Functions In Mitosis Research Articles

LIN9, a Subunit of the DREAM Complex, Regulates Mitotic Gene Expression and Proliferation of Embryonic Stem Cells

The DREAM complex plays an important role in regulation of gene expression during the cell cycle. We have previously shown that the DREAM subunit LIN9 is required for early embryonic development and for the maintenance of the inner cell mass in vitro. In this study we examined the effect of knocking down LIN9 on ESCs. We demonstrate that depletion of LIN9 alters the cell cycle distribution of ESCs and results in an accumulation of cells in G2 and M and in an increase of polyploid cells. Genome-wide expression studies showed that the depletion of LIN9 results in downregulation of mitotic genes and in upregulation of differentiation-specific genes. ChIP-on chip experiments showed that mitotic genes are direct targets of LIN9 while lineage specific markers are regulated indirectly. Importantly, depletion of LIN9 does not alter the expression of pluripotency markers SOX2, OCT4 and Nanog and LIN9 depleted ESCs retain alkaline phosphatase activity. We conclude that LIN9 is essential for proliferation and genome stability of ESCs by activating genes with important functions in mitosis and cytokinesis.

Abstract 4302: TACC3 promotes EMT through the up-regulation of PI3K/Akt and ERK signaling pathways.

Abstract Transforming acidic coiled-coil protein 3 (TACC3) is a member of TACC family, essential for the maintenance of centrosome integrity and the regulation of microtubule assembly and spindle stability during mitosis. In addition to its function in mitosis, TACC3 has been shown to be involved in controlling cell growth, differentiation and transcriptional regulation. Interestingly, deregulated expression of TACC3 has been found in many human cancers, including breast, ovary, bladder, glioblastoma and non-small cell lung cancer. Moreover, our previous microarray analysis suggested that overexpression of TACC3 may be associated with the progression, metastasis and chemoresistance of cervical cancer. Although these findings indicate that TACC3 may play an important role in human cancer, the fundamental molecular mechanisms underlying how TACC3 contributes to cancer initiation, progression and metastasis, and its clinical significance remain elusive. Here, we explored the significance of TACC3 in cervical cancer. Using cervical cancer tissue microarrays, we found that TACC3 is overexpressed in cervical cancer compared with normal cervix. By modulating the expression of TACC3, we found that overexpression of TACC3 leads to changes in cell morphology, proliferation, transforming capability, migratory/invasive behavior as well as the expression of epithelial-mesenchymal transition (EMT)-related markers. Moreover, phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated protein kinases (ERKs) signaling pathways are critical for TACC3-mediated EMT process. Notably, depletion of TACC3 is sufficient to suppress EMT phenotype. Our findings, therefore, identify a novel mechanism that promotes EMT, possibly leading to a more malignant and metastatic tumor phenotype as well as a potential biomarker for diagnosis, prognosis and therapy for cervical cancer. Citation Format: Geun-Hyoung Ha, Jong-Sup Park, Eun-Kyoung Y. Breuer. TACC3 promotes EMT through the up-regulation of PI3K/Akt and ERK signaling pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4302. doi:10.1158/1538-7445.AM2013-4302

Aurora B kinase is required for cytokinesis through effecting spindle structure

The spindle, composed of microtubules and associated proteins, serves as major machinery for cell division. Aurora B is a chromosome passenger protein that has important functions in mitosis. Its dynamic distribution at mitosis goes along with spindle structure and dynamics. We used the siRNA technique to knockdown protein expression and immunofluorescence technique to follow Aurora B during mitosis. Aurora B regulates microtubule plus ends as mitosis progresses, including both kinetochore and polar microtubules. Interactions between Aurora B and polar microtubule plus ends lead to failure in cytokinesis and abnormal midbody structure. We think Aurora B may not only play a role as kinase, but regulate microtubule plus ends.

Promoter hijack reveals pericentrin functions in mitosis and the DNA damage response

Centrosomes, the principal microtubule-organizing centers of animal somatic cells, consist of two centrioles embedded in the pericentriolar material (PCM). Pericentrin is a large PCM protein that is required for normal PCM assembly. Mutations in PCNT cause primordial dwarfism. Pericentrin has also been implicated in the control of DNA damage responses. To test how pericentrin is involved in cell cycle control after genotoxic stress, we disrupted the Pcnt locus in chicken DT40 cells. Pericentrin-deficient cells proceeded through mitosis more slowly, with a high level of monopolar spindles, and were more sensitive to spindle poisons than controls. Centriole structures appeared normal by light and electron microscopy, but the PCM did not recruit γ-tubulin efficiently. Cell cycle delays after ionizing radiation (IR) treatment were normal in pericentrin-deficient cells. However, pericentrin disruption in Mcph1−/− cells abrogated centrosome hyperamplification after IR. We conclude that pericentrin controls genomic stability by both ensuring appropriate mitotic spindle activity and centrosome regulation.

Time-Lapse Imaging of Neuroblastoma Cells to Determine Cell Fate upon Gene Knockdown

Neuroblastoma is the most common extra-cranial solid tumor of early childhood. Standard therapies are not effective in case of poor prognosis and chemotherapy resistance. To improve drug therapy, it is imperative to discover new targets that play a substantial role in tumorigenesis of neuroblastoma. The mitotic machinery is an attractive target for therapeutic interventions and inhibitors can be developed to target mitotic entry, spindle apparatus, spindle activation checkpoint, and mitotic exit. We present an elaborate analysis pipeline to determine cancer specific therapeutic targets by first performing a focused gene expression analysis to select genes followed by a gene knockdown screening assay of live cells. We interrogated gene expression studies of neuroblastoma tumors and selected 240 genes relevant for tumorigenesis and cell cycle. With these genes we performed time-lapse screening of gene knockdowns in neuroblastoma cells. We classified cellular phenotypes and used the temporal context of the perturbation effect to determine the sequence of events, particularly the mitotic entry preceding cell death. Based upon this phenotype kinetics from the gene knockdown screening, we inferred dynamic gene functions in mitosis and cell proliferation. We identified six genes (DLGAP5, DSCC1, SMO, SNRPD1, SSBP1, and UBE2C) with a vital role in mitosis and these are promising therapeutic targets for neuroblastoma. Images and movies of every time point of all screened genes are available at https://ichip.bioquant.uni-heidelberg.de.

Phosphoinositide 3-kinase β regulates chromosome segregation in mitosis

Class I(A) phosphoinositide 3-kinases (PI3K) are enzymes composed of a p85 regulatory and a p110 catalytic subunit that control formation of 3-poly-phosphoinositides (PIP(3)). The PI3K pathway regulates cell survival, migration, and division, and is mutated in approximately half of human tumors. For this reason, it is important to define the function of the ubiquitous PI3K subunits, p110α and p110β. Whereas p110α is activated at G1-phase entry and promotes protein synthesis and gene expression, p110β activity peaks in S phase and regulates DNA synthesis. PI3K activity also increases at the onset of mitosis, but the isoform activated is unknown; we have examined p110α and p110β function in mitosis. p110α was activated at mitosis entry and regulated early mitotic events, such as PIP(3) generation, prometaphase progression, and spindle orientation. In contrast, p110β was activated near metaphase and controlled dynein/dynactin and Aurora B activities in kinetochores, chromosome segregation, and optimal function of the spindle checkpoint. These results reveal a p110β function in preserving genomic stability during mitosis.

The Arabidopsis SYN3 cohesin protein is important for early meiotic events

α-Kleisins are core components of meiotic and mitotic cohesin complexes. Arabidopsis contains four genes that encode α-kleisin proteins: SYN1, SYN2, SYN3 and SYN4. SYN1, a REC8 ortholog, is essential for meiosis, while SYN2 and SYN4 appear to be SCC1 orthologs and function in mitosis. SYN3 is essential for megagametogenesis and is enriched in the nucleolus of meiotic and mitotic cells. In this study the role of SYN3 during meiosis was investigated by characterization of plants that express SYN3-RNAi constructs from either meiotic DMC1, native SYN3, or inducible PX7 promoters. Reduction of SYN3 caused defects in homologous chromosome synapsis and synaptonemal complex (SC) formation during male and female meiosis. Consistent with this observation, relatively little signal for the SC component ZYP1 was detected on the chromosomes of SYN3-RNAi plants. ZYP1 transcript levels were relatively normal, but several transcripts for genes that encode proteins involved in meiotic recombination were altered, which suggested that a reduction in SYN3 may inhibit meiotic progression by alteration of meiotic gene expression.

CRAF Has MEK-Independent Functions in Mitosis and Tumor Progression

Abstract Major finding: CRAF has a direct role in mitosis that does not require the RAF effector MEK. Mechanism: CRAF interacts with PLK1 and promotes its activity and accumulation at kinetochores. Impact: Allosteric RAF inhibitors that block phospho-Ser338 CRAF may be effective cancer therapies.

A Non-Motor Microtubule Binding Site Is Essential for the High Processivity and Mitotic Function of Kinesin-8 Kif18A

BackgroundMembers of the kinesin-8 subfamily are plus end-directed molecular motors that accumulate at the plus-ends of kinetochore-microtubules (kt-MTs) where they regulate MT dynamics. Loss of vertebrate kinesin-8 function induces hyperstable MTs and elongated mitotic spindles accompanied by severe chromosome congression defects. It has been reported that the motility of human kinesin-8, Kif18A, is required for its accumulation at the plus tips of kt-MTs.Methodology/FindingsHere, we investigate how Kif18A localizes to the plus-ends of kt-MTs. We find that Kif18A lacking its C-terminus does not accumulate on the tips of kt-MTs and fails to fulfill its mitotic function. In vitro studies reveal that Kif18A possesses a non-motor MT binding site located within its C-proximal 121 residues. Using single molecule measurements we find that Kif18A is a highly processive motor and, furthermore, that the C-terminal tail is essential for the high processivity of Kif18A.Conclusion/SignificanceThese results show that Kif18A like its yeast orthologue is a highly processive motor. The ability of Kif18A to walk on MTs for a long distance without dissociating depends on a non-motor MT binding site located at the C-terminus of Kif18A. This C-proximal tail of Kif18A is essential for its plus-end accumulation and mitotic function. These findings advance our understanding of how Kif18A accumulates at the tips of kt-MTs to fulfill its function in mitosis.

Chromatin maintenance by a molecular motor protein

The kinesin motor protein KIF4 performs essential functions in mitosis. Like other mitotic kinesins, loss of KIF4 causes spindle defects, aneuploidy, genomic instability and ultimately tumor formation. However, KIF4 is unique among molecular motors in that it resides in the cell nucleus throughout interphase, suggesting a non-mitotic function as well. Here we identify a novel cellular function for a molecular motor protein by demonstrating that KIF4 acts as a modulator of large-scale chromatin architecture during interphase. KIF4 binds globally to chromatin and its absence leads to chromatin decondensation and loss of heterochromatin domains. KIF4-dependent chromatin decondensation has functional consequences by causing replication defects and global mis-regulation of gene expression programs. KIF4 exerts its function in chromatin architecture via regulation of ADP-ribosylation of core and linker histones and by physical interaction and recruitment of chromatin assembly proteins during S-phase. These observations document a novel function for a molecular motor protein in establishment and maintenance of higher order chromatin structure.

STIM1 Function in Mitosis and Meiosis

Abstract The endoplasmic reticulum (ER) functions as a storehouse for intracellular Calcium. STIM1, a Calcium sensor localizes mostly to the ER membrane in interphase under resting conditions. Following Ca2+ store depletion, STIM1 forms puncta that localize to the cortical ER and binds Orai1, a Ca2+ channel to allow Ca2+ influx. This mechanism of Ca2+ influx is termed Store-Operated Calcium Entry (SOCE). Orai1 is internalized during meiosis and STIM1 fails to aggregate even when intracellular Ca2+ stores are depleted in both mitosis and meiosis. This causes inhibition of SOCE during mitosis and meiosis. This is an important mechanism to prevent sporadic Ca2+ influx that could disrupt its specialized functions during the cell cycle. We are interested in elucidating the mechanisms that allow STIM1 to cluster during interphase but not during M-phase. We have taken a proteomics approach to identify proteins that function in hSTIM1 clustering following store depletion, and to determine what makes mitotic cells...

Bulk Cytoplasmic Actin and Its Functions in Meiosis and Mitosis

Discussions of actin cell biology generally focus on the cortex, a thin, actin-rich layer of cytoplasm under the plasma membrane. Here we review the much less studied biology of actin filaments deeper in the cytoplasm and their recently revealed functions in mitosis and meiosis that are most prominent in large oocyte, egg and early embryo cells. The cellular functions of cytoplasmic actin range from the assembly and positioning of meiotic spindles to the prevention of cytoplasmic streaming. We discuss the possible use of evolutionarily conserved mechanisms to nucleate and organize actin filaments to achieve these diverse cellular functions, the cell-cycle regulation of these functions, and the many unanswered questions about this largely unexplored mechanism of cytoplasmic organization.

Phosphorylation of Right Open Reading Frame 2 (Rio2) Protein Kinase by Polo-like Kinase 1 Regulates Mitotic Progression

Polo-like kinase 1 (Plk1) plays essential roles during multiple stages of mitosis by phosphorylating a number of substrates. Here, we report that the atypical protein kinase Rio2 is a novel substrate of Plk1 and can be phosphorylated by Plk1 at Ser-335, Ser-380, and Ser-548. Overexpression of Rio2 causes a prolonged mitotic exit whereas knockdown of Rio2 accelerates mitotic progression, suggesting that Rio2 is required for the proper mitotic progression. Overexpression of phospho-mimicking mutant Rio2 S3D but not the nonphosphorylatable mutant Rio2 S3A displays a profile similar to that of wild-type Rio2. These results indicate that the phosphorylation status of Rio2 correlates with its function in mitosis. Furthermore, time-lapse imaging data show that overexpression of Rio2 but not Rio2 S3A results in a slowed metaphase-anaphase transition. Collectively, these findings strongly indicate that the Plk1-mediated phosphorylation of Rio2 regulates metaphase-anaphase transition during mitotic progression.

MoDUO1, a Duo1-like gene, is required for full virulence of the rice blast fungus Magnaporthe oryzae

Duo1, a major component of the Dam1 complex which has been found in two species of yeast (the budding yeast Saccharomyces cerevisae and the fission yeast Schizosaccharomyces pombe), is involved in mitosis-related chromosome segregation, while its relevance to pathogenicity in filamentous fungi remains unclear. This report elucidated this very fact in the case of the rice blast fungus Magnaporthe oryzae. A gene designated MoDUO1 that encodes a Duo1-like homolog (MoDuo1) was discovered in the M. oryzae genome. Two types of MoDUO1 mutants were obtained using genetic approaches of Agrobacterium-mediated gene disruption and homologous recombination. Both disruption and deletion of MoDUO1 can exert profound effects on the formation pattern of conidiophores and conidial morphology, such as abnormal nucleic numbers in conidia and delayed extension of infectious hyphae. Intriguingly, plant infection assays demonstrated that inactivation of MoDUO1 significantly attenuates the virulence in its natural host rice leaves, and functional complementation can restore it. Subcellular localization assays showed that MoDuo1 is mainly distributed in the cytosol of fungal cells. Proteomics-based investigation revealed that the expression of four mitosis-related proteins is shut down in the MoDUO1 mutant, suggesting that MoDuo1 may have a function in mitosis. In light of the fact that Duo1 orthologs are widespread in plant and human fungal pathogens, our finding may represent a common mechanism underlying fungal virulence. To the best of our knowledge, this is the first example of linking a Duo1-like homolog to the pathogenesis of a pathogenic fungus, which might provide clues to additional studies on the role of Dam1 complex in M. oryzae and its interaction with rice.

A mad partner for Shugoshin in meiosis

The centromeric cohesin protector Shugoshin (Sgo) and the spindle checkpoint protein Mad2 are known to act in distinct pathways for ensuring accurate chromosome segregation during eukaryotic cell division. In this issue of The EMBO Journal , [Orth et al (2011)][1] describe an unexpected physical interaction between vertebrate Sgo and Mad2, as well as indications of their potential mutual regulation in meiosis. [1]: #ref-7

A Positive Feedback Loop Involving Haspin and Aurora B Promotes CPC Accumulation at Centromeres in Mitosis

Haspin phosphorylates histone H3 at Thr3 (H3T3ph) during mitosis [1, 2], providing a chromatin binding site for the chromosomal passenger complex (CPC) at centromeres to regulate chromosome segregation [3-5]. H3T3ph becomes increasingly focused at inner centromeres during prometaphase [1, 2], but little is known about how its level or location and the consequent chromosomal localization of the CPC are regulated. In addition, CPC binding to shugoshin proteins contributes to centromeric Aurora B localization [5, 6]. Recruitment of the shugoshins to centromeres requires the phosphorylation of histone H2A at Thr120 (H2AT120ph) by the kinetochore kinase Bub1 [7], but the molecular basis for the collaboration of this pathway with H3T3ph has been unclear. Here, we show that Aurora B phosphorylates Haspin to promote generation of H3T3ph and that Aurora B kinase activity is required for normal chromosomal localization of the CPC, indicating an intimate linkage between Aurora B and Haspin functions in mitosis. We propose that Aurora B activity triggers a CPC-Haspin-H3T3ph feedback loop that promotes generation of H3T3ph on chromatin. We also provide evidence that the Bub1-shugoshin-CPC pathway supplies a signal that boosts the CPC-Haspin-H3T3ph feedback loop specifically at centromeres to produce the well-known accumulation of the CPC in these regions.

Reversal of endocrine resistance in breast cancer: interrelationships among 14-3-3ζ, FOXM1, and a gene signature associated with mitosis

IntroductionDespite the benefits of estrogen receptor (ER)-targeted endocrine therapies in breast cancer, many tumors develop resistance. 14-3-3 ζ/YWHAZ, a member of the 14-3-3 family of conserved proteins, is over-expressed in several types of cancer, and our previous work showed that high expression of 14-3-3ζ in ER-positive breast cancers was associated with a poor clinical outcome for women on tamoxifen. Therefore, we now probe the role of 14-3-3ζ in endocrine resistance, and we examine the functional dimensions and molecular basis that underlie 14-3-3ζ activities.MethodsFrom analyses of four independent breast cancer microarray datasets from nearly 400 women, we characterized a gene signature that correlated strongly with high expression of 14-3-3ζ in breast tumors and examined its association with breast cancer molecular subtypes and clinical-pathological features. We investigated the effects of altering 14-3-3ζ levels in ER-positive, endocrine sensitive and resistant breast cancer cells on the regulation of 14-3-3ζ signature genes, and on cellular signaling pathways and cell phenotypic properties.ResultsThe gene signature associated with high 14-3-3ζ levels in breast tumors encompassed many with functions in mitosis and cytokinesis, including aurora kinase-B, polo-like kinase-1, CDC25B, and BIRC5/survivin. The gene signature correlated with early recurrence and risk of metastasis, and was found predominantly in luminal B breast cancers, the more aggressive ER-positive molecular subtype. The expression of the signature genes was significantly decreased or increased upon reduction or overexpression of 14-3-3ζ in ER-positive breast cancer cells, indicating their coregulation. 14-3-3ζ also played a critical role in the regulation of FOXM1, with 14-3-3ζ acting upstream of FOXM1 to regulate cell division-signature genes. Depletion of 14-3-3ζ markedly increased apoptosis, reduced proliferation and receptor tyrosine kinase (HER2 and EGFR) signaling, and, importantly, reversed endocrine resistance.ConclusionsThis study reveals that 14-3-3ζ is a key predictive marker for risk of failure on endocrine therapy and serves a pivotal role impacting growth factor signaling, and promoting cell survival and resistance to endocrine therapies. Targeting 14-3-3ζ and its coregulated proteins, such as FOXM1, should prove valuable in restoring endocrine sensitivity and reducing risk of breast cancer recurrence.

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.

A TACC3/ch-TOG/clathrin complex stabilises kinetochore fibres by inter-microtubule bridging

Kinetochore fibres (K-fibres) of the spindle apparatus move chromosomes during mitosis. These fibres are discrete bundles of parallel microtubules (MTs) that are crosslinked by inter-MT 'bridges' that are thought to improve fibre stability during chromosomal movement. The identity of these bridges is unknown. Clathrin is a multimeric protein that has been shown to stabilise K-fibres during early mitosis by a mechanism independent of its role in membrane trafficking. In this study, we show that clathrin at the mitotic spindle is in a transforming acidic colied-coil protein 3 (TACC3)/colonic, hepatic tumour overexpressed gene (ch-TOG)/clathrin complex. The complex is anchored to the spindle by TACC3 and ch-TOG. Ultrastructural analysis of clathrin-depleted K-fibres revealed a selective loss of a population of short inter-MT bridges and a general loss of MTs. A similar loss of short inter-MT bridges was observed in TACC3-depleted K-fibres. Finally, immunogold labelling confirmed that inter-MT bridges in K-fibres contain clathrin. Our results suggest that the TACC3/ch-TOG/clathrin complex is an inter-MT bridge that stabilises K-fibres by physical crosslinking and by reducing rates of MT catastrophe.

Probing CENP-E function in chromosome dynamics using small molecule inhibitor syntelin

Probing CENP-E function in chromosome dynamics using small molecule inhibitor syntelin