Ran-GTP regulates Kinetochore Attachment in Somatic Cells

The Ran GTPase controls multiple mitotic processes in Xenopus egg extracts, including mitotic checkpoints, spindle assembly and post-mitotic nuclear envelope reassembly. We have analyzed Ran’s role in somatic cells. We uncovered a novel mitotic role of Ran-GTP, involving the Crm1 nuclear export receptor. This pathway is an important mode of Ran-GTP function during mitosis in mammalian somatic cells, whichmediates the recruitment of the RanGAP1/RanBP2 complex to kinetochores and maintains the microtubule-based fibers connecting kinetochores to spindle poles (kfibers). Here we discuss potential implications of these findings for normal k-fiber assembly.     

Targeting of RCC1 to chromosomes is required for proper mitotic spindle assembly in human cells

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. Here, we have examined the localization and function of RCC1 in mitotic human cells. We show that 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.     

Loading and Unloading: Orchestrating Centrosome Duplication and Spindle Assembly by Ran/Crm1

The cell cycle is an intricate process of DNA replication and cell division thatconcludes with the formation of two genetically equivalent daughter cells. In thisprogression, the centrosome is duplicated once and only once during the G1/S transitionto produce the bipolar spindle and ensure proper chromosome segregation. The presenceof multiple centrosomes in cancer cells suggests that this process is mis-regulated duringcarcinogenesis. This suggests that certain factors exist that license the progression ofcentrosome duplication and serve to inhibit further duplications during a single cell cycle.Recent studies suggest that the Ran/Crm1 complex not only regulates nucleocytoplasmictransport but is also independently involved in mitotic spindle assembly. Factors that arecapable of interacting with Ran/Crm1 through their nuclear export sequences, such ascyclins/cdks, p53 and Brca1/2, may potentially function as centrosome checkpoints akinto the G1/S and G2/M checkpoints of the cell cycle. Our recent findings indicate thatnucleophosmin, a protein whose trafficking is mediated by the Ran/Crm1 network, is oneof such checkpoint factors for maintaining proper centrosome duplication. We proposethat Ran/Crm1 may act as a ‘loading dock’ to coordinate various checkpoint factors inregulating the fidelity of centrosome duplication during cell cycle progression, and thedisruption of these processes may lead to genomic instability and an acceleration ofoncogenesis.     

Identification of an NTF2-related factor that binds Ran-GTP and regulates nuclear protein export

Active transport of macromolecules between the nucleus and cytoplasm requires signals for import and export and their recognition by shuttling receptors. Each class of macromolecule is thought to have a distinct receptor that mediates the transport reaction. Assembly and disassembly reactions of receptor-substrate complexes are coordinated by Ran, a GTP-binding protein whose nucleotide state is regulated catalytically by effector proteins. Ran function is modulated in a noncatalytic fashion by NTF2, a protein that mediates nuclear import of Ran-GDP. Here we characterize a novel component of the Ran system that is 26% identical to NTF2, which based on its function we refer to as NTF2-related export protein 1 (NXT1). In contrast to NTF2, NXT1 preferentially binds Ran-GTP, and it colocalizes with the nuclear pore complex (NPC) in mammalian cells. These properties, together with the fact that NXT1 shuttles between the nucleus and the cytoplasm, suggest an active role in nuclear transport. Indeed, NXT1 stimulates nuclear protein export of the NES-containing protein PKI in vitro. The export function of NXT1 is blocked by the addition of leptomycin B, a compound that selectively inhibits the NES receptor Crm1. Thus, NXT1 regulates the Crm1-dependent export pathway through its direct interaction with Ran-GTP.     

Localized RanGTP accumulation promotes microtubule nucleation at kinetochores in somatic mammalian cells

Centrosomes are the major sites for microtubule nucleation in mammalian cells, although both chromatin- and kinetochore-mediated microtubule nucleation have been observed during spindle assembly. As yet, it is still unclear whether these pathways are coregulated, and the molecular requirements for microtubule nucleation at kinetochore are not fully understood. This work demonstrates that kinetochores are initial sites for microtubule nucleation during spindle reassembly after nocodazole. This process requires local RanGTP accumulation concomitant with delocalization from kinetochores of the hydrolysis factor RanGAP1. Kinetochore-driven microtubule nucleation is also activated after cold-induced microtubule disassembly when centrosome nucleation is impaired, e.g., after Polo-like kinase 1 depletion, indicating that dominant centrosome activity normally masks the kinetochore-driven pathway. In cells with unperturbed centrosome nucleation, defective RanGAP1 recruitment at kinetochores after treatment with the Crm1 inhibitor leptomycin B activates kinetochore microtubule nucleation after cold. Finally, nascent microtubules associate with the RanGTP-regulated microtubule-stabilizing protein HURP in both cold- and nocodazole-treated cells. These data support a model for spindle assembly in which RanGTP-dependent abundance of nucleation/stabilization factors at centrosomes and kinetochores orchestrates the contribution of the two spindle assembly pathways in mammalian cells. The complex of RanGTP, the export receptor Crm1, and nuclear export signal-bearing proteins regulates microtubule nucleation at kinetochores.     

The Nup107-160 nucleoporin complex is required for correct bipolar spindle assembly

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, we stained human cells 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. We conclude 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.     

Centromere localization of INCENP-Aurora B is sufficient to support spindle checkpoint function

During mitosis, the chromosomal passenger complex (CPC) comprising the Aurora B kinase, INCENP, survivin and borealin is essential for correcting non-bipolar chromosome attachments and for cytokinesis. In addition, the CPC might fullfil a role in the mitotic spindle assembly checkpoint (SAC), but this activity might be related to its role in correcting non-bipolar chromosome attachments. Here, we demonstrate that treatment of mitotic cells with the antibiotic actinomycin D causes a displacement of an intact and active CPC from centromeres onto chromosome arms, which results in chromosome misalignment, cytokinesis failure and SAC override, but still preserves histone H3 phosphorylation on chromosome arms. This surprising and unique scenario allows the reconstitution of endogenous Aurora B at centromeres/inner kinetochores by expressing a Cenp-B-INCENP fusion protein. We find that although the selective recruitment of endogenous Aurora B to centromeres/inner kinetochores is not sufficient to restore chromosome alignment and cytokinesis, it can restore Cenp-A phosphorylation at kinetochores, BubR1 recruitment to kinetochores and SAC activity after spindle disruption. These results indicate that INCENP-Aurora B localized at centromeres/inner kinetochores is sufficient to mediate SAC activity upon spindle disruption.     

A survivin-ran complex regulates spindle formation in tumor cells

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, survivin is a novel effector of Ran signaling, and this pathway may be preferentially exploited for spindle assembly in tumor cells.     

Ran-GTP coordinates regulation of microtubule nucleation and dynamics during mitotic-spindle assembly

It was recently reported that 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. Here we examine the effects of Ran-GTP on microtubule nucleation and dynamics and show that Ran-GTP has independent effects on both the nucleation activity of centrosomes and the stability of centrosomal microtubules. We also show 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.     

The methylated N-terminal tail of RCC1 is required for stabilisation of its interaction with chromatin by Ran in live cells

Background  

Regulator of chromosome condensation 1 (RCC1) is the guanine nucleotide exchange factor for Ran GTPase. Localised generation of Ran-GTP by RCC1 on chromatin is critical for nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Both the N-terminal tail of RCC1 and its association with Ran are important for its interaction with chromatin in cells. In vitro, the association of Ran with RCC1 induces a conformational change in the N-terminal tail that promotes its interaction with DNA.     

The mammalian passenger protein TD-60 is an RCC1 family member with an essential role in prometaphase to metaphase progression

Passenger proteins migrate from inner centromeres to the spindle midzone during late mitosis, and those described to date are essential both for proper chromosome segregation and for completion of cell cleavage. We have purified and cloned the human passenger protein TD-60, and we here report that it is a member of the RCC1 family and that it binds preferentially the nucleotide-free form of the small G protein Rac1. Using siRNA, we further demonstrate that the absence of TD-60 substantially suppresses overall spindle assembly, blocks cells in prometaphase, and activates the spindle assembly checkpoint. These defects suggest TD-60 may have a role in global spindle assembly or may be specifically required to integrate kinetochores into the mitotic spindle. The latter is consistent with a TD-60 requirement for recruitment of the passenger proteins survivin and Aurora B, and suggests that like other passenger proteins, TD-60 is involved in regulation of cell cleavage.     

Roles of Ran-GTP and Ran-GDP in precursor vesicle recruitment and fusion during nuclear envelope assembly in a human cell-free system

The molecular mechanism of nuclear envelope (NE) assembly is poorly understood, but in a cell-free system made from Xenopus eggs NE assembly is controlled by the small GTPase Ran [1,2]. In this system, Sepharose beads coated with Ran induce the formation of functional NEs in the absence of chromatin [1]. Both generation of Ran-GTP by the guanine nucleotide exchange factor RCC1 and GTP hydrolysis by Ran are required for NE assembly, although the roles of the GDP- and GTP-bound forms of Ran in the recruitment of precursor vesicles and their fusion have been unclear. We now show that beads coated with either Ran-GDP or Ran-GTP assemble functional nuclear envelopes in a cell-free system derived from mitotic human cells, forming pseudo-nuclei that actively transport proteins across the NE. Both RCC1 and the GTPase-activating protein RanGAP1 are recruited to the beads, allowing interconversion between Ran-GDP and Ran-GTP. However, addition of antibodies to RCC1 and RanGAP1 shows that Ran-GDP must be converted to Ran-GTP by RCC1 before precursor vesicles are recruited, whereas GTP hydrolysis by Ran stimulated by RanGAP1 promotes vesicle recruitment and is necessary for vesicle fusion to form an intact envelope. Thus, the GTP-GDP cycle of Ran controls both the recruitment of vesicles and their fusion to form NEs.     

From the Nuclear Pore to the Fibrous Corona: A MAD Journey to Preserve Genome Stability

The relationship between kinetochores and nuclear pore complexes (NPCs) is intimate but poorly understood. Several NPC components and associated proteins are relocated to mitotic kinetochores to assist in different activities that ensure faithful chromosome segregation. Such is the case of the Mad1-c-Mad2 complex, the catalytic core of the spindle assembly checkpoint (SAC), a surveillance pathway that delays anaphase until all kinetochores are attached to spindle microtubules. Mad1-c-Mad2 is recruited to discrete domains of unattached kinetochores from where it promotes the rate-limiting step in the assembly of anaphase-inhibitory complexes. SAC proficiency further requires Mad1-c-Mad2 to be anchored at NPCs during interphase. However, the mechanistic relevance of this arrangement for SAC function remains ill-defined. Recent studies uncover the molecular underpinnings that coordinate the release of Mad1-c-Mad2 from NPCs with its prompt recruitment to kinetochores. Here, current knowledge on Mad1-c-Mad2 function and spatiotemporal regulation is reviewed and the critical questions that remain unanswered are highlighted.     

Induced ectopic kinetochore assembly bypasses the requirement for CENP-A nucleosomes

Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. Although prior work identified the centromeric histone H3-variant CENP-A as the important upstream factor necessary for centromere specification, in human cells CENP-A is not sufficient for kinetochore assembly. Here, we demonstrate that two constitutive DNA-binding kinetochore components, CENP-C and CENP-T, function to direct kinetochore formation. Replacing the DNA-binding regions of CENP-C and CENP-T with alternate chromosome-targeting domains recruits these proteins to ectopic loci, resulting in CENP-A-independent kinetochore assembly. These ectopic kinetochore-like foci are functional based on the stoichiometric assembly of multiple kinetochore components, including the microtubule-binding KMN network, the presence of microtubule attachments, the microtubule-sensitive recruitment of the spindle checkpoint protein Mad2, and the segregation behavior of foci-containing chromosomes. We additionally find that CENP-T phosphorylation regulates the mitotic assembly of both endogenous and ectopic kinetochores. Thus, CENP-C and CENP-T form a critical regulated platform for vertebrate kinetochore assembly.     

GTP酶Ran在细胞核运输、核分裂与重建中的作用

小分子的单体G蛋白Ran具有鸟苷三磷酸酶活性,其结合形式Ran-GTP作为区分间期细胞的核质和胞质的一个分子标记,并参与调控核质运输、指导纺锤体形成以及引导核膜解体与装配。现就Ran在真核细胞核质运输、有丝分裂纺锤体组装与核膜动力学中的功能作一综述。     

Mutual Dependence of Mob1 and the Chromosomal Passenger Complex for Localization during Mitosis

The spatial and temporal coordination of chromosome segregation with cytokinesis is essential to ensure that each daughter cell receives the correct complement of chromosomal and cytoplasmic material. In yeast, mitotic exit and cytokinesis are coordinated by signaling cascades whose terminal components include a nuclear Dbf2-related family kinase and a noncatalytic subunit, Mps one binding (Mob) 1. There are five human Mob1 isoforms, all of which display redundant localization patterns at the spindle poles and kinetochores in early mitosis, and the spindle midzone during cytokinesis. Mob1 shares similar localization patterns to Polo-like kinase (Plk1) and the chromosomal passenger complex (CPC), and although depletion of Plk1 resulted in a loss of Mob1 from the spindle poles, Mob1 recruitment to kinetochores was unaffected. Conversely, disruption of CPC signaling resulted in a loss of Mob1 from kinetochores without disrupting recruitment to the spindle poles. In Mob1-depleted cells, the relocalization of the CPC and mitotic kinesin-like protein (MKLP) 2 to the spindle midzone was delayed during early anaphase, and as a consequence, the midzone recruitment of MKLP1 also was affected. Together, these results suggest that Mob1 and the other mammalian orthologues of the mitotic exit network regulate mitotic progression by facilitating the timely mobilization of the CPC to the spindle midzone.     

The GTPase Ran regulates chromosome positioning and nuclear envelope assembly in vivo

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. Here we 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.     

The small GTPase Ran: interpreting the signs

The small GTPase Ran has roles in nuclear transport, mitotic spindle assembly and nuclear envelope assembly. During the past three years, it has become clear that many of these processes rely on conserved molecular mechanisms involving Ran-GTP-binding proteins of the importin-beta superfamily. Moreover, recent experimental evidence has documented the distribution of Ran-GTP within cells and supported the notion that Ran plays a central role in the spatial and temporal organization of the eukaryotic cell.     

NXT1 is necessary for the terminal step of Crm1-mediated nuclear export

Soluble factors are required to mediate nuclear export of protein and RNA through the nuclear pore complex (NPC). These soluble factors include receptors that bind directly to the transport substrate and regulators that determine the assembly state of receptor-substrate complexes. We recently reported the identification of NXT1, an NTF2-related export factor that stimulates nuclear protein export in permeabilized cells and undergoes nucleocytoplasmic shuttling in vivo (Black, B.E., L. Lévesque, J.M. Holaska, T.C. Wood, and B.M. Paschal. 1999. Mol. Cell. Biol. 19:8616-8624). Here, we describe the molecular characterization of NXT1 in the context of the Crm1-dependent export pathway. We find that NXT1 binds directly to Crm1, and that the interaction is sensitive to the presence of Ran-GTP. Moreover, mutations in NXT1 that reduce binding to Crm1 inhibit the activity of NXT1 in nuclear export assays. We show that recombinant Crm1 and Ran are sufficient to reconstitute nuclear translocation of a Rev reporter protein from the nucleolus to an antibody accessible site on the cytoplasmic side of the NPC. Further progress on the export pathway, including the terminal step of Crm1 and Rev reporter protein release, requires NXT1. We propose that NXT1 engages with the export complex in the nucleoplasm, and that it facilitates delivery of the export complex to a site on the cytoplasmic side of NPC where the receptor and substrate are released into the cytoplasm.     

Phosphorylation regulates the dynamic interaction of RCC1 with chromosomes during mitosis

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