosage of a particular modifier locus is increased. Both manipulations result in substantial nuclear accumulation of RanGAP. Distortion can be suppressed by overexpression of Ran or Ran guanine nucleotide exchange factor (RanGEF) in the male germ line, indicating that the primary consequence of nuclear mislocalization of RanGAP is reduction of intranuclear RanGTP levels. These results prove that segregation distortion does not depend on any unique properties of the mutant RanGAP encoded by Sd and provide a unifying explanation for the occurrence of distortion in a variety of experimental situations (Kusano, 2002).

A fundamental principle of Mendelian genetics is the equal transmission of both homologues or alleles from a heterozygous pair. Nonetheless, meiotic drive systems, in which this principle is regularly violated by the preferential transmission of a particular chromosome or allele at the expense of its partner, exist in nature. Segregation distorter (SD) is a naturally occurring meiotic drive system located on the second chromosome of Drosophila melanogaster. SD/SD⁺ males transmit the SD chromosome to almost 100% of the progeny. Distortion at full strength requires not only the primary locus, Sd, but also several upward modifiers including Enhancer of SD [E(SD)], Modifier of SD [M(SD)], and Stabilizer of SD [St(SD)]. The target of Sd and the upward modifiers is the Responder (Rsp) locus: chromosomes that carry a sensitive (Rsp^s) or supersensitive (Rsp^ss) allele of Responder are sensitive to the action of SD, whereas those carrying an insensitive allele (Rspⁱ) are resistant. The basic mechanism of distortion is sperm dysfunction, which involves a failure of chromatin condensation in SD⁺-bearing spermatids, leading to subsequent defects in spermatid elongation and maturation (Kusano, 2002 and references therein),

Sd, a dominant neomorphic mutation, encodes a truncated RanGTPase-activating protein (RanGAP) lacking 234 aa at the C terminus (Sd-RanGAP; Merrill, 1999). Ran is a small GTPase located predominantly in the nucleus. Along with its cofactors, RanGAP and RanGEF (Ran guanine nucleotide exchange factor), Ran is essential for nuclear transport (Görlich, 1999) as well as for other nuclear functions, including cell cycle regulation, mitotic spindle formation, and postmitotic nuclear envelope assembly. The cytoplasmic localization of RanGAP and the nuclear localization of RanGEF establish a concentration gradient of RanGTP across the nuclear envelope that is critical for proper function of the Ran signaling pathway. Sd-RanGAP retains essentially normal enzymatic activity but is mislocalized to nuclei. Both enzymatic activity and nuclear localization of Sd-RanGAP are required for distortion (Kusano, 2001). Overexpression of either Ran or RanGEF in the male germ line suppresses distortion, suggesting that Sd-RanGAP causes distortion by diminishing the concentration of nuclear RanGTP, thereby disrupting Ran-dependent functions. In particular, nuclear transport may be impaired (Kusano, 2001; Kusano, 2002).

By analogy with results of recent studies on yeast RanGAP (Feng, 1999), nuclear mislocalization of Sd-RanGAP can be explained if the usual cytoplasmic distribution of RanGAP is the outcome of a dynamic mechanism in which RanGAP shuttles in and out of nuclei. Normally, this process favors nuclear export over nuclear localization. Shuttling of yeast RanGAP is mediated by a nuclear localization signal (NLS) and two nuclear export signals (NESs) that are located in evolutionarily conserved regions of the protein. Amino acid alignment between yeast and Drosophila RanGAP suggests that these NLSs and NESs are conserved in Drosophila RanGAP. The C-terminal deletion in Sd-RanGAP removes one of the putative NESs, which could shift the equilibrium in favor of a predominant nuclear localization (Kusano, 1999). If this idea is correct, then there are no intrinsic functional differences between Sd-RanGAP and wild-type RanGAP other than their contrasting subcellular distributions. Thus, even wild-type RanGAP might be capable of causing segregation distortion if its subcellular distribution were perturbed such that there was significant accumulation inside nuclei. Alternatively, distortion could depend on entirely novel attributes of the truncated Sd-RanGAP that could not be mimicked in any simple way by the wild-type protein (Kusano, 2002).

This study demonstrates that wild-type RanGAP is capable of causing segregation distortion when it is overexpressed in the male germ line, a manipulation that results in aberrant nuclear accumulation of this enzyme. Moreover, presence of an extra dose of E(SD) causes segregation distortion even when Sd is absent. In this case, too, there is substantial accumulation of wild-type RanGAP inside nuclei. Distortion in these cases can be suppressed by overexpression of Ran or RanGEF in the male germ line, arguing that, as in the case of SD, a reduction in the level of intranuclear RanGTP is the primary cause of distortion. These results prove that nuclear localization of RanGAP activity is sufficient to cause distortion and provide a unifying explanation for the occurrence of distortion in a variety of experimental situations (Kusano, 2002).

Thus, overexpression of wild-type RanGAP in the male germ line causes strong segregation distortion. This distortion exhibits all of the properties characteristic of that associated with native SD chromosomes: it requires RanGAP enzymatic activity, it depends on the presence of upward modifiers and a sensitive Responder (Rsp) target, and it is eliminated by known suppressors of segregation distortion as well as by overexpression of Ran or RanGEF in the male germ line. Most strikingly, overexpression of wild-type RanGAP results in substantial mislocalization of the protein to nuclei in primary spermatocytes, similar to what has been observed for normal levels of expression of Sd-RanGAP. This result suggests that wild-type RanGAP is capable of entering nuclei and most likely does so to some degree even at endogenous levels of expression. Although this conclusion is contrary to the prevalent view that RanGAP is localized exclusively in the cytoplasm, it is in agreement with studies in yeast indicating that the normal cytoplasmic localization of RanGAP in the outcome of a dynamic equilibrium in which RanGAP shuttles in and out of the nucleus (Feng, 1999). This process is thought to be mediated by the activities of an NLS and two NESs that are located in evolutionarily conserved portions of the protein (Feng, 1999). Amino acid alignments with Drosophila RanGAP suggest that these elements are also present in the Drosophila protein. It has been argued that the truncated RanGAP encoded by Sd deletes one of the NESs, thereby biasing the subcellular distribution from cytoplasm to nucleus. In the present case, 10- to 20-fold overexpression of wild-type RanGAP is apparently sufficient to alter the usual equilibrium such that an excessive amount of RanGAP accumulates inside nuclei (Kusano, 2002).

The notion that nuclear mislocalization of enzymatically active RanGAP is responsible for distortion can also account for the distortion caused by two doses of E(SD) (Temin, 1991). Increased dosage of the modifier element E(SD) results in a marked accumulation of wild-type RanGAP inside spermatocyte nuclei even though the protein is expressed at endogenous levels. On the basis of this observation, it seems likely that E(SD) encodes a factor that facilitates nuclear import of RanGAP, inhibits its export, or in some other way enhances the nuclear localization of RanGAP. Because distortion associated with wild-type RanGAP is elicited by two doses of E(SD) but not one, it is imagined that the physiological effect of this protein is dose dependent and that a certain threshold amount is required to accumulate a sufficient level of wild-type RanGAP in germ-line nuclei to produce distortion. A single dose of E(SD) substantially increases the strength of distortion caused by Sd-RanGAP (Ganetzky, 1977; Merrill, 1999). The results suggest that E(SD) may do so by further increasing the propensity of Sd-RanGAP to localize to nuclei. Because E(SD) behaves as a dominant neomorphic mutation, what function is normally subserved by the wild-type protein remains an intriguing question. Unfortunately, the location of E(SD) within the heterochromatin of the second chromosome will greatly complicate molecular identification and characterization of this gene (Kusano, 2002).

The results argue that, aside from its altered subcellular distribution, there is nothing intrinsically unusual about the truncated Sd-RanGAP compared with the wild-type enzyme. Although the physical basis for nuclear mislocalization of RanGAP differs for Sd-RanGAP, ß2-RanGAP, and increased dosage of E(SD), the underlying mechanism of distortion is apparently the same in all of these cases. In the appropriate genetic background, all that is required to cause segregation distortion in Drosophila is an excess of enzymatically active RanGAP in nuclei of the male germ line. The information gained from this study provides a unifying explanation for the occurrence of distortion and will be essential in elucidating the remaining mechanistic details of this phenomenon (Kusano, 2002).

The organization and evolution of the Responder satellite in species of the Drosophila melanogaster group: dynamic evolution of a target of meiotic drive

Satellite DNA can make up a substantial fraction of eukaryotic genomes and has roles in genome structure and chromosome segregation. The rapid evolution of satellite DNA can contribute to genomic instability and genetic incompatibilities between species. Despite its ubiquity and its contribution to genome evolution, little about the dynamics of satellite DNA evolution. The Responder (Rsp) satellite DNA family is found in the pericentric heterochromatin of chromosome 2 of Drosophila melanogaster. Rsp is well-known for being the target of Segregation Distorter (SD), an autosomal meiotic drive system in D. melanogaster. This paper presents an evolutionary genetic analysis of the Rsp family of repeats in D. melanogaster and its closely-related species in the melanogaster group (D. simulans, D. sechellia, D. mauritiana, D. erecta, and D. yakuba) using a combination of available BAC sequences, whole genome shotgun Sanger reads, Illumina short read deep sequencing, and fluorescence in situ hybridization. Rsp repeats were shown to have euchromatic locations throughout the D. melanogaster genome, that Rsp arrays show evidence for concerted evolution, and that Rsp repeats exist outside of D. melanogaster, in the melanogaster group. The repeats in these species are considerably diverged at the sequence level compared to D. melanogaster, and have a strikingly different genomic distribution, even between closely-related sister taxa. It is concluded that the genomic organization of the Rsp repeat in the D. melanogaster genome is complex. It consists of large blocks of tandem repeats in the heterochromatin and small blocks of tandem repeats in the euchromatin. The discovery of heterochromatic Rsp-like sequences outside of D. melanogaster suggests that SD evolved after its target satellite and that the evolution of the Rsp satellite family is highly dynamic over a short evolutionary time scale (<240,000 years) (Larracuente, 2014: PubMed).

Mutations to the piRNA pathway component aubergine enhance meiotic drive of segregation distorter in Drosophila melanogaster

Diploid sexual reproduction involves segregation of allelic pairs, ensuring equal representation of genotypes in the gamete pool. Some genes, however, are able to 'cheat' the system by promoting their own transmission. The Segregation distorter (Sd) locus in Drosophila melanogaster males is one of the best-studied examples of this type of phenomenon. In this system the presence of Sd on one copy of chromosome 2 results in dysfunction of the non-Sd-bearing (Sd⁺) sperm and almost exclusive transmission of Sd to the next generation. The mechanism by which Sd wreaks such selective havoc has remained elusive. However, its effect requires a target locus on chromosome 2 known as Responder (Rsp). The Rsp locus comprises repeated copies of a satellite DNA sequence and Rsp copy number correlates with sensitivity to Sd. Under distorting conditions during spermatogenesis, nuclei with chromosomes containing greater than several hundred Rsp repeats fail to condense chromatin and are eliminated. Recently, Rsp sequences were found as small RNAs in association with Argonaute family proteins Aubergine (Aub) and Argonaute3 (AGO3). These proteins are involved in a germline-specific RNAi mechanism known as the Piwi-interacting RNA (piRNA) pathway, which specifically suppresses transposon activation in the germline. This study evaluated the role of piRNAs in segregation distortion by testing the effects of mutations to piRNA pathway components on distortion. Further, mutations to the aub locus of a Segregation Distorter (SD) chromosome were specifically targeted using ends-out homologous recombination. The data in this study demonstrate that mutations to piRNA pathway components act as enhancers of SD (Gell, 2013).

Tamo selectively modulates nuclear import in Drosophila

The NF-kappaB/Rel pathway functions in the establishment of dorsal-ventral polarity and in the innate humoral and cellular immune response in Drosophila. An important aspect of all NF-kappaB/Rel pathways is the translocation of the Rel proteins from the cytoplasm to the nucleus, where they function as transcription factors. A new protein, Tamo, has been identified that binds to Drosophila Rel protein Dorsal, but not to Dorsal lacking the nuclear localization sequence. Tamo does not bind to the other Drosophila Rel proteins, Dif and Relish. The Tamo-Dorsal complex forms in the cytoplasm and Tamo also interacts with a cytoplasmically orientated nucleoporin. In addition Tamo binds the Ras family small GTPase, Ran. Tamo functions during oogenesis and, based on phenotypic analysis, controls the levels of nuclear Dorsal in early embryos. It further regulates the accumulation of Dorsal in the nucleus after immune challenge. It is concluded that Tamo has an essential function during oogenesis. Tamo interacts with Dorsal and proteins that are part of the nuclear import machinery. It is proposed that tamo modulates the levels of import of Dorsal and other proteins (Minakhina, 2003).

The Drosophila RCC1 homolog, Bj1, regulates nucleocytoplasmic transport and neural differentiation during Drosophila development

The Bj1 gene encodes the Drosophila homolog of RCC1, the guanine-nucleotide exchange factor for RanGTPase. This study provides the first phenotypic characterization of a RCC1 homolog in a developmental model system. Bj1 (dRCC1) was identified in a genetic screen to identify mutations that alter central nervous system development. Zygotic dRCC1 mutant embryos exhibit specific defects in the development and differentiation of lateral CNS neurons although cell division and the cell cycle appear grossly normal. dRCC1 mutant nerve cords contain abnormally large cells with compartmentalized nuclei and exhibit increased transcription in the lateral CNS. Since RCC1 is an important component of the nucleocytoplasmic transport machinery, it was found that dRCC1 function is required for nuclear import of nuclear localization signal sequence (NLS)-carrying cargo molecules. Finally, it was shown that dRCC1 is required for cell proliferation and/or survival during germline, eye and wing development and that dRCC1 appears to facilitate apoptosis (Shia, 2004).

The importin-ß P446L dominant-negative mutant protein loses RanGTP binding ability and blocks the formation of intact nuclear envelope

Three of the four independently induced Ketel^D dominant-negative female sterile mutations that identify the Drosophila importin-ß gene, originated from a C4114-->T transition and the concurrent replacement of Pro446 by Leu (P446L). CD spectroscopy of representative peptides with Pro or Leu in the crucial position revealed that upon the Pro-->Leu exchange the P446L mutant protein loses flexibility and attains most likely an open conformation. The P446L mutation abolishes RanGTP binding of the P446L mutant form of importin-ß protein and results in increased RanGDP binding ability. Notably, the P446L mutant importin-ß does not exert its dominant-negative effect on nuclear protein import and has no effect on mitotic spindle-related functions and chromosome segregation. However, it interferes with nuclear envelope formation during mitosis-to-interphase transition, revealing a novel function of importin-ß (Timinszky, 2002).

The fact that in three of the four independently isolated Ketel^D mutations the same C-->T transition and the concurrent replacement of Pro446 by Leu is the basis of dominant female sterility underlines the importance of Pro446 in importin-ß function. It is assumed that during a nuclear import cycle, whereas importin-ß interacts with the NLS containing protein (directly or through importin-ß), nucleoporins and RanGTP, the conformation of importin-ß changes significantly. In fact the region around HEAT repeat 10 was suggested, based on X-ray crystallography, to be a flexible point during switching between the IBB- and the Ran-bound forms. Pro446 resides in the linker region connecting HEAT repeats 10 and 11 and, as described in this study, plays a crucial role in enduring flexibility of importin-ß. CD spectra of model peptides representing the wild-type and the P446L mutant proteins reveal loss of flexibility upon Pro446-->Leu replacement. The lost flexibility is most likely the consequence of fusion of the small alpha-helix in the linker region with the alpha-helix of HEAT 10B. Computer 3D modeling of the P446L protein structure, based on results of CD spectroscopy, shows altered molecular structure: the P446L molecule takes on an open conformation such that its inner hydrophobic surface becomes exposed to water, explaining the reduced hydrophilic nature of the P446L protein (Timinszky, 2002).

The significant conformational change due to the exchange of Pro446 to Leu in the Ketel protein is further supported by the S317T suppressor mutation that restores Ketel gene function. In human importin-ß the corresponding Ser311 (in the linker region between HEAT repeats 7 and 8) and Pro441 are 32.5 Å apart and yet the Ser-->Thr exchange in the Drosophila homologue restores function of importin-ß. The 10 Å area surrounding serine is hydrophobic. The stronger hydrophobicity of threonine compared with serine does perhaps increase apolar interactions and bend the molecule back to its functional structure (Timinszky, 2002).

Experiments with digitonin-permeabilized HeLa cells show that, to a reduced extent, the P446L proteins do participate in formation of the nuclear import complexes and in their docking to the cytoplasmic surface of the NE; however, they do not support import of the complexes into the nuclei in the presence of Ran, energy source, RanGAP and RanBP1. In fact, the import complexes do not form upon the addition of the latter components. Apparently the main structural domains of the P446L protein are intact (binds importin-alpha, NPC and Ran) but the interaction with Ran is altered. Indeed, the binding of wild-type and P446L Ketel proteins to Ran are very different: the P446L cannot bind to RanGTP, to which the wild-type importin-ß binds strongly, but shows elevated affinity to RanGDP, to which the wild-type protein shows very little affinity. It is noteworthy that a single amino acid exchange outside the classical Ran-binding domain can change Ran binding ability dramatically. The change in Ran-binding ability is most likely the source of the Ketel^D-associated dominant female sterility. However, the Ketel^D-associated dominant-negative effect is not manifested via nuclear protein import but rather through the prevention of cleavage nuclei formation: revealing a novel importin-ß function (Timinszky, 2002).

Injection experiments into wild-type cleavage embryos revealed that the P446L mutant protein does not inhibit nuclear protein import: when co-injected with P446L, a fluorescent nuclear substrate readily enteres the nuclei. Furthermore, although the cleavage nuclei enter mitosis and the chromosomes segregate normally, intact NE never forms in the presence of P446L mutant importin-ß. Failure of NE assembly in the presence of P446L is revealed by the following observations; 1) the homogenous distribution of a fluorescent nuclear substrate, the high molecular weight dextrane and the GFP-tubulin; 2) the absence of the nuclear lamina lining. Thus the mutant P446L importin-ß reveals a novel importin-ß function required during the mitosis-to-interphase transition, a function distinct from the already known functions of importin-ß in nuclear protein import and in mitotic spindle assembly (Timinszky, 2002).

The P446L mutant importin-ß possesses altered Ran-binding properties: it does not bind RanGTP but shows elevated affinity to RanGDP. A series of experiments showed that altered RanGTP-RanGDP balance leads to a similar phenotype in yeast (i.e., arrest in mitosis-to-interphase transition). Results of enzyme assays described in the present paper show that the altered Ran-binding ability of P446L importin-ß does not interfere with the GTP hydrolysis and nucleotide exchange on Ran and thus it is unlikely that the Ketel^D-related defects are consequences of distorted Ran metabolism. Most probably importin-ß is a downstream effector of Ran during mitosis-to-interphase transition, as in nuclear protein import and mitotic spindle assembly (Timinszky, 2002).

Although several functions of Ran and importin-ß during the cell cycle have been described, the exact molecular mechanisms are still missing. This study describes a novel function of Drosophila importin-ß during mitosis-to-interphase transition where it is involved in the formation of intact nuclear envelope (NE). There seem to be three feasible explanations for the P446L-associated defects. First, since the P446L importin-ß shows higher affinity to RanGDP than wild-type importin-ß, a possible explanation may be the depletion of significant amounts of RanGDP that is required for NE reassembly at the end of mitosis. Removal of RanGDP by P446L may lead to the failure of cleavage nuclei formation. This explanation is thought not to be very likely for the following reasons. (1) Binding and nucleotide exchange assays revealed that the affinity of the P446L to RanGDP is low and hence depletion of a significant fraction of Ran from the cytoplasm is rather unlikely. (2) Interestingly, defects do not evolve in nuclear protein import or in spindle formation and chromosome segregation following injection of P446L despite the fact that both nuclear protein import and spindle formation have been shown to be Ran dependent. Ran's involvement in NE assembly has also been described but since none of the aforementioned Ran-related processes were disturbed, the P446L protein does not seem to disturb the Ran cycle. A second possible explanation of the P446L-related defects is perhaps the inability of the P446L protein to bind RanGTP and, consequently, the inability to release factors required for proper chromatin decondensation and/or NE assembly. In this case the role of importin-ß in the above processes would resemble its function in mitotic spindle formation, where it is thought to be required for the release of factors needed for spindle assembly [e.g. NuMA, TPX2. A third possibility is that the P446L-related defects are not associated with the change in Ran-binding ability. The P446L mutation may disturb the association of thus far unidentified factors (e.g. nucleoporins). In the case of the second and third possibilities the factor(s) required for the newly described importin-ß-related functions remain to be identified (Timinszky, 2002).

Anillin-mediated targeting of Peanut to pseudocleavage furrows is regulated by the GTPase Ran

During early development in Drosophila, pseudocleavage furrows in the syncytial embryo prevent contact between neighboring spindles, thereby ensuring proper chromosome segregation. This study demonstrates that the GTPase Ran regulates pseudocleavage furrow organization. Ran can exert control on pseudocleavage furrows independently of its role in regulating the microtubule cytoskeleton. Disruption of the Ran pathway prevents pseudocleavage furrow formation and restricted the depth and duration of furrow ingression of those pseudocleavage furrows that form. Ran is required for the localization of the septin Peanut to the pseudocleavage furrow, but not anillin or actin. Biochemical assays revealed that the direct binding of the nuclear transport receptors importin α and β to anillin prevents the binding of Peanut to anillin. Furthermore, RanGTP reverses the inhibitory action of importin α and β. On expression of a mutant form of anillin that lacks an importin α and β binding site, inhibition of Ran no longer restricts the depth and duration of furrow ingression in those pseudocleavage furrows that form. These data suggest that anillin and Peanut are involved in pseudocleavage furrow ingression in syncytial embryos and that this process is regulated by Ran (Silverman-Gavrila, 2008).

During cytokinesis, the ingressing plasma membrane physically divides the mother cell into two daughter cells. Membrane ingression during cell division is both temporally and spatially regulated, ensuring that membrane scission occurs (1) only after the chromosomes have fully segregated and (2) between the two chromosomal masses. The signals within the cell that determine cytokinetic furrow positioning are complex, reflecting the strict control needed to ensure that cytokinesis is successful. Signals from astral microtubules, the spindle midbody, the nucleus, and the membrane itself direct the assembly of the contractile ring to the equatorial cortex of the plasma membrane. The contractile ring is an actomyosin-based structure that constricts and generates the force needed to drive membrane ingression. As the membrane ingresses, it is remodeled and stabilized (Silverman-Gavrila, 2008).

Other membrane ingression events share many of the same features and involve many of the same proteins as cytokinetic furrows. In the syncytial Drosophila embryo before cellularization, up to 6000 closely packed nuclei exist in a common cytosol close to the cortex. To ensure faithful chromosome segregation during the rapid nuclear divisions, nuclei are isolated from one another to prevent neighboring spindles from contacting and fusing. To achieve this, plasma membrane ingressions form transiently between nuclei during the rapid nuclear cycles before cellularization. These membrane ingressions, termed pseudocleavage or metaphase furrows, are organized by the actin cytoskeleton and bear a close resemblance to cytokinetic cleavage furrows. First actin caps form at the plasma membrane above each nucleus. Then during interphase, as the centrosomes migrate to either side of the nucleus, the actin caps expand correspondingly. In prophase the cap reorganizes to drive membrane ingression into the embryo such that nuclei and newly forming spindles are separated from one another. Toward the end of metaphase, the furrows begin to retract and dissipate by anaphase. This process is repeated from the tenth through the thirteenth nuclear cycles. During the fourteenth nuclear cycle, the syncytial embryo cellularizes to form 6000 columnar epithelial cells. In this instance the cleavage furrows extend down into the embryo, before growing transversally and fusing to form a single layer of nucleated cells (Silverman-Gavrila, 2008).

Most components required for furrow ingression are conserved between cytokinetic furrows (during conventional mitosis) and pseudocleavage furrows. However, there are some differences. Notably pseudocleavage furrows are membrane ingressions that do not meet and therefore do not lead to membrane fusion. Instead they extend into the embryo, perpendicular to the cortex, and then retract back toward the embryo cortex after the chromosomes have begun to segregate. In addition, there is a difference in the stage of the cell cycle when the furrow components assemble. Although the cytokinetic furrow begins to assemble during anaphase and is required to divide a cell in two, the syncytial embryo pseudocleavage furrows begin to assemble in prophase and serve to prevent neighboring spindles from contacting one another (Silverman-Gavrila, 2008).

A key protein involved in cytokinetic furrow function is anillin, which has multiple domains allowing it to bind and bundle actin filaments, target septins to the plasma membrane, and interact with components of the microtubule-bound centralspindilin complex (Gregory, 2008). Consequently anillin is thought to act as a scaffold for the correct assembly of the contractile ring (Piekny, 2007). It is not fully understood how the role of anillin in cytokinesis is regulated. However, its role in remodeling the actomyosin contractile ring in somatic cells is in part regulated by its differential spatial positioning in the cell during the cell cycle. In interphase anillin localizes to the nucleus where it cannot interact with actin and myosin at the plasma membrane. However, in mitosis upon nuclear envelope breakdown, anillin is released from the nucleus and is targeted to the cortex of the plasma membrane and later to the equatorial cortex of the plasma membrane in a RhoGTP-dependent manner. The spatial regulation of anillin during the cell cycle contributes to the restriction of its function to mitosis. However, in Drosophila syncytial embryos anillin is cytosolic, localizing to pseudocleavage furrows throughout the nuclear cycle, suggesting that it may be regulated by other mechanisms (Silverman-Gavrila, 2008).

One function of anillin is to target septins to the contractile ring. Septins are a family of GTP-binding proteins that can assemble into filaments. Septins have been attributed multiple roles: as membrane diffusion barriers, as stabilizers of the furrow, in membrane trafficking, and as a scaffold. In Drosophila there are five septins: Peanut, Sep1, Sep2, Sep4, and Sep5. Peanut, Sep1 and Sep2 have been isolated as a stoichiometric complex that in vitro can polymerize into filaments. In contrast, Xenopus laevis Sept2 can self assemble into filaments, suggesting that septins may function independently (Silverman-Gavrila, 2008 and references therein).

The GTPase Ran is a key positive regulator of mitosis (Ciciarello, 2007). RanGTP regulates a number of mitotic factors that are sequestered in the nucleus by nuclear transport receptors during interphase. In mitosis RanGTP antagonizes the binding of nuclear transport receptors to these proteins and thereby promotes their activity. RanGTP is at its highest concentration around the chromosomes, where RCC1 the nucleotide exchange factor for Ran is localized. Consequently, RanGTP has been proposed to act as a spatial cue by only activating these mitotic proteins close to the chromosomes (Caudron, 2005; Kalab, 2006). In so doing RanGTP is thought to specify where certain mitotic processes occur in the cell. For example, it could specify that spindle assembly only occurs around chromosomes. The full extent to which this mechanism regulates the mitotic cell is not known and continues to expand (Silverman-Gavrila, 2008).

This study demonstrates a new role for Ran in regulating pseudocleavage furrow ingression, a membrane invagination process in early Drosophila embryos. The Ran pathway regulates the interaction between anillin and the septin Peanut, thereby regulating furrow stability (Silverman-Gavrila, 2008).

A cytological screen was carried out to identify mitotic processes regulated by the Ran pathway. Inhibitors of the Ran pathway were injected into GFP-α-tubulin-expressing embryos just before mitotic entry, and then microtubule organization was monitored by time-lapse microscopy. One phenotype, the fusion of neighboring spindles, occurred more frequently upon the injection of inhibitors of the Ran pathway compared with control injections. In control injected embryos 0.2% of observed spindles fused to a neighboring spindle. In contrast, inhibition of the Ran pathway by injecting either the dominant negative allele of Ran, RanT24N, or importin α resulted in 8.4 and 7.8% of observed spindles fusing to neighboring spindles, respectively (Silverman-Gavrila, 2008).

Peanut is recruited to ingressing furrows by anillin, a multifunctional protein required for cytokinesis that interacts with myosin II, actin, and septins. Septins bind to the carboxy-terminus of anillin, which includes a pleckstrin homology (PH) domain. Drosophila anillin has three potential nuclear localization signals (NLS) that could bind to the nuclear transport receptors importin α and β. Two of the NLS motifs are located in or directly adjacent to the PH domain (Silverman-Gavrila, 2008).

To determine if the carboxy-terminus of anillin could bind to importin α and β, a fusion was constructed between GST and the carboxy-terminus of anillin (amino acids 815-1201, anillin-CT, and its ability to bind to recombinant importin α and β was analyzed. Both importin α and β bound to anillin-CT, and this binding was reversed in the presence of RanQ69L, a point mutant of Ran locked in the GTP-bound state. Of the two potential NLS motifs, the one located between amino acid residues 989 and 999, bares the closest resemblance to an archetypal bipartite NLS and is found in the same region of human anillin (amino acids 887-898). Mutation of lysines 997-999 to alanine (3A-anillin-CT) abrogate both importin α and β binding to this region of anillin, suggesting that amino acids 989-999 constitute a nuclear transport receptor-binding site (Silverman-Gavrila, 2008).

It was next asked if the anillin-CT could interact with Peanut. GST-anillin-CT was incubated with 0-3-h Drosophila embryo extract and then isolated using glutathione agarose beads. Anillin-CT copurified with Peanut and another septin, Sep2. However, the addition of exogenous importin α and importin β inhibited the binding of Peanut to anillin-CT in a concentration- and NLS-dependent manner. This inhibition was specific to Peanut, because Sep2 binding to anillin-CT was not inhibited by importins (Silverman-Gavrila, 2008).

To determine if the in vivo targeting of Peanut and Sep2 to the pseudocleavage furrows is differentially regulated, importin α was injected into syncytial embryos and GFP-Sep2 localization was determined by time-lapse microscopy. Consistent with in vitro results, GFP-Sep2 localization was not perturbed upon interfering with the Ran pathway. Furthermore, in fixed GFP-Sep2-expressing embryos in which the Ran pathway has been perturbed, Peanut fails to localize to nascent furrows, whereas GFP-Sep2 does localize to nascent furrows. These data suggest that Peanut and Sep2 are differentially regulated by Ran and that Sep2 can localize to pseudocleavage furrows independently of Peanut (Silverman-Gavrila, 2008).

This study has identified RanGTP as a regulator of the interaction between Peanut and anillin. This mechanism operates directly and independently of Ran's well-characterized role in regulating the mitotic microtubule cytoskeleton (Silverman-Gavrila, 2008).

Studies suggest that anillin is required for the recruitment of septins to the furrow. By perturbing the Ran pathway, this study has demonstrated that the recruitment of the septins Peanut and Sep2 is differentially regulated, consistent with previous observations that Sep1 recruitment to furrows is dependent on Peanut but Sep2 is not. Anillin lacking the importin binding site between residues 997 and 999 can bind to Peanut in the presence of importins, suggesting that importins directly block the anillin-Peanut interaction rather than disrupting the Peanut, Sep1, and Sep2 complex. These data suggest that although Peanut, Sep1, and Sep2 can exist in a single complex, they may be able to function independently of one another as has been demonstrated in vitro for a Xenopus septin (Silverman-Gavrila, 2008).

Perturbing the Ran pathway destabilizes pseudocleavage furrows. One mechanism for this is through the regulation of the anillin-Peanut interaction. In embryos that expressed an anillin mutant lacking the importin-binding site, Peanut recruitment to pseudocleavage furrows occurs even in the presence of exogenous importins, and furrows demonstrate wild-type dynamics. These data suggest that Peanut is required for pseudocleavage furrow stability. This role for anillin and Peanut is consistent with the observed role for these proteins in stabilizing the cellularization furrow later in Drosophila development. These findings may at first appear to contradict those studies, in which embryos lacking Peanut protein progressed through the syncytial nuclear divisions only showing the first defects during cellularization. However, these studies only analyzed syncytial furrows from the top, apical view and not from the lateral view to observe ingression dynamics. Therefore, these studies would not have detected changes in furrow ingression dynamics that were observed upon inhibition of Ran, which correlated with a failure to recruit Peanut to the furrow (Silverman-Gavrila, 2008).

The Ran pathway regulates pseudocleavage furrow ingression directly by regulating importin binding to anillin. It was previously shown that in Drosophila syncytial embryos the importin β, whose injection causes similar effects as importin α, is released from the nucleus upon nuclear envelope breakdown and becomes diffuse throughout the cytosol during the rest of mitosis. During this period pseudocleavage furrows begin to retract. Therefore, as importin β is cytosolic during metaphase and anaphase it could act to prevent the interaction of Peanut and anillin. In turn this would lead to furrow instability and retraction (Silverman-Gavrila, 2008).

It cannot unequivocally be ruled out that some of the defects caused by perturbing the Ran pathway are due to a disruption of microtubule cytoskeleton. Indeed, one microtubule-dependent furrow phenotype, the formation of pseudocleavage furrows that encompassed a small area of cytosol around a nucleus, was observed. This phenotype has also seen in another study upon depolymerization of microtubules in embryos. However, microtubule depolymerization when instigated in interphase does not cause a failure in pseudocleavage furrow formation, a finding consistent with a previous study (Silverman-Gavrila, 2008).

Another mechanism through which Ran could affect pseudocleavage furrows is by disrupting nuclear trafficking. Indeed it was observed that nuclear trafficking can be reduced by up to 50% upon disruption of the Ran pathway. However, it seems unlikely that the changes in nuclear import kinetics in these experiments disrupted the function of anillin because anillin is a cytosolic protein in the syncytial embryo and localizes to the leading edge of the ingressing furrow during interphase. It is not understood how anillin is retained in the cytoplasm of syncytial embryos because it is imported into nuclei in other developmental stages. However, this phenomenon is not unique to anillin and is also exhibited by the kinesin Pavarotti, another protein involved in pseudocleavage furrow organization (Silverman-Gavrila, 2008).

These studies suggest that Ran regulates multiple factors involved in pseudocleavage furrow ingression, because embryos expressing the mutant anillin still exhibit a failure to form all the expected pseudocleavage furrows. Failure to fully suppress the phenotype could be due to the continued presence of endogenous anillin or reflect that other Ran pathway-sensitive factors are involved in pseudocleavage furrow formation. Regulation through the Ran pathway could define a spatial cue concentrated around chromosomes and extending to the cortex. Such a spatiotemporal regulatory mechanism could be involved in promoting cytokinetic furrows in other cells. A recent study in oocytes finds that Ran regulates myosin II, whose activity is required for cytokinetic cleavage furrows. In addition importin α is required for ring canal organization during oogenesis. Ring canals form as a result of incomplete cytokinesis, and many proteins involved in cytokinesis both localize to and are required for their formation, including anillin and septins (Silverman-Gavrila, 2008).

The data suggest that the anillin-Peanut interaction, which is inhibited by importins must occur in regions of the cell where there are low levels of importins or high levels of RanGTP. Recent studies have visualized a RanGTP-importin β gradient and found that it persists from the chromosomes to the centrosomes, a distance similar to that between the metaphase plate and the cortex. Thus, RanGTP could play an important role in positioning the plane of cleavage by defining on the cell cortex where furrow proteins interact (Silverman-Gavrila, 2008).

Although there are clear differences between cytokinetic and pseudocleavage furrows, anillin and septins are involved in both. Therefore, this study suggests that Ran could also have a role in regulating cytokinetic furrows. Whether chromosomes play a significant role in cytokinesis remains controversial. However, studies where nuclei or chromosomes are asymmetrically positioned within a cell show that furrow ingression coincided with the region of the cell that contained the chromosomes, suggesting that signals from the nucleus and in particular the chromosomes had a role in specifying furrow ingression. Similarly, enucleated sea urchin eggs are able to duplicate their centrosomes and generate astral arrays of microtubules, but fail to form stable cleavage furrows. The current study proposes a molecular mechanism to explain, at least in part, these observations, suggesting that RanGTP generated around the chromosomes is a diffusible signal that facilitates multiple processes required for furrow formation. Whether RanGTP is required early in cytokinesis to 'prime' the cortex for a future ingression or acts directly later during the ingression process is unclear. Testing these hypotheses is not straightforward, since Ran is also required for organizing the mitotic microtubule cytoskeleton, which is required for cytokinesis. Taken together these findings suggest an additional mechanism involved in regulating cytokinesis that is dependent on signals from chromosomes in addition to those stemming from the different organizational states of the mitotic microtubule cytoskeleton (Silverman-Gavrila, 2008).

Canoe binds RanGTP to promote Pins^TPR/Mud-mediated spindle orientation

Regulated spindle orientation maintains epithelial tissue integrity and stem cell asymmetric cell division. In Drosophila neural stem cells (neuroblasts), the scaffolding protein Canoe (Afadin/Af-6 in mammals) regulates spindle orientation, but its protein interaction partners and mechanism of action are unknown. This paper uses a recently developed induced cell polarity system to dissect the molecular mechanism of Canoe-mediated spindle orientation. A previously uncharacterized portion of Canoe was shown to directly bind the Partner of Inscuteable (Pins) tetratricopeptide repeat (TPR) domain. The Canoe-Pins^TPR interaction recruits Canoe to the cell cortex and is required for activation of the Pins(TPR)-Mud (nuclear mitotic apparatus in mammals) spindle orientation pathway. The Canoe Ras-association (RA) domains directly bind RanGTP, and both the Canoe^RA domains and RanGTP are required to recruit Mud to the cortex and activate the Pins/Mud/dynein spindle orientation pathway (Wee, 2011).

Spindle orientation is essential to maintain epithelial integrity; planar spindle orientation results in both daughter cells maintaining apical junctions and remaining part of the epithelium, whereas apical/basal spindle orientation can lead to the loss of the basal daughter cell from the epithelium. Spindle orientation is also important during asymmetric cell division of stem, progenitor, and embryonic cells; when the spindle orients along an axis of intrinsic or extrinsic polarity, it will generate two different daughter cells, but, when the spindle aligns perpendicular to the axis of polarity, it will generate two identical daughter cells. Proper spindle orientation may even be necessary to prevent tumorigenesis. Thus, it is essential to understand the molecular mechanisms that regulate spindle orientation, particularly those that use evolutionarily conserved proteins and pathways, to help direct stem cell lineages and potentially treat pathological conditions caused by aberrant spindle orientation (Wee, 2011).

Drosophila neuroblasts provide an excellent system for studying spindle orientation during asymmetric cell division. Neuroblasts have an apical/basal polarity and orient their mitotic spindle along this cortical polarity axis to generate distinct apical and basal daughter cells. The apical neuroblast inherits fate determinants responsible for neuroblast self-renewal, whereas the basal daughter cell inherits fate determinants responsible for neuronal/glial differentiation. Genetic studies have identified proteins that regulate spindle orientation during asymmetric cell division, including the apically localized proteins Inscuteable, Partner of Inscuteable (Pins; LGN/AGS-3 in mammals), Mushroom body defect (Mud; nuclear mitotic apparatus [NuMA] in mammals), Discs large (Dlg), and Ga_i. In addition, many proteins that are not asymmetrically localized are required for spindle orientation, including the dynein complex and the Aurora A and Polo kinases (Wee, 2011).

An induced cell polarity/spindle orientation system has been developed using the normally apolar S2 cell line to biochemically dissect Drosophila and vertebrate spindle orientation (Johnston, 2009; Ségalen, 2010). Using this system to characterize Drosophila spindle orientation, it was shown that cortical Pins nucleates two spindle orientation pathways: (1) the Pins^LINKER domain is phosphorylated by Aurora A, which allows recruitment of Dlg, which interacts with the kinesin Khc-73 to promote partial spindle orientation; and (2) the Pins tetratricopeptide repeat (TPR) domain (Pins^TPR) binds Mud, which promotes dynein-dynactin complex-mediated spindle orientation (Johnston, 2009). This induced cell polarity system was used to characterize Dishevelled-mediated spindle orientation in the zebrafish embryo and in Drosophila sensory organ precursor cells, identifying a Dishevelled domain that is necessary and sufficient to bind Mud and regulate spindle orientation in both cell types (Wee, 2011).

The scaffolding protein Canoe has been shown to regulate spindle orientation and cell polarity in Drosophila neuroblasts (Speicher, 2008), although the mechanisms involved remain unknown. Canoe contains two Ras-association (RA) domains, a Forkhead domain, a myosin-like Dilute domain, and a PSD-95, Dlg, and ZO-1 (PDZ) domain. In addition to regulating neuroblast cell polarity and spindle orientation, it integrates Notch, Ras, and Wnt pathways during Drosophila muscle progenitor specification and serves as a Rap1 effector within the Jun N-terminal kinase pathway during dorsal closure of the Drosophila embryo, and the mammalian orthologue Afadin links cadherins to the actin cytoskeleton at adherens junctions. This study mapped direct Pins/Canoe and Canoe/RanGTP-binding domains and used the induced cell polarity/spindle orientation system to show that Canoe/RanGTP is required for Pins to recruit Mud and activate the Pins/Mud/dynein spindle orientation pathway (Wee, 2011).

How might Canoe/RanGTP promote Mud recruitment to the Pins cortical domain? One model is that Ran sequesters importin-a/β away from the Mud NLS, thereby allowing Mud to interact with Pins. This model is based on the observation that RanGTP inhibits binding of importin-β to the NLS of NuMA (the mammalian orthologue of Mud), increasing the pool of NuMA available to promote spindle formation. The model predicts that Mud can bind importin-a/β and that this binding prevents Mud/Pins association. Consistent with the model, importin-β/Mud were coimmunoprecipitated from S2 cell lysates, and a GST:Mud fragment containing the adjacent Mud TPR-interacting peptide (TIP)-NLS domains (GST:Mud^TIP-NLS) could bind purified importin-β in the presence of importin-a. However, it was found that increasing the concentration of purified importin-a/β did not effect the amount of Pins pulled down with GST:Mud^TIP-NLS, which does not support a model in which Ran must sequester importin-a/β to allow Pins/Mud binding. Furthermore, a GFP-tagged Mud^TIP-NLS fragment localized to Ed:Pins^TPR+LINKER crescents independently of the Canoe/Ran pathway, showing that the Mud NLS is not involved in the Canoe/Ran-regulated localization mechanism. Interestingly, Canoe/RanGTP regulation is required for recruitment of full-length endogenous Mud but not for the recruitment of the smaller Mud^TIP-NLS fragment; this indicates that Canoe/RanGTP normally functions by blocking an unknown inhibitor of the Mud-Pins^TPR interaction (Wee, 2011).

In conclusion, this study has characterized the molecular mechanism by which Canoe regulates spindle orientation. A region of Canoe (amino acids 1,755-1,950) was identified that directly interacts with the Pins^TPR domain, and it was showm that these domains are necessary and sufficient for Canoe-Pins association. It was shown that the Canoe RA domains bind directly to RanGTP, that both the Canoe RA domains and Ran are necessary for the Pins^TPR/Mud spindle orientation pathway, and that Canoe/RanGTP acts by promoting Mud recruitment to the cortical Pins domain. All of the proteins in the Pins/Canoe/Ran/Mud pathway are conserved from flies to mammals, suggesting that this pathway could be widely used to regulate spindle orientation (Wee, 2011).

Chromatin-mediated microtubule nucleation in Drosophila syncytial embryos

Upon entry into mitosis, many microtubules are nucleated that coordinately integrate into a stable, yet dynamic, mitotic spindle apparatus. Recent work has examined microtubule-generating pathways within the Drosophila syncytial embryo. It was found that, following depolymerisation of metaphase spindle microtubules by cold treatment, spindles regenerate predominantly from microtubules nucleated within the vicinity of chromatin. This chromatin-mediated microtubule nucleation is mediated by the Drosophila homolog of a vertebrate spindle assembly factor (SAF), HURP and is dependent on the conserved microtubule amplifying protein complex, Augmin. The investigation was expanded into Drosophila SAFs, providing evidence that, in vitro, both D-HURP and D-TPX2 are able to bind to and stabilize microtubules. GFP-D-HURP purified from embryos interacts with Importin-beta and Augmin and, consistent with this, it was demonstrated that the underlying basis of chromatin-mediated microtubule nucleation in Drosophila syncytial embryos is dependent on Ran-GTP (Hayward, 2014).

The RanGEF Bj1 promotes Prospero nuclear export and neuroblast self-renewal

Drosophila larval neuroblasts are a model system for studying stem cell self-renewal and differentiation. This study report a novel role for the Drosophila gene Bj1 in promoting larval neuroblast self-renewal. Bj1 is the guanine-nucleotide exchange factor for Ran GTPase, which regulates nuclear import/export. Bj1 transcripts are highly enriched in larval brain neuroblasts (in both central brain and optic lobe), while Bj1 protein is detected in both neuroblasts and their neuronal progeny. Loss of Bj1 using both mutants or RNAi causes a progressive loss of larval neuroblasts, showing that Bj1 is required to maintain neuroblast numbers. Loss of Bj1 does not result in neuroblast apoptosis, but rather leads to abnormal nuclear accumulation of the differentiation factor Prospero, and premature neuroblast differentiation. It is concluded that the Bj1 RanGEF promotes Prospero nuclear export and neuroblast self-renewal (Joy, 2014).

DEVELOPMENTAL BIOLOGY

Embryonic

As of 2001, a total of 89 ran expressed sequence tags (ESTs) have been reported in the Berkeley Drosophila Genome Project database while only a single ran-like EST has been identified. The ran like EST (GH25818) was isolated from an adult head cDNA library. In comparison, ran ESTs have been isolated from both embryo and adult tissues (61, embryo; 18, adult head; and 1 cell culture). The differences in the number of reported ran and ran-like ESTs suggest that the two genes may have different spatial/temporal expression dynamics during development. To test this hypothesis, their embryonic expression patterns were tested on 0- to 18-h embryo collections via whole-mount in situ hybridizations (Koizumi, 2001).

Localization of ran mRNA during embryonic development revealed two temporal phases of expression. Initially, ran expression is found in the pre-cellular blastoderm, suggesting that it is supplied maternally. Localization of ran transcripts in progressively older embryos revealed that shortly after the onset of gastrulation, there is a significant and rapid loss of ran mRNA throughout the embryo. By embryonic stage 7, no expression was observed. The second temporal phase of ran expression occurs in CNS neuroblasts but only during their late sublineage-forming mitotic divisions. No expression was observed in NBs or any other cell during stages 8-11 indicating that ran gene expression is restricted from NBs during the formation of their early sublineages. During germ-band contraction (stage 12), ran transcripts were detected in large-diameter cells that line the cephalic lobes and the ventral/ventral-lateral surface of the developing ventral cord ganglia. The size and location of ran-expressing cells suggest that they are predominantly NBs. By stage 16, ran transcripts were detected in only a few putative neural precursor cells (NBs and/or ganglion mother cells). The fact that ran CNS expression is progressively reduced, starting at early stage 14, further suggests that its expression is predominately restricted to NBs and ganglion mother cells and not to post-mitotic neurons or glia. In situ hybridization of third instar larval tissues with ran -specific probes reveals that ran is also expressed ubiquitously, albeit at low levels, in both larval tissues and imaginal discs (Koizumi, 2001).

ran-like in situ hybridization analysis of embryos, obtained from overnight collections, also identified a maternal and zygotic pattern of gene expression. Similar to ran, ran-like transcripts were detected uniformly distributed in syncytial blastoderm embryos, and starting just after cellularization there was a rapid decline in the ran-like in situ signal. By stage 5, little or no ran-like in situ staining was observed. However, differing from the neuronal precursor gene expression pattern of ran, zygotic ran-like expression was detected in salivary gland cells and in the epithelial cells lining the tracheal tubular network (Koizumi, 2001).

The GTPase Ran regulates multiple cellular functions throughout the cell cycle, including nucleocytoplasmic transport, nuclear membrane assembly, and spindle assembly. Ran mediates spindle assembly by affecting multiple spindle assembly pathways: microtubule dynamics, microtubule motor activity, and spindle pole assembly. Ran is predicted to facilitate spindle assembly by remaining in the GTP-bound state around the chromatin in mitosis. This study directly tests the central tenet of this hypothesis in vivo by determining the cellular localization of Ran pathway components in Drosophila embryos. During mitosis, RCC1, the nucleotide exchange factor for Ran, is associated with chromatin, while Ran and RanL43E, an allele locked in the GTP-bound state, localizes around the spindle. In contrast, nuclear proteins redistribute throughout the embryo upon nuclear envelope breakdown (NEB). Thus, in vivo RanGTP has the correct spatial localization within the cell to modulate spindle assembly (Trieselmann, 2002).

EFFECTS OF MUTATION

Targeted misexpression of Drosophila ran

To investigate the significance of ran-restricted CNS expression, its misexpression was targeted to different temporal windows of CNS development. In addition, a dominant- negative mutant form of ran was targeted to the developing CNS and to the larval eye/antenna imaginal disc to assess the role of ran-dependent functions. Embryonic CNS misexpression of the mutant, but not wild-type, ran results in larval death. Neither wild-type nor mutant ran misexpression had any detectable effect on embryonic CNS lineage specification, nuclear transport of a number of CNS-specific transcription factors or axonal guidance. However, expression of the dominant-negative mutant ran in the developing eye/antenna disc did result in a severe adult eye phenotype marked by apoptosis of photoreceptor, cone and pigment cells (Koizumi, 2001).

Blocking Ran function(s) in mammalian cultured cells has been achieved via the misexpression of different dominant-negative mutant forms of Ran. The mutant Ran proteins have single or double amino acid substitutions (G19V and/or Q69L) that alter the Ran protein in such a way that it can bind but not hydrolyze GTP (Coutavas, 1993; Ren, 1994; Lounsbury, 1996). These dominant-negative mutant alleles have been shown to have profound effects on multiple nuclear events, including progression of the cell cycle, RNA transport, and DNA replication (Ren, 1994; Melchior, 1998).

The restricted expression of ran to CNS NBs undergoing late lineage-generating divisions and its absence from post-mitotic neurons or glia suggests that ran may be required for a temporally defined event in neural development. Alternatively, ran may be required in all neural cells, but due to the high rate of NB mitotic divisions, the maternal supply is insufficient for CNS stem cells to complete their lineage development (Koizumi, 2001).

As an initial step toward determining the significance of the temporally restricted CNS expression, the effects of ran misexpression outside the temporal boundaries of its wild-type expression window was studied. The effects of the dominant-negative ran mutant, ranG19V;Q69L, on different aspects of embryonic development and on adult eye development was studied. The targeted ectopic expression was accomplished by using the yeast Gal4/UAS system. Both wild-type and mutant ran ORFs were inserted into the P-element based UAS vector to create the following constructs: P[UAS.ran^wild-type] and P[UAS.ranG19V;Q69L], respectively. Multiple independent transformant lines were generated with the above P-element constructs with integration sites on either the second or third chromosomes. For misexpression in the developing nervous system, Gal4 drivers were used that can activate the expression of the UAS transgenes in either NBs or post-mitotic neurons. For NB expression, pros.Gal4 and pdm.Gal4 drivers were used; for post-mitotic neurons an elav.Gal4 driver was used. Pan-neural embryonic transgene expression was also achieved using scabrous. Gal4. The effects of UAS.ran^wild-type and UAS.ranG19V;Q69L misexpression on early segmentation was also studied using a hairy.Gal4 driver. The effects of wild-type and mutant ran misexpression were assessed by three criteria: viability, changes in the sub-cellular localization of nuclear factors, and defects/changes in nervous system structure as assessed by axonal patterning. To confirm that these Gal4 drivers activated the different UAS.ran transgenes, misexpression was monitored via in situ hybridization (Koizumi, 2001).

The misexpression of wild-type ran with the Gal4 drivers used in this study did not result in any detectable abnormal phenotype in either the CNS or in other aspects of Drosophila biology. However, expression of UAS.ranG19V;Q69L under the control of each of the above Gal4 drivers, with the exception of the scabrous driver, resulted in either late first, early second instar larval or pupal mortality. No abnormal behavioral phenotypes were detected in the first instar larva harboring any of the different promoter-driven mutant ran transgene combinations. After hatching from the egg case, larva exhibited wildtype motility and eating behavior. The prosperoGal4/UAS.ranG19V;Q69L transformant adults failed to exit the pupa case and inspection of the pharate adults failed to identify any morphological defects. The scabrous.Gal4/UAS.ranG19V;Q69L transgene combination proved to be viable (Koizumi, 2001).

To determine if ectopic expression of the wild-type or mutant ran transgenes leads to defects in the subcellular distribution of developmentally regulated nuclear proteins, embryos were stained with antibodies to the Castor, Pdm1, Hb, Prospero and Even-skipped transcription factors. Immunostains of all Gal4/UAS combinations (both wild-type and mutant Ran) failed to reveal any defects in the subcellular distribution of these proteins. To assess if other aspects of neural development were affected, such as axonal outgrowth, axon markers (monoclonal antibodies BP102 and 22C10) were employed to immunostain embryos containing the different Gal4/UAS combinations. Again, within the resolution of this analysis, no defects in axonal outgrowth or axon fascicle development during embryonic development were detected in any of the Gal4/UAS combinations tested. However, the possibility can not be ruled out that misexpression of mutant ran triggers an embryonic defect, albeit subtle, which ultimately results in larval lethality (Koizumi, 2001).

Given the lack of detectable abnormal phenotype accompanying the wild-type or mutant ran transgene expression, the possibility exists that the levels of transgene expression were not sufficient to trigger cellular or developmental embryonic defects. To address this possibility, transformants were generated that contained multiple wild-type or mutant Ran Gal4/UAS transgene combinations. Analysis of these lines revealed no detectable differences between embryos and larva harboring single or multiple copies of the Gal4/UAS combinations. Nevertheless, embryonic expression of Ran G19V;Q69L does produce larval and pupal lethality, confirming that the Gal4/UAS.ranG19V;Q69L transgene combinations did in fact work. The data suggest that larval cells and/or postmitotic neurons may have a greater requirement for Ran dependent processes. Currently it is not known why the effects of the ectopic expression are delayed. Perhaps the high levels of endogenous Ran expression are sufficient to overcome the immediate effects of the different Gal4 driven UAS.ranG19V;Q69L combinations. It is also possible that other ran -independent nucleo-cytoplasmic transport mechanisms may compensate for a deficiency in ran function during embryonic development. Ran-independent nuclear transport has been observed in other systems (Nakielny, 1997; Fagotto, 1998; Sachdev, 2000; Koizumi, 2001 and references therein).

During embryonic development, perdurance of Ran function from the maternal expression of both ran genes may be sufficient to over-ride the misexpression of the dominant-negative mutant Ran. To avoid this possibility, we assessed the effects of ran misexpression during larval development in imaginal cells that should have little or no maternal contribution. To accomplish this, the misexpression of wild-type and mutant ran was targeted to different stages of adult eye development. The targeted misexpression of UAS.ran transgenes to the eye-imaginal disc takes advantage of the fact that the eye is not essential for viability and that disruption of the ommatidial organization is a sensitive indicator of phenotypic effects. In addition, different Gal4 drivers are available to target misexpression within different temporal windows of eye development. Three different spatial/temporal eye disc Gal4 drivers were employed: (1) dpp^disc-.Gal4 to target misexpression to undifferentiated cells within the morphogenetic furrow, (2) pGMR.Gal4 to target all cells behind the morphogenetic furrow, and (3) sevenless.Gal4 to drive misexpression in a subset of differentiating photoreceptor cells and non-neuronal cone and pigment cells. As in the embryo, the ectopic misexpression of wildtype ran during eye development resulted in no detectable mutant phenotype. In all cases the ommatidial organization of the various Gal4/UAS.ran combinations was indistinguishable from wild-type eyes. In addition, the misexpression of mutant ran in retinal precursors within the morphogenetic furrow, P[dpp^disc.Gal4]/P[UAS.ranG19V;Q69L], did not induce detectable changes in eye development. Expression of Ran G19V;Q69L in developing photoreceptors using a sevenless promoter resulted in an eye phenotype: close examination of eye cross-sections showed a disrupted ommatidial structure with absence of photoreceptor cells including the R7 cell. Expression of mutant ran behind the morphogenetic furrow via the P[GMR.Gal4] transgene resulted in a severe eye phenotype characterized by a complete loss of photoreceptor, cone and pigment cells. The severe phenotype was partially reverted in the presence of a constitutively active p35 genetic background. p35 has been shown to prevent programmed cell death in Drosophila, and acts by binding to and inhibiting the proteolytic activity of caspases. The partial rescue suggests that the loss of ommatidial structures is caused by programmed cell death. However, misexpression of mutant ran via the GMR promoter did not appear to affect the development of interommatidial bristles (Koizumi, 2001).

Interommatidial bristles are mechanosensory organs composed of four cells that are derived from a single sensory organ precursor (SOP). Unlike the photoreceptor and cone cells, the appearance of SOPs is not related to the distance from the morphogenetic furrow. The final cell divisions generating the interommatidial bristles take place between 6 h and 15 h after puparium formation. Photoreceptor cell determination occurs behind the furrow, during third instar development. Because all eye lineages come from equivalent precursors, cell death induction due to Ran G19V; Q69L expression occurs after the transit of the morphogenetic furrow and subsequent to or parallel to the induction of interommatidial bristles. It is not clear from these results whether the bristles cells are more resistant to RanG19V;Q69L or whether Ran is specifically required for a transport step in photoreceptor cells that does not take place in bristle cells (Koizumi, 2001).

RanGTP is required for meiotic spindle organization and the initiation of embryonic development in Drosophila

RanGTP is important for chromosome-dependent spindle assembly in Xenopus extracts. This study reports on experiments to determine the role of the Ran pathway on microtubule dynamics in Drosophila oocytes and embryos. Females expressing a dominant-negative form of Ran have fertility defects, suggesting that RanGTP is required for normal fertility. This is not, however, because of a defect in acentrosomal meiotic spindle assembly. Therefore, RanGTP does not appear to be essential or sufficient for the formation of the acentrosomal spindle. Instead, the most important function of the Ran pathway in spindle assembly appears to be in the tapering of microtubules at the spindle poles, which might be through regulation of proteins such as TACC and the HURP homolog, Mars. One consequence of this spindle organization defect is an increase in the nondisjunction of achiasmate chromosomes. However, the meiotic defects are not severe enough to cause the decreased fertility. Reductions in fertility occur because RanGTP has a role in microtubule assembly that is not directly nucleated by the chromosomes. This includes microtubules nucleated from the sperm aster, which are required for pronuclear fusion. It is proposed that following nuclear envelope breakdown, RanGTP is released from the nucleus and creates a cytoplasm that is activated for assembling microtubules, which is important for processes such as pronuclear fusion. Around the chromosomes, however, RanGTP might be redundant with other factors such as the chromosome passenger complex (Cesario, 2011).

The Ran pathway has a variety of targets, which leads to effects on kinetochores, centrosomes and microtubule-associated proteins(Kalab, 2008). RanGTP is potentially an important molecule for spindle assembly in acentrosomal oocytes because it has been identifiedas a key factor for chromatin-induced spindle formation in Xenopus extracts. Surprisingly, the current results suggest that RanGTP might be more important for microtubule assembly in other circumstances, such as when centrosomes are present or when microtubules assemble without direct contact with the chromosomes (Cesario, 2011).

Diffusion of RanGTP from its source, the chromatin, into the cytoplasm, where it is converted into RanGDP, can create a gradient that regulates microtubule organization. Drosophila oocytes contain two key regulators of the Ran pathway in distinct locations. RCC1, as expected, is located tightly around the karyosome in mature oocytes. RanGAP localization is more complex than expected because it is present in many clusters, possibly vesicles, within the oocyte, suggesting that conversion of RanGTP to RanGDP might be regulated and only occur in certain locations. This could mean that a gradient of RanGTP is not established in the oocyte. A candidate protein responsible for generating the concentrations of RanGAP is Ran binding protein 2 (RanBP2; also known as Nup358). This protein is found within the nuclear envelope and binds to RanGAP (Hutten, 2008). Following NEB, RanGAP could be anchored to RanBP2-containing cytoplasmic vesicles (Cesario, 2011).

Ran has an unusual localization pattern in oocytes; concentrating around the outside of the spindle. By contrast, Ran overlaps with the spindle in Drosophila mitotic cells. It has not been determined whether these concentrations of Ran are in the GDP or GTP state. However, it the basis of the localization patterns of wild-type and mutant proteins is open to speculation. From this type of evidence, it has been suggested that the bulk of Ran on the embryonic spindle is in the GTP state. Similarly, the bulk of the Ran localized around the outside of the meiotic spindle might be in the GTP form. The pattern of mutant Ran^Q69L staining suggests it enters RanGAP-containing vesicles but does not leave because it is not hydrolyzed. Thus, the wild-type Ran that localizes adjacent to the clusters of RanGAP could be the GDP form of the protein that has left RanGAP-containing vesicles (Cesario, 2011).

RCC1 and RanGTP were found to be required for chromatin-induced spindle assembly in Xenopus extracts. In such extracts, expression of Ran^T24N blocks spindle assembly and high concentrations of RCC1 or expression of a GTP-locked form of Ran leads to spindle formation in the absence of chromosomes and centrosomes (Cesario, 2011).

This analysis of RanGTP function in Drosophila oocytes is based on these and numerous other studies in which expression of the ran^T24N mutation effectively reduces the concentration of RanGTP. It is believed that the ran^T24N mutant had the desired effect of reducing RanGTP production, for four reasons. First, expression of Ran^T24N in somatic cells caused embryonic or early larval lethality. Second, Ran^T24N localized tightly to the meiotic chromosomes, consistent with the expectation that this form of Ran remains bound to RCC1 because it has a low rate of GTP exchange. The high affinity of Ran^T24N for RCC1 causes a reduction in the production of RanGTP. Third, the spindle organization defects observed in ran^T24N oocytes were similar to defects seen in mars¹ mutant oocytes, a protein known to be regulated by the Ran pathway. Fourth, ran^T24N caused dramatic disruptions in chromosome-independent microtubule assembly assays, such as pronuclear fusion. Because these chromosome-dependent (meiotic spindle) and -independent functions occur in the same cytoplasm, it is concluded that the reduction in RanGTP levels sufficient to block pronuclear fusion were not sufficient to block acentrosomal spindle assembly (Cesario, 2011).

Unlike the results in Xenopus extracts, expression of the dominant-negative GDP-locked variant of Ran had relatively mild effects on Drosophila oocyte spindle assembly and karyosome organization. Meiosis I spindles were bipolar in ran^T24N oocytes. Indeed, reducing the RanGTP concentration in the oocyte was not sufficient to severely affect either meiotic division, because meiosis II spindles and female meiotic products could be seen in the embryos. The most important defect was that the meiosis I spindle often had non-tapered poles, and was associated with the abnormal localization of proteins necessary for pole formation such as Mars or TACC (Cullen, 2001). These results are consistent with experiments in embryos that found depletion of RanGTP causes defects in spindle pole organization and chromosome organization and congression (Silverman-Gavrila, 2006). Abnormal spindle morphology in oocytes could be the reason for the disorganized karyosome phenotype and nondisjunction of achiasmate chromosomes. Loss of RanGTP could result in a failure to activate Aurora A, which phosphorylates TACC. In embryos, TACC localization to the centrosomes depends on phosphorylation by Aurora A (Barros, 2005). TACC initially binds all microtubules, but as the spindle matures, TACC is phosphorylated and localizes to the poles. Expression of ran^T24N in oocytes might cause a reduction in Aurora A activity, resulting in a failure to phosphorylate TACC and localize it to the poles. Further studies are needed, however, because the role of Aurora A in Drosophila female meiosis is not known. In addition, Mars might have a role in promoting the dephosphorylation of TACC (Tan, 2008). Overall, RanGTP might have a specific role in organizing spindle poles but might not be required for chromosome-promoted spindle assembly in oocytes (Cesario, 2011).

Expression of ran^T24N did not block spindle assembly. Conversely, expression of the GTP-locked mutant, ran^Q69L, did not induce an uncoupling between spindle assembly and the chromosomes, as it does in Xenopus oocytes. Thus, RanGTP might not be sufficient to initiate spindle assembly in Drosophila oocytes. Surprisingly, ran^Q69L oocytes showed loss-of-function spindle phenotypes similar to ran^T24N mutant oocytes. Interestingly, manipulation of RanGTP levels with T24N or Q69L mutations in mammals has similar phenotypes. For example, the expression of either form of Ran in mouse oocytes resulted in similar meiosis II spindle phenotypes. These results suggest that the effects of manipulating RanGTP levels in an intact oocyte are not easily predicted by experiments in Xenopus extracts. Other factors such as protein localization might play important roles in regulating the Ran pathway. Also it cannot be ruled out that the Ran pathway functions differently in oocyte meiosis, such as if active Ran is not GTP dependent (Cesario, 2011).

It is suggested that there could be two reasons for the similarity of the ran^Q69L and ran^T24N phenotypes. First, expressing the GTP-locked ran^Q69L mutation can inhibit the binding of RCC1 to the chromatin, causing a reduction in RanGTP near the chromatin. Alternatively, the phenotypes of the ran^Q69L mutant oocyte might be associated with defects in the organization of membranes or vesicles. For example, expression of the ran^Q69L mutation caused Lamin, RanGAP and Ran^Q69L to colocalize in globular structures throughout the oocyte. The transmembrane protein Axs has been proposed to be a component of a membranous structure surrounding the meiotic spindle. With the caveat that the link between membranous structures and spindle assembly is not known, the ran^Q69L mutation might cause defects in membranous structures that have a role in spindle organization. It was also found that Ran and Axs are closely associated, although at the light microscope level it is difficult to determine if Ran is inside or outside the Axs staining (Cesario, 2011).

Similar to the oocytes, the phenotype of the ran^Q69L mutant zygotes might be associated with defects in membrane structure. In ran^Q69L mutants, a single cluster of DNA and microtubules could be observed in the center of the zygote. A strikingly similar phenotype has been observed in dominant-negative Ketel mutants; Ketel is the Drosophila homolog of importin-β. In the Ketel dominant mutants, meiosis I and II occur and the female and male pronuclei come together, but they interact abnormally because of defects in the nuclear envelopes. Subsequently, the first mitotic division fails and the chromosomes disintegrate within a large aggregate of microtubules. Similar to the Ketel mutant, ran^Q69L could cause abnormal interactions among nuclear envelope proteins in the zygote, causing a failure in the first mitotic division (Cesario, 2011).

There are two chromosome segregation mechanisms in Drosophila females. The first is the segregation of bivalents connected by chiasmata, which is how most chromosomes segregate. The second is of the chromosomes that lack chiasmata. This includes the small fourth chromosome, which always lacks crossovers, and larger chromosomes, which lack a crossover in approximately 5% of meioses. Homologous pairs can be forced into the achiasmate system with balancers that suppress crossing over. In all these cases, homologous chromosomes segregate correctly even though they are not connected by chiasmata. Expression of ran^T24N had only mild effects on chiasmate segregation, but had a severe effect on the segregation of achiasmate X-chromosomes. These results suggest that the spindle pole organization defects caused by low RanGTP levels affect chromosome segregation (Cesario, 2011).

Unlike assembly of the meiosis I spindle, expression of ran^T24N blocked two other types of microtubule assembly. First, ran^T24N mutants had a defect in the fusion of the female and male pronuclei. This was the most probable cause of the fertility defect in ran^T24N mutants. Several genes with roles in microtubule assembly are also required for pronuclear fusion, including subito. This process depends on the assembly of a microtubule array that is nucleated by the centrosome donated by the sperm, and acts to draw the female pronucleus towards the male pronucleus. Second, ran^T24N suppressed the formation of the ectopic spindles that form in a neomorphic subito mutant (sub^?NT) . The formation of these spindles occurs after NEB, consistent with a dependence on release of RanGTP from the nucleus. Both of these examples involve assembly of microtubules without direct interaction with the chromosomes and suggest that the assembly and bundling of microtubules in the oocyte cytoplasm depend on RanGTP (Cesario, 2011).

One characteristic of the ectopic spindles in sub^δNT mutants is that they form in discrete clusters within the oocyte. Because RanGAP appears in clusters, it is possible that RanGTP is not in a gradient or distributed evenly in the cytoplasm. Thus, an interesting possibility is that the regions containing ectopic spindles are where the concentration of RanGTP-dependent spindle assembly factors are at their highest (Cesario, 2011).

These experiments revealed a surprising mutual suppression by the sub^?NT and ran^T24N mutations. Although both mutants have decreased fertility, the double mutant is fertile. One interpretation is that RanGTP regulates Subito, and the sub^δNT mutation bypasses the dependence on RanGTP. However, this study found that ran mutations did not affect Subito localization or the formation of the central spindle. A more probable explanation is that there are two independent spindle assembly pathways in the oocyte and the loss of spindle assembly factors in ran^T24N zygotes is balanced by the enhanced spindle assembly activity present in the sub^?NT mutant. Expression of ran^T24N might suppress the sterility phenotype of sub^?NT by abolishing ectopic spindles, whereas sub^?NT might suppress the reduced fertility phenotype of ran^T24N by overcoming the defects in microtubule assembly needed for processes such as pro-nuclear fusion (Cesario, 2011).

Using dominant-negative mutations, this study has found that RanGTP is required for the fertility of Drosophila females. No evidence was found that RanGTP is required, or sufficient, for the initiation of acentrosomal spindle assembly in Drosophila oocytes. A role was detected in organizing the spindle poles that could be explained by RanGTP regulation of proteins such as Mars/Hurp, TACC and Aurora A. These defects, however, would not be expected to have a severe effect on fertility. A similar conclusion was drawn from expressing a dominant-negative form of Ran in mouse oocytes or when RCC1 was depleted from Xenopus oocytes. By analyzing mutants similar to the ones used in this study, only mild defects in meiosis I spindle assembly were found, such as a delay establishing bipolarity. The failure to observe evidence supporting a role for RanGTP in acentrosomal spindle assembly might be explained by a predominant chromosome-dependent pathway in oocytes involving the chromosome passenger complex (CPC). The CPC is required for chromosome-dependent spindle assembly in Xenopus egg extracts (Cesario, 2011).

These results suggest that, compared with chromosome-mediated spindle assembly, RanGTP has a greater role in microtubule organization when centrosomes are present or when at a distance from the chromosomes. It is suggested that NEB preceding the assembly of the meiosis I spindle releases RanGTP into the cytoplasm, which results in a cytoplasm enriched for active spindle assembly factors. The restriction of RanGAP to vesicle-like structures could leave a considerable amount of RanGTP in the cytoplasm. This activity has only a minor role in meiotic spindle assembly, but is crucial for the early events of embryogenesis. The microtubule array facilitating pronuclear fusion assembles while the nuclear envelope is intact. Therefore, the oocyte might accumulate and store RanGTP during the meiotic divisions when there is no nuclear envelope in order to support pronuclear fusion, when the nuclear envelope is intact (Cesario, 2011).

EVOLUTIONARY HOMOLOGS

Tissue-specific expression of Ran isoforms

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).

Ran protein interactions

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).

A code for RanGDP binding in ankyrin repeats defines a nuclear import pathway

Regulation of nuclear import is fundamental to eukaryotic biology. The majority of nuclear import pathways are mediated by importin-cargo interactions. Yet not all nuclear proteins interact with importins, necessitating the identification of a general importin-independent nuclear import pathway. This study identified a code that determines importin-independent nuclear import of ankyrin repeats (ARs), a structural motif found in over 250 human proteins with diverse functions. AR-containing proteins (ARPs) with a hydrophobic residue at the 13th position of two consecutive ARs bind RanGDP efficiently, and consequently enter the nucleus. This code, experimentally tested in 17 ARPs, predicts the nuclear-cytoplasmic localization of over 150 annotated human ARPs with high accuracy and is acquired by the most common familial melanoma-associated CDKN2A mutation, leading to nuclear accumulation of mutant p16ink4a. The RaDAR (RanGDP/AR) pathway represents a general importin-independent nuclear import pathway and is frequently used by AR-containing transcriptional regulators, especially those regulating NF-kappaB/p53 (Lu, 2014).

CHD4 is a RanGTP-dependent MAP that stabilizes microtubules and regulates bipolar spindle formation

Production of the GTP-bound form of the Ran GTPase (RanGTP) around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)-containing proteins. Several NLS proteins have been identified as spindle assembly factors, but the complexity of the process led to search for additional proteins with distinct roles in spindle assembly. This study identified a chromatin-remodeling ATPase, CHD4 (45% identical to Drosophila Mi2), as a RanGTP-dependent microtubule (MT)-associated protein (MAP). MT binding occurs via the region containing an NLS and chromatin-binding domains. In Xenopus egg extracts and cultured cells, CHD4 largely dissociates from mitotic chromosomes and partially localizes to the spindle. Immunodepletion of CHD4 from egg extracts significantly reduces the quantity of MTs produced around chromatin and prevents spindle assembly. CHD4 RNAi in both HeLa and Drosophila S2 cells induces defects in spindle assembly and chromosome alignment in early mitosis, leading to chromosome missegregation. Further analysis in egg extracts and in HeLa cells reveals that CHD4 is a RanGTP-dependent MT stabilizer. Moreover, the CHD4-containing NuRD complex promotes organization of MTs into bipolar spindles in egg extracts. Importantly, this function of CHD4 is independent of chromatin remodeling. These results uncover a new role for CHD4 as a MAP required for MT stabilization and involved in generating spindle bipolarity (Yokoyama, 2013).

Ran mutation

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 (see Drosophila ketel), 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).

Ran and nuclear transport

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).

Stimulation of nuclear export and inhibition of nuclear import by a Ran mutant deficient in binding to Ran-binding protein 1

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).

Carrier-independent nuclear import of the transcription factor PU.1 via RanGTP-stimulated binding to Nup153

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).

Ran pathway and import of integral inner nuclear membrane proteins

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).

Ran function in yeast

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).

RAN and the Regulated trafficking of the MSP/Eph receptor during oocyte meiotic maturation in C. elegans

In C. elegans, a sperm-sensing mechanism regulates oocyte meiotic maturation and ovulation, tightly coordinating sperm availability and embryo production; sperm release the major sperm protein (MSP) signal to trigger meiotic resumption. Meiotic arrest depends on the parallel function of the oocyte VAB-1 MSP/Eph receptor and somatic G protein signaling. MSP promotes meiotic maturation by antagonizing Eph receptor signaling and counteracting inhibitory inputs from the gonadal sheath cells. This study presents evidence suggesting that in the absence of the MSP ligand, the VAB-1 Eph receptor inhibits meiotic maturation while either in or in transit to the endocytic-recycling compartment. VAB-1::GFP localization to the RAB-11-positive endocytic-recycling compartment is independent of ephrins but is antagonized by MSP signaling. Two negative regulators of oocyte meiotic maturation, DAB-1/Disabled and RAN-1, interact with the VAB-1 receptor and are required for its accumulation in the endocytic-recycling compartment in the absence of MSP or sperm (hereafter referred to as MSP/sperm). Inactivation of the endosomal recycling regulators rme-1 or rab-11.1 causes a vab-1-dependent reduction in the meiotic-maturation rate in the presence of MSP/sperm. Further, Gαs signaling in the gonadal sheath cells, which is required for meiotic maturation in the presence of MSP/sperm, affects VAB-1::GFP trafficking in oocytes. It is concluded that regulated endocytic trafficking of the VAB-1 MSP/Eph receptor contributes to the control of oocyte meiotic maturation in C. elegans. Eph receptor trafficking in other systems may be influenced by the conserved proteins DAB-1/Disabled and RAN-1 and by crosstalk with G protein signaling in neighboring cells (Cheng, 2008).

The Ran-GTP gradient in vertebrates

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).

Ran function in chromosome positioning and nuclear envelope assembly in C. elegans

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 and microtubule spindle assembly

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).

Formation of a bipolar mitotic spindle in somatic cells requires the cooperation of two assembly pathways, one based on kinetochore capture by centrosomal microtubules, the other on RanGTP-mediated microtubule organization in the vicinity of chromosomes. How RanGTP regulates kinetochore-microtubule (K-fiber) formation is not presently understood. This study identifies the mitotic spindle protein HURP as a novel target of RanGTP. HURP is a direct cargo of importin beta and in interphase cells, it shuttles between cytoplasm and nucleus. During mitosis, HURP localizes predominantly to kinetochore microtubules in the vicinity of chromosomes. Overexpression of importin beta or RanT24N (resulting in low RanGTP) negatively regulates its spindle localization, whereas overexpression of RanQ69L (mimicking high RanGTP) enhances HURP association with the spindle. Thus, RanGTP levels control HURP localization to the mitotic spindle in vivo, a conclusion supported by the analysis of tsBN2 cells (mutant in RCC1). Upon depletion of HURP, K-fiber stabilization is impaired and chromosome congression is delayed. Nevertheless, cells eventually align their chromosomes, progress into anaphase, and exit mitosis. HURP is able to bundle microtubules and, in vitro, this function is abolished upon complex formation with importin beta and regulated by Ran. These data indicate that HURP stabilizes K-fibers by virtue of its ability to bind and bundle microtubules. In conclusion this study identifies HURP as a novel component of the Ran-importin beta-regulated spindle assembly pathway, supporting the conclusion that K-fiber formation and stabilization involves both the centrosome-dependent microtubule search and capture mechanism and the RanGTP pathway (Sillje, 2006).

Ran and mitotic spindle assembly; kinetochore localization of checkpoint regulators

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).

RanBP1 governs spindle assembly by defining mitotic Ran-GTP production

Accurate control of the Ras-related nuclear protein (Ran) GTPase cycle depends on the regulated activity of regulator of chromosome condensation 1 (RCC1; see Drosophila RCC1), Ran's nucleotide exchange factor. RanBP1 has been characterized as a coactivator of the Ran GTPase-activating protein RanGAP1. RanBP1 can also form a stable complex with Ran and RCC1, although the dynamics and function of this complex remain poorly understood. This study shows that formation of the heterotrimeric RCC1/Ran/RanBP1 complex in M phase Xenopus egg extracts controls both RCC1's enzymatic activity and partitioning between the chromatin-bound and soluble pools of RCC1. This mechanism is critical for spatial control of Ran-guanosine triphosphate (GTP) gradients that guide mitotic spindle assembly. Moreover, phosphorylation of RanBP1 drives changes in the dynamics of chromatin-bound RCC1 pools at the metaphase-anaphase transition. These findings reveal an important mitotic role for RanBP1, controlling the spatial distribution and magnitude of mitotic Ran-GTP production and thereby ensuring accurate execution of Ran-dependent mitotic events (Zhang, 2014).

The Ras-related nuclear protein (Ran) GTPase plays critical roles in multiple cellular processes including nucleocytoplasmic transport, nuclear envelope (NE) assembly, and mitotic spindle assembly (Clarke, 2008). In interphase, guanosine triphosphate (GTP)-bound Ran (Ran-GTP) is concentrated within the nucleus, while guanosine diphosphate (GDP)-bound Ran (Ran-GDP) is predominant in the cytoplasm. This asymmetrical distribution drives transport between the nucleus and cytoplasm by regulating cargo binding and release of a family of Ran-GTP-binding transport receptors that are collectively called karyopherins. After mitotic NE breakdown, Ran-GTP is concentrated near mitotic chromatin, while the majority of Ran distal to chromosomes is GDP bound. The presence of such a chromatin-centered Ran-GTP gradient has been visualized in both M-phase Xenopus laevis egg extracts (XEEs) and mitotic somatic cells. The mitotic Ran-GTP gradient guides mitotic spindle assembly by releasing spindle assembly factors from karyopherins in a spatially regulated manner (Zhang, 2014).

The conversion of Ran-GDP to Ran-GTP is catalyzed by a Ran-specific guanine exchange factor (RanGEF), called regulator of chromosome condensation 1 (RCC1), whose binding to chromatin determines the asymmetrical distribution of Ran-GTP throughout the cell cycle (Nemergut, 2001). The association of RCC1 to chromatin changes dramatically as XEE progresses through mitosis, with large increases in the amount of chromatin-bound RCC1 shortly after the metaphase-anaphase transition (Arnaoutov, 2003). Mammalian RCC1 also shows changes in chromatin binding during the metaphase-anaphase window. The mechanisms underlying altered association of RCC1 to chromatin during anaphase are poorly understood. Nevertheless, the fact that elevated levels of RCC1 can disrupt kinetochore structures and spindle assembly checkpoint signaling (Arnaoutov, 2003) suggests that the dynamics of RCC1 have important functional consequences (Zhang, 2014).

RanBP1 is a Ran-GTP-binding protein whose in vivo function has been obscure. While RanBP1 is conserved between yeast and vertebrates, it is not found in some invertebrate species, such as flies and worms (Dasso, 2002). RanBP1 stimulates the enzymatic activity of Ran's GTPase-activating protein RanGAP1, roughly 10-fold within in vitro assays using purified proteins. In addition, karyopherins bind to Ran-GTP in a way that prevents its interaction with RanGAP1, but RanBP1 can release karyopherin binding and thereby allow RanGAP1-activated GTP hydrolysis on Ran. RanBP1 also forms a stable heterotrimeric complex with Ran and RCC1 in vitro, strongly inhibiting RCC1's RanGEF activity. The dynamics and potential functions of this RCC1/Ran/RanBP1 heterotrimeric complex (hereafter called the RRR complex) remain unresolved. Notably, RanBP1 is excluded from nuclei, preventing RRR complex formation within nucleoplasm and reducing RanBP1's capacity to inhibit RCC1 during interphase. No such barrier prevents RRR complex formation in mitosis (Zhang, 2014).

This study has investigated the mitotic function and regulation of the RRR complex using XEE, a well-established ex vivo model system for cell-cycle studies. Chromatin-based spindle assembly was defective when RCC1 was present without RanBP1, in a manner that could be corrected by restoration of RanBP1 to physiological levels. The interaction between RanBP1 and RCC1 within XEE determined the partitioning of RCC1 between its chromatin-bound and unbound forms, as well as the level of RanGEF activity. Notably, RanBP1 was phosphorylated in a cell-cycle-dependent manner, peaking in early anaphase. The mitotically phosphorylated residue of RanBP1 was mapped, and whether this modification might control the mitotic dynamics of RCC1 was tested. Consistent with this idea, a phosphomimetic mutation of this site disrupted RRR complex assembly and promoted increased loading of RCC1 onto mitotic chromosomes, while a nonphosphorylatable RanBP1 mutant suppressed the normal fluctuations of RCC1-chromatin binding at anaphase onset. Together, these findings reveal an important mitotic role for RanBP1, controlling the spatial distribution and magnitude of the Ran-GTP gradient and thereby ensuring accurate execution of Ran-dependent mitotic events (Zhang, 2014).

It has been known for nearly two decades that RCC1, Ran, and RanBP1 form a stable heterotrimeric complex (RRR complex), which inhibits RCC1's RanGEF activity in vitro. The function of the RRR complex has remained mysterious, however, because RanBP1 is physically separated from RCC1 during interphase and the formation of this complex has been presumed to have little role in regulating nuclear transport, if any. This study has demonstrated that the RRR complex forms readily in M-phase CSF-XEEs. RCC1 binding to chromatin and RRR complex assembly were mutually exclusive, so that promoting RRR complex formation through the addition of recombinant xRanBP1 sequestered RCC1 away from chromatin. Moreover, RRR complex assembly inhibited RCC1's RanGEF activity in CSF-XEE, in agreement with earlier in vitro observations. Together, these findings suggest that the RRR complex plays a pivotal mitotic role in determining the partitioning of RCC1 between its active chromatin-bound and inactive soluble states, thereby setting both the location and magnitude of Ran-GTP production during mitosis (Zhang, 2014).

These observations suggest that RCC1 inhibition through RRR complex assembly helps to organize Ran-GTP production and limit it to the close vicinity of mitotic chromatin. Inappropriate Ran-GTP production in the absence of RanBP1 can lead to the assembly of defective spindles and ectopic MT assembly in XEE. This mitotic function of RanBP1 may be particularly critical in Xenopus embryos and extracts, given the high concentration of RCC1 relative to chromatin binding sites within the early embryo (Dasso, 1992). Nevertheless, there is evidence that RanBP1 may play a similar role in somatic systems. For example, tissue culture cells depleted of RanBP1 show disorganized MT asters, hyperstable spindle MTs, and defective chromosome segregation. Notably, RanBP1 is downregulated in aging cells, which show relatively shallow Ran-GTP gradients. Finally, RanBP1 expression is misregulated in a variety of human cancers. These results suggest the possibility that this misregulation may contribute toward mitotic defects in these cells (Zhang, 2014).

Furthermore, it has been well established that RCC1's association to mitotic chromatin is dynamic and that there are particularly large changes as cycling XEE (Arnaoutov, 2003) or mammalian tissue culture cells pass through the metaphase-to-anaphase window. While a number of posttranslational modifications of RCC1 have been described previously, none of these appear to be responsible for control of RCC1 at anaphase onset. In particular, mitotic cdk1-dependent phosphorylation of RCC1 is not well correlated temporally to changes in RCC1-chromatin association, since it occurs much earlier in mitosis. It is also notable that the residues of human RCC1 that have been found as the primary targets of cdk1-dependent phosphorylation (serine resides 2 and 11) are not conserved in Xenopus laevis, so that their modifications could not modulate RCC1 association to chromatin in XEE. This study observed that RanBP1 is phosphorylated during anaphase in cycling XEE and that this modification disrupts RRR complex assembly. The data suggest that modification of RanBP1 drives changes in RCC1 binding to chromatin and thereby indirectly alters the rate of Ran-GTP production in anaphase (Zhang, 2014).

It is speculated that this regulation may also contribute to Ran pathway organization in early interphase. Enhanced activity of RCC1 on anaphase chromatin should help provide high levels of Ran-GTP to facilitate NE reassembly. Moreover, physical separation of RCC1 and RanBP1 after RRR complex dissociation would allow RCC1 sequestration to chromatin within reforming nuclei while excluding RanBP1 into the early interphase cytosol. It is expected that one detrimental effect of interphase NE rupture would be the loss of RanBP1 exclusion from nucleoplasm, resulting in RRR complex formation and decreased levels of active, chromatin-bound RCC1, which in turn could decrease Ran-GTP production and contribute to the general loss of compartmental identity that has been observed under these circumstances (Zhang, 2014).

The conservation of the Ser60 residue is consistent with the idea that phosphorylation of RanBP1 may play an important role in mitotic regulation of the Ran pathway in many vertebrate species. On the other hand, the absence of this residue in budding and fission yeast suggests that although RanBP1 may have mitotic roles in fungi, those functions are controlled through different mechanisms. This divergence may reflect the fact that yeast do not undergo NE breakdown during mitosis and thus do not have complete mixing of their nuclear and cytoplasmic contents. More interestingly, Caenorhabditis elegans and Drosophila melanogaster apparently not only lack this regulatory pathway but also appear to be devoid of RanBP1 homologs altogether, although they still possess potential homologs of RanBP2 and RanBP3 (Dasso, 2002). It is notable that the worm and fly RCC1 homologs possess extended N- or C-terminal domains, respectively, that are not found in vertebrate and fungal RCC1 homologs. In the case of Drosophila, this additional domain shares sequence homology with embryonic linker histones. It is speculated that worm and fly RCC1 may interact with chromatin in a different fashion than vertebrate RCC1 proteins, thus bypassing any requirement for RanBP1. Testing this idea in the future will be of considerable interest and will be important for understanding how the use of the Ran pathway has evolved in different organisms (Zhang, 2014).

In summary, this study has documented an important mitotic role of the RanBP1 protein in controlling the localization and activity of Ran's nucleotide exchange factor RCC1. Phosphorylation of RanBP1 during anaphase drives changes in RCC1 dynamics and allows increased Ran-GTP production. These findings resolve important and long-standing questions within the Ran field regarding the function of the heterotrimeric RRR complex and its dynamics (Zhang, 2014).

Ran pathway regulates centrosome function

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).

Ran triggers distinct steps of nuclear pore complex assembly

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).

Ran-regulated microtubule binding of the kinesin

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).

Crosstalk between the actin cytoskeleton and Ran-mediated nuclear transport

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 2^nd 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 2^nd and 3^rd chromosomal deficiency stocks (deficiency/balancer) uncovering 70% to 80% of the two autosomes, or about 60% of the Drosophila genome, were crossed with ntf-2^P7/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-NES^P12-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-NES^P12-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-NES^P12-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-NES^P12-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-2^P7 and ntf-2^P49 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 (acu^E636). 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 1^st 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 Ran GTPase mediates chromatin signaling to control cortical polarity during polar body extrusion in mouse oocytes

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).

Localized regulation of axonal RanGTPase controls retrograde injury signaling in peripheral nerve

Peripheral sensory neurons respond to axon injury by activating an importin-dependent retrograde signaling mechanism. How is this mechanism regulated? This study shows that Ran GTPase and its associated effectors RanBP1 and RanGAP regulate the formation of importin signaling complexes in injured axons. A gradient of nuclear RanGTP versus cytoplasmic RanGDP is thought to be fundamental for the organization of eukaryotic cells. Surprisingly, RanGTP is found in sciatic nerve axoplasm, distant from neuronal cell bodies and nuclei, and in association with dynein and importin-alpha. Following injury, localized translation of RanBP1 stimulates RanGTP dissociation from importins and subsequent hydrolysis, thereby allowing binding of newly synthesized importin-beta to importin-alpha and dynein. Perturbation of RanGTP hydrolysis or RanBP1 blockade at axonal injury sites reduces the neuronal conditioning lesion response. Thus, neurons employ localized mechanisms of Ran regulation to control retrograde injury signaling in peripheral nerve (Yudin, 2008).

While this study has established a role for importins in the transport of injury signals from axonal lesion sites to the cell body in injured peripheral sensory neurons, others have demonstrated roles for importins in cytoplasmic transport of synaptic signals in central neurons, and in tiling of photoreceptor axons in development of the Drosophila visual system. In addition, a switch in the subtypes of importin-α expressed in embryonic stem cells may be critical for neuronal differentiation, since the importin-α switch may dictate changes in the spectrum of cargo transcription factors imported into the nucleus. The Ran interactor RanBPM also influences neuronal differentiation in conjunction with TAF4 in embryonic cortical neural stem cells. Most recently, RanBPM was implicated in axon guidance as a modulator of semaphorin 3A signaling via an interaction with Plexin-A receptors. Intriguingly, Ran is known to regulate microtubule dynamics and organization during spindle formation in mitosis, thus RanBPM and Ran might link axon guidance receptors to microtubule functions in growth cones and in axons. Clearly, Ran and its associated effectors and interactors are involved in neuronal physiology from the tip of the growth cone to the nuclear center of the cell body. It is suspected that axonal regulation of the GTP-bound state of Ran will prove to be critical for the development and maintenance of neuronal projections under normal conditions as well as in response to injury (Yudin, 2008).

Identification of neural outgrowth genes using genome-wide RNAi

While genetic screens have identified many genes essential for neurite outgrowth, they have been limited in their ability to identify neural genes that also have earlier critical roles in the gastrula, or neural genes for which maternally contributed RNA compensates for gene mutations in the zygote. To address this, methods were developed to screen the Drosophila genome using RNA-interference (RNAi) on primary neural cells, and the results of the first full-genome RNAi screen in neurons are presented in this study. Live-cell imaging and quantitative image analysis were used to characterize the morphological phenotypes of fluorescently labelled primary neurons and glia in response to RNAi-mediated gene knockdown. From the full genome screen, analysis focused on 104 evolutionarily conserved genes that when downregulated by RNAi, have morphological defects such as reduced axon extension, excessive branching, loss of fasciculation, and blebbing. To assist in the phenotypic analysis of the large data sets, image analysis algorithms were generated that could assess the statistical significance of the mutant phenotypes. The algorithms were essential for the analysis of the thousands of images generated by the screening process and will become a valuable tool for future genome-wide screens in primary neurons. The analysis revealed unexpected, essential roles in neurite outgrowth for genes representing a wide range of functional categories including signalling molecules, enzymes, channels, receptors, and cytoskeletal proteins. It was also found that genes known to be involved in protein and vesicle trafficking showed similar RNAi phenotypes. Phenotypes of the protein trafficking genes Sec61alpha and Ran GTPase were confirmed using Drosophila embryo and mouse embryonic cerebral cortical neurons, respectively. Collectively, these results showed that RNAi phenotypes in primary neural culture can parallel in vivo phenotypes, and the screening technique can be used to identify many new genes that have important functions in the nervous system (Sepp, 2008).

Since a large number of genes identified in the RNAi screen have close homologs to vertebrate genes, one of these genes was evaluated in embryonic mouse brains. Ran GTPase was a highly conserved gene identified in the full genome screen that showed dramatic effects on neurite outgrowth when knocked down. Ran GTPase is a member of the Ras superfamily that is involved in a variety of cellular process, including nucleo-cytoplasmic transport and mitosis. The Drosophila Ran GTPase protein has 87% similarity to mouse and human Ran. Ran binds to the human AR receptor protein, which shows a polyglutamine expansion in Kennedy's Disease, a neurodegenerative disorder, but the role of Ran in Kennedy's disease, or in neurodevelopment is not known. In Drosophila, Ran transcripts are maternally deposited into the embryo. During later embryonic development Ran becomes zygotically expressed specifically in the CNS at stage 12, which corresponds to a time of rapid neural cell division and migration (Sepp, 2008).

Ran is known to be expressed in the mouse brain at early embryonic stages, and is thus a good gene candidate to characterize in mouse brain development using RNAi. Ran was labeled in dissociated cortical neurons and high levels of expression were found in the nuclei of these cells. Furthermore, Ran immunolabeling can be detected in the processes, suggesting a role for Ran in neurite outgrowth, as well as in nuclear import. To analyze the role of Ran in mouse development, Ran RNAi constructs were transfected into the lateral ventricles of the embryonic day 14 (E14) mouse brains using microinjection and electroporation techniques. The transfected cortices were dissected and cultured as explants or dissociated cultures. To test the efficacy of the Ran RNAi constructs (1 and 2) in reducing the levels of Ran protein, nih-3T3 cells were transfected with RNAi constructs at 70% transfection efficency. Western blot analysis of total protein from transfected and untransfected cells showed a 64% knockdown of Ran in the presence of Ran RNAi construct number. Ran RNAi electroporated neurons showed processes with abnormal blebbing compared to the normal appearing processes in the vector control. Only 0.7% of control neurons presented blebs while 65.6% of the Ran RNAi neurons showed blebs. To ensure that the blebs present in Ran RNAi neurons were not due to the cell death the explants were analyzed with an apoptosis marker, anti-Cleaved Caspase3. It was found that GFP-labeled neurons in the Ran RNAi explants did not colocalize with Cleaved Caspase3. Thus, the blebbing phenotype was probably due to defects in neurite outgrowth. In addition to the blebbing phenotype, Ran-deficient neurons showed an increase in branch arborization as compared to the normal branch morphology seen in the control. The number of branching points per neuron increased significantly (from 3.2 in the control to 11.5). The effect of Ran deficiency in vivo was analyzed using explant cultures. Analysis of 3D reconstruction of control and Ran RNAi showed that the branching and blebbing phenotype is also present in the in vivo situation. Quantitation of the number of blebs per nuclei in explant sections showed a very significant increase in Ran RNAi deficient explants. This increased arborization phenotype observed upon knockdown of Ran protein partly resembles the effect of Rac GTPase loss-of-function in mouse and Drosophila neurons. Rac GTPases are major regulators of the actin cytoskeleton while the Ran GTPase is a major regulator of the microtubule cytoskeleton. The interplay of both the actin and microtubule cytoskeletons is known to be important for axonal branching (Sepp, 2008).

To further characterize the role of Ran in neural development, sections from Ran RNAi-transfected brains were immunolabeled with various neuronal markers. In some explants only a few cell bodies of Ran RNAi neurons were present in the intermediate zone (IZ), compared to the control-transfected cells that were observed to be closer to the IZ and in the cortical plate (CP). In other cases, an apparent increase was noted in Ran RNAi electroporated cells close to the subventricular and ventricular zone (SVZ/VZ) compared to control explants in TAU1 (axonal marker) immunolabeled explants. The distribution of GFP-positive cells was analyzed in the explants by dividing the image in Bins I through VI. Three different planes were analyzed per explant. Quantitation of GFP-cell distribution in the explants suggested that upon Ran knockdown the distribution of cells might shift towards the lower areas (Bin IV-VI) compared to the controls. Immunolabeling for the axonal marker TuJ1 colocalized with processes from Ran RNAi transfected cells similar to the control transfected cells. Interestingly, analysis of MAP2 staining showed in some cases higher colocalization of MAP2 and GFP in the control neurons compared to the Ran RNAi neurons in the explant cultures. Together, these results suggest an essential and novel role for Ran GTPase primarily in regulation of neurite extension, which in turn could potentially affect neuronal polarity and migration (Sepp, 2008).

Structural basis for nuclear import of splicing factors by human Transportin 3

Transportin 3 (Tnpo3, Transportin-SR2) is implicated in nuclear import of splicing factors and HIV-1 replication. This study shows that the majority of cellular Tnpo3 binding partners contain arginine-serine (RS) repeat domains and present crystal structures of human Tnpo3 in its free as well as GTPase Ran- and alternative splicing factor/splicing factor 2 (ASF/SF2)-bound forms. The flexible beta-karyopherin fold of Tnpo3 embraces the RNA recognition motif and RS domains of the cargo. A constellation of charged residues on and around the arginine-rich helix of Tnpo3 HEAT repeat 15 engage the phosphorylated RS domain and are critical for the recognition and nuclear import of ASF/SF2. Mutations in the same region of Tnpo3 impair its interaction with the cleavage and polyadenylation specificity factor 6 (CPSF6) and its ability to support HIV-1 replication. Steric incompatibility of the RS domain and RanGTP engagement by Tnpo3 provides the mechanism for cargo release in the nucleus. These results elucidate the structural bases for nuclear import of splicing factors and the Tnpo3-CPSF6 nexus in HIV-1 biology (Maertens, 2014).

REFERENCES

Search PubMed for articles about Drosophila Ran

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date revised: 10 June 2023

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