Differential regulation of centrosome integrity by DNA damage response proteins

MDC1 and BRIT1 have been shown to function as key regulators in response to DNA damage. However, their roles in centrosomal regulation haven’t been elucidated. In this study, we demonstrated the novel functions of these two molecules in regulating centrosome duplication and mitosis. We found that MDC1 and BRIT1 were integral components of the centrosome that colocalize with γ-tubulin. Depletion of either protein led to centrosome amplification. However, the mechanisms that allow them to maintain centrosome integrity are different. MDC1-depleted cells exhibited centrosome overduplication, leading to multipolar mitosis, chromosome missegregation, and aneuploidy, whereas BRIT1 depletion led to misaligned spindles and/or lagging chromosomes with defective spindle checkpoint activation that resulted in defective cytokinesis and polyploidy. We further illustrated that both MDC1 and BRIT1 were negative regulators of Aurora A and Plk1, two centrosomal kinases involved in centrosome maturation and spindle assembly. Moreover, the levels of MDC1 and BRIT1 inversely correlated with centrosome amplification, defective mitosis, and cancer metastasis in human breast cancer. Together, MDC1 and BRIT1 may function as tumor-suppressor genes, at least in part by orchestrating proper centrosome duplication and mitotic spindle assembly.     

Mad2-independent spindle assembly checkpoint activation and controlled metaphase-anaphase transition in Drosophila S2 cells

The spindle assembly checkpoint is essential to maintain genomic stability during cell division. We analyzed the role of the putative Drosophila Mad2 homologue in the spindle assembly checkpoint and mitotic progression. Depletion of Mad2 by RNAi from S2 cells shows that it is essential to prevent mitotic exit after spindle damage, demonstrating its conserved role. Mad2-depleted cells also show accelerated transit through prometaphase and premature sister chromatid separation, fail to form metaphases, and exit mitosis soon after nuclear envelope breakdown with extensive chromatin bridges that result in severe aneuploidy. Interestingly, preventing Mad2-depleted cells from exiting mitosis by a checkpoint-independent arrest allows congression of normally condensed chromosomes. More importantly, a transient mitotic arrest is sufficient for Mad2-depleted cells to exit mitosis with normal patterns of chromosome segregation, suggesting that all the associated phenotypes result from a highly accelerated exit from mitosis. Surprisingly, if Mad2-depleted cells are blocked transiently in mitosis and then released into a media containing a microtubule poison, they arrest with high levels of kinetochore-associated BubR1, properly localized cohesin complex and fail to exit mitosis revealing normal spindle assembly checkpoint activity. This behavior is specific for Mad2 because BubR1-depleted cells fail to arrest in mitosis under these experimental conditions. Taken together our results strongly suggest that Mad2 is exclusively required to delay progression through early stages of prometaphase so that cells have time to fully engage the spindle assembly checkpoint, allowing a controlled metaphase-anaphase transition and normal patterns of chromosome segregation.     

Sirt3 controls chromosome alignment by regulating spindle dynamics during mitosis

Sirt3, one of mammalian sirtuins is a prominent mitochondrial deacetylase that controls mitochondrial oxidative pathways and the rate of reactive oxygen species. Sirt3 also regulates energy metabolism by deacetylating enzymes involved in the metabolic pathway related with lifespan. We report here a novel function of Sirt3 which was found to be involved in mitosis. Depletion of the Sirt3 protein generated unaligned chromosomes in metaphase which caused mitotic arrest by activating spindle assembly checkpoint (SAC). Furthermore, the shape and the amount of the spindles in Sirt3 depleted cells were abnormal. Microtubule (MT) polymerization also increased in Sirt3 depleted cells, suggesting that Sirt3 is involved in spindle dynamics. However, the level of acetylated tubulin was not increased significantly in Sirt3 depleted cells. The findings collectively suggest that Sirt3 is not a tubulin deacetylase but regulates the attachment of spindle MTs to the kinetochore and the subsequent chromosome alignment by increasing spindle dynamics.     

A gene required for the separation of chromosomes on the spindle apparatus in yeast

We describe the phenotypes caused by a cold-sensitive lethal mutation (ndc1-1) that defines the NDC1 gene of yeast. Incubation of ndc1-1 at a nonpermissive temperature causes failure of chromosome separation in mitosis but does not block the cell cycle. This defect results in an asymmetric cell division in which one daughter cell doubles in ploidy and the other inherits no chromosomes. The spindle poles are properly segregated to the two daughter cells. The primary visible defect is that the chromosomes remain associated with only one pole, and are thus delivered to one daughter cell. Meiosis II, but not meiosis I, is sensitive to the ndc1-1 defect, suggesting that NDC1 is required for some feature common to mitosis and meiosis II. ndc1-1 appears to define a new class of cell cycle gene required for the attachment of chromosomes to the spindle pole.     

Mitotic spindle pole separation

Eukaryotic cells utilize a microtubular spindle to segregate chromosomes during mitosis. Chromosome segragation requires the timely separation of the mitotic spindle poles to which the chromosomes are attached. Recent studies at the molecular and cellular levels have provided new insights into the mechanism and regulation of this process. On the one hand, the process now seems more complex, as redundant mechanisms apparently overlap in function during cell division. On the other hand, some of these processes may be acting continuously during the various stages of spindle pole separation, suggesting an underlying simplicity.     

Timely anaphase onset requires a novel spindle and kinetochore complex comprising Ska1 and Ska2

Chromosome segregation during mitosis requires chromosomes to undergo bipolar attachment on spindle microtubules (MTs) and subsequent silencing of the spindle checkpoint. Here, we describe the identification and characterisation of a novel spindle and kinetochore (KT)-associated complex that is required for timely anaphase onset. The complex comprises at least two proteins, termed Ska1 (Spindle and KT Associated 1) and Ska2. Ska1 associates with KTs following MT attachment during prometaphase. Ska1 and Ska2 interact with each other and Ska1 is required for Ska2 stability in vivo. Depletion of either Ska1 or Ska2 by small interfering RNA results in the loss of both proteins from the KT. The absence of Ska proteins does not disrupt overall KT structure, but KT fibres show an increased cold-sensitivity. Most strikingly, Ska-depleted cells undergo a prolonged checkpoint-dependent delay in a metaphase-like state. This delay is characterised by the recruitment of Mad2 protein to a few KTs and the occasional loss of individual chromosomes from the metaphase plate. These data suggest that the Ska1/2 complex plays a critical role in the maintenance of the metaphase plate and/or spindle checkpoint silencing.     

Human Cdc14B promotes progression through mitosis by dephosphorylating Cdc25 and regulating Cdk1/cyclin B activity

Entry into and progression through mitosis depends on phosphorylation and dephosphorylation of key substrates. In yeast, the nucleolar phosphatase Cdc14 is pivotal for exit from mitosis counteracting Cdk1-dependent phosphorylations. Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known. Here we show that hCdc14B serves a critical role in regulating progression through mitosis, which is distinct from hCdc14A. Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis. Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation. The results demonstrate that hCdc14B-dependent modulation of Cdc25 phosphatase and Cdk1/cyclin B activity is tightly linked to correct chromosome segregation and bipolar spindle formation, processes that are required for proper progression through mitosis and maintenance of genomic stability.     

AKAP95 interacts with nucleoporin TPR in mitosis and is important for the spindle assembly checkpoint

Faithful chromosome segregation during mitosis relies on a proofreading mechanism that monitors proper kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) is based on the concerted action of numerous components that maintain a repressive signal inhibiting transition into anaphase until all chromosomes are attached. Here we show that A-Kinase Anchoring Protein 95 (AKAP95) is necessary for proper SAC function. AKAP95-depleted HeLa cells show micronuclei formed from lagging chromosomes at mitosis. Using a BioID proximity-based proteomic screen, we identify the nuclear pore complex protein TPR as a novel AKAP95 binding partner. We show interaction between AKAP95 and TPR in mitosis, and an AKAP95-dependent enrichment of TPR in the spindle microtubule area in metaphase, then later in the spindle midzone area. AKAP95-depleted cells display faster prometaphase to anaphase transition, escape from nocodazole-induced mitotic arrest and show a partial delocalization from kinetochores of the SAC component MAD1. Our results demonstrate an involvement of AKAP95 in proper SAC function likely through its interaction with TPR.     

The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint

The proper segregation of sister chromatids in mitosis depends on bipolar attachment of all chromosomes to the mitotic spindle. We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation. Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B. Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous monooriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment). Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h. Together, our data suggest that Aurora B is required to generate unattached kinetochores on monooriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling.     

Phospho-regulation of DDA3 function in mitosis

DDA3 is a microtubule-associated protein that controls chromosome congression and segregation by regulating the mitotic spindle. Depletion of DDA3 alters spindle structure, generates unaligned chromosomes at metaphase, and delays the mitotic progression. Through a mass spectrometry analysis, we found that DDA3 is phosphorylated on Ser225 during mitosis. Phosphorylation of this residue is important for the mitotic function of DDA3, as the phospho-mimicking DDA3-S225D variant, but not the nonphosphorable DDA3-S225A mutant, rescues the DDA3-knockdown phenotype. We conclude that the mitotic function of DDA3 is regulated by phosphorylation on the Ser225 residue.     

Identification of two novel components of the human NDC80 kinetochore complex

Proper kinetochore function is essential for the accurate segregation of chromosomes during mitosis. Kinetochores provide the attachment sites for spindle microtubules and are required for the alignment of chromosomes at the metaphase plate (chromosome congression). Components of the conserved NDC80 complex are required for chromosome congression, and their disruption results in mitotic arrest accompanied by multiple spindle aberrations. To better understand the function of the NDC80 complex, we have identified two novel subunits of the human NDC80 complex, termed human SPC25 (hSPC25) and human SPC24 (hSPC24), using an immunoaffinity approach. hSPC25 interacted with HEC1 (human homolog of yeast Ndc80) throughout the cell cycle and localized to kinetochores during mitosis. RNA interference-mediated depletion of hSPC25 in HeLa cells caused aberrant mitosis, followed by cell death, a phenotype similar to that of cells depleted of HEC1. Loss of hSPC25 also caused multiple spindle aberrations, including elongated, multipolar, and fractured spindles. In the absence of hSPC25, MAD1 and HEC1 failed to localize to kinetochores during mitosis, whereas the kinetochore localization of BUB1 and BUBR1 was largely unaffected. Interestingly, the kinetochore localization of MAD1 in cells with a compromised NDC80 function was restored upon microtubule depolymerization. Thus, hSPC25 is an essential kinetochore component that plays a significant role in proper execution of mitotic events.     

Cohesin is needed for bipolar mitosis in human cells

Multi-polar mitosis is strongly linked with aggressive cancers and it is a histological diagnostic of tumor-grade. However, factors that cause chromosomes to segregate to more than two spindle poles are not well understood. Here we show that cohesins Rad21, Smc1 and Smc3 are required for bipolar mitosis in human cells. After Rad21 depletion, chromosomes align at the metaphase plate and bipolar spindles assemble in most cases, but in anaphase the separated chromatids segregate to multiple poles. Time-lapse microscopy revealed that the spindle poles often become split in Rad21-depleted metaphase cells. Interestingly, exogenous expression of non-cleavable Rad21 results in multi-polar anaphase. Since cohesins are present at the spindle poles in mitosis, these data are consistent with a non-chromosomal function of cohesin.     

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.     

Caffeine promotes apoptosis in mitotic spindle checkpoint-arrested cells

The spindle assembly checkpoint arrests cells in mitosis when defects in mitotic spindle assembly or partitioning of the replicated genome are detected. This checkpoint blocks exit from mitosis until the defect is rectified or the cell initiates apoptosis. In this study we have used caffeine to identify components of the mechanism that signals apoptosis in mitotic checkpoint-arrested cells. Addition of caffeine to spindle checkpoint-arrested cells induced >40% apoptosis within 5 h. It also caused proteasome-mediated destruction of cyclin B1, a corresponding reduction in cyclin B1/cdk1 activity, and reduction in MPM-2 reactivity. However, cells retained MAD2 staining at the kinetochores, an indication of continued spindle checkpoint function. Blocking proteasome activity did not block apoptosis, but continued spindle checkpoint function was essential for apoptosis. After systematically eliminating all known targets, we have identified p21-activated kinase PAK1, which has an anti-apoptotic function in spindle checkpoint-arrested cells, as a target for caffeine inhibition. Knockdown of PAK1 also increased apoptosis in spindle checkpoint-arrested cells. This study demonstrates that the spindle checkpoint not only regulates mitotic exit but apoptosis in mitosis through the activity of PAK1.     

The fission yeast dma1 gene is a component of the spindle assembly checkpoint, required to prevent septum formation and premature exit from mitosis if spindle function is compromised.

Premature initiation of cytokinesis can lead to loss of chromosomes, and 'cutting' of the nucleus. Therefore, the proper spatial and temporal co-ordination of mitosis and cytokinesis is essential for maintaining the integrity of the genome. The fission yeast cdc16 gene is implicated both in the spindle assembly checkpoint and control of septum formation. To identify other proteins involved in these controls, we have isolated multicopy suppressors of the cdc16-116 mutation, and the characterization of one of these, dma1 (defective in mitotic arrest), is presented here. dma1 is not an essential gene, but in a dma1 null background (dma1-D1) the function of the spindle assembly checkpoint is compromised. If assembly of the spindle is prevented, dma1-D1 cells do not arrest, the activity of cdc2 kinase decays and cells form a division septum without completing a normal mitosis. dma1-D1 cells also show an increased rate of chromosome loss during exponential growth. Upon ectopic expression from an inducible promoter, dma1p delays progress through mitosis and inhibits septum formation, giving rise to elongated, multinucleate cells. We propose that dma1 is a component of the spindle assembly checkpoint, required to prevent septum formation and premature exit from mitosis if spindle function is impaired.     

ISWI is a RanGTP-dependent MAP required for chromosome segregation

Production of RanGTP around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)–containing factors. Here, we show that the NLS protein ISWI, a known chromatin-remodeling ATPase, is a RanGTP-dependent microtubule (MT)-associated protein. Recombinant ISWI induces MT nucleation, stabilization, and bundling in vitro. In Xenopus culture cells and egg extract, ISWI localizes within the nucleus in interphase and on spindles during mitosis. Depletion of ISWI in egg extracts does not affect spindle assembly, but in anaphase spindle MTs disappear and chromosomes do not segregate. We show directly that ISWI is required for the RanGTP-dependent stabilization of MTs during anaphase independently of its effect on chromosomes. ISWI depletion in Drosophila S2 cells induces defects in spindle MTs and chromosome segregation in anaphase, and the cells eventually stop growing. Our results demonstrate that distinctly from its role in spindle assembly, RanGTP maintains spindle MTs in anaphase through the local activation of ISWI and that this is essential for proper chromosome segregation.     

Rad21 is required for centrosome integrity in human cells independently of its role in chromosome cohesion

Classically, chromosomal functions in DNA repair and sister chromatid association have been assigned to the cohesin proteins. More recent studies have provided evidence that cohesins also localize to the centrosomes, which organize the bipolar spindle during mitosis. Depletion of cohesin proteins is associated with multi-polar mitosis in which spindle pole integrity is compromised. However, the spindle pole defects after cohesin depletion could be an indirect consequence of a chromosomal cohesion defect which might impact centrosome integrity via alterations to the spindle microtubule network. Here we show that the cohesin Rad21 is required for centrosome integrity independently of its role as a chromosomal cohesin. Thus, Rad21 may promote accurate chromosome transmission not only by virtue of its function as a chromosomal cohesin, but also because it is required for centrosome function.     

A Role for Mitogen-activated Protein Kinase in the Spindle Assembly Checkpoint in XTC Cells

The spindle assembly checkpoint prevents cells whose spindles are defective or chromosomes are misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the Erk2 mitogen-activated protein kinase (MAP kinase) in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic progression. Here we have investigated whether MAP kinase function is required for mitotic progression or the spindle assembly checkpoint in vivo in Xenopus tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase and metaphase, consistent with the idea that MAP kinase might regulate or monitor the status of the spindle. Next, we microinjected purified recombinant XCL100, a Xenopus MAP kinase phosphatase, into XTC cells in various stages of mitosis to interfere with MAP kinase activation. We found that mitotic progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole—the chromosomes decondensed and the nuclear envelope re-formed—whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells.     

Characterization of the cytotoxic mechanism of Mana-Hox, an analog of manzamine alkaloids

Mana-Hox is a synthetic analog of manzamines, which are beta-carboline alkaloids isolated from marine sponges. Mana-Hox exhibited cytotoxicity against various tumor cell lines with the IC(50) range from 1 to 5 microM. Cell cycle synchronization and flow cytometric analysis showed that Mana-Hox delayed cell cycle progression at mitosis. At the concentration that delayed mitotic progression, bipolar spindle with lagged chromosomes and multipolar spindle with disorganized chromosomes were detected. The presence of such aberrant mitotic cells accompanied by the activation of spindle checkpoint that delayed cells exit from mitosis. However, after a short delay, lagged chromosomes were able to display in the abnormal metaphase plates, and subsequent cell division resulting in chromosome missegregation. Furthermore, the aberrant mitotic cells showed lower viability, indicating that Mana-Hox-induced cell death resulting from chromosome missegregation. This study is the first to explore cytotoxic mechanism of a manzamine-related compound and understand its potential as a lead compound for the development of future anticancer agents.     

DDA3 recruits microtubule depolymerase Kif2a to spindle poles and controls spindle dynamics and mitotic chromosome movement

Dynamic turnover of the spindle is a driving force for chromosome congression and segregation in mitosis. Through a functional genomic analysis, we identify DDA3 as a previously unknown regulator of spindle dynamics that is essential for mitotic progression. DDA3 depletion results in a high frequency of unaligned chromosomes, a substantial reduction in tension across sister kinetochores at metaphase, and a decrease in the velocity of chromosome segregation at anaphase. DDA3 associates with the mitotic spindle and controls microtubule (MT) dynamics. Mechanistically, DDA3 interacts with the MT depolymerase Kif2a in an MT-dependent manner and recruits Kif2a to the mitotic spindle and spindle poles. Depletion of DDA3 increases the steady-state levels of spindle MTs by reducing the turnover rate of the mitotic spindle and by increasing the rate of MT polymerization, which phenocopies the effects of partial knockdown of Kif2a. Thus, DDA3 represents a new class of MT-destabilizing protein that controls spindle dynamics and mitotic progression by regulating MT depolymerases.