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.     

A Rae1-containing ribonucleoprotein complex is required for mitotic spindle assembly

Centrosome-independent microtubule polymerization around chromosomes has been shown to require a local gradient of RanGTP, which discharges mitotic cargoes from the nuclear import receptor importin beta. Here, we have used an activity-based assay in Xenopus egg extracts to purify the mRNA export protein Rae1 as a spindle assembly factor regulated by this pathway. Rae1 is a microtubule-associated protein that binds directly to importin beta. Depletion of Rae1 from extracts or cells severely inhibits mitotic spindle assembly. A purified Rae1 complex stabilizes microtubules in egg extracts in a RanGTP/importin beta-regulated manner. Interestingly, Rae1 exists in a large ribonucleoprotein complex, which requires RNA for its activity to control microtubule dynamics in vitro. Furthermore, we provide evidence that RNA associates with the mitotic spindle and that it plays a direct, translation-independent role in spindle assembly. Our studies reveal an unexpected function for RNA in spindle morphogenesis.     

The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts

Chromosome condensation is required for the physical resolution and segregation of sister chromatids during cell division, but the precise role of higher order chromatin structure in mitotic chromosome functions is unclear. Here, we address the role of the major condensation machinery, the condensin complex, in spindle assembly and function in Xenopus laevis egg extracts. Immunodepletion of condensin inhibited microtubule growth and organization around chromosomes, reducing the percentage of sperm nuclei capable of forming spindles, and causing dramatic defects in anaphase chromosome segregation. Although the motor CENP-E was recruited to kinetochores pulled poleward during anaphase, the disorganized chromosome mass was not resolved. Inhibition of condensin function during anaphase also inhibited chromosome segregation, indicating its continuous requirement. Spindle assembly around DNA-coated beads in the absence of kinetochores was also impaired upon condensin inhibition. These results support an important role for condensin in establishing chromosomal architecture necessary for proper spindle assembly and chromosome segregation.     

Nek7 kinase is enriched at the centrosome, and is required for proper spindle assembly and mitotic progression

Members of the NIMA-related kinases (NRK) family are recently emerging as central regulators of various aspects of the cell cycle. However, the cellular roles of the mammalian NRK, Nek7, remain obscure. We show here that the endogenous Nek7 protein is enriched at the centrosome in a microtubule-independent manner. Overexpression of wt or kinase-defective Nek7 resulted in cells of rounder appearance, and higher proportions of multinuclear and apoptotic cells. Down-regulation of Nek7 using a small interfering RNA approach resulted in a significant increase in mitotic cells presenting multipolar spindle phenotype. These results suggest a role for Nek7 in regulating proper spindle assembly and mitotic progression.     

Human Cep192 is required for mitotic centrosome and spindle assembly

As cells enter mitosis, centrosomes dramatically increase in size and ability to nucleate microtubules. This process, termed centrosome maturation, is driven by the accumulation and activation of gamma-tubulin and other proteins that form the pericentriolar material on centrosomes during G2/prophase. Here, we show that the human centrosomal protein, Cep192 (centrosomal protein of 192 kDa), is an essential component of the maturation machinery. Specifically, we have found that siRNA depletion of Cep192 results in a complete loss of functional centrosomes in mitotic but not interphase cells. In mitotic cells lacking Cep192, microtubules become organized around chromosomes but rarely acquire stable bipolar configurations. These cells contain normal numbers of centrioles but cannot assemble gamma-tubulin, pericentrin, or other pericentriolar proteins into an organized PCM. Alternatively, overexpression of Cep192 results in the formation of multiple, extracentriolar foci of gamma-tubulin and pericentrin. Together, our findings support the hypothesis that Cep192 stimulates the formation of the scaffolding upon which gamma-tubulin ring complexes and other proteins involved in microtubule nucleation and spindle assembly become functional during mitosis.     

Nucleophosmin is required for chromosome congression, proper mitotic spindle formation, and kinetochore-microtubule attachment in HeLa cells

Nucleophosmin (NPM) is an abundantly expressed multifunctional nucleolar phosphoprotein. Here we show that depletion of NPM by RNA interference causes defects in cell division, followed by an arrest of DNA synthesis due to activation of a p53-dependent checkpoint response in HeLa cells. Depletion of NPM leads to mitotic arrest due to spindle checkpoint activation. The mitotic cells arrested by NPM depletion have defects in chromosome congression, proper mitotic spindle and centrosome formation, as well as defects in kinetochore-microtubule attachments. Loss of NPM thus causes severe mitotic defects and delayed mitotic progression. These findings indicate that NPM is essential for mitotic progression and cell proliferation.     

Kinesin-related proteins required for assembly of the mitotic spindle

We identified two new Saccharomyces cerevisiae kinesin-related genes, KIP1 and KIP2, using polymerase chain reaction primers corresponding to highly conserved regions of the kinesin motor domain. Both KIP proteins are expressed in vivo, but deletion mutations conferred no phenotype. Moreover, kip1 kip2 double mutants and a triple mutant with kinesin-related kar3 had no synthetic phenotype. Using a genetic screen for mutations that make KIP1 essential, we identified another gene, KSL2, which proved to be another kinesin-related gene, CIN8. KIP1 and CIN8 are functionally redundant: double mutants arrested in mitosis whereas the single mutants did not. The microtubule organizing centers of arrested cells were duplicated but unseparated, indicating that KIP1 or CIN8 is required for mitotic spindle assembly. Consistent with this role, KIP1 protein was found to colocalize with the mitotic spindle.     

MISP is a novel Plk1 substrate required for proper spindle orientation and mitotic progression

Precise positioning of the mitotic spindle determines the correct cell division axis and is crucial for organism development. Spindle positioning is mediated through a cortical machinery by capturing astral microtubules, thereby generating pushing/pulling forces at the cell cortex. However, the molecular link between these two structures remains elusive. Here we describe a previously uncharacterized protein, MISP (C19orf21), as a substrate of Plk1 that is required for correct mitotic spindle positioning. MISP is an actin-associated protein throughout the cell cycle. MISP depletion led to an impaired metaphase-to-anaphase transition, which depended on phosphorylation by Plk1. Loss of MISP induced mitotic defects including spindle misorientation accompanied by shortened astral microtubules. Furthermore, we find that MISP formed a complex with and regulated the cortical distribution of the +TIP binding protein p150^glued, a subunit of the dynein–dynactin complex. We propose that Plk1 phosphorylates MISP, thus stabilizing cortical and astral microtubule attachments required for proper mitotic spindle positioning.     

The transition from meiotic to mitotic spindle assembly is gradual during early mammalian development

The transition from meiosis to mitosis, classically defined by fertilization, is a fundamental process in development. However, its mechanism remains largely unexplored. In this paper, we report a surprising gradual transition from meiosis to mitosis over the first eight divisions of the mouse embryo. The first cleavages still largely share the mechanism of spindle formation with meiosis, during which the spindle is self-assembled from randomly distributed microtubule-organizing centers (MTOCs) without centrioles, because of the concerted activity of dynein and kinesin-5. During preimplantation development, the number of cellular MTOCs progressively decreased, the spindle pole gradually became more focused, and spindle length progressively scaled down with cell size. The typical mitotic spindle with centrin-, odf2-, kinesin-12-, and CP110-positive centrosomes was established only in the blastocyst. Overall, the transition from meiosis to mitosis progresses gradually throughout the preimplantation stage in the mouse embryo, thus providing a unique system to study the mechanism of centrosome biogenesis in vivo.     

The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation

During cell division, kinetochores form the primary chromosomal attachment sites for spindle microtubules. We previously identified a network of 10 interacting kinetochore proteins conserved between Caenorhabditis elegans and humans. In this study, we investigate three proteins in the human network (hDsn1Q9H410, hNnf1PMF1, and hNsl1DC31). Using coexpression in bacteria and fractionation of mitotic extracts, we demonstrate that these proteins form a stable complex with the conserved kinetochore component hMis12. Human or chicken cells depleted of Mis12 complex subunits are delayed in mitosis with misaligned chromosomes and defects in chromosome biorientation. Aligned chromosomes exhibited reduced centromere stretch and diminished kinetochore microtubule bundles. Consistent with this, localization of the outer plate constituent Ndc80HEC1 was severely reduced. The checkpoint protein BubR1, the fibrous corona component centromere protein (CENP) E, and the inner kinetochore proteins CENP-A and CENP-H also failed to accumulate to wild-type levels in depleted cells. These results indicate that a four-subunit Mis12 complex plays an essential role in chromosome segregation in vertebrates and contributes to mitotic kinetochore assembly.     

The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast

Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation. In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome. In the yeast Saccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked. We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome. Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome. Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well. In the doa4Delta mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase. Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Delta cells. This loss of viability and several other defects of doa4Delta cells are rescued by provision of additional ubiquitin. Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells. Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis. We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole.     

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.     

Myosin II-dependent cortical movement is required for centrosome separation and positioning during mitotic spindle assembly

The role of myosin II in mitosis is generally thought to be restricted to cytokinesis. We present surprising new evidence that cortical myosin II is also required for spindle assembly in cells. Drug- or RNAi-mediated disruption of myosin II in cells interferes with normal spindle assembly and positioning. Time-lapse movies reveal that these treatments block the separation and positioning of duplicated centrosomes after nuclear envelope breakdown (NEBD), thereby preventing the migration of the microtubule asters to opposite sides of chromosomes. Immobilization of cortical movement with tetravalent lectins produces similar spindle defects to myosin II disruption and suggests that myosin II activity is required within the cortex. Latex beads bound to the cell surface move in a myosin II-dependent manner in the direction of the separating asters. We propose that after NEBD, completion of centrosome separation and positioning around chromosomes depends on astral microtubule connections to a moving cell cortex.     

Drosophila EB1 is important for proper assembly,dynamics, and positioning of the mitotic spindle

EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 cell line that promotes cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the cell cortex.     

The chromokinesin Kid is required for maintenance of proper metaphase spindle size

The human chromokinesin Kid/kinesin-10, a plus end-directed microtubule (MT)-based motor with both microtubule- and DNA-binding domains, is required for proper chromosome alignment at the metaphase plate. Here, we performed RNA interference experiments to deplete endogenous Kid from HeLa cells and confirmed defects in metaphase chromosome arm alignment in Kid-depleted cells. In addition, we noted a shortening of the spindle length, resulting in a pole-to-pole distance only 80% of wild type. The spindle microtubule-bundles with which Kid normally colocalize became less robust. Rescue of the two Kid deficiency phenotypes-imprecise chromosome alignment at metaphase and shortened spindles- exhibited distinct requirements. Mutants lacking either the DNA-binding domain or the MT motor ATPase failed to rescue the former defect, whereas rescue of the shortened spindle phenotype required neither activity. Kid also exhibits microtubule bundling activity in vitro, and rescue of the shortened spindle phenotype and the bundling activity displayed similar domain requirements, except that rescue required a coiled-coil domain not needed for bundling. These results suggest that distinct from its role in chromosome movement, Kid contributes to spindle morphogenesis by mediating spindle microtubules stabilization.     

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

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

BRCA1 is required for meiotic spindle assembly and spindle assembly checkpoint activation in mouse oocytes

BRCA1 as a tumor suppressor has been widely investigated in mitosis, but its functions in meiosis are unclear. In the present study, we examined the expression, localization, and function of BRCA1 during mouse oocyte meiotic maturation. We found that expression level of BRCA1 was increased progressively from germinal vesicle to metaphase I stage, and then remained stable until metaphase II stage. Immunofluorescent analysis showed that BRCA1 was localized to the spindle poles at metaphase I and metaphase II stages, colocalizing with centrosomal protein gamma-tubulin. Taxol treatment resulted in the presence of BRCA1 onto the spindle microtubule fibers, whereas nocodazole treatment induced the localization of BRCA1 onto the chromosomes. Depletion of BRCA1 by both antibody injection and siRNA injection caused severely impaired spindles and misaligned chromosomes. Furthermore, BRCA1-depleted oocytes could not arrest at the metaphase I in the presence of low-dose nocodazole, suggesting that the spindle checkpoint is defective. Also, in BRCA1-depleted oocytes, gamma-tubulin dissociated from spindle poles and MAD2L1 failed to rebind to the kinetochores when exposed to nocodazole at metaphase I stage. Collectively, these data indicate that BRCA1 regulates not only meiotic spindle assembly, but also spindle assembly checkpoint, implying a link between BRCA1 deficiency and aneuploid embryos.     

Amphiastral mitotic spindle assembly in vertebrate cells lacking centrosomes

The role of centrosomes and centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays--termed an amphiaster ("a star on both sides")--that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB). Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC reassembled, and, prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ~35% of aMTOCs failed to split and separate before NEB, and these entered mitosis with persistent monastral spindles. Chromatin-associated RAN-GTP--the small GTPase Ran in its GTP bound state--could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but, in its absence, the fidelity of bipolar spindle assembly is highly compromised.     

Gamma-tubulin is required for bipolar spindle assembly and for proper kinetochore microtubule attachments during prometaphase I in Drosophila oocytes

In many animal species the meiosis I spindle in oocytes is anastral and lacks centrosomes. Previous studies of Drosophila oocytes failed to detect the native form of the germline-specific γ-tubulin (γTub37C) in meiosis I spindles, and genetic studies have yielded conflicting data regarding the role of γTub37C in the formation of bipolar spindles at meiosis I. Our examination of living and fixed oocytes carrying either a null allele or strong missense mutation in the γtub37C gene demonstrates a role for γTub37C in the positioning of the oocyte nucleus during late prophase, as well as in the formation and maintenance of bipolar spindles in Drosophila oocytes. Prometaphase I spindles in γtub37C mutant oocytes showed wide, non-tapered spindle poles and disrupted positioning. Additionally, chromosomes failed to align properly on the spindle and showed morphological defects. The kinetochores failed to properly co-orient and often lacked proper attachments to the microtubule bundles, suggesting that γTub37C is required to stabilize kinetochore microtubule attachments in anastral spindles. Although spindle bipolarity was sometimes achieved by metaphase I in both γtub37C mutants, the resulting chromosome masses displayed highly disrupted chromosome alignment. Therefore, our data conclusively demonstrate a role for γTub37C in both the formation of the anastral meiosis I spindle and in the proper attachment of kinetochore microtubules. Finally, multispectral imaging demonstrates the presences of native γTub37C along the length of wild-type meiosis I spindles.