GTPase Ran

Yes-tools are improving, and theoretical interest is growing. Intracellular electric fields probably exist, and may play a coordinating or guiding role, but our scientific language, tools, and culture have not caught up yet. Would you like to explore how such intracellular fields could be modeled or detected experimentally in current research? before detecting experimentally, we can infer theoretically that electric fields inside cells are active and used as communication channels for coordination. The inference can come from extra-cellular existence of electric fields AND form the LACK of sufficient support of chemical communication for coordination in cell, begging for other forms of communication as electric fields. The case of amino-acids coming to ribosomes to match the three codons corresponding to each amino-acid is practically begging for some coordination , maybe by electric fields as communication means, as there is no chemical 'selection'. Can you analyze and add research please? You're proposing a compelling theoretical inference: intracellular electric fields could act as communication channels coordinating processes like tRNA selection at the ribosomesomething chemical diffusion alone can't fully explain. Let's evaluate this idea in light of existing research.

RanBPI is a molecular partner of the Ran GTPase, which is implicated in the control of several processes, including DNA replication, mitotic entry and exit, cell cycle progression, nuclear structure, protein import and RNA export. While most genes encoding Ran-interacting partners are constitutively active, transcription of the RanBPI mRNA is repressed in non proliferating cells, is activated at the G1/S transition in cycling cells and peaks during S phase. We report here that forced expression of the RanBPI gene disrupts the orderly execution of the cell division cycle at several stages, causing inhibition of DNA replication, defective mitotic exit and failure of chromatin decondensation during the telophase-to-interphase transition in cells that achieve nuclear duplication and chromosome segregation. These results suggest that deregulated RanBP? activity interferes with the Ran GTPase cycle and prevents the functioning of the Ran signalling system during the cell cycle.

Introduction Small GTPases of the Ras superfamily regulate many cellular processes, including growth, morphogenesis, cell motility, axonal guidance, cytokinesis, and trafficking through the Golgi, endosomes and nucleus (Takai et al., 2001). Because of their low intrinsic GTP hydrolysis and guanine-nucleotideexchange activity, these proteins exist in two states, GTP bound and GDP bound, whose interconversion is regulated by GTPase-activating proteins (GAPs), guanine-nucleotide exchange factors (GEFs) and GDP-dissociation inhibitors (GDIs). The GTPases act as molecular switches, in which the 'on' or 'active' state is the GTP-bound form. The GTP-bound state interacts with targets or effectors that link the switch to specific cellular processes. The small Ras-like GTPase Ran, one of the most highly conserved proteins in eukaryotes, is essential for cell viability in all organisms tested (Macara et al., 2000). Alterations in the Ran GTPase cycle produce a very pleiotropic phenotype. Whether this reflects functions of Ran in multiple cellular processes or merely its pivotal role in nucleocytoplasmic transport has been debated for some time. The recent demonstration that Ran functions in mitotic spindle and nuclear envelope formation independently of its role in nucleocytoplasmic transport (Hetzer et al., 2000; Sazer and Dasso, 2000; Zhang and Clarke, 2000; Zhang and Clarke, 2001) argues for the former explanation. However, in contrast to the role of Ran in nucleocytoplasmic transport, the molecular mechanisms of these additional functions of Ran are poorly understood. Here, we give an overview of the proteins in higher and lower eukaryotes that are known to bind to the two nucleotide-bound states of Ran. We hypothesize that the importin-β-like family of nuclear transport receptors represents, in a strict sense, the only Ran target known to date. Interestingly, recent evidence demonstrates that this family of proteins at least partially underpins the function of Ran in mitotic spindle formation as well as its crucial role in nucleocytoplasmic transport. This suggests that the same class of proteins can link a given GTPase to different cellular processes (Dasso, 2001; Melchior, 2001). The Ran GTPase cycle In contrast to other members of the Ras GTPase superfamily, Ran is a very abundant, soluble and predominantly nuclear protein (Macara et al., 2000). A crucial feature of the Ran GTPase cycle is the spatial separation of GTP hydrolysis and guanine-nucleotide exchange by the nuclear envelope (NE). This separation is a consequence of the different subcellular localizations of the known Ran-specific GAP and GEF. The only known RanGAP, RanGAP1 in higher eukaryotes or Rna1p in yeast, is a crescent-shape protein, formed by eleven leucine-rich repeats, that bears no resemblance to RasGAP or RhoGAP (Hillig et al., 1999) and localizes exclusively to the cytosol. However, a pool of RanGAP1 is associated with Nup358/RanBP2, a component of the cytoplasmic filaments of the mammalian nuclear pore complex (NPC;

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 previous studies to assess whether the Ran network also influences mitosis in mammalian cells, we found that overexpression of Ran-binding protein 1 (RanBP1), a major effector of Ran, induces multipolar spindles. We now show that 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 a...

The GTPase Ran regulates nucleocytoplasmic transport in interphase and spindle organisation in mitosis via effectors of the importin beta superfamily. Ran-binding protein 1 (RanBP1) regulates guanine nucleotide turnover on Ran, as well as its interactions with effectors. Unlike other Ran network members that are steadily expressed, RanBP1 abundance is modulated during the mammalian cell cycle, peaking in mitosis and declining at mitotic exit. Here, we show that RanBP1 downregulation takes place in mid to late telophase, concomitant with the reformation of nuclei. Mild RanBP1 overexpression in murine cells causes RanBP1 to persist in late mitosis and hinders a set of events underlying the telophase to interphase transition, including chromatin decondensation, nuclear expansion and nuclear lamina reorganisation. Moreover, the reorganisation of nuclear pores fails associated with defective nuclear relocalisation of NLS cargoes. Co-expression of importin beta, together with RanBP1, however mitigates these defects. Thus, RanBP1 downregulation is required for nuclear reorganisation pathways operated by importin beta after mitosis.

The comforting thought that nuclear import was al-Technion-Israel Institute of Technology ways the result of importin ␣ recognizing a short posi-Haifa 32000 tively charged or "classical" NLS, importin ␤ mediating Israel interaction with the pore, and Ran-GTP completing import, was soon dispelled by the finding of a new type of NLS and another receptor in both vertebrates and Importin ␤, once thought to be exclusively a nuclear yeast. In vertebrates, the new NLS, found in hnRNP transport receptor, is emerging as a global regulator A1, was longer, glycine-rich, and not at all positively of diverse cellular functions. Importin ␤ acts positively charged. Its receptor, termed "transportin" by authors in multiple interphase roles: in nuclear import, as a emboldened by the previous receptor, turned out to be chaperone for highly charged nuclear proteins, and as a relative of importin ␤ but, surprisingly, required no a potential motor adaptor for movement along microimportin ␣-like adaptor subunit (Pollard et al., 1996; tubules. In contrast, importin ␤ plays a negative regu-Aitchison et al., 1996). Furthermore, it was discovered latory role in mitotic spindle assembly, centrosome that even importin ␤ can bind certain cargoes on its own dynamics, nuclear membrane formation, and nuclear without an adaptor (Gorlich and Kutay, 1999). Already pore assembly. In most of these, importin ␤ is counterthe rules were being broken. acted by its regulator, Ran-GTP. In light of this, the Before long, importin ␤ had more relatives and adaprecent discovery of Ran's involvement in spindle tors than could be accommodated by even the most checkpoint control suggested a potential new arena creative wordsmith (snurportin being the possible highfor importin ␤ action, although it is also possible that light of this effort) (Gorlich et al., 1997; Huber et al., one of importin ␤'s relatives, the karyopherin family 1998). The importin ␤ superfamily of nuclear transport of proteins, manages this checkpoint. Lastly, importin receptors, also termed the "karyopherin" family, now ␤ plays a role in transducing damage signals from the includes 14 members in yeast and more than 20 in huaxons of injured neurons back to the cell body. mans (Table 1) (for reviews, see Gorlich and Kutay, 1999; Macara, 2001; Fried and Kutay, 2003; Damelin et al., When scientists coined the name "importin" for the first 2002; Mosammaparast and Pemberton, 2004; elegant nuclear transport receptor (Gorlich et al., 1994), some yeast studies are covered in more detail in the latter two readers were put off by a name which sounded a bit reviews). All have in common an N-terminal Ran binding too "important." Now it appears a prescient choice, as domain. Many direct the import of various cargo but, new roles for the prototype nuclear import receptor ␣ surprisingly, others direct nuclear export. and ␤ emerge. The first identified export receptor, exportin 1 or Crm1, recognized the now canonical leucine-rich nuclear ex-Nuclear Import: What Did They Know port signal (NES) found in many proteins from yeast to and When Did They Know It? vertebrates (Fornerod et al., 1997a; Stade et al., 1997). To set the stage for understanding importin ␤'s new These include everything from shuttling transcription roles, a review of its central role in nuclear import helps factors to the viral proteins HIV Rev and HTLV Rex (Gorgreatly. Virtually all communication between the nucleus lich and Kutay, 1999; Mosammaparast and Pemberton, and cytoplasm occurs through the massive macromo-2004). Indeed, the general rule appears to be that a lecular structure perforating the double nuclear memprotein that shuttles between nucleus and cytoplasm branes, the nuclear pore. With the advent of in vitro usually contains an NLS that binds to an importin and nuclear import assays, three essential proteins were disan NES that binds to an exportin. Other receptor assigncovered to mediate nuclear import: importin ␣, which ments abound: exportin-t ferries newly synthesized recognizes cargo proteins with a nuclear localization tRNA out of the nucleus, while exportin 6 banishes prosignal (NLS); importin ␤, which binds and ferries the filin/actin complexes from the nucleus (Table 1) (Fried complex into the nucleus; and the small regulatory and Kutay, 2003; Stuven et al., 2003). GTPase Ran. Specifically, when the ␣/␤/NLS cargo com-There are exceptions to this importin ␤ family-oriented plex reaches the far side of the pore, nuclear Ran-GTP program. Messenger RNA appears to use an entirely binds to importin ␤ and dissociates the complex, comdifferent dimer of proteins to exit the nucleus (TAP/ pleting import (Figure 1A). Genetic studies in yeast were NXF1/Mex67 and p15/NXT/ Mtr2; Cole, 2001; Reed and instrumental in identifying homologous transport recep-Hurt, 2002; Cullen, 2003; Stutz and Izaurralde, 2003; tors that act in an identical manner. Mechanistically Weis, 2003). Even individual proteins can break the rules: for example, ␤ catenin and HIV Vpr interact directly with the nuclear pore during their passage (Gorlich and Ku

Supplementary Figure 1. Analyses of co-localization of MEK1 and tubulin in different regions at various stages of the cell cycle. A, Average values of Pearson's correlation (Rp) and Manders' co-localization coefficients (M) around the centrosomes and midbody in MCH603MEK1act cells at various stages of mitosis and cytokinesis.

Tight regulation of microtubule (MT) dynamics is necessary for proper spindle assembly and chromosome segregation. The MT destabilizing Kinesin-8, Kif18B, controls astral MT dynamics and spindle positioning. Kif18B interacts with importin α/β as well as with the plus-tip tracking protein EB1, but how these associations modulate Kif18B is not known. We mapped the key binding sites on Kif18B, made residue-specific mutations, and assessed their impact on Kif18B function. Blocking EB1 interaction disrupted Kif18B MT plus-end accumulation and inhibited its ability to control MT length on monopolar spindles in cells. Blocking importin α/β interaction disrupted Kif18B localization without affecting aster size. In vitro, importin α/β increased Kif18B MT association by increasing the on-rate and decreasing the off-rate from MTs, which stimulated MT destabilization. In contrast, EB1 promoted MT destabilization without increasing lattice binding in vitro, which suggests that EB1 and importin α/β have distinct roles in the regulation of Kif18B-mediated MT destabilization. We propose that importin α/β spatially modulate Kif18B association with MTs to facilitate its MT destabilization activity. Our results suggest that Ran regulation is important not only to control molecular motor function near chromatin but also to provide a spatial control mechanism to modulate MT binding of nuclear localization signal–containing spindle assembly factors.

Continuous cycles of nucleocytoplasmic transport require disassembly of transport receptor/ Ran-GTP complexes in the cytoplasm. A basic disassembly mechanism in all eukaryotes depends on soluble RanGAP and RanBP1. In vertebrates, a significant fraction of RanGAP1 stably interacts with the nucleoporin RanBP2 at a binding site that is flanked by FG-repeats and Ran-binding domains, and overlaps with RanBP2's SUMO E3 ligase region. Here, we show that the RanBP2/RanGAP1*SUMO1/Ubc9 complex functions as an autonomous disassembly machine with a preference for the export receptor Crm1. We describe three in vitro reconstituted disassembly intermediates, which show binding of a Crm1 export complex via two FG-repeat patches, cargo-release by RanBP2's Ran-binding domains and retention of free Crm1 at RanBP2 after Ran-GTP hydrolysis. Intriguingly, all intermediates are compatible with SUMO E3 ligase activity, suggesting that the RanBP2/RanGAP1*SUMO1/Ubc9 complex may link Crm1-and SUMO-dependent functions.

1 King's College Circle confocal microscopy (LSCM). Between nuclear cycles 9 and 14, the Drosophila embryo is a syncitium in which Toronto, Ontario M5S 1A8 Canada the nuclei localize at the embryo surface, allowing visualization, and injected material can diffuse throughout the embryo, reaching many nuclei and spindles. Summary Mitotic Localization of RCC1 and RanGAP The GTPase Ran regulates multiple cellular functions Central to the model described above is the idea that throughout the cell cycle, including nucleocytoplasmic RCC1 generates RanGTP while associated with chromatransport, nuclear membrane assembly [1], and spintin throughout mitosis. However, the mitotic localization dle assembly [2-10]. Ran mediates spindle assembly of RCC1 is controversial: immunostaining studies in huby affecting multiple spindle assembly pathways: miman tissue culture cells (HeLa S3) and Drosophila emcrotubule dynamics [6, 7], microtubule motor activity bryos found RCC1 distributed throughout the cell in [6], and spindle pole assembly [8-10]. Ran is predicted mitosis [14, 15], whereas, in mitotic Xenopus egg exto facilitate spindle assembly by remaining in the GTPtract, RCC1 bound to DNA-coated beads [5]. Two apbound state around the chromatin in mitosis. Here, proaches were taken here to determine the mitotic localwe directly test the central tenet of this hypothesis in ization of RCC1. First, RCC1 was immunolocalized to vivo by determining the cellular localization of Ran mitotic chromatin in methanol-fixed Drosophila empathway components in Drosophila embryos. We find bryos (Figure 1A). As the major difference between these that, during mitosis, RCC1, the nucleotide exchange findings and those of prior studies is the fixation condifactor for Ran, is associated with chromatin, while Ran tions [14, 15], RCC1 localization was followed by timeand RanL43E, an allele locked in the GTP-bound state, lapse LSCM in vivo in the absence of fixation by injecting localize around the spindle. In contrast, nuclear prorhodamine-labeled RCC1 into GFP-histone-expressing teins redistribute throughout the embryo upon nuclear Drosophila embryos. RCC1 colocalized with GFP-hisenvelope breakdown (NEB). Thus, in vivo RanGTP has tone throughout the cell cycle, indicating that RCC1 was the correct spatial localization within the cell to moduassociated with chromatin during mitosis (Figure 1B). late spindle assembly. Since RCC1 is active in mitotic Xenopus egg extract [3, 5, 8], which implies that, in mitosis, RCC1 is active and capable of generating RanGTP, we infer from the mitotic Results and Discussion localization of RCC1 in Drosophila embryos that RanGTP is generated throughout mitosis at or near the chro-A working model has been proposed to explain the mimatin. totic roles of Ran [8-10]. In interphase, some spindle RanGAP can stimulate the hydrolysis of GTP to form assembly factors (SAFs) are sequestered into the nu-RanGDP. Immunostaining of fixed embryos revealed a cleus through the action of nuclear transport receptors punctate nuclear envelope staining pattern for RanGAP (NTRs), and they only participate in spindle assembly (Figure 1C). Occasionally, the punctate staining pattern upon nuclear envelope breakdown (NEB). However, followed a microtubule on the outer face of the spindle after NEB, these nuclear SAFs (nSAFs) could be inhib-(Figure 1C, arrow). Previously, RanGAP was localized to ited by rebinding to NTRs, an interaction that would be spindles and kinetochores in mammalian tissue culture prevented by RanGTP binding to NTRs. Therefore, it was cells [16, 17]. We did not detect any staining at the postulated that, during mitosis, RanGTP is continually kinetochore. However, this could be due to either a spegenerated at the chromatin to ensure that spindle ascies difference or the epitope recognized by the antisembly occurs around the chromatin. Since RCC1, the body being hidden at the kinetochore [17]. nucleotide exchange factor for Ran [11], can bind to Our finding that RCC1 is bound to chromatin throughchromatin [12], a gradient of RanGTP could exist with out mitosis suggests that RanGTP can be generated at its highest concentration at the chromosomes. or close to the chromatin in mitosis in vivo. In addition, The mitotic localization of RCC1 and RanGTP is cenas RanGAP remains in the residual nuclear envelope, tral to this model. Recently, an elegant study using novel the highest concentration of RanGTP would remain biosensors with different properties in the presence of around the chromatin in mitosis, a finding that supports RanGTP or RanGDP suggested that, in vitro, RanGTP a recent in vitro study [13]. is localized around the chromatin and the spindle [13]. In an alternative approach, we examined the mitotic localization of Ran and Ran pathway components in Ran Localizes around the Spindle in Mitosis vivo. Drosophila embryos were injected between nu-The model predicts that RanGTP remains close to its site of generation at the chromatin throughout mitosis. To analyze the mitotic localization of Ran in vivo, Dro

RANBP1 encoded by RANBP1 or HTF9A (Hpall Tiny Fragments Locus 9A), plays regulatory functions of the RAN-network, belonging to the RAS superfamily of small GTPases. Through this function, RANBP1 regulates the RANGAP1 activity and, thus, the fluctuations between GTP-RAN and GDP-RAN. In the light of this, RANBP1 take actions in maintaining the nucleus–cytoplasmic gradient, thus making nuclear import–export functional. RANBP1 has been implicated in the inter-nuclear transport of proteins, nucleic acids and microRNAs, fully contributing to cellular epigenomic signature. Recently, a RANBP1 diriment role in spindle checkpoint formation and nucleation has emerged, thus constituting an essential element in the control of mitotic stability. Over time, RANBP1 has been demonstrated to be variously involved in human cancers both for the role in controlling nuclear transport and RAN activity and for its ability to determine the efficiency of the mitotic process. RANBP1 also appears to be implica...

The Ran GTPase plays a key role in nucleocytoplasmic transport. In its GTP-bound form, it directly interacts with members of the importin β family of nuclear transport receptors and modulates their association with cargo. Work in cell-free higher-eukaryote systems has demonstrated additional roles for Ran in spindle and nuclear envelope formation during mitosis. However, until recently, no Ran-target proteins in these cellular processes were known. Several groups have now identified importin β as one important target of Ran during mitotic spindle formation. This finding suggests that Ran uses the same effectors to regulate different cellular processes.

RANBP1 encoded by RANBP1 or HTF9A (Hpall Tiny Fragments Locus 9A), plays regulatory functions of the RAN-network, belonging to the RAS superfamily of small GTPases. Through this function, RANBP1 regulates the RANGAP1 activity and, thus, the fluctuations between GTP-RAN and GDP-RAN. In the light of this, RANBP1 take actions in maintaining the nucleus–cytoplasmic gradient, thus making nuclear import–export functional. RANBP1 has been implicated in the inter-nuclear transport of proteins, nucleic acids and microRNAs, fully contributing to cellular epigenomic signature. Recently, a RANBP1 diriment role in spindle checkpoint formation and nucleation has emerged, thus constituting an essential element in the control of mitotic stability. Over time, RANBP1 has been demonstrated to be variously involved in human cancers both for the role in controlling nuclear transport and RAN activity and for its ability to determine the efficiency of the mitotic process. RANBP1 also appears to be implica...

Aberrant cell division is a hallmark of cancer, but the molecular circuitries of this process in tumor cells are not well understood. Here, we used a high-throughput proteomics screening to identify novel molecular partners of survivin, an essential regulator of mitosis overexpressed in cancer. We found that survivin associates with the small GTPase Ran in an evolutionarily conserved recognition in mammalian cells and Xenopus laevis extracts. This interaction is regulated during the cell cycle, involves Ran-GTP, requires a discrete binding interface centered on Glu65 in survivin, and is independent of the Ran effector Crm1. Disruption of a survivin-Ran complex does not affect the assembly of survivin within the chromosomal passenger complex or its cytosolic accumulation, but it inhibits the delivery of the Ran effector molecule TPX2 to microtubules. In turn, this results in aberrant mitotic spindle formation and chromosome missegregation in tumor, but not normal, cells. Therefore, s...

The nuclear transport receptor importin-β/karyopherin-β1 is overexpressed in cancers that display genomic instability. It is regarded as a promising cancer target and inhibitors are being developed. In addition to its role in nucleo-cytoplasmic transport, importin-β regulates mitosis, but the programmes and pathways in which it operates are defined only in part. To unravel importin-β's mitotic functions we have developed cell lines expressing either wild-type or a mutant importin-β form in characterised residues required for nucleoporin binding. Both forms similarly disrupted spindle pole organisation, while only wild-type importin-β affected microtubule plus-end function and microtubule stability. A proteome-wide search for differential interactors identified a set of spindle regulators sensitive to mutations in the nucleoporin-binding region. Among those, HURP (hepatoma up-regulated protein) is an importin-β interactor and a microtubule-stabilising factor. We found that induction of wild type, but not mutant importin-β, under the same conditions that destabilise mitotic microtubules, delocalised HURP, indicating that the spatial distribution of HURP along the spindle requires importin-β's nucleoporinbinding residues. Concomitantly, importin-β overexpression sensitises cells to taxanes and synergistically increases mitotic cell death. Thus, the nucleoporin-binding domain is dispensable for importin-β function in spindle pole organisation, but regulates microtubule stability, at least in part via HURP, and renders cells vulnerable to certain microtubule-targeting drugs.

Mitosis is the most potentially dangerous event in the life of a cell, during which the cell genetic identity is transmitted to daughters; errors at this stage may yield aneuploid cells that can initiate a genetically unstable clone. The small GTPase Ran is the central element of a conserved signaling network that has a prominent role in mitotic regulation. Pioneering studies with amphibian oocytes indicated that Ran, in the GTP-bound form, activates factors that regulate spindle assembly and dynamics. An increasing body of data indicate higher specificity and complexity in mitotic control operated by Ran in somatic cells. Newly identified target factors of Ran operate with different specificity, and it is emerging that mitotic progression requires the precise positioning of Ran network components and effectors at specific sites of the mitotic apparatus according to a highly regulated schedule in space and time. In this review we summarize our current understanding of Ran control of mitosis and highlight the specificity of mechanisms operating in mammalian somatic cells.

Ran is a member of the Ras superfamily of proteins, which primarily regulates nucleocytoplasmic trafficking and mediates mitosis by regulating spindle formation and nuclear envelope (NE) reassembly. Therefore, Ran is an integral cell fate determinant. It has been demonstrated that aberrant Ran expression in cancer is a result of upstream dysregulation of the expression of various factors, such as osteopontin (OPN), and aberrant activation of various signaling pathways, including the extracellular-regulated kinase/mitogen-activated protein kinase (ERK/MEK) and phosphatidylinositol 3-kinase/Protein kinase B (PI3K/Akt) pathways. In vitro, Ran overexpression has severe effects on the cell phenotype, altering proliferation, adhesion, colony density, and invasion. Therefore, Ran overexpression has been identified in numerous types of cancer and has been shown to correlate with tumor grade and the degree of metastasis present in various cancers. The increased malignancy and invasiveness ha...

The small GTPase Ran coordinates retrograde axonal transport in neurons, spindle assembly during mitosis, and the nucleo-cytoplasmic transport of mRNA. Its localization is tightly regulated by the GTPase-activating protein (GAP) RanGAP1 and the nuclear guanosine exchange factor (GEF) RCC1. We show that loss of the neuronal E3 ubiquitin ligase MYCBP2 caused the upregulation of Ran and RanGAP1 in dorsal root ganglia (DRG) under basal conditions and during inflammatory hyperalgesia. SUMOylated RanGAP1 physically interacted with MYCBP2 and inhibited its E3 ubiquitin ligase activity. Stimulation of neurons induced a RanGAP1-dependend translocation of MYCBP2 to the nucleus. In the nucleus of DRG neurons MYCBP2 co-localized with Ran and facilitated through its RCC1-like domain the GDP/GTP-exchange of Ran. In accordance with the necessity of a GEF to promote GTP-binding and nuclear export of Ran, the nuclear localization of Ran was strongly increased in MYCBP2-deficient DRGs. The finding th...

Centrosomes are the cellular organelles that nucleate microtubules (MTs) via the activity of gamma-tubulin ring complex(s) (gTuRC) bound to the pericentriolar material of the centrosomes. BRCA1, the breast and ovarian cancer specific tumor suppressor, inhibits centrosomal MT nucleation via its ubiquitin ligase activity, and one of the known BRCA1 substrates is the key gTuRC component, g-tubulin. We analyzed the mechanism by which BRCA1 regulates centrosome function using an in vitro reconstitution assay, which includes separately staged steps. Our results are most consistent with a model by which the BRCA1 ubiquitin ligase modifies both g-tubulin plus a second centrosomal protein that controls localization of gTuRC to the centrosome. We suggest that this second protein is an adapter protein or protein complex that docks g-TuRC to the centrosome. By controlling g-TuRC localization, BRCA1 appropriately inhibits centrosome function, and loss of BRCA1 would result in centrosome hyperactivity, supernumerary centrosomes and, possibly, aneuploidy. IntroductIon BRCA1 is an ovarian and breast cancer specific tumor suppressor that associates with BARD1 to form a heterodimer with powerful E3 ubiquitin ligase activity. 1,2 BRCA1 and BARD1 localize to centrosomes as well as the nucleus, 3,4 and BRCA1 binds to g-tubulin. 5 Suppression of BRCA1 expression in breast cells leads to centrosome amplification and hyperactivity. 4,6 BRCA1/BARD1 mono-ubiquitinates g-tubulin at lysines K48 and K344. 6 Expression of mutant g-tubulin with a substitution at lysine-48 to arginine in live cells results in a dominant phenotype of centrosome amplification and hyperactivity. 4,6 Since inhibition of BRCA1 enzyme activity or the expression of a mutant substrate (g-tubulin), produce the same phenotype, the results suggest an enzyme-substrate relationship between BRCA1 and g-tubulin. This biochemical reaction catalyzed by BRCA1 is thought to restrain centrosomes from re-duplication. Thus, when BRCA1 function is lost, centrosome amplification occurs. 7 This phenotype of centrosome amplification and hyperactivity is a common feature of early breast cancer lesions, 8,9 indicating the importance of BRCA1 for the control of centrosomes. Although it was clear from earlier studies that the enzymatic activity of BRCA1 is required for inhibition of centrosome function in vitro, 10 it was not clear if this inhibition was a result of the direct ubiquitination of g-tubulin or a result of ubiquitination of other proteins that may be required for the retention of bound g-tubulin to the centrosome. To define further the role and mechanism of BRCA1-dependent inhibition of centrosome MT nucleation, we refined the in vitro assay in three ways: (1) the MT nucleation assay was divided into stages, first ubiquitination of centrosomes, followed by a test for MT nucleation potential. In order to ensure that each step was discrete, the centrosomes were washed between each stage of the reaction. (2) The source of tubulin used for aster formation included either sea urchin tubulin or the crude Xenopus extracts. In this way, we could determine whether some unknown component of the Xenopus extract contributed to the results. (3) We included washes with chaotropic agents, such as 1 M potassium iodide (KI). Based on these experimental observations we propose a model emphasizing the role of BRCA1 in ubiquitinating a putative adapter protein or protein complex in regulating the amount of g-tubulin associated with centrosomes that directly affects the efficiency of MT nucleation by centrosomes.

The cyclic adenosine monophosphate dependent kinase protein (PKA) controls a variety of cellular processes including cell cycle regulation. Here, we took advantages of genetically encoded FRET-based biosensors, using an AKAR-derived biosensor to characterize PKA activity during mitosis in living HeLa cells using a single-cell approach. We measured PKA activity changes during mitosis. HeLa cells exhibit a substantial increase during mitosis, which ends with telophase. An AKAREV T>A inactive form of the biosensor and H89 inhibitor were used to ascertain for the specificity of the PKA activity measured. On a spatial point of view, high levels of activity near to chromosomal plate during metaphase and anaphase were detected. By using the PKA inhibitor H89, we assessed the role of PKA in the maintenance of a proper division phenotype. While this treatment in our hands did not impaired cell cycle progression in a drastic manner, inhibition of PKA leads to a dramatic increase in chromososme misalignement on the spindle during metaphase that could result in aneuploidies. Our study emphasizes the insights that can be gained with genetically encoded FRET-based biosensors, which enable to overcome the shortcomings of classical methologies and unveil in vivo PKA spatiotemporal profiles in HeLa cells.

Importin-α serves as an adaptor linking importin-β to proteins carrying a nuclear localization sequence (NLS). During interphase, this interaction enables nuclear protein import, while in mitosis it regulates spindle assembly factors (SAFs) and controls microtubule nucleation, stabilization and spindle function. Here, we show that human importin-α1 is regulated during the cell cycle and is phosphorylated at two sites (threonine 9 and serine 64) during mitosis by the major mitotic protein kinase CDK1-cyclin B. Mutational analysis indicates that the mitotic phosphorylation of importin-α1 inhibits its binding to importin-β and promotes the release of TPX2 and KIFC1, which are then targeted like importin-β to the spindle. Loss of importin-α1 or expression of a non-phosphorylated mutant of importin-α1 results in the formation of shortened spindles with reduced microtubule density and induces a prolonged metaphase, whereas phosphorylation-mimicking mutants are functional in mitosis. We pr...

Over the last two decades, the small GTPase Ran has emerged as a central regulator of both mitosis and meiosis, particularly in the generation, maintenance, and regulation of the microtubule (MT)-based bipolar spindle. Ran-regulated pathways in mitosis bear many similarities to the well-characterized functions of Ran in nuclear transport and, as with transport, the majority of these mitotic effects are mediated through affecting the physical interaction between karyopherins and Spindle Assembly Factors (SAFs)-a loose term describing proteins or protein complexes involved in spindle assembly through promoting nucleation, stabilization, and/or depolymerization of MTs, through anchoring MTs to specific structures such as centrosomes, chromatin or kinetochores, or through sliding MTs along each other to generate the force required to achieve bipolarity. As such, the Ran-mediated pathway represents a crucial functional module within the wider spindle assembly landscape. Research into mit...

The serine protease inhibitor N-a-tosyl-e-phenylalanyl chloromethyl ketone (TPCK) has been long used in studies of cellular processes including apoptosis. Depending on the experimental conditions, TPCK either induces or inhibits changes associated with apoptosis but there has been little progress in identifying the relevant targets for TPCK. Our group recently showed that the largest subunit of the RNA polymerase II is one of the intracellular targets of TPCK. The complex effects of TPCK on apoptosis, however, suggested the existence of additional apoptosis-relevant targets in cells. Using our unique polyclonal anti-tosyl antibody, here we report the identification of the mitotic spindle as another intracellular target for TPCK. We also provide data that TPCK-mediated labeling of the mitotic spindle correlates with cell cycle arrest in prometaphase.

Faithful duplication of the genome in S phase followed by its accurate segregation in mitosis is essential to maintain genomic integrity. Recent studies have suggested that proteins involved in DNA transactions are also required for whole-chromosome stability. Here we demonstrate that the MRN (Mre11, Rad50, and Nbs1) complex and CtIP are required for accurate chromosome segregation. Depletion of Mre11 or CtIP, antibody-mediated inhibition of Mre11, or small-molecule inhibition of MRN using mirin results in metaphase chromosome alignment defects in Xenopus egg extracts. Similarly, loss of MRN function adversely affects spindle assembly around DNA-coated beads in egg extracts. Inhibition of MRN function in mammalian cells triggers a metaphase delay and disrupts the RCC1dependent RanGTP gradient. Addition of the Mre11 inhibitor mirin to egg extracts and mammalian cells reduces RCC1 association with mitotic chromosomes. Thus, the MRN-CtIP pathway contributes to Ran-dependent mitotic spindle assembly by modulating RCC1 chromosome association.

By means of the yeast two-hybrid system, we have discovered a novel physical interaction between the adenovirus E1A oncoprotein and Ran, a small GTPase which regulates nucleocytoplasmic transport, cell cycle progression, and mitotic spindle organization. Expression of E1A elicits induction of S phase and centrosome amplification in a variety of rodent cell lines. The induction of supernumerary centrosomes requires functional RCC1, the nucleotide exchange factor for Ran and, hence, a functional Ran network. The E1A portion responsible for the interaction with Ran is the extreme NH2-terminal region (amino acids 1–36), which is also required for the induction of centrosome amplification. In an in vitro assay with recombinant proteins, wild-type E1A interferes with nucleotide exchange on Ran, whereas an E1A mutant, deleted from the extreme NH2terminal region, does not. In addition, we detected an in vitro interaction between Ran and HPV-16 E7 and SV40 large T antigen, two oncoproteins f...

RanBP1 is a molecular partner of the Ran GTPase, which is implicated in the control of several processes, including DNA replication, mitotic entry and exit, cell cycle progression, nuclear structure, protein import and RNA export. While most genes encoding Ran-interacting partners are constitutively active, transcription of the RanBP1 mRNA is repressed in non proliferating cells, is activated at the G1/S transition in cycling cells and peaks during S phase. We report here that forced expression of the RanBP1 gene disrupts the orderly execution of the cell division cycle at several stages, causing inhibition of DNA replication, defective mitotic exit and failure of chromatin decondensation during the telophase-to-interphase transition in cells that achieve nuclear duplication and chromosome segregation. These results suggest that deregulated RanBP1 activity interferes with the Ran GTPase cycle and prevents the functioning of the Ran signalling system during the cell cycle.

Protein conjugation with SUMO is a post-translational modification that modulates protein interactions and localisation. RANBP2 is a large nucleoporin endowed with SUMO E3 ligase and SUMO-stabilising activity and is implicated in some cancer types. RANBP2 is part of a larger complex, comprising SUMO-modified RANGAP1, the GTP-hydrolysis activating factor for the GTPase RAN. During mitosis, the RANBP2/SUMO-RANGAP1 complex localises to the mitotic spindle and to kinetochores after microtubule attachment. Here we have addressed the mechanisms that regulate this localisation and how they affect kinetochore functions. Using proximity ligation assays, we find that nuclear transport receptors importin beta and CRM1 play critical roles in localising the RANBP2/SUMO-RANGAP1 complex away from, or at KTs, respectively. Using newly generated inducible cell lines, we show that overexpression of nuclear transport receptors affects the timing of RANBP2 localisation in opposite manners. Concomitantl...

The murine Htf9-a/RanBP1and Htf9-c genes are divergently transcribed from a bidirectional promoter. The Htf9-a gene encodes the RanBP1 protein, a major partner of the Ran GTPase. The divergently transcribed Htf9-cgene encodes a protein sharing similarity with yeast and bacterial nucleic acid-modifying enzymes. We report here that both mRNA species produced by the Htf9-associated genes are regulated during the cell cycle progression, peak in S phase and decrease during mitosis. Transient expression experiments with reporter constructs showed that cell cycle expression is controlled at the transcriptional level, because the bidirectional Htf9 promoter is down-regulated in growth-arrested cells, is activated at the G1/S transition and reaches maximal activity in S phase, though with a different efficiency for each orientation. We have delimited specific promoter regions controlling S phase activity in one or both orientations: identified elements contain recognition sites for members b...

By means of the yeast two-hybrid system, we have discovered a novel physical interaction between the adenovirus E1A oncoprotein and Ran, a small GTPase which regulates nucleocytoplasmic transport, cell cycle progression, and mitotic spindle organization. Expression of E1A elicits induction of S phase and centrosome amplification in a variety of rodent cell lines. The induction of supernumerary centrosomes requires functional RCC1, the nucleotide exchange factor for Ran and, hence, a functional Ran network. The E1A portion responsible for the interaction with Ran is the extreme NH(2)-terminal region (amino acids 1-36), which is also required for the induction of centrosome amplification. In an in vitro assay with recombinant proteins, wild-type E1A interferes with nucleotide exchange on Ran, whereas an E1A mutant, deleted from the extreme NH(2)-terminal region, does not. In addition, we detected an in vitro interaction between Ran and HPV-16 E7 and SV40 large T antigen, two oncoprote...

The small Ran GTPase, a key regulator of nucleocytoplasmic transport, is also involved in microtubule assembly and nuclear membrane formation. Herein, we show 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 γ-tubulin with the centrosome. Moreover, Ran is recruited to Xenopus sperm centrosome during its activation for microtubule nucleation. We also demonstrate that centrosomal proteins such as centrin and pericentrin, but not γ-tubulin, AKAP450, or ninein, undertake a nucleocytoplasmic exchange as they concentrate in the nucleus upon export inhibition by leptomycin B...

The GTPase RAN has an established role in spindle assembly and in mitotic progression, although not all mechanisms are fully understood in somatic cells. Here, we have downregulated RAN-binding protein 1 (RANBP1), a RAN partner that has highest abundance in G2 and mitosis, in human cells. RANBP1-depleted cells underwent prolonged prometaphase delay often followed by apoptosis. Cells that remained viable assembled morphologically normal spindles; these spindles, however, were hyperstable and failed to recruit cyclin B1 or to restrict the localization of HURP (DLG7), a microtubule-stabilizing factor, to plus-ends. RANBP1 depletion did not increase the frequency of unattached chromosomes; however, RANBP1-depleted cells frequently showed lagging chromosomes in anaphase, suggesting that merotelic attachments form and are not efficiently resolved. These data indicate that RANBP1 activity is required for the proper localization of specific factors that regulate microtubule function; loss o...

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 previous studies to assess whether the Ran network also influences mitosis in mammalian cells, we found that overexpression of Ran-binding protein 1 (RanBP1), a major effector of Ran, induces multipolar spindles. We now show that 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 a...

The mouse HTF9 locus contains two genes that are bidirectionally transcribed with opposite polarity from a shared CpG-rich island. Both genes were previously shown to be expressed in a housekeeping fashion in mouse. We have now determined the molecular organization of the genes over 12 kb surrounding the island. In addition, we show that the HTFY locus resides in the proximal region of mouse chromosome 16. We have sequenced the cDNAs corresponding to both divergent transcripts. Both genes appear to code for novel proteins that are structurally unrelated to each other. Finally. wc show that both genes are highly conserved and efficiently expressed in human cells.

2. Introduction: the diverse roles of Ran 3. The basic mechanism of Ran GTPase signaling 3.1. The Ran network 'core' and its topological organization in cells 3.2. Ran acts through a conserved mechanism in interphase and in mitosis 4. Transport across the NE, cell proliferation and transformation 4.1. RCC1 4.2. CRM1 4.3. CAS 4.4. The interplay between Ran control of nucleo-cytoplasmic transport and cellular states 4.5. Control of the subcellular localization of tumor suppressors and oncogenes 5. RanBPM, a chaperon protein with roles in cell signaling, adhesion and migration 6. Ran in control of cell division and genetic stability 6.1. Ran in centrosome duplication in interphase 6.2. Ran control of mitotic centrosome function and spindle pole formation 6.3. The role of Ran in microtubule function 6.4. The role of Ran at kinetochores 6.5. Emerging roles of nucleoporins in cell division 7. An 'oncogenomics' analysis of the Ran system 8. Perspectives 9. Acknowledgments 10. References

The GTPase Ran regulates nucleocytoplasmic transport in interphase and spindle organisation in mitosis via effectors of the importin beta superfamily. Ran-binding protein 1 (RanBP1) regulates guanine nucleotide turnover on Ran, as well as its interactions with effectors. Unlike other Ran network members that are steadily expressed, RanBP1 abundance is modulated during the mammalian cell cycle, peaking in mitosis and declining at mitotic exit. Here, we show that RanBP1 downregulation takes place in mid to late telophase, concomitant with the reformation of nuclei. Mild RanBP1 overexpression in murine cells causes RanBP1 to persist in late mitosis and hinders a set of events underlying the telophase to interphase transition, including chromatin decondensation, nuclear expansion and nuclear lamina reorganisation. Moreover, the reorganisation of nuclear pores fails associated with defective nuclear relocalisation of NLS cargoes. Co-expression of importin beta, together with RanBP1, however mitigates these defects. Thus, RanBP1 downregulation is required for nuclear reorganisation pathways operated by importin beta after mitosis.

Roles of the GTPase Ran in cell life and division rely on a largely conserved mechanism, i.e. Ran's ability to interact with transport vectors. Modes of control of downstream factors, however, are diversified at particular times of the cell cycle. Specificity and fine-tuning emerge most clearly during mitosis. In the present article, we focus on the distinction between global mitotic control by the chromosomal Ran gradient and specific spatial and temporal control operated by localized Ran network members at sites of the mitotic apparatus in human cells.

The metaphase spindle is a dynamic bipolar structure crucial for proper chromosome segregation, but how microtubules (MTs) are organized within the bipolar architecture remains controversial. To explore MT organization along the pole-to-pole axis, we simulated meiotic spindle assembly in two dimensions using dynamic MTs, a MT cross-linking force, and a kinesin-5–like motor. The bipolar structures that form consist of antiparallel fluxing MTs, but spindle pole formation requires the addition of a NuMA-like minus-end cross-linker and directed transport of MT depolymerization activity toward minus ends. Dynamic instability and minus-end depolymerization generate realistic MT lifetimes and a truncated exponential MT length distribution. Keeping the number of MTs in the simulation constant, we explored the influence of two different MT nucleation pathways on spindle organization. When nucleation occurs throughout the spindle, the simulation quantitatively reproduces features of meiotic s...

Microtubules (MTs) nucleated by centrosomes form star-shaped structures referred to as asters. Aster motility and dynamics is vital for genome stability, cell division, polarization and differentiation. Asters move either towards the cell center or away from it. Here, we focus on the centering mechanism in a membrane independent system of Xenopus cytoplasmic egg extracts. Using live microscopy and single particle tracking, we find that asters move towards chromatinized DNA structures. The velocity and directionality profiles suggest a random walk with drift directed towards DNA. We have developed a theoretical model that can explain this movement as a result of a gradient of MT length dynamics and MT gliding on immobilized dynein motors. In simulations, the antagonistic action of the motor species on the radial array of MTs leads to a tug-of-war purely due to geometric considerations and aster motility resembles a directed random-walk. Additionally our model predicts that aster velocities do not change greatly with varying initial distance from DNA. The movement of asymmetric asters becomes increasingly super-diffusive with increasing motor density, but for symmetric asters it becomes less super-diffusive. The transition of symmetric asters from superdiffusive to diffusive mobility is the result of number fluctuations in bound motors in the tug-of-war. Overall, our model is in good agreement with experimental data in Xenopus cytoplasmic extracts and predicts novel features of the collective effects of motor-MT interactions.

The formation of the nuclear envelope and the subsequent compartmentalization of the genome is a defining feature of eukaryotes. Traditionally, the nuclear envelope was purely viewed as a physical barrier to preserve genetic material in eukaryotic cells. However, in the last few decades, it has been revealed to be a critical cellular component in controlling gene expression and has been implicated in several human diseases. In cancer, the relevance of the cell nucleus was first reported in the mid-1800s when an altered nuclear morphology was observed in tumor cells. This review aims to give a current and comprehensive view of the role of the nuclear envelope on cancer first by recapitulating the changes of the nuclear envelope during cell division, second, by reviewing the role of the nuclear envelope in cell cycle regulation, signaling, and the regulation of the genome, and finally, by addressing the nuclear envelope link to cell migration and metastasis and its use in cancer progn...

Recent reports show that, after nuclear envelope breakdown, lamin-B, a component of the nuclear lamina in interphase, localizes around the mitotic spindle as a membranous network. How this process occurs, however, and how it influences mitotic spindle morphogenesis is unclear. Here, we develop a computational model based on a continuum description to represent the abundance and location of various molecular species involved during mitosis, and use the model to test a number of hypotheses regarding the formation of the mitotic matrix. Our model illustrates that freely diffusible nuclear proteins can be captured and transported to the spindle poles by minus-end-directed microtubule (MT) motors. Moreover, simulations show that these proteins can be used to build a shell-like region that envelopes the mitotic spindle, which helps to improve the focusing of the mitotic spindle by spatially restricting MT polymerization and limiting the effective diffusion of the free MTs. Simulations als...

The comforting thought that nuclear import was al-Technion-Israel Institute of Technology ways the result of importin ␣ recognizing a short posi-Haifa 32000 tively charged or "classical" NLS, importin ␤ mediating Israel interaction with the pore, and Ran-GTP completing import, was soon dispelled by the finding of a new type of NLS and another receptor in both vertebrates and Importin ␤, once thought to be exclusively a nuclear yeast. In vertebrates, the new NLS, found in hnRNP transport receptor, is emerging as a global regulator A1, was longer, glycine-rich, and not at all positively of diverse cellular functions. Importin ␤ acts positively charged. Its receptor, termed "transportin" by authors in multiple interphase roles: in nuclear import, as a emboldened by the previous receptor, turned out to be chaperone for highly charged nuclear proteins, and as a relative of importin ␤ but, surprisingly, required no a potential motor adaptor for movement along microimportin ␣-like adaptor subunit (Pollard et al., 1996; tubules. In contrast, importin ␤ plays a negative regu-Aitchison et al., 1996). Furthermore, it was discovered latory role in mitotic spindle assembly, centrosome that even importin ␤ can bind certain cargoes on its own dynamics, nuclear membrane formation, and nuclear without an adaptor (Gorlich and Kutay, 1999). Already pore assembly. In most of these, importin ␤ is counterthe rules were being broken. acted by its regulator, Ran-GTP. In light of this, the Before long, importin ␤ had more relatives and adaprecent discovery of Ran's involvement in spindle tors than could be accommodated by even the most checkpoint control suggested a potential new arena creative wordsmith (snurportin being the possible highfor importin ␤ action, although it is also possible that light of this effort) (Gorlich et al., 1997; Huber et al., one of importin ␤'s relatives, the karyopherin family 1998). The importin ␤ superfamily of nuclear transport of proteins, manages this checkpoint. Lastly, importin receptors, also termed the "karyopherin" family, now ␤ plays a role in transducing damage signals from the includes 14 members in yeast and more than 20 in huaxons of injured neurons back to the cell body. mans (Table 1) (for reviews, see Gorlich and Kutay, 1999; Macara, 2001; Fried and Kutay, 2003; Damelin et al., When scientists coined the name "importin" for the first 2002; Mosammaparast and Pemberton, 2004; elegant nuclear transport receptor (Gorlich et al., 1994), some yeast studies are covered in more detail in the latter two readers were put off by a name which sounded a bit reviews). All have in common an N-terminal Ran binding too "important." Now it appears a prescient choice, as domain. Many direct the import of various cargo but, new roles for the prototype nuclear import receptor ␣ surprisingly, others direct nuclear export. and ␤ emerge. The first identified export receptor, exportin 1 or Crm1, recognized the now canonical leucine-rich nuclear ex-Nuclear Import: What Did They Know port signal (NES) found in many proteins from yeast to and When Did They Know It? vertebrates (Fornerod et al., 1997a; Stade et al., 1997). To set the stage for understanding importin ␤'s new These include everything from shuttling transcription roles, a review of its central role in nuclear import helps factors to the viral proteins HIV Rev and HTLV Rex (Gorgreatly. Virtually all communication between the nucleus lich and Kutay, 1999; Mosammaparast and Pemberton, and cytoplasm occurs through the massive macromo-2004). Indeed, the general rule appears to be that a lecular structure perforating the double nuclear memprotein that shuttles between nucleus and cytoplasm branes, the nuclear pore. With the advent of in vitro usually contains an NLS that binds to an importin and nuclear import assays, three essential proteins were disan NES that binds to an exportin. Other receptor assigncovered to mediate nuclear import: importin ␣, which ments abound: exportin-t ferries newly synthesized recognizes cargo proteins with a nuclear localization tRNA out of the nucleus, while exportin 6 banishes prosignal (NLS); importin ␤, which binds and ferries the filin/actin complexes from the nucleus (Table 1) (Fried complex into the nucleus; and the small regulatory and Kutay, 2003; Stuven et al., 2003). GTPase Ran. Specifically, when the ␣/␤/NLS cargo com-There are exceptions to this importin ␤ family-oriented plex reaches the far side of the pore, nuclear Ran-GTP program. Messenger RNA appears to use an entirely binds to importin ␤ and dissociates the complex, comdifferent dimer of proteins to exit the nucleus (TAP/ pleting import (Figure 1A). Genetic studies in yeast were NXF1/Mex67 and p15/NXT/ Mtr2; Cole, 2001; Reed and instrumental in identifying homologous transport recep-Hurt, 2002; Cullen, 2003; Stutz and Izaurralde, 2003; tors that act in an identical manner. Mechanistically Weis, 2003). Even individual proteins can break the rules: for example, ␤ catenin and HIV Vpr interact directly with the nuclear pore during their passage (Gorlich and Ku

By means of the yeast two-hybrid system, we have discovered a novel physical interaction between the adenovirus E1A oncoprotein and Ran, a small GTPase which regulates nucleocytoplasmic transport, cell cycle progression, and mitotic spindle organization. Expression of E1A elicits induction of S phase and centrosome amplification in a variety of rodent cell lines. The induction of supernumerary centrosomes requires functional RCC1, the nucleotide exchange factor for Ran and, hence, a functional Ran network. The E1A portion responsible for the interaction with Ran is the extreme NH2-terminal region (amino acids 1–36), which is also required for the induction of centrosome amplification. In an in vitro assay with recombinant proteins, wild-type E1A interferes with nucleotide exchange on Ran, whereas an E1A mutant, deleted from the extreme NH2terminal region, does not. In addition, we detected an in vitro interaction between Ran and HPV-16 E7 and SV40 large T antigen, two oncoproteins f...