Micromechanical-biochemical studies of mitotic chromosome elasticity and structure

NASA Astrophysics Data System (ADS)

Poirier, Michael Guy

The structure of mitotic chromosomes was studied by combining micromechanical force measurements with microfluidic biochemical exposures. Our method is to use glass micropipettes attached to either end of a single chromosome to do mechanical experiments in the extracellular buffer. A third pipette can be used to locally 'spray' reactants so as to carry out dynamical mechanical-chemical experiments. The following elastic properties of mitotic chromosomes are found: Young's modulus, Y = 300 Pa; Poisson ratio, sigma = 0.1; Bending rigidity, B = 1 x 10 -22 J·m; Internal viscosity, eta' = 100 kg/m·sec; Volume fraction, ϕ = 0.7; Extensions of less than 3 times the relaxed length are linear and reversible; Extensions beyond 30 fold exhibit a force plateau at 15 nN and convert the chromosome to a disperse ghost-like state with little change in chromatin structure; Mitotic chromosomes are relatively isotropic; dsDNA cuts of at least every 3 kb cause the a mitotic chromosomes to fall apart; dsDNA cuts less frequently than every 50 kb do not affect mitotic chromosome structure. These results lead to the conclusion that mitotic chromosomes are a network crosslinked every 50 kb between which chromatin is fold by chromatin folding proteins, which are likely to be condensins.

Regulating positioning and orientation of mitotic spindles via cell size and shape

NASA Astrophysics Data System (ADS)

Li, Jingchen; Jiang, Hongyuan

2018-01-01

Proper location of the mitotic spindle is critical for chromosome segregation and the selection of the cell division plane. However, how mitotic spindles sense cell size and shape to regulate their own position and orientation is still largely unclear. To investigate this question systematically, we used a general model by considering chromosomes, microtubule dynamics, and forces of various molecular motors. Our results show that in cells of various sizes and shapes, spindles can always be centered and oriented along the long axis robustly in the absence of other specified mechanisms. We found that the characteristic time of positioning and orientation processes increases with cell size. Spindles sense the cell size mainly by the cortical force in small cells and by the cytoplasmic force in large cells. In addition to the cell size, the cell shape mainly influences the orientation process. We found that more slender cells have a faster orientation process, and the final orientation is not necessarily along the longest axis but is determined by the radial profile and the symmetry of the cell shape. Finally, our model also reproduces the separation and repositioning of the spindle poles during the anaphase. Therefore, our work provides a general tool for studying the mitotic spindle across the whole mitotic phase.

The NIMA Kinase Is Required To Execute Stage-Specific Mitotic Functions after Initiation of Mitosis

PubMed Central

Govindaraghavan, Meera; Lad, Alisha A.

2014-01-01

The G2-M transition in Aspergillus nidulans requires the NIMA kinase, the founding member of the Nek kinase family. Inactivation of NIMA results in a late G2 arrest, while overexpression of NIMA is sufficient to promote mitotic events independently of cell cycle phase. Endogenously tagged NIMA-GFP has dynamic mitotic localizations appearing first at the spindle pole body and then at nuclear pore complexes before transitioning to within nuclei and the mitotic spindle and back at the spindle pole bodies at mitotic exit, suggesting that it functions sequentially at these locations. Since NIMA is indispensable for mitotic entry, it has been difficult to determine the requirement of NIMA for subaspects of mitosis. We show here that when NIMA is partially inactivated, although mitosis can be initiated, a proportion of cells fail to successfully generate two daughter nuclei. We further define the mitotic defects to show that normal NIMA function is required for the formation of a bipolar spindle, nuclear pore complex disassembly, completion of chromatin segregation, and the normal structural rearrangements of the nuclear envelope required to generate two nuclei from one. In the remaining population of cells that enter mitosis with inadequate NIMA, two daughter nuclei are generated in a manner dependent on the spindle assembly checkpoint, indicating highly penetrant defects in mitotic progression without sufficient NIMA activity. This study shows that NIMA is required not only for mitotic entry but also sequentially for successful completion of stage-specific mitotic events. PMID:24186954

The Physics of the Metaphase Spindle.

PubMed

Oriola, David; Needleman, Daniel J; Brugués, Jan

2018-05-20

The assembly of the mitotic spindle and the subsequent segregation of sister chromatids are based on the self-organized action of microtubule filaments, motor proteins, and other microtubule-associated proteins, which constitute the fundamental force-generating elements in the system. Many of the components in the spindle have been identified, but until recently it remained unclear how their collective behaviors resulted in such a robust bipolar structure. Here, we review the current understanding of the physics of the metaphase spindle that is only now starting to emerge.

Cytoplasmic flows as signatures for the mechanics of mitotic positioning

PubMed Central

Nazockdast, Ehssan; Rahimian, Abtin; Needleman, Daniel; Shelley, Michael

2017-01-01

The proper positioning of mitotic spindle in the single-cell Caenorhabditis elegans embryo is achieved initially by the migration and rotation of the pronuclear complex (PNC) and its two associated astral microtubules (MTs). Pronuclear migration produces global cytoplasmic flows that couple the mechanics of all MTs, the PNC, and the cell periphery with each other through their hydrodynamic interactions (HIs). We present the first computational study that explicitly accounts for detailed HIs between the cytoskeletal components and demonstrate the key consequences of HIs for the mechanics of pronuclear migration. First, we show that, because of HIs between the MTs, the cytoplasm-filled astral MTs behave like a porous medium, with its permeability decreasing with increasing the number of MTs. We then directly study the dynamics of PNC migration under various force-transduction models, including the pushing or pulling of MTs at the cortex and the pulling of MTs by cytoplasmically bound force generators. Although achieving proper position and orientation on reasonable time scales does not uniquely choose a model, we find that each model produces a different signature in its induced cytoplasmic flow. We suggest that cytoplasmic flows can be used to differentiate between mechanisms. PMID:28331070

Dietary flavonoid fisetin induces a forced exit from mitosis by targeting the mitotic spindle checkpoint

PubMed Central

Salmela, Anna-Leena; Pouwels, Jeroen; Varis, Asta; Kukkonen, Anu M.; Toivonen, Pauliina; Halonen, Pasi K.; Perälä, Merja; Kallioniemi, Olli; Gorbsky, Gary J.; Kallio, Marko J.

2009-01-01

Fisetin is a natural flavonol present in edible vegetables, fruits and wine at 2–160 μg/g concentrations and an ingredient in nutritional supplements with much higher concentrations. The compound has been reported to exert anticarcinogenic effects as well as antioxidant and anti-inflammatory activity via its ability to act as an inhibitor of cell proliferation and free radical scavenger, respectively. Our cell-based high-throughput screen for small molecules that override chemically induced mitotic arrest identified fisetin as an antimitotic compound. Fisetin rapidly compromised microtubule drug-induced mitotic block in a proteasome-dependent manner in several human cell lines. Moreover, in unperturbed human cancer cells fisetin caused premature initiation of chromosome segregation and exit from mitosis without normal cytokinesis. To understand the molecular mechanism behind these mitotic errors, we analyzed the consequences of fisetin treatment on the localization and phoshorylation of several mitotic proteins. Aurora B, Bub1, BubR1 and Cenp-F rapidly lost their kinetochore/centromere localization and others became dephosphorylated upon addition of fisetin to the culture medium. Finally, we identified Aurora B kinase as a novel direct target of fisetin. The activity of Aurora B was significantly reduced by fisetin in vitro and in cells, an effect that can explain the observed forced mitotic exit, failure of cytokinesis and decreased cell viability. In conclusion, our data propose that fisetin perturbs spindle checkpoint signaling, which may contribute to the antiproliferative effects of the compound. PMID:19395653

Self-Organization and Forces in the Mitotic Spindle.

PubMed

Pavin, Nenad; Tolić, Iva M

2016-07-05

At the onset of division, the cell forms a spindle, a precise self-constructed micromachine composed of microtubules and the associated proteins, which divides the chromosomes between the two nascent daughter cells. The spindle arises from self-organization of microtubules and chromosomes, whose different types of motion help them explore the space and eventually approach and interact with each other. Once the interactions between the chromosomes and the microtubules have been established, the chromosomes are moved to the equatorial plane of the spindle and ultimately toward the opposite spindle poles. These transport processes rely on directed forces that are precisely regulated in space and time. In this review, we discuss how microtubule dynamics and their rotational movement drive spindle self-organization, as well as how the forces acting in the spindle are generated, balanced, and regulated.

Hunting the mechanisms of self-renewal of immortal cell populations by means of real-time imaging of living cells.

PubMed

Kvitko, O V; Koneva, I I; Sheiko, Y I; Anisovich, M V

2005-12-01

The causes of the indefinite propagation of immortalized cell populations remain insufficiently understood, that hinders the research of such fundamental processes as ageing and cancer. In this study the interrelations between clonal proliferation and abnormalities of mitotic divisions in the immortalized cell line established from the mouse embryo were investigated with the aid of computerized microscopy of living cells. 3 mitoses with three daughter cells and 7 asymmetric mitoses which generated two daughter cells of conspicuously different sizes were registered among 71 mitotic divisions in the individual cell genealogy. Abnormal mitotic divisions either did not slow the proliferation in cell clones compared with progenies of cells that divided by means of normal mitoses or were followed by the acceleration of divisions in consecutive cell generations. These data suggest that abnormal mitotic divisions may contribute to the maintenance of the immortalized state of cell populations by means of generating chromosomal instability.

Micromechanics of human mitotic chromosomes

NASA Astrophysics Data System (ADS)

Sun, Mingxuan; Kawamura, Ryo; Marko, John F.

2011-02-01

Eukaryote cells dramatically reorganize their long chromosomal DNAs to facilitate their physical segregation during mitosis. The internal organization of folded mitotic chromosomes remains a basic mystery of cell biology; its understanding would likely shed light on how chromosomes are separated from one another as well as into chromosome structure between cell divisions. We report biophysical experiments on single mitotic chromosomes from human cells, where we combine micromanipulation, nano-Newton-scale force measurement and biochemical treatments to study chromosome connectivity and topology. Results are in accord with previous experiments on amphibian chromosomes and support the 'chromatin network' model of mitotic chromosome structure. Prospects for studies of chromosome-organizing proteins using siRNA expression knockdowns, as well as for differential studies of chromosomes with and without mutations associated with genetic diseases, are also discussed.

Optical volume and mass measurements show that mammalian cells swell during mitosis

PubMed Central

Zlotek-Zlotkiewicz, Ewa; Monnier, Sylvain; Cappello, Giovanni; Le Berre, Mael

2015-01-01

The extent, mechanism, and function of cell volume changes during specific cellular events, such as cell migration and cell division, have been poorly studied, mostly because of a lack of adequate techniques. Here we unambiguously report that a large range of mammalian cell types display a significant increase in volume during mitosis (up to 30%). We further show that this increase in volume is tightly linked to the mitotic state of the cell and not to its spread or rounded shape and is independent of the presence of an intact actomyosin cortex. Importantly, this volume increase is not accompanied by an increase in dry mass and thus corresponds to a decrease in cell density. This mitotic swelling might have important consequences for mitotic progression: it might contribute to produce strong pushing forces, allowing mitotic cells to round up; it might also, by lowering cytoplasmic density, contribute to the large change of physicochemical properties observed in mitotic cells. PMID:26598614

A mitotic SKAP isoform regulates spindle positioning at astral microtubule plus ends

PubMed Central

Kern, David M.; Nicholls, Peter K.; Page, David C.

2016-01-01

The Astrin/SKAP complex plays important roles in mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, we demonstrate that SKAP is expressed as two distinct isoforms in mammals: a longer, testis-specific isoform that was used for the previous studies in mitotic cells and a novel, shorter mitotic isoform. Unlike the long isoform, short SKAP rescues SKAP depletion in mitosis and displays robust microtubule plus-end tracking, including localization to astral microtubules. Eliminating SKAP microtubule binding results in severe chromosome segregation defects. In contrast, SKAP mutants specifically defective for plus-end tracking facilitate proper chromosome segregation but display spindle positioning defects. Cells lacking SKAP plus-end tracking have reduced Clasp1 localization at microtubule plus ends and display increased lateral microtubule contacts with the cell cortex, which we propose results in unbalanced dynein-dependent cortical pulling forces. Our work reveals an unappreciated role for the Astrin/SKAP complex as an astral microtubule mediator of mitotic spindle positioning. PMID:27138257

Perturbation of Incenp function impedes anaphase chromatid movements and chromosomal passenger protein flux at centromeres

PubMed Central

Ahonen, Leena J.; Kukkonen, Anu M.; Pouwels, Jeroen; Bolton, Margaret A.; Jingle, Christopher D.; Stukenberg, P. Todd; Kallio, Marko J.

2012-01-01

Incenp is an essential mitotic protein that, together with Aurora B, Survivin, and Borealin, forms the core of the chromosomal passenger protein complex (CPC). The CPC regulates various mitotic processes and functions to maintain genomic stability. The proper subcellular localization of the CPC and its full catalytic activity require the presence of each core subunit in the complex. We have investigated the mitotic tasks of the CPC using a function blocking antibody against Incenp microinjected into cells at different mitotic phases. This method allowed temporal analysis of CPC functions without perturbation of complex assembly or activity prior to injection. We have also studied the dynamic properties of Incenp and Aurora B using fusion protein photobleaching. We found that in early mitotic cells, Incenp and Aurora B exhibit dynamic turnover at centromeres, which is prevented by the anti-Incenp antibody. In these cells, the loss of centromeric CPC turnover is accompanied by forced mitotic exit without the execution of cytokinesis. Introduction of anti-Incenp antibody into early anaphase cells causes abnormalities in sister chromatid separation through defects in anaphase spindle functions. In summary, our data uncovers new mitotic roles for the CPC in anaphase and proposes that CPC turnover at centromeres modulates spindle assembly checkpoint signaling. PMID:18784935

Perturbation of Incenp function impedes anaphase chromatid movements and chromosomal passenger protein flux at centromeres.

PubMed

Ahonen, Leena J; Kukkonen, Anu M; Pouwels, Jeroen; Bolton, Margaret A; Jingle, Christopher D; Stukenberg, P Todd; Kallio, Marko J

2009-02-01

Incenp is an essential mitotic protein that, together with Aurora B, Survivin, and Borealin, forms the core of the chromosomal passenger protein complex (CPC). The CPC regulates various mitotic processes and functions to maintain genomic stability. The proper subcellular localization of the CPC and its full catalytic activity require the presence of each core subunit in the complex. We have investigated the mitotic tasks of the CPC using a function blocking antibody against Incenp microinjected into cells at different mitotic phases. This method allowed temporal analysis of CPC functions without perturbation of complex assembly or activity prior to injection. We have also studied the dynamic properties of Incenp and Aurora B using fusion protein photobleaching. We found that in early mitotic cells, Incenp and Aurora B exhibit dynamic turnover at centromeres, which is prevented by the anti-Incenp antibody. In these cells, the loss of centromeric CPC turnover is accompanied by forced mitotic exit without the execution of cytokinesis. Introduction of anti-Incenp antibody into early anaphase cells causes abnormalities in sister chromatid separation through defects in anaphase spindle functions. In summary, our data uncovers new mitotic roles for the CPC in anaphase and proposes that CPC turnover at centromeres modulates spindle assembly checkpoint signaling.

Kinesin-5-independent mitotic spindle assembly requires the antiparallel microtubule crosslinker Ase1 in fission yeast

PubMed Central

Rincon, Sergio A.; Lamson, Adam; Blackwell, Robert; Syrovatkina, Viktoriya; Fraisier, Vincent; Paoletti, Anne; Betterton, Meredith D.; Tran, Phong T.

2017-01-01

Bipolar spindle assembly requires a balance of forces where kinesin-5 produces outward pushing forces to antagonize the inward pulling forces from kinesin-14 or dynein. Accordingly, Kinesin-5 inactivation results in force imbalance leading to monopolar spindle and chromosome segregation failure. In fission yeast, force balance is restored when both kinesin-5 Cut7 and kinesin-14 Pkl1 are deleted, restoring spindle bipolarity. Here we show that the cut7Δpkl1Δ spindle is fully competent for chromosome segregation independently of motor activity, except for kinesin-6 Klp9, which is required for anaphase spindle elongation. We demonstrate that cut7Δpkl1Δ spindle bipolarity requires the microtubule antiparallel bundler PRC1/Ase1 to recruit CLASP/Cls1 to stabilize microtubules. Brownian dynamics-kinetic Monte Carlo simulations show that Ase1 and Cls1 activity are sufficient for initial bipolar spindle formation. We conclude that pushing forces generated by microtubule polymerization are sufficient to promote spindle pole separation and the assembly of bipolar spindle in the absence of molecular motors. PMID:28513584

Exotic mitotic mechanisms

PubMed Central

Drechsler, Hauke; McAinsh, Andrew D.

2012-01-01

The emergence of eukaryotes around two billion years ago provided new challenges for the chromosome segregation machineries: the physical separation of multiple large and linear chromosomes from the microtubule-organizing centres by the nuclear envelope. In this review, we set out the diverse solutions that eukaryotic cells use to solve this problem, and show how stepping away from ‘mainstream’ mitosis can teach us much about the mechanisms and mechanics that can drive chromosome segregation. We discuss the evidence for a close functional and physical relationship between membranes, nuclear pores and kinetochores in generating the forces necessary for chromosome segregation during mitosis. PMID:23271831

NuMA-microtubule interactions are critical for spindle orientation and the morphogenesis of diverse epidermal structures

PubMed Central

Seldin, Lindsey; Muroyama, Andrew; Lechler, Terry

2016-01-01

Mitotic spindle orientation is used to generate cell fate diversity and drive proper tissue morphogenesis. A complex of NuMA and dynein/dynactin is required for robust spindle orientation in a number of cell types. Previous research proposed that cortical dynein/dynactin was sufficient to generate forces on astral microtubules (MTs) to orient the spindle, with NuMA acting as a passive tether. In this study, we demonstrate that dynein/dynactin is insufficient for spindle orientation establishment in keratinocytes and that NuMA’s MT-binding domain, which targets MT tips, is also required. Loss of NuMA-MT interactions in skin caused defects in spindle orientation and epidermal differentiation, leading to neonatal lethality. In addition, we show that NuMA-MT interactions are also required in adult mice for hair follicle morphogenesis and spindle orientation within the transit-amplifying cells of the matrix. Loss of spindle orientation in matrix cells results in defective differentiation of matrix-derived lineages. Our results reveal an additional and direct function of NuMA during mitotic spindle positioning, as well as a reiterative use of spindle orientation in the skin to build diverse structures. DOI: http://dx.doi.org/10.7554/eLife.12504.001 PMID:26765568

Phospho-Regulation of DDA3 Function in Mitosis

PubMed Central

Jang, Chang-Young; Coppinger, Judith A.; Yates, John R.; Fang, Guowei

2010-01-01

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. PMID:20117088

Comparative effects of 60Co gamma-rays and neon and helium ions on cycle duration and division probability of EMT 6 cells. A time-lapse cinematography study.

PubMed

Collyn-d'Hooghe, M; Hemon, D; Gilet, R; Curtis, S B; Valleron, A J; Malaise, E P

1981-03-01

Exponentially growing cultures of EMT 6 cells were irradiated in vitro with neon ions, helium ions or 60Co gamma-rays. Time-lapse cinematography allowed the determination, for individual cells, of cycle duration, success of the mitotic division and the age of the cell at the moment of irradiation. Irradiation induced a significant mitotic delay increasing proportionally with the delivered dose. Using mitotic delay as an endpoint, the r.b.e. for neon ions with respect to 60Co gamma-rays was 3.3 +/- 0.2 while for helium ions it was 1.2 +/- 0.1. Mitotic delay was greatest in those cells that had progressed furthest in their cycle at the time of irradiation. No significant mitotic delay was observed in the post-irradiation generation. Division probability was significantly reduced by irradiation both in the irradiated and in the post-irradiated generation. The reduction in division probability obtained with 3 Gy of neon ions was similar to that obtained after irradiation with 6 Gy of helium ions or 60Co gamma-rays.

Loops determine the mechanical properties of mitotic chromosomes

NASA Astrophysics Data System (ADS)

Zhang, Yang; Heermann, Dieter W.

2013-03-01

In mitosis, chromosomes undergo a condensation into highly compacted, rod-like objects. Many models have been put forward for the higher-order organization of mitotic chromosomes including radial loop and hierarchical folding models. Additionally, mechanical properties of mitotic chromosomes under different conditions were measured. However, the internal organization of mitotic chromosomes still remains unclear. Here we present a polymer model for mitotic chromosomes and show how chromatin loops play a major role for their mechanical properties. The key assumption of the model is the ability of the chromatin fibre to dynamically form loops with the help of binding proteins. Our results show that looping leads to a tight compaction and significantly increases the bending rigidity of chromosomes. Moreover, our qualitative prediction of the force elongation behaviour is close to experimental findings. This indicates that the internal structure of mitotic chromosomes is based on self-organization of the chromatin fibre. We also demonstrate how number and size of loops have a strong influence on the mechanical properties. We suggest that changes in the mechanical characteristics of chromosomes can be explained by an altered internal loop structure. YZ gratefully appreciates funding by the German National Academic Foundation (Studienstiftung des deutschen Volkes) and support by the Heidelberg Graduate School for Mathematical and Computational Methods in the Sciences (HGS MathComp).

The Notch pathway regulates the Second Mitotic Wave cell cycle independently of bHLH proteins.

PubMed

Bhattacharya, Abhishek; Li, Ke; Quiquand, Manon; Rimesso, Gerard; Baker, Nicholas E

2017-11-15

Notch regulates both neurogenesis and cell cycle activity to coordinate precursor cell generation in the differentiating Drosophila eye. Mosaic analysis with mitotic clones mutant for Notch components was used to identify the pathway of Notch signaling that regulates the cell cycle in the Second Mitotic Wave. Although S phase entry depends on Notch signaling and on the transcription factor Su(H), the transcriptional co-activator Mam and the bHLH repressor genes of the E(spl)-Complex were not essential, although these are Su(H) coactivators and targets during the regulation of neurogenesis. The Second Mitotic Wave showed little dependence on ubiquitin ligases neuralized or mindbomb, and although the ligand Delta is required non-autonomously, partial cell cycle activity occurred in the absence of known Notch ligands. We found that myc was not essential for the Second Mitotic Wave. The Second Mitotic Wave did not require the HLH protein Extra macrochaetae, and the bHLH protein Daughterless was required only cell-nonautonomously. Similar cell cycle phenotypes for Daughterless and Atonal were consistent with requirement for neuronal differentiation to stimulate Delta expression, affecting Notch activity in the Second Mitotic Wave indirectly. Therefore Notch signaling acts to regulate the Second Mitotic Wave without activating bHLH gene targets. Copyright © 2017 Elsevier Inc. All rights reserved.

An automated fluorescence videomicroscopy assay for the detection of mitotic catastrophe

PubMed Central

Rello-Varona, S; Kepp, O; Vitale, I; Michaud, M; Senovilla, L; Jemaà, M; Joza, N; Galluzzi, L; Castedo, M; Kroemer, G

2010-01-01

Mitotic catastrophe can be defined as a cell death mode that occurs during or shortly after a prolonged/aberrant mitosis, and can show apoptotic or necrotic features. However, conventional procedures for the detection of apoptosis or necrosis, including biochemical bulk assays and cytofluorometric techniques, cannot discriminate among pre-mitotic, mitotic and post-mitotic death, and hence are inappropriate to monitor mitotic catastrophe. To address this issue, we generated isogenic human colon carcinoma cell lines that differ in ploidy and p53 status, yet express similar amounts of fluorescent biosensors that allow for the visualization of chromatin (histone H2B coupled to green fluorescent protein (GFP)) and centrosomes (centrin coupled to the Discosoma striata red fluorescent protein (DsRed)). By combining high-resolution fluorescence videomicroscopy and automated image analysis, we established protocols and settings for the simultaneous assessment of ploidy, mitosis, centrosome number and cell death (which in our model system occurs mainly by apoptosis). Time-lapse videomicroscopy showed that this approach can be used for the high-throughput detection of mitotic catastrophe induced by three mechanistically distinct anti-mitotic agents (dimethylenastron (DIMEN), nocodazole (NDZ) and paclitaxel (PTX)), and – in this context – revealed an important role of p53 in the control of centrosome number. PMID:21364633

JMJD5 (Jumonji Domain-containing 5) Associates with Spindle Microtubules and Is Required for Proper Mitosis.

PubMed

He, Zhimin; Wu, Junyu; Su, Xiaonan; Zhang, Ye; Pan, Lixia; Wei, Huimin; Fang, Qiang; Li, Haitao; Wang, Da-Liang; Sun, Fang-Lin

2016-02-26

Precise mitotic spindle assembly is a guarantee of proper chromosome segregation during mitosis. Chromosome instability caused by disturbed mitosis is one of the major features of various types of cancer. JMJD5 has been reported to be involved in epigenetic regulation of gene expression in the nucleus, but little is known about its function in mitotic process. Here we report the unexpected localization and function of JMJD5 in mitotic progression. JMJD5 partially accumulates on mitotic spindles during mitosis, and depletion of JMJD5 results in significant mitotic arrest, spindle assembly defects, and sustained activation of the spindle assembly checkpoint (SAC). Inactivating SAC can efficiently reverse the mitotic arrest caused by JMJD5 depletion. Moreover, JMJD5 is found to interact with tubulin proteins and associate with microtubules during mitosis. JMJD5-depleted cells show a significant reduction of α-tubulin acetylation level on mitotic spindles and fail to generate enough interkinetochore tension to satisfy the SAC. Further, JMJD5 depletion also increases the susceptibility of HeLa cells to the antimicrotubule agent. Taken together, these results suggest that JMJD5 plays an important role in regulating mitotic progression, probably by modulating the stability of spindle microtubules. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mitotic Recombination and Genetic Changes in Saccharomyces cerevisiae during Wine Fermentation

PubMed Central

Puig, Sergi; Querol, Amparo; Barrio, Eladio; Pérez-Ortín, José E.

2000-01-01

Natural strains of Saccharomyces cerevisiae are prototrophic homothallic yeasts that sporulate poorly, are often heterozygous, and may be aneuploid. This genomic constitution may confer selective advantages in some environments. Different mechanisms of recombination, such as meiosis or mitotic rearrangement of chromosomes, have been proposed for wine strains. We studied the stability of the URA3 locus of a URA3/ura3 wine yeast in consecutive grape must fermentations. ura3/ura3 homozygotes were detected at a rate of 1 × 10−5 to 3 × 10−5 per generation, and mitotic rearrangements for chromosomes VIII and XII appeared after 30 mitotic divisions. We used the karyotype as a meiotic marker and determined that sporulation was not involved in this process. Thus, we propose a hypothesis for the genome changes in wine yeasts during vinification. This putative mechanism involves mitotic recombination between homologous sequences and does not necessarily imply meiosis. PMID:10788381

Brownian dynamics simulation of fission yeast mitotic spindle formation

NASA Astrophysics Data System (ADS)

Edelmaier, Christopher

2014-03-01

The mitotic spindle segregates chromosomes during mitosis. The dynamics that establish bipolar spindle formation are not well understood. We have developed a computational model of fission-yeast mitotic spindle formation using Brownian dynamics and kinetic Monte Carlo methods. Our model includes rigid, dynamic microtubules, a spherical nuclear envelope, spindle pole bodies anchored in the nuclear envelope, and crosslinkers and crosslinking motor proteins. Crosslinkers and crosslinking motor proteins attach and detach in a grand canonical ensemble, and exert forces and torques on the attached microtubules. We have modeled increased affinity for crosslinking motor attachment to antiparallel microtubule pairs, and stabilization of microtubules in the interpolar bundle. We study parameters controlling the stability of the interpolar bundle and assembly of a bipolar spindle from initially adjacent spindle-pole bodies.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang Bo; Huang Bo; School of Public Health, University of South China, Hengyang, Hunan 421001

Mitotic catastrophe, a form of cell death resulting from abnormal mitosis, is a cytotoxic death pathway as well as an appealing mechanistic strategy for the development of anti-cancer drugs. In this study, 6-bromine-5-hydroxy-4-methoxybenzaldehyde was demonstrated to induce DNA double-strand break, multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human hepatoma HepG2 cells. We used proteomic profiling to identify the differentially expressed proteins underlying mitotic catastrophe. A total of 137 differentially expressed proteins (76 upregulated and 61 downregulated proteins) were identified. Some of the changed proteins have previously been associated with mitotic catastrophe,more » such as DNA-PKcs, FoxM1, RCC1, cyclin E, PLK1-pT210, 14-3-3{sigma} and HSP70. Multiple isoforms of 14-3-3, heat-shock proteins and tubulin were upregulated. Analysis of functional significance revealed that the 14-3-3-mediated signaling network was the most significantly enriched for the differentially expressed proteins. The modulated proteins were found to be involved in macromolecule complex assembly, cell death, cell cycle, chromatin remodeling and DNA repair, tubulin and cytoskeletal organization. These findings revealed the overall molecular events and functional signaling networks associated with spindle disruption and mitotic catastrophe. - Graphical abstract: Display Omitted Research highlights: > 6-bromoisovanillin induced spindle disruption and sustained mitotic arrest, consequently resulted in mitotic catastrophe. > Proteomic profiling identified 137 differentially expressed proteins associated mitotic catastrophe. > The 14-3-3-mediated signaling network was the most significantly enriched for the altered proteins. > The macromolecule complex assembly, cell cycle, chromatin remodeling and DNA repair, tubulin organization were also shown involved in mitotic catastrophe.« less

Identification of Mitosis-Specific Phosphorylation in Mitotic Chromosome-Associated Proteins.

PubMed

Ohta, Shinya; Kimura, Michiko; Takagi, Shunsuke; Toramoto, Iyo; Ishihama, Yasushi

2016-09-02

During mitosis, phosphorylation of chromosome-associated proteins is a key regulatory mechanism. Mass spectrometry has been successfully applied to determine the complete protein composition of mitotic chromosomes, but not to identify post-translational modifications. Here, we quantitatively compared the phosphoproteome of isolated mitotic chromosomes with that of chromosomes in nonsynchronized cells. We identified 4274 total phosphorylation sites and 350 mitosis-specific phosphorylation sites in mitotic chromosome-associated proteins. Significant mitosis-specific phosphorylation in centromere/kinetochore proteins was detected, although the chromosomal association of these proteins did not change throughout the cell cycle. This mitosis-specific phosphorylation might play a key role in regulation of mitosis. Further analysis revealed strong dependency of phosphorylation dynamics on kinase consensus patterns, thus linking the identified phosphorylation sites to known key mitotic kinases. Remarkably, chromosomal axial proteins such as non-SMC subunits of condensin, TopoIIα, and Kif4A, together with the chromosomal periphery protein Ki67 involved in the establishment of the mitotic chromosomal structure, demonstrated high phosphorylation during mitosis. These findings suggest a novel mechanism for regulation of chromosome restructuring in mitosis via protein phosphorylation. Our study generated a large quantitative database on protein phosphorylation in mitotic and nonmitotic chromosomes, thus providing insights into the dynamics of chromatin protein phosphorylation at mitosis onset.

Suspension of Mitotic Activity in Dentate Gyrus of the Hibernating Ground Squirrel

PubMed Central

Popov, Victor I.; Kraev, Igor V.; Ignat'ev, Dmitri A.; Stewart, Michael G.

2011-01-01

Neurogenesis occurs in the adult mammalian hippocampus, a region of the brain important for learning and memory. Hibernation in Siberian ground squirrels provides a natural model to study mitosis as the rapid fall in body temperature in 24 h (from 35-36°C to +4–6°C) permits accumulation of mitotic cells at different stages of the cell cycle. Histological methods used to study adult neurogenesis are limited largely to fixed tissue, and the mitotic state elucidated depends on the specific phase of mitosis at the time of day. However, using an immunohistochemical study of doublecortin (DCX) and BrdU-labelled neurons, we demonstrate that the dentate gyrus of the ground squirrel hippocampus contains a population of immature cells which appear to possess mitotic activity. Our data suggest that doublecortin-labelled immature cells exist in a mitotic state and may represent a renewable pool for generation of new neurons within the dentate gyrus. PMID:21773054

Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast

PubMed Central

Gergely, Zachary R.; Crapo, Ammon; Hough, Loren E.; McIntosh, J. Richard; Betterton, Meredith D.

2016-01-01

Kinesin-8 motor proteins destabilize microtubules. Their absence during cell division is associated with disorganized mitotic chromosome movements and chromosome loss. Despite recent work studying effects of kinesin-8s on microtubule dynamics, it remains unclear whether the kinesin-8 mitotic phenotypes are consequences of their effect on microtubule dynamics, their well-established motor activity, or additional, unknown functions. To better understand the role of kinesin-8 proteins in mitosis, we studied the effects of deletion of the fission yeast kinesin-8 proteins Klp5 and Klp6 on chromosome movements and spindle length dynamics. Aberrant microtubule-driven kinetochore pushing movements and tripolar mitotic spindles occurred in cells lacking Klp5 but not Klp6. Kinesin-8–deletion strains showed large fluctuations in metaphase spindle length, suggesting a disruption of spindle length stabilization. Comparison of our results from light microscopy with a mathematical model suggests that kinesin-8–induced effects on microtubule dynamics, kinetochore attachment stability, and sliding force in the spindle can explain the aberrant chromosome movements and spindle length fluctuations seen. PMID:27146110

Nuclear Chk1 prevents premature mitotic entry.

PubMed

Matsuyama, Makoto; Goto, Hidemasa; Kasahara, Kousuke; Kawakami, Yoshitaka; Nakanishi, Makoto; Kiyono, Tohru; Goshima, Naoki; Inagaki, Masaki

2011-07-01

Chk1 inhibits the premature activation of the cyclin-B1-Cdk1. However, it remains controversial whether Chk1 inhibits Cdk1 in the centrosome or in the nucleus before the G2-M transition. In this study, we examined the specificity of the mouse monoclonal anti-Chk1 antibody DCS-310, with which the centrosome was stained. Conditional Chk1 knockout in mouse embryonic fibroblasts reduced nuclear but not centrosomal staining with DCS-310. In Chk1(+/myc) human colon adenocarcinoma (DLD-1) cells, Chk1 was detected in the nucleus but not in the centrosome using an anti-Myc antibody. Through the combination of protein array and RNAi technologies, we identified Ccdc-151 as a protein that crossreacted with DCS-310 on the centrosome. Mitotic entry was delayed by expression of the Chk1 mutant that localized in the nucleus, although forced immobilization of Chk1 to the centrosome had little impact on the timing of mitotic entry. These results suggest that nuclear but not centrosomal Chk1 contributes to correct timing of mitotic entry.

Two-phase deep convolutional neural network for reducing class skewness in histopathological images based breast cancer detection.

PubMed

Wahab, Noorul; Khan, Asifullah; Lee, Yeon Soo

2017-06-01

Different types of breast cancer are affecting lives of women across the world. Common types include Ductal carcinoma in situ (DCIS), Invasive ductal carcinoma (IDC), Tubular carcinoma, Medullary carcinoma, and Invasive lobular carcinoma (ILC). While detecting cancer, one important factor is mitotic count - showing how rapidly the cells are dividing. But the class imbalance problem, due to the small number of mitotic nuclei in comparison to the overwhelming number of non-mitotic nuclei, affects the performance of classification models. This work presents a two-phase model to mitigate the class biasness issue while classifying mitotic and non-mitotic nuclei in breast cancer histopathology images through a deep convolutional neural network (CNN). First, nuclei are segmented out using blue ratio and global binary thresholding. In Phase-1 a CNN is then trained on the segmented out 80×80 pixel patches based on a standard dataset. Hard non-mitotic examples are identified and augmented; mitotic examples are oversampled by rotation and flipping; whereas non-mitotic examples are undersampled by blue ratio histogram based k-means clustering. Based on this information from Phase-1, the dataset is modified for Phase-2 in order to reduce the effects of class imbalance. The proposed CNN architecture and data balancing technique yielded an F-measure of 0.79, and outperformed all the methods relying on specific handcrafted features, as well as those using a combination of handcrafted and CNN-generated features. Copyright © 2017 Elsevier Ltd. All rights reserved.

Stronger net posterior cortical forces and asymmetric microtubule arrays produce simultaneous centration and rotation of the pronuclear complex in the early Caenorhabditis elegans embryo

PubMed Central

Coffman, Valerie C.; McDermott, Matthew B. A.; Shtylla, Blerta; Dawes, Adriana T.

2016-01-01

Positioning of microtubule-organizing centers (MTOCs) incorporates biochemical and mechanical cues for proper alignment of the mitotic spindle and cell division site. Current experimental and theoretical studies in the early Caenorhabditis elegans embryo assume remarkable changes in the origin and polarity of forces acting on the MTOCs. These changes must occur over a few minutes, between initial centration and rotation of the pronuclear complex and entry into mitosis, and the models do not replicate in vivo timing of centration and rotation. Here we propose a model that incorporates asymmetry in the microtubule arrays generated by each MTOC, which we demonstrate with in vivo measurements, and a similar asymmetric force profile to that required for posterior-directed spindle displacement during mitosis. We find that these asymmetries are capable of and important for recapitulating the simultaneous centration and rotation of the pronuclear complex observed in vivo. The combination of theoretical and experimental evidence provided here offers a unified framework for the spatial organization and forces needed for pronuclear centration, rotation, and spindle displacement in the early C. elegans embryo. PMID:27733624

DOE Office of Scientific and Technical Information (OSTI.GOV)

Era, Saho; Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501; Abe, Takuya

Highlights: Black-Right-Pointing-Pointer SENP1 knockout chicken DT40 cells are hypersensitive to spindle poisons. Black-Right-Pointing-Pointer Spindle poison treatment of SENP1{sup -/-} cells leads to increased mitotic slippage. Black-Right-Pointing-Pointer Mitotic slippage in SENP1{sup -/-} cells associates with apoptosis and endoreplication. Black-Right-Pointing-Pointer SENP1 counteracts sister chromatid separation during mitotic arrest. Black-Right-Pointing-Pointer Plk1-mediated cohesion down-regulation is involved in colcemid cytotoxicity. -- Abstract: SUMO conjugation is a reversible posttranslational modification that regulates protein function. SENP1 is one of the six SUMO-specific proteases present in vertebrate cells and its altered expression is observed in several carcinomas. To characterize SENP1 role in genome integrity, we generated Senp1 knockoutmore » chicken DT40 cells. SENP1{sup -/-} cells show normal proliferation, but are sensitive to spindle poisons. This hypersensitivity correlates with increased sister chromatid separation, mitotic slippage, and apoptosis. To test whether the cohesion defect had a causal relationship with the observed mitotic events, we restored the cohesive status of sister chromatids by introducing the TOP2{alpha}{sup +/-} mutation, which leads to increased catenation, or by inhibiting Plk1 and Aurora B kinases that promote cohesin release from chromosomes during prolonged mitotic arrest. Although TOP2{alpha} is SUMOylated during mitosis, the TOP2{alpha}{sup +/-} mutation had no obvious effect. By contrast, inhibition of Plk1 or Aurora B rescued the hypersensitivity of SENP1{sup -/-} cells to colcemid. In conclusion, we identify SENP1 as a novel factor required for mitotic arrest and cohesion maintenance during prolonged mitotic arrest induced by spindle poisons.« less

Microtubule dynamics in cell division: exploring living cells with polarized light microscopy.

PubMed

Inoué, Shinya

2008-01-01

This Perspective is an account of my early experience while I studied the dynamic organization and behavior of the mitotic spindle and its submicroscopic filaments using polarized light microscopy. The birefringence of spindle filaments in normally dividing plant and animal cells, and those treated by various agents, revealed (a) the reality of spindle fibers and fibrils in healthy living cells; (b) the labile, dynamic nature of the molecular filaments making up the spindle fibers; (c) the mode of fibrogenesis and action of orienting centers; and (d) force-generating properties based on the disassembly and assembly of the fibrils. These studies, which were carried out directly on living cells using improved polarizing microscopes, in fact predicted the reversible assembly properties of microtubules.

Misato Controls Mitotic Microtubule Generation by Stabilizing the TCP-1 Tubulin Chaperone Complex [corrected].

PubMed

Palumbo, Valeria; Pellacani, Claudia; Heesom, Kate J; Rogala, Kacper B; Deane, Charlotte M; Mottier-Pavie, Violaine; Gatti, Maurizio; Bonaccorsi, Silvia; Wakefield, James G

2015-06-29

Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies, but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1) induces G2/M arrest and mitotic catastrophe in human leukemia HL-60 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, Mei-Hua; Liu, Chin-Yu; Lin, Chiao-Min

2012-03-01

2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1), a 2-phenyl-1,8-naphthyridin-4-one (2-PN) derivative, was synthesized and evaluated as an effective antimitotic agent in our laboratory. However, the molecular mechanisms are uncertain. In this study, HKL-1 was demonstrated to induce multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human leukemia HL-60 cells. Western blotting showed that HKL-1 induces mitotic catastrophe in HL-60 cells through regulating mitotic phase-specific kinases (down-regulating CDK1, cyclin B1, CENP-E, and aurora B) and regulating the expression of Bcl-2 family proteins (down-regulating Bcl-2 and up-regulating Bax and Bak), followed by caspase-9/-3 cleavage. These findings suggest that HKL-1more » appears to exert its cytotoxicity toward HL-60 cells in culture by inducing mitotic catastrophe. Highlights: ► HKL-1 is a potential antimitotic agent against HL-60 cells. ► HKL-1 induces spindle disruption and sustained resulted in mitotic catastrophe. ► CENP-E and aurora B protein expressions significantly reduced. ► Bcl-2 family protein expressions altered and caspase-9/-3 activation. ► HKL-1 is an attractive candidate for possible use as a novel antimitotic agent.« less

Effects of the phosphatase inhibitors, okadaic acid, ATPgammaS, and calyculin A on the dividing sand dollar egg.

PubMed

Hamaguchi, Yukihisa; Kuriyama, Ryoko

2002-06-01

The effects of the phosphatase inhibitors, okadaic acid (OA), adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), and calyculin A (CL-A) on anaphase chromosome movement, cytokinesis, and cytoskeletal structures at cell division were examined by being microinjected into mitotic sand dollar eggs. When OA was injected, chromosome movement was inhibited and, moreover, chromosomes were ejected from the polar regions of the mitotic apparatus. By immunofluorescence, microtubules were observed to be severed in the OA-injected eggs, causing the smooth cell surface to be changed to an irregular surface. When ATPgammaS and CL-A were injected, the effect on cell shape was remarkable: In dividing eggs, furrowing stopped within several seconds after injection, small blebs appeared on the cell surface and became large, spherical or dumbbell cell shapes then changed to irregular forms, and subsequently cytoplasmic flow occurred. Microfilament detection revealed that actin accumulation in the cortex, which was not limited to the furrow cortex, occurred shortly after injection. Cortical accumulation of actin is thought to induce force generation and random cortical contraction, and accordingly to result in bleb extrusion from the cortex. Consequently, the phosphatase inhibitors inhibited the transition from mitosis to interphase by mediating cortical accumulation of actin filaments and/or fragmentation of microtubules.

Direct Microtubule-Binding by Myosin-10 Orients Centrosomes toward Retraction Fibers and Subcortical Actin Clouds.

PubMed

Kwon, Mijung; Bagonis, Maria; Danuser, Gaudenz; Pellman, David

2015-08-10

Positioning of centrosomes is vital for cell division and development. In metazoan cells, spindle positioning is controlled by a dynamic pool of subcortical actin that organizes in response to the position of retraction fibers. These actin "clouds" are proposed to generate pulling forces on centrosomes and mediate spindle orientation. However, the motors that pull astral microtubules toward these actin structures are not known. Here, we report that the unconventional myosin, Myo10, couples actin-dependent forces from retraction fibers and subcortical actin clouds to centrosomes. Myo10-mediated centrosome positioning requires its direct microtubule binding. Computational image analysis of large microtubule populations reveals a direct effect of Myo10 on microtubule dynamics and microtubule-cortex interactions. Myo10's role in centrosome positioning is distinct from, but overlaps with, that of dynein. Thus, Myo10 plays a key role in integrating the actin and microtubule cytoskeletons to position centrosomes and mitotic spindles. Copyright © 2015 Elsevier Inc. All rights reserved.

Direct Microtubule-Binding by Myosin-10 Orients Centrosomes toward Retraction Fibers and Subcortical Actin Clouds

PubMed Central

Kwon, Mijung; Bagonis, Maria; Danuser, Gaudenz; Pellman, David

2015-01-01

SUMMARY Positioning of centrosomes is vital for cell division and development. In metazoan cells, spindle positioning is controlled by a dynamic pool of subcortical actin that organizes in response to the position of retraction fibers. These actin “clouds” are proposed to generate pulling forces on centrosomes and mediate spindle orientation. However, the motors that pull astral microtubules toward these actin structures are not known. Here, we report that the unconventional myosin, Myo10, couples actin-dependent forces from retraction fibers and subcortical actin clouds to centrosomes. Myo10-mediated centrosome positioning requires its direct microtubule binding. Computational image analysis of large microtubule populations reveals a direct effect of Myo10 on microtubule dynamics and microtubule-cortex interactions. Myo10’s role in centrosome positioning is distinct from, but overlaps with, that of dynein. Thus, Myo10 plays a key role in integrating the actin and microtubule cytoskeletons to position centrosomes and mitotic spindles. PMID:26235048

Assembly and positioning of actomyosin rings by contractility and planar cell polarity

PubMed Central

Sehring, Ivonne M; Recho, Pierre; Denker, Elsa; Kourakis, Matthew; Mathiesen, Birthe; Hannezo, Edouard; Dong, Bo; Jiang, Di

2015-01-01

The actomyosin cytoskeleton is a primary force-generating mechanism in morphogenesis, thus a robust spatial control of cytoskeletal positioning is essential. In this report, we demonstrate that actomyosin contractility and planar cell polarity (PCP) interact in post-mitotic Ciona notochord cells to self-assemble and reposition actomyosin rings, which play an essential role for cell elongation. Intriguingly, rings always form at the cells′ anterior edge before migrating towards the center as contractility increases, reflecting a novel dynamical property of the cortex. Our drug and genetic manipulations uncover a tug-of-war between contractility, which localizes cortical flows toward the equator and PCP, which tries to reposition them. We develop a simple model of the physical forces underlying this tug-of-war, which quantitatively reproduces our results. We thus propose a quantitative framework for dissecting the relative contribution of contractility and PCP to the self-assembly and repositioning of cytoskeletal structures, which should be applicable to other morphogenetic events. DOI: http://dx.doi.org/10.7554/eLife.09206.001 PMID:26486861

Oocyte formation by mitotically-active germ cells purified from ovaries of reproductive age women

PubMed Central

White, Yvonne A. R.; Woods, Dori C.; Takai, Yasushi; Ishihara, Osamu; Seki, Hiroyuki; Tilly, Jonathan L.

2012-01-01

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a FACS-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically-active cells that exhibit a gene expression profile consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and spontaneously generate 35–50 µm oocytes, as determined by morphology, gene expression and attainment of haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1–2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, like adult mice, possess rare mitotically-active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo. PMID:22366948

Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women.

PubMed

White, Yvonne A R; Woods, Dori C; Takai, Yasushi; Ishihara, Osamu; Seki, Hiroyuki; Tilly, Jonathan L

2012-02-26

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1-2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.

Pericentromere tension is self-regulated by spindle structure in metaphase

PubMed Central

Chacón, Jeremy M.; Mukherjee, Soumya; Schuster, Breanna M.; Clarke, Duncan J.

2014-01-01

During cell division, a mitotic spindle is built by the cell and acts to align and stretch duplicated sister chromosomes before their ultimate segregation into daughter cells. Stretching of the pericentromeric chromatin during metaphase is thought to generate a tension-based signal that promotes proper chromosome segregation. However, it is not known whether the mitotic spindle actively maintains a set point tension magnitude for properly attached sister chromosomes to facilitate robust mechanochemical checkpoint signaling. By imaging and tracking the thermal movements of pericentromeric fluorescent markers in Saccharomyces cerevisiae, we measured pericentromere stiffness and then used the stiffness measurements to quantitatively evaluate the tension generated by pericentromere stretch during metaphase in wild-type cells and in mutants with disrupted chromosome structure. We found that pericentromere tension in yeast is substantial (4–6 pN) and is tightly self-regulated by the mitotic spindle: through adjustments in spindle structure, the cell maintains wild-type tension magnitudes even when pericentromere stiffness is disrupted. PMID:24821839

Pericentromere tension is self-regulated by spindle structure in metaphase.

PubMed

Chacón, Jeremy M; Mukherjee, Soumya; Schuster, Breanna M; Clarke, Duncan J; Gardner, Melissa K

2014-05-12

During cell division, a mitotic spindle is built by the cell and acts to align and stretch duplicated sister chromosomes before their ultimate segregation into daughter cells. Stretching of the pericentromeric chromatin during metaphase is thought to generate a tension-based signal that promotes proper chromosome segregation. However, it is not known whether the mitotic spindle actively maintains a set point tension magnitude for properly attached sister chromosomes to facilitate robust mechanochemical checkpoint signaling. By imaging and tracking the thermal movements of pericentromeric fluorescent markers in Saccharomyces cerevisiae, we measured pericentromere stiffness and then used the stiffness measurements to quantitatively evaluate the tension generated by pericentromere stretch during metaphase in wild-type cells and in mutants with disrupted chromosome structure. We found that pericentromere tension in yeast is substantial (4-6 pN) and is tightly self-regulated by the mitotic spindle: through adjustments in spindle structure, the cell maintains wild-type tension magnitudes even when pericentromere stiffness is disrupted.

High-Resolution Mapping of Two Types of Spontaneous Mitotic Gene Conversion Events in Saccharomyces cerevisiae

PubMed Central

Yim, Eunice; O’Connell, Karen E.; St. Charles, Jordan; Petes, Thomas D.

2014-01-01

Gene conversions and crossovers are related products of the repair of double-stranded DNA breaks by homologous recombination. Most previous studies of mitotic gene conversion events have been restricted to measuring conversion tracts that are <5 kb. Using a genetic assay in which the lengths of very long gene conversion tracts can be measured, we detected two types of conversions: those with a median size of ∼6 kb and those with a median size of >50 kb. The unusually long tracts are initiated at a naturally occurring recombination hotspot formed by two inverted Ty elements. We suggest that these long gene conversion events may be generated by a mechanism (break-induced replication or repair of a double-stranded DNA gap) different from the short conversion tracts that likely reflect heteroduplex formation followed by DNA mismatch repair. Both the short and long mitotic conversion tracts are considerably longer than those observed in meiosis. Since mitotic crossovers in a diploid can result in a heterozygous recessive deleterious mutation becoming homozygous, it has been suggested that the repair of DNA breaks by mitotic recombination involves gene conversion events that are unassociated with crossing over. In contrast to this prediction, we found that ∼40% of the conversion tracts are associated with crossovers. Spontaneous mitotic crossover events in yeast are frequent enough to be an important factor in genome evolution. PMID:24990991

Molecular networks linked by Moesin drive remodeling of the cell cortex during mitosis

PubMed Central

Roubinet, Chantal; Decelle, Barbara; Chicanne, Gaëtan; Dorn, Jonas F.; Payrastre, Bernard; Payre, François; Carreno, Sébastien

2011-01-01

The cortical mechanisms that drive the series of mitotic cell shape transformations remain elusive. In this paper, we identify two novel networks that collectively control the dynamic reorganization of the mitotic cortex. We demonstrate that Moesin, an actin/membrane linker, integrates these two networks to synergize the cortical forces that drive mitotic cell shape transformations. We find that the Pp1-87B phosphatase restricts high Moesin activity to early mitosis and down-regulates Moesin at the polar cortex, after anaphase onset. Overactivation of Moesin at the polar cortex impairs cell elongation and thus cytokinesis, whereas a transient recruitment of Moesin is required to retract polar blebs that allow cortical relaxation and dissipation of intracellular pressure. This fine balance of Moesin activity is further adjusted by Skittles and Pten, two enzymes that locally produce phosphoinositol 4,5-bisphosphate and thereby, regulate Moesin cortical association. These complementary pathways provide a spatiotemporal framework to explain how the cell cortex is remodeled throughout cell division. PMID:21969469

The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles

PubMed Central

Levesque, Aime A.; Compton, Duane A.

2001-01-01

Chromokinesins have been postulated to provide the polar ejection force needed for chromosome congression during mitosis. We have evaluated that possibility by monitoring chromosome movement in vertebrate-cultured cells using time-lapse differential interference contrast microscopy after microinjection with antibodies specific for the chromokinesin Kid. 17.5% of cells injected with Kid-specific antibodies have one or more chromosomes that remain closely opposed to a spindle pole and fail to enter anaphase. In contrast, 82.5% of injected cells align chromosomes in metaphase, progress to anaphase, and display chromosome velocities not significantly different from control cells. However, injected cells lack chromosome oscillations, and chromosome orientation is atypical because chromosome arms extend toward spindle poles during both congression and metaphase. Furthermore, chromosomes cluster into a mass and fail to oscillate when Kid is perturbed in cells containing monopolar spindles. These data indicate that Kid generates the polar ejection force that pushes chromosome arms away from spindle poles in vertebrate-cultured cells. This force increases the efficiency with which chromosomes make bipolar spindle attachments and regulates kinetochore activities necessary for chromosome oscillation, but is not essential for chromosome congression. PMID:11564754

The flavonoid eupatorin inactivates the mitotic checkpoint leading to polyploidy and apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salmela, Anna-Leena; Turku Graduate School of Biomedical Sciences, Turku; Turku Centre for Biotechnology, P.O. Box 123, University of Turku

The spindle assembly checkpoint (SAC) is a conserved mechanism that ensures the fidelity of chromosome distribution in mitosis by preventing anaphase onset until the correct bipolar microtubule-kinetochore attachments are formed. Errors in SAC function may contribute to tumorigenesis by inducing numerical chromosome anomalies (aneuploidy). On the other hand, total disruption of SAC can lead to massive genomic imbalance followed by cell death, a phenomena that has therapeutic potency. We performed a cell-based high-throughput screen with a compound library of 2000 bioactives for novel SAC inhibitors and discovered a plant-derived phenolic compound eupatorin (3 Prime ,5-dihydroxy-4 Prime ,6,7-trimethoxyflavone) as an anti-mitoticmore » flavonoid. The premature override of the microtubule drug-imposed mitotic arrest by eupatorin is dependent on microtubule-kinetochore attachments but not interkinetochore tension. Aurora B kinase activity, which is essential for maintenance of normal SAC signaling, is diminished by eupatorin in cells and in vitro providing a mechanistic explanation for the observed forced mitotic exit. Eupatorin likely has additional targets since eupatorin treatment of pre-mitotic cells causes spindle anomalies triggering a transient M phase delay followed by impaired cytokinesis and polyploidy. Finally, eupatorin potently induces apoptosis in multiple cancer cell lines and suppresses cancer cell proliferation in organotypic 3D cell culture model.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seo, Jae Sung; Kim, Ha Na; Kim, Sun-Jick

Highlights: •NuMA is modified by SUMO-1 in a cell cycle-dependent manner. •NuMA lysine 1766 is the primary target site for SUMOylation. •SUMOylation-deficient NuMA induces multiple spindle poles during mitosis. •SUMOylated NuMA induces microtubule bundling. -- Abstract: Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugationmore » to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis.« less

Biophysical Aspects of Spindle Evolution

NASA Astrophysics Data System (ADS)

Farhadifar, Reza; Baer, Charlie; Needleman, Daniel

2011-03-01

The continual propagation of genetic material from one generation to the next is one of the most basic characteristics of all organisms. In eukaryotes, DNA is segregated into the two daughter cells by a highly dynamic, self-organizing structure called the mitotic spindle. Mitotic spindles can show remarkable variability between tissues and organisms, but there is currently little understanding of the biophysical and evolutionary basis of this diversity. We are studying how spontaneous mutations modify cell division during nematode development. By comparing the mutational variation - the raw material of evolution - with the variation present in nature, we are investigating how the mitotic spindle is shaped over the course of evolution. This combination of quantitative genetics and cellular biophysics gives insight into how the structure and dynamics of the spindle is formed through selection, drift, and biophysical constraints.

Argonaute-1 functions as a mitotic regulator by controlling Cyclin B during Drosophila early embryogenesis.

PubMed

Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Bag, Indira; Hunt, Clayton R; Ramaiah, M Janaki; Pandita, Tej K; Bhadra, Utpal; Pal-Bhadra, Manika

2014-02-01

The role of Ago-1 in microRNA (miRNA) biogenesis has been thoroughly studied, but little is known about its involvement in mitotic cell cycle progression. In this study, we established evidence of the regulatory role of Ago-1 in cell cycle control in association with the G2/M cyclin, cyclin B. Immunostaining of early embryos revealed that the maternal effect gene Ago-1 is essential for proper chromosome segregation, mitotic cell division, and spindle fiber assembly during early embryonic development. Ago-1 mutation resulted in the up-regulation of cyclin B-Cdk1 activity and down-regulation of p53, grp, mei-41, and wee1. The increased expression of cyclin B in Ago-1 mutants caused less stable microtubules and probably does not produce enough force to push the nuclei to the cortex, resulting in a decreased number of pole cells. The role of cyclin B in mitotic defects was further confirmed by suppressing the defects in the presence of one mutant copy of cyclin B. We identified involvement of 2 novel embryonic miRNAs--miR-981 and miR--317-for spatiotemporal regulation of cyclin B. In summary, our results demonstrate that the haploinsufficiency of maternal Ago-1 disrupts mitotic chromosome segregation and spindle fiber assembly via miRNA-guided control during early embryogenesis in Drosophila. The increased expression of cyclin B-Cdk1 and decreased activity of the Cdk1 inhibitor and cell cycle checkpoint proteins (mei-41 and grp) in Ago-1 mutant embryos allow the nuclei to enter into mitosis prematurely, even before completion of DNA replication. Thus, our results have established a novel role of Ago-1 as a regulator of the cell cycle.

Mechanism of APC/CCDC20 activation by mitotic phosphorylation.

PubMed

Qiao, Renping; Weissmann, Florian; Yamaguchi, Masaya; Brown, Nicholas G; VanderLinden, Ryan; Imre, Richard; Jarvis, Marc A; Brunner, Michael R; Davidson, Iain F; Litos, Gabriele; Haselbach, David; Mechtler, Karl; Stark, Holger; Schulman, Brenda A; Peters, Jan-Michael

2016-05-10

Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/C(CDC20) activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/C(CDC20) activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/C(CDC20) activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis.

Mechanism of APC/CCDC20 activation by mitotic phosphorylation

PubMed Central

Qiao, Renping; Weissmann, Florian; Yamaguchi, Masaya; Brown, Nicholas G.; VanderLinden, Ryan; Imre, Richard; Jarvis, Marc A.; Brunner, Michael R.; Davidson, Iain F.; Litos, Gabriele; Haselbach, David; Mechtler, Karl; Stark, Holger; Schulman, Brenda A.; Peters, Jan-Michael

2016-01-01

Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/CCDC20 activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/CCDC20 activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/CCDC20 activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis. PMID:27114510

Cutting edge science: Laser surgery illuminates viscoelasticity of merotelic kinetochores

PubMed Central

Cabello, Simon

2016-01-01

Increasing evidence in eukaryotic cells suggests that mechanical forces are essential for building a robust mitotic apparatus and correcting inappropriate chromosome attachments. In this issue, Cojoc et al. (2016. J. Cell Biol., http://dx.doi.org/10.1083/jcb.201506011) use laser microsurgery in vivo to measure and study the viscoelastic properties of kinetochores. PMID:27002164

Cutting edge science: Laser surgery illuminates viscoelasticity of merotelic kinetochores.

PubMed

Cabello, Simon; Gachet, Yannick; Tournier, Sylvie

2016-03-28

Increasing evidence in eukaryotic cells suggests that mechanical forces are essential for building a robust mitotic apparatus and correcting inappropriate chromosome attachments. In this issue, Cojoc et al. (2016. J. Cell Biol., http://dx.doi.org/10.1083/jcb.201506011) use laser microsurgery in vivo to measure and study the viscoelastic properties of kinetochores. © 2016 Cabello.

Sds22 regulates aurora B activity and microtubule–kinetochore interactions at mitosis

PubMed Central

Posch, Markus; Khoudoli, Guennadi A.; Swift, Sam; King, Emma M.; DeLuca, Jennifer G.

2010-01-01

We have studied Sds22, a conserved regulator of protein phosphatase 1 (PP1) activity, and determined its role in modulating the activity of aurora B kinase and kinetochore–microtubule interactions. Sds22 is required for proper progression through mitosis and localization of PP1 to mitotic kinetochores. Depletion of Sds22 increases aurora B T-loop phosphorylation and the rate of recovery from monastrol arrest. Phospho–aurora B accumulates at kinetochores in Sds22-depleted cells juxtaposed to critical kinetochore substrates. Sds22 modulates sister kinetochore distance and the interaction between Hec1 and the microtubule lattice and, thus, the activation of the spindle assembly checkpoint. These results demonstrate that Sds22 specifically defines PP1 function and localization in mitosis. Sds22 regulates PP1 targeting to the kinetochore, accumulation of phospho–aurora B, and force generation at the kinetochore–microtubule interface. PMID:20921135

Measuring mitotic forces.

PubMed

Ye, Anna A; Maresca, Thomas J

2018-01-01

Productive chromosome movements require that a large multiprotein complex called the kinetochore assemble on sister centromeres. The kinetochore fulfills two critical functions as (1) the physical linkage between chromosomes and spindle microtubules and (2) a mechanomolecular sensor that relays a spindle assembly checkpoint signal delaying anaphase onset until chromosomes are attached to spindle microtubules and bioriented. Given its central roles in such a vital process, the kinetochore is one of the most important force-transducing structures in cells; yet it has been technically challenging to measure kinetochore forces. Barriers to measuring cellular forces have begun to be broken by the development of fluorescence-based tension sensors. In this chapter, two methods will be described for measuring kinetochore forces in living cells and strategies for applying these sensors to other force-transducing processes and molecules will be discussed. © 2018 Elsevier Inc. All rights reserved.

Quantification of surface tension and internal pressure generated by single mitotic cells

NASA Astrophysics Data System (ADS)

Fischer-Friedrich, Elisabeth; Hyman, Anthony A.; Jülicher, Frank; Müller, Daniel J.; Helenius, Jonne

2014-08-01

During mitosis, adherent cells round up, by increasing the tension of the contractile actomyosin cortex while increasing the internal hydrostatic pressure. In the simple scenario of a liquid cell interior, the surface tension is related to the local curvature and the hydrostatic pressure difference by Laplace's law. However, verification of this scenario for cells requires accurate measurements of cell shape. Here, we use wedged micro-cantilevers to uniaxially confine single cells and determine confinement forces while concurrently determining cell shape using confocal microscopy. We fit experimentally measured confined cell shapes to shapes obeying Laplace's law with uniform surface tension and find quantitative agreement. Geometrical parameters derived from fitting the cell shape, and the measured force were used to calculate hydrostatic pressure excess and surface tension of cells. We find that HeLa cells increase their internal hydrostatic pressure excess and surface tension from ~ 40 Pa and 0.2 mNm-1 during interphase to ~ 400 Pa and 1.6 mNm-1 during metaphase. The method introduced provides a means to determine internal pressure excess and surface tension of rounded cells accurately and with minimal cellular perturbation, and should be applicable to characterize the mechanical properties of various cellular systems.

A link between mitotic entry and membrane growth suggests a novel model for cell size control

PubMed Central

Anastasia, Steph D.; Nguyen, Duy Linh; Thai, Vu; Meloy, Melissa; MacDonough, Tracy

2012-01-01

Addition of new membrane to the cell surface by membrane trafficking is necessary for cell growth. In this paper, we report that blocking membrane traffic causes a mitotic checkpoint arrest via Wee1-dependent inhibitory phosphorylation of Cdk1. Checkpoint signals are relayed by the Rho1 GTPase, protein kinase C (Pkc1), and a specific form of protein phosphatase 2A (PP2ACdc55). Signaling via this pathway is dependent on membrane traffic and appears to increase gradually during polar bud growth. We hypothesize that delivery of vesicles to the site of bud growth generates a signal that is proportional to the extent of polarized membrane growth and that the strength of the signal is read by downstream components to determine when sufficient growth has occurred for initiation of mitosis. Growth-dependent signaling could explain how membrane growth is integrated with cell cycle progression. It could also control both cell size and morphogenesis, thereby reconciling divergent models for mitotic checkpoint function. PMID:22451696

PLK1 regulation of PCNT cleavage ensures fidelity of centriole separation during mitotic exit.

PubMed

Kim, Jaeyoun; Lee, Kwanwoo; Rhee, Kunsoo

2015-12-09

Centrioles are duplicated and segregated in close link to the cell cycle. During mitosis, daughter centrioles are disengaged and eventually separated from mother centrioles. New daughter centrioles may be generated only after centriole separation. Therefore, centriole separation is considered a licensing step for centriole duplication. It was previously known that separase specifically cleaves pericentrin (PCNT) during mitotic exit. Here we report that PCNT has to be phosphorylated by PLK1 to be a suitable substrate of separase. Phospho-resistant mutants of PCNT are not cleaved by separase and eventually inhibit centriole separation. Furthermore, phospho-mimetic PCNT mutants rescue centriole separation even in the presence of a PLK1 inhibitor. On the basis on these results, we propose that PLK1 phosphorylation is a priming step for separase-mediated cleavage of PCNT and eventually for centriole separation. PLK1 phosphorylation of PCNT provides an additional layer of regulatory mechanism to ensure the fidelity of centriole separation during mitotic exit.

A link between mitotic entry and membrane growth suggests a novel model for cell size control.

PubMed

Anastasia, Steph D; Nguyen, Duy Linh; Thai, Vu; Meloy, Melissa; MacDonough, Tracy; Kellogg, Douglas R

2012-04-02

Addition of new membrane to the cell surface by membrane trafficking is necessary for cell growth. In this paper, we report that blocking membrane traffic causes a mitotic checkpoint arrest via Wee1-dependent inhibitory phosphorylation of Cdk1. Checkpoint signals are relayed by the Rho1 GTPase, protein kinase C (Pkc1), and a specific form of protein phosphatase 2A (PP2A(Cdc55)). Signaling via this pathway is dependent on membrane traffic and appears to increase gradually during polar bud growth. We hypothesize that delivery of vesicles to the site of bud growth generates a signal that is proportional to the extent of polarized membrane growth and that the strength of the signal is read by downstream components to determine when sufficient growth has occurred for initiation of mitosis. Growth-dependent signaling could explain how membrane growth is integrated with cell cycle progression. It could also control both cell size and morphogenesis, thereby reconciling divergent models for mitotic checkpoint function.

Microtubule Flux and Sliding in Mitotic Spindles of Drosophila EmbryosV⃞

PubMed Central

Brust-Mascher, Ingrid; Scholey, Jonathan M.

2002-01-01

We proposed that spindle morphogenesis in Drosophila embryos involves progression through four transient isometric structures in which a constant spacing of the spindle poles is maintained by a balance of forces generated by multiple microtubule (MT) motors and that tipping this balance drives pole-pole separation. Here we used fluorescent speckle microscopy to evaluate the influence of MT dynamics on the isometric state that persists through metaphase and anaphase A and on pole-pole separation in anaphase B. During metaphase and anaphase A, fluorescent punctae on kinetochore and interpolar MTs flux toward the poles at 0.03 μm/s, too slow to drive chromatid-to-pole motion at 0.11 μm/s, and during anaphase B, fluorescent punctae on interpolar MTs move away from the spindle equator at the same rate as the poles, consistent with MT-MT sliding. Loss of Ncd, a candidate flux motor or brake, did not affect flux in the metaphase/anaphase A isometric state or MT sliding in anaphase B but decreased the duration of the isometric state. Our results suggest that, throughout this isometric state, an outward force exerted on the spindle poles by MT sliding motors is balanced by flux, and that suppression of flux could tip the balance of forces at the onset of anaphase B, allowing MT sliding and polymerization to push the poles apart. PMID:12429839

Cytotoxicity of Spent Pot Liner on Allium cepa root tip cells: A comparative analysis in meristematic cell type on toxicity bioassays.

PubMed

Palmieri, Marcel José; Andrade-Vieira, Larissa Fonseca; Campos, José Marcello Salabert; Dos Santos Gedraite, Leonardo; Davide, Lisete Chamma

2016-11-01

Spent Pot Liner (SPL) is a waste generated during the production of aluminum. It is comprised of a mixture of substances most of which, like cyanide, aluminum and fluoride, are toxic. Previous studies indicate the highly toxic nature of SPL. However studies using cells of the differentiation/elongation zone of the root meristem (referred as M2 cells in this study) after a proper recovery period in water were never considered. Using these cells could be useful to further understanding the toxicity mechanisms of SPL. A comparative approach between the effects on M2 cells and meristematic cells of the proximal meristem zone (referred as M1 cells in this study) could lead to understanding how DNA damage caused by SPL behaves on successive generations of cells. Allium cepa cells were exposed to 4 different concentrations of SPL (2.5, 5, 7.5 and 10gL(-1)) mixed with soil and diluted in a CaCl2 0.01M to simulate the ionic forces naturally encountered on the environment. A solution containing only soil diluted on CaCl2 0.01M was used as control. M1 and M2 cells were evaluated separately, taking into account four different parameters: (1) mitotic alterations (MA); (2) presence of condensed nuclei (CN); (3) mitotic index (MI); (4) presence of micronucleus (MCN). Significant differences were observed between M1 and M2 roots tip cells for these four parameters accessed. M1 cells was more prompt to reveal citogenotoxicity through the higher frequency of MA observed. Meanwhile, for M2 cells higher frequencies of MCN and CN was noticed, followed by a reduction of MI. Also, it was possible to detect significant differences between the tested treatments and the control on every case. These results indicate SPL toxic effects carries on to future cells generations. This emphasizes the need to properly manage this waste. Joint evaluation of cells from both M1 and M2 regions was proven valuable for the evaluation of a series of parameters on all toxicity tests. Copyright © 2016. Published by Elsevier Inc.

Cell biology: scaling and the emergence of evolutionary cell biology.

PubMed

Phillips, Patrick C; Bowerman, Bruce

2015-03-16

A new study investigating the origins of diversity in the structure of the mitotic spindle in nematode embryos, at timescales spanning a few generations to hundreds of millions of years, finds that most features of the spindle evolve via a scaling relationship generated by natural selection acting directly upon embryo size. Copyright © 2015 Elsevier Ltd. All rights reserved.

Interlaboratory assessment of mitotic index by flow cytometry confirms superior reproducibility relative to microscopic scoring.

PubMed

Roberts, D J; Spellman, R A; Sanok, K; Chen, H; Chan, M; Yurt, P; Thakur, A K; DeVito, G L; Murli, H; Stankowski, L F

2012-05-01

A flow cytometric procedure for determining mitotic index (MI) as part of the metaphase chromosome aberrations assay, developed and utilized routinely at Pfizer as part of their standard assay design, has been adopted successfully by Covance laboratories. This method, using antibodies against phosphorylated histone tails (H3PS10) and nucleic acid stain, has been evaluated by the two independent test sites and compared to manual scoring. Primary human lymphocytes were treated with cyclophosphamide, mitomycin C, benzo(a)pyrene, and etoposide at concentrations inducing dose-dependent cytotoxicity. Deming regression analysis indicates that the results generated via flow cytometry (FCM) were more consistent between sites than those generated via microscopy. Further analysis using the Bland-Altman modification of the Tukey mean difference method supports this finding, as the standard deviations (SDs) of differences in MI generated by FCM were less than half of those generated manually. Decreases in scoring variability owing to the objective nature of FCM, and the greater number of cells analyzed, make FCM a superior method for MI determination. In addition, the FCM method has proven to be transferable and easily integrated into standard genetic toxicology laboratory operations. Copyright © 2012 Wiley Periodicals, Inc.

SKAP, an outer kinetochore protein, is required for mouse germ cell development

PubMed Central

Grey, Corinne; Espeut, Julien; Ametsitsi, Rachel; Kumar, Rajeev; Luksza, Malgorzata; Brun, Christine; Verlhac, Marie-Hélene; Suja, José Angél; de Massy, Bernard

2016-01-01

In sexually reproducing organisms, accurate gametogenesis is crucial for the transmission of genetic material from one generation to the next. This requires the faithful segregation of chromosomes during mitotic and meiotic divisions. One of the main players in this process is the kinetochore, a large multi-protein complex that forms at the interface of centromeres and microtubules. Here, we analyzed the expression profile and function of small kinetochore-associated protein (SKAP) in the mouse. We found that two distinct SKAP isoforms are specifically expressed in the germline: a smaller isoform, which is detected in spermatogonia and spermatocytes and localized in the outer mitotic and meiotic kinetochores from metaphase to telophase, and a larger isoform, which is expressed in the cytoplasm of elongating spermatids. We generated SKAP-deficient mice and found that testis size and sperm production were severely reduced in mutant males. This phenotype was partially caused by defects during spermatogonia proliferation before entry into meiosis. We conclude that mouse SKAP, while being dispensable for somatic cell divisions, has an important role in the successful outcome of male gametogenesis. In germ cells, analogous to what has been suggested in studies using immortalized cells, SKAP most likely stabilizes the interaction between kinetochores and microtubules, where it might be needed as an extra safeguard to ensure the correct segregation of mitotic and meiotic chromosomes. PMID:26667018

Antiproliferative Fate of the Tetraploid Formed after Mitotic Slippage and Its Promotion; A Novel Target for Cancer Therapy Based on Microtubule Poisons.

PubMed

Nakayama, Yuji; Inoue, Toshiaki

2016-05-19

Microtubule poisons inhibit spindle function, leading to activation of spindle assembly checkpoint (SAC) and mitotic arrest. Cell death occurring in prolonged mitosis is the first target of microtubule poisons in cancer therapies. However, even in the presence of microtubule poisons, SAC and mitotic arrest are not permanent, and the surviving cells exit the mitosis without cytokinesis (mitotic slippage), becoming tetraploid. Another target of microtubule poisons-based cancer therapy is antiproliferative fate after mitotic slippage. The ultimate goal of both the microtubule poisons-based cancer therapies involves the induction of a mechanism defined as mitotic catastrophe, which is a bona fide intrinsic oncosuppressive mechanism that senses mitotic failure and responds by driving a cell to an irreversible antiproliferative fate of death or senescence. This mechanism of antiproliferative fate after mitotic slippage is not as well understood. We provide an overview of mitotic catastrophe, and explain new insights underscoring a causal association between basal autophagy levels and antiproliferative fate after mitotic slippage, and propose possible improved strategies. Additionally, we discuss nuclear alterations characterizing the mitotic catastrophe (micronuclei, multinuclei) after mitotic slippage, and a possible new type of nuclear alteration (clustered micronuclei).

Identification and purification of a soluble region of BubR1: a critical component of the mitotic checkpoint complex.

PubMed

Yoon, Jongchul; Kang, Yup; Kim, Kyunggon; Park, Jungeun; Kim, Youngsoo

2005-11-01

The mitotic checkpoint complex (MCC) ensures the fidelity of chromosomal segregation, by delaying the onset of anaphase until all sister chromatids have been properly attached to the mitotic spindle. In essence, this MCC-induced delay is achieved via the inhibition of the anaphase-promoting complex (APC). Among the components of the MCC, BubR1 plays two major roles in the functions of the mitotic checkpoint. First, BubR1 is able to inhibit APC activity, either by itself or as a component of the MCC, by sequestering a APC coactivator, known as Cdc20. Second, BubR1 activates mitotic checkpoint signaling cascades by binding to the centromere-associated protein E, a microtubule motor protein. Obtaining highly soluble BubR1 is a prerequisite for the study of its structure. BubR1 is a multi-domain protein, which includes a KEN box motif, a mad3-like region, a Bub3 binding domain, and a kinase domain. We obtained a soluble BubR1 construct using a three-step expression strategy. First, we obtained two constructs from BLAST sequence homology searches, both of which were expressed abundantly in the inclusion bodies. We then adjusted the lengths of the two constructs by secondary structure prediction, thereby generating partially soluble constructs. Third, we optimized the solubility of the two constructs by either chopping or adding a few residues at the C-terminus. Finally, we obtained a highly soluble BubR1 construct via the Escherichia coli expression system, which allowed for a yield of 10.8 mg/L culture. This report may provide insight into the design of highly soluble constructs of insoluble multi-domain proteins.

Belinostat and vincristine demonstrate mutually synergistic cytotoxicity associated with mitotic arrest and inhibition of polyploidy in a preclinical model of aggressive diffuse large B cell lymphoma.

PubMed

Havas, Aaron P; Rodrigues, Kameron B; Bhakta, Anvi; Demirjian, Joseph A; Hahn, Seongmin; Tran, Jack; Scavello, Margarethakay; Tula-Sanchez, Ana A; Zeng, Yi; Schmelz, Monika; Smith, Catharine L

2016-12-01

Diffuse Large B-cell lymphoma (DLBCL) is an aggressive malignancy that has a 60 percent 5-year survival rate, highlighting a need for new therapeutic approaches. Histone deacetylase inhibitors (HDACi) are novel therapeutics being clinically-evaluated in combination with a variety of other drugs. However, rational selection of companion therapeutics for HDACi is difficult due to their poorly-understood, cell-type specific mechanisms of action. To address this, we developed a pre-clinical model system of sensitivity and resistance to the HDACi belinostat using DLBCL cell lines. In the current study, we demonstrate that cell lines sensitive to the cytotoxic effects of HDACi undergo early mitotic arrest prior to apoptosis. In contrast, HDACi-resistant cell lines complete mitosis after a short delay and arrest in G1. To force mitotic arrest in HDACi-resistant cell lines, we used low dose vincristine or paclitaxel in combination with belinostat and observed synergistic cytotoxicity. Belinostat curtails vincristine-induced mitotic arrest and triggers a strong apoptotic response associated with downregulated MCL-1 expression and upregulated BIM expression. Resistance to microtubule targeting agents (MTAs) has been associated with their propensity to induce polyploidy and thereby increase the probability of genomic instability that enables cancer progression. Co-treatment with belinostat effectively eliminated a vincristine-induced, actively cycling polyploid cell population. Our study demonstrates that vincristine sensitizes DLBCL cells to the cytotoxic effects of belinostat and that belinostat prevents polyploidy that could cause vincristine resistance. Our findings provide a rationale for using low dose MTAs in conjunction with HDACi as a potential therapeutic strategy for treatment of aggressive DLBCL.

Human Nek7-interactor RGS2 is required for mitotic spindle organization.

PubMed

de Souza, Edmarcia Elisa; Hehnly, Heidi; Perez, Arina Marina; Meirelles, Gabriela Vaz; Smetana, Juliana Helena Costa; Doxsey, Stephen; Kobarg, Jörg

2015-01-01

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.

Prognostic value of mitotic counts in breast cancer of Saudi Arabian patients.

PubMed

Buhmeida, Abdelbaset; Al-Maghrabi, Jaudah; Merdad, Adnan; Al-Thubaity, Fatima; Chaudhary, Adeel; Gari, Mamdooh; Abuzenadah, Adel; Collan, Yrjö; Syrjänen, Kari; Al-Qahtani, Mohammed

2011-01-01

Quantitative methods in combination with other objective prognostic criteria can improve the evaluation of a cancer patient's prognosis, and possibly predict response to therapy. One of the important prognostic and predictive markers is the mitotic count, which has proven valuable in many aspects. In this study, the prognostic value of the mitotic count was assessed in breast cancer (BC) patients in Saudi Arabia. The study comprised a series of 87 patients diagnosed and treated for breast cancer at the Departments of Surgery and Oncology, King Abdul-Aziz University Hospital, between 2000 and 2008. Mitotic counts were carried out using a standard laboratory microscope (objective, × 40; field diameter, 420 μm). The number of mitotic figures in 10 consecutive high-power fields (hpf) from the most cellular area of the sample gave the mitotic activity index (MAI, mitotic figures/10 hpf). The standardized mitotic index (SMI) recorded the mitotic count as the number of mitotic figures by area of the neoplastic tissue in the microscopic field, thus the number of mitoses in 10 consecutive fields was corrected for the volume fraction and field size (mitotic figures/mm²). The means of MAI and SMI of the tumors in the entire series of 87 patients were 15 mitotic figures/10 hpf (range 4-45) and 4 mitotic figures/mm² (range 1-9), respectively. The mitotic counts were higher in advanced stages than in early cancer (p < 0.04). The mitotic counts were significantly larger in patients with high-grade tumor (p < 0.004) and in cases with tumor metastasis (p < 0.004). The mitotic counts were also significantly larger in the recurrent cases than in non-recurrent ones (p < 0.02). The quantitatively measurable mitotic counts of cancer cell nuclei are of significant prognostic value in invasive ductal carcinoma of the breast in Saudi Arabia and the mean cut-off values of MAI and SMI can be applied as objective (quantitative) criteria to distinguish breast cancer patients into groups with favorable and less favorable prognosis.

Pyridine-pyrimidine amides that prevent HGF-induced epithelial scattering by two distinct mechanisms.

PubMed

Siddiqui-Jain, Adam; Hoj, Jacob P; Hargiss, J Blade; Hoj, Taylor H; Payne, Carter J; Ritchie, Collin A; Herron, Steven R; Quinn, Colette; Schuler, Jeffrey T; Hansen, Marc D H

2017-09-01

Stimulation of cultured epithelial cells with scatter factor/hepatocyte growth factor (HGF) results in individual cells detaching and assuming a migratory and invasive phenotype. Epithelial scattering recapitulates cancer progression and studies have implicated HGF signaling as a driver of cancer metastasis. Inhibitors of HGF signaling have been proposed to act as anti-cancer agents. We previously screened a small molecule library for compounds that block HGF-induced epithelial scattering. Most hits identified in this screen exhibit anti-mitotic properties. Here we assess the biological mechanism of a compound that blocks HGF-induced scattering with limited anti-mitotic activity. Analogs of this compound have one of two distinct activities: inhibiting either cell migration or cell proliferation with cell cycle arrest in G2/M. Each activity bears unique structure-activity relationships. The mechanism of action of anti-mitotic compounds is by inhibition of microtubule polymerization; these compounds entropically and enthalpically bind tubulin in the colchicine binding site, generating a conformational change in the tubulin dimer. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mathematical modeling and numerical simulation of the mitotic spindle orientation system.

PubMed

Ibrahim, Bashar

2018-05-21

The mitotic spindle orientation and position is crucial for the fidelity of chromosome segregation during asymmetric cell division to generate daughter cells with different sizes or fates. This mechanism is best understood in the budding yeast Saccharomyces cerevisiae, named the spindle position checkpoint (SPOC). The SPOC inhibits cells from exiting mitosis until the mitotic spindle is properly oriented along the mother-daughter polarity axis. Despite many experimental studies, the mechanisms underlying SPOC regulation remains elusive and unexplored theoretically. Here, a minimal mathematical is developed to describe SPOC activation and silencing having autocatalytic feedback-loop. Numerical simulations of the nonlinear ordinary differential equations (ODEs) model accurately reproduce the phenotype of SPOC mechanism. Bifurcation analysis of the nonlinear ODEs reveals the orientation dependency on spindle pole bodies, and how this dependence is altered by parameter values. These results provide for systems understanding on the molecular organization of spindle orientation system via mathematical modeling. The presented mathematical model is easy to understand and, within the above mentioned context, can be used as a base for further development of quantitative models in asymmetric cell-division. Copyright © 2018. Published by Elsevier Inc.

Regulators of alternative polyadenylation operate at the transition from mitosis to meiosis.

PubMed

Shan, Lingjuan; Wu, Chan; Chen, Di; Hou, Lei; Li, Xin; Wang, Lixia; Chu, Xiao; Hou, Yifeng; Wang, Zhaohui

2017-02-20

In the sexually reproductive organisms, gametes are produced by meiosis following a limited mitotic amplification. However, the intrinsic program switching cells from mitotic to meiotic cycle is unclear. Alternative polyadenylation (APA) is a highly conserved means of gene regulation and is achieved by the RNA 3'-processing machinery to generate diverse 3'UTR profiles. In Drosophila spermatogenesis, we observed distinct profiles of transcriptome-wide 3'UTR between mitotic and meiotic cells. In mutant germ cells stuck in mitosis, 3'UTRs of hundreds of genes were consistently shifted. Remarkably, altering the levels of multiple 3'-processing factors disrupted germline's progression to meiosis, indicative of APA's active role in this transition. An RNA-binding protein (RBP) Tut could directly bind 3'UTRs of 3'-processing factors whose expressions were repressed in the presence of Tut-containing complex. Further, we demonstrated that this RBP complex could execute the repression post-transcriptionally by recruiting CCR4/Twin of deadenylation complex. Thus, we propose that an RBP complex regulates the dynamic APA profile to promote the mitosis-to-meiosis transition. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Arsenite inhibits mitotic division and perturbs spindle dynamics in HeLa S3 cells.

PubMed

Huang, S C; Lee, T C

1998-05-01

Arsenical compounds, known to be human carcinogens, were shown to disturb cell cycle progression and induce cytogenetic alterations in a variety of cell systems. We report here that a 24 h treatment of arsenite induced mitotic accumulation in human cell lines. HeLa S3 and KB cells were most susceptible: 35% of the total cell population was arrested at the mitotic stage after treatment with 5 microM sodium arsenite in HeLa S3 cells and after 10 microM in KB cells. Under a microscope, we observed abnormal mitotic figures in arsenite-arrested mitotic cells, including deranged chromosome congression, elongated polar distance of mitotic spindle, and enhanced microtubule immunofluorescence. The spindle microtubules of arsenite-arrested mitotic cells were more resistant to nocodazole-induced dissolution than those of control mitotic cells. According to turbidity assay, arsenite at concentrations below 100 microM significantly enhanced polymerization of tubulins. Since spindle dynamics play a crucial role in mitotic progression, our results suggest that arsenite-induced mitotic arrest may be due to arsenite's effects on attenuation of spindle dynamics.

Loss of function of Saccharomyces cerevisiae kinesin-related CIN8 and KIP1 is suppressed by KAR3 motor domain mutations.

PubMed

Hoyt, M A; He, L; Totis, L; Saunders, W S

1993-09-01

The kinesin-related products of the CIN8 and KIP1 genes of Saccharomyces cerevisiae redundantly perform an essential function in mitosis. The action of either gene-product is required for an outwardly directed force that acts upon the spindle poles. We have selected mutations that suppress the temperature-sensitivity of a cin8-temperature-sensitive kip1-delta strain. The extragenic suppressors analyzed were all found to be alleles of the KAR3 gene. KAR3 encodes a distinct kinesin-related protein whose action antagonizes Cin8p/Kip1p function. All seven alleles analyzed were altered within the region of KAR3 that encodes the putative force-generating (or "motor") domain. These mutations also suppressed the inviability associated with the cin8-delta kip1-delta genotype, a property not shared by a deletion of KAR3. Other properties of the suppressing alleles revealed that they were not null for function. Six of the seven were unaffected for the essential karyogamy and meiosis properties of KAR3 and the seventh was dominant for the suppressing trait. Our findings suggest that despite an antagonistic relationship between Cin8p/Kip1p and Kar3p, aspects of their mitotic roles may be similar.

Measuring APC/C-Dependent Ubiquitylation In Vitro.

PubMed

Jarvis, Marc A; Brown, Nicholas G; Watson, Edmond R; VanderLinden, Ryan; Schulman, Brenda A; Peters, Jan-Michael

2016-01-01

The anaphase-promoting complex/cyclosome (APC/C) is a 1.2 MDa ubiquitin ligase complex with important functions in both proliferating and post-mitotic differentiated cells. In proliferating cells, APC/C controls cell cycle progression by targeting inhibitors of chromosome segregation and mitotic exit for degradation by the 26S proteasome. To understand how APC/C recruits and ubiquitylates its substrate proteins and how these processes are controlled, it is essential to analyze APC/C activity in vitro. In the past, such experiments have been limited by the fact that large quantities of purified APC/C were difficult to obtain and that mutated versions of the APC/C could not be easily generated. In this chapter we review recent advances in generating and purifying recombinant forms of the human APC/C and its co-activators, using methods that are scalable and compatible with mutagenesis. We also describe a method that allows the quantitative analysis of APC/C activity using fluorescently labeled substrate proteins.

Genome co-amplification upregulates a mitotic gene network activity that predicts outcome and response to mitotic protein inhibitors in breast cancer

DOE PAGES

Hu, Zhi; Mao, Jian-Hua; Curtis, Christina; ...

2016-07-01

Background: High mitotic activity is associated with the genesis and progression of many cancers. Small molecule inhibitors of mitotic apparatus proteins are now being developed and evaluated clinically as anticancer agents. With clinical trials of several of these experimental compounds underway, it is important to understand the molecular mechanisms that determine high mitotic activity, identify tumor subtypes that carry molecular aberrations that confer high mitotic activity, and to develop molecular markers that distinguish which tumors will be most responsive to mitotic apparatus inhibitors. Methods: We identified a coordinately regulated mitotic apparatus network by analyzing gene expression profiles for 53 malignantmore » and non-malignant human breast cancer cell lines and two separate primary breast tumor datasets. We defined the mitotic network activity index (MNAI) as the sum of the transcriptional levels of the 54 coordinately regulated mitotic apparatus genes. The effect of those genes on cell growth was evaluated by small interfering RNA (siRNA). Results: High MNAI was enriched in basal-like breast tumors and was associated with reduced survival duration and preferential sensitivity to i nhibitors of the mitotic apparatus proteins, polo-like kinase, centromere associated protein E and aurora kinase designated GSK462364, GSK923295 and GSK1070916, respectively. Co-amplification of regions of chromosomes 8q24, 10p15-p12, 12p13, and 17q24-q25 was associated with the transcriptional upregulation of this network of 54 mitotic apparatus genes, and we identify transcription factors that localize to these regions and putatively regulate mitotic activity. Knockdown of the mitotic network by siRNA identified 22 genes that might be considered as additional therapeutic targets for this clinically relevant patient subgroup. Conclusions: We define a molecular signature which may guide therapeutic approaches for tumors with high mitotic network activity.« less

Genome co-amplification upregulates a mitotic gene network activity that predicts outcome and response to mitotic protein inhibitors in breast cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Zhi; Mao, Jian-Hua; Curtis, Christina

Background: High mitotic activity is associated with the genesis and progression of many cancers. Small molecule inhibitors of mitotic apparatus proteins are now being developed and evaluated clinically as anticancer agents. With clinical trials of several of these experimental compounds underway, it is important to understand the molecular mechanisms that determine high mitotic activity, identify tumor subtypes that carry molecular aberrations that confer high mitotic activity, and to develop molecular markers that distinguish which tumors will be most responsive to mitotic apparatus inhibitors. Methods: We identified a coordinately regulated mitotic apparatus network by analyzing gene expression profiles for 53 malignantmore » and non-malignant human breast cancer cell lines and two separate primary breast tumor datasets. We defined the mitotic network activity index (MNAI) as the sum of the transcriptional levels of the 54 coordinately regulated mitotic apparatus genes. The effect of those genes on cell growth was evaluated by small interfering RNA (siRNA). Results: High MNAI was enriched in basal-like breast tumors and was associated with reduced survival duration and preferential sensitivity to i nhibitors of the mitotic apparatus proteins, polo-like kinase, centromere associated protein E and aurora kinase designated GSK462364, GSK923295 and GSK1070916, respectively. Co-amplification of regions of chromosomes 8q24, 10p15-p12, 12p13, and 17q24-q25 was associated with the transcriptional upregulation of this network of 54 mitotic apparatus genes, and we identify transcription factors that localize to these regions and putatively regulate mitotic activity. Knockdown of the mitotic network by siRNA identified 22 genes that might be considered as additional therapeutic targets for this clinically relevant patient subgroup. Conclusions: We define a molecular signature which may guide therapeutic approaches for tumors with high mitotic network activity.« less

Human Nek7-interactor RGS2 is required for mitotic spindle organization

PubMed Central

de Souza, Edmarcia Elisa; Hehnly, Heidi; Perez, Arina Marina; Meirelles, Gabriela Vaz; Smetana, Juliana Helena Costa; Doxsey, Stephen; Kobarg, Jörg

2015-01-01

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization. PMID:25664600

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Chundong; Zhang, Ying; Li, Yi

Recently, we have demonstrated that proline-rich protein 11 (PRR11) is a novel tumor-related gene product likely implicated in the regulation of cell cycle progression as well as lung cancer development. However, its precise role in cell cycle progression remains unclear. In the present study, we have further investigated the expression pattern and functional implication of PRR11 during cell cycle in detail in human lung carcinoma-derived H1299 cells. According to our immunofluorescence study, PRR11 was expressed largely in cytoplasm, the amount of PRR11 started to increase in the late S phase, and was retained until just before mitotic telophase. Consistent withmore » those observations, siRNA-mediated knockdown of PRR11 caused a significant cell cycle arrest in the late S phase. Intriguingly, the treatment with dNTPs further augmented PRR11 silencing-mediated S phase arrest. Moreover, knockdown of PRR11 also resulted in a remarkable retardation of G2/M progression, and PRR11-knockdown cells subsequently underwent G2 phase cell cycle arrest accompanied by obvious mitotic defects such as multipolar spindles and multiple nuclei. In addition, forced expression of PRR11 promoted the premature Chromatin condensation (PCC), and then proliferation of PRR11-expressing cells was massively attenuated and induced apoptosis. Taken together, our current observations strongly suggest that PRR11, which is strictly regulated during cell cycle progression, plays a pivotal role in the regulation of accurate cell cycle progression through the late S phase to mitosis. - Highlights: • PRR11 started to increase in the late S phase and was retained until just before mitotic telophase. • PRR11-knockdown caused a significant cell cycle arrest in the late S phase and G2 phase. • The treatment with dNTPs further augmented PRR11 silencing-mediated S phase arrest. • PRR11-knockdown led to multipolar spindles and multiple nuclei. • Forced expression of PRR11 promoted the PCC and inhibited cell proliferation.« less

Phospholipase D function in Saccharomyces cerevisiae.

PubMed

Mendonsa, Rima; Engebrecht, JoAnne

2009-09-01

Phosphatidylinositol 4,5-bisphosphate-regulated phosphatidylcholine-specific phospholipase D is conserved from yeast to man. The essential role of this enzyme in yeast is to mediate the fusion of Golgi and endosome-derived vesicles to generate the prospore membrane during the developmental program of sporulation, through the production of the fusogenic lipid phosphatidic acid. In addition to recruiting proteins required for fusion, phosphatidic acid is believed to lower the energy barrier to stimulate membrane curvature. During mitotic growth, phospholipase D activity is dispensable unless the major phosphatidylinositol/phosphatidylcholine transfer protein is absent; it also appears to play a nonessential role in the mating signal transduction pathway. The regulation of phospholipase D activity during both sporulation and mitotic growth is still not fully understood and awaits further characterization.

Loss of p53 induces M-phase retardation following G2 DNA damage checkpoint abrogation.

PubMed

Minemoto, Yuzuru; Uchida, Sanae; Ohtsubo, Motoaki; Shimura, Mari; Sasagawa, Toshiyuki; Hirata, Masato; Nakagama, Hitoshi; Ishizaka, Yukihito; Yamashita, Katsumi

2003-04-01

Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.

Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties.

PubMed

Burkhart, Annette; Thomsen, Louiza Bohn; Thomsen, Maj Schneider; Lichota, Jacek; Fazakas, Csilla; Krizbai, István; Moos, Torben

2015-08-07

Primary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity. Primary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation. The cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly, transfection of BCECs exhibiting BBB characteristics did not alter the integrity of the BCECs cell layer. The data clearly indicate that non-viral gene therapy of BCECs is possible in primary culture conditions with an intact BBB.

Electron tomography of the microtubule cytoskeleton in multinucleated hyphae of Ashbya gossypii.

PubMed

Gibeaux, Romain; Lang, Claudia; Politi, Antonio Z; Jaspersen, Sue L; Philippsen, Peter; Antony, Claude

2012-12-01

We report the mechanistic basis guiding the migration pattern of multiple nuclei in hyphae of Ashbya gossypii. Using electron tomography, we reconstructed the cytoplasmic microtubule (cMT) cytoskeleton in three tip regions with a total of 13 nuclei and also the spindle microtubules of four mitotic nuclei. Each spindle pole body (SPB) nucleates three cMTs and most cMTs above a certain length grow according to their plus-end structure. Long cMTs closely align for several microns along the cortex, presumably marking regions where dynein generates pulling forces on nuclei. Close proximity between cMTs emanating from adjacent nuclei was not observed. The majority of nuclei carry duplicated side-by-side SPBs, which together emanate an average of six cMTs, in most cases in opposite orientation with respect to the hyphal growth axis. Such cMT arrays explain why many nuclei undergo short-range back and forth movements. Only occasionally do all six cMTs orient in one direction, a precondition for long-range nuclear bypassing. Following mitosis, daughter nuclei carry a single SPB with three cMTs. The increased probability that all three cMTs orient in one direction explains the high rate of nuclear bypassing observed in these nuclei. The A. gossypii mitotic spindle was found to be structurally similar to that of Saccharomyces cerevisiae in terms of nuclear microtubule (nMT) number, length distribution and three-dimensional organization even though the two organisms differ significantly in chromosome number. Our results suggest that two nMTs attach to each kinetochore in A. gossypii and not only one nMT like in S. cerevisiae.

Genome instability in Novel Lolium multiflorum x L. arundinaceum hybrids

USDA-ARS?s Scientific Manuscript database

We have identified a method whereby Lolium multiflorum (Lm) or L. arundinaceum (Fa) genomes are preferentially eliminated through a mitotic loss behavior in interspecific Lm x Fa F1 hybrids,generating either dihaploid Lm lines or Fa lines. The genome instability has been visualized phenotypically an...

Multi-scale computational study of the mechanical regulation of cell mitotic rounding in epithelia

PubMed Central

Xu, Zhiliang; Zartman, Jeremiah J.; Alber, Mark

2017-01-01

Mitotic rounding during cell division is critical for preventing daughter cells from inheriting an abnormal number of chromosomes, a condition that occurs frequently in cancer cells. Cells must significantly expand their apical area and transition from a polygonal to circular apical shape to achieve robust mitotic rounding in epithelial tissues, which is where most cancers initiate. However, how cells mechanically regulate robust mitotic rounding within packed tissues is unknown. Here, we analyze mitotic rounding using a newly developed multi-scale subcellular element computational model that is calibrated using experimental data. Novel biologically relevant features of the model include separate representations of the sub-cellular components including the apical membrane and cytoplasm of the cell at the tissue scale level as well as detailed description of cell properties during mitotic rounding. Regression analysis of predictive model simulation results reveals the relative contributions of osmotic pressure, cell-cell adhesion and cortical stiffness to mitotic rounding. Mitotic area expansion is largely driven by regulation of cytoplasmic pressure. Surprisingly, mitotic shape roundness within physiological ranges is most sensitive to variation in cell-cell adhesivity and stiffness. An understanding of how perturbed mechanical properties impact mitotic rounding has important potential implications on, amongst others, how tumors progressively become more genetically unstable due to increased chromosomal aneuploidy and more aggressive. PMID:28531187

Functional reprogramming of polyploidization in megakaryocytes.

PubMed

Trakala, Marianna; Rodríguez-Acebes, Sara; Maroto, María; Symonds, Catherine E; Santamaría, David; Ortega, Sagrario; Barbacid, Mariano; Méndez, Juan; Malumbres, Marcos

2015-01-26

Polyploidization is a natural process that frequently accompanies differentiation; its deregulation is linked to genomic instability and cancer. Despite its relevance, why cells select different polyploidization mechanisms is unknown. Here we report a systematic genetic analysis of endomitosis, a process in which megakaryocytes become polyploid by entering mitosis but aborting anaphase. Whereas ablation of the APC/C cofactor Cdc20 results in mitotic arrest and severe thrombocytopenia, lack of the kinases Aurora-B, Cdk1, or Cdk2 does not affect megakaryocyte polyploidization or platelet levels. Ablation of Cdk1 forces a switch to endocycles without mitosis, whereas polyploidization in the absence of Cdk1 and Cdk2 occurs in the presence of aberrant re-replication events. Importantly, ablation of these kinases rescues the defects in Cdc20 null megakaryocytes. These findings suggest that endomitosis can be functionally replaced by alternative polyploidization mechanisms in vivo and provide the cellular basis for therapeutic approaches aimed to discriminate mitotic and polyploid cells. Copyright © 2015 Elsevier Inc. All rights reserved.

A Regulatory Switch Alters Chromosome Motions at the Metaphase to Anaphase Transition

PubMed Central

Su, Kuan-Chung; Barry, Zachary; Schweizer, Nina; Maiato, Helder; Bathe, Mark; Cheeseman, Iain McPherson

2016-01-01

Summary To achieve chromosome segregation during mitosis, sister chromatids must undergo a dramatic change in their behavior to switch from balanced oscillations at the metaphase plate to directed poleward motion during anaphase. However, the factors that alter chromosome behavior at the metaphase-to-anaphase transition remain incompletely understood. Here, we perform time-lapse imaging to analyze anaphase chromosome dynamics in human cells. Using multiple directed biochemical, genetic, and physical perturbations, our results demonstrate that differences in the global phosphorylation states between metaphase and anaphase are the major determinant of chromosome motion dynamics. Indeed, causing a mitotic phosphorylation state to persist into anaphase produces dramatic metaphase-like oscillations. These induced oscillations depend on both kinetochore-derived and polar ejection forces that oppose poleward motion. Thus, our analysis of anaphase chromosome motion reveals that dephosphorylation of multiple mitotic substrates is required to suppress metaphase chromosome oscillatory motions and achieve directed poleward motion for successful chromosome segregation. PMID:27829144

Xanthium strumarium extract inhibits mammalian cell proliferation through mitotic spindle disruption mediated by xanthatin.

PubMed

Sánchez-Lamar, Angel; Piloto-Ferrer, Janet; Fiore, Mario; Stano, Pasquale; Cozzi, Renata; Tofani, Daniela; Cundari, Enrico; Francisco, Marbelis; Romero, Aylema; González, Maria L; Degrassi, Francesca

2016-12-24

Xanthium strumarium L. is a member of the Asteraceae family popularly used with multiple therapeutic purposes. Whole extracts of this plant have shown anti-mitotic activity in vitro suggesting that some components could induce mitotic arrest in proliferating cells. Aim of the present work was to characterize the anti-mitotic properties of the X. strumarium whole extract and to isolate and purify active molecule(s). The capacity of the whole extract to inhibit mitotic progression in mammalian cultured cells was investigated to identify its anti-mitotic activity. Isolation of active component(s) was performed using a bioassay-guided multistep separation procedure in which whole extract was submitted to a progressive process of fractionation and fractions were challenged for their anti-mitotic activity. Our results show for the first time that X. strumarium whole extract inhibits assembly of the mitotic spindle and spindle-pole separation, thereby heavily affecting mitosis, impairing the metaphase to anaphase transition and inducing apoptosis. The purification procedure led to a fraction with an anti-mitotic activity comparable to that of the whole extract. Chemical analysis of this fraction showed that its major component was xanthatin. The present work shows a new activity of X. strumarium extract, i.e. the alteration of the mitotic apparatus in cultured cells that may be responsible for the anti-proliferative activity of the extract. Anti-mitotic activity is shown to be mainly exerted by xanthatin. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Roles of CDK and DDK in Genome Duplication and Maintenance: Meiotic Singularities.

PubMed

Gómez-Escoda, Blanca; Wu, Pei-Yun Jenny

2017-03-20

Cells reproduce using two types of divisions: mitosis, which generates two daughter cells each with the same genomic content as the mother cell, and meiosis, which reduces the number of chromosomes of the parent cell by half and gives rise to four gametes. The mechanisms that promote the proper progression of the mitotic and meiotic cycles are highly conserved and controlled. They require the activities of two types of serine-threonine kinases, the cyclin-dependent kinases (CDKs) and the Dbf4-dependent kinase (DDK). CDK and DDK are essential for genome duplication and maintenance in both mitotic and meiotic divisions. In this review, we aim to highlight how these kinases cooperate to orchestrate diverse processes during cellular reproduction, focusing on meiosis-specific adaptions of their regulation and functions in DNA metabolism.

Inhibition of the Ras-ERK pathway in mitotic COS7 cells is due to the inability of EGFR/Raf to transduce EGF signaling to downstream proteins.

PubMed

Shi, Huaiping; Zhang, Tianying; Yi, Yongqing; Ma, Yue

2016-06-01

Although previous studies have shown that Ras-ERK signaling in mitosis is closed due to the inhibition of signal transduction, the events involved in the molecular mechanisms are still unclear. In the present study, we investigated the Ras-ERK signaling pathway in mitotic COS7 cells. The results demonstrated that treatment with epidermal growth factor (EGF) failed to increase the endocytosis of EGF-EGFR (EGF receptor) complexes in mitotic COS7 cells, although a large amount of endosomes were found in asynchronous COS7 cells. Clathrin expression levels in mitotic COS7 cells were inhibited whereas caveolin expression levels in mitotic COS7 cells were almost unaffected. Y1068 and Y1086 residues of EGFR in the mitotic COS7 cells were activated. However, Grb2 and Shc in the mitotic COS7 cells did not bind to activated EGFR. Ras activity was inhibited in the mitotic COS7 cells whereas its downstream protein, Raf, was obviously phosphorylated by EGF in mitosis. Treatment with phorbol 12-myristate 13-acetate (PMA) also increased the phosphorylation levels of Raf in the mitotic COS7 cells. Nevertheless, Raf phosphorylation in mitosis was significantly inhibited by AG1478. Lastly, activation of EGF-mediated MEK and ERK in the mitotic COS7 cells was obviously inhibited. In summary, our results suggest that the Ras-ERK pathway is inhibited in mitotic COS7 cells which may be the dual result of the difficulty in the transduction of EGF signaling by EGFR or Raf to downstream proteins.

Cell Death During Crisis Is Mediated by Mitotic Telomere Deprotection

PubMed Central

Hayashi, Makoto T.; Cesare, Anthony J.; Rivera, Teresa; Karlseder, Jan

2015-01-01

Tumour formation is blocked by two barriers, replicative senescence and crisis1. Senescence is triggered by short telomeres and is bypassed by disruption of tumour suppressive pathways. After senescence bypass, cells undergo crisis, during which almost all of the cells in the population die. Cells that escape crisis harbor unstable genomes and other parameters of transformation. The mechanism of cell death during crisis remained elusive. We show that cells in crisis undergo spontaneous mitotic arrest, resulting in death during mitosis or in the following cell cycle. The phenotype was induced by loss of p53 function, and suppressed by telomerase overexpression. Telomere fusions triggered mitotic arrest in p53-compromised non-crisis cells, indicating such fusions as the underlying cause. Exacerbation of mitotic telomere deprotection by partial TRF2 knockdown2 increased the ratio of cells that died during mitotic arrest and sensitized cancer cells to mitotic poisons. We propose a crisis pathway wherein chromosome fusions induce mitotic arrest, resulting in mitotic telomere deprotection and cell death, thereby eliminating precancerous cells from the population. PMID:26108857

Salt-inducible kinase 3 is a novel mitotic regulator and a target for enhancing antimitotic therapeutic-mediated cell death

PubMed Central

Chen, H; Huang, S; Han, X; Zhang, J; Shan, C; Tsang, Y H; Ma, H T; Poon, R Y C

2014-01-01

Many mitotic kinases are both critical for maintaining genome stability and are important targets for anticancer therapies. We provide evidence that SIK3 (salt-inducible kinase 3), an AMP-activated protein kinase-related kinase, is important for mitosis to occur properly in mammalian cells. Downregulation of SIK3 resulted in an extension of mitosis in both mouse and human cells but did not affect the DNA damage checkpoint. Time-lapse microscopy and other approaches indicated that mitotic exit but not mitotic entry was delayed. Although repression of SIK3 alone simply delayed mitotic exit, it was able to sensitize cells to various antimitotic chemicals. Both mitotic arrest and cell death caused by spindle poisons were enhanced after SIK3 depletion. Likewise, the antimitotic effects due to pharmacological inhibition of mitotic kinases including Aurora A, Aurora B, and polo-like kinase 1 were enhanced in the absence of SIK3. Finally, in addition to promoting the sensitivity of a small-molecule inhibitor of the mitotic kinesin Eg5, SIK3 depletion was able to overcome cells that developed drug resistance. These results establish the importance of SIK3 as a mitotic regulator and underscore the potential of SIK3 as a druggable antimitotic target. PMID:24743732

ATP depletion during mitotic arrest induces mitotic slippage and APC/CCdh1-dependent cyclin B1 degradation.

PubMed

Park, Yun Yeon; Ahn, Ju-Hyun; Cho, Min-Guk; Lee, Jae-Ho

2018-04-27

ATP depletion inhibits cell cycle progression, especially during the G1 phase and the G2 to M transition. However, the effect of ATP depletion on mitotic progression remains unclear. We observed that the reduction of ATP after prometaphase by simultaneous treatment with 2-deoxyglucose and NaN 3 did not arrest mitotic progression. Interestingly, ATP depletion during nocodazole-induced prometaphase arrest resulted in mitotic slippage, as indicated by a reduction in mitotic cells, APC/C-dependent degradation of cyclin B1, increased cell attachment, and increased nuclear membrane reassembly. Additionally, cells successfully progressed through the cell cycle after mitotic slippage, as indicated by EdU incorporation and time-lapse imaging. Although degradation of cyclin B during normal mitotic progression is primarily regulated by APC/C Cdc20 , we observed an unexpected decrease in Cdc20 prior to degradation of cyclin B during mitotic slippage. This decrease in Cdc20 was followed by a change in the binding partner preference of APC/C from Cdc20 to Cdh1; consequently, APC/C Cdh1 , but not APC/C Cdc20 , facilitated cyclin B degradation following ATP depletion. Pulse-chase analysis revealed that ATP depletion significantly abrogated global translation, including the translation of Cdc20 and Cdh1. Additionally, the half-life of Cdh1 was much longer than that of Cdc20. These data suggest that ATP depletion during mitotic arrest induces mitotic slippage facilitated by APC/C Cdh1 -dependent cyclin B degradation, which follows a decrease in Cdc20 resulting from reduced global translation and the differences in the half-lives of the Cdc20 and Cdh1 proteins.

Identification and characterization of novel NuMA isoforms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Jin, E-mail: petersdu2112@hotmail.com; Xu, Zhe; Core Laboratory for Clinical Medical Research, Beijing Tiantan Hospital, Capital Medical University, Beijing

2014-11-21

Highlights: • Seven NuMA isoforms generated by alternative splicing were categorized into 3 groups: long, middle and short. • Both exons 15 and 16 in long NuMA were “hotspot” for alternative splicing. • Lower expression of short NuMA was observed in cancer cells compared with nonneoplastic controls. • Distinct localization pattern of short isoforms indicated different function from that of long and middle NuMA. - Abstract: The large nuclear mitotic apparatus (NuMA) has been investigated for over 30 years with functions related to the formation and maintenance of mitotic spindle poles during mitosis. However, the existence and functions of NuMAmore » isoforms generated by alternative splicing remains unclear. In the present work, we show that at least seven NuMA isoforms (categorized into long, middle and short groups) generated by alternative splicing from a common NuMA mRNA precursor were discovered in HeLa cells and these isoforms differ mainly at the carboxyl terminus and the coiled-coil domains. Two “hotspot” exons with molecular mass of 3366-nt and 42-nt tend to be spliced during alternative splicing in long and middle groups. Furthermore, full-length coding sequences of long and middle NuMA obtained by using fusion PCR were constructed into GFP-tagged vector to illustrate their cellular localization. Long NuMA mainly localized in the nucleus with absence from nucleoli during interphase and translocated to the spindle poles in mitosis. Middle NuMA displayed the similar cell cycle-dependent distribution pattern as long NuMA. However, expression of NuMA short isoforms revealed a distinct subcellular localization. Short NuMA were present in the cytosol during the whole cycle, without colocalization with mitotic apparatus. These results have allowed us tentatively to explore a new research direction for NuMA’s various functions.« less

Nuclear movement in fungi.

PubMed

Xiang, Xin

2017-12-11

Nuclear movement within a cell occurs in a variety of eukaryotic organisms including yeasts and filamentous fungi. Fungal molecular genetic studies identified the minus-end-directed microtubule motor cytoplasmic dynein as a critical protein for nuclear movement or orientation of the mitotic spindle contained in the nucleus. Studies in the budding yeast first indicated that dynein anchored at the cortex via its anchoring protein Num1 exerts pulling force on an astral microtubule to orient the anaphase spindle across the mother-daughter axis before nuclear division. Prior to anaphase, myosin V interacts with the plus end of an astral microtubule via Kar9-Bim1/EB1 and pulls the plus end along the actin cables to move the nucleus/spindle close to the bud neck. In addition, pushing or pulling forces generated from cortex-linked polymerization or depolymerization of microtubules drive nuclear movements in yeasts and possibly also in filamentous fungi. In filamentous fungi, multiple nuclei within a hyphal segment undergo dynein-dependent back-and-forth movements and their positioning is also influenced by cytoplasmic streaming toward the hyphal tip. In addition, nuclear movement occurs at various stages of fungal development and fungal infection of plant tissues. This review discusses our current understanding on the mechanisms of nuclear movement in fungal organisms, the importance of nuclear positioning and the regulatory strategies that ensure the proper positioning of nucleus/spindle. Published by Elsevier Ltd.

A hybrid computational model to explore the topological characteristics of epithelial tissues.

PubMed

González-Valverde, Ismael; García-Aznar, José Manuel

2017-11-01

Epithelial tissues show a particular topology where cells resemble a polygon-like shape, but some biological processes can alter this tissue topology. During cell proliferation, mitotic cell dilation deforms the tissue and modifies the tissue topology. Additionally, cells are reorganized in the epithelial layer and these rearrangements also alter the polygon distribution. We present here a computer-based hybrid framework focused on the simulation of epithelial layer dynamics that combines discrete and continuum numerical models. In this framework, we consider topological and mechanical aspects of the epithelial tissue. Individual cells in the tissue are simulated by an off-lattice agent-based model, which keeps the information of each cell. In addition, we model the cell-cell interaction forces and the cell cycle. Otherwise, we simulate the passive mechanical behaviour of the cell monolayer using a material that approximates the mechanical properties of the cell. This continuum approach is solved by the finite element method, which uses a dynamic mesh generated by the triangulation of cell polygons. Forces generated by cell-cell interaction in the agent-based model are also applied on the finite element mesh. Cell movement in the agent-based model is driven by the displacements obtained from the deformed finite element mesh of the continuum mechanical approach. We successfully compare the results of our simulations with some experiments about the topology of proliferating epithelial tissues in Drosophila. Our framework is able to model the emergent behaviour of the cell monolayer that is due to local cell-cell interactions, which have a direct influence on the dynamics of the epithelial tissue. Copyright © 2017 John Wiley & Sons, Ltd.

Kif18A and chromokinesins confine centromere movements via microtubule growth suppression and spatial control of kinetochore tension

PubMed Central

Stumpff, Jason; Wagenbach, Michael; Franck, Andrew; Asbury, Charles L.; Wordeman, Linda

2012-01-01

Summary Alignment of chromosomes at the metaphase plate is a signature of cell division in metazoan cells, yet the mechanisms controlling this process remain ambiguous. Here we use a combination of quantitative live cell imaging and reconstituted dynamic microtubule assays to investigate the molecular control of mitotic centromere movements. We establish that Kif18A (kinesin-8) attenuates centromere movement by directly promoting microtubule pausing in a concentration-dependent manner. This activity provides the dominant mechanism for restricting centromere movement to the spindle midzone. Furthermore, polar ejection forces spatially confine chromosomes via position-dependent regulation of kinetochore tension and centromere switch rates. We demonstrate that polar ejection forces are antagonistically modulated by chromokinesins. These pushing forces depend on Kid (kinesin-10) activity and are antagonized by Kif4A (kinesin-4), which functions to directly suppress microtubule growth. These data support a model in which Kif18A and polar ejection forces synergistically promote centromere alignment via spatial control of kinetochore-microtubule dynamics. PMID:22595673

Timely Endocytosis of Cytokinetic Enzymes Prevents Premature Spindle Breakage during Mitotic Exit

PubMed Central

Onishi, Masayuki; Yeong, Foong May

2016-01-01

Cytokinesis requires the spatio-temporal coordination of membrane deposition and primary septum (PS) formation at the division site to drive acto-myosin ring (AMR) constriction. It has been demonstrated that AMR constriction invariably occurs only after the mitotic spindle disassembly. It has also been established that Chitin Synthase II (Chs2p) neck localization precedes mitotic spindle disassembly during mitotic exit. As AMR constriction depends upon PS formation, the question arises as to how chitin deposition is regulated so as to prevent premature AMR constriction and mitotic spindle breakage. In this study, we propose that cells regulate the coordination between spindle disassembly and AMR constriction via timely endocytosis of cytokinetic enzymes, Chs2p, Chs3p, and Fks1p. Inhibition of endocytosis leads to over accumulation of cytokinetic enzymes during mitotic exit, which accelerates the constriction of the AMR, and causes spindle breakage that eventually could contribute to monopolar spindle formation in the subsequent round of cell division. Intriguingly, the mitotic spindle breakage observed in endocytosis mutants can be rescued either by deleting or inhibiting the activities of, CHS2, CHS3 and FKS1, which are involved in septum formation. The findings from our study highlight the importance of timely endocytosis of cytokinetic enzymes at the division site in safeguarding mitotic spindle integrity during mitotic exit. PMID:27447488

Cellular Tug-of-War: Forces at Work and DNA Stretching in Mitosis

NASA Astrophysics Data System (ADS)

Griffin, Brian; Kilfoil, Maria L.

2013-03-01

In the microscopic world of the cell dominated by thermal noise, a cell must be able to successfully segregate its DNA with high fidelity in order to pass its genetic information on to its progeny. In this process of mitosis in eukaryotes, driving forces act on the cytoskeleton-based architecture called the mitotic spindle to promote this division. Our preliminary data demonstrates that the dynamics of this process in yeast cells is universal. Moreover, the dynamics suggest an increasing load as the chromosomes are pulled apart. To investigate this, we use three-dimensional imaging to track the dynamics of the poles of this architecture and the points of attachment to chromosomes simultaneously and with high spatial resolution. We analyze the relative motions of chromosomes as they are organized before segregation and as they are pulled apart, using this data to investigate the force-response behavior of this cytoskeleton-chromosome polymer system.

Utilization of flow cytometry to identify chimeral sectors in leaf tissue of Lolium multiflorum x L. arundinaceum hybrids

USDA-ARS?s Scientific Manuscript database

We have identified a method whereby Lolium multiflorum (Lm) or L. arundinaceum (Fa) genomes are preferentially eliminated through a mitotic loss behavior in interspecific Lm x Fa F1 hybrids, generating either dihaploid Lm lines or Fa lines. Flow cytometry, a method for rapidly characterizing optical...

The radiation hypersensitivity of cells at mitosis.

PubMed

Stobbe, C C; Park, S J; Chapman, J D

2002-12-01

Mitotic cells are hypersensitive to ionizing radiation, exhibiting single-hit inactivation coefficients near to those of repair deficient cell lines and lymphocytes. To elucidate possible mechanisms for this hypersensitivity, the kinetics of oxygen radiosensitization, the proportion of indirect effect by OH radicals and the kinetics of radiation-induced DNA strand breakage in the chromatin of mitotic cells were investigated. Synchronized populations of >90% mitotic HT-29 cells were obtained by the mitotic shake-off method. Cells were irradiated at < or =4 degrees C with (137)Cs gamma-rays. Cellular oxygen concentration was varied by gassing cell suspensions prior to and during irradiation with mixtures of pure N(2) that contained 5% CO(2) and measured quantities of O(2). The indirect effect of OH radicals was investigated with the radical scavenger, DMSO. DNA strand breakage was measured by the comet assay. Mitotic HT-29 cell inactivation is well described by a single-hit inactivation coefficient (alpha) of 1.14 +/- 0.06 Gy(-1). The oxygen enhancement ratio of mitotic cells (at 10% survival) was found to be approximately 2.0, significantly lower than the value of 2.8 measured for interphase (asynchronous) cells. More than 60% of mitotic cell killing was eliminated when the media contained 2 M DMSO, indicating that indirect effect is as important in the killing of mitotic cells as it is for interphase cells. The chromatin in mitotic cells was found to be ~2.8 times more sensitive to radiation-induced DNA single-strand breakage than the chromatin of interphase cells. The alpha-inactivation coefficient of mitotic HT-29 cells was ~30 times larger than that of interphase cells. Mitotic cell chromatin appears to contain intrinsic DNA breaks that are not lethal. In addition, chromatin in mitotic cells was found to be more susceptible to radiation-induced DNA strand-breakage than the dispersed chromatin of interphase cells. How the enhanced production of these simple DNA lesions (that are usually reparable) translates into the lethal (non-reparable) events associated with alpha-inactivation is not known. The compaction/dispersion status of DNA throughout the cell cycle appears to be an important factor for determining intrinsic cell radiosensitivity and might be manipulated for radiotherapeutic advantage.

Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress.

PubMed

Wilhelm, Therese; Ragu, Sandrine; Magdalou, Indiana; Machon, Christelle; Dardillac, Elodie; Técher, Hervé; Guitton, Jérôme; Debatisse, Michelle; Lopez, Bernard S

2016-05-01

Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation, and emphasize the importance of homologous recombination as a barrier against spontaneous genetic instability triggered by the endogenous oxidative/replication stress axis.

The p90 ribosomal S6 kinase 2 specifically affects mitotic progression by regulating the basal level, distribution and stability of mitotic spindles

PubMed Central

Park, Yun Yeon; Nam, Hyun-Ja; Do, Mihyang; Lee, Jae-Ho

2016-01-01

RSK2, also known as RPS6KA3 (ribosomal protein S6 kinase, 90 kDa, polypeptide 3), is a downstream kinase of the mitogen-activated protein kinase (MAPK) pathway, which is important in regulating survival, transcription, growth and proliferation. However, its biological role in mitotic progression is not well understood. In this study, we examined the potential involvement of RSK2 in the regulation of mitotic progression. Interestingly, depletion of RSK2, but not RSK1, caused the accumulation of mitotic cells. Time-lapse analysis revealed that mitotic duration, particularly the duration for metaphase-to-anaphase transition was prolonged in RSK2-depleted cells, suggesting activation of spindle assembly checkpoint (SAC). Indeed, more BubR1 (Bub1-related kinase) was present on metaphase plate kinetochores in RSK2-depleted cells, and depletion of BubR1 abolished the mitotic accumulation caused by RSK2 depletion, confirming BubR1-dependent SAC activation. Along with the shortening of inter-kinetochore distance, these data suggested that weakening of the tension across sister kinetochores by RSK2 depletion led to the activation of SAC. To test this, we analyzed the RSK2 effects on the stability of kinetochore–microtubule interactions, and found that RSK2-depleted cells formed less kinetochore–microtubule fibers. Moreover, RSK2 depletion resulted in the decrease of basal level of microtubule as well as an irregular distribution of mitotic spindles, which might lead to observed several mitotic progression defects such as increase in unaligned chromosomes, defects in chromosome congression and a decrease in pole-to-pole distance in these cells. Taken together, our data reveal that RSK2 affects mitotic progression by regulating the distribution, basal level and the stability of mitotic spindles. PMID:27491410

DOE Office of Scientific and Technical Information (OSTI.GOV)

Galán-Malo, Patricia; Vela, Laura; Gonzalo, Oscar

Microtubule poisons and other anti-mitotic drugs induce tumor death but the molecular events linking mitotic arrest to cell death are still not fully understood. We have analyzed cell fate after mitotic arrest produced by the microtubule-destabilizing drug vincristine in a panel of human tumor cell lines showing different response to vincristine. In Jurkat, RPMI 8226 and HeLa cells, apoptosis was triggered shortly after vincristine-induced mitotic arrest. However, A549 cells, which express a great amount of Bcl-x{sub L} and undetectable amounts of Bak, underwent mitotic slippage prior to cell death. However, when Bcl-x{sub L} gene was silenced in A549 cells, vincristinemore » induced apoptosis during mitotic arrest. Another different behavior was found in MiaPaca2 cells, where vincristine caused death by mitotic catastrophe that switched to apoptosis when cyclin B1 degradation was prevented by proteasome inhibition. Overexpression of Bcl-x{sub L} or silencing Bax and Bak expression delayed the onset of apoptosis in Jurkat and RPMI 8226 cells, enabling mitotic slippage and endoreduplication. In HeLa cells, overexpression of Bcl-x{sub L} switched cell death from apoptosis to mitotic catastrophe. Mcl-1 offered limited protection to vincristine-induced cell death and Mcl-1 degradation was not essential for vincristine-induced death. All these results, taken together, indicate that the Bcl-x{sub L}/Bak ratio and the ability to degrade cyclin B1 determine cell fate after mitotic arrest in the different tumor cell types. Highlights: ► Vincristine induces cell death by apoptosis or mitotic catastrophe. ► Apoptosis-proficient cells die by apoptosis during mitosis upon vincristine treatment. ► p53wt apoptosis-deficient cells undergo apoptosis from a G1-like tetraploid state. ► p53mt apoptosis-deficient cells can survive and divide giving rise to 8N cells.« less

Physical determinants of bipolar mitotic spindle assembly and stability in fission yeast

PubMed Central

Blackwell, Robert; Edelmaier, Christopher; Sweezy-Schindler, Oliver; Lamson, Adam; Gergely, Zachary R.; O’Toole, Eileen; Crapo, Ammon; Hough, Loren E.; McIntosh, J. Richard; Glaser, Matthew A.; Betterton, Meredith D.

2017-01-01

Mitotic spindles use an elegant bipolar architecture to segregate duplicated chromosomes with high fidelity. Bipolar spindles form from a monopolar initial condition; this is the most fundamental construction problem that the spindle must solve. Microtubules, motors, and cross-linkers are important for bipolarity, but the mechanisms necessary and sufficient for spindle assembly remain unknown. We describe a physical model that exhibits de novo bipolar spindle formation. We began with physical properties of fission-yeast spindle pole body size and microtubule number, kinesin-5 motors, kinesin-14 motors, and passive cross-linkers. Our model results agree quantitatively with our experiments in fission yeast, thereby establishing a minimal system with which to interrogate collective self-assembly. By varying the features of our model, we identify a set of functions essential for the generation and stability of spindle bipolarity. When kinesin-5 motors are present, their bidirectionality is essential, but spindles can form in the presence of passive cross-linkers alone. We also identify characteristic failed states of spindle assembly—the persistent monopole, X spindle, separated asters, and short spindle, which are avoided by the creation and maintenance of antiparallel microtubule overlaps. Our model can guide the identification of new, multifaceted strategies to induce mitotic catastrophes; these would constitute novel strategies for cancer chemotherapy. PMID:28116355

Discovery of Supernumerary B Chromosomes in Drosophila melanogaster

PubMed Central

Bauerly, Elisabeth; Hughes, Stacie E.; Vietti, Dana R.; Miller, Danny E.; McDowell, William; Hawley, R. Scott

2014-01-01

B chromosomes are small, heterochromatic chromosomes that are transmitted in a non-Mendelian manner. We have identified a stock of Drosophila melanogaster that recently (within the last decade) acquired an average of 10 B chromosomes per fly. These B chromosomes are transmitted by both males and females and can be maintained for multiple generations in a wild-type genetic background despite the fact that they cause high levels of 4th chromosome meiotic nondisjunction in females. Most curiously, these B chromosomes are mitotically unstable, suggesting either the absence of critical chromosomal sites or the inability of the meiotic or mitotic systems to cope with many additional chromosomes. These B chromosomes also contain centromeres and are primarily composed of the heterochromatic AATAT satellite sequence. Although the AATAT sequence comprises the majority of the 4th chromosome heterochromatin, the B chromosomes lack most, if not all, 4th chromosome euchromatin. Presumably as a consequence of their heterochromatic content, these B chromosomes significantly modify position-effect variegation in two separate reporter systems, acting as enhancers of variegation in one case and suppressors in the other. The identification of B chromosomes in a genetically tractable organism like D. melanogaster will facilitate studies of chromosome evolution and the analysis of the mechanisms by which meiotic and mitotic processes cope with additional chromosomes. PMID:24478336

The Drosophila Microtubule-Associated Protein Mars Stabilizes Mitotic Spindles by Crosslinking Microtubules through Its N-Terminal Region

PubMed Central

Zhang, Gang; Beati, Hamze; Nilsson, Jakob; Wodarz, Andreas

2013-01-01

Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs) are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs) have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs. PMID:23593258

The Differential Roles of Budding Yeast Tem1p, Cdc15p, and Bub2p Protein Dynamics in Mitotic ExitD⃞V⃞

PubMed Central

Molk, Jeffrey N.; Schuyler, Scott C.; Liu, Jenny Y.; Evans, James G.; Salmon, E. D.; Pellman, David; Bloom, Kerry

2004-01-01

In the budding yeast Saccharomyces cerevisiae the mitotic spindle must be positioned along the mother-bud axis to activate the mitotic exit network (MEN) in anaphase. To examine MEN proteins during mitotic exit, we imaged the MEN activators Tem1p and Cdc15p and the MEN regulator Bub2p in vivo. Quantitative live cell fluorescence microscopy demonstrated the spindle pole body that segregated into the daughter cell (dSPB) signaled mitotic exit upon penetration into the bud. Activation of mitotic exit was associated with an increased abundance of Tem1p-GFP and the localization of Cdc15p-GFP on the dSPB. In contrast, Bub2p-GFP fluorescence intensity decreased in mid-to-late anaphase on the dSPB. Therefore, MEN protein localization fluctuates to switch from Bub2p inhibition of mitotic exit to Cdc15p activation of mitotic exit. The mechanism that elevates Tem1p-GFP abundance in anaphase is specific to dSPB penetration into the bud and Dhc1p and Lte1p promote Tem1p-GFP localization. Finally, fluorescence recovery after photobleaching (FRAP) measurements revealed Tem1p-GFP is dynamic at the dSPB in late anaphase. These data suggest spindle pole penetration into the bud activates mitotic exit, resulting in Tem1p and Cdc15p persistence at the dSPB to initiate the MEN signal cascade. PMID:14718561

Mitotic rate is associated with positive lymph nodes in patients with thin melanomas.

PubMed

Wheless, Lee; Isom, Chelsea A; Hooks, Mary A; Kauffmann, Rondi M

2018-05-01

The American Joint Commission on Cancer will remove mitotic rate from its staging guidelines in 2018. Using a large nationally representative cohort, we examined the association between mitotic rate and lymph node positivity among thin melanomas. A total of 149,273 thin melanomas in the National Cancer Database were examined for their association of high-risk features of mitotic rate, ulceration, and Breslow depth with lymph node status. Among 17,204 patients with thin melanomas with data on Breslow depth, ulceration, and mitotic rate who underwent a lymph node biopsy, there was a strong linear relationship between odds of having a positive lymph node and mitotic rate (R 2  = 0.96, P < .0001, β = 3.31). The odds of having a positive node increased by 19% with each 1-point increase in mitotic rate (odds ratio, 1.19; 95% confidence interval, 1.17-1.21). Cases with negative nodes had a mean mitotic rate of 1.54 plus or minus 2.07 mitoses/mm 2 compared with 3.30 plus or minus 3.54 mitoses/mm 2 for those with positive nodes (P < .0001). The data collected do not allow for survival analyses. Mitotic rate was strongly associated with the odds of having a positive lymph node and should continue to be reported on pathology reports. Copyright © 2017 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

Cell cycle-dependent regulation of Greatwall kinase by protein phosphatase 1 and regulatory subunit 3B.

PubMed

Ren, Dapeng; Fisher, Laura A; Zhao, Jing; Wang, Ling; Williams, Byron C; Goldberg, Michael L; Peng, Aimin

2017-06-16

Greatwall (Gwl) kinase plays an essential role in the regulation of mitotic entry and progression. Mitotic activation of Gwl requires both cyclin-dependent kinase 1 (CDK1)-dependent phosphorylation and its autophosphorylation at an evolutionarily conserved serine residue near the carboxyl terminus (Ser-883 in Xenopus ). In this study we show that Gwl associates with protein phosphatase 1 (PP1), particularly PP1γ, which mediates the dephosphorylation of Gwl Ser-883. Consistent with the mitotic activation of Gwl, its association with PP1 is disrupted in mitotic cells and egg extracts. During mitotic exit, PP1-dependent dephosphorylation of Gwl Ser-883 occurs prior to dephosphorylation of other mitotic substrates; replacing endogenous Gwl with a phosphomimetic S883E mutant blocks mitotic exit. Moreover, we identified PP1 regulatory subunit 3B (PPP1R3B) as a targeting subunit that can direct PP1 activity toward Gwl. PPP1R3B bridges PP1 and Gwl association and promotes Gwl Ser-883 dephosphorylation. Consistent with the cell cycle-dependent association of Gwl and PP1, Gwl and PPP1R3B dissociate in M phase. Interestingly, up-regulation of PPP1R3B facilitates mitotic exit and blocks mitotic entry. Thus, our study suggests PPP1R3B as a new cell cycle regulator that functions by governing Gwl dephosphorylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Spatial Reorganization of the Endoplasmic Reticulum during Mitosis Relies on Mitotic Kinase Cyclin A in the Early Drosophila Embryo

PubMed Central

Bergman, Zane J.; Mclaurin, Justin D.; Eritano, Anthony S.; Johnson, Brittany M.; Sims, Amanda Q.; Riggs, Blake

2015-01-01

Mitotic cyclin-dependent kinase with their cyclin partners (cyclin:Cdks) are the master regulators of cell cycle progression responsible for regulating a host of activities during mitosis. Nuclear mitotic events, including chromosome condensation and segregation have been directly linked to Cdk activity. However, the regulation and timing of cytoplasmic mitotic events by cyclin:Cdks is poorly understood. In order to examine these mitotic cytoplasmic events, we looked at the dramatic changes in the endoplasmic reticulum (ER) during mitosis in the early Drosophila embryo. The dynamic changes of the ER can be arrested in an interphase state by inhibition of either DNA or protein synthesis. Here we show that this block can be alleviated by micro-injection of Cyclin A (CycA) in which defined mitotic ER clusters gathered at the spindle poles. Conversely, micro-injection of Cyclin B (CycB) did not affect spatial reorganization of the ER, suggesting CycA possesses the ability to initiate mitotic ER events in the cytoplasm. Additionally, RNAi-mediated simultaneous inhibition of all 3 mitotic cyclins (A, B and B3) blocked spatial reorganization of the ER. Our results suggest that mitotic ER reorganization events rely on CycA and that control and timing of nuclear and cytoplasmic events during mitosis may be defined by release of CycA from the nucleus as a consequence of breakdown of the nuclear envelope. PMID:25689737

DOE Office of Scientific and Technical Information (OSTI.GOV)

Komura, Jun-ichiro, E-mail: junkom@med.tohoku.ac.jp; Ikehata, Hironobu; Mori, Toshio

During mitosis, chromatin is highly condensed, and activities such as transcription and semiconservative replication do not occur. Consequently, the condensed condition of mitotic chromatin is assumed to inhibit DNA metabolism by impeding the access of DNA-transacting proteins. However, about 40 years ago, several researchers observed unscheduled DNA synthesis in UV-irradiated mitotic chromosomes, suggesting the presence of excision repair. We re-examined this subject by directly measuring the removal of UV-induced DNA lesions by an ELISA and by a Southern-based technique in HeLa cells arrested at mitosis. We observed that the removal of (6-4) photoproducts from the overall genome in mitotic cellsmore » was as efficient as in interphase cells. This suggests that global genome repair of (6-4) photoproducts is fully functional during mitosis, and that the DNA in mitotic chromatin is accessible to proteins involved in this mode of DNA repair. Nevertheless, not all modes of DNA repair seem fully functional during mitosis. We also observed that the removal of cyclobutane pyrimidine dimers from the dihydrofolate reductase and c-MYC genes in mitotic cells was very slow. This suggests that transcription-coupled repair of cyclobutane pyrimidine dimers is compromised or non-functional during mitosis, which is probably the consequence of mitotic transcriptional repression. -- Highlights: Black-Right-Pointing-Pointer Global genome repair of (6-4) photoproducts is fully active in mitotic cells. Black-Right-Pointing-Pointer DNA in condensed mitotic chromatin does not seem inaccessible or inert. Black-Right-Pointing-Pointer Mitotic transcriptional repression may impair transcription-coupled repair.« less

Phosphorylation of histone H3 on Ser-10 by Aurora B is essential for chromosome condensation in porcine embryos during the first mitotic division.

PubMed

Chen, Changchao; Zhang, Zixiao; Cui, Panpan; Liao, Yaya; Zhang, Yue; Yao, Lingyun; Rui, Rong; Ju, Shiqiang

2017-07-01

Phosphorylation of histone H3 on Ser-10 (H3S10ph) is involved in regulating mitotic chromosome condensation and decondensation, which plays an important regulatory role during mitotic cell cycle progression in mammalian cells. However, whether H3S10ph plays a similar role in early porcine embryos during the first mitotic division remains uncertain. In this study, the subcellular localization and possible roles of H3S10ph were evaluated in the first mitotic cell cycle progression of porcine embryos using western blot, indirect immunofluorescence and barasertib (H3S10ph upstream regulator Aurora-B inhibitor) treatments. H3S10ph exhibited a dynamic localization pattern and was localized to chromosomes from prometaphase to anaphase stages. Treatment of porcine embryos with barasertib inhibited mitotic division at the prophase stage and was associated with a defect in chromosome condensation accompanied by the reduction of H3S10ph. These results indicated that H3S10ph is involved in the first mitotic division in porcine embryos through its regulatory function in chromosome condensation, which further affects porcine embryo cell cycle progression during mitotic division.

Loss of Adult Cardiac Myocyte GSK-3 Leads to Mitotic Catastrophe Resulting in Fatal Dilated Cardiomyopathy

PubMed Central

Zhou, Jibin; Ahmad, Firdos; Parikh, Shan; Hoffman, Nichole E.; Rajan, Sudarsan; Verma, Vipin K.; Song, Jianliang; Yuan, Ancai; Shanmughapriya, Santhanam; Guo, Yuanjun; Gao, Erhe; Koch, Walter; Woodgett, James R.; Muniswamy, Madesh; Kishore, Raj; Lal, Hind; Force, Thomas

2016-01-01

Rationale Cardiac myocyte-specific deletion of either Glycogen Synthase Kinase (GSK)3A or GSK3B leads to cardiac protection following myocardial infarction, suggesting that deletion of both isoforms may provide synergistic protection. This is an important consideration due to the fact that all GSK-3–targeted drugs including the drugs already in clinical trial target both isoforms of GSK-3 and none are isoform specific. Objective To identify the consequences of combined deletion of cardiac myocyte GSK3A and GSK3B in heart function. Methods and Results We generated tamoxifen-inducible cardiac myocyte-specific mice lacking both GSK-3 isoforms (double knockout, DKO). We unexpectedly found that cardiac myocyte GSK-3 is essential for cardiac homeostasis and overall survival. Serial echocardiographic analysis reveals that within 2 weeks of tamoxifen treatment, DKO hearts leads to excessive dilatative remodeling and ventricular dysfunction. Further experimentation with isolated adult cardiac myocytes and fibroblasts from DKO implicated cardiac myocytes intrinsic factors responsible for observed phenotype. Mechanistically, loss of GSK-3 in adult cardiac myocytes resulted in induction of mitotic catastrophe, a previously unreported event in cardiac myocytes. DKO cardiac myocytes showed cell cycle progression resulting in increased DNA content and multi-nucleation. However, increased cell cycle activity was rivaled by marked activation of DNA damage, cell cycle checkpoint activation, and mitotic catastrophe induced apoptotic cell death. Importantly, mitotic catastrophe was also confirmed in isolated adult cardiac myocytes. Conclusion Together, our findings suggest that cardiac myocyte GSK-3 is required to maintain normal cardiac homeostasis and its loss is incompatible with life due to cell cycle dysregulation that ultimately results in a severe fatal dilated cardiomyopathy. PMID:26976650

Mammalian neurogenesis requires Treacle-Plk1 for precise control of spindle orientation, mitotic progression, and maintenance of neural progenitor cells.

PubMed

Sakai, Daisuke; Dixon, Jill; Dixon, Michael J; Trainor, Paul A

2012-01-01

The cerebral cortex is a specialized region of the brain that processes cognitive, motor, somatosensory, auditory, and visual functions. Its characteristic architecture and size is dependent upon the number of neurons generated during embryogenesis and has been postulated to be governed by symmetric versus asymmetric cell divisions, which mediate the balance between progenitor cell maintenance and neuron differentiation, respectively. The mechanistic importance of spindle orientation remains controversial, hence there is considerable interest in understanding how neural progenitor cell mitosis is controlled during neurogenesis. We discovered that Treacle, which is encoded by the Tcof1 gene, is a novel centrosome- and kinetochore-associated protein that is critical for spindle fidelity and mitotic progression. Tcof1/Treacle loss-of-function disrupts spindle orientation and cell cycle progression, which perturbs the maintenance, proliferation, and localization of neural progenitors during cortical neurogenesis. Consistent with this, Tcof1(+/-) mice exhibit reduced brain size as a consequence of defects in neural progenitor maintenance. We determined that Treacle elicits its effect via a direct interaction with Polo-like kinase1 (Plk1), and furthermore we discovered novel in vivo roles for Plk1 in governing mitotic progression and spindle orientation in the developing mammalian cortex. Increased asymmetric cell division, however, did not promote increased neuronal differentiation. Collectively our research has therefore identified Treacle and Plk1 as novel in vivo regulators of spindle fidelity, mitotic progression, and proliferation in the maintenance and localization of neural progenitor cells. Together, Treacle and Plk1 are critically required for proper cortical neurogenesis, which has important implications in the regulation of mammalian brain size and the pathogenesis of congenital neurodevelopmental disorders such as microcephaly.

Mammalian Neurogenesis Requires Treacle-Plk1 for Precise Control of Spindle Orientation, Mitotic Progression, and Maintenance of Neural Progenitor Cells

PubMed Central

Sakai, Daisuke; Dixon, Jill; Dixon, Michael J.; Trainor, Paul A.

2012-01-01

The cerebral cortex is a specialized region of the brain that processes cognitive, motor, somatosensory, auditory, and visual functions. Its characteristic architecture and size is dependent upon the number of neurons generated during embryogenesis and has been postulated to be governed by symmetric versus asymmetric cell divisions, which mediate the balance between progenitor cell maintenance and neuron differentiation, respectively. The mechanistic importance of spindle orientation remains controversial, hence there is considerable interest in understanding how neural progenitor cell mitosis is controlled during neurogenesis. We discovered that Treacle, which is encoded by the Tcof1 gene, is a novel centrosome- and kinetochore-associated protein that is critical for spindle fidelity and mitotic progression. Tcof1/Treacle loss-of-function disrupts spindle orientation and cell cycle progression, which perturbs the maintenance, proliferation, and localization of neural progenitors during cortical neurogenesis. Consistent with this, Tcof1 +/− mice exhibit reduced brain size as a consequence of defects in neural progenitor maintenance. We determined that Treacle elicits its effect via a direct interaction with Polo-like kinase1 (Plk1), and furthermore we discovered novel in vivo roles for Plk1 in governing mitotic progression and spindle orientation in the developing mammalian cortex. Increased asymmetric cell division, however, did not promote increased neuronal differentiation. Collectively our research has therefore identified Treacle and Plk1 as novel in vivo regulators of spindle fidelity, mitotic progression, and proliferation in the maintenance and localization of neural progenitor cells. Together, Treacle and Plk1 are critically required for proper cortical neurogenesis, which has important implications in the regulation of mammalian brain size and the pathogenesis of congenital neurodevelopmental disorders such as microcephaly. PMID:22479190

The Multiplicity of the Mitotic Centers and the Time-Course of Their Duplication and Separation

PubMed Central

Mazia, Daniel; Harris, Patricia J.; Bibring, Thomas

1960-01-01

In this study, the reproduction of the mitotic centers in the eggs of a sea urchin, Strongylocentrotus purpuratus and a sand dollar Dendraster excentricus has been studied by means of experimental designs that do not depend on the actual visualization of centrioles. The centers are defined in operational terms as potential poles. Blockage of mitosis by mercaptoethanol, it was found, inhibits the duplication of the centers, but does not inhibit the splitting and separation of centers that have already duplicated and thus potential poles could be realized as actual poles in multipolar divisions. At all times, the center is at least a duplex structure; that is, it contains two potential poles. The actual duplication process is the earliest event in a given mitotic cycle, taking place at very early interphase or in late telophase of the previous division. The splitting of the centers following duplication is a distinct process, dissociable from the duplication as such. Duplication and splitting normally occur at about the same time in the mitotic cycle, with a precession of the former. That is, as the two members of a pair of "old" centers split, each one gives rise to a new one, which remains associated with it until the next phase of splitting and duplication occurs. The results are consistent with what is termed a "generative" model of the self-reproduction of an intracellular body. According to this, the body does not immediately produce a full-fledged copy of itself, with simultaneous fission, but the primary duplication event involves only a part of the parent structure. This gives rise to a "germ" or "seed" which then grows to be equivalent to the parent body, and finally splits from it. PMID:19866563

Alterations and Abnormal Mitosis of Wheat Chromosomes Induced by Wheat-Rye Monosomic Addition Lines

PubMed Central

Fu, Shulan; Yang, Manyu; Fei, Yunyan; Tan, Feiquan; Ren, Zhenglong; Yan, Benju; Zhang, Huaiyu; Tang, Zongxiang

2013-01-01

Background Wheat-rye addition lines are an old topic. However, the alterations and abnormal mitotic behaviours of wheat chromosomes caused by wheat-rye monosomic addition lines are seldom reported. Methodology/Principal Findings Octoploid triticale was derived from common wheat T. aestivum L. ‘Mianyang11’×rye S. cereale L. ‘Kustro’ and some progeny were obtained by the controlled backcrossing of triticale with ‘Mianyang11’ followed by self-fertilization. Genomic in situ hybridization (GISH) using rye genomic DNA and fluorescence in situ hybridization (FISH) using repetitive sequences pAs1 and pSc119.2 as probes were used to analyze the mitotic chromosomes of these progeny. Strong pSc119.2 FISH signals could be observed at the telomeric regions of 3DS arms in ‘Mianyang11’. However, the pSc119.2 FISH signals were disappeared from the selfed progeny of 4R monosomic addition line and the changed 3D chromosomes could be transmitted to next generation stably. In one of the selfed progeny of 7R monosomic addition line, one 2D chromosome was broken and three 4A chromosomes were observed. In the selfed progeny of 6R monosomic addition line, structural variation and abnormal mitotic behaviour of 3D chromosome were detected. Additionally, 1A and 4B chromosomes were eliminated from some of the progeny of 6R monosomic addition line. Conclusions/Significance These results indicated that single rye chromosome added to wheat might cause alterations and abnormal mitotic behaviours of wheat chromosomes and it is possible that the stress caused by single alien chromosome might be one of the factors that induced karyotype alteration of wheat. PMID:23936213

Loss of Adult Cardiac Myocyte GSK-3 Leads to Mitotic Catastrophe Resulting in Fatal Dilated Cardiomyopathy.

PubMed

Zhou, Jibin; Ahmad, Firdos; Parikh, Shan; Hoffman, Nichole E; Rajan, Sudarsan; Verma, Vipin K; Song, Jianliang; Yuan, Ancai; Shanmughapriya, Santhanam; Guo, Yuanjun; Gao, Erhe; Koch, Walter; Woodgett, James R; Madesh, Muniswamy; Kishore, Raj; Lal, Hind; Force, Thomas

2016-04-15

Cardiac myocyte-specific deletion of either glycogen synthase kinase (GSK)-3α and GSK-3β leads to cardiac protection after myocardial infarction, suggesting that deletion of both isoforms may provide synergistic protection. This is an important consideration because of the fact that all GSK-3-targeted drugs, including the drugs already in clinical trial target both isoforms of GSK-3, and none are isoform specific. To identify the consequences of combined deletion of cardiac myocyte GSK-3α and GSK-3β in heart function. We generated tamoxifen-inducible cardiac myocyte-specific mice lacking both GSK-3 isoforms (double knockout). We unexpectedly found that cardiac myocyte GSK-3 is essential for cardiac homeostasis and overall survival. Serial echocardiographic analysis reveals that within 2 weeks of tamoxifen treatment, double-knockout hearts leads to excessive dilatative remodeling and ventricular dysfunction. Further experimentation with isolated adult cardiac myocytes and fibroblasts from double-knockout implicated cardiac myocytes intrinsic factors responsible for observed phenotype. Mechanistically, loss of GSK-3 in adult cardiac myocytes resulted in induction of mitotic catastrophe, a previously unreported event in cardiac myocytes. Double-knockout cardiac myocytes showed cell cycle progression resulting in increased DNA content and multinucleation. However, increased cell cycle activity was rivaled by marked activation of DNA damage, cell cycle checkpoint activation, and mitotic catastrophe-induced apoptotic cell death. Importantly, mitotic catastrophe was also confirmed in isolated adult cardiac myocytes. Together, our findings suggest that cardiac myocyte GSK-3 is required to maintain normal cardiac homeostasis, and its loss is incompatible with life because of cell cycle dysregulation that ultimately results in a severe fatal dilated cardiomyopathy. © 2016 American Heart Association, Inc.

Revealing the micromechanics driving cellular division: optical manipulation of force-bearing substructure in mitotic cells

NASA Astrophysics Data System (ADS)

Ono, Matthew; Preece, Daryl; Duquette, Michelle; Forer, Arthur; Berns, Michael

2017-08-01

During the anaphase stage of mitosis, a motility force transports genetic material in the form of chromosomes to the poles of the cell. Chromosome deformations during anaphase transport have largely been attributed to viscous drag force, however LaFountain et. al. found that a physical tether connects separating chromosome ends in crane-fly spermatocytes such that a backwards tethering force elongates the separating chromosomes. In the presented study laser microsurgery was used to deduce the mechanistic basis of chromosome elongation in rat-kangaroo cells. In half of tested chromosome pairs, laser microsurgery between separating chromosome ends reduced elongation by 7+/-3% suggesting a source of chromosome strain independent of viscous drag. When microsurgery was used to sever chromosomes during transport, kinetochore attached fragments continued poleward travel while half of end fragments traveled towards the opposite pole and the remaining fragments either did not move or segregated to the proper pole. Microsurgery directed between chromosome ends always ceased cross-polar fragment travel suggesting the laser severed a physical tether transferring force to the fragment. Optical trapping of fragments moving towards the opposite pole estimates an upper boundary on the tethering force of 1.5 pN.

Effects of hypergravity on rat liver regeneration

NASA Technical Reports Server (NTRS)

Feller, D. D.

1982-01-01

The effects of centrifugation on liver regrowth were examined by measuring mitotic activity. The results indicate that the increased gravity caused a delay in the onset of mitotic activity and a significant decrease in overall mitotic activity.

Analysis of mitosis and antimitotic drug responses in tumors by in vivo microscopy and single-cell pharmacodynamics.

PubMed

Orth, James D; Kohler, Rainer H; Foijer, Floris; Sorger, Peter K; Weissleder, Ralph; Mitchison, Timothy J

2011-07-01

Cancer relies upon frequent or abnormal cell division, but how the tumor microenvironment affects mitotic processes in vivo remains unclear, largely due to the technical challenges of optical access, spatial resolution, and motion. We developed high-resolution in vivo microscopy methods to visualize mitosis in a murine xenograft model of human cancer. Using these methods, we determined whether the single-cell response to the antimitotic drug paclitaxel (Ptx) was the same in tumors as in cell culture, observed the impact of Ptx on the tumor response as a whole, and evaluated the single-cell pharmacodynamics (PD) of Ptx (by in vivo PD microscopy). Mitotic initiation was generally less frequent in tumors than in cell culture, but subsequently it proceeded normally. Ptx treatment caused spindle assembly defects and mitotic arrest, followed by slippage from mitotic arrest, multinucleation, and apoptosis. Compared with cell culture, the peak mitotic index in tumors exposed to Ptx was lower and the tumor cells survived longer after mitotic arrest, becoming multinucleated rather than dying directly from mitotic arrest. Thus, the tumor microenvironment was much less proapoptotic than cell culture. The morphologies associated with mitotic arrest were dose and time dependent, thereby providing a semiquantitative, single-cell measure of PD. Although many tumor cells did not progress through Ptx-induced mitotic arrest, tumor significantly regressed in the model. Our findings show that in vivo microscopy offers a useful tool to visualize mitosis during tumor progression, drug responses, and cell fate at the single-cell level. ©2011 AACR.

Tripolar acytokinetic mitosis and formation of feto-maternal syncytia in the bovine placentome: different modes of the generation of multinuclear cells.

PubMed

Klisch, K; Pfarrer, C; Schuler, G; Hoffmann, B; Leiser, R

1999-08-01

The vast majority of trophoblast giant cells in the ruminant placenta are binuclear and are believed to derive from mononuclear trophoblastic cells by a single acytokinetic mitosis. There is no satisfactory explanation for the generation of the small proportion of trophoblast giant cells with one, three, or more nuclei. In this light-and electronmicroscopic study of bovine placentomal tissue from the second half of gestation, developmental stages of the trophoblast giant cells are investigated. Large mitotic figures indicate mitotic polyploidization, which is proposed to be due to two subsequent acytokinetic mitoses. Tripolar mitoses offer an explanation for the development of trinucleate trophoblast giant cells. Measurements of nuclear volumes in a series of semithin sections revealed that three size classes of trophoblast giant cells occur. The approximately doubling of nuclear volume between each class is thought to reflect different levels of DNA content that result from polyploidization in this cell type. Although trinuclear feto-maternal hybrid cells are the standard outcome of the fusion of binuclear trophoblast giant cells with uterine epithelial cells, some syncytia with at least five nuclei were observed in the uterine epithelium.

Acidosis overrides oxygen deprivation to maintain mitochondrial function and cell survival

PubMed Central

Khacho, Mireille; Tarabay, Michelle; Patten, David; Khacho, Pamela; MacLaurin, Jason G.; Guadagno, Jennifer; Bergeron, Richard; Cregan, Sean P.; Harper, Mary-Ellen; Park, David S.; Slack, Ruth S.

2014-01-01

Sustained cellular function and viability of high-energy demanding post-mitotic cells rely on the continuous supply of ATP. The utilization of mitochondrial oxidative phosphorylation for efficient ATP generation is a function of oxygen levels. As such, oxygen deprivation, in physiological or pathological settings, has profound effects on cell metabolism and survival. Here we show that mild extracellular acidosis, a physiological consequence of anaerobic metabolism, can reprogramme the mitochondrial metabolic pathway to preserve efficient ATP production regardless of oxygen levels. Acidosis initiates a rapid and reversible homeostatic programme that restructures mitochondria, by regulating mitochondrial dynamics and cristae architecture, to reconfigure mitochondrial efficiency, maintain mitochondrial function and cell survival. Preventing mitochondrial remodelling results in mitochondrial dysfunction, fragmentation and cell death. Our findings challenge the notion that oxygen availability is a key limiting factor in oxidative metabolism and brings forth the concept that mitochondrial morphology can dictate the bioenergetic status of post-mitotic cells. PMID:24686499

The shape of things to come: regulation of shape changes in endoplasmic reticulum.

PubMed

Paiement, J; Bergeron, J

2001-01-01

Shape changes in the endoplasmic reticulum control fundamental cell processes including nuclear envelope assembly in mitotic cells, calcium homeostasis in cytoplasmic domains of secreting and motile cells, and membrane traffic in the early secretion apparatus between the endoplasmic reticulum and Golgi. Opposing forces of assembly (membrane fusion) and disassembly (membrane fragmentation) ultimately determine the size and shape of this organelle. This review examines some of the regulatory mechanisms involved in these processes and how they occur at specific sites or subcompartments of the endoplasmic reticulum.

Epigenetic Characteristics of the Mitotic Chromosome in 1D and 3D

PubMed Central

Oomen, Marlies E.; Dekker, Job

2017-01-01

While chromatin characteristics in interphase are widely studied, characteristics of mitotic chromatin and their inheritance through mitosis are still poorly understood. During mitosis chromatin undergoes dramatic changes: Transcription stalls, chromatin binding factors leave the chromatin, histone modifications change and chromatin becomes highly condensed. Many key insights into mitotic chromosome state and conformation have come from extensive microscopy studies over the last century. Over the last decade the development of 3C-based techniques has enabled the study of higher order chromosome organization during mitosis in a genome-wide manner. During mitosis chromosomes lose their cell type specific and locus-dependent chromatin organization that characterizes interphase chromatin and fold into randomly positioned loop arrays. Upon exit of mitosis cells are capable of quickly rearranging the chromosome conformation to form the cell type specific interphase organization again. The information that enables this rearrangement after mitotic exit is thought to be encoded at least in part in mitotic bookmarks, e.g. histone modifications and variants, histone remodelers, chromatin factors and non-coding RNA. Here we give an overview of the chromosomal organization and epigenetic characteristics of the interphase and mitotic chromatin in vertebrates. Second, we describe different ways in which mitotic bookmarking enables epigenetic memory of the features of the interphase chromatin through mitosis. And third, we explore the role of epigenetic modifications and mitotic bookmarking in cell differentiation. PMID:28228067

Repeated furrow formation from a single mitotic apparatus in cylindrical sand dollar eggs.

PubMed

Rappaport, R

1985-04-01

The methods used previously to demonstrate the ability of a single mitotic apparatus to elicit multiple furrows involved considerable cell distortion and did not permit the investigator to control the positioning of the parts or to observe satisfactorily the early stages of furrow development. In this investigation, Echinarachnius parma eggs were confined in 82 microns i.d. transparent, silicone rubber-walled capillaries, and the mitotic apparatus was moved by pushing the poles inward with 55-microns-diameter glass balls. When the mitotic apparatus was shifted immediately after the furrow first appeared, a new furrow appeared in the normal relation to the new position in 1-2 minutes. The same mitotic apparatus could elicit up to 13 furrows as it was shifted back and forth by alternately pushing in the poles. The previous furrow regressed as the new furrow developed. The operations protracted the furrow establishment period to as long as 24.5 minutes after establishment of the first furrow. The characteristics of furrow regression were related to the distance the mitotic apparatus was moved. It is unlikely that regression was caused either by stress imposed on the surface or the removal of the mitotic apparatus from the vicinity of the furrow.

The Clathrin-dependent Spindle Proteome*

PubMed Central

Rao, Sushma R.; Flores-Rodriguez, Neftali; Page, Scott L.; Wong, Chin; Robinson, Phillip J.; Chircop, Megan

2016-01-01

The mitotic spindle is required for chromosome congression and subsequent equal segregation of sister chromatids. These processes involve a complex network of signaling molecules located at the spindle. The endocytic protein, clathrin, has a “moonlighting” role during mitosis, whereby it stabilizes the mitotic spindle. The signaling pathways that clathrin participates in to achieve mitotic spindle stability are unknown. Here, we assessed the mitotic spindle proteome and phosphoproteome in clathrin-depleted cells using quantitative MS/MS (data are available via ProteomeXchange with identifier PXD001603). We report a spindle proteome that consists of 3046 proteins and a spindle phosphoproteome consisting of 5157 phosphosites in 1641 phosphoproteins. Of these, 2908 (95.4%) proteins and 1636 (99.7%) phosphoproteins are known or predicted spindle-associated proteins. Clathrin-depletion from spindles resulted in dysregulation of 121 proteins and perturbed signaling to 47 phosphosites. The majority of these proteins increased in mitotic spindle abundance and six of these were validated by immunofluorescence microscopy. Functional pathway analysis confirmed the reported role of clathrin in mitotic spindle stabilization for chromosome alignment and highlighted possible new mechanisms of clathrin action. The data also revealed a novel second mitotic role for clathrin in bipolar spindle formation. PMID:27174698

The Clathrin-dependent Spindle Proteome.

PubMed

Rao, Sushma R; Flores-Rodriguez, Neftali; Page, Scott L; Wong, Chin; Robinson, Phillip J; Chircop, Megan

2016-08-01

The mitotic spindle is required for chromosome congression and subsequent equal segregation of sister chromatids. These processes involve a complex network of signaling molecules located at the spindle. The endocytic protein, clathrin, has a "moonlighting" role during mitosis, whereby it stabilizes the mitotic spindle. The signaling pathways that clathrin participates in to achieve mitotic spindle stability are unknown. Here, we assessed the mitotic spindle proteome and phosphoproteome in clathrin-depleted cells using quantitative MS/MS (data are available via ProteomeXchange with identifier PXD001603). We report a spindle proteome that consists of 3046 proteins and a spindle phosphoproteome consisting of 5157 phosphosites in 1641 phosphoproteins. Of these, 2908 (95.4%) proteins and 1636 (99.7%) phosphoproteins are known or predicted spindle-associated proteins. Clathrin-depletion from spindles resulted in dysregulation of 121 proteins and perturbed signaling to 47 phosphosites. The majority of these proteins increased in mitotic spindle abundance and six of these were validated by immunofluorescence microscopy. Functional pathway analysis confirmed the reported role of clathrin in mitotic spindle stabilization for chromosome alignment and highlighted possible new mechanisms of clathrin action. The data also revealed a novel second mitotic role for clathrin in bipolar spindle formation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Simple method for culture of peripheral blood lymphocytes of Testudinidae.

PubMed

Silva, T L; Silva, M I A; Venancio, L P R; Zago, C E S; Moscheta, V A G; Lima, A V B; Vizotto, L D; Santos, J R; Bonini-Domingos, C R; Azeredo-Oliveira, M T V

2011-12-06

We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.

[The effect of pemolin on the mitotic activity of Vicia faba L (author's transl)].

PubMed

Brabec, F; Röper, W

1976-02-01

The effect of diverse concentrations of 5-phenyl-2-imino-4-oxazolidone (PIO, pemolin, Tradon) on the mitotic activity in lateral roots of Vicia faba L. was studied by aerated and non-aerated hydrocultivation with and without mineral nutrition, respectively. With optimal conditions (aerated nutrient solution) weak PIO-concentrations, most significantly 10(-6) g/ml, effected a marked increase of the mitotic index. Contrarily, strong PIO-concentrations (10(-4) and 3 X 10(-4) g/ml = saturated solution) significantly decreased the mitotic index though simultaneously preserving the mitotic activity in long-term experiments, when on account of nutrient deficiency it had already collapsed in weak PIO-concentrations and the controls. The activating effect of weak PIO-concentrations compared with the controls is more significant in stress situations (nutrient deficiency, O2-deficiency) than under optimal conditions. Furthermore a slight acceleration of mid-mitotic phases (metaphase--anaphase) recognized by a marked decrease in percentage of these phases, can be stated with weak PIO-concentrations, again particularly so with 10(-6) g/ml. In total, dependent on concentration, pemolin presumably may either activate or suppress cell metabolism and particularly the mitotic cycle. The exact site of action of the substance is still unknown.

Evidence for a Transketolase-Mediated Metabolic Checkpoint Governing Biotrophic Growth in Rice Cells by the Blast Fungus Magnaporthe oryzae

PubMed Central

Fernandez, Jessie; Marroquin-Guzman, Margarita; Wilson, Richard A.

2014-01-01

The blast fungus Magnaporthe oryzae threatens global food security through the widespread destruction of cultivated rice. Foliar infection requires a specialized cell called an appressorium that generates turgor to force a thin penetration hypha through the rice cuticle and into the underlying epidermal cells, where the fungus grows for the first days of infection as a symptomless biotroph. Understanding what controls biotrophic growth could open new avenues for developing sustainable blast intervention programs. Here, using molecular genetics and live-cell imaging, we dismantled M. oryzae glucose-metabolizing pathways to reveal that the transketolase enzyme, encoded by TKL1, plays an essential role in facilitating host colonization during rice blast disease. In the absence of transketolase, Δtkl1 mutant strains formed functional appressoria that penetrated rice cuticles successfully and developed invasive hyphae (IH) in rice cells from primary hyphae. However, Δtkl1 could not undertake sustained biotrophic growth or cell-to-cell movement. Transcript data and observations using fluorescently labeled histone H1:RFP fusion proteins indicated Δtkl1 mutant strains were alive in host cells but were delayed in mitosis. Mitotic delay could be reversed and IH growth restored by the addition of exogenous ATP, a metabolite depleted in Δtkl1 mutant strains. We show that ATP might act via the TOR signaling pathway, and TOR is likely a downstream target of activation for TKL1. TKL1 is also involved in controlling the migration of appressorial nuclei into primary hyphae in host cells. When taken together, our results indicate transketolase has a novel role in mediating - via ATP and TOR signaling - an in planta-specific metabolic checkpoint that controls nuclear migration from appressoria into primary hyphae, prevents mitotic delay in early IH and promotes biotrophic growth. This work thus provides new information about the metabolic strategies employed by M. oryzae to enable rice cell colonization. PMID:25188286

Retention of Chs2p in the ER requires N-terminal CDK1-phosphorylation sites.

PubMed

Teh, Ee Mei; Chai, Chuan Chung; Yeong, Foong May

2009-09-15

In budding yeast, the secretory pathway is constitutively transporting cargoes such as invertase and alpha-factor throughout the cell division cycle. However, chitin synthase 2 (Chs2p), another cargo of the secretory pathway, is retained at the endoplasmic reticulum (ER) during mitosis when the mitotic kinase activity is high. Chs2p is exported from the ER to the mother-daughter neck only upon mitotic kinase destruction, indicating that the mitotic kinase activity is critical for the ER retention of Chs2p. However, a key question is whether the mitotic kinase acts directly upon Chs2p to prevent its ER export. We report here that mutation of Ser residues to Glu at 4 perfect CDK1-phosphorylation sites at the N-terminus of Chs2p leads to its retention in the ER when the mitotic kinase activity is absent. Conversely, Ser-to-Ala mutations result in the loss of Chs2p ER retention even when mitotic kinase activity is high. The mere overexpression of the non-destructible form of the mitotic cyclin in G(1) cells can confine the wild-type Chs2p but not the Ser-to-Ala mutant in the ER. Furthermore, overexpression of the Ser-to-Ala mutant kills cells. Time-lapsed imaging revealed that Chs2p is exported from the ER rapidly and synchronously to the Golgi upon metaphase release. Our data indicate that direct phosphorylation of Chs2p by the mitotic CDK1 helps restrain it in the ER during mitosis to prevent its rapid export in an untimely manner until after sister chromatid occurs and mitotic exit executed.

The structure of the mitotic spindle and nucleolus during mitosis in the amebo-flagellate Naegleria.

PubMed

Walsh, Charles J

2012-01-01

Mitosis in the amebo-flagellate Naegleria pringsheimi is acentrosomal and closed (the nuclear membrane does not break down). The large central nucleolus, which occupies about 20% of the nuclear volume, persists throughout the cell cycle. At mitosis, the nucleolus divides and moves to the poles in association with the chromosomes. The structure of the mitotic spindle and its relationship to the nucleolus are unknown. To identify the origin and structure of the mitotic spindle, its relationship to the nucleolus and to further understand the influence of persistent nucleoli on cellular division in acentriolar organisms like Naegleria, three-dimensional reconstructions of the mitotic spindle and nucleolus were carried out using confocal microscopy. Monoclonal antibodies against three different nucleolar regions and α-tubulin were used to image the nucleolus and mitotic spindle. Microtubules were restricted to the nucleolus beginning with the earliest prophase spindle microtubules. Early spindle microtubules were seen as short rods on the surface of the nucleolus. Elongation of the spindle microtubules resulted in a rough cage of microtubules surrounding the nucleolus. At metaphase, the mitotic spindle formed a broad band completely embedded within the nucleolus. The nucleolus separated into two discreet masses connected by a dense band of microtubules as the spindle elongated. At telophase, the distal ends of the mitotic spindle were still completely embedded within the daughter nucleoli. Pixel by pixel comparison of tubulin and nucleolar protein fluorescence showed 70% or more of tubulin co-localized with nucleolar proteins by early prophase. These observations suggest a model in which specific nucleolar binding sites for microtubules allow mitotic spindle formation and attachment. The fact that a significant mass of nucleolar material precedes the chromosomes as the mitotic spindle elongates suggests that spindle elongation drives nucleolar division.

The Utilization during Mitotic Cell Division of Loci Controlling Meiotic Recombination and Disjunction in DROSOPHILA MELANOGASTER

PubMed Central

Baker, Bruce S.; Carpenter, Adelaide T. C.; Ripoll, P.

1978-01-01

To inquire whether the loci identified by recombination-defective and disjunction-defective meiotic mutants in Drosophila are also utilized during mitotic cell division, the effects of 18 meiotic mutants (representing 13 loci) on mitotic chromosome stability have been examined genetically. To do this, meiotic-mutant-bearing flies heterozygous for recessive somatic cell markers were examined for the frequencies and types of spontaneous clones expressing the cell markers. In such flies, marked clones can arise via mitotic recombination, mutation, chromosome breakage, nondisjunction or chromosome loss, and clones from these different origins can be distinguished. In addition, meiotic mutants at nine loci have been examined for their effects on sensitivity to killing by UV and X rays.—Mutants at six of the seven recombination-defective loci examined (mei-9, mei-41, c(3)G, mei-W68, mei-S282, mei-352, mei-218) cause mitotic chromosome instability in both sexes, whereas mutants at one locus (mei-218) do not affect mitotic chromosome stability. Thus many of the loci utilized during meiotic recombination also function in the chromosomal economy of mitotic cells.—The chromosome instability produced by mei-41 alleles is the consequence of chromosome breakage, that of mei-9 alleles is primarily due to chromosome breakage and, to a lesser extent, to an elevated frequency of mitotic recombination, whereas no predominant mechanism responsible for the instability caused by c(3)G alleles is discernible. Since these three loci are defective in their responses to mutagen damage, their effects on chromosome stability in nonmutagenized cells are interpreted as resulting from an inability to repair spontaneous lesions. Both mei-W68 and mei-S282 increase mitotic recombination (and in mei-W68, to a lesser extent, chromosome loss) in the abdomen but not the wing. In the abdomen, the primary effect on chromosome stability occurs during the larval period when the abdominal histoblasts are in a nondividing (G2) state.—Mitotic recombination is at or above control levels in the presence of each of the recombination-defective meiotic mutants examined, suggesting that meiotic and mitotic recombination are under separate genetic control in Drosophila.—Of the six mutants examined that are defective in processes required for regular meiotic chromosome segregation, four (l(1)TW-6cs, cand, mei-S332, ord) affect mitotic chromosome behavior. At semi-restrictive temperatures, the cold sensitive lethal l(1)TW-6cs causes very frequent somatic spots, a substantial proportion of which are attributable to nondisjunction or loss. Thus, this locus specifies a function essential for chromosome segregation at mitosis as well as at the first meiotic division in females. The patterns of mitotic effects caused by cand, mei-S332, and ord suggest that they may be leaky alleles at essential loci that specify functions common to meiosis and mitosis. Mutants at the two remaining loci (nod, pal) do not affect mitotic chromosome stability. PMID:17248870

The influence of serotonin on the mitotic rate in the colonic crypt epithelium and in colonic adenocarcinoma in rats.

PubMed

Tutton, P J; Barkla, D H

1978-01-01

1. The mitotic rate in the crypts of Lieberkühn of the descending colon and in dimethylhydrazine-induced adenocarcinomata of the descending colon of rat was measured using a stathmokinetic technique. 2. Intraperitoneal injection of a small dose (10 microgram/kg) of serotonin resulted in an increase in the tumour cell mitotic rate. 3. Blockade of serotonin receptors by 2-bromolysergic acid diethylamide and depletion of tissue serotonin levels following injection of DL-6-fluorotryptophan both result in a decrease in the tumour cell mitotic rate. 4. Treatment with serotonin, 2-bromolysergic acid diethylamide and DL-6-fluorotryptophan were all without effect on the colonic crypt cell mitotic rate.

Aurora B potentiates Mps1 activation to ensure rapid checkpoint establishment at the onset of mitosis.

PubMed

Saurin, Adrian T; van der Waal, Maike S; Medema, René H; Lens, Susanne M A; Kops, Geert J P L

2011-01-01

The mitotic checkpoint prevents mitotic exit until all chromosomes are attached to spindle microtubules. Aurora B kinase indirectly invokes this checkpoint by destabilizing incorrect attachments; however, a more direct role remains controversial. In contrast, activity of the kinase Mps1 is indispensible for the mitotic checkpoint. Here we show that Aurora B and Hec1 are needed for efficient Mps1 recruitment to unattached kinetochores, allowing rapid Mps1 activation at the onset of mitosis. Live monitoring of cyclin B degradation reveals that this is essential to establish the mitotic checkpoint quickly at the start of mitosis. Delayed Mps1 activation and checkpoint establishment upon Aurora B inhibition or Hec1 depletion are rescued by tethering Mps1 to kinetochores, demonstrating that Mps1 recruitment is the primary role of Aurora B and Hec1 in mitotic checkpoint signalling. These data demonstrate a direct role for Aurora B in initiating the mitotic checkpoint rapidly at the onset of mitosis.

Mitosis can drive cell cannibalism through entosis

PubMed Central

Durgan, Joanne; Tseng, Yun-Yu; Hamann, Jens C; Domart, Marie-Charlotte; Collinson, Lucy; Overholtzer, Michael; Florey, Oliver

2017-01-01

Entosis is a form of epithelial cell cannibalism that is prevalent in human cancer, typically triggered by loss of matrix adhesion. Here, we report an alternative mechanism for entosis in human epithelial cells, driven by mitosis. Mitotic entosis is regulated by Cdc42, which controls mitotic morphology. Cdc42 depletion enhances mitotic deadhesion and rounding, and these biophysical changes, which depend on RhoA activation and are phenocopied by Rap1 inhibition, permit subsequent entosis. Mitotic entosis occurs constitutively in some human cancer cell lines and mitotic index correlates with cell cannibalism in primary human breast tumours. Adherent, wild-type cells can act efficiently as entotic hosts, suggesting that normal epithelia may engulf and kill aberrantly dividing neighbours. Finally, we report that Paclitaxel/taxol promotes mitotic rounding and subsequent entosis, revealing an unconventional activity of this drug. Together, our data uncover an intriguing link between cell division and cannibalism, of significance to both cancer and chemotherapy. DOI: http://dx.doi.org/10.7554/eLife.27134.001 PMID:28693721

A dynamic mode of mitotic bookmarking by transcription factors

PubMed Central

Teves, Sheila S; An, Luye; Hansen, Anders S; Xie, Liangqi; Darzacq, Xavier; Tjian, Robert

2016-01-01

During mitosis, transcription is shut off, chromatin condenses, and most transcription factors (TFs) are reported to be excluded from chromosomes. How do daughter cells re-establish the original transcription program? Recent discoveries that a select set of TFs remain bound on mitotic chromosomes suggest a potential mechanism for maintaining transcriptional programs through the cell cycle termed mitotic bookmarking. Here we report instead that many TFs remain associated with chromosomes in mouse embryonic stem cells, and that the exclusion previously described is largely a fixation artifact. In particular, most TFs we tested are significantly enriched on mitotic chromosomes. Studies with Sox2 reveal that this mitotic interaction is more dynamic than in interphase and is facilitated by both DNA binding and nuclear import. Furthermore, this dynamic mode results from lack of transcriptional activation rather than decreased accessibility of underlying DNA sequences in mitosis. The nature of the cross-linking artifact prompts careful re-examination of the role of TFs in mitotic bookmarking. DOI: http://dx.doi.org/10.7554/eLife.22280.001 PMID:27855781

Loading of PAX3 to Mitotic Chromosomes Is Mediated by Arginine Methylation and Associated with Waardenburg Syndrome*

PubMed Central

Wu, Tsu-Fang; Yao, Ya-Li; Lai, I-Lu; Lai, Chien-Chen; Lin, Pei-Lun; Yang, Wen-Ming

2015-01-01

PAX3 is a transcription factor critical to gene regulation in mammalian development. Mutations in PAX3 are associated with Waardenburg syndrome (WS), but the mechanism of how mutant PAX3 proteins cause WS remains unclear. Here, we found that PAX3 loads on mitotic chromosomes using its homeodomain. PAX3 WS mutants with mutations in homeodomain lose the ability to bind mitotic chromosomes. Moreover, loading of PAX3 on mitotic chromosomes requires arginine methylation, which is regulated by methyltransferase PRMT5 and demethylase JMJD6. Mutant PAX3 proteins that lose mitotic chromosome localization block cell proliferation and normal development of zebrafish. These results reveal the molecular mechanism of PAX3s loading on mitotic chromosomes and the importance of this localization pattern in normal development. Our findings suggest that PAX3 WS mutants interfere with the normal functions of PAX3 in a dominant negative manner, which is important to the understanding of the pathogenesis of Waardenburg syndrome. PMID:26149688

Neuregulin 1 Type II-ErbB Signaling Promotes Cell Divisions Generating Neurons from Neural Progenitor Cells in the Developing Zebrafish Brain.

PubMed

Sato, Tomomi; Sato, Fuminori; Kamezaki, Aosa; Sakaguchi, Kazuya; Tanigome, Ryoma; Kawakami, Koichi; Sehara-Fujisawa, Atsuko

2015-01-01

Post-mitotic neurons are generated from neural progenitor cells (NPCs) at the expense of their proliferation. Molecular and cellular mechanisms that regulate neuron production temporally and spatially should impact on the size and shape of the brain. While transcription factors such as neurogenin1 (neurog1) and neurod govern progression of neurogenesis as cell-intrinsic mechanisms, recent studies show regulatory roles of several cell-extrinsic or intercellular signaling molecules including Notch, FGF and Wnt in production of neurons/neural progenitor cells from neural stem cells/radial glial cells (NSCs/RGCs) in the ventricular zone (VZ). However, it remains elusive how production of post-mitotic neurons from neural progenitor cells is regulated in the sub-ventricular zone (SVZ). Here we show that newborn neurons accumulate in the basal-to-apical direction in the optic tectum (OT) of zebrafish embryos. While neural progenitor cells are amplified by mitoses in the apical ventricular zone, neurons are exclusively produced through mitoses of neural progenitor cells in the sub-basal zone, later in the sub-ventricular zone, and accumulate apically onto older neurons. This neurogenesis depends on Neuregulin 1 type II (NRG1-II)-ErbB signaling. Treatment with an ErbB inhibitor, AG1478 impairs mitoses in the sub-ventricular zone of the optic tectum. Removal of AG1478 resumes sub-ventricular mitoses without precedent mitoses in the apical ventricular zone prior to basal-to-apical accumulation of neurons, suggesting critical roles of ErbB signaling in mitoses for post-mitotic neuron production. Knockdown of NRG1-II impairs both mitoses in the sub-basal/sub-ventricular zone and the ventricular zone. Injection of soluble human NRG1 into the developing brain ameliorates neurogenesis of NRG1-II-knockdown embryos, suggesting a conserved role of NRG1 as a cell-extrinsic signal. From these results, we propose that NRG1-ErbB signaling stimulates cell divisions generating neurons from neural progenitor cells in the developing vertebrate brain.

Plk1 Inhibitors in Cancer Therapy: From Laboratory to Clinics.

PubMed

Gutteridge, Rosie Elizabeth Ann; Ndiaye, Mary Ann; Liu, Xiaoqi; Ahmad, Nihal

2016-07-01

Polo-like kinase 1 (Plk1) overexpression has been shown to occur in a wide range of tumors, prompting research and development of Plk1 inhibitors as a means of cancer treatment. This review discusses recent advances in the development of Plk1 inhibitors for cancer management. Plk1 inhibition has been shown to cause mitotic block and apoptosis of cells with higher mitotic index and therefore higher Plk1 expression. The potential of Plk1 inhibitors as cancer therapeutics has been widely investigated. However, a complete understanding of Plk1 biology/mechanism is yet to be fully achieved. Resistance to certain chemotherapeutic drugs has been linked to Plk1 overexpression, and Plk1-mediated mitotic events such as microtubule rearrangement have been found to reduce the efficacy of chemotherapeutic agents. The Plk1 inhibitor volasertib has shown considerable promise in clinical studies, having reached phase III trials. However, preclinical success with Plk1 inhibitors has not translated well into clinical success. In our view, combined therapies targeting other relevant pathways together with Plk1 may be vital to combat issues observed with monotherapy, especially resistance. In addition, research should also be directed toward understanding the mechanisms of Plk1 and designing additional next generations of specific, potent Plk1 inhibitors to target cancer. Mol Cancer Ther; 15(7); 1427-35. ©2016 AACR. ©2016 American Association for Cancer Research.

Probing Mitotic CENP-E Kinesin with the Tethered Cargo Motion Assay and Laser Tweezers.

PubMed

Gudimchuk, Nikita; Tarasovetc, Ekaterina V; Mustyatsa, Vadim; Drobyshev, Alexei L; Vitre, Benjamin; Cleveland, Don W; Ataullakhanov, Fazly I; Grishchuk, Ekaterina L

2018-06-05

Coiled-coil stalks of various kinesins differ significantly in predicted length and structure; this is an adaption that helps these motors carry out their specialized functions. However, little is known about the dynamic stalk configuration in moving motors. To gain insight into the conformational properties of the transporting motors, we developed a theoretical model to predict Brownian motion of a microbead tethered to the tail of a single, freely walking molecule. This approach, which we call the tethered cargo motion (TCM) assay, provides an accurate measure of the mechanical properties of motor-cargo tethering, verified using kinesin-1 conjugated to a microbead via DNA links in vitro. Applying the TCM assay to the mitotic kinesin CENP-E unexpectedly revealed that when walking along a microtubule track, this highly elongated molecule with a contour length of 230 nm formed a 20-nm-long tether. The stalk of a walking CENP-E could not be extended fully by application of sideways force with optical tweezers (up to 4 pN), implying that CENP-E carries its cargo in a compact configuration. Assisting force applied along the microtubule track accelerates CENP-E walking, but this increase does not depend on the presence of the CENP-E stalk. Our results suggest that the unusually large stalk of CENP-E has little role in regulating its function as a transporter. The adjustable stalk configuration may represent a regulatory mechanism for controlling the physical reach between kinetochore-bound CENP-E and spindle microtubules, or it may assist localizing various kinetochore regulators in the immediate vicinity of the kinetochore-embedded microtubule ends. The TCM assay and underlying theoretical framework will provide a general guide for determining the dynamic configurations of various molecular motors moving along their tracks, freely or under force. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Fe-S cluster coordination of the chromokinesin KIF4A alters its sub-cellular localization during mitosis.

PubMed

Ben-Shimon, Lilach; Paul, Viktoria D; David-Kadoch, Galit; Volpe, Marina; Stümpfig, Martin; Bill, Eckhard; Mühlenhoff, Ulrich; Lill, Roland; Ben-Aroya, Shay

2018-05-30

Fe-S clusters act as co-factors of proteins with diverse functions, e.g. in DNA repair. Down-regulation of the cytosolic iron-sulfur protein assembly (CIA) machinery promotes genomic instability by the inactivation of multiple DNA repair pathways. Furthermore, CIA deficiencies are associated with so far unexplained mitotic defects. Here, we show that CIA2B and MMS19, constituents of the CIA targeting complex involved in facilitating Fe-S cluster insertion into cytosolic and nuclear target proteins, co-localize with components of the mitotic machinery. Down-regulation of CIA2B and MMS19 impairs the mitotic cycle. We identify the chromokinesin KIF4A as a mitotic component involved in these effects. KIF4A binds a Fe-S cluster in vitro through its conserved cysteine-rich domain. We demonstrate in vivo that this domain is required for the mitosis-related KIF4A localization and for the mitotic defects associated with KIF4A knockout. KIF4A is the first identified mitotic component carrying such a post-translational modification. These findings suggest that the lack of Fe-S clusters in KIF4A upon down-regulation of the CIA targeting complex contributes to the mitotic defects. © 2018. Published by The Company of Biologists Ltd.

Immunodetection of phosphohistone H3 as a surrogate of mitotic figure count and clinical outcome in cutaneous melanoma.

PubMed

Tetzlaff, Michael T; Curry, Jonathan L; Ivan, Doina; Wang, Wei-Lien; Torres-Cabala, Carlos A; Bassett, Roland L; Valencia, Karla M; McLemore, Michael S; Ross, Merrick I; Prieto, Victor G

2013-09-01

In the American Joint Committee on Cancer (AJCC)-TNM (2009) staging system, the key prognostic factor in cutaneous melanoma is the depth of dermal invasion (Breslow thickness) with further refinement according to the presence of epidermal ulceration or dermal mitoses. Immunodetection of phosphohistone H3 has been shown to facilitate the identification of mitotic figures in various neoplasms. We selected 120 cases of primary cutaneous melanoma with completely annotated histopathologic parameters and clinical outcomes and performed double immunohistochemical staining for MLANA (Mart-1/Melan-A) and phosphohistone H3. One hundred and thirteen cases were amenable to antiphosphohistone H3 staining from 66 men and 47 women, with mean age of 64 years (9-93), including 61 superficial spreading type, 24 nodular, 6 lentigo maligna, 8 acral lentiginous, and 14 unclassified. The mean Breslow thickness was 2.53 mm (0.20-25), ulceration was present in 25/113 (22%) and the mean mitotic count was 3.2/mm(2) (<1-29/mm(2)). In 27/113 (24%) of the cases, antiphosphohistone H3 failed to highlight mitotic figures anywhere in the tissue (normal or tumor cell), whereas in 86/113 (76%) antiphosphohistone H3 detected at least one mitotic figure. Among the latter, antiphosphohistone H3 did not detect mitotic figures in dermal tumor cells in 37/86 cases (43%), whereas anti-PHH3 identified at least one melanocytic mitotic figure in the other 49/86 cases (57%; range: 1-66/mm(2)). The relationship between phosphohistone H3 and manual mitotic count was statistically significant (Pearson correlation=0.59, P<0.0001). Logistic regression analyses demonstrated an association between the development of subsequent metastatic disease and the following variables: mitotic figures (odds ratio (OR)=5.7; P=0.0001); phosphohistone H3-positive mitotic figures (OR=3.0; P=0.008); Breslow thickness (OR=4.0 per mm; P=0.0002); ulceration (OR=3.94; P=0.008). The application of phosphohistone H3 immunohistochemistry to the description of primary cutaneous melanoma is useful in identifying mitotic figures, improves upon the specificity of this designation when used together with MLANA, and correlates with an increased risk for metastasis in univariate analyses.

Cytoplasmic connection of sperm cells to the pollen vegetative cell nucleus: potential roles of the male germ unit revisited.

PubMed

McCue, Andrea D; Cresti, Mauro; Feijó, José A; Slotkin, R Keith

2011-03-01

The male germ cells of angiosperm plants are neither free-living nor flagellated and therefore are dependent on the unique structure of the pollen grain for fertilization. During angiosperm male gametogenesis, an asymmetric mitotic division produces the generative cell, which is completely enclosed within the cytoplasm of the larger pollen grain vegetative cell. Mitotic division of the generative cell generates two sperm cells that remain connected by a common extracellular matrix with potential intercellular connections. In addition, one sperm cell has a cytoplasmic projection in contact with the vegetative cell nucleus. The shared extracellular matrix of the two sperm cells and the physical association of one sperm cell to the vegetative cell nucleus forms a linkage of all the genetic material in the pollen grain, termed the male germ unit. Found in species representing both the monocot and eudicot lineages, the cytoplasmic projection is formed by vesicle formation and microtubule elongation shortly after the formation of the generative cell and tethers the male germ unit until just prior to fertilization. The cytoplasmic projection plays a structural role in linking the male germ unit, but potentially plays other important roles. Recently, it has been speculated that the cytoplasmic projection and the male germ unit may facilitate communication between the somatic vegetative cell nucleus and the germinal sperm cells, via RNA and/or protein transport. This review focuses on the nature of the sperm cell cytoplasmic projection and the potential communicative function of the male germ unit.

Live-cell imaging RNAi screen identifies PP2A–B55α and importin-β1 as key mitotic exit regulators in human cells

PubMed Central

Schmitz, Michael H. A.; Held, Michael; Janssens, Veerle; Hutchins, James R. A.; Hudecz, Otto; Ivanova, Elitsa; Goris, Jozef; Trinkle-Mulcahy, Laura; Lamond, Angus I.; Poser, Ina; Hyman, Anthony A.; Mechtler, Karl; Peters, Jan-Michael; Gerlich, Daniel W.

2013-01-01

When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells1. This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates2–4. Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit5,6, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we use a live-cell imaging assay and RNAi knockdown to screen a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identify a trimeric PP2A–B55α complex as a key factor in mitotic spindle breakdown and postmitotic reassembly of the nuclear envelope, Golgi apparatus and decondensed chromatin. Using a chemically induced mitotic exit assay, we find that PP2A–B55α functions downstream of Cdk1 inactivation. PP2A–B55α isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate, histone H1, and was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor importin-β1, and RNAi depletion of importin-β1 delayed mitotic exit synergistically with PP2A–B55α. This demonstrates that PP2A–B55α and importin-β1 cooperate in the regulation of postmitotic assembly mechanisms in human cells. PMID:20711181

AN INDIRECT METHOD TO ASSAY FOR MITOTIC CENTERS IN SAND DOLLAR (DENDRASTER EXCENTRICUS) EGGS

PubMed Central

Went, Hans A.

1966-01-01

It is possible consistently to induce sea urchin and sand dollar eggs to cleave directly from one cell into four cells. This is done by exposing the fertilized eggs to benzimidazole for 20 to 30 min beginning about early metaphase. The mitotic apparatus regresses, the cells do not cleave, and shortly after they are returned to normal sea water an early-prophase-appearing nucleus is present in each cell. Each cell then organizes a tetrapolar tetrahedral mitotic apparatus de novo, instead of transforming a bipolar mitotic apparatus into a tetrapolar figure, and cleaves one-to-four. In another type of experiment, it appears that sand dollar eggs exposed to mercaptoethanol during the first period of mitotic center duplication have only half as many centers by first cleavage metaphase as the normal controls. This is consistent with an earlier report by Mazia et al (1960). Using this same experimental technique, it was demonstrated that benzimidazole, on the contrary, does not interfere with mitotic center duplication in sand dollar eggs. A labeling experiment demonstrated that benzimidazole does not interfere markedly with the normal pattern of incorporation of C14-thymidine into the DNA of sea urchin eggs. The data reported here suggest that judicious treatment of sand dollar eggs (and probably sea urchin eggs, too) with benzimidazole can induce the eggs to cleave into as many cells as there were mitotic centers sometime earlier, for example at early metaphase of the first cleavage division. This provides a very useful tool for studies on the process of mitotic center duplication. PMID:6008198

An indirect method to assay for mitotic centers in sand dollar (Dendraster excentricus) eggs.

PubMed

Went, H A

1966-09-01

It is possible consistently to induce sea urchin and sand dollar eggs to cleave directly from one cell into four cells. This is done by exposing the fertilized eggs to benzimidazole for 20 to 30 min beginning about early metaphase. The mitotic apparatus regresses, the cells do not cleave, and shortly after they are returned to normal sea water an early-prophase-appearing nucleus is present in each cell. Each cell then organizes a tetrapolar tetrahedral mitotic apparatus de novo, instead of transforming a bipolar mitotic apparatus into a tetrapolar figure, and cleaves one-to-four. In another type of experiment, it appears that sand dollar eggs exposed to mercaptoethanol during the first period of mitotic center duplication have only half as many centers by first cleavage metaphase as the normal controls. This is consistent with an earlier report by Mazia et al (1960). Using this same experimental technique, it was demonstrated that benzimidazole, on the contrary, does not interfere with mitotic center duplication in sand dollar eggs. A labeling experiment demonstrated that benzimidazole does not interfere markedly with the normal pattern of incorporation of C(14)-thymidine into the DNA of sea urchin eggs. The data reported here suggest that judicious treatment of sand dollar eggs (and probably sea urchin eggs, too) with benzimidazole can induce the eggs to cleave into as many cells as there were mitotic centers sometime earlier, for example at early metaphase of the first cleavage division. This provides a very useful tool for studies on the process of mitotic center duplication.

Arsenite-induced mitotic death involves stress response and is independent of tubulin polymerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taylor, B. Frazier; McNeely, Samuel C.; Miller, Heather L.

2008-07-15

Arsenite, a known mitotic disruptor, causes cell cycle arrest and cell death at anaphase. The mechanism causing mitotic arrest is highly disputed. We compared arsenite to the spindle poisons nocodazole and paclitaxel. Immunofluorescence analysis of {alpha}-tubulin in interphase cells demonstrated that, while nocodazole and paclitaxel disrupt microtubule polymerization through destabilization and hyperpolymerization, respectively, microtubules in arsenite-treated cells remain comparable to untreated cells even at supra-therapeutic concentrations. Immunofluorescence analysis of {alpha}-tubulin in mitotic cells showed spindle formation in arsenite- and paclitaxel-treated cells but not in nocodazole-treated cells. Spindle formation in arsenite-treated cells appeared irregular and multi-polar. {gamma}-tubulin staining showed that cellsmore » treated with nocodazole and therapeutic concentrations of paclitaxel contained two centrosomes. In contrast, most arsenite-treated mitotic cells contained more than two centrosomes, similar to centrosome abnormalities induced by heat shock. Of the three drugs tested, only arsenite treatment increased expression of the inducible isoform of heat shock protein 70 (HSP70i). HSP70 and HSP90 proteins are intimately involved in centrosome regulation and mitotic spindle formation. HSP90 inhibitor 17-DMAG sensitized cells to arsenite treatment and increased arsenite-induced centrosome abnormalities. Combined treatment of 17-DMAG and arsenite resulted in a supra-additive effect on viability, mitotic arrest, and centrosome abnormalities. Thus, arsenite-induced abnormal centrosome amplification and subsequent mitotic arrest is independent of effects on tubulin polymerization and may be due to specific stresses that are protected against by HSP90 and HSP70.« less

The MiAge Calculator: a DNA methylation-based mitotic age calculator of human tissue types.

PubMed

Youn, Ahrim; Wang, Shuang

2018-01-01

Cell division is important in human aging and cancer. The estimation of the number of cell divisions (mitotic age) of a given tissue type in individuals is of great interest as it allows not only the study of biological aging (using a new molecular aging target) but also the stratification of prospective cancer risk. Here, we introduce the MiAge Calculator, a mitotic age calculator based on a novel statistical framework, the MiAge model. MiAge is designed to quantitatively estimate mitotic age (total number of lifetime cell divisions) of a tissue using the stochastic replication errors accumulated in the epigenetic inheritance process during cell divisions. With the MiAge model, the MiAge Calculator was built using the training data of DNA methylation measures of 4,020 tumor and adjacent normal tissue samples from eight TCGA cancer types and was tested using the testing data of DNA methylation measures of 2,221 tumor and adjacent normal tissue samples of five other TCGA cancer types. We showed that within each of the thirteen cancer types studied, the estimated mitotic age is universally accelerated in tumor tissues compared to adjacent normal tissues. Across the thirteen cancer types, we showed that worse cancer survivals are associated with more accelerated mitotic age in tumor tissues. Importantly, we demonstrated the utility of mitotic age by showing that the integration of mitotic age and clinical information leads to improved survival prediction in six out of the thirteen cancer types studied. The MiAge Calculator is available at http://www.columbia.edu/∼sw2206/softwares.htm .

Epithelial tricellular junctions act as interphase cell shape sensors to orient mitosis

PubMed Central

Bosveld, Floris; Markova, Olga; Guirao, Boris; Martin, Charlotte; Wang, Zhimin; Pierre, Anaëlle; Balakireva, Maria; Gaugue, Isabelle; Ainslie, Anna; Christophorou, Nicolas; Lubensky, David K.; Minc, Nicolas; Bellaïche, Yohanns

2017-01-01

The orientation of cell division along the interphase cell long-axis, the century old Hertwig’s rule, has profound roles in tissue proliferation, morphogenesis, architecture and mechanics1,2. In epithelial tissues, the shape of the interphase cell is influenced by cell adhesion, mechanical stress, neighbour topology, and planar polarity pathways3–12. At mitosis, epithelial cells usually round up to ensure faithful chromosome segregation and to promote morphogenesis1. The mechanisms underlying interphase cell shape sensing in tissues are therefore unknown. We found that in Drosophila epithelia, tricellular junctions (TCJ) localize microtubule force generators, orienting cell division via the Dynein associated protein Mud independently of the classical Pins/Gαi pathway. Moreover, as cells round up during mitosis, TCJs serve as spatial landmarks, encoding information about interphase cell shape anisotropy to orient division in the rounded mitotic cell. Finally, experimental and simulation data show that shape and mechanical strain sensing by the TCJ emerge from a general geometric property of TCJ distributions in epithelial tissues. Thus, in addition to their function as epithelial barrier structures, TCJs serve as polarity cues promoting geometry and mechanical sensing in epithelial tissues. PMID:26886796

Expansion and concatenation of nonmuscle myosin IIA filaments drive cellular contractile system formation during interphase and mitosis

PubMed Central

Fenix, Aidan M.; Taneja, Nilay; Buttler, Carmen A.; Lewis, John; Van Engelenburg, Schuyler B.; Ohi, Ryoma; Burnette, Dylan T.

2016-01-01

Cell movement and cytokinesis are facilitated by contractile forces generated by the molecular motor, nonmuscle myosin II (NMII). NMII molecules form a filament (NMII-F) through interactions of their C-terminal rod domains, positioning groups of N-terminal motor domains on opposite sides. The NMII motors then bind and pull actin filaments toward the NMII-F, thus driving contraction. Inside of crawling cells, NMIIA-Fs form large macromolecular ensembles (i.e., NMIIA-F stacks), but how this occurs is unknown. Here we show NMIIA-F stacks are formed through two non–mutually exclusive mechanisms: expansion and concatenation. During expansion, NMIIA molecules within the NMIIA-F spread out concurrent with addition of new NMIIA molecules. Concatenation occurs when multiple NMIIA-Fs/NMIIA-F stacks move together and align. We found that NMIIA-F stack formation was regulated by both motor activity and the availability of surrounding actin filaments. Furthermore, our data showed expansion and concatenation also formed the contractile ring in dividing cells. Thus interphase and mitotic cells share similar mechanisms for creating large contractile units, and these are likely to underlie how other myosin II–based contractile systems are assembled. PMID:26960797

Computational Models Reveal a Passive Mechanism for Cell Migration in the Crypt

PubMed Central

Dunn, Sara-Jane; Näthke, Inke S.; Osborne, James M.

2013-01-01

Cell migration in the intestinal crypt is essential for the regular renewal of the epithelium, and the continued upward movement of cells is a key characteristic of healthy crypt dynamics. However, the driving force behind this migration is unknown. Possibilities include mitotic pressure, active movement driven by motility cues, or negative pressure arising from cell loss at the crypt collar. It is possible that a combination of factors together coordinate migration. Here, three different computational models are used to provide insight into the mechanisms that underpin cell movement in the crypt, by examining the consequence of eliminating cell division on cell movement. Computational simulations agree with existing experimental results, confirming that migration can continue in the absence of mitosis. Importantly, however, simulations allow us to infer mechanisms that are sufficient to generate cell movement, which is not possible through experimental observation alone. The results produced by the three models agree and suggest that cell loss due to apoptosis and extrusion at the crypt collar relieves cell compression below, allowing cells to expand and move upwards. This finding suggests that future experiments should focus on the role of apoptosis and cell extrusion in controlling cell migration in the crypt. PMID:24260407

ASURA (PHB2) Is Required for Kinetochore Assembly and Subsequent Chromosome Congression

PubMed Central

Lee, Mei Hann; Lin, Linyen; Equilibrina, Ilma; Uchiyama, Susumu; Matsunaga, Sachihiro; Fukui, Kiichi

2011-01-01

ASURA (PHB2) knockdown has been known to cause premature loss of sister chromatid cohesion, and disrupt the localization of several outer plate proteins to the kinetochore. As a result, cells are arrested at mitotic phase and chromosomes fail to congress to the metaphase plate. In this study, we further clarified the mechanism underlying ASURA function on chromosome congression. Interestingly, ASURA is not specifically localized at the kinetochore during mitotic phase, unlike other kinetochore proteins which construct the kinetochore. Electron microscopy (EM) observation showed that ASURA is required for proper kinetochore formation. By the partial depletion of ASURA, kinetochore maturation is impaired, and kinetochores showing fibrillar balls without a well-defined outer plates are often observed. Moreover, even when the outer plates of kinetochores are constructed, most showed structures stretched and/or distended from the centromere, which resembled premature kinetochores at prometaphase, indicating that the constructed kinetochore plates are less rigid against tension derived from kinetochore microtubule pulling forces. We concluded that ASURA is an essential protein for complete kinetochore development, although ASURA is not being integrated to the kinetochore. These results highlight the uniqueness of ASURA as a kinetochore protein. PMID:22282585

Cdk1 Activates Pre-mitotic Nuclear Envelope Dynein Recruitment and Apical Nuclear Migration in Neural Stem Cells.

PubMed

Baffet, Alexandre D; Hu, Daniel J; Vallee, Richard B

2015-06-22

Dynein recruitment to the nuclear envelope is required for pre-mitotic nucleus-centrosome interactions in nonneuronal cells and for apical nuclear migration in neural stem cells. In each case, dynein is recruited to the nuclear envelope (NE) specifically during G2 via two nuclear pore-mediated mechanisms involving RanBP2-BicD2 and Nup133-CENP-F. The mechanisms responsible for cell-cycle control of this behavior are unknown. We now find that Cdk1 serves as a direct master controller for NE dynein recruitment in neural stem cells and HeLa cells. Cdk1 phosphorylates conserved sites within RanBP2 and activates BicD2 binding and early dynein recruitment. Late recruitment is triggered by a Cdk1-induced export of CENP-F from the nucleus. Forced NE targeting of BicD2 overrides Cdk1 inhibition, fully rescuing dynein recruitment and nuclear migration in neural stem cells. These results reveal how NE dynein recruitment is cell-cycle regulated and identify the trigger mechanism for apical nuclear migration in the brain. Copyright © 2015 Elsevier Inc. All rights reserved.

Proteasome inhibition enhances the efficacy of volasertib-induced mitotic arrest in AML in vitro and prolongs survival in vivo.

PubMed

Schnerch, Dominik; Schüler, Julia; Follo, Marie; Felthaus, Julia; Wider, Dagmar; Klingner, Kathrin; Greil, Christine; Duyster, Justus; Engelhardt, Monika; Wäsch, Ralph

2017-03-28

Elderly and frail patients, diagnosed with acute myeloid leukemia (AML) and ineligible to undergo intensive treatment, have a dismal prognosis. The small molecule inhibitor volasertib induces a mitotic block via inhibition of polo-like kinase 1 and has shown remarkable anti-leukemic activity when combined with low-dose cytarabine. We have demonstrated that AML cells are highly vulnerable to cell death in mitosis yet manage to escape a mitotic block through mitotic slippage by sustained proteasome-dependent slow degradation of cyclin B. Therefore, we tested whether interfering with mitotic slippage through proteasome inhibition arrests and kills AML cells more efficiently during mitosis. We show that therapeutic doses of bortezomib block the slow degradation of cyclin B during a volasertib-induced mitotic arrest in AML cell lines and patient-derived primary AML cells. In a xenotransplant mouse model of human AML, mice receiving volasertib in combination with bortezomib showed superior disease control compared to mice receiving volasertib alone, highlighting the potential therapeutic impact of this drug combination.

A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germ line stem cells

PubMed Central

Hehnly, Heidi; Canton, David; Bucko, Paula; Langeberg, Lorene K; Ogier, Leah; Gelman, Irwin; Santana, L Fernando; Wordeman, Linda; Scott, John D

2015-01-01

Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma. Depletion of Gravin correlates with an increased mitotic index and disorganization of seminiferous tubules. Biochemical, super-resolution imaging, and enzymology approaches establish that this Gravin scaffold accumulates at the mother spindle pole during metaphase. Manipulating elements of the Gravin-Aurora A-Plk1 axis prompts mitotic delay and prevents appropriate assembly of astral microtubules to promote spindle misorientation. These pathological responses are conserved in seminiferous tubules from Gravin−/− mice where an overabundance of Oct3/4 positive germ line stem cells displays randomized orientation of mitotic spindles. Thus, we propose that Gravin-mediated recruitment of Aurora A and Plk1 to the mother (oldest) spindle pole contributes to the fidelity of symmetric cell division. DOI: http://dx.doi.org/10.7554/eLife.09384.001 PMID:26406118

Loading of PAX3 to Mitotic Chromosomes Is Mediated by Arginine Methylation and Associated with Waardenburg Syndrome.

PubMed

Wu, Tsu-Fang; Yao, Ya-Li; Lai, I-Lu; Lai, Chien-Chen; Lin, Pei-Lun; Yang, Wen-Ming

2015-08-14

PAX3 is a transcription factor critical to gene regulation in mammalian development. Mutations in PAX3 are associated with Waardenburg syndrome (WS), but the mechanism of how mutant PAX3 proteins cause WS remains unclear. Here, we found that PAX3 loads on mitotic chromosomes using its homeodomain. PAX3 WS mutants with mutations in homeodomain lose the ability to bind mitotic chromosomes. Moreover, loading of PAX3 on mitotic chromosomes requires arginine methylation, which is regulated by methyltransferase PRMT5 and demethylase JMJD6. Mutant PAX3 proteins that lose mitotic chromosome localization block cell proliferation and normal development of zebrafish. These results reveal the molecular mechanism of PAX3s loading on mitotic chromosomes and the importance of this localization pattern in normal development. Our findings suggest that PAX3 WS mutants interfere with the normal functions of PAX3 in a dominant negative manner, which is important to the understanding of the pathogenesis of Waardenburg syndrome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

[Mechanism of mutant induction in the ade2 gene of diploid Saccharomyces cerevisiae yeasts by ultraviolet rays].

PubMed

Gordenin, D A; Inge-Vechtomov, S G

1981-01-01

Ultraviolet light (UV) at 3000 ergs/mm-2 induces ade2 mutants with a frequency about 10(-4) in wild-type haploid strains of yeast and about 10(-5) in diploid wild-type strains. UV irradiation effectively induced mitotic segregation of ade2 in the heterozygous diploid (the frequency of segregation is 6%). Interallelic complementation and localization spectra are similar for mutations induced both in haploids and diploids. The occurrence of ade2 mutants in diploids correlated with mitotic segregation of the marker his8 which is situated in the same arm of XY chromosome as ade2 is, distal to the centromere. Our data about the frequency of ade2 mutants in diploids and haploids, the frequency of ade2 mitotic segregation, mitotic segregation of other markers and genetic characteristics of ade2 mutations confirm the suggestion that the major mechanism of diploid ade2 mutants appearance is mutation in one of the two ADE2 alleles and consequent mitotic homozygotisation of mutation as a result of mitotic crossingover between ade2 and the centromere.

Regulation of spindle integrity and mitotic fidelity by BCCIP

PubMed Central

Huhn, S C; Liu, J; Ye, C; Lu, H; Jiang, X; Feng, X; Ganesan, S; White, E; Shen, Z

2017-01-01

Centrosomes together with the mitotic spindle ensure the faithful distribution of chromosomes between daughter cells, and spindle orientation is a major determinant of cell fate during tissue regeneration. Spindle defects are not only an impetus of chromosome instability but are also a cause of developmental disorders involving defective asymmetric cell division. In this work, we demonstrate BCCIP, especially BCCIPα, as a previously unidentified component of the mitotic spindle pole and the centrosome. We demonstrate that BCCIP localizes proximal to the mother centriole and participates in microtubule organization and then redistributes to the spindle pole to ensure faithful spindle architecture. We find that BCCIP depletion leads to morphological defects, disoriented mitotic spindles, chromosome congression defects and delayed mitotic progression. Our study identifies BCCIP as a novel factor critical for microtubule regulation and explicates a mechanism utilized by BCCIP in tumor suppression. PMID:28394342

Comparative analysis of mitotic aberrations induced by diethyl sulphate (DES) and sodium azide (SA) in Vicia faba L. (Fabaceae).

PubMed

Bhat, Tariq Ahmad; Sharma, Monika; Anis, M

2007-03-01

The present investigation provides a comparative account of different concentrations (0.01, 0.02, 0.03, 0.04, 0.05 and 0.06%) of diethylsulphate (DES) and Sodium Azide (SA) on mitotic aberrations, seed germination, seedling survival, plant height and mitotic index in Vicia faba L. variety major. The control plants were normal while as treated ones showed significant alterations. The mutagens caused dose dependent decrease in seed germination, seedling survival, plant height and mitotic index. All the parameters were found negatively affected and were positively correlated with mutagenic concentrations. The cytological study revealed various types of mitotic aberrations, among them the dominant were fragments, stickiness, precocious separation, c-metaphase, ring chromosomes, unequal separation, laggards, bridges, micronuclei, disturbed anaphase etc. Stickiness and fragments were more frequent as compared to other types.

B-chromosome systems in the greater glider (Petauroides volans volans) (Marsupialia:Petauridae). I. B-chromosome distribution.

PubMed

McQuade, L R

1985-01-01

Variations in diploid chromosome number, due to the presence of B chromosomes, are found within the distribution of P. v. volans. B chromosomes vary in number between one and eight per animal, are mitotically stable in various body tissues and, unlike the Y chromosome in male P. v. volans, are not eliminated from bone marrow cells. Animals possessing B chromosomes have a distinct distribution, and it appears that a stable equilibrium between the forces of B chromosome accumulation or elimination is operating in those populations possessing these chromosomes.

Hypermutable DNA chronicles the evolution of human colon cancer

PubMed Central

Naxerova, Kamila; Brachtel, Elena; Salk, Jesse J.; Seese, Aaron M.; Power, Karen; Abbasi, Bardia; Snuderl, Matija; Chiang, Sarah; Kasif, Simon; Jain, Rakesh K.

2014-01-01

Intratumor genetic heterogeneity reflects the evolutionary history of a cancer and is thought to influence treatment outcomes. Here we report that a simple PCR-based assay interrogating somatic variation in hypermutable polyguanine (poly-G) repeats can provide a rapid and reliable assessment of mitotic history and clonal architecture in human cancer. We use poly-G repeat genotyping to study the evolution of colon carcinoma. In a cohort of 22 patients, we detect poly-G variants in 91% of tumors. Patient age is positively correlated with somatic mutation frequency, suggesting that some poly-G variants accumulate before the onset of carcinogenesis during normal division in colonic stem cells. Poorly differentiated tumors have fewer mutations than well-differentiated tumors, possibly indicating a shorter mitotic history of the founder cell in these cancers. We generate poly-G mutation profiles of spatially separated samples from primary carcinomas and matched metastases to build well-supported phylogenetic trees that illuminate individual patients’ path of metastatic progression. Our results show varying degrees of intratumor heterogeneity among patients. Finally, we show that poly-G mutations can be found in other cancers than colon carcinoma. Our approach can generate reliable maps of intratumor heterogeneity in large numbers of patients with minimal time and cost expenditure. PMID:24753616

Regulation of mitosis by the NIMA kinase involves TINA and its newly discovered partner, An-WDR8, at spindle pole bodies

PubMed Central

Shen, Kuo-Fang; Osmani, Stephen A.

2013-01-01

The NIMA kinase is required for mitotic nuclear pore complex disassembly and potentially controls other mitotic-specific events. To investigate this possibility, we imaged NIMA–green fluorescent protein (GFP) using four-dimensional spinning disk confocal microscopy. At mitosis NIMA-GFP locates to spindle pole bodies (SPBs), which contain Cdk1/cyclin B, followed by Aurora, TINA, and the BimC kinesin. NIMA promotes NPC disassembly in a spatially regulated manner starting near SPBs. NIMA is also required for TINA, a NIMA-interacting protein, to locate to SPBs during initiation of mitosis, and TINA is then necessary for locating NIMA back to SPBs during mitotic progression. To help expand the NIMA-TINA pathway, we affinity purified TINA and found it to uniquely copurify with An-WDR8, a WD40-domain protein conserved from humans to plants. Like TINA, An-WDR8 accumulates within nuclei during G2 but disperses from nuclei before locating to mitotic SPBs. Without An-WDR8, TINA levels are greatly reduced, whereas TINA is necessary for mitotic targeting of An-WDR8. Finally, we show that TINA is required to anchor mitotic microtubules to SPBs and, in combination with An-WDR8, for successful mitosis. The findings provide new insights into SPB targeting and indicate that the mitotic microtubule-anchoring system at SPBs involves WDR8 in complex with TINA. PMID:24152731

Effect of HIV-1 Tat on the formation of the mitotic spindle by interaction with ribosomal protein S3.

PubMed

Kim, Jiyoung; Kim, Yeon-Soo

2018-06-06

Human immunodeficiency virus type 1 (HIV-1) Tat, an important regulator of viral transcription, interacts with diverse cellular proteins and promotes or inhibits cell proliferation. Here, we show that ribosomal protein S3 (RPS3) plays an important role in mitosis through an interaction with α-tubulin and that Tat binds to and inhibits the localization of RPS3 in the mitotic spindle during mitosis. RPS3 colocalized with α-tubulin around chromosomes in the mitotic spindle. Depletion of RPS3 promoted α-tubulin assembly, while overexpression of RPS3 impaired α-tubulin assembly. Depletion of RPS3 resulted in aberrant mitotic spindle formation, segregation failure, and defective abscission. Moreover, ectopic expression of RPS3 rescued the cell proliferation defect in RPS3-knockdown cells. HIV-1 Tat interacted with RPS3 through its basic domain and increased the level of RPS3 in the nucleus. Expression of Tat caused defects in mitotic spindle formation and chromosome assembly in mitosis. Moreover, the localization of RPS3 in the mitotic spindle was disrupted when HIV-1 Tat was expressed in HeLa and Jurkat cells. These results suggest that Tat inhibits cell proliferation via an interaction with RPS3 and thereby disrupts mitotic spindle formation during HIV-1 infection. These results might provide insight into the mechanism underlying lymphocyte pathogenesis during HIV-1 infection.

PIASy Mediates SUMO-2/3 Conjugation of Poly(ADP-ribose) Polymerase 1 (PARP1) on Mitotic Chromosomes*

PubMed Central

Ryu, Hyunju; Al-Ani, Gada; Deckert, Katelyn; Kirkpatrick, Donald; Gygi, Steven P.; Dasso, Mary; Azuma, Yoshiaki

2010-01-01

PIASy is a small ubiquitin-related modifier (SUMO) ligase that modifies chromosomal proteins in mitotic Xenopus egg extracts and plays an essential role in mitotic chromosome segregation. We have isolated a novel SUMO-2/3-modified mitotic chromosomal protein and identified it as poly(ADP-ribose) polymerase 1 (PARP1). PARP1 was robustly conjugated to SUMO-2/3 on mitotic chromosomes but not on interphase chromatin. PIASy promotes SUMOylation of PARP1 both in egg extracts and in vitro reconstituted SUMOylation assays. Through tandem mass spectrometry analysis of mitotically SUMOylated PARP1, we identified a residue within the BRCA1 C-terminal domain of PARP1 (lysine 482) as its primary SUMOylation site. Mutation of this residue significantly reduced PARP1 SUMOylation in egg extracts and enhanced the accumulation of species derived from modification of secondary lysine residues in assays using purified components. SUMOylation of PARP1 did not alter in vitro PARP1 enzyme activity, poly-ADP-ribosylation (PARylation), nor did inhibition of SUMOylation of PARP1 alter the accumulation of PARP1 on mitotic chromosomes, suggesting that SUMOylation regulates neither the intrinsic activity of PARP1 nor its localization. However, loss of SUMOylation increased PARP1-dependent PARylation on isolated chromosomes, indicating SUMOylation controls the capacity of PARP1 to modify other chromatin-associated proteins. PMID:20228053

Kif11 dependent cell cycle progression in radial glial cells is required for proper neurogenesis in the zebrafish neural tube.

PubMed

Johnson, Kimberly; Moriarty, Chelsea; Tania, Nessy; Ortman, Alissa; DiPietrantonio, Kristina; Edens, Brittany; Eisenman, Jean; Ok, Deborah; Krikorian, Sarah; Barragan, Jessica; Golé, Christophe; Barresi, Michael J F

2014-03-01

Radial glia serve as the resident neural stem cells in the embryonic vertebrate nervous system, and their proliferation must be tightly regulated to generate the correct number of neuronal and glial cell progeny in the neural tube. During a forward genetic screen, we recently identified a zebrafish mutant in the kif11 loci that displayed a significant increase in radial glial cell bodies at the ventricular zone of the spinal cord. Kif11, also known as Eg5, is a kinesin-related, plus-end directed motor protein responsible for stabilizing and separating the bipolar mitotic spindle. We show here that Gfap+ radial glial cells express kif11 in the ventricular zone and floor plate. Loss of Kif11 by mutation or pharmacological inhibition with S-trityl-L-cysteine (STLC) results in monoastral spindle formation in radial glial cells, which is characteristic of mitotic arrest. We show that M-phase radial glia accumulate over time at the ventricular zone in kif11 mutants and STLC treated embryos. Mathematical modeling of the radial glial accumulation in kif11 mutants not only confirmed an ~226× delay in mitotic exit (likely a mitotic arrest), but also predicted two modes of increased cell death. These modeling predictions were supported by an increase in the apoptosis marker, anti-activated Caspase-3, which was also found to be inversely proportional to a decrease in cell proliferation. In addition, treatment with STLC at different stages of neural development uncovered two critical periods that most significantly require Kif11 function for stem cell progression through mitosis. We also show that loss of Kif11 function causes specific reductions in oligodendroglia and secondary interneurons and motorneurons, suggesting these later born populations require proper radial glia division. Despite these alterations to cell cycle dynamics, survival, and neurogenesis, we document unchanged cell densities within the neural tube in kif11 mutants, suggesting that a mechanism of compensatory regulation may exist to maintain overall proportions in the neural tube. We propose a model in which Kif11 normally functions during mitotic spindle formation to facilitate the progression of radial glia through mitosis, which leads to the maturation of progeny into specific secondary neuronal and glial lineages in the developing neural tube. Copyright © 2014. Published by Elsevier Inc.

Kif11 dependent cell cycle progression in radial glial cells is required for proper neurogenesis in the zebrafish neural tube

PubMed Central

Johnson, Kimberly; Moriarty, Chelsea; Tania, Nessy; Ortman, Alissa; DiPietrantonio, Kristina; Edens, Brittany; Eisenman, Jean; Ok, Deborah; Krikorian, Sarah; Barragan, Jessica; Gole, Christophe; Barresi, Michael J.F.

2014-01-01

Radial glia serve as the resident neural stem cells in the embryonic vertebrate nervous system, and their proliferation must be tightly regulated to generate the correct number of neuronal and glial cell progeny in the neural tube. During a forward genetic screen, we recently identified a zebrafish mutant in the kif11 loci that displayed a significant increase in radial glial cell bodies at the ventricular zone of the spinal cord. Kif11, also known as Eg5, is a kinesin-related, plus-end directed motor protein responsible for stabilizing and separating the bipolar mitotic spindle. We show here that Gfap+ radial glial cells express kif11 in the ventricular zone and floor plate. Loss of Kif11 by mutation or pharmacological inhibition with S-trityl-L-cysteine (STLC) results in monoastral spindle formation in radial glial cells, which is characteristic of mitotic arrest. We show that M-phase radial glia accumulate over time at the ventricular zone in kif11 mutants and STLC treated embryos. Mathematical modeling of the radial glial accumulation in kif11 mutants not only confirmed an ~226x delay in mitotic exit (likely a mitotic arrest), but also predicted two modes of increased cell death. These modeling predictions were supported by an increase in the apoptosis marker, anti-activated Caspase-3, which was also found to be inversely proportional to a decrease in cell proliferation. In addition, treatment with STLC at different stages of neural development uncovered two critical periods that most significantly require Kif11 function for stem cell progression through mitosis. We also show that loss of Kif11 function causes specific reductions in oligodendroglia and secondary interneurons and motorneurons, suggesting these later born populations require proper radial glia division. Despite these alterations to cell cycle dynamics, survival, and neurogenesis, we document unchanged cell densities within the neural tube in kif11 mutants, suggesting that a mechanism of compensatory regulation may exist to maintain overall proportions in the neural tube. We propose a model in which Kif11 normally functions during mitotic spindle formation to facilitate the progression of radial glia through mitosis, which leads to the maturation of progeny into specific secondary neuronal and glial lineages in the developing neural tube. PMID:24370453

Curcumin-induced mitotic arrest is characterized by spindle abnormalities, defects in chromosomal congression and DNA damage

PubMed Central

Manson, Margaret M.

2013-01-01

The chemopreventive agent curcumin has anti-proliferative effects in many tumour types, but characterization of cell cycle arrest, particularly with physiologically relevant concentrations, is still incomplete. Following oral ingestion, the highest concentrations of curcumin are achievable in the gut. Although it has been established that curcumin induces arrest at the G2/M stage of the cell cycle in colorectal cancer lines, it is not clear whether arrest occurs at the G2/M transition or in mitosis. To elucidate the precise stage of arrest, we performed a direct comparison of the levels of curcumin-induced G2/M boundary and mitotic arrest in eight colorectal cancer lines (Caco-2, DLD-1, HCA-7, HCT116p53+/+, HCT116p53–/–, HCT116p21–/–, HT-29 and SW480). Flow cytometry confirmed that these lines underwent G2/M arrest following treatment for 12h with clinically relevant concentrations of curcumin (5–10 μM). In all eight lines, the majority of this arrest occurred at the G2/M transition, with a proportion of cells arresting in mitosis. Examination of the mitotic index using fluorescence microscopy showed that the HCT116 and Caco-2 lines exhibited the highest levels of curcumin-induced mitotic arrest. Image analysis revealed impaired mitotic progression in all lines, exemplified by mitotic spindle abnormalities and defects in chromosomal congression. Pre-treatment with inhibitors of the DNA damage signalling pathway abrogated curcumin-induced mitotic arrest, but had little effect at the G2/M boundary. Moreover, pH2A.X staining seen in mitotic, but not interphase, cells suggests that this aberrant mitosis results in DNA damage. PMID:23125222

Mcl-1 dynamics influence mitotic slippage and death in mitosis.

PubMed

Sloss, Olivia; Topham, Caroline; Diez, Maria; Taylor, Stephen

2016-02-02

Microtubule-binding drugs such as taxol are frontline treatments for a variety of cancers but exactly how they yield patient benefit is unclear. In cell culture, inhibiting microtubule dynamics prevents spindle assembly, leading to mitotic arrest followed by either apoptosis in mitosis or slippage, whereby a cell returns to interphase without dividing. Myeloid cell leukaemia-1 (Mcl-1), a pro-survival member of the Bcl-2 family central to the intrinsic apoptosis pathway, is degraded during a prolonged mitotic arrest and may therefore act as a mitotic death timer. Consistently, we show that blocking proteasome-mediated degradation inhibits taxol-induced mitotic apoptosis in a Mcl-1-dependent manner. However, this degradation does not require the activity of either APC/C-Cdc20, FBW7 or MULE, three separate E3 ubiquitin ligases implicated in targeting Mcl-1 for degradation. This therefore challenges the notion that Mcl-1 undergoes regulated degradation during mitosis. We also show that Mcl-1 is continuously synthesized during mitosis and that blocking protein synthesis accelerates taxol induced death-in-mitosis. Modulating Mcl-1 levels also influences slippage; overexpressing Mcl-1 extends the time from mitotic entry to mitotic exit in the presence of taxol, while inhibiting Mcl-1 accelerates it. We suggest that Mcl-1 competes with Cyclin B1 for binding to components of the proteolysis machinery, thereby slowing down the slow degradation of Cyclin B1 responsible for slippage. Thus, modulating Mcl-1 dynamics influences both death-in-mitosis and slippage. However, because mitotic degradation of Mcl-1 appears not to be under the control of an E3 ligase, we suggest that the notion of network crosstalk is used with caution.

Radmis, a Novel Mitotic Spindle Protein that Functions in Cell Division of Neural Progenitors

PubMed Central

Yumoto, Takahito; Nakadate, Kazuhiko; Nakamura, Yuki; Sugitani, Yoshinobu; Sugitani-Yoshida, Reiko; Ueda, Shuichi; Sakakibara, Shin-ichi

2013-01-01

Developmental dynamics of neural stem/progenitor cells (NSPCs) are crucial for embryonic and adult neurogenesis, but its regulatory factors are not fully understood. By differential subtractive screening with NSPCs versus their differentiated progenies, we identified the radmis (radial fiber and mitotic spindle)/ckap2l gene, a novel microtubule-associated protein (MAP) enriched in NSPCs. Radmis is a putative substrate for the E3-ubiquitin ligase, anaphase promoting complex/cyclosome (APC/C), and is degraded via the KEN box. Radmis was highly expressed in regions of active neurogenesis throughout life, and its distribution was dynamically regulated during NSPC division. In embryonic and perinatal brains, radmis localized to bipolar mitotic spindles and radial fibers (basal processes) of dividing NSPCs. As central nervous system development proceeded, radmis expression was lost in most brain regions, except for several neurogenic regions. In adult brain, radmis expression persisted in the mitotic spindles of both slowly-dividing stem cells and rapid amplifying progenitors. Overexpression of radmis in vitro induced hyper-stabilization of microtubules, severe defects in mitotic spindle formation, and mitotic arrest. In vivo gain-of-function using in utero electroporation revealed that radmis directed a reduction in NSPC proliferation and a concomitant increase in cell cycle exit, causing a reduction in the Tbr2-positive basal progenitor population and shrinkage of the embryonic subventricular zone. Besides, radmis loss-of-function by shRNAs induced the multipolar mitotic spindle structure, accompanied with the catastrophe of chromosome segregation including the long chromosome bridge between two separating daughter nuclei. These findings uncover the indispensable role of radmis in mitotic spindle formation and cell-cycle progression of NSPCs. PMID:24260314

Calibrated mitotic oscillator drives motile ciliogenesis.

PubMed

Al Jord, Adel; Shihavuddin, Asm; Servignat d'Aout, Raphaël; Faucourt, Marion; Genovesio, Auguste; Karaiskou, Anthi; Sobczak-Thépot, Joëlle; Spassky, Nathalie; Meunier, Alice

2017-11-10

Cell division and differentiation depend on massive and rapid organelle remodeling. The mitotic oscillator, centered on the cyclin-dependent kinase 1-anaphase-promoting complex/cyclosome (CDK1-APC/C) axis, spatiotemporally coordinates this reorganization in dividing cells. Here we discovered that nondividing cells could also implement this mitotic clocklike regulatory circuit to orchestrate subcellular reorganization associated with differentiation. We probed centriole amplification in differentiating mouse-brain multiciliated cells. These postmitotic progenitors fine-tuned mitotic oscillator activity to drive the orderly progression of centriole production, maturation, and motile ciliation while avoiding the mitosis commitment threshold. Insufficient CDK1 activity hindered differentiation, whereas excessive activity accelerated differentiation yet drove postmitotic progenitors into mitosis. Thus, postmitotic cells can redeploy and calibrate the mitotic oscillator to uncouple cytoplasmic from nuclear dynamics for organelle remodeling associated with differentiation. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

The bipolar assembly domain of the mitotic motor kinesin-5

PubMed Central

Acar, Seyda; Carlson, David B.; Budamagunta, Madhu S.; Yarov-Yarovoy, Vladimir; Correia, John J.; Niñonuevo, Milady R.; Jia, Weitao; Tao, Li; Leary, Julie A.; Voss, John C.; Evans, James E.; Scholey, Jonathan M.

2013-01-01

An outstanding unresolved question is how does the mitotic spindle utilize microtubules and mitotic motors to coordinate accurate chromosome segregation during mitosis? This process depends upon the mitotic motor, kinesin-5, whose unique bipolar architecture, with pairs of motor domains lying at opposite ends of a central rod, allows it to crosslink microtubules within the mitotic spindle and to coordinate their relative sliding during spindle assembly, maintenance and elongation. The structural basis of kinesin-5’s bipolarity is, however, unknown, as protein asymmetry has so far precluded its crystallization. Here we use electron microscopy of single molecules of kinesin-5 and its subfragments, combined with hydrodynamic analysis plus mass spectrometry, circular dichroism and site-directed spin label electron paramagnetic resonance spectroscopy, to show how a staggered antiparallel coiled-coil ‘BASS’ (bipolar assembly) domain directs the assembly of four kinesin-5 polypeptides into bipolar minifilaments. PMID:23299893

Loss of centrioles causes chromosomal instability in vertebrate somatic cells.

PubMed

Sir, Joo-Hee; Pütz, Monika; Daly, Owen; Morrison, Ciaran G; Dunning, Mark; Kilmartin, John V; Gergely, Fanni

2013-12-09

Most animal cells contain a centrosome, which comprises a pair of centrioles surrounded by an ordered pericentriolar matrix (PCM). Although the role of this organelle in organizing the mitotic spindle poles is well established, its precise contribution to cell division and cell survival remains a subject of debate. By genetically ablating key components of centriole biogenesis in chicken DT40 B cells, we generated multiple cell lines that lack centrioles. PCM components accumulated in acentriolar microtubule (MT)-organizing centers but failed to adopt a higher-order structure, as shown by three-dimensional structured illumination microscopy. Cells without centrioles exhibited both a delay in bipolar spindle assembly and a high rate of chromosomal instability. Collectively, our results expose a vital role for centrosomes in establishing a mitotic spindle geometry that facilitates correct kinetochore-MT attachments. We propose that centrosomes are essential in organisms in which rapid segregation of a large number of chromosomes needs to be attained with fidelity.

A single cyclin–CDK complex is sufficient for both mitotic and meiotic progression in fission yeast

PubMed Central

Gutiérrez-Escribano, Pilar; Nurse, Paul

2015-01-01

The dominant model for eukaryotic cell cycle control proposes that cell cycle progression is driven by a succession of CDK complexes with different substrate specificities. However, in fission yeast it has been shown that a single CDK complex generated by the fusion of the Cdc13 cyclin with the CDK protein Cdc2 can drive the mitotic cell cycle. Meiosis is a modified cell cycle programme in which a single S-phase is followed by two consecutive rounds of chromosome segregation. Here we systematically analyse the requirements of the different fission yeast cyclins for meiotic cell cycle progression. We also show that a single Cdc13–Cdc2 complex, in the absence of the other cyclins, can drive the meiotic cell cycle. We propose that qualitatively different CDK complexes are not absolutely required for cell cycle progression either during mitosis or meiosis, and that a single CDK complex can drive both cell cycle programmes. PMID:25891897

CDE-1 affects chromosome segregation through uridylation of CSR-1-bound siRNAs.

PubMed

van Wolfswinkel, Josien C; Claycomb, Julie M; Batista, Pedro J; Mello, Craig C; Berezikov, Eugene; Ketting, René F

2009-10-02

We have studied the function of a conserved germline-specific nucleotidyltransferase protein, CDE-1, in RNAi and chromosome segregation in C. elegans. CDE-1 localizes specifically to mitotic chromosomes in embryos. This localization requires the RdRP EGO-1, which physically interacts with CDE-1, and the Argonaute protein CSR-1. We found that CDE-1 is required for the uridylation of CSR-1 bound siRNAs, and that in the absence of CDE-1 these siRNAs accumulate to inappropriate levels, accompanied by defects in both meiotic and mitotic chromosome segregation. Elevated siRNA levels are associated with erroneous gene silencing, most likely through the inappropriate loading of CSR-1 siRNAs into other Argonaute proteins. We propose a model in which CDE-1 restricts specific EGO-1-generated siRNAs to the CSR-1 mediated, chromosome associated RNAi pathway, thus separating it from other endogenous RNAi pathways. The conserved nature of CDE-1 suggests that similar sorting mechanisms may operate in other animals, including mammals.

Nuclear dynamics during germination, conidiation, and hyphal fusion of Fusarium oxysporum.

PubMed

Ruiz-Roldán, M Carmen; Köhli, Michael; Roncero, M Isabel G; Philippsen, Peter; Di Pietro, Antonio; Espeso, Eduardo A

2010-08-01

In many fungal pathogens, infection is initiated by conidial germination. Subsequent stages involve germ tube elongation, conidiation, and vegetative hyphal fusion (anastomosis). Here, we used live-cell fluorescence to study the dynamics of green fluorescent protein (GFP)- and cherry fluorescent protein (ChFP)-labeled nuclei in the plant pathogen Fusarium oxysporum. Hyphae of F. oxysporum have uninucleated cells and exhibit an acropetal nuclear pedigree, where only the nucleus in the apical compartment is mitotically active. In contrast, conidiation follows a basopetal pattern, whereby mononucleated microconidia are generated by repeated mitotic cycles of the subapical nucleus in the phialide, followed by septation and cell abscission. Vegetative hyphal fusion is preceded by directed growth of the fusion hypha toward the receptor hypha and followed by a series of postfusion nuclear events, including mitosis of the apical nucleus of the fusion hypha, migration of a daughter nucleus into the receptor hypha, and degradation of the resident nucleus. These previously unreported patterns of nuclear dynamics in F. oxysporum could be intimately related to its pathogenic lifestyle.

A time-series method for automated measurement of changes in mitotic and interphase duration from time-lapse movies.

PubMed

Sigoillot, Frederic D; Huckins, Jeremy F; Li, Fuhai; Zhou, Xiaobo; Wong, Stephen T C; King, Randall W

2011-01-01

Automated time-lapse microscopy can visualize proliferation of large numbers of individual cells, enabling accurate measurement of the frequency of cell division and the duration of interphase and mitosis. However, extraction of quantitative information by manual inspection of time-lapse movies is too time-consuming to be useful for analysis of large experiments. Here we present an automated time-series approach that can measure changes in the duration of mitosis and interphase in individual cells expressing fluorescent histone 2B. The approach requires analysis of only 2 features, nuclear area and average intensity. Compared to supervised learning approaches, this method reduces processing time and does not require generation of training data sets. We demonstrate that this method is as sensitive as manual analysis in identifying small changes in interphase or mitotic duration induced by drug or siRNA treatment. This approach should facilitate automated analysis of high-throughput time-lapse data sets to identify small molecules or gene products that influence timing of cell division.

Triphenylbutanamines: Kinesin Spindle Protein Inhibitors with in Vivo Antitumor Activity†

PubMed Central

2012-01-01

The human mitotic kinesin Eg5 represents a novel mitotic spindle target for cancer chemotherapy. We previously identified S-trityl-l-cysteine (STLC) and related analogues as selective potent inhibitors of Eg5. We herein report on the development of a series of 4,4,4-triphenylbutan-1-amine inhibitors derived from the STLC scaffold. This new generation systematically improves on potency: the most potent C-trityl analogues exhibit Kiapp ≤ 10 nM and GI50 ≈ 50 nM, comparable to results from the phase II clinical benchmark ispinesib. Crystallographic studies reveal that they adopt the same overall binding configuration as S-trityl analogues at an allosteric site formed by loop L5 of Eg5. Evaluation of their druglike properties reveals favorable profiles for future development and, in the clinical candidate ispinesib, moderate hERG and CYP inhibition. One triphenylbutanamine analogue and ispinesib possess very good bioavailability (51% and 45%, respectively), with the former showing in vivo antitumor growth activity in nude mice xenograft studies. PMID:22248262

Experimental evidence showing that no mitotically active female germline progenitors exist in postnatal mouse ovaries.

PubMed

Zhang, Hua; Zheng, Wenjing; Shen, Yan; Adhikari, Deepak; Ueno, Hiroo; Liu, Kui

2012-07-31

It has been generally accepted for more than half a century that, in most mammalian species, oocytes cannot renew themselves in postnatal or adult life, and that the number of oocytes is already fixed in fetal or neonatal ovaries. This assumption, however, has been challenged over the past decade. In this study, we have taken an endogenous genetic approach to this question and generated a multiple fluorescent Rosa26(rbw/+);Ddx4-Cre germline reporter mouse model for in vivo and in vitro tracing of the development of female germline cell lineage. Through live cell imaging and de novo folliculogenesis experiments, we show that the Ddx4-expressing cells from postnatal mouse ovaries did not enter mitosis, nor did they contribute to oocytes during de novo folliculogenesis. Our results provide evidence that supports the traditional view that no postnatal follicular renewal occurs in mammals, and no mitotically active Ddx4-expressing female germline progenitors exist in postnatal mouse ovaries.

Caspase 2 in mitotic catastrophe: The terminator of aneuploid and tetraploid cells.

PubMed

Vitale, Ilio; Manic, Gwenola; Castedo, Maria; Kroemer, Guido

2017-01-01

Mitotic catastrophe is an oncosuppressive mechanism that targets cells experiencing defective mitoses via the activation of specific cell cycle checkpoints, regulated cell death pathways and/or cell senescence. This prevents the accumulation of karyotypic aberrations, which otherwise may drive oncogenesis and tumor progression. Here, we summarize experimental evidence confirming the role of caspase 2 (CASP2) as the main executor of mitotic catastrophe, and we discuss the signals that activate CASP2 in the presence of mitotic aberrations. In addition, we summarize the main p53-dependent and -independent effector pathways through which CASP2 limits chromosomal instability and non-diploidy, hence mediating robust oncosuppressive functions.

Spatiotemporal Regulation of the Anaphase-Promoting Complex in Mitosis

PubMed Central

Sivakumar, Sushama; Gorbsky, Gary J

2015-01-01

The appropriate timing of events that lead to chromosome segregation during mitosis and cytokinesis is essential to prevent aneuploidy, and defects in these processes can contribute to tumorigenesis. Key mitotic regulators are controlled through ubiquitylation and proteasome-mediated degradation. The Anaphase-Promoting Complex or Cyclosome (APC/C) is an E3 ubiquitin ligase that has a crucial function in the regulation of the mitotic cell cycle, particularly at the onset of anaphase and during mitotic exit. Co-activator proteins, inhibitor proteins, protein kinases and phosphatases interact with the APC/C to temporally and spatially control its activity and thus ensure accurate timing of mitotic events. PMID:25604195

Robust mitotic entry is ensured by a latching switch.

PubMed

Tuck, Chloe; Zhang, Tongli; Potapova, Tamara; Malumbres, Marcos; Novák, Béla

2013-01-01

Cell cycle events are driven by Cyclin dependent kinases (CDKs) and by their counter-acting phosphatases. Activation of the Cdk1:Cyclin B complex during mitotic entry is controlled by the Wee1/Myt1 inhibitory kinases and by Cdc25 activatory phosphatase, which are themselves regulated by Cdk1:Cyclin B within two positive circuits. Impairing these two feedbacks with chemical inhibitors induces a transient entry into M phase referred to as mitotic collapse. The pathology of mitotic collapse reveals that the positive circuits play a significant role in maintaining the M phase state. To better understand the function of these feedback loops during G2/M transition, we propose a simple model for mitotic entry in mammalian cells including spatial control over Greatwall kinase phosphorylation. After parameter calibration, the model is able to recapture the complex and non-intuitive molecular dynamics reported by Potapova et al. (Potapova et al., 2011). Moreover, it predicts the temporal patterns of other mitotic regulators which have not yet been experimentally tested and suggests a general design principle of cell cycle control: latching switches buffer the cellular stresses which accompany cell cycle processes to ensure that the transitions are smooth and robust.

Cdk1 phosphorylates SPAT-1/Bora to trigger PLK-1 activation and drive mitotic entry in C. elegans embryos

PubMed Central

Tavernier, Nicolas; Noatynska, Anna; Panbianco, Costanza; Martino, Lisa; Van Hove, Lucie; Schwager, Françoise; Léger, Thibaut

2015-01-01

The molecular mechanisms governing mitotic entry during animal development are incompletely understood. Here, we show that the mitotic kinase CDK-1 phosphorylates Suppressor of Par-Two 1 (SPAT-1)/Bora to regulate its interaction with PLK-1 and to trigger mitotic entry in early Caenorhabditis elegans embryos. Embryos expressing a SPAT-1 version that is nonphosphorylatable by CDK-1 and that is defective in PLK-1 binding in vitro present delays in mitotic entry, mimicking embryos lacking SPAT-1 or PLK-1 functions. We further show that phospho–SPAT-1 activates PLK-1 by triggering phosphorylation on its activator T loop in vitro by Aurora A. Likewise, we show that phosphorylation of human Bora by Cdk1 promotes phosphorylation of human Plk1 by Aurora A, suggesting that this mechanism is conserved in humans. Our results suggest that CDK-1 activates PLK-1 via SPAT-1 phosphorylation to promote entry into mitosis. We propose the existence of a positive feedback loop that connects Cdk1 and Plk1 activation to ensure a robust control of mitotic entry and cell division timing. PMID:25753036

UV-C irradiation delays mitotic progression by recruiting Mps1 to kinetochores.

PubMed

Zhang, Xiaojuan; Ling, Youguo; Wang, Wenjun; Zhang, Yanhong; Ma, Qingjun; Tan, Pingping; Song, Ting; Wei, Congwen; Li, Ping; Liu, Xuedong; Ma, Runlin Z; Zhong, Hui; Cao, Cheng; Xu, Quanbin

2013-04-15

The effect of UV irradiation on replicating cells during interphase has been studied extensively. However, how the mitotic cell responds to UV irradiation is less well defined. Herein, we found that UV-C irradiation (254 nm) increases recruitment of the spindle checkpoint proteins Mps1 and Mad2 to the kinetochore during metaphase, suggesting that the spindle assembly checkpoint (SAC) is reactivated. In accordance with this, cells exposed to UV-C showed delayed mitotic progression, characterized by a prolonged chromosomal alignment during metaphase. UV-C irradiation also induced the DNA damage response and caused a significant accumulation of γ-H2AX on mitotic chromosomes. Unexpectedly, the mitotic delay upon UV-C irradiation is not due to the DNA damage response but to the relocation of Mps1 to the kinetochore. Further, we found that UV-C irradiation activates Aurora B kinase. Importantly, the kinase activity of Aurora B is indispensable for full recruitment of Mps1 to the kinetochore during both prometaphase and metaphase. Taking these findings together, we propose that UV irradiation delays mitotic progression by evoking the Aurora B-Mps1 signaling cascade, which exerts its role through promoting the association of Mps1 with the kinetochore in metaphase.

Revised genetic requirements for the decatenation G2 checkpoint: the role of ATM

PubMed Central

Bower, Jacquelyn J.; Zhou, Yingchun; Zhou, Tong; Simpson, Dennis A.; Arlander, Sonnet J.; Paules, Richard S.; Cordeiro-Stone, Marila; Kaufmann, William K.

2010-01-01

The decatenation G2 checkpoint is proposed to delay cellular progression from G2 into mitosis when intertwined daughter chromatids are insufficiently decatenated. Previous studies indicated that the ATM- and Rad3-related (ATR) checkpoint kinase, but not the ataxia telangiectasia-mutated (ATM) kinase, was required for decatenation G2 checkpoint function. Here, we show that the method used to quantify decatenation G2 checkpoint function can influence the identification of genetic requirements for the checkpoint. Normal human diploid fibroblast (NHDF) lines responded to the topoisomerase II (topo II) catalytic inhibitor ICRF-193 with a stringent G2 arrest and a reduction in the mitotic index. While siRNA-mediated depletion of ATR and CHEK1 increased the mitotic index in ICRF-193 treated NHDF lines, depletion of these proteins did not affect the mitotic entry rate, indicating that the decatenation G2 checkpoint was functional. These results suggest that ATR and CHEK1 are not required for the decatenation G2 checkpoint, but may influence mitotic exit after inhibition of topo II. A re-evaluation of ataxia telangiectasia (AT) cell lines using the mitotic entry assay indicated that ATM was required for the decatenation G2 checkpoint. Three NHDF cell lines responded to ICRF-193 with a mean 98% inhibition of the mitotic entry rate. Examination of the mitotic entry rates in AT fibroblasts upon treatment with ICRF-193 revealed a significantly attenuated decatenation G2 checkpoint response, with a mean 59% inhibition of the mitotic entry rate. In addition, a normal lymphoblastoid line exhibited a 95% inhibition of the mitotic entry rate after incubation with ICRF-193, whereas two AT lymphoblastoid lines displayed only 36% and 20% inhibition of the mitotic entry rate. Stable depletion of ATM in normal human fibroblasts with short hairpin RNA also attenuated decatenation G2 checkpoint function by an average of 40%. Western immunoblot analysis demonstrated that treatment with ICRF-193 induced ATM autophosphorylation and ATM-dependent phosphorylation of Ser15-p53 and Thr68 in CHEK2, but no appreciable phosphorylation of Ser139 on H2AX. The results suggest that inhibition of topo II induces ATM to phosphorylate selected targets that contribute to a G2 arrest independently of DNA damage. PMID:20372057

Discovery of benzo[e]pyridoindolones as kinase inhibitors that disrupt mitosis exit while erasing AMPK-Thr172 phosphorylation on the spindle.

PubMed

Le, Ly-Thuy-Tram; Couvet, Morgane; Favier, Bertrand; Coll, Jean-Luc; Nguyen, Chi-Hung; Molla, Annie

2015-09-08

Aurora kinases play an essential role in mitotic progression and are attractive targets in cancer therapy. The first generation of benzo[e]pyridoindole exhibited powerful aurora kinase inhibition but their low solubility limited further development. Grafting a pyperidine-ethoxy group gives rise to a hydrosoluble inhibitor: compound C5M.C5M could efficiently inhibit the proliferation of cells from different origins. C5M prevented cell cycling, induced a strong mitotic arrest then, cells became polyploid and finally died. C5M did not impair the spindle checkpoint, the separation of the sister chromatids and the transfer of aurora B on the mid-zone. C5M prevented histone H3 phosphorylation at mitotic entry and erased AMPK-Thr172 phosphorylation in late mitosis. With this unique profile of inhibition, C5M could be useful for understanding the role of phospho-Thr172-AMPK in abscission and the relationship between the chromosomal complex and the energy sensing machinery.C5M is a multikinase inhibitor with interesting preclinical characteristics: high hydro-solubility and a good stability in plasma. A single dose prevents the expansion of multicellular spheroids. C5M can safely be injected to mice and reduces significantly the development of xenograft. The next step will be to define the protocol of treatment and the cancer therapeutic field of this new anti-proliferative drug.

Mitotic events in cerebellar granule progenitor cells that expand cerebellar surface area are critical for normal cerebellar cortical lamination in mice.

PubMed

Chang, Joshua C; Leung, Mark; Gokozan, Hamza Numan; Gygli, Patrick Edwin; Catacutan, Fay Patsy; Czeisler, Catherine; Otero, José Javier

2015-03-01

Late embryonic and postnatal cerebellar folial surface area expansion promotes cerebellar cortical cytoarchitectural lamination. We developed a streamlined sampling scheme to generate unbiased estimates of murine cerebellar surface area and volume using stereologic principles. We demonstrate that, during the proliferative phase of the external granular layer (EGL) and folial surface area expansion, EGL thickness does not change and thus is a topological proxy for progenitor self-renewal. The topological constraints indicate that, during proliferative phases, migration out of the EGL is balanced by self-renewal. Progenitor self-renewal must, therefore, include mitotic events yielding 2 cells in the same layer to increase surface area (β events) and mitotic events yielding 2 cells, with 1 cell in a superficial layer and 1 cell in a deeper layer (α events). As the cerebellum grows, therefore, β events lie upstream of α events. Using a mathematical model constrained by the measurements of volume and surface area, we could quantify intermitotic times for β events on a per-cell basis in postnatal mouse cerebellum. Furthermore, we found that loss of CCNA2, which decreases EGL proliferation and secondarily induces cerebellar cortical dyslamination, shows preserved α-type events. Thus, CCNA2-null cerebellar granule progenitor cells are capable of self-renewal of the EGL stem cell niche; this is concordant with prior findings of extensive apoptosis in CCNA2-null mice. Similar methodologies may provide another layer of depth to the interpretation of results from stereologic studies.

Image analysis assisted study of mitotic figures in oral epithelial dysplasia and squamous cell carcinoma using differential stains.

PubMed

Tandon, Ankita; Singh, Narendra Nath; Brave, V R; Sreedhar, Gadiputi

2016-11-01

Mitosis is a process of cell division resulting in two genetically equivalent daughter cells. Excessive proliferation of cells due to mitosis is the hallmark in pre cancer and cancer. This study was conducted to count the number of mitotic figures in normal oral mucosa, oral epithelial dysplasia and squamous cell carcinoma in both Hematoxylin and Eosin (H&E) and Crystal Violet stained sections. Also the overall number of mitotic figures with both stains were compared along with the evaluation of staining efficacy of both the stains. The present study was conducted on 20 specimens each of the three categories. These were further divided into two groups for staining with H&E and with 1% Crystal Violet respectively. Images were captured and analyzed using image analysis software Dewinter Biowizard 4.1. Comparison of mitotic figure count in three categories in sections stained with both stains showed statistically significant difference ( p  < 0.001). The mean number of mitotic figures seen in Crystal Violet reagent were significantly higher as seen in H&E stain ( p  < 0.001). The overall diagnostic efficacy of Crystal Violet was 87.6%. Crystal Violet scored over H&E stain and also helped to better appreciate metaphases in Squamous cell carcinoma and telophases in dysplasia. Number of mitotic figures progressively increase with the advancement of the pathology. Use of 1% Crystal Violet provides better appreciation of mitotic figures and can be employed as a selective stain in routine histopathology.

Grading system for blood vessel tumor emboli of invasive ductal carcinoma of the breast.

PubMed

Sugiyama, Michiko; Hasebe, Takahiro; Shimada, Hiroko; Takeuchi, Hideki; Shimizu, Kyoko; Shimizu, Michio; Yasuda, Masanori; Ueda, Shigeto; Shigekawa, Takashi; Osaki, Akihiko; Saeki, Toshiaki

2015-06-01

We previously reported that the number of mitotic and apoptotic figures in tumor cells in blood vessel tumor emboli had the greatest significant power for the accurate prediction of the outcome of patients with invasive ductal carcinoma of the breast. The purpose of the present study was to devise a grading system for blood vessel tumor emboli based on the mitotic and apoptotic figures of tumor cells in blood vessel tumor emboli, enabling accurate prediction of the outcome of patients with invasive ductal carcinoma of the breast. We classified 263 invasive ductal carcinomas into the following 3 grades according to the numbers of mitotic and apoptotic figures in tumor cells located in blood vessels within 1 high-power field: grade 0, no blood vessel invasion; grade 1, absence of mitotic figures and presence of any number of apoptotic figures, or 1 mitotic figure and 0 to 2 apoptotic figures; and grade 2, 1 mitotic figure and 3 or more apoptotic figures, or 2 or more mitotic figures and 1 or more apoptotic figures. Multivariate analyses with well-known prognostic factors demonstrated that grade 2 blood vessel tumor emboli significantly increased the hazard ratios for tumor recurrence independent of the nodal status, pathological TNM stage, hormone receptor status, or HER2 status. The presently reported grading system for blood vessel tumor emboli is the strongest histologic factor for accurate prediction of the outcome of patients with invasive ductal carcinoma of the breast. Copyright © 2015 Elsevier Inc. All rights reserved.

Sister acts: coordinating DNA replication and cohesion establishment

PubMed Central

Sherwood, Rebecca; Takahashi, Tatsuro S.; Jallepalli, Prasad V.

2010-01-01

The ring-shaped cohesin complex links sister chromatids and plays crucial roles in homologous recombination and mitotic chromosome segregation. In cycling cells, cohesin's ability to generate cohesive linkages is restricted to S phase and depends on loading and establishment factors that are intimately connected to DNA replication. Here we review how cohesin is regulated by the replication machinery, as well as recent evidence that cohesin itself influences how chromosomes are replicated. PMID:21159813

[Induced autotetraploid grape mutants].

PubMed

Kuliev, V M

2011-01-01

The methods of experimental mitotic and meiotic polyploidy in grapes are represented in the article. Results of cytological, histo-anatomical, biomorphological researches of induced autotetraploids are shown. Genetic characteristics, parameters of generative organs, quantitative and structural genome changes were studied. Comparative quantitative changes in the content of chloroplast and mitochondrion DNAs and RNAs in diploids and autotetraploids were defined. Also are shown. The biology-economic evaluation of autotetraploids on comparison with the initial grape variety is represented.

Comparison of staining of mitotic figures by haematoxylin and eosin-and crystal violet stains, in oral epithelial dysplasia and squamous cell carcinoma.

PubMed

Ankle, Madhuri R; Kale, Alka D; Charantimath, Seema

2007-01-01

Mitosis of cells gives rise to tissue integrity. Defects during mitosis bring about abnormalities. Excessive proliferation of cells due to increased mitosis is one such outcome, which is the hallmark in precancer and cancer. The localization of proliferating cells or their precursors may not be obvious and easy. Establishing an easy way to distinguish these mitotic cells will help in grading and understanding their biological potential. Although immunohistochemistry is an advanced method in use, the cost and time factor makes it less feasible for many laboratories. Selective histochemical stains like toluidine blue, giemsa and crystal violet have been used in tissues including the developing brain, neural tissue and skin. 1) To compare the staining of mitotic cells in haematoxylin and eosin with that in crystal violet. 2) To compare the number of mitotic figures present in normal oral mucosa, epithelial dysplasia and oral squamous cell carcinoma in crystal violet-stained sections with that in H and E-stained sections. Ten tissues of normal oral mucosa and 15 tissues each of oral epithelial dysplasia seen in tobacco-associated leukoplakia and squamous cell carcinoma were studied to evaluate the selectivity of 1% crystal violet for mitotic figures. The staining was compared with standard H and E staining. Statistical analysis was done using Mann-Whitney U test. A statistically significant increase in the mean mitotic count was observed in crystal violet-stained sections of epithelial dysplasia as compared to the H and E-stained sections (p=0.0327). A similar increase in the mitotic counts was noted in crystal violet-stained sections of oral squamous cell carcinoma as compared to the H and E-stained sections.(p=0.0443). No significant difference was found in the mitotic counts determined in dysplasia or carcinoma by either the crystal violet (p=0.4429) or the H and E-staining techniques (p=0.2717). One per cent crystal violet provides a definite advantage over the H and E-stained sections in selectively staining the mitotic figures.

Targeting Alp7/TACC to the spindle pole body is essential for mitotic spindle assembly in fission yeast

PubMed Central

Tang, Ngang Heok; Okada, Naoyuki; Fong, Chii Shyang; Arai, Kunio; Sato, Masamitsu; Toda, Takashi

2014-01-01

The conserved TACC protein family localises to the centrosome (the spindle pole body, SPB in fungi) and mitotic spindles, thereby playing a crucial role in bipolar spindle assembly. However, it remains elusive how TACC proteins are recruited to the centrosome/SPB. Here, using fission yeast Alp7/TACC, we have determined clustered five amino acid residues within the TACC domain required for SPB localisation. Critically, these sequences are essential for the functions of Alp7, including proper spindle formation and mitotic progression. Moreover, we have identified pericentrin-like Pcp1 as a loading factor to the mitotic SPB, although Pcp1 is not a sole platform. PMID:24937146

Mio depletion links mTOR regulation to Aurora A and Plk1 activation at mitotic centrosomes

PubMed Central

Trinkle-Mulcahy, Laura; Porter, Michael; Jeyaprakash, A. Arockia

2015-01-01

Coordination of cell growth and proliferation in response to nutrient supply is mediated by mammalian target of rapamycin (mTOR) signaling. In this study, we report that Mio, a highly conserved member of the SEACAT/GATOR2 complex necessary for the activation of mTORC1 kinase, plays a critical role in mitotic spindle formation and subsequent chromosome segregation by regulating the proper concentration of active key mitotic kinases Plk1 and Aurora A at centrosomes and spindle poles. Mio-depleted cells showed reduced activation of Plk1 and Aurora A kinase at spindle poles and an impaired localization of MCAK and HURP, two key regulators of mitotic spindle formation and known substrates of Aurora A kinase, resulting in spindle assembly and cytokinesis defects. Our results indicate that a major function of Mio in mitosis is to regulate the activation/deactivation of Plk1 and Aurora A, possibly by linking them to mTOR signaling in a pathway to promote faithful mitotic progression. PMID:26124292

Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

PubMed Central

Whigham, Arlene; Clarke, Rosemary; Brenes-Murillo, Alejandro J; Estes, Brett; Madhessian, Diana; Lundberg, Emma; Wadsworth, Patricia

2017-01-01

The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al., 2014, 2015). Here, we show that by using specific intracellular immunolabelling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabelled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, termed ‘early risers’. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide the first a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase. PMID:29052541

O-Linked N-Acetylglucosamine Cycling Regulates Mitotic Spindle Organization*

PubMed Central

Tan, Ee Phie; Caro, Sarah; Potnis, Anish; Lanza, Christopher; Slawson, Chad

2013-01-01

Any defects in the correct formation of the mitotic spindle will lead to chromosomal segregation errors, mitotic arrest, or aneuploidy. We demonstrate that O-linked N-acetylglucosamine (O-GlcNAc), a post-translational modification of serine and threonine residues in nuclear and cytoplasmic proteins, regulates spindle function. In O-GlcNAc transferase or O-GlcNAcase gain of function cells, the mitotic spindle is incorrectly assembled. Chromosome condensation and centrosome assembly is impaired in these cells. The disruption in spindle architecture is due to a reduction in histone H3 phosphorylation by Aurora kinase B. However, gain of function cells treated with the O-GlcNAcase inhibitor Thiamet-G restored the assembly of the spindle and partially rescued histone phosphorylation. Together, these data suggest that the coordinated addition and removal of O-GlcNAc, termed O-GlcNAc cycling, regulates mitotic spindle organization and provides a potential new perspective on how O-GlcNAc regulates cellular events. PMID:23946484

Anti-mitotic agents: Are they emerging molecules for cancer treatment?

PubMed

Penna, Larissa Siqueira; Henriques, João Antonio Pêgas; Bonatto, Diego

2017-05-01

Mutations in cancer cells frequently result in cell cycle alterations that lead to unrestricted growth compared to normal cells. Considering this phenomenon, many drugs have been developed to inhibit different cell-cycle phases. Mitotic phase targeting disturbs mitosis in tumor cells, triggers the spindle assembly checkpoint and frequently results in cell death. The first anti-mitotics to enter clinical trials aimed to target tubulin. Although these drugs improved the treatment of certain cancers, and many anti-microtubule compounds are already approved for clinical use, severe adverse events such as neuropathies were observed. Since then, efforts have been focused on the development of drugs that also target kinases, motor proteins and multi-protein complexes involved in mitosis. In this review, we summarize the major proteins involved in the mitotic phase that can also be targeted for cancer treatment. Finally, we address the activity of anti-mitotic drugs tested in clinical trials in recent years. Copyright © 2017 Elsevier Inc. All rights reserved.

Human papillomavirus type 16 E7 oncoprotein engages but does not abrogate the mitotic spindle assembly checkpoint

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Yueyang; Munger, Karl, E-mail: kmunger@rics.bwh.harvard.edu

2012-10-10

The mitotic spindle assembly checkpoint (SAC) ensures faithful chromosome segregation during mitosis by censoring kinetochore-microtubule interactions. It is frequently rendered dysfunctional during carcinogenesis causing chromosome missegregation and genomic instability. There are conflicting reports whether the HPV16 E7 oncoprotein drives chromosomal instability by abolishing the SAC. Here we report that degradation of mitotic cyclins is impaired in cells with HPV16 E7 expression. RNAi-mediated depletion of Mad2 or BubR1 indicated the involvement of the SAC, suggesting that HPV16 E7 expression causes sustained SAC engagement. Mutational analyses revealed that HPV16 E7 sequences that are necessary for retinoblastoma tumor suppressor protein binding as wellmore » as sequences previously implicated in binding the nuclear and mitotic apparatus (NuMA) protein and in delocalizing dynein from the mitotic spindle contribute to SAC engagement. Importantly, however, HPV16 E7 does not markedly compromise the SAC response to microtubule poisons.« less

Kinesin spindle protein (KSP) inhibitors. Part 4: Structure-based design of 5-alkylamino-3,5-diaryl-4,5-dihydropyrazoles as potent, water-soluble inhibitors of the mitotic kinesin KSP.

PubMed

Cox, Christopher D; Torrent, Maricel; Breslin, Michael J; Mariano, Brenda J; Whitman, David B; Coleman, Paul J; Buser, Carolyn A; Walsh, Eileen S; Hamilton, Kelly; Schaber, Michael D; Lobell, Robert B; Tao, Weikang; South, Vicki J; Kohl, Nancy E; Yan, Youwei; Kuo, Lawrence C; Prueksaritanont, Thomayant; Slaughter, Donald E; Li, Chunze; Mahan, Elizabeth; Lu, Bing; Hartman, George D

2006-06-15

Molecular modeling in combination with X-ray crystallographic information was employed to identify a region of the kinesin spindle protein (KSP) binding site not fully utilized by our first generation inhibitors. We discovered that by appending a propylamine substituent at the C5 carbon of a dihydropyrazole core, we could effectively fill this unoccupied region of space and engage in a hydrogen-bonding interaction with the enzyme backbone. This change led to a second generation compound with increased potency, a 400-fold enhancement in aqueous solubility at pH 4, and improved dog pharmacokinetics relative to the first generation compound.

Heat shock protein inhibitors, 17-DMAG and KNK437, enhance arsenic trioxide-induced mitotic apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu Yichen; Yen Wenyen; Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan

2009-04-15

Arsenic trioxide (ATO) has recently emerged as a promising therapeutic agent in leukemia because of its ability to induce apoptosis. However, there is no sufficient evidence to support its therapeutic use for other types of cancers. In this study, we investigated if, and how, 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG), an antagonist of heat shock protein 90 (HSP90), and KNK437, a HSP synthesis inhibitor, potentiated the cytotoxic effect of ATO. Our results showed that cotreatment with ATO and either 17-DMAG or KNK437 significantly increased ATO-induced cell death and apoptosis. siRNA-mediated attenuation of the expression of the inducible isoform of HSP70 (HSP70i) or HSP90{alpha}/{beta} alsomore » enhanced ATO-induced apoptosis. In addition, cotreatment with ATO and 17-DMAG or KNK437 significantly increased ATO-induced mitotic arrest and ATO-induced BUBR1 phosphorylation and PDS1 accumulation. Cotreatment also significantly increased the percentage of mitotic cells with abnormal mitotic spindles and promoted metaphase arrest as compared to ATO treatment alone. These results indicated that 17-DMAG or KNK437 may enhance ATO cytotoxicity by potentiating mitotic arrest and mitotic apoptosis possibly through increased activation of the spindle checkpoint.« less

Relationship between DNA ploidy and proliferative cell nuclear antigen index in canine hemangiopericytoma.

PubMed

Kang, Seong-Kwi; Park, Nam-Yong; Cho, Ho-Sung; Shin, Sung-Shik; Kang, Mun-Il; Kim, Sang-Ki; Hyun, Changbaig; Park, In-Chul; Kim, Jong-Tack; Jeong, Cheol; Park, Sung-Hee; Park, Su-Jin; Jeong, Jae-Ho; Kim, You-Jung; Ochiai, Kenji; Umemura, Takashi; Cho, Kyoung-Oh

2006-03-01

The mitotic index is reported to be correlated with recurrence, mean patient survival, and metastasis of canine hemangiopericytoma (CHP). However, to the authors' knowledge, studies investigating the parameters that can predict recurrence or metastasis of CHP with low mitotic index have not been done. To evaluate growth kinetics of CHP with low mitotic index, a retrospective analysis of the proliferative activity by antiproliferative cell nuclear antigen monoclonal antibody and DNA contents by flow cytometry (FCM) was performed with 21 formalin-fixed and paraffin-embedded CHP samples. Of the 21 tumors evaluated by FCM, 6 (26.6%) were aneuploid tumors, and 15 (71.4%) were diploid tumors. There was significant correlation between the PCNA index and ploidy pattern. The diploid group had 39.1 +/- 9.2 PCNA index, whereas the aneuploid group's proliferative cell nuclear antigen (PCNA) index was 63.1 +/- 8.2. The diploid group had mean mitotic index value of 1.140 +/- 0.855, and the aneuploid group had a mean value of 1.067 +/- 0.767. From these results, the CHP samples with low mitotic index were classified into either the aneuploid group with higher PCNA index or the diploid group with lower PCNA index, suggesting that DNA ploidy and proliferative activity may give an indication about malignancy of CHPs with a low mitotic index.

Wnt activation followed by Notch inhibition promotes mitotic hair cell regeneration in the postnatal mouse cochlea

PubMed Central

Li, Wenyan; Chen, Yan; Zhang, Shasha; Tang, Mingliang; Sun, Shan; Chai, Renjie; Li, Huawei

2016-01-01

Hair cell (HC) loss is the main cause of permanent hearing loss in mammals. Previous studies have reported that in neonatal mice cochleae, Wnt activation promotes supporting cell (SC) proliferation and Notch inhibition promotes the trans-differentiation of SCs into HCs. However, Wnt activation alone fails to regenerate significant amounts of new HCs, Notch inhibition alone regenerates the HCs at the cost of exhausting the SC population, which leads to the death of the newly regenerated HCs. Mitotic HC regeneration might preserve the SC number while regenerating the HCs, which could be a better approach for long-term HC regeneration. We present a two-step gene manipulation, Wnt activation followed by Notch inhibition, to accomplish mitotic regeneration of HCs while partially preserving the SC number. We show that Wnt activation followed by Notch inhibition strongly promotes the mitotic regeneration of new HCs in both normal and neomycin-damaged cochleae while partially preserving the SC number. Lineage tracing shows that the majority of the mitotically regenerated HCs are derived specifically from the Lgr5+ progenitors with or without HC damage. Our findings suggest that the co-regulation of Wnt and Notch signaling might provide a better approach to mitotically regenerate HCs from Lgr5+ progenitor cells. PMID:27564256

Sulforaphane induces reactive oxygen species-mediated mitotic arrest and subsequent apoptosis in human bladder cancer 5637 cells.

PubMed

Park, Hyun Soo; Han, Min Ho; Kim, Gi-Young; Moon, Sung-Kwon; Kim, Wun-Jae; Hwang, Hye Jin; Park, Kun Young; Choi, Yung Hyun

2014-02-01

The present study was undertaken to determine whether sulforaphane-derived reactive oxygen species (ROS) might cause growth arrest and apoptosis in human bladder cancer 5637 cells. Our results show that the reduced viability of 5637 cells by sulforaphane is due to mitotic arrest, but not the G2 phase. The sulforaphane-induced mitotic arrest correlated with an induction of cyclin B1 and phosphorylation of Cdk1, as well as a concomitant increased complex between cyclin B1 and Cdk1. Sulforaphane-induced apoptosis was associated with the activation of caspase-8 and -9, the initiators caspases of the extrinsic and intrinsic apoptotic pathways, respectively, and activation of effector caspase-3 and cleavage of poly (ADP-ribose) polymerase. However, blockage of caspase activation inhibited apoptosis and abrogated growth inhibition in sulforaphane-treated 5637 cells. This study further investigated the roles of ROS with respect to mitotic arrest and the apoptotic effect of sulforaphane, and the maximum level of ROS accumulation was observed 3h after sulforaphane treatment. However, a ROS scavenger, N-acetyl-L-cysteine, notably attenuated sulforaphane-mediated apoptosis as well as mitotic arrest. Overall, these results suggest that sulforaphane induces mitotic arrest and apoptosis of 5637 cells via a ROS-dependent pathway. Copyright © 2013 Elsevier Ltd. All rights reserved.

UV-C irradiation delays mitotic progression by recruiting Mps1 to kinetochores

PubMed Central

Zhang, Xiaojuan; Ling, Youguo; Wang, Wenjun; Zhang, Yanhong; Ma, Qingjun; Tan, Pingping; Song, Ting; Wei, Congwen; Li, Ping; Liu, Xuedong; Ma, Runlin Z.; Zhong, Hui; Cao, Cheng; Xu, Quanbin

2013-01-01

The effect of UV irradiation on replicating cells during interphase has been studied extensively. However, how the mitotic cell responds to UV irradiation is less well defined. Herein, we found that UV-C irradiation (254 nm) increases recruitment of the spindle checkpoint proteins Mps1 and Mad2 to the kinetochore during metaphase, suggesting that the spindle assembly checkpoint (SAC) is reactivated. In accordance with this, cells exposed to UV-C showed delayed mitotic progression, characterized by a prolonged chromosomal alignment during metaphase. UV-C irradiation also induced the DNA damage response and caused a significant accumulation of γ-H2AX on mitotic chromosomes. Unexpectedly, the mitotic delay upon UV-C irradiation is not due to the DNA damage response but to the relocation of Mps1 to the kinetochore. Further, we found that UV-C irradiation activates Aurora B kinase. Importantly, the kinase activity of Aurora B is indispensable for full recruitment of Mps1 to the kinetochore during both prometaphase and metaphase. Taking these findings together, we propose that UV irradiation delays mitotic progression by evoking the Aurora B-Mps1 signaling cascade, which exerts its role through promoting the association of Mps1 with the kinetochore in metaphase. PMID:23531678

Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity

PubMed Central

Dewey, Evan B.; Johnston, Christopher A.

2017-01-01

Proper assembly and orientation of the bipolar mitotic spindle is critical to the fidelity of cell division. Mitotic precision fundamentally contributes to cell fate specification, tissue development and homeostasis, and chromosome distribution within daughter cells. Defects in these events are thought to contribute to several human diseases. The underlying mechanisms that function in spindle morphogenesis and positioning remain incompletely defined, however. Here we describe diverse roles for the actin-microtubule cross-linker Shortstop (Shot) in mitotic spindle function in Drosophila. Shot localizes to mitotic spindle poles, and its knockdown results in an unfocused spindle pole morphology and a disruption of proper spindle orientation. Loss of Shot also leads to chromosome congression defects, cell cycle progression delay, and defective chromosome segregation during anaphase. These mitotic errors trigger apoptosis in Drosophila epithelial tissue, and blocking this apoptotic response results in a marked induction of the epithelial–mesenchymal transition marker MMP-1. The actin-binding domain of Shot directly interacts with Actin-related protein-1 (Arp-1), a key component of the Dynein/Dynactin complex. Knockdown of Arp-1 phenocopies Shot loss universally, whereas chemical disruption of F-actin does so selectively. Our work highlights novel roles for Shot in mitosis and suggests a mechanism involving Dynein/Dynactin activation. PMID:28747439

Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rahayu, Retno; Ohsaki, Eriko; Omori, Hiroko

In latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), viral gene expression is extremely limited and copy numbers of viral genomes remain constant. Latency-associated nuclear antigen (LANA) is known to have a role in maintaining viral genome copy numbers in growing cells. Several studies have shown that LANA is localized in particular regions on mitotic chromosomes, such as centromeres/pericentromeres. We independently examined the distinct localization of LANA on mitotic chromosomes during mitosis, using super-resolution laser confocal microscopy and correlative fluorescence microscopy–electron microscopy (FM-EM) analyses. We found that the majority of LANA were not localized at particular regions such as telomeres/peritelomeres, centromeres/pericentromeres,more » and cohesion sites, but at the bodies of condensed chromosomes. Thus, LANA may undergo various interactions with the host factors on the condensed chromosomes in order to tether the viral genome to mitotic chromosomes and realize faithful viral genome segregation during cell division. - Highlights: • This is the first report showing LANA dots on mitotic chromosomes by fluorescent microscopy followed by electron microscopy. • LANA dots localized randomly on condensed chromosomes other than centromere/pericentromere and telomere/peritelomre. • Cellular mitotic checkpoint should not be always involved in the segregation of KSHV genomes in the latency.« less

Protective effects of mito-TEMPO against doxorubicin cardiotoxicity in mice.

PubMed

Rocha, Viviane Costa Junqueira; França, Luciana Souza de Aragão; de Araújo, Cintia Figueiredo; Ng, Ayling Martins; de Andrade, Candace Machado; Andrade, André Cronemberger; Santos, Emanuelle de Souza; Borges-Silva, Mariana da Cruz; Macambira, Simone Garcia; Noronha-Dutra, Alberto Augusto; Pontes-de-Carvalho, Lain Carlos

2016-03-01

Doxorubicin (DOX) is a chemotherapeutic that is widely used for the treatment of many human tumors. However, the development of cardiotoxicity has limited its use. The aim of the present study was to evaluate the possible efficacy of mito-TEMPO (mito-T) as a protective agent against DOX-induced cardiotoxicity in mice. C57BL/6 mice were treated twice with mito-T at low (5 mg/kg body weight) or high (20 mg/kg body weight) dose and once with DOX (24 mg/kg body weight) or saline (0.1 mL/20 g body weight) by means of intraperitoneal injections. The levels of malondialdehyde (MLDA), a marker of lipid peroxidation, and serum levels of creatine kinase were evaluated 48 h after the injection of DOX. DOX induced lipid peroxidation in heart mitochondria (p < 0.001), and DOX-treated mice receiving mito-T at low dose had levels of MLDA significantly lower than the mice that received only DOX (p < 0.01). Furthermore, administration of mito-T alone did not cause any significant changes from control values. Additionally, DOX-treated mice treated with mito-T at high dose showed decrease in serum levels of total CK compared to mice treated with DOX alone (p < 0.05). Our results indicate that mito-T protects mice against DOX-induced cardiotoxicity.

Division of constricted and urethane-treated sand dollar eggs: a test of the polar stimulation hypothesis.

PubMed

Rappaport, R; Rappaport, B N

1984-07-01

In spherical cells with a central mitotic apparatus, the centers of the asters are closer to the poles than to the equator. This circumstance is basic to several hypothetical explanations of the way in which the mitotic apparatus establishes the division mechanism. This investigation was designed to determine whether that geometrical relationship is necessary for division. Fertilized, mechanically denuded sand dollar eggs were inserted into glass loops, which reduced the diameter in the constriction plane from the normal 142 to 78-80 microns and partly constricted the cell into equal parts. The mitotic apparatus straddled the constriction, and its length was not significantly changed. The manipulation increased the distance from the astral centers to the poles and decreased the distance from the astral centers to the equator to a degree that reversed the normal distance relations. These cells divided normally. Ethyl urethane (0.06 M) reduces the size of the mitotic apparatus and blocks cleavage in spherical cells. When treated cells are confined in 80-microns i.d. capillaries, they divide. Treated cells also divide when they are constricted by an 80-microns i.d. glass loop if the mitotic apparatus straddles the constriction. An equal degree of constriction in the subfurrow and subpolar areas did not reverse the effect of urethane. The results demonstrate that cleavage does not depend on the normal distance relation between the mitotic apparatus and the poles, and that the urethane effect can be remedied only by reducing the distance between the mitotic apparatus and the equatorial surface. Both findings are inconsistent with the polar stimulation hypothesis.

The Spo12 protein of Saccharomyces cerevisiae: a regulator of mitotic exit whose cell cycle-dependent degradation is mediated by the anaphase-promoting complex.

PubMed Central

Shah, R; Jensen, S; Frenz, L M; Johnson, A L; Johnston, L H

2001-01-01

The Spo12 protein plays a regulatory role in two of the most fundamental processes of biology, mitosis and meiosis, and yet its biochemical function remains elusive. In this study we concentrate on the genetic and biochemical analysis of its mitotic function. Since high-copy SPO12 is able to suppress a wide variety of mitotic exit mutants, all of which arrest with high Clb-Cdc28 activity, we speculated whether SPO12 is able to facilitate exit from mitosis when overexpressed by antagonizing mitotic kinase activity. We show, however, that Spo12 is not a potent regulator of Clb-Cdc28 activity and can function independently of either the cyclin-dependent kinase inhibitor (CDKi), Sic1, or the anaphase-promoting complex (APC) regulator, Hct1. Spo12 protein level is regulated by the APC and the protein is degraded in G1 by an Hct1-dependent mechanism. We also demonstrate that in addition to localizing to the nucleus Spo12 is a nucleolar protein. We propose a model where overexpression of Spo12 may lead to the delocalization of a small amount of Cdc14 from the nucleolus, resulting in a sufficient lowering of mitotic kinase levels to facilitate mitotic exit. Finally, site-directed mutagenesis of highly conserved residues in the Spo12 protein sequence abolishes both its mitotic suppressor activity as well as its meiotic function. This result is the first indication that Spo12 may carry out the same biochemical function in mitosis as it does in meiosis. PMID:11729145

Phosphohistone-H3 (PHH3) is prognostic relevant in Merkel cell carcinomas but Merkel cell polyomavirus is a more powerful prognostic factor than AJCC clinical stage, PHH3, Ki-67 or mitotic indices.

PubMed

Iwasaki, Takeshi; Matsushita, Michiko; Nonaka, Daisuke; Kato, Masako; Nagata, Keiko; Murakami, Ichiro; Hayashi, Kazuhiko

2015-08-01

Merkel cell carcinomas (MCCs) associated with Merkel cell polyomavirus (MCPyV) have better prognosis than those without MCPyV. The relationship between mitotic index (MI) and MCC outcome has remained elusive because of the difficulty in differentiating mitotic cells from apoptotic ones. We evaluated the role of phosphohistone-H3 (PHH3) (Ser10), a new mitotic count biomarker, in MCPyV-positive or -negative MCC patients, and assessed its prognostic value in comparison to Ki-67 labeling index or MI using hematoxylin and eosin (HE) staining. We compared the prognostic value of PHH3 mitotic index with that of MI by HE in 19 MCPyV-positive and 9 MCPyV-negative MCC patients. PHH3-positive immunoreactivity was mostly observed in mitotic figures. Multivariate analysis significantly showed that MCPyV status (HR, 0.004; 95% CI 0.0003-0.058) and the American Joint Committee of Cancer (AJCC) stage (HR, 5.02; 95% CI 1.23-20.51) were observed as significantly independent prognostic factors for OS. PHH3-positive cell counts/10 HPF was a slightly significant independent prognostic factor for OS (HR, 4.96; 95% CI 0.93-26.55). PHH3-positive MI and MCPyV status in MCC patients are useful in prognostication, although MCPyV-infection is a more powerful prognostic factor in MCCs than the AJCC scheme on proliferation or mitotic indices. © 2015 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

Mitotic figure counts are significantly overestimated in resection specimens of invasive breast carcinomas.

PubMed

Lehr, Hans-Anton; Rochat, Candice; Schaper, Cornelia; Nobile, Antoine; Shanouda, Sherien; Vijgen, Sandrine; Gauthier, Arnaud; Obermann, Ellen; Leuba, Susana; Schmidt, Marcus; C, Curzio Ruegg; Delaloye, Jean-Francois; Simiantonaki, Nectaria; Schaefer, Stephan C

2013-03-01

Several authors have demonstrated an increased number of mitotic figures in breast cancer resection specimen when compared with biopsy material. This has been ascribed to a sampling artifact where biopsies are (i) either too small to allow formal mitotic figure counting or (ii) not necessarily taken form the proliferating tumor periphery. Herein, we propose a different explanation for this phenomenon. Biopsy and resection material of 52 invasive ductal carcinomas was studied. We counted mitotic figures in 10 representative high power fields and quantified MIB-1 immunohistochemistry by visual estimation, counting and image analysis. We found that mitotic figures were elevated by more than three-fold on average in resection specimen over biopsy material from the same tumors (20±6 vs 6±2 mitoses per 10 high power fields, P=0.008), and that this resulted in a relative diminution of post-metaphase figures (anaphase/telophase), which made up 7% of all mitotic figures in biopsies but only 3% in resection specimen (P<0.005). At the same time, the percentages of MIB-1 immunostained tumor cells among total tumor cells were comparable in biopsy and resection material, irrespective of the mode of MIB-1 quantification. Finally, we found no association between the size of the biopsy material and the relative increase of mitotic figures in resection specimen. We propose that the increase in mitotic figures in resection specimen and the significant shift towards metaphase figures is not due to a sampling artifact, but reflects ongoing cell cycle activity in the resected tumor tissue due to fixation delay. The dwindling energy supply will eventually arrest tumor cells in metaphase, where they are readily identified by the diagnostic pathologist. Taken together, we suggest that the rapidly fixed biopsy material better represents true tumor biology and should be privileged as predictive marker of putative response to cytotoxic chemotherapy.

Evidence of Selection against Complex Mitotic-Origin Aneuploidy during Preimplantation Development

PubMed Central

McCoy, Rajiv C.; Demko, Zachary P.; Ryan, Allison; Banjevic, Milena; Hill, Matthew; Sigurjonsson, Styrmir; Rabinowitz, Matthew; Petrov, Dmitri A.

2015-01-01

Whole-chromosome imbalances affect over half of early human embryos and are the leading cause of pregnancy loss. While these errors frequently arise in oocyte meiosis, many such whole-chromosome abnormalities affecting cleavage-stage embryos are the result of chromosome missegregation occurring during the initial mitotic cell divisions. The first wave of zygotic genome activation at the 4–8 cell stage results in the arrest of a large proportion of embryos, the vast majority of which contain whole-chromosome abnormalities. Thus, the full spectrum of meiotic and mitotic errors can only be detected by sampling after the initial cell divisions, but prior to this selective filter. Here, we apply 24-chromosome preimplantation genetic screening (PGS) to 28,052 single-cell day-3 blastomere biopsies and 18,387 multi-cell day-5 trophectoderm biopsies from 6,366 in vitro fertilization (IVF) cycles. We precisely characterize the rates and patterns of whole-chromosome abnormalities at each developmental stage and distinguish errors of meiotic and mitotic origin without embryo disaggregation, based on informative chromosomal signatures. We show that mitotic errors frequently involve multiple chromosome losses that are not biased toward maternal or paternal homologs. This outcome is characteristic of spindle abnormalities and chaotic cell division detected in previous studies. In contrast to meiotic errors, our data also show that mitotic errors are not significantly associated with maternal age. PGS patients referred due to previous IVF failure had elevated rates of mitotic error, while patients referred due to recurrent pregnancy loss had elevated rates of meiotic error, controlling for maternal age. These results support the conclusion that mitotic error is the predominant mechanism contributing to pregnancy losses occurring prior to blastocyst formation. This high-resolution view of the full spectrum of whole-chromosome abnormalities affecting early embryos provides insight into the cytogenetic mechanisms underlying their formation and the consequences for human fertility. PMID:26491874

Dynein-mediated pulling forces drive rapid mitotic spindle elongation in Ustilago maydis

PubMed Central

Fink, Gero; Schuchardt, Isabel; Colombelli, Julien; Stelzer, Ernst; Steinberg, Gero

2006-01-01

Spindle elongation segregates chromosomes and occurs in anaphase, an essential step in mitosis. Dynein-mediated pulling forces position the spindle, but their role in anaphase is a matter of debate. Here, we demonstrate that dynein is responsible for rapid spindle elongation in the model fungus Ustilago maydis. We show that initial slow elongation is supported by kinesin-5, which is located in the spindle mid-zone. When the spindle reaches ∼2 μm in length, the elongation rate increases four-fold. This coincides with the appearance of long and less-dynamic microtubules (MTs) at each pole that accumulate dynein at their tips. Laser-mediated nanosurgery revealed that these MTs exert pulling forces in control cells, but not in dynein mutants. In addition, dynein mutants undergo initial slow anaphase, but fail to establish less-dynamic MTs and do not perform rapid spindle elongation, suggesting that dynein drives anaphase B. This is most likely mediated by cortical sliding of astral MTs along stationary dynein, which is off-loaded from the MT plus-end to the cortex. PMID:17024185

The Kinesin-Related Protein, Hset, Opposes the Activity of Eg5 and Cross-Links Microtubules in the Mammalian Mitotic Spindle

PubMed Central

Mountain, Vicki; Simerly, Calvin; Howard, Louisa; Ando, Asako; Schatten, Gerald; Compton, Duane A.

1999-01-01

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes. PMID:10525540

The Msd1–Wdr8–Pkl1 complex anchors microtubule minus ends to fission yeast spindle pole bodies

PubMed Central

Yukawa, Masashi; Ikebe, Chiho

2015-01-01

The minus ends of spindle microtubules are anchored to a microtubule-organizing center. The conserved Msd1/SSX2IP proteins are localized to the spindle pole body (SPB) and the centrosome in fission yeast and humans, respectively, and play a critical role in microtubule anchoring. In this paper, we show that fission yeast Msd1 forms a ternary complex with another conserved protein, Wdr8, and the minus end–directed Pkl1/kinesin-14. Individual deletion mutants displayed the identical spindle-protrusion phenotypes. Msd1 and Wdr8 were delivered by Pkl1 to mitotic SPBs, where Pkl1 was tethered through Msd1–Wdr8. The spindle-anchoring defect imposed by msd1/wdr8/pkl1 deletions was suppressed by a mutation of the plus end–directed Cut7/kinesin-5, which was shown to be mutual. Intriguingly, Pkl1 motor activity was not required for its anchoring role once targeted to the SPB. Therefore, spindle anchoring through Msd1–Wdr8–Pkl1 is crucial for balancing the Cut7/kinesin-5–mediated outward force at the SPB. Our analysis provides mechanistic insight into the spatiotemporal regulation of two opposing kinesins to ensure mitotic spindle bipolarity. PMID:25987607

Elucidating cdc25’s Oncogenic Mechanism in Breast Cancer Using Pin1, a Negative Mitotic Regulator

DTIC Science & Technology

2000-07-01

inhibitor aphidicolin. This defect in replication checkpoint function was reversed after addition of recombinant wild type Pin 1, but not an isomerase... inhibitor , aphidicolin. Mock-depleted extracts effectively postponed mitotic entry in response to replication inhibition, while depletion of Pin 1 from...fail to haft mitotic entry in response to the DNA polymerase inhibitor , aphidicolin. The addition of recombinant Pin1 restores the appropriate G2

Hippo Signaling in Mitosis: An Updated View in Light of the MEN Pathway.

PubMed

Hergovich, Alexander

2017-01-01

The Hippo pathway is an essential tumor suppressor signaling network that coordinates cell proliferation, death, and differentiation in higher eukaryotes. Intriguingly, the core components of the Hippo pathway are conserved from yeast to man, with the yeast analogs of mammalian MST1/2 (fly Hippo), MOB1 (fly Mats), LATS1/2 (fly Warts), and NDR1/2 (fly Tricornered) functioning as essential components of the mitotic exit network (MEN). Here, we update our previous summary of mitotic functions of Hippo core components in Drosophila melanogaster and mammals, with particular emphasis on similarities between the yeast MEN pathway and mitotic Hippo signaling. Mitotic functions of YAP and TAZ, the two main effectors of Hippo signaling, are also discussed.

Inhibition of intra-Golgi transport in vitro by mitotic kinase.

PubMed

Stuart, R A; Mackay, D; Adamczewski, J; Warren, G

1993-02-25

It has previously been shown that exocytic and endocytic membrane traffic are inhibited in mitotic mammalian cells. Here we have used a cell-free intra-Golgi transport assay supplemented with heterologous cytosols to mimic this effect in vitro. Cytosols with high histone kinase activity, made either from mitotic cells or by cyclin A treatment of interphase cells, inhibited intra-Golgi transport by up to 75%. Inhibition of transport was reversed by the kinase inhibitor staurosporine or by reduction in ATP levels leading to inactivation of histone kinase. The data indicate that cell cycle control of intra-Golgi transport is due to a reversible modification of cytosol, and this assay system may be used to study the molecular mechanism of mitotic transport inhibition in mammalian cells.

Effects of caffeine on mitotic index, mitotic aberrations and bimitosis with and without aeration.

PubMed

Röper, W

1977-07-01

The effects of 1 to 3 h 0.2% caffeine treatment on mitosis in lateral roots of Vicia faba with and without aeration have been investigated. During the treatment a marked decrease of the mitotic index followed by strong deviations and changing phase indices can be stated. By means of aeration the number of mitotic aberrations increases with time of treatment, while it decreases without aeration until 3 h treatment. Tetraploid cells are supposed to be formed by spindle aberrations at early anaphase. The number of binucleate and tetraploid cells is affected by aeration during caffeine treatment. During division of the binucleate cells tetraploid nuclei are formed by fusions, so the population of binucleate cells may become smaller.

Mononuclear Cells from Dedifferentiation of Mouse Myotubes display Remarkable Regenerative Capability

PubMed Central

Yang, Zhong; Liu, Qiang; Mannix, Robert J.; Xu, Xiaoyin; Li, Hongli; Ma, Zhiyuan; Ingber, Donald E.; Allen, Paul D.; Wang, Yaming

2015-01-01

Certain lower organisms achieve organ regeneration by reverting differentiated cells into tissue-specific progenitors that re-enter embryonic programs. During muscle regeneration in the urodele amphibian, post-mitotic multinucleated skeletal myofibers transform into mononucleated proliferating cells upon injury, and a transcription factor-msx1 plays a role in their reprograming. Whether this powerful regeneration strategy can be leveraged in mammals remains unknown, as it has not been demonstrated that the dedifferentiated progenitor cells arising from muscle cells overexpressing Msx1 are lineage-specific and possess the same potent regenerative capability as their amphibian counterparts. Here we show that ectopic expression of Msx1 reprograms post-mitotic, multinucleated, primary mouse myotubes to become proliferating mononuclear cells. These dedifferentiated cells reactivate genes expressed by embryonic muscle progenitor cells and generate only muscle tissue in vivo both in an ectopic location and inside existing muscle. More importantly, distinct from adult muscle satellite cells, these cells appear both to fuse with existing fibers and to regenerate myofibers in a robust and time-dependent manner. Upon transplantation into a degenerating muscle, these dedifferentiated cells generated a large number of myofibers that increased over time and replenished almost half of the cross-sectional area of the muscle in only 12 weeks. Our study demonstrates that mammals can harness a muscle regeneration strategy used by lower organisms when the same molecular pathway is activated. PMID:24916688

Sensitivity and usefulness of anti-phosphohistone-H3 antibody immunostaining for counting mitotic figures in meningioma cases.

PubMed

Fukushima, Shintaro; Terasaki, Mizuhiko; Sakata, Kiyohiko; Miyagi, Naohisa; Kato, Seiya; Sugita, Yasuo; Shigemori, Minoru

2009-01-01

According to current World Health Organization (WHO) criteria, counting mitotic figures (MF), which is equal to the mitotic index (MI), on paraffin sections stained with hematoxylin and eosin (HE) is one of the recognized classification methods for meningiomas. However, it is not always easy to find the area of highest mitotic activity, and there are different perspectives among pathologists with regard to differentiating MF from non-MF, i.e., which are apoptotic figures and which are crushed or distorted cells. Moreover, there is an issue of overgrading in meningiomas with preoperative feeder embolization. Recently, anti-phosphohistone-H3 (PHH3) antibody has been reported as a mitosis-specific marker for meningioma grading. In this study, we attempted PHH3 immunostaining for our meningioma cases and verified not only the sensitivity of PHH3 immunostaining but also that of its usefulness in grading meningiomas. Forty-five initial histologically confirmed meningiomas (37 benign, 7 atypical, and 1 anaplastic) were reviewed according to current WHO criteria based on counting MF on HE-stained slides. PHH3-immunostained MF were counted in the same way, and the MIB-1 labeling index (LI) was calculated for each sample. PHH3-labeled MF were easily identified and permitted rapid recognition of the areas of highest mitotic activity. As a result, significant increase of PHH3 mitotic index (PHH3-MI) in comparison with HE mitotic index (HE-MI) and strong correlations with HE-MI to PHH3-MI as well as PHH3-MI to MIB-1 LI were demonstrated. Furthermore, no significant differences of PHH3-MI between cases with and without feeder embolization were demonstrated. As such, PHH3 may be a sensitive and useful marker for meningioma grading as based on the MF.

Live-cell imaging visualizes frequent mitotic skipping during senescence-like growth arrest in mammary carcinoma cells exposed to ionizing radiation.

PubMed

Suzuki, Masatoshi; Yamauchi, Motohiro; Oka, Yasuyoshi; Suzuki, Keiji; Yamashita, Shunichi

2012-06-01

Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO(2)-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ß-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation. Copyright © 2012 Elsevier Inc. All rights reserved.

[Genetic control of mitotic crossing-over in yeasts. III. Induction by 8-methoxypsoralen and long-wave UV irradiation (lambda=365 nm)].

PubMed

Fedorova, I V; Marfin, S V

1982-02-01

The lethal effect of 8-methoxypsoralen (8-MOP) plus 365 nm light has been studied in haploid radiosensitive strains of Saccharomyces cerevisiae. The diploid of wild type and the diploid homozygous for the rad2 mutation (this mutation blocks the excision of UV-induced pyrimidine dimers) were more resistant to the lethal effect of 8-MOP plus 365 nm light than the haploid of wild type and rad2 haploid, respectively. The diploid homozygous for rad54 mutation (the mutation blocks the repair of double-strand breaks in DNA) was more sensitive than haploid rad54. The method of repeated irradiation allowed to study the capacity of radiosensitive diploids to remove monoadducts induced by 8-MOP in DNA. This process was very effective in diploids of wild type and in the rad54 rad54 diploid, while the rad2 rad2 diploid was characterized by nearly complete absence of monoadduct excision. The study of mitotic crossing over and mitotic segregation in yeast diploids, containing a pair of complementing alleles of the ade2 gene (red/pink) has shown a very high recombinogenic effect of 8-MOP plus 365 nm light. The rad2 mutation slightly increased the frequency of mitotic segregation and mitotic crossing over. The rad54 mutation decreased the frequency of mitotic segregation and entirely suppressed mitotic crossing over. The method of repeated irradiation showed that the cross-links, but not monoadducts, are the main cause of high recombinogenic effect of 8-MOP plus 365 nm light. The possible participation of different repair systems in recombinational processes induced by 8-MOP in yeast cells is discussed.

A study of directional instability during mitotic chromosome movement

NASA Astrophysics Data System (ADS)

Joglekar, Ajit P.

Mitotic chromosome movements are responsible for the correct segregation of duplicated chromosomes into the daughter cells. Errors in this process are known to play a role in some of the serious diseases such as cancer, and the little understood process of aging. A thorough comprehension of the physical basis of this process is therefore necessary. An intriguing aspect of chromosome movements during mitosis is "directional instability": runs with approximately constant speed punctuated by abrupt reversal in direction of motion. I have constructed a mechanistic model that views chromosome movement as a result of interplay between poleward and antipoleward or polar ejection forces (PEF) on a chromosome; and microtubule (MT) depolymerization-coupled movement of the chromosome. Computer simulations based on this model using a single set of parameters accurately and quantitatively predict: the force, character, speed, and duration of chromosome movements, oscillations of chromosomes associated with only one spindle pole, the larger force during anaphase, the effect of MT-depolymerizing drugs on chromosome movements, and the decreased turnover of kinetochore-MTs during anaphase. The model also predicts how chromosome behavior should respond to perturbations of the PEF. These predictions could be unequivocally tested if it were possible to destroy structures smaller than the light resolution limit with minimal collateral damage. To address these requirements, I developed a methodology for ultrahigh resolution microsurgery with tightly-focused, ultrafast lasers pulses. This entailed an in-depth study of optical breakdown in dielectrics. Characterization of the single pulse damage in test dielectric materials ranging from silicon and glass to cell walls and membranes has shown that in the target regions where the laser intensity exceeds critical intensity, optical breakdown proceeds by tunneling ionization followed by a runaway avalanche ionization that ends with the ionization of all the valence electrons. Highly reproducible features on the nanometer size-scale indicate that the valence electron density is the central factor determining the critical intensity, implying that high precision can be maintained in a wide range of solids. Along with the new understanding optical breakdown, this technique will find potential applications in diverse fields ranging from MEMS fabrication to nano-fluidics, as well as cellular nanosurgery.

Evaluation of efficacy of 1% Crystal Violet & Nuclear Fast Red stain compared to Haematoxyline & Eosin stain for assessing mitotic figures in oral premalignant and malignant lesions.

PubMed

Motiwale, Gauri; Jaiswal, Shradha; Vikey, Ashok; Motiwale, Tejas; Bagulkar, Bhupesh; Bhat, Atul; Kapoor, Prakhar

2016-07-01

Various chromosomal arrangements in cells undergoing division are referred to as Mitotic figure (MF). The abnormal excess of mitotic figures is commonly seen in oral epithelial dysplasia (ED) and oral squamous cell carcinoma (OSCC). In present study, we compared the number of mitotic figures in normal oral mucosa, epithelial dysplasia & OSCC sections with haematoxyline & eosine (H&E) and 1%Crystal Violet & Nuclear Fast Red (CV&NFR) stain, also the efficacy of the CV&NFR stain as compared to H & E stain. We investigated the correlation between the number of mitotic figures & grades of OSCC. Study sample comprised of two serial sections of archival blocks of normal oral mucosa & diagnosed cases of epithelial dysplasia & OSCC. One slide stained with H& E & the other one with 1% CV & NFR. Mitotic figures were counted with the grid eyepiece. There was significant increase in number of MFs in oral ED and OSCC in comparison with normal oral mucosa. There was a highly significant increase in number of MFs in CV&NFR stained tissue sections when compared with H & E stain. Metaphase is the most commonly observed phase of mitosis. In summary, our study proposes the use of Crystal violet & Nuclear fast red stain as a selective stain for better contrast & easy identification MFs. © 2016 Old City Publishing, Inc.

Less understood issues: p21(Cip1) in mitosis and its therapeutic potential.

PubMed

Kreis, N-N; Louwen, F; Yuan, J

2015-04-02

p21(Cip1) is a multifunctional protein and a key player in regulating different cellular processes. The transcription of p21 is regulated by p53-dependent and -independent pathways. The expression of p21 is increased in response to various cellular stresses to arrest the cell cycle and ensure genomic stability. p21 has been shown to be a tumor suppressor and an oncogene as well. The function of p21 in mitosis has been proposed but not systematically studied. We have recently shown that p21 binds to and inhibits the activity of Cdk1/cyclin B1, and is important for a fine-tuned mitotic progression. Loss of p21 prolongs the duration of mitosis and results in severe mitotic defects like chromosome segregation and cytokinesis failures promoting consequently genomic instability. Moreover, p21 is dramatically stabilized in mitotic tumor cells upon treatment with mitotic agents like paclitaxel or mitotic kinase inhibitors. Increased p21 is mainly localized in the cytoplasm and associates with cell survival indicating a crucial role of p21 in susceptibility to mitotic agents in tumor cells. In this review we will briefly summarize the structure and general physiological functions as well as regulation of p21, discuss in detail its role in mitosis and its potential to serve as a therapeutic target.

Partitioning of the Golgi Apparatus during Mitosis in Living HeLa Cells

PubMed Central

Shima, David T.; Haldar, Kasturi; Pepperkok, Rainer; Watson, Rose; Warren, Graham

1997-01-01

The Golgi apparatus of HeLa cells was fluorescently tagged with a green fluorescent protein (GFP), localized by attachment to the NH2-terminal retention signal of N-acetylglucosaminyltransferase I (NAGT I). The location was confirmed by immunogold and immunofluorescence microscopy using a variety of Golgi markers. The behavior of the fluorescent Golgi marker was observed in fixed and living mitotic cells using confocal microscopy. By metaphase, cells contained a constant number of Golgi fragments dispersed throughout the cytoplasm. Conventional and cryoimmunoelectron microscopy showed that the NAGT I–GFP chimera (NAGFP)-positive fragments were tubulo-vesicular mitotic Golgi clusters. Mitotic conversion of Golgi stacks into mitotic clusters had surprisingly little effect on the polarity of Golgi membrane markers at the level of fluorescence microscopy. In living cells, there was little self-directed movement of the clusters in the period from metaphase to early telophase. In late telophase, the Golgi ribbon began to be reformed by a dynamic process of congregation and tubulation of the newly inherited Golgi fragments. The accuracy of partitioning the NAGFP-tagged Golgi was found to exceed that expected for a stochastic partitioning process. The results provide direct evidence for mitotic clusters as the unit of partitioning and suggest that precise regulation of the number, position, and compartmentation of mitotic membranes is a critical feature for the ordered inheritance of the Golgi apparatus. PMID:9182657

Immunolocalization of dually phosphorylated MAPKs in dividing root meristem cells of Vicia faba, Pisum sativum, Lupinus luteus and Lycopersicon esculentum.

PubMed

Winnicki, Konrad; Żabka, Aneta; Bernasińska, Joanna; Matczak, Karolina; Maszewski, Janusz

2015-06-01

In plants, phosphorylated MAPKs display constitutive nuclear localization; however, not all studied plant species show co-localization of activated MAPKs to mitotic microtubules. The mitogen-activated protein kinase (MAPK) signaling pathway is involved not only in the cellular response to biotic and abiotic stress but also in the regulation of cell cycle and plant development. The role of MAPKs in the formation of a mitotic spindle has been widely studied and the MAPK signaling pathway was found to be indispensable for the unperturbed course of cell division. Here we show cellular localization of activated MAPKs (dually phosphorylated at their TXY motifs) in both interphase and mitotic root meristem cells of Lupinus luteus, Pisum sativum, Vicia faba (Fabaceae) and Lycopersicon esculentum (Solanaceae). Nuclear localization of activated MAPKs has been found in all species. Co-localization of these kinases to mitotic microtubules was most evident in L. esculentum, while only about 50% of mitotic cells in the root meristems of P. sativum and V. faba displayed activated MAPKs localized to microtubules during mitosis. Unexpectedly, no evident immunofluorescence signals at spindle microtubules and phragmoplast were noted in L. luteus. Considering immunocytochemical analyses and studies on the impact of FR180204 (an inhibitor of animal ERK1/2) on mitotic cells, we hypothesize that MAPKs may not play prominent role in the regulation of microtubule dynamics in all plant species.

LIS1 controls mitosis and mitotic spindle organization via the LIS1–NDEL1–dynein complex

PubMed Central

Moon, Hyang Mi; Youn, Yong Ha; Pemble, Hayley; Yingling, Jessica; Wittmann, Torsten; Wynshaw-Boris, Anthony

2014-01-01

Heterozygous LIS1 mutations are responsible for the human neuronal migration disorder lissencephaly. Mitotic functions of LIS1 have been suggested from many organisms throughout evolution. However, the cellular functions of LIS1 at distinct intracellular compartments such as the centrosome and the cell cortex have not been well defined especially during mitotic cell division. Here, we used detailed cellular approaches and time-lapse live cell imaging of mitosis from Lis1 mutant mouse embryonic fibroblasts to reveal critical roles of LIS1 in mitotic spindle regulation. We found that LIS1 is required for the tight control of chromosome congression and segregation to dictate kinetochore–microtubule (MT) interactions and anaphase progression. In addition, LIS1 is essential for the establishment of mitotic spindle pole integrity by maintaining normal centrosome number. Moreover, LIS1 plays crucial roles in mitotic spindle orientation by increasing the density of astral MT plus-end movements toward the cell cortex, which enhances cortical targeting of LIS1–dynein complex. Overexpression of NDEL1–dynein and MT stabilization rescues spindle orientation defects in Lis1 mutants, demonstrating that mouse LIS1 acts via the LIS1–NDEL1–dynein complex to regulate astral MT plus-ends dynamics and establish proper contacts of MTs with the cell cortex to ensure precise cell division. PMID:24030547

Ki-67 acts as a biological surfactant to disperse mitotic chromosomes

PubMed Central

Cuylen, Sara; Blaukopf, Claudia; Politi, Antonio Z.; Müller-Reichert, Thomas; Neumann, Beate; Poser, Ina; Ellenberg, Jan; Hyman, Anthony A.; Gerlich, Daniel W.

2016-01-01

Summary Eukaryotic genomes are partitioned into chromosomes, which during mitosis form compact and spatially well-separated mechanical bodies1–3.This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes4 and the discovery of proteins at the chromosome surface3,5,6, the molecular and biophysical basis of mitotic chromosome individuality have remained unclear. We report that Ki-67, a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of Ki-67 is not confined within a specific protein domain but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrical barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-color labeling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic for polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery and suggests that natural proteins can function as surfactants in intracellular compartmentalization. PMID:27362226

The role of DNA double-strand breaks in spontaneous homologous recombination in S. cerevisiae.

PubMed

Lettier, Gaëlle; Feng, Qi; de Mayolo, Adriana Antúnez; Erdeniz, Naz; Reid, Robert J D; Lisby, Michael; Mortensen, Uffe H; Rothstein, Rodney

2006-11-10

Homologous recombination (HR) is a source of genomic instability and the loss of heterozygosity in mitotic cells. Since these events pose a severe health risk, it is important to understand the molecular events that cause spontaneous HR. In eukaryotes, high levels of HR are a normal feature of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs). By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Here we provide the first direct evidence that most spontaneous mitotic HR in Saccharomyces cerevisiae is initiated by DNA lesions other than DSBs. Specifically, we describe a class of rad52 mutants that are fully proficient in inter- and intra-chromosomal mitotic HR, yet at the same time fail to repair DNA DSBs. The conclusions are drawn from genetic analyses, evaluation of the consequences of DSB repair failure at the DNA level, and examination of the cellular re-localization of Rad51 and mutant Rad52 proteins after introduction of specific DSBs. In further support of our conclusions, we show that, as in wild-type strains, UV-irradiation induces HR in these rad52 mutants, supporting the view that DNA nicks and single-stranded gaps, rather than DSBs, are major sources of spontaneous HR in mitotic yeast cells.

MASTL is essential for anaphase entry of proliferating primordial germ cells and establishment of female germ cells in mice

PubMed Central

Risal, Sanjiv; Zhang, Jingjing; Adhikari, Deepak; Liu, Xiaoman; Shao, Jingchen; Hu, Mengwen; Busayavalasa, Kiran; Tu, Zhaowei; Chen, Zijiang; Kaldis, Philipp; Liu, Kui

2017-01-01

In mammals, primordial germ cells (PGCs) are the embryonic cell population that serve as germ cell precursors in both females and males. During mouse embryonic development, the majority of PGCs are arrested at the G2 phase when they migrate into the hindgut at 7.75–8.75 dpc (days post coitum). It is after 9.5 dpc that the PGCs undergo proliferation with a doubling time of 12.6 h. The molecular mechanisms underlying PGC proliferation are however not well studied. In this work. Here we studied how MASTL (microtubule-associated serine/threonine kinase-like)/Greatwall kinase regulates the rapid proliferation of PGCs. We generated a mouse model where we specifically deleted Mastl in PGCs and found a significant loss of PGCs before the onset of meiosis in female PGCs. We further revealed that the deletion of Mastl in PGCs did not prevent mitotic entry, but led to a failure of the cells to proceed beyond metaphase-like stage, indicating that MASTL-mediated molecular events are indispensable for anaphase entry in PGCs. These mitotic defects further led to the death of Mastl-null PGCs by 12.5 dpc. Moreover, the defect in mitotic progression observed in the Mastl-null PGCs was rescued by simultaneous deletion of Ppp2r1a (α subunit of PP2A). Thus, our results demonstrate that MASTL, PP2A, and therefore regulated phosphatase activity have a fundamental role in establishing female germ cell population in gonads by controlling PGC proliferation during embryogenesis. PMID:28224044

Elevating the frequency of chromosome mis-segregation as a strategy to kill tumor cells

PubMed Central

Janssen, Aniek; Kops, Geert J. P. L.; Medema, René H.

2009-01-01

The mitotic checkpoint has evolved to prevent chromosome mis-segregations by delaying mitosis when unattached chromosomes are present. Inducing severe chromosome segregation errors by ablating the mitotic checkpoint causes cell death. Here we have analyzed the consequences of gradual increases in chromosome segregation errors on the viability of tumor cells and normal human fibroblasts. Partial reduction of essential mitotic checkpoint components in four tumor cell lines caused mild chromosome mis-segregations, but no lethality. These cells were, however, remarkably more sensitive to low doses of taxol, which enhanced the amount and severity of chromosome segregation errors. Sensitization to taxol was achieved by reducing levels of Mps1 or BubR1, proteins having dual roles in checkpoint activation and chromosome alignment, but not by reducing Mad2, functioning solely in the mitotic checkpoint. Moreover, we find that untransformed human fibroblasts with reduced Mps1 levels could not be sensitized to sublethal doses of taxol. Thus, targeting the mitotic checkpoint and chromosome alignment simultaneously may selectively kill tumor cells by enhancing chromosome mis-segregations. PMID:19855003

A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division

PubMed Central

Li, Yubing; Liu, Dianyi; López-Paz, Cristina; Olson, Bradley JSC; Umen, James G

2016-01-01

Proliferating cells actively control their size by mechanisms that are poorly understood. The unicellular green alga Chlamydomonas reinhardtii divides by multiple fission, wherein a ‘counting’ mechanism couples mother cell-size to cell division number allowing production of uniform-sized daughters. We identified a sizer protein, CDKG1, that acts through the retinoblastoma (RB) tumor suppressor pathway as a D-cyclin-dependent RB kinase to regulate mitotic counting. Loss of CDKG1 leads to fewer mitotic divisions and large daughters, while mis-expression of CDKG1 causes supernumerous mitotic divisions and small daughters. The concentration of nuclear-localized CDKG1 in pre-mitotic cells is set by mother cell size, and its progressive dilution and degradation with each round of cell division may provide a link between mother cell-size and mitotic division number. Cell-size-dependent accumulation of limiting cell cycle regulators such as CDKG1 is a potentially general mechanism for size control. DOI: http://dx.doi.org/10.7554/eLife.10767.001 PMID:27015111

Localization of phosphorylated forms of Bcl-2 in mitosis: co-localization with Ki-67 and nucleolin in nuclear structures and on mitotic chromosomes.

PubMed

Barboule, Nadia; Truchet, Isabelle; Valette, Annie

2005-04-01

Bcl-2 phosphorylation is a normal physiological process occurring at mitosis or during mitotic arrest induced by microtubule damaging agents. The consequences of Bcl-2 phosphorylation on its function are still controversial. To better understand the role of Bcl-2 phosphorylation in mitosis, we studied the subcellular localization of phosphorylated forms of Bcl-2. Immunofluorescence experiments performed in synchronized HeLa cells indicate for the first time that mitotic phosphorylated forms of Bcl-2 can be detected in nuclear structures in prophase cells together with nucleolin and Ki-67. In later mitotic stages, as previously described, phosphorylated forms of Bcl-2 are localized on mitotic chromosomes. In addition, we demonstrate that Bcl-2 in these structures is at least in part phosphorylated on the T56 residue. Then, coimmunoprecipitation experiments reveal that, in cells synchronized at the onset of mitosis, Bcl-2 is present in a complex with nucleolin, cdc2 kinase and PP1 phosphatase. Taken together, these data further support the idea that Bcl-2 could have a new function at mitosis.

Developmental alterations in centrosome integrity contribute to the post-mitotic state of mammalian cardiomyocytes

PubMed Central

Zebrowski, David C; Vergarajauregui, Silvia; Wu, Chi-Chung; Piatkowski, Tanja; Becker, Robert; Leone, Marina; Hirth, Sofia; Ricciardi, Filomena; Falk, Nathalie; Giessl, Andreas; Just, Steffen; Braun, Thomas; Weidinger, Gilbert; Engel, Felix B

2015-01-01

Mammalian cardiomyocytes become post-mitotic shortly after birth. Understanding how this occurs is highly relevant to cardiac regenerative therapy. Yet, how cardiomyocytes achieve and maintain a post-mitotic state is unknown. Here, we show that cardiomyocyte centrosome integrity is lost shortly after birth. This is coupled with relocalization of various centrosome proteins to the nuclear envelope. Consequently, postnatal cardiomyocytes are unable to undergo ciliogenesis and the nuclear envelope adopts the function as cellular microtubule organizing center. Loss of centrosome integrity is associated with, and can promote, cardiomyocyte G0/G1 cell cycle arrest suggesting that centrosome disassembly is developmentally utilized to achieve the post-mitotic state in mammalian cardiomyocytes. Adult cardiomyocytes of zebrafish and newt, which are able to proliferate, maintain centrosome integrity. Collectively, our data provide a novel mechanism underlying the post-mitotic state of mammalian cardiomyocytes as well as a potential explanation for why zebrafish and newts, but not mammals, can regenerate their heart. DOI: http://dx.doi.org/10.7554/eLife.05563.001 PMID:26247711

Breast cancer mitosis detection in histopathological images with spatial feature extraction

NASA Astrophysics Data System (ADS)

Albayrak, Abdülkadir; Bilgin, Gökhan

2013-12-01

In this work, cellular mitosis detection in histopathological images has been investigated. Mitosis detection is very expensive and time consuming process. Development of digital imaging in pathology has enabled reasonable and effective solution to this problem. Segmentation of digital images provides easier analysis of cell structures in histopathological data. To differentiate normal and mitotic cells in histopathological images, feature extraction step is very crucial step for the system accuracy. A mitotic cell has more distinctive textural dissimilarities than the other normal cells. Hence, it is important to incorporate spatial information in feature extraction or in post-processing steps. As a main part of this study, Haralick texture descriptor has been proposed with different spatial window sizes in RGB and La*b* color spaces. So, spatial dependencies of normal and mitotic cellular pixels can be evaluated within different pixel neighborhoods. Extracted features are compared with various sample sizes by Support Vector Machines using k-fold cross validation method. According to the represented results, it has been shown that separation accuracy on mitotic and non-mitotic cellular pixels gets better with the increasing size of spatial window.

Anti-mitotic potential of 7-diethylamino-3(2 Prime -benzoxazolyl)-coumarin in 5-fluorouracil-resistant human gastric cancer cell line SNU620/5-FU

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Nam Hyun; Kim, Su-Nam; Oh, Joa Sub

2012-02-24

Highlights: Black-Right-Pointing-Pointer DBC exerts antiproliferative potential against 5FU-resistant human gastric cancer cells. Black-Right-Pointing-Pointer This effect is mediated by destabilization of microtubules and subsequent mitotic arrest. Black-Right-Pointing-Pointer DBC enhances apoptosis via caspase activation and downregulation of antiapoptotic genes. -- Abstract: In this study, we investigate an anti-mitotic potential of the novel synthetic coumarin-based compound, 7-diethylamino-3(2 Prime -benzoxazolyl)-coumarin, in 5-fluorouracil-resistant human gastric cancer cell line SNU-620-5FU and its parental cell SNU-620. It exerts the anti-proliferative effects with similar potencies against both cancer cells, which is mediated by destabilization of microtubules and subsequent mitotic arrest. Furthermore, this compound enhances caspase-dependent apoptotic cell deathmore » via decreased expression of anti-apoptotic genes. Taken together, our data strongly support anti-mitotic potential of 7-diethylamino-3(2 Prime -benzoxazolyl)-coumarin against drug-resistant cancer cells which will prompt us to further develop as a novel microtubule inhibitor for drug-resistant cancer chemotherapy.« less

Mechanisms and Regulation of Mitotic Recombination in Saccharomyces cerevisiae

PubMed Central

Symington, Lorraine S.; Rothstein, Rodney; Lisby, Michael

2014-01-01

Homology-dependent exchange of genetic information between DNA molecules has a profound impact on the maintenance of genome integrity by facilitating error-free DNA repair, replication, and chromosome segregation during cell division as well as programmed cell developmental events. This chapter will focus on homologous mitotic recombination in budding yeast Saccharomyces cerevisiae. However, there is an important link between mitotic and meiotic recombination (covered in the forthcoming chapter by Hunter et al. 2015) and many of the functions are evolutionarily conserved. Here we will discuss several models that have been proposed to explain the mechanism of mitotic recombination, the genes and proteins involved in various pathways, the genetic and physical assays used to discover and study these genes, and the roles of many of these proteins inside the cell. PMID:25381364

Mto2 multisite phosphorylation inactivates non-spindle microtubule nucleation complexes during mitosis

PubMed Central

Borek, Weronika E.; Groocock, Lynda M.; Samejima, Itaru; Zou, Juan; de Lima Alves, Flavia; Rappsilber, Juri; Sawin, Kenneth E.

2015-01-01

Microtubule nucleation is highly regulated during the eukaryotic cell cycle, but the underlying molecular mechanisms are largely unknown. During mitosis in fission yeast Schizosaccharomyces pombe, cytoplasmic microtubule nucleation ceases simultaneously with intranuclear mitotic spindle assembly. Cytoplasmic nucleation depends on the Mto1/2 complex, which binds and activates the γ-tubulin complex and also recruits the γ-tubulin complex to both centrosomal (spindle pole body) and non-centrosomal sites. Here we show that the Mto1/2 complex disassembles during mitosis, coincident with hyperphosphorylation of Mto2 protein. By mapping and mutating multiple Mto2 phosphorylation sites, we generate mto2-phosphomutant strains with enhanced Mto1/2 complex stability, interaction with the γ-tubulin complex and microtubule nucleation activity. A mutant with 24 phosphorylation sites mutated to alanine, mto2[24A], retains interphase-like behaviour even in mitotic cells. This provides a molecular-level understanding of how phosphorylation ‘switches off' microtubule nucleation complexes during the cell cycle and, more broadly, illuminates mechanisms regulating non-centrosomal microtubule nucleation. PMID:26243668

Chronic centrosome amplification without tumorigenesis

PubMed Central

Vitre, Benjamin; Holland, Andrew J.; Kulukian, Anita; Shoshani, Ofer; Hirai, Maretoshi; Wang, Yin; Maldonado, Marcus; Cho, Thomas; Boubaker, Jihane; Swing, Deborah A.; Tessarollo, Lino; Evans, Sylvia M.; Fuchs, Elaine; Cleveland, Don W.

2015-01-01

Centrosomes are microtubule-organizing centers that facilitate bipolar mitotic spindle assembly and chromosome segregation. Recognizing that centrosome amplification is a common feature of aneuploid cancer cells, we tested whether supernumerary centrosomes are sufficient to drive tumor development. To do this, we constructed and analyzed mice in which centrosome amplification can be induced by a Cre-recombinase–mediated increase in expression of Polo-like kinase 4 (Plk4). Elevated Plk4 in mouse fibroblasts produced supernumerary centrosomes and enhanced the expected mitotic errors, but proliferation continued only after inactivation of the p53 tumor suppressor. Increasing Plk4 levels in mice with functional p53 produced centrosome amplification in liver and skin, but this did not promote spontaneous tumor development in these tissues or enhance the growth of chemically induced skin tumors. In the absence of p53, Plk4 overexpression generated widespread centrosome amplification, but did not drive additional tumors or affect development of the fatal thymic lymphomas that arise in animals lacking p53. We conclude that, independent of p53 status, supernumerary centrosomes are not sufficient to drive tumor formation. PMID:26578792

Asymmetric Distribution of GFAP in Glioma Multipotent Cells

PubMed Central

Guichet, Pierre-Olivier; Guelfi, Sophie; Ripoll, Chantal; Teigell, Marisa; Sabourin, Jean-Charles; Bauchet, Luc; Rigau, Valérie; Rothhut, Bernard; Hugnot, Jean-Philippe

2016-01-01

Asymmetric division (AD) is a fundamental mechanism whereby unequal inheritance of various cellular compounds during mitosis generates unequal fate in the two daughter cells. Unequal repartitions of transcription factors, receptors as well as mRNA have been abundantly described in AD. In contrast, the involvement of intermediate filaments in this process is still largely unknown. AD occurs in stem cells during development but was also recently observed in cancer stem cells. Here, we demonstrate the asymmetric distribution of the main astrocytic intermediate filament, namely the glial fibrillary acid protein (GFAP), in mitotic glioma multipotent cells isolated from glioblastoma (GBM), the most frequent type of brain tumor. Unequal mitotic repartition of GFAP was also observed in mice non-tumoral neural stem cells indicating that this process occurs across species and is not restricted to cancerous cells. Immunofluorescence and videomicroscopy were used to capture these rare and transient events. Considering the role of intermediate filaments in cytoplasm organization and cell signaling, we propose that asymmetric distribution of GFAP could possibly participate in the regulation of normal and cancerous neural stem cell fate. PMID:26953813

Systems cell biology of the mitotic spindle.

PubMed

Saleem, Ramsey A; Aitchison, John D

2010-01-11

Cell division depends critically on the temporally controlled assembly of mitotic spindles, which are responsible for the distribution of duplicated chromosomes to each of the two daughter cells. To gain insight into the process, Vizeacoumar et al., in this issue (Vizeacoumar et al. 2010. J. Cell Biol. doi:10.1083/jcb.200909013), have combined systems genetics with high-throughput and high-content imaging to comprehensively identify and classify novel components that contribute to the morphology and function of the mitotic spindle.

Mitotic Recombination in the Heterochromatin of the Sex Chromosomes of DROSOPHILA MELANOGASTER

PubMed Central

Ripoll, P.; Garcia-Bellido, A.

1978-01-01

The frequency of spontaneous and X-ray-induced mitotic recombination involving the Y chromosome has been studied in individuals with a marked Y chromosome arm and different XY compound chromosomes. The genotypes used include X chromosomes with different amounts of X heterochromatin and either or both arms of the Y chromosome attached to either side of the centromere. Individuals with two Y chromosomes have also been studied. The results show that the bulk of mitotic recombination takes place between homologous regions. PMID:100372

Effects of polyamines and polyamine biosynthetic inhibitors on mitotic activity of Allium cepa root tips.

PubMed

Unal, Meral; Palavan-Unsal, Narcin; Tufekci, M A

2008-03-01

The genotoxic and cytotoxic effects of exogenous polyamines (PAs), putrescine (Put), spermidine (Spd), spermine (Spm) and PA biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO), cyclohexilamine (CHA), methylglioxal bis-(guanylhydrazone) (MGBG) were investigated in the root meristems of Allium cepa L. The reduction of mitotic index and the induction of chromosomal aberrations such as bridges, stickiness, c-mitotic anaphases, micronuclei, endoredupliction by PAs and PA biosynthetic inhibitors were observed and these were used as evidence of genotoxicity and cytotoxicity.

Mitotic and apoptotic activity in colorectal neoplasia.

PubMed

Kohoutova, Darina; Pejchal, Jaroslav; Bures, Jan

2018-05-18

Colorectal cancer (CRC) is third most commonly diagnosed cancer worldwide. The aim of the prospective study was to evaluate mitosis and apoptosis of epithelial cells at each stage of colorectal neoplasia. A total of 61 persons were enrolled into the study: 18 patients with non-advanced colorectal adenoma (non-a-A), 13 patients with advanced colorectal adenoma (a-A), 13 patients with CRC and 17 controls: individuals with normal findings on colonoscopy. Biopsy samples were taken from pathology (patients) and healthy mucosa (patients and healthy controls). Samples were formalin-fixed paraffin-embedded and stained with haematoxylin-eosin. Mitotic and apoptotic activity were evaluated in lower and upper part of the crypts and in the superficial compartment. Apoptotic activity was also assessed using detection of activated caspase-3. In controls, mitotic activity was present in lower part of crypts, accompanied with low apoptotic activity. Mitotic and apoptotic activity decreased (to almost zero) in upper part of crypts. In superficial compartment, increase in apoptotic activity was observed. Transformation of healthy mucosa into non-a-A was associated with significant increase of mitotic activity in lower and upper part of the crypts and with significant increase of apoptotic activity in all three compartments; p < 0.05. Transformation of non-a-A into a-A did not lead to any further significant increase in apoptotic activity, but was related to significant increase in mitotic activity in upper part of crypts and superficial compartment. A significant decrease in apoptotic activity was detected in all three comparments of CRC samples compared to a-A; p < 0.05. No differences in mitotic and apoptotic activity between biopsies in healthy controls and biopsy samples from healthy mucosa in patients with colorectal neoplasia were observed. Detection of activated caspase-3 confirmed the above findings in apoptotic activity. Significant dysregulation of mitosis and apoptosis during the progression of colorectal neoplasia, corresponding with histology, was confirmed. In patients with sporadic colorectal neoplasia, healthy mucosa does not display different mitotic and apoptotic activity compared to mucosa in healthy controls and therefore adequate endoscopic/surgical removal of colorectal neoplasia is sufficient.

Inference of genetic network of Xenopus frog egg: improved genetic algorithm.

PubMed

Wu, Shinq-Jen; Chou, Chia-Hsien; Wu, Cheng-Tao; Lee, Tsu-Tian

2006-01-01

An improved genetic algorithm (IGA) is proposed to achieve S-system gene network modeling of Xenopus frog egg. Via the time-courses training datasets from Michaelis-Menten model, the optimal parameters are learned. The S-system can clearly describe activative and inhibitory interaction between genes as generating and consuming process. We concern the mitotic control in cell-cycle of Xenopus frog egg to realize cyclin-Cdc2 and Cdc25 for MPF activity. The proposed IGA can achieve global search with migration and keep the best chromosome with elitism operation. The generated gene regulatory networks can provide biological researchers for further experiments in Xenopus frog egg cell cycle control.

Evaluation of allelopathic, decomposition and cytogenetic activities of Jasminum officinale L. f. var. grandiflorum (L.) Kob. on bioassay plants.

PubMed

Teerarak, Montinee; Laosinwattana, Chamroon; Charoenying, Patchanee

2010-07-01

Methanolic extracts prepared from dried leaves of Jasminum officinale f. var. grandiflorum (L.) Kob. (Spanish jasmine) inhibited seed germination and stunted both root and shoot length of the weeds Echinochloa crus-galli (L.) Beauv. and Phaseolus lathyroides L. The main active compound was isolated and determined by spectral data as a secoiridoid glucoside named oleuropein. In addition, a decrease in allelopathic efficacy appeared as the decomposition periods increased. The mitotic index in treated onion root tips decreased with increasing concentrations of the extracts and longer periods of treatment. Likewise, the mitotic phase index was altered in onion incubated with crude extract. Furthermore, crude extract produced mitotic abnormalities resulting from its action on chromatin organization and mitotic spindle. Copyright (c)2010 Elsevier Ltd. All rights reserved.

The magic of numbers: malignant melanoma between science and pseudoscience.

PubMed

Weyers, Wolfgang

2011-06-01

In 2009, a new system for staging and classification of malignant melanoma has been proposed by the American Joint Committee on Cancer (AJCC). The AJCC recommends that staging of primary melanoma be based on 3 criteria, namely, thickness, ulceration, and mitotic rate, the latter substituting Clark levels in the previous classification. In melanomas measuring ≤1 mm in thickness, ulceration or finding of single mitotic figure in the dermis defines stage T1b. According to the AJCC, sentinel lymph node dissection should be considered for those melanomas because of a significantly impaired prognosis. As with other prognostic parameters, however, assessment of mitotic rate, with one mitotic figure being the cutoff point, is highly unreliable, and statistics based on such data lack validity. Despite the large database being employed, they may be pseudoscience rather than science.

Effects of intracellular pH on the mitotic apparatus and mitotic stage in the sand dollar egg.

PubMed

Watanabe, K; Hamaguchi, M S; Hamaguchi, Y

1997-01-01

The effect of change in intracellular pH (pHi) on mitosis was investigated in the sand dollar egg. The pHi in the fertilized egg of Scaphechinus mirabilis and Clypeaster japonicus, which was 7.34 and 7.31, respectively, changed by means of treating the egg at nuclear envelope breakdown with sea water containing acetate and/or ammonia at various values of pH. The mitotic apparatus at pHi 6.70 became larger than that of normal fertilized eggs; that is, the mitotic spindle had the maximal size, especially in length at pHi 6.70. The spindle length linearly decreased when pHi increased from 6.70 to 7.84. By polarization microscopy, the increase in birefringence retardation was detected at slightly acidic pHi, suggesting that the increase in size of the spindle is caused by the increase in the amount of microtubules in the spindle. At pHi 6.30, the organization of the mitotic apparatus was inhibited. Furthermore, slightly acidic pHi caused cleavage retardation or inhibition. By counting the number of the eggs at various mitotic stages with time after treating them with the media, it is found that metaphase was persistent and most of the S. mirabilis eggs were arrested at metaphase under the condition of pHi 6.70. It is concluded that at slightly acidic pH, the microtubules in the spindle are stabilized and more microtubules assembled than those in the normal eggs.

The histone acetyltransferase component TRRAP is targeted for destruction during the cell cycle.

PubMed

Ichim, G; Mola, M; Finkbeiner, M G; Cros, M-P; Herceg, Z; Hernandez-Vargas, H

2014-01-09

Chromosomes are dynamic structures that must be reversibly condensed and unfolded to accommodate mitotic division and chromosome segregation. Histone modifications are involved in the striking chromatin reconfiguration taking place during mitosis. However, the mechanisms that regulate activity and function of histone-modifying factors as cells enter and exit mitosis are poorly understood. Here, we show that the anaphase-promoting complex or cyclosome (APC/C) is involved in the mitotic turnover of TRRAP (TRansformation/tRanscription domain-Associated Protein), a common component of histone acetyltransferase (HAT) complexes, and that the pre-mitotic degradation of TRRAP is mediated by the APC/C ubiquitin ligase activators Cdc20 and Cdh1. Ectopic expression of both Cdh1 and Cdc20 reduced the levels of coexpressed TRRAP protein and induced its ubiquitination. TRRAP overexpression or stabilization induces multiple mitotic defects, including lagging chromosomes, chromosome bridges and multipolar spindles. In addition, lack of sister chromatid cohesion and impaired chromosome condensation were found after TRRAP overexpression or stabilization. By using a truncated form of TRRAP, we show that mitotic delay is associated with a global histone H4 hyperacetylation induced by TRRAP overexpression. These results demonstrate that the chromatin modifier TRRAP is targeted for destruction in a cell cycle-dependent fashion. They also suggest that degradation of TRRAP by the APC/C is necessary for a proper condensation of chromatin and proper chromosome segregation. Chromatin compaction mediated by histone modifiers may represent a fundamental arm for APC/C orchestration of the mitotic machinery.

Modulation of vinblastine cytotoxicity by dilantin (phenytoin) or the protein phosphatase inhibitor okadaic acid involves the potentiation of anti-mitotic effects and induction of apoptosis in human tumour cells.

PubMed Central

Kawamura, K. I.; Grabowski, D.; Weizer, K.; Bukowski, R.; Ganapathi, R.

1996-01-01

Cellular insensitivity to vinca alkaloids is suggested to be primarily due to drug efflux by P-glycoprotein (P-gp). The anti-epileptic phenytoin (DPH), which does not bind to P-gp, can selectively enhance vincristine (VCR) cytotoxicity in wild-type (WT) or multidrug-resistant (MDR) cells. We now demonstrate that the protein phosphatase inhibitor okadaic acid (OKA) can mimic the effect of DPH by selectively enhancing cytotoxicity of vinblastine (VBL), but not taxol and doxorubicin, in human leukaemia HL-60 cells. Both DPH and OKA potentiate the anti-mitotic effects of VBL by enhanced damage to the mitotic spindle, resulting in prolonged growth arrest. Also, unlike VBL alone, in human leukaemia or non-small-cell lung carcinoma cells treated with VBL plus DPH, recovery from damage to the mitotic spindle is compromised in drug-free medium and cell death by apoptosis in interphase ensues. Since protein phosphatases are involved with the regulation of metaphase to anaphase transit of cells during the mitotic cycle, enhanced VBL cytotoxicity in the presence of DPH or OKA may involve effects during metaphase on the mitotic spindle tubulin leading to growth arrest and apoptosis in interphase. These novel results suggest that DPH or OKA could be powerful tools to study cellular effects of vinca alkaloids and possibly for the development of novel therapeutic strategies. Images Figure 6 PMID:8546904

Molecular Memory of Morphologies by Septins during Neuron Generation Allows Early Polarity Inheritance.

PubMed

Boubakar, Leila; Falk, Julien; Ducuing, Hugo; Thoinet, Karine; Reynaud, Florie; Derrington, Edmund; Castellani, Valérie

2017-08-16

Transmission of polarity established early during cell lineage history is emerging as a key process guiding cell differentiation. Highly polarized neurons provide a fascinating model to study inheritance of polarity over cell generations and across morphological transitions. Neural crest cells (NCCs) migrate to the dorsal root ganglia to generate neurons directly or after cell divisions in situ. Using live imaging of vertebrate embryo slices, we found that bipolar NCC progenitors lose their polarity, retracting their processes to round for division, but generate neurons with bipolar morphology by emitting processes from the same locations as the progenitor. Monitoring the dynamics of Septins, which play key roles in yeast polarity, indicates that Septin 7 tags process sites for re-initiation of process growth following mitosis. Interfering with Septins blocks this mechanism. Thus, Septins store polarity features during mitotic rounding so that daughters can reconstitute the initial progenitor polarity. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanical design principles of a mitotic spindle.

PubMed

Ward, Jonathan J; Roque, Hélio; Antony, Claude; Nédélec, François

2014-12-18

An organised spindle is crucial to the fidelity of chromosome segregation, but the relationship between spindle structure and function is not well understood in any cell type. The anaphase B spindle in fission yeast has a slender morphology and must elongate against compressive forces. This 'pushing' mode of chromosome transport renders the spindle susceptible to breakage, as observed in cells with a variety of defects. Here we perform electron tomographic analyses of the spindle, which suggest that it organises a limited supply of structural components to increase its compressive strength. Structural integrity is maintained throughout the spindle's fourfold elongation by organising microtubules into a rigid transverse array, preserving correct microtubule number and dynamically rescaling microtubule length.

Impact of the 2009 AJCC staging guidelines for melanoma on the number of mitotic figures reported by dermatopathologists at one institution.

PubMed

Larson, Allison R; Rothschild, Brian; Walls, Andrew C; Granter, Scott R; Qureshi, Abrar A; Murphy, George F; Laga, Alvaro C

2015-08-01

In 2009 the revised seventh staging system for melanoma recommended the use of mitotic count to separate stage T1a from T1b. However, careful scrutiny of cases may lead to an inadvertent selection effect, with consequent increased reporting of mitotic counts. We investigated whether there is a significant increase in mitotic counts reported since 2009 for melanomas with a Breslow thickness of 1.0 mm or less. We conducted a retrospective, case-controlled study examining invasive melanoma cases at a large academic center. Mitotic counts were compared between pathology reports before 2009 (n = 61) and after 2009 (n = 125), with a subset of slides re-examined in a blinded fashion. Before the 2009 staging guidelines, 51% of cases had one or more mitosis reported compared to 38% after 2009 (p = 0.113). Blinded re-counting did not yield a significant difference when compared with the original pathology reports in either group. There was not a significant difference in the number of mitoses reported after the implementation of the new guidelines. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Interkinetic nuclear migration and basal tethering facilitates post-mitotic daughter separation in intestinal organoids

PubMed Central

Carroll, Thomas D.; Langlands, Alistair J.; Osborne, James M.; Newton, Ian P.; Appleton, Paul L.

2017-01-01

ABSTRACT Homeostasis of renewing tissues requires balanced proliferation, differentiation and movement. This is particularly important in the intestinal epithelium where lineage tracing suggests that stochastic differentiation choices are intricately coupled to the position of a cell relative to a niche. To determine how position is achieved, we followed proliferating cells in intestinal organoids and discovered that the behaviour of mitotic sisters predicted long-term positioning. We found that, normally, 70% of sisters remain neighbours, while 30% lose contact and separate after cytokinesis. These post-mitotic placements predict longer term differences in positions assumed by sisters: adjacent sisters reach similar positions over time; in a pair of separating sisters, one remains close to its birthplace while the other is displaced upward. Computationally modelling crypt dynamics confirmed that post-mitotic separation leads to sisters reaching different compartments. We show that interkinetic nuclear migration, cell size and asymmetric tethering by a process extending from the basal side of cells contribute to separations. These processes are altered in adenomatous polyposis coli (Apc) mutant epithelia where separation is lost. We conclude that post-mitotic placement contributes to stochastic niche exit and, when defective, supports the clonal expansion of Apc mutant cells. PMID:28982714

Clinicopathological relevance of tumour grading in canine osteosarcoma.

PubMed

Loukopoulos, P; Robinson, W F

2007-01-01

Tumour grading assesses biological aggressiveness and is of prognostic significance in many malignancies. The clinicopathological features of 140 primary canine osteosarcomas and their metastases were analysed, and the interrelations between them and an established grading system and its constituent parameters (mitotic index, necrosis, pleomorphism) were examined. Of these tumours, 35% were grade III (high-grade), 37% grade II and 28% grade I. Primary tumours that had metastasized were of significantly higher grade than non-metastatic osteosarcomas. Osteosarcomas belonging to the osteoblastic minimally productive subtype, but not chondroblastic or telangiectatic subtypes, differed from fibroblastic osteosarcomas in being associated with a significantly higher number of high-grade cases. Dogs younger than 4 years of age had osteosarcomas with higher grade, score and mitotic index than did older animals. Appendicular differed from axial tumours in having a higher mitotic index; distal differed from proximal tumours in being of higher grade; cranial tumours differed from tumours in most other sites in being of lower grade and lower mitotic index. Rib osteosarcomas showed a particularly high degree of necrosis. The mitotic index varied widely between tumour locations. Pleomorphism did not have prognostic merit when examined separately, as most osteosarcomas were highly pleomorphic.

Defects in chromosome congression and mitotic progression in KIF18A-deficient cells are partly mediated through impaired functions of CENP-E.

PubMed

Huang, Ying; Yao, Yixin; Xu, Han-Zhang; Wang, Zhu-Gang; Lu, Luo; Dai, Wei

2009-08-15

KIF18A, a molecular motor, is an essential component in the regulation of orderly chromosome congression by attenuation of the kinetochore oscillation amplitude at the midzone during mitosis in vertebrate cells. Here we report that KIF18A depletion resulted in mitotic arrest which was accompanied by the presence of unaligned chromosomes in HeLa cells. This resembles the phenotype induced by an impaired function of CENP-E, also a mitotic kinesin essential for the formation of the mitotic spindles. Our further analysis showed that KIF18A depletion caused specific downregulation of CENP-E. Downregulation of CENP-E as the result of KIF18A silencing was not due to reduced transcription but primarily due to the enhanced protein degradation. Co-immunoprecipitation revealed that KIF18A physically interacted with CENP-E and BubR1 during mitosis. Ectopic expression of the wild-type tail domain of CENP-E, but not a corresponding mutant, significantly suppressed chromosome congression defects in mitotic cells. Together, our studies strongly suggest that chromosome congression defects as the result of KIF18A depletion is at least in part mediated through destabilizing kinetochore CENP-E.

Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Feng; Camp, David G.; Gritsenko, Marina A.

2007-11-16

The chromosomal passenger complex (CPC) is a critical regulator of chromosome, cytoskeleton and membrane dynamics during mitosis. Here, we identified phosphopeptides and phosphoprotein complexes recognized by a phosphorylation specific antibody that labels the CPC using liquid chromatography coupled to mass spectrometry. A mitotic phosphorylation motif (PX{G/T/S}{L/M}[pS]P or WGL[pS]P) was identified in 11 proteins including Fzr/Cdh1 and RIC-8, two proteins with potential links to the CPC. Phosphoprotein complexes contained known CPC components INCENP, Aurora-B and TD-60, as well as SMAD2, 14-3-3 proteins, PP2A, and Cdk1, a likely kinase for this motif. Protein sequence analysis identified phosphorylation motifs in additional proteins includingmore » SMAD2, Plk3 and INCENP. Mitotic SMAD2 and Plk3 phosphorylation was confirmed using phosphorylation specific antibodies, and in the case of Plk3, phosphorylation correlates with its localization to the mitotic apparatus. A mutagenesis approach was used to show INCENP phosphorylation is required for midbody localization. These results provide evidence for a shared phosphorylation event that regulates localization of critical proteins during mitosis.« less

The Sin3p PAH Domains Provide Separate Functions Repressing Meiotic Gene Transcription in Saccharomyces cerevisiae ▿

PubMed Central

Mallory, Michael J.; Law, Michael J.; Buckingham, Lela E.; Strich, Randy

2010-01-01

Meiotic genes in budding yeast are repressed during vegetative growth but are transiently induced during specific stages of meiosis. Sin3p represses the early meiotic gene (EMG) by bridging the DNA binding protein Ume6p to the histone deacetylase Rpd3p. Sin3p contains four paired amphipathic helix (PAH) domains, one of which (PAH3) is required for repressing several genes expressed during mitotic cell division. This report examines the roles of the PAH domains in mediating EMG repression during mitotic cell division and following meiotic induction. PAH2 and PAH3 are required for mitotic EMG repression, while electrophoretic mobility shift assays indicate that only PAH2 is required for stable Ume6p-promoter interaction. Unlike mitotic repression, reestablishing EMG repression following transient meiotic induction requires PAH3 and PAH4. In addition, the role of Sin3p in reestablishing repression is expanded to include additional loci that it does not control during vegetative growth. These findings indicate that mitotic and postinduction EMG repressions are mediated by two separate systems that utilize different Sin3p domains. PMID:20971827

Real-time fluorescence imaging of the DNA damage repair response during mitosis.

PubMed

Miwa, Shinji; Yano, Shuya; Yamamoto, Mako; Matsumoto, Yasunori; Uehara, Fuminari; Hiroshima, Yukihiko; Toneri, Makoto; Murakami, Takashi; Kimura, Hiroaki; Hayashi, Katsuhiro; Yamamoto, Norio; Efimova, Elena V; Tsuchiya, Hiroyuki; Hoffman, Robert M

2015-04-01

The response to DNA damage during mitosis was visualized using real-time fluorescence imaging of focus formation by the DNA-damage repair (DDR) response protein 53BP1 linked to green fluorescent protein (GFP) (53BP1-GFP) in the MiaPaCa-2(Tet-On) pancreatic cancer cell line. To observe 53BP1-GFP foci during mitosis, MiaPaCa-2(Tet-On) 53BP1-GFP cells were imaged every 30 min by confocal microscopy. Time-lapse imaging demonstrated that 11.4 ± 2.1% of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells had increased focus formation over time. Non-mitotic cells did not have an increase in 53BP1-GFP focus formation over time. Some of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells with focus formation became apoptotic. The results of the present report suggest that DNA strand breaks occur during mitosis and undergo repair, which may cause some of the mitotic cells to enter apoptosis in a phenomenon possibly related to mitotic catastrophe. © 2014 Wiley Periodicals, Inc.

Mediator can regulate mitotic entry and direct periodic transcription in fission yeast.

PubMed

Banyai, Gabor; Lopez, Marcela Davila; Szilagyi, Zsolt; Gustafsson, Claes M

2014-11-01

Cdk8 is required for correct timing of mitotic progression in fission yeast. How the activity of Cdk8 is regulated is unclear, since the kinase is not activated by T-loop phosphorylation and its partner, CycC, does not oscillate. Cdk8 is, however, a component of the multiprotein Mediator complex, a conserved coregulator of eukaryotic transcription that is connected to a number of intracellular signaling pathways. We demonstrate here that other Mediator components regulate the activity of Cdk8 in vivo and thereby direct the timing of mitotic entry. Deletion of Mediator components Med12 and Med13 leads to higher cellular Cdk8 protein levels, premature phosphorylation of the Cdk8 target Fkh2, and earlier entry into mitosis. We also demonstrate that Mediator is recruited to clusters of mitotic genes in a periodic fashion and that the complex is required for the transcription of these genes. We suggest that Mediator functions as a hub for coordinated regulation of mitotic progression and cell cycle-dependent transcription. The many signaling pathways and activator proteins shown to function via Mediator may influence the timing of these cell cycle events. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase-2

PubMed Central

Andersen, Joshua L; Johnson, Carrie E; Freel, Christopher D; Parrish, Amanda B; Day, Jennifer L; Buchakjian, Marisa R; Nutt, Leta K; Thompson, J Will; Moseley, M Arthur; Kornbluth, Sally

2009-01-01

The apoptotic initiator caspase-2 has been implicated in oocyte death, in DNA damage- and heat shock-induced death, and in mitotic catastrophe. We show here that the mitosis-promoting kinase, cdk1–cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase-2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase-2 interdomain, prevents caspase-2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase-2 detected during interphase was lost in mitosis. Expression of S340A non-phosphorylatable caspase-2 abrogated mitotic suppression of caspase-2 and apoptosis in various settings, including oocytes induced to undergo cdk1-dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase-2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase-2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase-2 and suggest that under conditions of mitotic arrest, cdk1–cyclin B1 activity must be overcome for apoptosis to occur. PMID:19730412

Greatwall is essential to prevent mitotic collapse after nuclear envelope breakdown in mammals.

PubMed

Álvarez-Fernández, Mónica; Sánchez-Martínez, Ruth; Sanz-Castillo, Belén; Gan, Pei Pei; Sanz-Flores, María; Trakala, Marianna; Ruiz-Torres, Miguel; Lorca, Thierry; Castro, Anna; Malumbres, Marcos

2013-10-22

Greatwall is a protein kinase involved in the inhibition of protein phosphatase 2 (PP2A)-B55 complexes to maintain the mitotic state. Although its biochemical activity has been deeply characterized in Xenopus, its specific relevance during the progression of mitosis is not fully understood. By using a conditional knockout of the mouse ortholog, Mastl, we show here that mammalian Greatwall is essential for mouse embryonic development and cell cycle progression. Yet, Greatwall-null cells enter into mitosis with normal kinetics. However, these cells display mitotic collapse after nuclear envelope breakdown (NEB) characterized by defective chromosome condensation and prometaphase arrest. Intriguingly, Greatwall is exported from the nucleus to the cytoplasm in a CRM1-dependent manner before NEB. This export occurs after the nuclear import of cyclin B-Cdk1 complexes, requires the kinase activity of Greatwall, and is mediated by Cdk-, but not Polo-like kinase 1-dependent phosphorylation. The mitotic collapse observed in Greatwall-deficient cells is partially rescued after concomitant depletion of B55 regulatory subunits, which are mostly cytoplasmic before NEB. These data suggest that Greatwall is an essential protein in mammals required to prevent mitotic collapse after NEB.

Impaired mitotic progression and preimplantation lethality in mice lacking OMCG1, a new evolutionarily conserved nuclear protein.

PubMed

Artus, Jérôme; Vandormael-Pournin, Sandrine; Frödin, Morten; Nacerddine, Karim; Babinet, Charles; Cohen-Tannoudji, Michel

2005-07-01

While highly conserved through evolution, the cell cycle has been extensively modified to adapt to new developmental programs. Recently, analyses of mouse mutants revealed that several important cell cycle regulators are either dispensable for development or have a tissue- or cell-type-specific function, indicating that many aspects of cell cycle regulation during mammalian embryo development remain to be elucidated. Here, we report on the characterization of a new gene, Omcg1, which codes for a nuclear zinc finger protein. Embryos lacking Omcg1 die by the end of preimplantation development. In vitro cultured Omcg1-null blastocysts exhibit a dramatic reduction in the total cell number, a high mitotic index, and the presence of abnormal mitotic figures. Importantly, we found that Omcg1 disruption results in the lengthening of M phase rather than in a mitotic block. We show that the mitotic delay in Omcg1-/- embryos is associated with neither a dysfunction of the spindle checkpoint nor abnormal global histone modifications. Taken together, these results suggest that Omcg1 is an important regulator of the cell cycle in the preimplantation embryo.

Impaired Mitotic Progression and Preimplantation Lethality in Mice Lacking OMCG1, a New Evolutionarily Conserved Nuclear Protein†

PubMed Central

Artus, Jérôme; Vandormael-Pournin, Sandrine; Frödin, Morten; Nacerddine, Karim; Babinet, Charles; Cohen-Tannoudji, Michel

2005-01-01

While highly conserved through evolution, the cell cycle has been extensively modified to adapt to new developmental programs. Recently, analyses of mouse mutants revealed that several important cell cycle regulators are either dispensable for development or have a tissue- or cell-type-specific function, indicating that many aspects of cell cycle regulation during mammalian embryo development remain to be elucidated. Here, we report on the characterization of a new gene, Omcg1, which codes for a nuclear zinc finger protein. Embryos lacking Omcg1 die by the end of preimplantation development. In vitro cultured Omcg1-null blastocysts exhibit a dramatic reduction in the total cell number, a high mitotic index, and the presence of abnormal mitotic figures. Importantly, we found that Omcg1 disruption results in the lengthening of M phase rather than in a mitotic block. We show that the mitotic delay in Omcg1−/− embryos is associated with neither a dysfunction of the spindle checkpoint nor abnormal global histone modifications. Taken together, these results suggest that Omcg1 is an important regulator of the cell cycle in the preimplantation embryo. PMID:15988037

Ultrastructural study on dynamics of plastids and mitochondria during microgametogenesis in watermelon.

PubMed

Liu, Lin

2012-02-01

Dynamics of plastids and mitochondria during microgametogenesis in watermelon were examined by means of transmission electron microscopy. Plastids are present as proplastids in the microspore and as amyloplasts in the vegetative cell of the bicellular pollen grain, whereas the generative cell is completely devoid of plastids, suggesting that microspore plastids are excluded from the generative cell during the microspore mitotic division. Therefore, watermelon is classified as Lycopersicon type, where plastids exclusion from the generative cell leads to purely maternal plastid inheritance. Mitochondria in the generative cell show noticeable alterations in size and cristae during microgametogenesis. The diameter of mitochondria is about 0.5 μm in the newly born generative cell, while only about 0.16 μm in the spindle-shaped generative cell. Numerous cristae are present in mitochondria in the spherical generative cell, but, in contrast, mere two or three cristae retain in the spindle-shaped generative cell in the mature pollen grain. In conclusion, the size and cristae number of mitochondria in the generative cell are reduced significantly during microgametogenesis in watermelon. Copyright © 2011 Elsevier Ltd. All rights reserved.

Value of counting positive PHH3 cells in the diagnosis of uterine smooth muscle tumors

PubMed Central

Pang, Shu-Jie; Li, Cheng-Cheng; Shen, Yan; Liu, Yian-Zhu; Shi, Yi-Quan; Liu, Yi-Xin

2015-01-01

The diagnosis of uterine smooth muscle tumors including leiomyosarcomas (LMS), smooth muscle tumors of uncertain malignant potential (STUMP), bizarre (atypical) leiomyoma (BLM), mitotically active leiomyoma (MAL) and leiomyoma (LM) depends on a combination of microscopic features, such as mitoses, cytologic atypia, and coagulative tumor cell necrosis. However, a small number of these tumors still pose difficult diagnostic challenges. The assessment of accurate mitotic figures (MF) is one of the major parameters in the proper classification of uterine smooth muscle tumors. This assessment can be hampered by the presence of increased number of apoptotic bodies or pyknotic nuclei, which frequently mimic mitoses. Phospho-histone H3 (PHH3) is a recently described immunomarker specific for cells undergoing mitoses. In our study, we collected 132 cases of uterine smooth muscle tumors, including 26 LMSs, 16 STUMPs, 30 BLMs, 30 MALs and 30 LMs. We used mitosis specific marker PHH3 to count mitotic indexes (MI) of uterine smooth muscle tumors and compared with the mitotic indexes of hematoxylin and eosin (H&E). There is a positive correlation with the number of mitotic figures in H&E-stained sections and PHH3-stained sections (r=0.944, P<0.05). The ratio of PHH3-MI to H&E-MI has no statistically significant difference in each group except for LMs (P>0.05). The counting value of PHH3 in LMSs have significantly higher than STUMPs, BLMs, MALs and LMs (P<0.001) and the counting value of PHH3 is 1.5±0.5 times of the number of mitotic indexes in H&E. To conclude, our results show that counting PHH3 is a useful index in the diagnosis of uterine smooth muscle tumors and it can provide a more accurate index instead of the time-honored mitotic figure counts at a certain ratio. PMID:26191133

Genetic effects of methyl benzimidazole-2-yl-carbamate on Saccharomyces cerevisiae.

PubMed Central

Wood, J S

1982-01-01

The genetic effects of the mitotic inhibitor methyl benzimidazole-2-yl-carbamate (MBC) have been studied in Saccharomyces cerevisiae. MBC had little or no effect on the frequency of mutation. In some experiments MBC caused an increase in the frequency of mitotic recombination; however, this effect was small and not reproducible. The primary genetic effect of MBC was to induce mitotic chromosome loss at a high frequency. Chromosome loss occurred at equal frequencies for all chromosomes tested (13 of 16). Cells which had lost multiple chromosomes were found more frequently than predicted if individual chromosome loss events were independent. The probability of loss for a particular chromosome increased with length of time cells were incubated with MBC. MBC treatment also increased the frequency at which polyploid cells were found. These results suggested that MBC acted to disrupt the structure or function of the mitotic spindle and cause chromosome nondisjunction. PMID:6757720

EGF Induced Centrosome Separation Promotes Mitotic Progression and Cell Survival

PubMed Central

Mardin, Balca R.; Isokane, Mayumi; Cosenza, Marco R.; Krämer, Alwin; Ellenberg, Jan; Fry, Andrew M.; Schiebel, Elmar

2014-01-01

Summary Timely and accurate assembly of the mitotic spindle is critical for the faithful segregation of chromosomes and centrosome separation is a key step in this process. The timing of centrosome separation varies dramatically between cell types; however, the mechanisms responsible for these differences and its significance are unclear. Here, we show that activation of epidermal growth factor receptor (EGFR) signaling determines the timing of centrosome separation. Premature separation of centrosomes decreases the requirement for the major mitotic kinesin Eg5 for spindle assembly, accelerates mitosis and decreases the rate of chromosome missegregation. Importantly, EGF stimulation impacts upon centrosome separation and mitotic progression to different degrees in different cell lines. Cells with high EGFR levels fail to arrest in mitosis upon Eg5 inhibition. This has important implications for cancer therapy since cells with high centrosomal response to EGF are more susceptible to combinatorial inhibition of EGFR and Eg5. PMID:23643362

A nontranscriptional role for Oct4 in the regulation of mitotic entry

PubMed Central

Zhao, Rui; Deibler, Richard W.; Lerou, Paul H.; Ballabeni, Andrea; Heffner, Garrett C.; Cahan, Patrick; Unternaehrer, Juli J.; Kirschner, Marc W.; Daley, George Q.

2014-01-01

Rapid progression through the cell cycle and a very short G1 phase are defining characteristics of embryonic stem cells. This distinct cell cycle is driven by a positive feedback loop involving Rb inactivation and reduced oscillations of cyclins and cyclin-dependent kinase (Cdk) activity. In this setting, we inquired how ES cells avoid the potentially deleterious consequences of premature mitotic entry. We found that the pluripotency transcription factor Oct4 (octamer-binding transcription factor 4) plays an unappreciated role in the ES cell cycle by forming a complex with cyclin–Cdk1 and inhibiting Cdk1 activation. Ectopic expression of Oct4 or a mutant lacking transcriptional activity recapitulated delayed mitotic entry in HeLa cells. Reduction of Oct4 levels in ES cells accelerated G2 progression, which led to increased chromosomal missegregation and apoptosis. Our data demonstrate an unexpected nontranscriptional function of Oct4 in the regulation of mitotic entry. PMID:25324523

Disappearance of nucleosome positioning in mitotic chromatin in vivo.

PubMed

Komura, Jun-ichiro; Ono, Tetsuya

2005-04-15

During mitosis, transcription is silenced and most transcription factors are displaced from their recognition sequences. By in vivo footprinting analysis, we have confirmed and extended previous studies showing loss of transcription factors from an RNA polymerase II promoter (c-FOS) and, for the first time, an RNA polymerase III promoter (U6) in HeLa cells. Because little was known about nucleosomal organization in mitotic chromosomes, we performed footprinting analysis for nucleosomes on these promoters in interphase and mitotic cells. During interphase, each of the promoters had a positioned nucleosome in the region intervening between proximal promoter elements and distal enhancer elements, but the strong nucleosome positioning disappeared during mitosis. Thus, the nucleosomal organization that appears to facilitate transcription in interphase cells may be lost in mitotic cells, and nucleosome positioning during mitosis does not seem to be a major component of the epigenetic mechanisms to mark genes for rapid reactivation after this phase.

Joint effects of microwave and chromium trioxide on root tip cells of Vicia faba *

PubMed Central

Qian, Xiao-Wei; Luo, Wei-Hua; Zheng, Ou-Xiang

2006-01-01

The mutagenic effects of microwave and chromium trioxide (CrO3) on Vicia faba root tip were studied. Micronucleus assay and chromosomal aberration assay were used to determine the mitotic index, the micronucleus frequency and chromosomal aberration frequency of Vicia faba root tip cells induced by microwave and CrO3. The results showed that the micronucleus frequency decreased, and that the mitotic index and chromosomal aberration frequency showed linear dose responses to CrO3, in treatment of microwave for 5 s. In microwave of 25 s, the mitotic index decreased, the micronucleus frequency and chromosomal aberration frequency increased with increase of CrO3 concentration. We concluded that microwave and CrO3 had antagonistic effect on the mitotic index of Vicia faba root tip cells, but had synergetic effect on micronucleus frequency and chromosomal aberration frequency of Vicia faba root tip cells. PMID:16502510

Joint effects of microwave and chromium trioxide on root tip cells of Vicia faba.

PubMed

Qian, Xiao-wei; Luo, Wei-hua; Zheng, Ou-xiang

2006-03-01

The mutagenic effects of microwave and chromium trioxide (CrO(3)) on Vicia faba root tip were studied. Micronucleus assay and chromosomal aberration assay were used to determine the mitotic index, the micronucleus frequency and chromosomal aberration frequency of Vicia faba root tip cells induced by microwave and CrO(3). The results showed that the micronucleus frequency decreased, and that the mitotic index and chromosomal aberration frequency showed linear dose responses to CrO(3), in treatment of microwave for 5 s. In microwave of 25 s, the mitotic index decreased, the micronucleus frequency and chromosomal aberration frequency increased with increase of CrO(3) concentration. We concluded that microwave and CrO(3) had antagonistic effect on the mitotic index of Vicia faba root tip cells, but had synergetic effect on micronucleus frequency and chromosomal aberration frequency of Vicia faba root tip cells.

The pso4-1 mutation reduces spontaneous mitotic gene conversion and reciprocal recombination in Saccharomyces cerevisiae.

PubMed

Meira, L B; Fonseca, M B; Averbeck, D; Schenberg, A C; Henriques, J A

1992-11-01

Spontaneous mitotic recombination was examined in the haploid pso4-1 mutant of Saccharomyces cerevisiae and in the corresponding wild-type strain. Using a genetic system involving a duplication of the his4 gene it was shown that the pso4-1 mutation decreases at least fourfold the spontaneous rate of mitotic recombination. The frequency of spontaneous recombination was reduced tenfold in pso4-1 strains, as previously observed in the rad52-1 mutant. However, whereas the rad52-1 mutation specifically reduces gene conversion, the pso4-1 mutation reduces both gene conversion and reciprocal recombination. Induced mitotic recombination was also studied in pso4-1 mutant and wild-type strains after treatment with 8-methoxypsoralen plus UVA and 254 nm UV irradiation. Consistent with previous results, the pso4-1 mutation was found strongly to affect recombination induction.

Mitotic Evolution of Plasmodium falciparum Shows a Stable Core Genome but Recombination in Antigen Families

PubMed Central

Bopp, Selina E. R.; Manary, Micah J.; Bright, A. Taylor; Johnston, Geoffrey L.; Dharia, Neekesh V.; Luna, Fabio L.; McCormack, Susan; Plouffe, David; McNamara, Case W.; Walker, John R.; Fidock, David A.; Denchi, Eros Lazzerini; Winzeler, Elizabeth A.

2013-01-01

Malaria parasites elude eradication attempts both within the human host and across nations. At the individual level, parasites evade the host immune responses through antigenic variation. At the global level, parasites escape drug pressure through single nucleotide variants and gene copy amplification events conferring drug resistance. Despite their importance to global health, the rates at which these genomic alterations emerge have not been determined. We studied the complete genomes of different Plasmodium falciparum clones that had been propagated asexually over one year in the presence and absence of drug pressure. A combination of whole-genome microarray analysis and next-generation deep resequencing (totaling 14 terabases) revealed a stable core genome with only 38 novel single nucleotide variants appearing in seventeen evolved clones (avg. 5.4 per clone). In clones exposed to atovaquone, we found cytochrome b mutations as well as an amplification event encompassing the P. falciparum multidrug resistance associated protein (mrp1) on chromosome 1. We observed 18 large-scale (>1 kb on average) deletions of telomere-proximal regions encoding multigene families, involved in immune evasion (9.5×10−6 structural variants per base pair per generation). Six of these deletions were associated with chromosomal crossovers generated during mitosis. We found only minor differences in rates between genetically distinct strains and between parasites cultured in the presence or absence of drug. Using these derived mutation rates for P. falciparum (1.0–9.7×10−9 mutations per base pair per generation), we can now model the frequency at which drug or immune resistance alleles will emerge under a well-defined set of assumptions. Further, the detection of mitotic recombination events in var gene families illustrates how multigene families can arise and change over time in P. falciparum. These results will help improve our understanding of how P. falciparum evolves to evade control efforts within both the individual hosts and large populations. PMID:23408914

Use of DEAD-box polypeptide-4 (Ddx4) gene promoter-driven fluorescent reporter mice to identify mitotically active germ cells in post-natal mouse ovaries

PubMed Central

Park, Eun-Sil; Tilly, Jonathan L.

2015-01-01

Several laboratories have independently isolated mitotically active germ cells, termed female germline stem cells or oogonial stem cells (OSCs), from adult mouse ovaries. However, a recent study using Ddx4-Cre;Rosa26 reporter mice concluded that such germ cells do not exist. Given the disparity in conclusions drawn in this study compared with others, we felt it was important to re-assess the utility of Ddx4-Cre;Rosa26 reporter mice for identification of OSCs in adult mouse ovaries. Transgenic Ddx4-Cre mice were crossed with Rosa26tdTm/tdTm mice to drive restricted tomato red (tdTm) gene expression in cells in which the Ddx4 gene promoter has been activated. Crude dispersion of ovaries from recombined offspring generated cell fractions containing tdTm-positive immature oocytes, which are incapable of proliferation and thus probably represent the uncharacterized reporter-positive ovarian cells identified in the paper Zhang et al. (2012) as being mitotically inactive. Dispersed ovaries further subjected to fluorescence-activated cell sorting yielded a large population of non-germline tdTm-positive cells, indicative of promoter ‘leakiness’ in the Ddx4-Cre mouse line. Nonetheless, a small percentage of these tdTm-positive cells exhibited externalized (extracellular, ec) expression of Ddx4 protein (ecDdx4-positive), expressed markers of primitive germ cells but not of oocytes, and actively proliferated in culture, all of which are characteristic features of OSCs. Thus, crude dispersion of ovaries collected from Ddx4 gene promoter-driven reporter mice is not, by itself, a reliable approach to identify OSCs, whereas the same ovarian dispersates further subjected to cell sorting strategies yield purified OSCs that can be expanded in culture. PMID:25147160

Tripolar chromosome segregation drives the association between maternal genotype at variants spanning PLK4 and aneuploidy in human preimplantation embryos.

PubMed

McCoy, Rajiv C; Newnham, Louise J; Ottolini, Christian S; Hoffmann, Eva R; Chatzimeletiou, Katerina; Cornejo, Omar E; Zhan, Qiansheng; Zaninovic, Nikica; Rosenwaks, Zev; Petrov, Dmitri A; Demko, Zachary P; Sigurjonsson, Styrmir; Handyside, Alan H

2018-04-24

Aneuploidy is prevalent in human embryos and is the leading cause of pregnancy loss. Many aneuploidies arise during oogenesis, increasing with maternal age. Superimposed on these meiotic aneuploidies are frequent errors occurring during early mitotic divisions, contributing to widespread chromosomal mosaicism. Here we reanalyzed a published dataset comprising preimplantation genetic testing for aneuploidy in 24,653 blastomere biopsies from day-3 cleavage-stage embryos, as well as 17,051 trophectoderm biopsies from day-5 blastocysts. We focused on complex abnormalities that affected multiple chromosomes simultaneously, seeking insights into their formation. In addition to well-described patterns such as triploidy and haploidy, we identified 4.7% of blastomeres possessing characteristic hypodiploid karyotypes. We inferred this signature to have arisen from tripolar chromosome segregation in normally-fertilized diploid zygotes or their descendant diploid cells. This could occur via segregation on a tripolar mitotic spindle or by rapid sequential bipolar mitoses without an intervening S-phase. Both models are consistent with time-lapse data from an intersecting set of 77 cleavage-stage embryos, which were enriched for the tripolar signature among embryos exhibiting abnormal cleavage. The tripolar signature was strongly associated with common maternal genetic variants spanning the centrosomal regulator PLK4, driving the association we previously reported with overall mitotic errors. Our findings are consistent with the known capacity of PLK4 to induce tripolar mitosis or precocious M-phase upon dysregulation. Together, our data support tripolar chromosome segregation as a key mechanism generating complex aneuploidy in cleavage-stage embryos and implicate maternal genotype at a quantitative trait locus spanning PLK4 as a factor influencing its occurrence.

Trivalent dimethylarsenic compound induces histone H3 phosphorylation and abnormal localization of Aurora B kinase in HepG2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki, Toshihide, E-mail: toshi-su@pharm.teikyo-u.ac.j; Miyazaki, Koichi; Kita, Kayoko

2009-12-15

Trivalent dimethylarsinous acid [DMA(III)] has been shown to induce mitotic abnormalities, such as centrosome abnormality, multipolar spindles, multipolar division, and aneuploidy, in several cell lines. In order to elucidate the mechanisms underlying these mitotic abnormalities, we investigated DMA(III)-mediated changes in histone H3 phosphorylation and localization of Aurora B kinase, which is a key molecule in cell mitosis. DMA(III) caused the phosphorylation of histone H3 (ser10) and was distributed predominantly in mitotic cells, especially in prometaphase cells. By contrast, most of the phospho-histone H3 was found to be localized in interphase cells after treatment with inorganic arsenite [iAs(III)], suggesting the involvementmore » of a different pathway in phosphorylation. DMA(III) activated Aurora B kinase and slightly activated ERK MAP kinase. Phosphorylation of histone H3 by DMA(III) was effectively reduced by ZM447439 (Aurora kinase inhibitor) and slightly reduced by U0126 (MEK inhibitor). By contrast, iAs(III)-dependent histone H3 phosphorylation was markedly reduced by U0126. Aurora B kinase is generally localized in the midbody during telophase and plays an important role in cytokinesis. However, in some cells treated with DMA(III), Aurora B was not localized in the midbody of telophase cells. These findings suggested that DMA(III) induced a spindle abnormality, thereby activating the spindle assembly checkpoint (SAC) through the Aurora B kinase pathway. In addition, cytokinesis was not completed because of the abnormal localization of Aurora B kinase by DMA(III), thereby resulting in the generation of multinucleated cells. These results provide insight into the mechanism of arsenic tumorigenesis.« less

Oxidative Damage to Rhesus Macaque Spermatozoa Results in Mitotic Arrest and Transcript Abundance Changes in Early Embryos1

PubMed Central

Burruel, Victoria; Klooster, Katie L.; Chitwood, James; Ross, Pablo J.; Meyers, Stuart A.

2013-01-01

ABSTRACT Our objective was to determine whether oxidative damage of rhesus macaque sperm induced by reactive oxygen species (ROS) in vitro would affect embryo development following intracytoplasmic sperm injection (ICSI) of metaphase II (MII) oocytes. Fresh rhesus macaque spermatozoa were treated with ROS as follows: 1 mM xanthine and 0.1 U/ml xanthine oxidase (XXO) at 37°C and 5% CO2 in air for 2.25 h. Sperm were then assessed for motility, viability, and lipid peroxidation. Motile ROS-treated and control sperm were used for ICSI of MII oocytes. Embryo culture was evaluated for 3 days for development to the eight-cell stage. Embryos were fixed and stained for signs of cytoplasmic and nuclear abnormalities. Gene expression was analyzed by RNA-Seq in two-cell embryos from control and treated groups. Exposure of sperm to XXO resulted in increased lipid peroxidation and decreased sperm motility. ICSI of MII oocytes with motile sperm induced similar rates of fertilization and cleavage between treatments. Development to four- and eight-cell stage was significantly lower for embryos generated with ROS-treated sperm than for controls. All embryos produced from ROS-treated sperm demonstrated permanent embryonic arrest and varying degrees of degeneration and nuclear fragmentation, changes that are suggestive of prolonged senescence or apoptotic cell death. RNA-Seq analysis of two-cell embryos showed changes in transcript abundance resulting from sperm treatment with ROS. Differentially expressed genes were enriched for processes associated with cytoskeletal organization, cell adhesion, and protein phosphorylation. ROS-induced damage to sperm adversely affects embryo development by contributing to mitotic arrest after ICSI of MII rhesus oocytes. Changes in transcript abundance in embryos destined for mitotic arrest is evident at the two-cell stage of development. PMID:23904511

Reprogramming of fibroblasts from older women with pelvic floor disorders alters cellular behavior associated with donor age.

PubMed

Wen, Yan; Wani, Prachi; Zhou, Lu; Baer, Tom; Phadnis, Smruti Madan; Reijo Pera, Renee A; Chen, Bertha

2013-02-01

We aimed to derive induced pluripotent stem cell (iPSC) lines from vaginal fibroblasts from older women with pelvic organ prolapse. We examined the effect of donor age on iPSCs and on the cells redifferentiated from these iPSCs. Vaginal fibroblasts were isolated from younger and older subjects for reprogramming. iPSCs were generated simultaneously using an excisable polycistronic lentiviral vector expressing Oct4, Klf4, Sox2, and cMyc. The pluripotent markers of iPSCs were confirmed by immunocytochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Spectral karyotyping was performed. The ability of the iPSCs to differentiate into three germ layers was confirmed by embryoid body and teratoma formation. Senescence marker (p21, p53, and Bax) expressions were determined by qRT-PCR and Western blot. The iPSCs were redifferentiated to fibroblasts and were evaluated with senescence-associated β-galactosidase (SA) activity and mitotic index using time-lapse dark-field microscopy. iPSCs derived from both the younger and older subjects expressed pluripotency markers and showed normal karyotype and positive teratoma assays. There was no significant difference in expression of senescence and apoptosis markers (p21, p53, and Bax) in iPSCs derived from the younger subject compared with the older subject. Furthermore, fibroblasts redifferentiated from these iPSCs did not differ in SA activity or mitotic index. We report successful derivation of iPSCs from women with pelvic organ prolapse. Older age did not interfere with successful reprogramming. Donor age differences were not observed in these iPSCs using standard senescence markers, and donor age did not appear to affect cell mitotic activity in fibroblasts redifferentiated from iPSCs.

Genetic variation in mitotic regulatory pathway genes is associated with breast tumor grade

PubMed Central

Purrington, Kristen S.; Slettedahl, Seth; Bolla, Manjeet K.; Michailidou, Kyriaki; Czene, Kamila; Nevanlinna, Heli; Bojesen, Stig E.; Andrulis, Irene L.; Cox, Angela; Hall, Per; Carpenter, Jane; Yannoukakos, Drakoulis; Haiman, Christopher A.; Fasching, Peter A.; Mannermaa, Arto; Winqvist, Robert; Brenner, Hermann; Lindblom, Annika; Chenevix-Trench, Georgia; Benitez, Javier; Swerdlow, Anthony; Kristensen, Vessela; Guénel, Pascal; Meindl, Alfons; Darabi, Hatef; Eriksson, Mikael; Fagerholm, Rainer; Aittomäki, Kristiina; Blomqvist, Carl; Nordestgaard, Børge G.; Nielsen, Sune F.; Flyger, Henrik; Wang, Xianshu; Olswold, Curtis; Olson, Janet E.; Mulligan, Anna Marie; Knight, Julia A.; Tchatchou, Sandrine; Reed, Malcolm W.R.; Cross, Simon S.; Liu, Jianjun; Li, Jingmei; Humphreys, Keith; Clarke, Christine; Scott, Rodney; Fostira, Florentia; Fountzilas, George; Konstantopoulou, Irene; Henderson, Brian E.; Schumacher, Fredrick; Le Marchand, Loic; Ekici, Arif B.; Hartmann, Arndt; Beckmann, Matthias W.; Hartikainen, Jaana M.; Kosma, Veli-Matti; Kataja, Vesa; Jukkola-Vuorinen, Arja; Pylkäs, Katri; Kauppila, Saila; Dieffenbach, Aida Karina; Stegmaier, Christa; Arndt, Volker; Margolin, Sara; Balleine, Rosemary; Arias Perez, Jose Ignacio; Pilar Zamora, M.; Menéndez, Primitiva; Ashworth, Alan; Jones, Michael; Orr, Nick; Arveux, Patrick; Kerbrat, Pierre; Truong, Thérèse; Bugert, Peter; Toland, Amanda E.; Ambrosone, Christine B.; Labrèche, France; Goldberg, Mark S.; Dumont, Martine; Ziogas, Argyrios; Lee, Eunjung; Dite, Gillian S.; Apicella, Carmel; Southey, Melissa C.; Long, Jirong; Shrubsole, Martha; Deming-Halverson, Sandra; Ficarazzi, Filomena; Barile, Monica; Peterlongo, Paolo; Durda, Katarzyna; Jaworska-Bieniek, Katarzyna; Tollenaar, Robert A.E.M.; Seynaeve, Caroline; Brüning, Thomas; Ko, Yon-Dschun; Van Deurzen, Carolien H.M.; Martens, John W.M.; Kriege, Mieke; Figueroa, Jonine D.; Chanock, Stephen J.; Lissowska, Jolanta; Tomlinson, Ian; Kerin, Michael J.; Miller, Nicola; Schneeweiss, Andreas; Tapper, William J.; Gerty, Susan M.; Durcan, Lorraine; Mclean, Catriona; Milne, Roger L.; Baglietto, Laura; dos Santos Silva, Isabel; Fletcher, Olivia; Johnson, Nichola; Van'T Veer, Laura J.; Cornelissen, Sten; Försti, Asta; Torres, Diana; Rüdiger, Thomas; Rudolph, Anja; Flesch-Janys, Dieter; Nickels, Stefan; Weltens, Caroline; Floris, Giuseppe; Moisse, Matthieu; Dennis, Joe; Wang, Qin; Dunning, Alison M.; Shah, Mitul; Brown, Judith; Simard, Jacques; Anton-Culver, Hoda; Neuhausen, Susan L.; Hopper, John L.; Bogdanova, Natalia; Dörk, Thilo; Zheng, Wei; Radice, Paolo; Jakubowska, Anna; Lubinski, Jan; Devillee, Peter; Brauch, Hiltrud; Hooning, Maartje; García-Closas, Montserrat; Sawyer, Elinor; Burwinkel, Barbara; Marmee, Frederick; Eccles, Diana M.; Giles, Graham G.; Peto, Julian; Schmidt, Marjanka; Broeks, Annegien; Hamann, Ute; Chang-Claude, Jenny; Lambrechts, Diether; Pharoah, Paul D.P.; Easton, Douglas; Pankratz, V. Shane; Slager, Susan; Vachon, Celine M.; Couch, Fergus J.

2014-01-01

Mitotic index is an important component of histologic grade and has an etiologic role in breast tumorigenesis. Several small candidate gene studies have reported associations between variation in mitotic genes and breast cancer risk. We measured associations between 2156 single nucleotide polymorphisms (SNPs) from 194 mitotic genes and breast cancer risk, overall and by histologic grade, in the Breast Cancer Association Consortium (BCAC) iCOGS study (n = 39 067 cases; n = 42 106 controls). SNPs in TACC2 [rs17550038: odds ratio (OR) = 1.24, 95% confidence interval (CI) 1.16–1.33, P = 4.2 × 10−10) and EIF3H (rs799890: OR = 1.07, 95% CI 1.04–1.11, P = 8.7 × 10−6) were significantly associated with risk of low-grade breast cancer. The TACC2 signal was retained (rs17550038: OR = 1.15, 95% CI 1.07–1.23, P = 7.9 × 10−5) after adjustment for breast cancer risk SNPs in the nearby FGFR2 gene, suggesting that TACC2 is a novel, independent genome-wide significant genetic risk locus for low-grade breast cancer. While no SNPs were individually associated with high-grade disease, a pathway-level gene set analysis showed that variation across the 194 mitotic genes was associated with high-grade breast cancer risk (P = 2.1 × 10−3). These observations will provide insight into the contribution of mitotic defects to histological grade and the etiology of breast cancer. PMID:24927736

Mitotic control of human papillomavirus genome-containing cells is regulated by the function of the PDZ-binding motif of the E6 oncoprotein.

PubMed

Marsh, Elizabeth K; Delury, Craig P; Davies, Nicholas J; Weston, Christopher J; Miah, Mohammed A L; Banks, Lawrence; Parish, Joanna L; Higgs, Martin R; Roberts, Sally

2017-03-21

The function of a conserved PDS95/DLG1/ZO1 (PDZ) binding motif (E6 PBM) at the C-termini of E6 oncoproteins of high-risk human papillomavirus (HPV) types contributes to the development of HPV-associated malignancies. Here, using a primary human keratinocyte-based model of the high-risk HPV18 life cycle, we identify a novel link between the E6 PBM and mitotic stability. In cultures containing a mutant genome in which the E6 PBM was deleted there was an increase in the frequency of abnormal mitoses, including multinucleation, compared to cells harboring the wild type HPV18 genome. The loss of the E6 PBM was associated with a significant increase in the frequency of mitotic spindle defects associated with anaphase and telophase. Furthermore, cells carrying this mutant genome had increased chromosome segregation defects and they also exhibited greater levels of genomic instability, as shown by an elevated level of centromere-positive micronuclei. In wild type HPV18 genome-containing organotypic cultures, the majority of mitotic cells reside in the suprabasal layers, in keeping with the hyperplastic morphology of the structures. However, in mutant genome-containing structures a greater proportion of mitotic cells were retained in the basal layer, which were often of undefined polarity, thus correlating with their reduced thickness. We conclude that the ability of E6 to target cellular PDZ proteins plays a critical role in maintaining mitotic stability of HPV infected cells, ensuring stable episome persistence and vegetative amplification.

The relative effect of citral on mitotic microtubules in wheat roots and BY2 cells.

PubMed

Chaimovitsh, D; Rogovoy Stelmakh, O; Altshuler, O; Belausov, E; Abu-Abied, M; Rubin, B; Sadot, E; Dudai, N

2012-03-01

The plant volatile monoterpene citral is a highly active compound with suggested allelopathic traits. Seed germination and seedling development are inhibited in the presence of citral, and it disrupts microtubules in both plant and animal cells in interphase. We addressed the following additional questions: can citral interfere with cell division; what is the relative effect of citral on mitotic microtubules compared to interphase cortical microtubules; what is its effect on newly formed cell plates; and how does it affect the association of microtubules with γ-tubulin? In wheat seedlings, citral led to inhibition of root elongation, curvature of newly formed cell walls and deformation of microtubule arrays. Citral's effect on microtubules was both dose- and time-dependent, with mitotic microtubules appearing to be more sensitive to citral than cortical microtubules. Association of γ-tubulin with microtubules was more sensitive to citral than were the microtubules themselves. To reveal the role of disrupted mitotic microtubules in dictating aberrations in cell plates in the presence of citral, we used tobacco BY2 cells expressing GFP-Tua6. Citral disrupted mitotic microtubules, inhibited the cell cycle and increased the frequency of asymmetric cell plates in these cells. The time scale of citral's effect in BY2 cells suggested a direct influence on cell plates during their formation. Taken together, we suggest that at lower concentrations, citral interferes with cell division by disrupting mitotic microtubules and cell plates, and at higher concentrations it inhibits cell elongation by disrupting cortical microtubules. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

Relationship between plant growth and cytological effect in root apical meristem after exposure of wheat dry seeds to carbon ion beams

NASA Astrophysics Data System (ADS)

Liu, Qingfang; Wang, Zhuanzi; Zhou, Libin; Qu, Ying; Lu, Dong; Yu, Lixia; Du, Yan; Jin, Wenjie; Li, Wenjian

2013-06-01

In order to analyze the relationship between plant growth and cytological effects, wheat dry seeds were exposed to various doses of 12C6+ beams and the biological endpoints reflecting plant growth and root apical meristem (RAM) activities were investigated. The results showed that most of the seeds were able to germinate normally within all dose range, while the plant survival rate descended at higher doses. The seedling growth including root length and seedling height also decreased significantly at higher doses. Mitotic index (MI) in RAM had no changes at 10 and 20 Gy and decreased obviously at higher doses and the proportion of prophase cells had the same trend with MI. These data suggested that RAM cells experienced cell cycle arrest, which should be responsible for the inhibition of root growth after exposure to higher doses irradiation. Moreover, various types of chromosome aberrations (CAs) were observed in the mitotic cells. The frequencies of mitotic cells with lagging chromosomes and these with anaphase bridges peaked around 60 Gy, while the frequencies of these with fragments increased as the irradiation doses increased up to 200 Gy. The total frequencies of mitotic cells with CAs induced by irradiation increased significantly with the increasing doses. The serious damage of mitotic chromosomes maybe caused cell cycle arrest or cell death. These findings suggested that the influences of 12C6+ beams irradiation on plant growth were related to the alternation of mitotic activities and the chromosomal damages in RAM.

TC Mps1 12, a novel Mps1 inhibitor, suppresses the growth of hepatocellular carcinoma cells via the accumulation of chromosomal instability.

PubMed

Choi, Minji; Min, Yoo Hong; Pyo, Jaehyuk; Lee, Chang-Woo; Jang, Chang-Young; Kim, Ja-Eun

2017-06-01

Chromosomal instability is not only a hallmark of cancer but also an attractive therapeutic target. A diverse set of mitotic kinases maintains chromosomal stability. One of these is monopolar spindle 1 (Mps1, also known as TTK), which is essential for chromosome alignment and for the spindle assembly checkpoint (SAC). Pharmacological inhibition of Mps1 has been suggested as a cancer therapeutic; however, despite the existence of a novel Mps1 inhibitor, TC Mps1 12, no such studies have been performed. The effects of TC Mps1 12 on cell viability, chromosome alignment, centrosome number, mitotic duration, apoptosis and SAC were determined in hepatocellular carcinoma (HCC) cells. In addition, the association of Mps1 expression with the overall survival of HCC patients was analysed. Treatment of human HCC cells with TC Mps1 12 led to chromosome misalignment and missegregation, and disorganization of centrosomes. Even in the presence of these errors, TC Mps1 12-treated cells overrode the SAC, resulting in a shortened mitotic duration and mitotic slippage. This mitotic catastrophe triggered apoptosis and, finally, inhibited the growth of HCC cells. In addition, the expression of the Mps1-encoding TTK gene was associated with poor overall survival of HCC patients. TC Mps1 12 results in the accumulation of chromosomal instabilities and mitotic catastrophe in HCC cells. Overall, these data demonstrate that the inhibition of Mps1 kinase using TC Mps1 12 is a promising therapeutic approach for liver cancer. © 2017 The British Pharmacological Society.

High throughput screening of natural products for anti-mitotic effects in MDA-MB-231 human breast carcinoma cells

PubMed Central

Mazzio, E; Badisa, R; Mack, N; Deiab, S; Soliman, KFA

2013-01-01

Some of the most effective anti-mitotic microtubule-binding agents, such as paclitaxel (Taxus brevifolia) were originally discovered through robust NCI botanical screenings. In this study, a high-through microarray format was utilized to screen 897 aqueous extracts of commonly used natural products (0.00015–0.5 mg/ml) relative to paclitaxel for anti-mitotic effects (independent of toxicity) on proliferation of MDA-MB-231 cells. The data obtained showed that less than 1.34 % tested showed inhibitory growth (IG50) properties <0.0183 mg/ml. The most potent anti-mitotics (independent of toxicity) were Mandrake root (Podophyllum peltatum), Truja Twigs (Thuja occidentalis), Colorado desert mistletoe (Phoradendron flavescens), Tou Gu Cao Speranskia Herb (Speranskia tuberculata), Bentonite Clay, Bunge Root (Pulsatilla chinensis), Brucea Fruit (Brucea javanica), Madder Root (Rubia tinctorum), Gallnut of Chinese Sumac (Melaphis chinensis), Elecampane Root (Inula Helenium), Yuan Zhi Root (Polygala tenuifolia), Pagoda Tree Fruit (Melia Toosendan), Stone Root (Collinsonia Canadensis) and others such as American Witchhazel, Arjun and Bladderwrack. The strongest tumoricidal herbs identified from amongst the subset evaluated for anti-mitotic properties were wild yam (Dioscorea villosa), beth-root (Trillium Pendulum) and alkanet-root (Lithospermum canescens). Additional data was obtained on a lesser-recognized herb: (Speranskia tuberculata) which showed growth inhibition on BT-474 (human ductal breast carcinoma) and Ishikawa (human endometrial adenocarcinoma) cells with ability to block replicative DNA synthesis leading to G2 arrest in MDA-MB-231 cells. In conclusion, these findings present relative potency of natural anti-mitotic resources effective against human breast carcinoma MDA-MB-231 cell division. PMID:24105850

Genome accessibility is widely preserved and locally modulated during mitosis

PubMed Central

Hsiung, Chris C.-S.; Morrissey, Christapher S.; Udugama, Maheshi; Frank, Christopher L.; Keller, Cheryl A.; Baek, Songjoon; Giardine, Belinda; Crawford, Gregory E.; Sung, Myong-Hee; Hardison, Ross C.

2015-01-01

Mitosis entails global alterations to chromosome structure and nuclear architecture, concomitant with transient silencing of transcription. How cells transmit transcriptional states through mitosis remains incompletely understood. While many nuclear factors dissociate from mitotic chromosomes, the observation that certain nuclear factors and chromatin features remain associated with individual loci during mitosis originated the hypothesis that such mitotically retained molecular signatures could provide transcriptional memory through mitosis. To understand the role of chromatin structure in mitotic memory, we performed the first genome-wide comparison of DNase I sensitivity of chromatin in mitosis and interphase, using a murine erythroblast model. Despite chromosome condensation during mitosis visible by microscopy, the landscape of chromatin accessibility at the macromolecular level is largely unaltered. However, mitotic chromatin accessibility is locally dynamic, with individual loci maintaining none, some, or all of their interphase accessibility. Mitotic reduction in accessibility occurs primarily within narrow, highly DNase hypersensitive sites that frequently coincide with transcription factor binding sites, whereas broader domains of moderate accessibility tend to be more stable. In mitosis, proximal promoters generally maintain their accessibility more strongly, whereas distal regulatory elements tend to lose accessibility. Large domains of DNA hypomethylation mark a subset of promoters that retain accessibility during mitosis and across many cell types in interphase. Erythroid transcription factor GATA1 exerts site-specific changes in interphase accessibility that are most pronounced at distal regulatory elements, but has little influence on mitotic accessibility. We conclude that features of open chromatin are remarkably stable through mitosis, but are modulated at the level of individual genes and regulatory elements. PMID:25373146

Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity.

PubMed

Dewey, Evan B; Johnston, Christopher A

2017-09-15

Proper assembly and orientation of the bipolar mitotic spindle is critical to the fidelity of cell division. Mitotic precision fundamentally contributes to cell fate specification, tissue development and homeostasis, and chromosome distribution within daughter cells. Defects in these events are thought to contribute to several human diseases. The underlying mechanisms that function in spindle morphogenesis and positioning remain incompletely defined, however. Here we describe diverse roles for the actin-microtubule cross-linker Shortstop (Shot) in mitotic spindle function in Drosophila Shot localizes to mitotic spindle poles, and its knockdown results in an unfocused spindle pole morphology and a disruption of proper spindle orientation. Loss of Shot also leads to chromosome congression defects, cell cycle progression delay, and defective chromosome segregation during anaphase. These mitotic errors trigger apoptosis in Drosophila epithelial tissue, and blocking this apoptotic response results in a marked induction of the epithelial-mesenchymal transition marker MMP-1. The actin-binding domain of Shot directly interacts with Actin-related protein-1 (Arp-1), a key component of the Dynein/Dynactin complex. Knockdown of Arp-1 phenocopies Shot loss universally, whereas chemical disruption of F-actin does so selectively. Our work highlights novel roles for Shot in mitosis and suggests a mechanism involving Dynein/Dynactin activation. © 2017 Dewey and Johnston. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Two Bistable Switches Govern M Phase Entry.

PubMed

Mochida, Satoru; Rata, Scott; Hino, Hirotsugu; Nagai, Takeharu; Novák, Béla

2016-12-19

The abrupt and irreversible transition from interphase to M phase is essential to separate DNA replication from chromosome segregation. This transition requires the switch-like phosphorylation of hundreds of proteins by the cyclin-dependent kinase 1 (Cdk1):cyclin B (CycB) complex. Previous studies have ascribed these switch-like phosphorylations to the auto-activation of Cdk1:CycB through the removal of inhibitory phosphorylations on Cdk1-Tyr15 [1, 2]. The positive feedback in Cdk1 activation creates a bistable switch that makes mitotic commitment irreversible [2-4]. Here, we surprisingly find that Cdk1 auto-activation is dispensable for irreversible, switch-like mitotic entry due to a second mechanism, whereby Cdk1:CycB inhibits its counteracting phosphatase (PP2A:B55). We show that the PP2A:B55-inhibiting Greatwall (Gwl)-endosulfine (ENSA) pathway is both necessary and sufficient for switch-like phosphorylations of mitotic substrates. Using purified components of the Gwl-ENSA pathway in a reconstituted system, we found a sharp Cdk1 threshold for phosphorylation of a luminescent mitotic substrate. The Cdk1 threshold to induce mitotic phosphorylation is distinctly higher than the Cdk1 threshold required to maintain these phosphorylations-evidence for bistability. A combination of mathematical modeling and biochemical reconstitution show that the bistable behavior of the Gwl-ENSA pathway emerges from its mutual antagonism with PP2A:B55. Our results demonstrate that two interlinked bistable mechanisms provide a robust solution for irreversible and switch-like mitotic entry. Copyright © 2016 Elsevier Ltd. All rights reserved.

CENP-W Plays a Role in Maintaining Bipolar Spindle Structure

PubMed Central

Kaczmarczyk, Agnieszka; Sullivan, Kevin F.

2014-01-01

The CENP-W/T complex was previously reported to be required for mitosis. HeLa cells depleted of CENP-W displayed profound mitotic defects, with mitotic timing delay, disorganized prometaphases and multipolar spindles as major phenotypic consequences. In this study, we examined the process of multipolar spindle formation induced by CENP-W depletion. Depletion of CENP-W in HeLa cells labeled with histone H2B and tubulin fluorescent proteins induced rapid fragmentation of originally bipolar spindles in a high proportion of cells. CENP-W depletion was associated with depletion of Hec1 at kinetochores. The possibility of promiscuous centrosomal duplication was ruled out by immunofluorescent examination of centrioles. However, centrioles were frequently observed to be abnormally split. In addition, a large proportion of the supernumerary poles lacked centrioles, but were positively stained with different centrosomal markers. These observations suggested that perturbation in spindle force distribution caused by defective kinetochores could contribute to a mechanical mechanism for spindle pole disruption. ‘Spindle free’ nocodazole arrested cells did not exhibit pole fragmentation after CENP-W depletion, showing that pole fragmentation is microtubule dependent. Inhibition of centrosome separation by monastrol reduced the incidence of spindle pole fragmentation, indicating that Eg5 plays a role in spindle pole disruption. Surprisingly, CENP-W depletion rescued the monopolar spindle phenotype of monastrol treatment, with an increased frequency of bipolar spindles observed after CENP-W RNAi. We overexpressed the microtubule cross-linking protein TPX2 to create spindle poles stabilized by the microtubule cross-linking activity of TPX2. Spindle pole fragmentation was suppressed in a TPX2-dependent fashion. We propose that CENP-W, by influencing proper kinetochore assembly, particularly microtubule docking sites, can confer spindle pole resistance to traction forces exerted by motor proteins during chromosome congression. Taken together, our findings are consistent with a model in which centrosome integrity is controlled by the pathways regulating kinetochore-microtubule attachment stability. PMID:25329824

Identification of Phosphohistone H3 Cutoff Values Corresponding to Original WHO Grades but Distinguishable in Well-Differentiated Gastrointestinal Neuroendocrine Tumors.

PubMed

Kim, Min Jeong; Kwon, Mi Jung; Kang, Ho Suk; Choi, Kyung Chan; Nam, Eun Sook; Cho, Seong Jin; Park, Hye-Rim; Min, Soo Kee; Seo, Jinwon; Choe, Ji-Young; Park, Hyoung-Chul

2018-01-01

Mitotic counts in the World Health Organization (WHO) grading system have narrow cutoff values. True mitotic figures, however, are not always distinguishable from apoptotic bodies and darkly stained nuclei, complicating the ability of the WHO grading system to diagnose well-differentiated neuroendocrine tumors (NETs). The mitosis-specific marker phosphohistone H3 (PHH3) can identify true mitoses and grade tumors reliably. The aim of this study was to investigate the correspondence of tumor grades, as determined by PHH3 mitotic index (MI) and mitotic counts according to WHO criteria, and to determine the clinically relevant cutoffs of PHH3 MI in rectal and nonrectal gastrointestinal NETs. Mitotic counts correlated with both the Ki-67 labeling index and PHH3 MI, but the correlation with PHH3 MI was slightly higher. The PHH3 MI cutoff ≥4 correlated most closely with original WHO grades for both rectal NETs. A PHH3 MI cutoff ≥4, which could distinguish between G1 and G2 tumors, was associated with disease-free survival in patients with rectal NETs, whereas that cutoff value showed marginal significance for overall survival in patient with rectal NETs. In conclusion, the use of PHH3 ≥4 correlated most closely with original WHO grades.

Identification of Phosphohistone H3 Cutoff Values Corresponding to Original WHO Grades but Distinguishable in Well-Differentiated Gastrointestinal Neuroendocrine Tumors

PubMed Central

Kim, Min Jeong; Kang, Ho Suk; Choi, Kyung Chan; Nam, Eun Sook; Cho, Seong Jin; Park, Hye-Rim; Seo, Jinwon; Choe, Ji-Young

2018-01-01

Mitotic counts in the World Health Organization (WHO) grading system have narrow cutoff values. True mitotic figures, however, are not always distinguishable from apoptotic bodies and darkly stained nuclei, complicating the ability of the WHO grading system to diagnose well-differentiated neuroendocrine tumors (NETs). The mitosis-specific marker phosphohistone H3 (PHH3) can identify true mitoses and grade tumors reliably. The aim of this study was to investigate the correspondence of tumor grades, as determined by PHH3 mitotic index (MI) and mitotic counts according to WHO criteria, and to determine the clinically relevant cutoffs of PHH3 MI in rectal and nonrectal gastrointestinal NETs. Mitotic counts correlated with both the Ki-67 labeling index and PHH3 MI, but the correlation with PHH3 MI was slightly higher. The PHH3 MI cutoff ≥4 correlated most closely with original WHO grades for both rectal NETs. A PHH3 MI cutoff ≥4, which could distinguish between G1 and G2 tumors, was associated with disease-free survival in patients with rectal NETs, whereas that cutoff value showed marginal significance for overall survival in patient with rectal NETs. In conclusion, the use of PHH3 ≥4 correlated most closely with original WHO grades. PMID:29780816

Picropodophyllin causes mitotic arrest and catastrophe by depolymerizing microtubules via Insulin-like growth factor-1 receptor-independent mechanism

PubMed Central

Waraky, Ahmed; Akopyan, Karen; Parrow, Vendela; Strömberg, Thomas; Axelson, Magnus; Abrahmsén, Lars; Lindqvist, Arne; Larsson, Olle; Aleem, Eiman

2014-01-01

Picropodophyllin (PPP) is an anticancer drug undergoing clinical development in NSCLC. PPP has been shown to suppress IGF-1R signaling and to induce a G2/M cell cycle phase arrest but the exact mechanisms remain to be elucidated. The present study identified an IGF-1-independent mechanism of PPP leading to pro-metaphase arrest. The mitotic block was induced in human cancer cell lines and in an A549 xenograft mouse but did not occur in normal hepatocytes/mouse tissues. Cell cycle arrest by PPP occurred in vitro and in vivo accompanied by prominent CDK1 activation, and was IGF-1R-independent since it occurred also in IGF-1R-depleted and null cells. The tumor cells were not arrested in G2/M but in mitosis. Centrosome separation was prevented during mitotic entry, resulting in a monopolar mitotic spindle with subsequent prometaphase-arrest, independent of Plk1/Aurora A or Eg5, and leading to cell features of mitotic catastrophe. PPP also increased soluble tubulin and decreased spindle-associated tubulin within minutes, indicating that it interfered with microtubule dynamics. These results provide a novel IGF-1R-independent mechanism of antitumor effects of PPP. PMID:25268741

Cyclin K dependent regulation of Aurora B affects apoptosis and proliferation by induction of mitotic catastrophe in prostate cancer.

PubMed

Schecher, Sabrina; Walter, Britta; Falkenstein, Michael; Macher-Goeppinger, Stephan; Stenzel, Philipp; Krümpelmann, Kristina; Hadaschik, Boris; Perner, Sven; Kristiansen, Glen; Duensing, Stefan; Roth, Wilfried; Tagscherer, Katrin E

2017-10-15

Cyclin K plays a critical role in transcriptional regulation as well as cell development. However, the role of Cyclin K in prostate cancer is unknown. Here, we describe the impact of Cyclin K on prostate cancer cells and examine the clinical relevance of Cyclin K as a biomarker for patients with prostate cancer. We show that Cyclin K depletion in prostate cancer cells induces apoptosis and inhibits proliferation accompanied by an accumulation of cells in the G2/M phase. Moreover, knockdown of Cyclin K causes mitotic catastrophe displayed by multinucleation and spindle multipolarity. Furthermore, we demonstrate a Cyclin K dependent regulation of the mitotic kinase Aurora B and provide evidence for an Aurora B dependent induction of mitotic catastrophe. In addition, we show that Cyclin K expression is associated with poor biochemical recurrence-free survival in patients with prostate cancer treated with an adjuvant therapy. In conclusion, targeting Cyclin K represents a novel, promising anti-cancer strategy to induce cell cycle arrest and apoptotic cell death through induction of mitotic catastrophe in prostate cancer cells. Moreover, our results indicate that Cyclin K is a putative predictive biomarker for clinical outcome and therapy response for patients with prostate cancer. © 2017 UICC.

Cell Cycle Synchronization of HeLa Cells to Assay EGFR Pathway Activation.

PubMed

Wee, Ping; Wang, Zhixiang

2017-01-01

Progression through the cell cycle causes changes in the cell's signaling pathways that can alter EGFR signal transduction. Here, we describe drug-derived protocols to synchronize HeLa cells in various phases of the cell cycle, including G1 phase, S phase, G2 phase, and mitosis, specifically in the mitotic stages of prometaphase, metaphase, and anaphase/telophase. The synchronization procedures are designed to allow synchronized cells to be treated for EGF and collected for the purpose of Western blotting for EGFR signal transduction components.S phase synchronization is performed by thymidine block, G2 phase with roscovitine, prometaphase with nocodazole, metaphase with MG132, and anaphase/telophase with blebbistatin. G1 phase synchronization is performed by culturing synchronized mitotic cells obtained by mitotic shake-off. We also provide methods to validate the synchronization methods. For validation by Western blotting, we provide the temporal expression of various cell cycle markers that are used to check the quality of the synchronization. For validation of mitotic synchronization by microscopy, we provide a guide that describes the physical properties of each mitotic stage, using their cellular morphology and DNA appearance. For validation by flow cytometry, we describe the use of imaging flow cytometry to distinguish between the phases of the cell cycle, including between each stage of mitosis.

The Stil protein regulates centrosome integrity and mitosis through suppression of Chfr

PubMed Central

Castiel, Asher; Danieli, Michal Mark; David, Ahuvit; Moshkovitz, Sharon; Aplan, Peter D.; Kirsch, Ilan R.; Brandeis, Michael; Krämer, Alwin; Izraeli, Shai

2011-01-01

Stil (Sil, SCL/TAL1 interrupting locus) is a cytosolic and centrosomal protein expressed in proliferating cells that is required for mouse and zebrafish neural development and is mutated in familial microcephaly. Recently the Drosophila melanogaster ortholog of Stil was found to be important for centriole duplication. Consistent with this finding, we report here that mouse embryonic fibroblasts lacking Stil are characterized by slow growth, low mitotic index and absence of clear centrosomes. We hypothesized that Stil regulates mitosis through the tumor suppressor Chfr, an E3 ligase that blocks mitotic entry in response to mitotic stress. Mouse fibroblasts lacking Stil by genomic or RNA interference approaches, as well as E9.5 Stil−/− embryos, express high levels of the Chfr protein and reduced levels of the Chfr substrate Plk1. Exogenous expression of Stil, knockdown of Chfr or overexpression of Plk1 reverse the abnormal mitotic phenotypes of fibroblasts lacking Stil. We further demonstrate that Stil increases Chfr auto-ubiquitination and reduces its protein stability. Thus, Stil is required for centrosome organization, entry into mitosis and cell proliferation, and these functions are at least partially mediated by Chfr and its targets. This is the first identification of a negative regulator of the Chfr mitotic checkpoint. PMID:21245198

Controlling the response to DNA damage by the APC/C-Cdh1.

PubMed

de Boer, H Rudolf; Guerrero Llobet, S; van Vugt, Marcel A T M

2016-03-01

Proper cell cycle progression is safeguarded by the oscillating activities of cyclin/cyclin-dependent kinase complexes. An important player in the regulation of mitotic cyclins is the anaphase-promoting complex/cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase. Prior to entry into mitosis, the APC/C remains inactive, which allows the accumulation of mitotic regulators. APC/C activation requires binding to either the Cdc20 or Cdh1 adaptor protein, which sequentially bind the APC/C and facilitate targeting of multiple mitotic regulators for proteasomal destruction, including Securin and Cyclin B, to ensure proper chromosome segregation and mitotic exit. Emerging data have indicated that the APC/C, particularly in association with Cdh1, also functions prior to mitotic entry. Specifically, the APC/C-Cdh1 is activated in response to DNA damage in G2 phase cells. These observations are in line with in vitro and in vivo genetic studies, in which cells lacking Cdh1 expression display various defects, including impaired DNA repair and aberrant cell cycle checkpoints. In this review, we summarize the current literature on APC/C regulation in response to DNA damage, the functions of APC/C-Cdh1 activation upon DNA damage, and speculate how APC/C-Cdh1 can control cell fate in the context of persistent DNA damage.

Physical determinants of bipolar mitotic spindle assembly and stability in fission yeast

NASA Astrophysics Data System (ADS)

Betterton, Meredith; Blackwell, Robert; Edelmaier, Christopher; Sweezy-Schindler, Oliver; Lamson, Adam; Gergely, Zachary; O'Toole, Eileen; Crapo, Ammon; Hough, Loren; McIntosh, J. Richard; Glaser, Matthew

Mitotic spindles use an elegant bipolar architecture to segregate duplicated chromosomes with high fidelity. Bipolar spindles form from a monopolar initial condition; this is the most fundamental construction problem that the spindle must solve. Microtubules, motors, and crosslinkers are important for bipolarity, but the mechanisms necessary and sufficient for spindle assembly remain unknown. Here we describe a physical model that exhibits de novo bipolar spindle formation. We began with previously published data on fission-yeast spindle-pole-body size and microtubule number, kinesin-5 motors, kinesin-14 motors, and passive crosslinkers. Our model results agree quantitatively with our experiments in fission yeast, thereby establishing a minimal system with which to interrogate collective self assembly. By varying features of our model, we identify a set of functions essential for the generation and stability of spindle bipolarity. When kinesin-5 motors are present, their bidirectionality is essential, but spindles can form in the presence of passive crosslinkers alone. We also identify characteristic failed states of spindle assembly, which are avoided by creation and maintenance of antiparallel microtubule overlaps. DMR-0847685, DMR-1551095, DMR-1420736, K25GM110486, R01GM104976, R01GM033787.

Bmi1 regulates murine intestinal stem cell proliferation and self-renewal downstream of Notch.

PubMed

López-Arribillaga, Erika; Rodilla, Verónica; Pellegrinet, Luca; Guiu, Jordi; Iglesias, Mar; Roman, Angel Carlos; Gutarra, Susana; González, Susana; Muñoz-Cánoves, Pura; Fernández-Salguero, Pedro; Radtke, Freddy; Bigas, Anna; Espinosa, Lluís

2015-01-01

Genetic data indicate that abrogation of Notch-Rbpj or Wnt-β-catenin pathways results in the loss of the intestinal stem cells (ISCs). However, whether the effect of Notch is direct or due to the aberrant differentiation of the transit-amplifying cells into post-mitotic goblet cells is unknown. To address this issue, we have generated composite tamoxifen-inducible intestine-specific genetic mouse models and analyzed the expression of intestinal differentiation markers. Importantly, we found that activation of β-catenin partially rescues the differentiation phenotype of Rbpj deletion mutants, but not the loss of the ISC compartment. Moreover, we identified Bmi1, which is expressed in the ISC and progenitor compartments, as a gene that is co-regulated by Notch and β-catenin. Loss of Bmi1 resulted in reduced proliferation in the ISC compartment accompanied by p16(INK4a) and p19(ARF) (splice variants of Cdkn2a) accumulation, and increased differentiation to the post-mitotic goblet cell lineage that partially mimics Notch loss-of-function defects. Finally, we provide evidence that Bmi1 contributes to ISC self-renewal. © 2015. Published by The Company of Biologists Ltd.

A novel snRNA-like transcript affects amyloidogenesis and cell cycle progression through perturbation of Fe65L1 (APBB2) alternative splicing.

PubMed

Penna, Ilaria; Vassallo, Irene; Nizzari, Mario; Russo, Debora; Costa, Delfina; Menichini, Paola; Poggi, Alessandro; Russo, Claudio; Dieci, Giorgio; Florio, Tullio; Cancedda, Ranieri; Pagano, Aldo

2013-06-01

FE65 proteins constitute a family of adaptors which modulates the processing of amyloid precursor protein and the consequent amyloid β production. Thus, they have been involved in the complex and partially unknown cascade of reactions at the base of Alzheimer's disease etiology. However, FE65 and FE65-like proteins may be linked to neurodegeneration through the regulation of cell cycle in post-mitotic neurons. In this work we disclose novel molecular mechanisms by which APBB2 can modulate APP processing. We show that APBB2 mRNA splicing, driven by the over-expression of a novel non-coding RNA named 45A, allow the generation of alternative protein forms endowed with differential effects on Aβ production, cell cycle control, and DNA damage response. 45A overexpression also favors cell transformation and tumorigenesis leading to a marked increase of malignancy of neuroblastoma cells. Therefore, our results highlight a novel regulatory pathway of considerable interest linking APP processing with cell cycle regulation and DNA-surveillance systems, that may represent a molecular mechanism to induce neurodegeneration in post-mitotic neurons. Copyright © 2013 Elsevier B.V. All rights reserved.

Polyploidization and cell fusion contribute to wound healing in the adult Drosophila epithelium

PubMed Central

Losick, Vicki P.; Fox, Donald T.; Spradling, Allan C.

2014-01-01

Summary Background Re-establishing epithelial integrity and biosynthetic capacity is critically important following tissue damage. The adult Drosophila abdominal epithelium provides an attractive new system to address how post-mitotic diploid cells contribute to repair. Results Puncture wounds to the adult Drosophila epidermis close initially by forming a melanized scab. We found that epithelial cells near the wound site fuse to form a giant syncytium, which sends lamellae under the scab to re-epithelialize the damaged site. Other large cells arise more peripherally by initiating endocycles and becoming polyploid, or by cell fusion. Rac GTPase activity is needed for syncytium formation, while the Hippo signaling effector Yorkie modulates both polyploidization and cell fusion. Large cell formation is functionally important because when both polyploidization and fusion are blocked, wounds do not re-epithelialize. Conclusions Our observations indicate that cell mass lost upon wounding can be replaced by polyploidization instead of mitotic proliferation. We propose that large cells generated by polyploidization or cell fusion are essential because they are better able than diploid cells to mechanically stabilize wounds, especially those containing permanent acellular structures, such as scar tissue. PMID:24184101

Antisense expression of an Arabidopsis ran binding protein renders transgenic roots hypersensitive to auxin and alters auxin-induced root growth and development by arresting mitotic progress

NASA Technical Reports Server (NTRS)

Kim, S. H.; Arnold, D.; Lloyd, A.; Roux, S. J.

2001-01-01

We cloned a cDNA encoding an Arabidopsis Ran binding protein, AtRanBP1c, and generated transgenic Arabidopsis expressing the antisense strand of the AtRanBP1c gene to understand the in vivo functions of the Ran/RanBP signal pathway. The transgenic plants showed enhanced primary root growth but suppressed growth of lateral roots. Auxin significantly increased lateral root initiation and inhibited primary root growth in the transformants at 10 pM, several orders of magnitude lower than required to induce these responses in wild-type roots. This induction was followed by a blockage of mitosis in both newly emerged lateral roots and in the primary root, ultimately resulting in the selective death of cells in the tips of both lateral and primary roots. Given the established role of Ran binding proteins in the transport of proteins into the nucleus, these findings are consistent with a model in which AtRanBP1c plays a key role in the nuclear delivery of proteins that suppress auxin action and that regulate mitotic progress in root tips.

TopoIIα prevents telomere fragility and formation of ultra thin DNA bridges during mitosis through TRF1-dependent binding to telomeres.

PubMed

d'Alcontres, Martina Stagno; Palacios, Jose Alejandro; Mejias, Diego; Blasco, Maria A

2014-01-01

Telomeres are repetitive nucleoprotein structures at the ends of chromosomes. Like most genomic regions consisting of repetitive DNA, telomeres are fragile sites prone to replication fork stalling and generation of chromosomal instability. In particular, abrogation of the TRF1 telomere binding protein leads to stalled replication forks and aberrant telomere structures known as "multitelomeric signals". Here, we report that TRF1 deficiency also leads to the formation of "ultra-fine bridges" (UFB) during mitosis, and to an increased time to complete mitosis mediated by the spindle assembly checkpoint proteins (SAC). We find that topoisomerase IIα (TopoIIα), an enzyme essential for resolution of DNA replication intermediates, binds telomeres in a TRF1-mediated manner. Indeed, similar to TRF1 abrogation, TopoIIα downregulation leads to telomere fragility and UFB, suggesting that these phenotypes are due to decreased TopoIIα at telomeres. We find that SAC proteins bind telomeres in vivo, and that this is disrupted upon TRF1 deletion. These findings suggest that TRF1 links TopoIIα and SAC proteins in a pathway that ensures correct telomere replication and mitotic segregation, unveiling how TRF1 protects from telomere fragility and mitotic defects.

Diploid yeast cells yield homozygous spontaneous mutations

NASA Technical Reports Server (NTRS)

Esposito, M. S.; Bruschi, C. V.; Brushi, C. V. (Principal Investigator)

1993-01-01

A leucine-requiring hybrid of Saccharomyces cerevisiae, homoallelic at the LEU1 locus (leu1-12/leu1-12) and heterozygous for three chromosome-VII genetic markers distal to the LEU1 locus, was employed to inquire: (1) whether spontaneous gene mutation and mitotic segregation of heterozygous markers occur in positive nonrandom association and (2) whether homozygous LEU1/LEU1 mutant diploids are generated. The results demonstrate that gene mutation of leu1-12 to LEU1 and mitotic segregation of heterozygous chromosome-VII markers occur in strong positive nonrandom association, suggesting that the stimulatory DNA lesion is both mutagenic and recombinogenic. In addition, genetic analysis of diploid Leu+ revertants revealed that approximately 3% of mutations of leu1-12 to LEU1 result in LEU1/LEU1 homozygotes. Red-white sectored Leu+ colonies exhibit genotypes that implicate post-replicational chromatid breakage and exchange near the site of leu1-12 reversion, chromosome loss, and subsequent restitution of diploidy, in the sequence of events leading to mutational homozygosis. By analogy, diploid cell populations can yield variants homozygous for novel recessive gene mutations at biologically significant rates. Mutational homozygosis may be relevant to both carcinogenesis and the evolution of asexual diploid organisms.

A signature inferred from Drosophila mitotic genes predicts survival of breast cancer patients.

PubMed

Damasco, Christian; Lembo, Antonio; Somma, Maria Patrizia; Gatti, Maurizio; Di Cunto, Ferdinando; Provero, Paolo

2011-02-28

The classification of breast cancer patients into risk groups provides a powerful tool for the identification of patients who will benefit from aggressive systemic therapy. The analysis of microarray data has generated several gene expression signatures that improve diagnosis and allow risk assessment. There is also evidence that cell proliferation-related genes have a high predictive power within these signatures. We thus constructed a gene expression signature (the DM signature) using the human orthologues of 108 Drosophila melanogaster genes required for either the maintenance of chromosome integrity (36 genes) or mitotic division (72 genes). The DM signature has minimal overlap with the extant signatures and is highly predictive of survival in 5 large breast cancer datasets. In addition, we show that the DM signature outperforms many widely used breast cancer signatures in predictive power, and performs comparably to other proliferation-based signatures. For most genes of the DM signature, an increased expression is negatively correlated with patient survival. The genes that provide the highest contribution to the predictive power of the DM signature are those involved in cytokinesis. This finding highlights cytokinesis as an important marker in breast cancer prognosis and as a possible target for antimitotic therapies.

Mitotic rate in primary melanoma: interobserver and intraobserver reliability, analyzed using H&E sections and immunohistochemistry.

PubMed

Garbe, Claus; Eigentler, Thomas K; Bauer, Jürgen; Blödorn-Schlicht, Norbert; Cerroni, Lorenzo; Fend, Falko; Hantschke, Markus; Kurschat, Peter; Kutzner, Heinz; Metze, Dieter; Mielke, Volker; Preßler, Harald; Reusch, Michael; Reusch, Ursula; Stadler, Rudolf; Tronnier, Michael; Yazdi, Amir; Metzler, Gisela

2016-09-01

In 2009, the AJCC issued a revised melanoma staging system. In addition to tumor thickness and ulceration, the mitotic rate was introduced as the third major prognostic parameter for the classification of primary cutaneous melanoma. Given that, according to the 2009 AJCC classification, the detection of one or more dermal tumor mitoses leads to an upstaging - from stage Ia to Ib - of melanomas with a tumor thickness of ≤ 1.0 mm, we set out to investigate the reproducibility of this new parameter. In order to assess interobserver reliability, 17 dermatopathologists und pathologists - all well versed in the diagnosis of cutaneous melanoma - analyzed the mitotic rate in 15 thin primary cutaneous melanomas (mean tumor thickness 0.91 mm) using identical slides. Mitotic rates were determined on H&E and phosphohistone H3 (Ser10)-stained samples. Without knowledge of their previous assessment, five of the aforementioned examiners reevaluated the samples after more than one year in order to ascertain intraobserver reliability. Interobserver reliability of the mitotic rate in thin primary melanomas is disappointing and independent of whether H&E or immunohistochemically stained samples are used (kappa value: 0.088 [H&E], 0.154 [IH], respectively). Kappa values improved to 0.345 (H&E) and 0.403 (IH) when using a cutoff of 0/1 vs. 2+ mitoses. Similarly unsatisfactory, kappa values for intraobserver reliability ranged from 0.18 and 0.348, depending on the individual examiner. Given the unsatisfactory reproducibility and large variations in assessing the mitotic rate, it remains a matter of debate whether this diagnostic parameter should play a role in therapeutic decisions. © 2016 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd.

Distribution of fluorescently labeled tubulin injected into sand dollar eggs from fertilization through cleavage.

PubMed

Hamaguchi, Y; Toriyama, M; Sakai, H; Hiramoto, Y

1985-04-01

Porcine brain tubulin labeled with fluorescein isothiocyanate (FITC) was able to polymerize by itself and co-polymerize with tubulin purified from starfish sperm flagella. When we injected the FITC-labeled tubulin into unfertilized eggs of the sand dollar, Clypeaster japonicus, and the eggs were then fertilized, the labeled tubulin was incorporated into the sperm aster. When injected into fertilized eggs at streak stage, the tubulin was quickly incorporated into each central region of growing asters. It was clearly visualized that the labeled tubulin, upon reaching metaphase, accumulated in the mitotic apparatus and later disappeared over the cytoplasm during interphase. The accumulation of the fluorescence in the mitotic apparatus was observed repeatedly at successive cleavage. After lysis of the fertilized eggs with a microtubule-stabilizing solution, fluorescent fibrous structures around the nucleus and those of the sperm aster and the mitotic apparatus were preserved and coincided with the fibrous structures observed by polarization and differential interference microscopy. We found the FITC-labeled tubulin to be incorporated into the entire mitotic apparatus within 20-30 s when injected into the eggs at metaphase or anaphase. This rapid incorporation of the labeled tubulin into the mitotic apparatus suggests that the equilibrium between mitotic microtubules and tubulin is attained very rapidly in the living eggs. Axonemal tubulin purified from starfish sperm flagella and labeled with FITC was also incorporated into microtubular structures in the same fashion as the FITC-labeled brain tubulin. These results suggest that even FITC-labeled heterogeneous tubulins undergo spatial and stage-specific regulation of assembly-disassembly in the same manner as does sand dollar egg tubulin.

Distribution of fluorescently labeled tubulin injected into sand dollar eggs from fertilization through cleavage

PubMed Central

1985-01-01

Porcine brain tubulin labeled with fluorescein isothiocyanate (FITC) was able to polymerize by itself and co-polymerize with tubulin purified from starfish sperm flagella. When we injected the FITC- labeled tubulin into unfertilized eggs of the sand dollar, Clypeaster japonicus, and the eggs were then fertilized, the labeled tubulin was incorporated into the sperm aster. When injected into fertilized eggs at streak stage, the tubulin was quickly incorporated into each central region of growing asters. It was clearly visualized that the labeled tubulin, upon reaching metaphase, accumulated in the mitotic apparatus and later disappeared over the cytoplasm during interphase. The accumulation of the fluorescence in the mitotic apparatus was observed repeatedly at successive cleavage. After lysis of the fertilized eggs with a microtubule-stabilizing solution, fluorescent fibrous structures around the nucleus and those of the sperm aster and the mitotic apparatus were preserved and coincided with the fibrous structures observed by polarization and differential interference microscopy. We found the FITC-labeled tubulin to be incorporated into the entire mitotic apparatus within 20-30 s when injected into the eggs at metaphase or anaphase. This rapid incorporation of the labeled tubulin into the mitotic apparatus suggests that the equilibrium between mitotic microtubules and tubulin is attained very rapidly in the living eggs. Axonemal tubulin purified from starfish sperm flagella and labeled with FITC was also incorporated into microtubular structures in the same fashion as the FITC-labeled brain tubulin. These results suggest that even FITC-labeled heterogeneous tubulins undergo spatial and stage-specific regulation of assembly-disassembly in the same manner as does sand dollar egg tubulin. PMID:3920225

Genotoxicity of multi-walled carbon nanotubes at occupationally relevant doses

PubMed Central

2014-01-01

Carbon nanotubes are commercially-important products of nanotechnology; however, their low density and small size makes carbon nanotube respiratory exposures likely during their production or processing. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to single-walled carbon nanotubes (SWCNT). In this study, we examined whether multi-walled carbon nanotubes (MWCNT) cause mitotic spindle damage in cultured cells at doses equivalent to 34 years of exposure at the NIOSH Recommended Exposure Limit (REL). MWCNT induced a dose responsive increase in disrupted centrosomes, abnormal mitotic spindles and aneuploid chromosome number 24 hours after exposure to 0.024, 0.24, 2.4 and 24 μg/cm2 MWCNT. Monopolar mitotic spindles comprised 95% of disrupted mitoses. Three-dimensional reconstructions of 0.1 μm optical sections showed carbon nanotubes integrated with microtubules, DNA and within the centrosome structure. Cell cycle analysis demonstrated a greater number of cells in S-phase and fewer cells in the G2 phase in MWCNT-treated compared to diluent control, indicating a G1/S block in the cell cycle. The monopolar phenotype of the disrupted mitotic spindles and the G1/S block in the cell cycle is in sharp contrast to the multi-polar spindle and G2 block in the cell cycle previously observed following exposure to SWCNT. One month following exposure to MWCNT there was a dramatic increase in both size and number of colonies compared to diluent control cultures, indicating a potential to pass the genetic damage to daughter cells. Our results demonstrate significant disruption of the mitotic spindle by MWCNT at occupationally relevant exposure levels. PMID:24479647

TC Mps1 12, a novel Mps1 inhibitor, suppresses the growth of hepatocellular carcinoma cells via the accumulation of chromosomal instability

PubMed Central

Choi, Minji; Min, Yoo Hong; Pyo, Jaehyuk; Lee, Chang‐Woo; Jang, Chang‐Young

2017-01-01

Background and Purpose Chromosomal instability is not only a hallmark of cancer but also an attractive therapeutic target. A diverse set of mitotic kinases maintains chromosomal stability. One of these is monopolar spindle 1 (Mps1, also known as TTK), which is essential for chromosome alignment and for the spindle assembly checkpoint (SAC). Pharmacological inhibition of Mps1 has been suggested as a cancer therapeutic; however, despite the existence of a novel Mps1 inhibitor, TC Mps1 12, no such studies have been performed. Experimental Approach The effects of TC Mps1 12 on cell viability, chromosome alignment, centrosome number, mitotic duration, apoptosis and SAC were determined in hepatocellular carcinoma (HCC) cells. In addition, the association of Mps1 expression with the overall survival of HCC patients was analysed. Key Results Treatment of human HCC cells with TC Mps1 12 led to chromosome misalignment and missegregation, and disorganization of centrosomes. Even in the presence of these errors, TC Mps1 12‐treated cells overrode the SAC, resulting in a shortened mitotic duration and mitotic slippage. This mitotic catastrophe triggered apoptosis and, finally, inhibited the growth of HCC cells. In addition, the expression of the Mps1‐encoding TTK gene was associated with poor overall survival of HCC patients. Conclusion and Implications TC Mps1 12 results in the accumulation of chromosomal instabilities and mitotic catastrophe in HCC cells. Overall, these data demonstrate that the inhibition of Mps1 kinase using TC Mps1 12 is a promising therapeutic approach for liver cancer. PMID:28299790

Effects of heat stress in the leaf mitotic cell cycle and chromosomes of four wine-producing grapevine varieties.

PubMed

Carvalho, Ana; Leal, Fernanda; Matos, Manuela; Lima-Brito, José

2018-05-22

Grapevine varieties respond differentially to heat stress (HS). HS ultimately reduces the photosynthesis and respiratory performance. However, the HS effects in the leaf nuclei and mitotic cells of grapevine are barely known. This work intends to evaluate the HS effects in the leaf mitotic cell cycle and chromosomes of four wine-producing varieties: Touriga Franca (TF), Touriga Nacional (TN), Rabigato, and Viosinho. In vitro plants with 11 months were used in a stepwise acclimation and recovery (SAR) experimental setup comprising different phases: heat acclimation period (3 h-32 °C), extreme HS (1 h-42 °C), and two recovery periods (3 h-32 °C and 24 h-25 °C), and compared to control plants (maintained in vitro at 25 °C). At the end of each SAR phase, leaves were collected, fixed, and used for cell suspensions and chromosome preparations. Normal and abnormal interphase and mitotic cells were observed, scored, and statistically analyzed in all varieties and treatments (control and SAR phases). Different types of chromosomal anomalies in all mitotic phases, treatments, and varieties were found. In all varieties, the percentage of dividing cells with anomalies (%DCA) after extreme HS increased relative to control. TF and Viosinho were considered the most tolerant to HS. TF showed a gradual MI reduction from heat acclimation to HS and the lowest %DCA after HS and 24 h of recovery. Only Viosinho reached the control values after the long recovery period. Extrapolating these data to the field, we hypothesize that during consecutive hot summer days, the grapevine plants will not have time or capacity to recover from the mitotic anomalies caused by high temperatures.

Genome accessibility is widely preserved and locally modulated during mitosis.

PubMed

Hsiung, Chris C-S; Morrissey, Christapher S; Udugama, Maheshi; Frank, Christopher L; Keller, Cheryl A; Baek, Songjoon; Giardine, Belinda; Crawford, Gregory E; Sung, Myong-Hee; Hardison, Ross C; Blobel, Gerd A

2015-02-01

Mitosis entails global alterations to chromosome structure and nuclear architecture, concomitant with transient silencing of transcription. How cells transmit transcriptional states through mitosis remains incompletely understood. While many nuclear factors dissociate from mitotic chromosomes, the observation that certain nuclear factors and chromatin features remain associated with individual loci during mitosis originated the hypothesis that such mitotically retained molecular signatures could provide transcriptional memory through mitosis. To understand the role of chromatin structure in mitotic memory, we performed the first genome-wide comparison of DNase I sensitivity of chromatin in mitosis and interphase, using a murine erythroblast model. Despite chromosome condensation during mitosis visible by microscopy, the landscape of chromatin accessibility at the macromolecular level is largely unaltered. However, mitotic chromatin accessibility is locally dynamic, with individual loci maintaining none, some, or all of their interphase accessibility. Mitotic reduction in accessibility occurs primarily within narrow, highly DNase hypersensitive sites that frequently coincide with transcription factor binding sites, whereas broader domains of moderate accessibility tend to be more stable. In mitosis, proximal promoters generally maintain their accessibility more strongly, whereas distal regulatory elements tend to lose accessibility. Large domains of DNA hypomethylation mark a subset of promoters that retain accessibility during mitosis and across many cell types in interphase. Erythroid transcription factor GATA1 exerts site-specific changes in interphase accessibility that are most pronounced at distal regulatory elements, but has little influence on mitotic accessibility. We conclude that features of open chromatin are remarkably stable through mitosis, but are modulated at the level of individual genes and regulatory elements. © 2015 Hsiung et al.; Published by Cold Spring Harbor Laboratory Press.

The phosphorylation-dependent regulation of nuclear SREBP1 during mitosis links lipid metabolism and cell growth

PubMed Central

Bengoechea-Alonso, Maria Teresa; Ericsson, Johan

2016-01-01

ABSTRACT The SREBP transcription factors are major regulators of lipid metabolism. Disturbances in lipid metabolism are at the core of several health issues facing modern society, including cardiovascular disease, obesity and diabetes. In addition, the role of lipid metabolism in cancer cell growth is receiving increased attention. Transcriptionally active SREBP molecules are unstable and rapidly degraded in a phosphorylation-dependent manner by Fbw7, a ubiquitin ligase that targets several cell cycle regulatory proteins for degradation. We have previously demonstrated that active SREBP1 is stabilized during mitosis. We have now delineated the mechanisms involved in the stabilization of SREBP1 in mitotic cells. This process is initiated by the phosphorylation of a specific serine residue in nuclear SREBP1 by the mitotic kinase Cdk1. The phosphorylation of this residue creates a docking site for a separate mitotic kinase, Plk1. Plk1 interacts with nuclear SREBP1 in mitotic cells and phosphorylates a number of residues in the C-terminal domain of the protein, including a threonine residue in close proximity of the Fbw7 docking site in SREBP1. The phosphorylation of these residues by Plk1 blocks the interaction between SREBP1 and Fbw7 and attenuates the Fbw7-dependent degradation of nuclear SREBP1 during cell division. Inactivation of SREBP1 results in a mitotic defect, suggesting that SREBP1 could regulate cell division. We propose that the mitotic phosphorylation and stabilization of nuclear SREBP1 during cell division provides a link between lipid metabolism and cell proliferation. Thus, the current study provides additional support for the emerging hypothesis that SREBP-dependent lipid metabolism may be important for cell growth. PMID:27579997

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki, Masatoshi, E-mail: msuzuki@nagasaki-u.ac.jp; Yamauchi, Motohiro; Oka, Yasuyoshi

Purpose: Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Methods and Materials: Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Results: Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% andmore » 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO{sub 2}-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ss-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. Conclusions: The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation.« less

Changes in basal cell mitosis and transepidermal water loss in skin cultures treated with vitamins C and E.

PubMed

Parish, W E; Read, J; Paterson, S E

2005-09-01

Three variants of the living skin equivalent cultures were compared in order to determine the most suitable to grow human differentiated epidermis to test beneficial properties of nutrients. Criteria of culture quality were mitotic index and transepidermal water loss (TEWL) assayed by means of a ServoMed Evaporimeter EP-2TM (ServoMed, Kinna, Sweden). Standards were donor skin mean mitotic index 11.1% and TEWL of living subjects mean 6.4 g/m(2)/h. Cultures (i) in 5% serum, 10 ng/ml of epidermal growth factor (EGF) at 37 degrees C and 95% relative humidity (RH); mitotic index on day 14, 19.2%, but on day 21, 1.8% and TEWL 9.5 g/m(2)/h on day 18. (ii) In 1% serum, no EGF, 33 degrees C and 95% RH, mitotic index on day 21, 9.1% and TEWL, 9.5% on day 18. (iii) Culture in same medium, 33 degrees C and 60% RH, mitotic index on day 28, 9.5% and TEWL 6.1 g/m(2)/h on day 18 as in vivo. Incubation in 60% RH was achieved using a novel chamber and dishes exposing only the corneum, sealing the medium. Vitamins C and E were used as model test nutrients. Culture conditions were 1% serum, no EGF at 33 degrees C and 95% RH. Vitamin C at 142 and 284 microM increased the mitotic index after 10- and 15-day treatment, but at 586 microM it was weakly toxic. Vitamin E at 20 and 40 microM did not. Both vitamins reduced TEWL providing functional data in support of previous reports on barrier properties. These are functional biomarkers of skin benefit relevant to skin in vivo.

Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis

PubMed Central

Pillai, Smitha; Nguyen, Jonathan; Johnson, Joseph; Haura, Eric; Coppola, Domenico; Chellappan, Srikumar

2015-01-01

TANK Binding Kinase 1 (TBK1) is a non-canonical IκB kinase that contributes to KRAS-driven lung cancer. Here we report that TBK1 plays essential roles in mammalian cell division. Specifically, levels of active phospho-TBK1 increase during mitosis and localize to centrosomes, mitotic spindles and midbody, and selective inhibition or silencing of TBK1 triggers defects in spindle assembly and prevents mitotic progression. TBK1 binds to the centrosomal protein CEP170 and to the mitotic apparatus protein NuMA, and both CEP170 and NuMA are TBK1 substrates. Further, TBK1 is necessary for CEP170 centrosomal localization and binding to the microtubule depolymerase Kif2b, and for NuMA binding to dynein. Finally, selective disruption of the TBK1–CEP170 complex augments microtubule stability and triggers defects in mitosis, suggesting that TBK1 functions as a mitotic kinase necessary for microtubule dynamics and mitosis. PMID:26656453

GAK, a regulator of clathrin-mediated membrane traffic, also controls centrosome integrity and chromosome congression.

PubMed

Shimizu, Hiroyuki; Nagamori, Ippei; Yabuta, Norikazu; Nojima, Hiroshi

2009-09-01

Cyclin G-associated kinase (GAK) is an association partner of clathrin heavy chain (CHC) and is essential for clathrin-mediated membrane trafficking. Here, we report two novel functions of GAK: maintenance of proper centrosome maturation and of mitotic chromosome congression. Indeed, GAK knockdown by siRNA caused cell-cycle arrest at metaphase, which indicates that GAK is required for proper mitotic progression. We found that this impaired mitotic progression was due to activation of the spindle-assembly checkpoint, which senses protruded, misaligned or abnormally condensed chromosomes in GAK-siRNA-treated cells. GAK knockdown also caused multi-aster formation, which was due to abnormal fragmentation of pericentriolar material, but not of the centrioles. Moreover, GAK and CHC cooperated in the same pathway and interacted in mitosis to regulate the formation of a functional spindle. Taken together, we conclude that GAK and clathrin function cooperatively not only in endocytosis, but also in mitotic progression.

ARHGEF17 is an essential spindle assembly checkpoint factor that targets Mps1 to kinetochores

PubMed Central

Isokane, Mayumi; Walter, Thomas; Mahen, Robert; Nijmeijer, Bianca; Hériché, Jean-Karim; Miura, Kota; Maffini, Stefano; Ivanov, Miroslav Penchev; Kitajima, Tomoya S.; Peters, Jan-Michael

2016-01-01

To prevent genome instability, mitotic exit is delayed until all chromosomes are properly attached to the mitotic spindle by the spindle assembly checkpoint (SAC). In this study, we characterized the function of ARHGEF17, identified in a genome-wide RNA interference screen for human mitosis genes. Through a series of quantitative imaging, biochemical, and biophysical experiments, we showed that ARHGEF17 is essential for SAC activity, because it is the major targeting factor that controls localization of the checkpoint kinase Mps1 to the kinetochore. This mitotic function is mediated by direct interaction of the central domain of ARHGEF17 with Mps1, which is autoregulated by the activity of Mps1 kinase, for which ARHGEF17 is a substrate. This mitosis-specific role is independent of ARHGEF17’s RhoGEF activity in interphase. Our study thus assigns a new mitotic function to ARHGEF17 and reveals the molecular mechanism for a key step in SAC establishment. PMID:26953350

ARHGEF17 is an essential spindle assembly checkpoint factor that targets Mps1 to kinetochores.

PubMed

Isokane, Mayumi; Walter, Thomas; Mahen, Robert; Nijmeijer, Bianca; Hériché, Jean-Karim; Miura, Kota; Maffini, Stefano; Ivanov, Miroslav Penchev; Kitajima, Tomoya S; Peters, Jan-Michael; Ellenberg, Jan

2016-03-14

To prevent genome instability, mitotic exit is delayed until all chromosomes are properly attached to the mitotic spindle by the spindle assembly checkpoint (SAC). In this study, we characterized the function of ARHGEF17, identified in a genome-wide RNA interference screen for human mitosis genes. Through a series of quantitative imaging, biochemical, and biophysical experiments, we showed that ARHGEF17 is essential for SAC activity, because it is the major targeting factor that controls localization of the checkpoint kinase Mps1 to the kinetochore. This mitotic function is mediated by direct interaction of the central domain of ARHGEF17 with Mps1, which is autoregulated by the activity of Mps1 kinase, for which ARHGEF17 is a substrate. This mitosis-specific role is independent of ARHGEF17's RhoGEF activity in interphase. Our study thus assigns a new mitotic function to ARHGEF17 and reveals the molecular mechanism for a key step in SAC establishment. © 2016 Isokane et al.

Release of Mps1 from kinetochores is crucial for timely anaphase onset.

PubMed

Jelluma, Nannette; Dansen, Tobias B; Sliedrecht, Tale; Kwiatkowski, Nicholas P; Kops, Geert J P L

2010-10-18

Mps1 kinase activity is required for proper chromosome segregation during mitosis through its involvements in microtubule-chromosome attachment error correction and the mitotic checkpoint. Mps1 dynamically exchanges on unattached kinetochores but is largely removed from kinetochores in metaphase. Here we show that Mps1 promotes its own turnover at kinetochores and that removal of Mps1 upon chromosome biorientation is a prerequisite for mitotic checkpoint silencing. Inhibition of Mps1 activity increases its half-time of recovery at unattached kinetochores and causes accumulation of Mps1 protein at these sites. Strikingly, preventing dissociation of active Mps1 from kinetochores delays anaphase onset despite normal chromosome attachment and alignment, and high interkinetochore tension. This delay is marked by continued recruitment of Mad1 and Mad2 to bioriented chromosomes and is attenuated by Mad2 depletion, indicating chronic engagement of the mitotic checkpoint in metaphase. We propose that release of Mps1 from kinetochores is essential for mitotic checkpoint silencing and a fast metaphase-to-anaphase transition.

SIRT6 deacetylates H3K18Ac at pericentric chromatin to prevent mitotic errors and cell senescence

PubMed Central

Tasselli, Luisa; Xi, Yuanxin; Zheng, Wei; Tennen, Ruth I.; Odrowaz, Zaneta; Simeoni, Federica; Li, Wei; Chua, Katrin F.

2018-01-01

Pericentric heterochromatin silencing at mammalian centromeres is essential for mitotic fidelity and genomic stability. Defective pericentric silencing is observed in senescent cells, aging tissues, and mammalian tumors, but the underlying mechanisms and functional consequences of these defects are unclear. Here, we uncover a pivotal role of the human SIRT6 enzyme in pericentric transcriptional silencing, and show that this function protects against mitotic defects, genomic instability, and cellular senescence. At pericentric heterochromatin, SIRT6 promotes deacetylation of a new substrate, histone H3 lysine K18 (H3K18), and inactivation of SIRT6 in cells leads to H3K18 hyperacetylation and aberrant accumulation of pericentric transcripts. Strikingly, RNAi-depletion of these transcripts rescues the mitotic and senescence phenotypes of SIRT6-deficient cells. Together, our findings reveal a new function for SIRT6 and H3K18Ac regulation at heterochromatin, and demonstrate the pathogenic role of de-regulated pericentric transcription in aging- and cancer- related cellular dysfunction. PMID:27043296

γH2AX foci formation in the absence of DNA damage: mitotic H2AX phosphorylation is mediated by the DNA-PKcs/CHK2 pathway.

PubMed

Tu, Wen-Zhi; Li, Bing; Huang, Bo; Wang, Yu; Liu, Xiao-Dan; Guan, Hua; Zhang, Shi-Meng; Tang, Yan; Rang, Wei-Qing; Zhou, Ping-Kun

2013-11-01

Phosphorylated H2AX is considered to be a biomarker for DNA double-strand breaks (DSB), but recent evidence suggests that γH2AX does not always indicate the presence of DSB. Here we demonstrate the bimodal dynamic of H2AX phosphorylation induced by ionizing radiation, with the second peak appearing when G2/M arrest is induced. An increased level of γH2AX occurred in mitotic cells, and this increase was attenuated by DNA-PKcs inactivation or Chk2 depletion, but not by ATM inhibition. The phosphorylation-mimic CHK2-T68D abrogated the attenuation of mitotic γH2AX induced by DNA-PKcs inactivation. Thus, the DNA-PKcs/CHK2 pathway mediates the mitotic phosphorylation of H2AX in the absence of DNA damage. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Inhibition of Bcl-xL sensitizes cells to mitotic blockers, but not mitotic drivers

PubMed Central

Bennett, Ailsa; Sloss, Olivia; Topham, Caroline; Nelson, Louisa; Tighe, Anthony

2016-01-01

Cell fate in response to an aberrant mitosis is governed by two competing networks: the spindle assembly checkpoint (SAC) and the intrinsic apoptosis pathway. The mechanistic interplay between these two networks is obscured by functional redundancy and the ability of cells to die either in mitosis or in the subsequent interphase. By coupling time-lapse microscopy with selective pharmacological agents, we systematically probe pro-survival Bcl-xL in response to various mitotic perturbations. Concentration matrices show that BH3-mimetic-mediated inhibition of Bcl-xL synergises with perturbations that induce an SAC-mediated mitotic block, including drugs that dampen microtubule dynamics, and inhibitors targeting kinesins and kinases required for spindle assembly. By contrast, Bcl-xL inhibition does not synergize with drugs which drive cells through an aberrant mitosis by overriding the SAC. This differential effect, which is explained by compensatory Mcl-1 function, provides opportunities for patient stratification and combination treatments in the context of cancer chemotherapy. PMID:27512141

Changes in Ect2 Localization Couple Actomyosin-Dependent Cell Shape Changes to Mitotic Progression

PubMed Central

Matthews, Helen K.; Delabre, Ulysse; Rohn, Jennifer L.; Guck, Jochen; Kunda, Patricia; Baum, Buzz

2012-01-01

Summary As they enter mitosis, animal cells undergo profound actin-dependent changes in shape to become round. Here we identify the Cdk1 substrate, Ect2, as a central regulator of mitotic rounding, thus uncovering a link between the cell-cycle machinery that drives mitotic entry and its accompanying actin remodeling. Ect2 is a RhoGEF that plays a well-established role in formation of the actomyosin contractile ring at mitotic exit, through the local activation of RhoA. We find that Ect2 first becomes active in prophase, when it is exported from the nucleus into the cytoplasm, activating RhoA to induce the formation of a mechanically stiff and rounded metaphase cortex. Then, at anaphase, binding to RacGAP1 at the spindle midzone repositions Ect2 to induce local actomyosin ring formation. Ect2 localization therefore defines the stage-specific changes in actin cortex organization critical for accurate cell division. PMID:22898780

Prolonged Mitosis of Neural Progenitors Alters Cell Fate in the Developing Brain.

PubMed

Pilaz, Louis-Jan; McMahon, John J; Miller, Emily E; Lennox, Ashley L; Suzuki, Aussie; Salmon, Edward; Silver, Debra L

2016-01-06

Embryonic neocortical development depends on balanced production of progenitors and neurons. Genetic mutations disrupting progenitor mitosis frequently impair neurogenesis; however, the link between altered mitosis and cell fate remains poorly understood. Here we demonstrate that prolonged mitosis of radial glial progenitors directly alters neuronal fate specification and progeny viability. Live imaging of progenitors from a neurogenesis mutant, Magoh(+/-), reveals that mitotic delay significantly correlates with preferential production of neurons instead of progenitors, as well as apoptotic progeny. Independently, two pharmacological approaches reveal a causal relationship between mitotic delay and progeny fate. As mitotic duration increases, progenitors produce substantially more apoptotic progeny or neurons. We show that apoptosis, but not differentiation, is p53 dependent, demonstrating that these are distinct outcomes of mitotic delay. Together our findings reveal that prolonged mitosis is sufficient to alter fates of radial glia progeny and define a new paradigm to understand how mitosis perturbations underlie brain size disorders such as microcephaly. Copyright © 2016 Elsevier Inc. All rights reserved.

Mitotic Transcriptional Activation: Clearance of Actively Engaged Pol II via Transcriptional Elongation Control in Mitosis.

PubMed

Liang, Kaiwei; Woodfin, Ashley R; Slaughter, Brian D; Unruh, Jay R; Box, Andrew C; Rickels, Ryan A; Gao, Xin; Haug, Jeffrey S; Jaspersen, Sue L; Shilatifard, Ali

2015-11-05

Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division. Copyright © 2015 Elsevier Inc. All rights reserved.

PLK1 has tumor-suppressive potential in APC-truncated colon cancer cells.

PubMed

Raab, Monika; Sanhaji, Mourad; Matthess, Yves; Hörlin, Albrecht; Lorenz, Ioana; Dötsch, Christina; Habbe, Nils; Waidmann, Oliver; Kurunci-Csacsko, Elisabeth; Firestein, Ron; Becker, Sven; Strebhardt, Klaus

2018-03-16

The spindle assembly checkpoint (SAC) acts as a molecular safeguard in ensuring faithful chromosome transmission during mitosis, which is regulated by a complex interplay between phosphatases and kinases including PLK1. Adenomatous polyposis coli (APC) germline mutations cause aneuploidy and are responsible for familial adenomatous polyposis (FAP). Here we study the role of PLK1 in colon cancer cells with chromosomal instability promoted by APC truncation (APC-ΔC). The expression of APC-ΔC in colon cells reduces the accumulation of mitotic cells upon PLK1 inhibition, accelerates mitotic exit and increases the survival of cells with enhanced chromosomal abnormalities. The inhibition of PLK1 in mitotic, APC-∆C-expressing cells reduces the kinetochore levels of Aurora B and hampers the recruitment of SAC component suggesting a compromised mitotic checkpoint. Furthermore, Plk1 inhibition (RNAi, pharmacological compounds) promotes the development of adenomatous polyps in two independent Apc Min/+ mouse models. High PLK1 expression increases the survival of colon cancer patients expressing a truncated APC significantly.

BRCA1 interaction of centrosomal protein Nlp is required for successful mitotic progression.

PubMed

Jin, Shunqian; Gao, Hua; Mazzacurati, Lucia; Wang, Yang; Fan, Wenhong; Chen, Qiang; Yu, Wei; Wang, Mingrong; Zhu, Xueliang; Zhang, Chuanmao; Zhan, Qimin

2009-08-21

Breast cancer susceptibility gene BRCA1 is implicated in the control of mitotic progression, although the underlying mechanism(s) remains to be further defined. Deficiency of BRCA1 function leads to disrupted mitotic machinery and genomic instability. Here, we show that BRCA1 physically interacts and colocalizes with Nlp, an important molecule involved in centrosome maturation and spindle formation. Interestingly, Nlp centrosomal localization and its protein stability are regulated by normal cellular BRCA1 function because cells containing BRCA1 mutations or silenced for endogenous BRCA1 exhibit disrupted Nlp colocalization to centrosomes and enhanced Nlp degradation. Its is likely that the BRCA1 regulation of Nlp stability involves Plk1 suppression. Inhibition of endogenous Nlp via the small interfering RNA approach results in aberrant spindle formation, aborted chromosomal segregation, and aneuploidy, which mimic the phenotypes of disrupted BRCA1. Thus, BRCA1 interaction of Nlp might be required for the successful mitotic progression, and abnormalities of Nlp lead to genomic instability.

BRCA1 Interaction of Centrosomal Protein Nlp Is Required for Successful Mitotic Progression*♦

PubMed Central

Jin, Shunqian; Gao, Hua; Mazzacurati, Lucia; Wang, Yang; Fan, Wenhong; Chen, Qiang; Yu, Wei; Wang, Mingrong; Zhu, Xueliang; Zhang, Chuanmao; Zhan, Qimin

2009-01-01

Breast cancer susceptibility gene BRCA1 is implicated in the control of mitotic progression, although the underlying mechanism(s) remains to be further defined. Deficiency of BRCA1 function leads to disrupted mitotic machinery and genomic instability. Here, we show that BRCA1 physically interacts and colocalizes with Nlp, an important molecule involved in centrosome maturation and spindle formation. Interestingly, Nlp centrosomal localization and its protein stability are regulated by normal cellular BRCA1 function because cells containing BRCA1 mutations or silenced for endogenous BRCA1 exhibit disrupted Nlp colocalization to centrosomes and enhanced Nlp degradation. Its is likely that the BRCA1 regulation of Nlp stability involves Plk1 suppression. Inhibition of endogenous Nlp via the small interfering RNA approach results in aberrant spindle formation, aborted chromosomal segregation, and aneuploidy, which mimic the phenotypes of disrupted BRCA1. Thus, BRCA1 interaction of Nlp might be required for the successful mitotic progression, and abnormalities of Nlp lead to genomic instability. PMID:19509300

The timing of synthesis of proteins required for mitotic spindle and phragmoplast in partially synchronized root meristems of Vicia faba L.

PubMed

Olszewska, M J; Marciniak, K; Kuran, H

1990-10-01

After cycloheximide treatment (1 h, 2.5 micrograms/ml) protein synthesis was decreased by 70% and was partially restored after 7 h of postincubation (still 20% decrease). In partially synchronized root meristems of Vicia faba L. treated with cycloheximide at middle G2, a strong decrease of the mitotic index was observed. Exposure to the drug at late G2 did not modify the mitotic index; the changes in the phase indices suggested that the course of mitosis was blocked at prophase-metaphase/anaphase-telophase transitions. The use of indirect immunocytochemical staining of tubulin (second antibody labeled with peroxidase) made it possible to show a decreased number of cells with preprophase bands in cycloheximide-treated meristems and the mitotic spindles and phragmoplasts containing a reduced number of shortened bands of microtubules. As a result of these structural and functional disturbances, binucleate cells and polyploid nuclei were observed.

Automatic digital image analysis for identification of mitotic cells in synchronous mammalian cell cultures.

PubMed

Eccles, B A; Klevecz, R R

1986-06-01

Mitotic frequency in a synchronous culture of mammalian cells was determined fully automatically and in real time using low-intensity phase-contrast microscopy and a newvicon video camera connected to an EyeCom III image processor. Image samples, at a frequency of one per minute for 50 hours, were analyzed by first extracting the high-frequency picture components, then thresholding and probing for annular objects indicative of putative mitotic cells. Both the extraction of high-frequency components and the recognition of rings of varying radii and discontinuities employed novel algorithms. Spatial and temporal relationships between annuli were examined to discern the occurrences of mitoses, and such events were recorded in a computer data file. At present, the automatic analysis is suited for random cell proliferation rate measurements or cell cycle studies. The automatic identification of mitotic cells as described here provides a measure of the average proliferative activity of the cell population as a whole and eliminates more than eight hours of manual review per time-lapse video recording.

Kinesin-5–dependent Poleward Flux and Spindle Length Control in Drosophila Embryo Mitosis

PubMed Central

Brust-Mascher, Ingrid; Sommi, Patrizia; Cheerambathur, Dhanya K.

2009-01-01

We used antibody microinjection and genetic manipulations to dissect the various roles of the homotetrameric kinesin-5, KLP61F, in astral, centrosome-controlled Drosophila embryo spindles and to test the hypothesis that it slides apart interpolar (ip) microtubules (MT), thereby controlling poleward flux and spindle length. In wild-type and Ncd null mutant embryos, anti-KLP61F dissociated the motor from spindles, producing a spatial gradient in the KLP61F content of different spindles, which was visible in KLP61F-GFP transgenic embryos. The resulting mitotic defects, supported by gene dosage experiments and time-lapse microscopy of living klp61f mutants, reveal that, after NEB, KLP61F drives persistent MT bundling and the outward sliding of antiparallel MTs, thereby contributing to several processes that all appear insensitive to cortical disruption. KLP61F activity contributes to the poleward flux of both ipMTs and kinetochore MTs and to the length of the metaphase spindle. KLP61F activity maintains the prometaphase spindle by antagonizing Ncd and another unknown force-generator and drives anaphase B, although the rate of spindle elongation is relatively insensitive to the motor's concentration. Finally, KLP61F activity contributes to normal chromosome congression, kinetochore spacing, and anaphase A rates. Thus, a KLP61F-driven sliding filament mechanism contributes to multiple aspects of mitosis in this system. PMID:19158379

Targeted interactomics reveals a complex core cell cycle machinery in Arabidopsis thaliana.

PubMed

Van Leene, Jelle; Hollunder, Jens; Eeckhout, Dominique; Persiau, Geert; Van De Slijke, Eveline; Stals, Hilde; Van Isterdael, Gert; Verkest, Aurine; Neirynck, Sandy; Buffel, Yelle; De Bodt, Stefanie; Maere, Steven; Laukens, Kris; Pharazyn, Anne; Ferreira, Paulo C G; Eloy, Nubia; Renne, Charlotte; Meyer, Christian; Faure, Jean-Denis; Steinbrenner, Jens; Beynon, Jim; Larkin, John C; Van de Peer, Yves; Hilson, Pierre; Kuiper, Martin; De Veylder, Lieven; Van Onckelen, Harry; Inzé, Dirk; Witters, Erwin; De Jaeger, Geert

2010-08-10

Cell proliferation is the main driving force for plant growth. Although genome sequence analysis revealed a high number of cell cycle genes in plants, little is known about the molecular complexes steering cell division. In a targeted proteomics approach, we mapped the core complex machinery at the heart of the Arabidopsis thaliana cell cycle control. Besides a central regulatory network of core complexes, we distinguished a peripheral network that links the core machinery to up- and downstream pathways. Over 100 new candidate cell cycle proteins were predicted and an in-depth biological interpretation demonstrated the hypothesis-generating power of the interaction data. The data set provided a comprehensive view on heterodimeric cyclin-dependent kinase (CDK)-cyclin complexes in plants. For the first time, inhibitory proteins of plant-specific B-type CDKs were discovered and the anaphase-promoting complex was characterized and extended. Important conclusions were that mitotic A- and B-type cyclins form complexes with the plant-specific B-type CDKs and not with CDKA;1, and that D-type cyclins and S-phase-specific A-type cyclins seem to be associated exclusively with CDKA;1. Furthermore, we could show that plants have evolved a combinatorial toolkit consisting of at least 92 different CDK-cyclin complex variants, which strongly underscores the functional diversification among the large family of cyclins and reflects the pivotal role of cell cycle regulation in the developmental plasticity of plants.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hitomi, Toshiaki; Habu, Toshiyuki; Kobayashi, Hatasu

Highlights: •Overexpression of RNF213 R4810K inhibited cell proliferation. •Overexpression of RNF213 R4810K had the time of mitosis 4-fold and mitotic failure. •R4810K formed a complex with MAD2 more readily than wild-type. •iPSECs from the MMD patients had elevated mitotic failure compared from the control. •RNF213 R4810K induced mitotic abnormality and increased risk of aneuploidy. -- Abstract: Moyamoya disease (MMD) is a cerebrovascular disease characterized by occlusive lesions in the Circle of Willis. The RNF213 R4810K polymorphism increases susceptibility to MMD. In the present study, we characterized phenotypes caused by overexpression of RNF213 wild type and R4810K variant in the cellmore » cycle to investigate the mechanism of proliferation inhibition. Overexpression of RNF213 R4810K in HeLa cells inhibited cell proliferation and extended the time of mitosis 4-fold. Ablation of spindle checkpoint by depletion of mitotic arrest deficiency 2 (MAD2) did not shorten the time of mitosis. Mitotic morphology in HeLa cells revealed that MAD2 colocalized with RNF213 R4810K. Immunoprecipitation revealed an RNF213/MAD2 complex: R4810K formed a complex with MAD2 more readily than RNF213 wild-type. Desynchronized localization of MAD2 was observed more frequently during mitosis in fibroblasts from patients (n = 3, 61.0 ± 8.2%) compared with wild-type subjects (n = 6, 13.1 ± 7.7%; p < 0.01). Aneuploidy was observed more frequently in fibroblasts (p < 0.01) and induced pluripotent stem cells (iPSCs) (p < 0.03) from patients than from wild-type subjects. Vascular endothelial cells differentiated from iPSCs (iPSECs) of patients and an unaffected carrier had a longer time from prometaphase to metaphase than those from controls (p < 0.05). iPSECs from the patients and unaffected carrier had significantly increased mitotic failure rates compared with controls (p < 0.05). Thus, RNF213 R4810K induced mitotic abnormalities and increased risk of genomic instability.« less

Characterization of novel MPS1 inhibitors with preclinical anticancer activity.

PubMed

Jemaà, M; Galluzzi, L; Kepp, O; Senovilla, L; Brands, M; Boemer, U; Koppitz, M; Lienau, P; Prechtl, S; Schulze, V; Siemeister, G; Wengner, A M; Mumberg, D; Ziegelbauer, K; Abrieu, A; Castedo, M; Vitale, I; Kroemer, G

2013-11-01

Monopolar spindle 1 (MPS1), a mitotic kinase that is overexpressed in several human cancers, contributes to the alignment of chromosomes to the metaphase plate as well as to the execution of the spindle assembly checkpoint (SAC). Here, we report the identification and functional characterization of three novel inhibitors of MPS1 of two independent structural classes, N-(4-{2-[(2-cyanophenyl)amino][1,2,4]triazolo[1,5-a]pyridin-6-yl}phenyl)-2-phenylacetamide (Mps-BAY1) (a triazolopyridine), N-cyclopropyl-4-{8-[(2-methylpropyl)amino]-6-(quinolin-5-yl)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2a) and N-cyclopropyl-4-{8-(isobutylamino)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2b) (two imidazopyrazines). By selectively inactivating MPS1, these small inhibitors can arrest the proliferation of cancer cells, causing their polyploidization and/or their demise. Cancer cells treated with Mps-BAY1 or Mps-BAY2a manifested multiple signs of mitotic perturbation including inefficient chromosomal congression during metaphase, unscheduled SAC inactivation and severe anaphase defects. Videomicroscopic cell fate profiling of histone 2B-green fluorescent protein-expressing cells revealed the capacity of MPS1 inhibitors to subvert the correct timing of mitosis as they induce a premature anaphase entry in the context of misaligned metaphase plates. Hence, in the presence of MPS1 inhibitors, cells either divided in a bipolar (but often asymmetric) manner or entered one or more rounds of abortive mitoses, generating gross aneuploidy and polyploidy, respectively. In both cases, cells ultimately succumbed to the mitotic catastrophe-induced activation of the mitochondrial pathway of apoptosis. Of note, low doses of MPS1 inhibitors and paclitaxel (a microtubular poison) synergized at increasing the frequency of chromosome misalignments and missegregations in the context of SAC inactivation. This resulted in massive polyploidization followed by the activation of mitotic catastrophe. A synergistic interaction between paclitaxel and MPS1 inhibitors could also be demonstrated in vivo, as the combination of these agents efficiently reduced the growth of tumor xenografts and exerted superior antineoplastic effects compared with either compound employed alone. Altogether, these results suggest that MPS1 inhibitors may exert robust anticancer activity, either as standalone therapeutic interventions or combined with microtubule-targeting chemicals.

The gammaTuRC Nanomachine Mechanism and Future Applications

NASA Astrophysics Data System (ADS)

Riehlman, Timothy D.

The complexity and precision of the eukaryotic cell's cytoskeletal network is unrivaled by any man-made systems, perfected by billions of years of evolution, mastering elegant processes of self-assembly, error correction, and self-repair. Understanding the capabilities of these networks will have important and far reaching applications in human medicine by aiding our understanding of developmental processes, cellular division, and disease mechanisms, and through biomimicry will provide insights for biosynthetic manufacturing at the nanoscale and across scales. My research utilizes cross species techniques from Human to the model organism of Fission Yeast to investigate the structure and mechanisms of the g-tubulin ring complex (gTuRC). The gTuRC is a highly conserved eukaryotic multiprotein complex serving as a microtubule organizing center (MTOC) responsible for microtubule nucleation through templating, regulation of dynamics, and establishment of microtubule polarity. Microtubules are 25 nm diameter dynamic flexible polymers of a/b-tubulin heterodimers that function as scaffolds, force generators, distributors, and intracellular highways. The microtubule cytoskeleton is essential for numerous fundamental cellular processes such as mitotic division of chromosomes and cell division, organelle distribution within the cell, cell signaling, and cell shape. This incredible diversity in functions is made possible in part due to molecular motor Kinesin-like proteins (Klps), which allow expansion into more specialized neural, immune, and ciliated cell functions. Combined, the MTOC, microtubules, and Klps represent ideal microtubule cytoskeleton protein (MCP) modular components for in vitro biomimicry towards generation of adaptable patterned networks for human designed applications. My research investigates the hypothesis that a mechanistic understanding of conserved MTOC gTuRC mechanisms will help us understand dynamic cellular nanomachines and their ability to self-assemble complex structures for applications in biomedicine and new roles in biomimetic nanotechnologies.

Comparison of cytotoxic and genotoxic effects of plutonium-239 alpha particles and mobile phone GSM 900 radiation in the Allium cepa test.

PubMed

Pesnya, Dmitry S; Romanovsky, Anton V

2013-01-20

The goal of this study was to compare the cytotoxic and genotoxic effects of plutonium-239 alpha particles and GSM 900 modulated mobile phone (model Sony Ericsson K550i) radiation in the Allium cepa test. Three groups of bulbs were exposed to mobile phone radiation during 0 (sham), 3 and 9h. A positive control group was treated during 20min with plutonium-239 alpha-radiation. Mitotic abnormalities, chromosome aberrations, micronuclei and mitotic index were analyzed. Exposure to alpha-radiation from plutonium-239 and exposure to modulated radiation from mobile phone during 3 and 9h significantly increased the mitotic index. GSM 900 mobile phone radiation as well as alpha-radiation from plutonium-239 induced both clastogenic and aneugenic effects. However, the aneugenic activity of mobile phone radiation was more pronounced. After 9h of exposure to mobile phone radiation, polyploid cells, three-groups metaphases, amitoses and some unspecified abnormalities were detected, which were not registered in the other experimental groups. Importantly, GSM 900 mobile phone radiation increased the mitotic index, the frequency of mitotic and chromosome abnormalities, and the micronucleus frequency in a time-dependent manner. Due to its sensitivity, the A. cepa test can be recommended as a useful cytogenetic assay to assess cytotoxic and genotoxic effects of radiofrequency electromagnetic fields. Copyright © 2012 Elsevier B.V. All rights reserved.

CDK-dependent potentiation of MPS1 kinase activity is essential to the mitotic checkpoint.

PubMed

Morin, Violeta; Prieto, Susana; Melines, Sabrina; Hem, Sonia; Rossignol, Michel; Lorca, Thierry; Espeut, Julien; Morin, Nathalie; Abrieu, Ariane

2012-02-21

Accurate chromosome segregation relies upon a mitotic checkpoint that monitors kinetochore attachment toward opposite spindle poles before enabling chromosome disjunction [1]. The MPS1/TTK protein kinase is a core component of the mitotic checkpoint that lies upstream of MAD2 and BubR1 both at the kinetochore and in the cytoplasm [2, 3]. To gain insight into the mechanisms underlying the regulation of MPS1 kinase, we undertook the identification of Xenopus MPS1 phosphorylation sites by mass spectrometry. We mapped several phosphorylation sites onto MPS1 and we show that phosphorylation of S283 in the noncatalytic region of MPS1 is required for full kinase activity. This phosphorylation potentiates MPS1 catalytic efficiency without impairing its affinity for the substrates. By using Xenopus egg extracts depleted of endogenous MPS1 and reconstituted with single point mutants, we show that phosphorylation of S283 is essential to activate the mitotic checkpoint. This phosphorylation does not regulate the localization of MPS1 to the kinetochore but is required for the recruitment of MAD1/MAD2, demonstrating its role at the kinetochore. Constitutive phosphorylation of S283 lowers the number of kinetochores required to hold the checkpoint, which suggests that CDK-dependent phosphorylation of MPS1 is essential to sustain the mitotic checkpoint when few kinetochores remain unattached. Copyright © 2012 Elsevier Ltd. All rights reserved.

900 MHz radiation does not induce micronucleus formation in different cell types.

PubMed

Hintzsche, Henning; Jastrow, Christian; Kleine-Ostmann, Thomas; Schrader, Thorsten; Stopper, Helga

2012-07-01

The exposure of the population to non-ionising electromagnetic radiation is still increasing, mainly due to mobile communication. Whether low-intensity electromagnetic fields can cause other effects apart from heating has been a subject of debate. One of the effects, which were proposed to be caused by mobile phone radiation, is the occurrence of mitotic disturbances. The aim of this study was to investigate possible consequences of these mitotic disturbances as manifest genomic damage, i.e. micronucleus induction. Cells were irradiated at a frequency of 900 MHz, which is located in one of the main frequency bands applied for mobile communication. Two cell types were used, HaCaT cells as human cells and A(L) cells (human-hamster hybrid cells), in which mitotic disturbances had been reported to occur. After different post-exposure incubation periods, cells were fixed and micronucleus frequencies were evaluated. Both cell types did not show any genomic damage after exposure. To adapt the protocol for the micronucleus test into the direction of the protocol for mitotic disturbances, the post-exposure incubation period was reduced and exposure time was extended to one cell cycle length. This did not result in any increase of the genomic damage. In conclusion, micronucleus induction was not observed as a consequence of exposure to non-ionising radiation, even though this agent was reported to cause mitotic disturbances under similar experimental conditions.

Deficiency of RITA results in multiple mitotic defects by affecting microtubule dynamics.

PubMed

Steinhäuser, K; Klöble, P; Kreis, N-N; Ritter, A; Friemel, A; Roth, S; Reichel, J M; Michaelis, J; Rieger, M A; Louwen, F; Oswald, F; Yuan, J

2017-04-01

Deregulation of mitotic microtubule (MT) dynamics results in defective spindle assembly and chromosome missegregation, leading further to chromosome instability, a hallmark of tumor cells. RBP-J interacting and tubulin-associated protein (RITA) has been identified as a negative regulator of the Notch signaling pathway. Intriguingly, deregulated RITA is involved in primary hepatocellular carcinoma and other malignant entities. We were interested in the potential molecular mechanisms behind its involvement. We show here that RITA binds to tubulin and localizes to various mitotic MT structures. RITA coats MTs and affects their structures in vitro as well as in vivo. Tumor cell lines deficient of RITA display increased acetylated α-tubulin, enhanced MT stability and reduced MT dynamics, accompanied by multiple mitotic defects, including chromosome misalignment and segregation errors. Re-expression of wild-type RITA, but not RITA Δtub ineffectively binding to tubulin, restores the phenotypes, suggesting that the role of RITA in MT modulation is mediated via its interaction with tubulin. Mechanistically, RITA interacts with tubulin/histone deacetylase 6 (HDAC6) and its suppression decreases the binding of the deacetylase HDAC6 to tubulin/MTs. Furthermore, the mitotic defects and increased MT stability are also observed in RITA -/- mouse embryonic fibroblasts. RITA has thus a novel role in modulating MT dynamics and its deregulation results in erroneous chromosome segregation, one of the major reasons for chromosome instability in tumor cells.

Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins.

PubMed

Colin, Didier J; Hain, Karolina O; Allan, Lindsey A; Clarke, Paul R

2015-03-01

Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.

Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins

PubMed Central

Colin, Didier J.; Hain, Karolina O.; Allan, Lindsey A.; Clarke, Paul R.

2015-01-01

Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies. PMID:25761368

Mammalian polo-like kinase 1-dependent regulation of the PBIP1-CENP-Q complex at kinetochores.

PubMed

Kang, Young H; Park, Chi Hoon; Kim, Tae-Sung; Soung, Nak-Kyun; Bang, Jeong K; Kim, Bo Y; Park, Jung-Eun; Lee, Kyung S

2011-06-03

Mammalian polo-like kinase 1 (Plk1) plays a pivotal role during M-phase progression. Plk1 localizes to specific subcellular structures through the targeting activity of the C-terminal polo-box domain (PBD). Disruption of the PBD function results in improper bipolar spindle formation, chromosome missegregation, and cytokinesis defect that ultimately lead to the generation of aneuploidy. It has been shown that Plk1 recruits itself to centromeres by phosphorylating and binding to a centromere scaffold, PBIP1 (also called MLF1IP and CENP-U[50]) through its PBD. However, how PBIP1 itself is targeted to centromeres and what roles it plays in the regulation of Plk1-dependent mitotic events remain unknown. Here, we demonstrated that PBIP1 directly interacts with CENP-Q, and this interaction was mutually required not only for their stability but also for their centromere localization. Plk1 did not appear to interact with CENP-Q directly. However, Plk1 formed a ternary complex with PBIP1 and CENP-Q through a self-generated p-T78 motif on PBIP1. This complex formation was central for Plk1-dependent phosphorylation of PBIP1-bound CENP-Q and delocalization of the PBIP1-CENP-Q complex from mitotic centromeres. This study reveals a unique mechanism of how PBIP1 mediates Plk1-dependent phosphorylation event onto a third protein, and provides new insights into the mechanism of how Plk1 and its recruitment scaffold, PBIP1-CENP-Q complex, are localized to and delocalized from centromeres.

Mammalian Polo-like Kinase 1-dependent Regulation of the PBIP1-CENP-Q Complex at Kinetochores*

PubMed Central

Kang, Young H.; Park, Chi Hoon; Kim, Tae-Sung; Soung, Nak-Kyun; Bang, Jeong K.; Kim, Bo Y.; Park, Jung-Eun; Lee, Kyung S.

2011-01-01

Mammalian polo-like kinase 1 (Plk1) plays a pivotal role during M-phase progression. Plk1 localizes to specific subcellular structures through the targeting activity of the C-terminal polo-box domain (PBD). Disruption of the PBD function results in improper bipolar spindle formation, chromosome missegregation, and cytokinesis defect that ultimately lead to the generation of aneuploidy. It has been shown that Plk1 recruits itself to centromeres by phosphorylating and binding to a centromere scaffold, PBIP1 (also called MLF1IP and CENP-U[50]) through its PBD. However, how PBIP1 itself is targeted to centromeres and what roles it plays in the regulation of Plk1-dependent mitotic events remain unknown. Here, we demonstrated that PBIP1 directly interacts with CENP-Q, and this interaction was mutually required not only for their stability but also for their centromere localization. Plk1 did not appear to interact with CENP-Q directly. However, Plk1 formed a ternary complex with PBIP1 and CENP-Q through a self-generated p-T78 motif on PBIP1. This complex formation was central for Plk1-dependent phosphorylation of PBIP1-bound CENP-Q and delocalization of the PBIP1-CENP-Q complex from mitotic centromeres. This study reveals a unique mechanism of how PBIP1 mediates Plk1-dependent phosphorylation event onto a third protein, and provides new insights into the mechanism of how Plk1 and its recruitment scaffold, PBIP1-CENP-Q complex, are localized to and delocalized from centromeres. PMID:21454580

Force Control Characteristics for Generation and Relaxation in the Lower Limb.

PubMed

Ohtaka, Chiaki; Fujiwara, Motoko

2018-05-29

We investigated the characteristics for force generation and relaxation using graded isometric contractions of the knee extensors. Participants performed the following tasks as quickly and accurately as possible. For the force generation task, force was increased from 0% to 20%, 40% and 60% of the maximal voluntary force (MVF). For the force relaxation task, force was decreased from 60% to 40%, 20% and 0%. The following parameters of the recorded force were calculated: error, time, and rate of force development. The error was consistently greater for force relaxation than generation. Reaction and adjustment times were independent of the tasks. The control strategy was markedly different for force relaxation and generation, this tendency was particularly evident for the lower limb compared to the upper limb.

The yeast SUMO isopeptidase Smt4/Ulp2 and the polo kinase Cdc5 act in an opposing fashion to regulate sumoylation in mitosis and cohesion at centromeres.

PubMed

Baldwin, Melissa L; Julius, Jeffrey A; Tang, Xianying; Wang, Yanchang; Bachant, Jeff

2009-10-15

Post-translation modification through the SUMO pathway is cell cycle regulated, with specific SUMO conjugates accumulating in mitotic cells. The basis for this regulation, however, and its functional significance remain poorly understood. We present evidence that in budding yeast sumoylation during mitosis may be controlled through the SUMO deconjugating enzyme Smt4/Ulp2. We isolated the polo kinase Cdc5 as an Ulp2-interacting protein, and find a C-terminal region of Ulp2 is phosphorylated during mitosis in a Cdc5-dependent manner. cdc5 mutants display reduced levels of mitotic SUMO conjugates, suggesting Cdc5 may negatively regulate Ulp2 to promote sumoylation. Previously, we found one phenotype associated with ulp2 mutants is an inability to maintain chromatid cohesion at centromere-proximal chromosomal regions. We now show this defect is rescued by inactivating Cdc5, indicating Ulp2 maintains cohesion by counter-acting Cdc5 activity. The cohesinregulator Pds5 is a likely target of this pathway, as Cdc5 overproduction forces Pds5 dissociation from chromosomes and Pds5 overproduction restores cohesion in ulp2 mutants. Overall, these observations reveal Cdc5 is a novel regulator of the SUMO pathway and suggest the outlines of a broader circuitry in which Ulp2 and Cdc5 act in a mutually antagonistic fashion to modulate maintenance and dissolution of cohesion at centromeres.

Estimation of mutagenic effect and modifications of mitosis by silver nanoparticles.

PubMed

Prokhorova, I M; Kibrik, B S; Pavlov, A V; Pesnya, D S

2013-12-01

We analyzed mutagenic and mitosis-modifying effects of silver nanoparticles (Allium test). Chromosome aberrations and laggings and micronuclei were simultaneously registered in the same sample. Mitotic and phase indexes were calculated. No mutagenic effects were detected after treatment with silver nanoparticles in doses of 1.0, 2.5, 5.0, and 50 mg/liter. Silver nanoparticles in a concentration of 50 mg/liter significantly increased the mitotic index. Nanoparticles in a dose of 5 mg/liter induced slight, but significant increase in mitotic index, but did not affect the ratio of phase indexes. Exposure to silver nanoparticles in concentrations of 1.0 and 2.5 mg/liter was not followed by modification of mitosis.

Antagonistic effects of pemoline to colchicine and caffeine.

PubMed

Röper, W

1975-10-15

Pemoline, the constituent of Tradon, is able to slow down the decrease of the mitotic index caused by 0.1% caffeine in roots of Vicia faba, and mitotic aberrations are reduced. With 0.005% colchicine and 3 x 10(-4) g/ml pemoline, no metaphase-accumulation can be observed, and anaphase-disorder is delayed.

Temporal Regulation of Lipin Activity Diverged to Account for Differences in Mitotic Programs

PubMed Central

Makarova, Maria; Gu, Ying; Chen, Jun-Song; Beckley, Janel Renée; Gould, Kathleen Louise; Oliferenko, Snezhana

2016-01-01

Summary Eukaryotes remodel the nucleus during mitosis using a variety of mechanisms that differ in the timing and the extent of nuclear envelope (NE) breakdown. Here, we probe the principles enabling this functional diversity by exploiting the natural divergence in NE management strategies between the related fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus [1, 2, 3]. We show that inactivation of Ned1, the phosphatidic acid phosphatase of the lipin family, by CDK phosphorylation is both necessary and sufficient to promote NE expansion required for “closed” mitosis in S. pombe. In contrast, Ned1 is not regulated during division in S. japonicus, thus limiting membrane availability and necessitating NE breakage. Interspecies gene swaps result in phenotypically normal divisions with the S. japonicus lipin acquiring an S. pombe-like mitotic phosphorylation pattern. Our results provide experimental evidence for the mitotic regulation of phosphatidic acid flux and suggest that the regulatory networks governing lipin activity diverged in evolution to give rise to strikingly dissimilar mitotic programs. PMID:26774782

The Aurora kinase A inhibitor TC-A2317 disrupts mitotic progression and inhibits cancer cell proliferation

PubMed Central

Min, Yoo Hong; Kim, Wootae; Kim, Ja-Eun

2016-01-01

Mitotic progression is crucial for the maintenance of chromosomal stability. A proper progression is ensured by the activities of multiple kinases. One of these enzymes, the serine/threonine kinase Aurora A, is required for proper mitosis through the regulation of centrosome and spindle assembly. In this study, we functionally characterized a newly developed Aurora kinase A inhibitor, TC-A2317. In human lung cancer cells, TC-A2317 slowed proliferation by causing aberrant formation of centrosome and microtubule spindles and prolonging the duration of mitosis. Abnormal mitotic progression led to accumulation of cells containing micronuclei or multinuclei. Furthermore, TC-A2317–treated cells underwent apoptosis, autophagy or senescence depending on cell type. In addition, TC-A2317 inactivated the spindle assembly checkpoint triggered by paclitaxel, thereby exacerbating mitotic catastrophe. Consistent with this, the expression level of Aurora A in tumors was inversely correlated with survival in lung cancer patients. Collectively, these data suggest that inhibition of Aurora kinase A using TC-A2317 is a promising target for anti-cancer therapeutics. PMID:27713168

Tripolar mitosis in human cells and embryos: occurrence, pathophysiology and medical implications.

PubMed

Kalatova, Beata; Jesenska, Renata; Hlinka, Daniel; Dudas, Marek

2015-01-01

Tripolar mitosis is a specific case of cell division driven by typical molecular mechanisms of mitosis, but resulting in three daughter cells instead of the usual count of two. Other variants of multipolar mitosis show even more mitotic poles and are relatively rare. In nature, this phenomenon was frequently observed or suspected in multiple common cancers, infected cells, the placenta, and in early human embryos with impaired pregnancy-yielding potential. Artificial causes include radiation and various toxins. Here we combine several pieces of the most recent evidence for the existence of different types of multipolar mitosis in preimplantation embryos together with a detailed review of the literature. The related molecular and cellular mechanisms are discussed, including the regulation of centriole duplication, mitotic spindle biology, centromere functions, cell cycle checkpoints, mitotic autocorrection mechanisms, and the related complicating factors in healthy and affected cells, including post-mitotic cell-cell fusion often associated with multipolar cell division. Clinical relevance for oncology and embryo selection in assisted reproduction is also briefly discussed in this context. Copyright © 2014 Elsevier GmbH. All rights reserved.

Tumor Environmental Factors Glucose Deprivation and Lactic Acidosis Induce Mitotic Chromosomal Instability – An Implication in Aneuploid Human Tumors

PubMed Central

Zhu, Chunpeng; Hu, Xun

2013-01-01

Mitotic chromosomal instability (CIN) plays important roles in tumor progression, but what causes CIN is incompletely understood. In general, tumor CIN arises from abnormal mitosis, which is caused by either intrinsic or extrinsic factors. While intrinsic factors such as mitotic checkpoint genes have been intensively studied, the impact of tumor microenvironmental factors on tumor CIN is largely unknown. We investigate if glucose deprivation and lactic acidosis – two tumor microenvironmental factors – could induce cancer cell CIN. We show that glucose deprivation with lactic acidosis significantly increases CIN in 4T1, MCF-7 and HCT116 scored by micronuclei, or aneuploidy, or abnormal mitosis, potentially via damaging DNA, up-regulating mitotic checkpoint genes, and/or amplifying centrosome. Of note, the feature of CIN induced by glucose deprivation with lactic acidosis is similar to that of aneuploid human tumors. We conclude that tumor environmental factors glucose deprivation and lactic acidosis can induce tumor CIN and propose that they are potentially responsible for human tumor aneuploidy. PMID:23675453

A TPR domain–containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B

PubMed Central

Nijenhuis, Wilco; von Castelmur, Eleonore; Littler, Dene; De Marco, Valeria; Tromer, Eelco; Vleugel, Mathijs; van Osch, Maria H.J.; Snel, Berend

2013-01-01

The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B–dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization. PMID:23569217

A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B.

PubMed

Nijenhuis, Wilco; von Castelmur, Eleonore; Littler, Dene; De Marco, Valeria; Tromer, Eelco; Vleugel, Mathijs; van Osch, Maria H J; Snel, Berend; Perrakis, Anastassis; Kops, Geert J P L

2013-04-15

The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.

Axin localizes to mitotic spindles and centrosomes in mitotic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Shi-Mun; Choi, Eun-Jin; Song, Ki-Joon

2009-04-01

Wnt signaling plays critical roles in cell proliferation and carcinogenesis. In addition, numerous recent studies have shown that various Wnt signaling components are involved in mitosis and chromosomal instability. However, the role of Axin, a negative regulator of Wnt signaling, in mitosis has remained unclear. Using monoclonal antibodies against Axin, we found that Axin localizes to the centrosome and along mitotic spindles. This localization was suppressed by siRNA specific for Aurora A kinase and by Aurora kinase inhibitor. Interestingly, Axin over-expression altered the subcellular distribution of Plk1 and of phosphorylated glycogen synthase kinase (GSK3{beta}) without producing any notable changes inmore » cellular phenotype. In the presence of Aurora kinase inhibitor, Axin over-expression induced the formation of cleavage furrow-like structures and of prominent astral microtubules lacking midbody formation in a subset of cells. Our results suggest that Axin modulates distribution of Axin-associated proteins such as Plk1 and GSK3{beta} in an expression level-dependent manner and these interactions affect the mitotic process, including cytokinesis under certain conditions, such as in the presence of Aurora kinase inhibitor.« less

Mitosis, double strand break repair, and telomeres: a view from the end: how telomeres and the DNA damage response cooperate during mitosis to maintain genome stability.

PubMed

Cesare, Anthony J

2014-11-01

Double strand break (DSB) repair is suppressed during mitosis because RNF8 and downstream DNA damage response (DDR) factors, including 53BP1, do not localize to mitotic chromatin. Discovery of the mitotic kinase-dependent mechanism that inhibits DSB repair during cell division was recently reported. It was shown that restoring mitotic DSB repair was detrimental, resulting in repair dependent genome instability and covalent telomere fusions. The telomere DDR that occurs naturally during cellular aging and in cancer is known to be refractory to G2/M checkpoint activation. Such DDR-positive telomeres, and those that occur as part of the telomere-dependent prolonged mitotic arrest checkpoint, normally pass through mitosis without covalent ligation, but result in cell growth arrest in G1 phase. The discovery that suppressing DSB repair during mitosis may function primarily to protect DDR-positive telomeres from fusing during cell division reinforces the unique cooperation between telomeres and the DDR to mediate tumor suppression. © 2014 The Author. Bioessays published by WILEY Periodicals, Inc.

Phospho-H1 Decorates the Inter-chromatid Axis and Is Evicted along with Shugoshin by SET during Mitosis.

PubMed

Krishnan, Swathi; Smits, Arne H; Vermeulen, Michiel; Reinberg, Danny

2017-08-17

Precise control of sister chromatid separation during mitosis is pivotal to maintaining genomic integrity. Yet, the regulatory mechanisms involved are not well understood. Remarkably, we discovered that linker histone H1 phosphorylated at S/T18 decorated the inter-chromatid axial DNA on mitotic chromosomes. Sister chromatid resolution during mitosis required the eviction of such H1S/T18ph by the chaperone SET, with this process being independent of and most likely downstream of arm-cohesin dissociation. SET also directed the disassembly of Shugoshins in a polo-like kinase 1-augmented manner, aiding centromere resolution. SET ablation compromised mitotic fidelity as evidenced by unresolved sister chromatids with marked accumulation of H1S/T18ph and centromeric Shugoshin. Thus, chaperone-assisted eviction of linker histones and Shugoshins is a fundamental step in mammalian mitotic progression. Our findings also elucidate the functional implications of the decades-old observation of mitotic linker histone phosphorylation, serving as a paradigm to explore the role of linker histones in bio-signaling processes. Copyright © 2017 Elsevier Inc. All rights reserved.

PLK1 Activation in Late G2 Sets Up Commitment to Mitosis.

PubMed

Gheghiani, Lilia; Loew, Damarys; Lombard, Bérangère; Mansfeld, Jörg; Gavet, Olivier

2017-06-06

Commitment to mitosis must be tightly coordinated with DNA replication to preserve genome integrity. While we have previously established that the timely activation of CyclinB1-Cdk1 in late G2 triggers mitotic entry, the upstream regulatory mechanisms remain unclear. Here, we report that Polo-like kinase 1 (Plk1) is required for entry into mitosis during an unperturbed cell cycle and is rapidly activated shortly before CyclinB1-Cdk1. We determine that Plk1 associates with the Cdc25C1 phosphatase and induces its phosphorylation before mitotic entry. Plk1-dependent Cdc25C1 phosphosites are sufficient to promote mitotic entry, even when Plk1 activity is inhibited. Furthermore, we find that activation of Plk1 during G2 relies on CyclinA2-Cdk activity levels. Our findings thus elucidate a critical role for Plk1 in CyclinB1-Cdk1 activation and mitotic entry and outline how CyclinA2-Cdk, an S-promoting factor, poises cells for commitment to mitosis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells.

PubMed

Rusin, Scott F; Schlosser, Kate A; Adamo, Mark E; Kettenbach, Arminja N

2015-10-13

Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry-based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c-dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2-dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. Copyright © 2015, American Association for the Advancement of Science.

Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells

PubMed Central

Rusin, Scott F.; Schlosser, Kate A.; Adamo, Mark E.; Kettenbach, Arminja N.

2017-01-01

Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry–based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c–dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2–dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. PMID:26462736

Functional organization of mitotic microtubules. Physical chemistry of the in vivo equilibrium system.

PubMed Central

Inoué, S; Fuseler, J; Salmon, E D; Ellis, G W

1975-01-01

Equilibrium between mitotic microtubules and tubulin is analyzed, using birefringence of mitotic spindle to measure microtubule concentration in vivo. A newly designed temperature-controlled slide and miniature, thermostated hydrostatic pressure chamber permit rapid alteration of temperature and of pressure. Stress birefringence of the windows is minimized, and a system for rapid recording of compensation is incorporated, so that birefringence can be measured to 0.1 nm retardation every few seconds. Both temperature and pressure data yield thermodynamic values (delta H similar to 35 kcal/mol, delta S similar to 120 entropy units [eu], delta V similar to 400 ml/mol of subunit polymerized) consistent with the explanation that polymerization of tubulin is entropy driven and mediated by hydrophobic interactions. Kinetic data suggest pseudo-zero-order polymerization and depolymerization following rapid temperature shifts, and a pseudo-first-order depolymerization during anaphase at constant temperature. The equilibrium properties of the in vivo mitotic microtubules are compared with properties of isolated brain tubules. Images FIGURE 1 FIGURE 2 FIGURE 5 FIGURE 12 FIGURE 13 FIGURE 14 FIGURE 19 PMID:1139037

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Linlin; Sun, Xiaodong; Xie, Songbo

Highlights: • DIMEN displays higher anti-proliferative activity than enastron. • DIMEN induced mitotic arrest and apoptosis more significantly than enastron. • DIMEN blocked the conformational change of ADP-binding pocket more effectively. • DIMEN hindered ADP release more potently than enastron. - Abstract: Eg5 is a mitotic kinesin that plays a crucial role in the formation of bipolar mitotic spindles, by hydrolyzing ATP to push apart anti-parallel microtubules. Dimethylenastron is potent specific small molecule inhibitor of Eg5. The mechanism by which dimethylenastron inhibits Eg5 function remains unclear. By comparing with enastron, here we report that dimethylenastron prevents the growth of pancreaticmore » and lung cancer cells more effectively, by halting mitotic progression and triggering apoptosis. We analyze their interactions with ADP-bound Eg5 crystal structure, and find that dimethylenastron binds Eg5 motor domain with higher affinity. In addition, dimethylenastron allosterically blocks the conformational change of the “sandwich”-like ADP-binding pocket more effectively. We subsequently use biochemical approach to reveal that dimethylenastron slows ADP release more significantly than enastron. These data thus provide biological, structural and mechanistic insights into the potent inhibitory activity of dimethylenastron.« less

Force sensor

DOEpatents

Grahn, A.R.

1993-05-11

A force sensor and related method for determining force components is described. The force sensor includes a deformable medium having a contact surface against which a force can be applied, a signal generator for generating signals that travel through the deformable medium to the contact surface, a signal receptor for receiving the signal reflected from the contact surface, a generation controller, a reception controller, and a force determination apparatus. The signal generator has one or more signal generation regions for generating the signals. The generation controller selects and activates the signal generation regions. The signal receptor has one or more signal reception regions for receiving signals and for generating detections signals in response thereto. The reception controller selects signal reception regions and detects the detection signals. The force determination apparatus measures signal transit time by timing activation and detection and, optionally, determines force components for selected cross-field intersections. The timer which times by activation and detection can be any means for measuring signal transit time. A cross-field intersection is defined by the overlap of a signal generation region and a signal reception region.

Force sensor

DOEpatents

Grahn, Allen R.

1993-01-01

A force sensor and related method for determining force components. The force sensor includes a deformable medium having a contact surface against which a force can be applied, a signal generator for generating signals that travel through the deformable medium to the contact surface, a signal receptor for receiving the signal reflected from the contact surface, a generation controller, a reception controller, and a force determination apparatus. The signal generator has one or more signal generation regions for generating the signals. The generation controller selects and activates the signal generation regions. The signal receptor has one or more signal reception regions for receiving signals and for generating detections signals in response thereto. The reception controller selects signal reception regions and detects the detection signals. The force determination apparatus measures signal transit time by timing activation and detection and, optionally, determines force components for selected cross-field intersections. The timer which times by activation and detection can be any means for measuring signal transit time. A cross-field intersection is defined by the overlap of a signal generation region and a signal reception region.

Precommitment low-level Neurog3 expression defines a long-lived mitotic endocrine-biased progenitor pool that drives production of endocrine-committed cells

PubMed Central

Bechard, Matthew E.; Bankaitis, Eric D.; Hipkens, Susan B.; Ustione, Alessandro; Piston, David W.; Yang, Yu-Ping; Magnuson, Mark A.; Wright, Christopher V.E.

2016-01-01

The current model for endocrine cell specification in the pancreas invokes high-level production of the transcription factor Neurogenin 3 (Neurog3) in Sox9+ bipotent epithelial cells as the trigger for endocrine commitment, cell cycle exit, and rapid delamination toward proto-islet clusters. This model posits a transient Neurog3 expression state and short epithelial residence period. We show, however, that a Neurog3TA.LO cell population, defined as Neurog3 transcriptionally active and Sox9+ and often containing nonimmunodetectable Neurog3 protein, has a relatively high mitotic index and prolonged epithelial residency. We propose that this endocrine-biased mitotic progenitor state is functionally separated from a pro-ductal pool and endows them with long-term capacity to make endocrine fate-directed progeny. A novel BAC transgenic Neurog3 reporter detected two types of mitotic behavior in Sox9+ Neurog3TA.LO progenitors, associated with progenitor pool maintenance or derivation of endocrine-committed Neurog3HI cells, respectively. Moreover, limiting Neurog3 expression dramatically increased the proportional representation of Sox9+ Neurog3TA.LO progenitors, with a doubling of its mitotic index relative to normal Neurog3 expression, suggesting that low Neurog3 expression is a defining feature of this cycling endocrine-biased state. We propose that Sox9+ Neurog3TA.LO endocrine-biased progenitors feed production of Neurog3HI endocrine-committed cells during pancreas organogenesis. PMID:27585590

Curcumin-treated cancer cells show mitotic disturbances leading to growth arrest and induction of senescence phenotype.

PubMed

Mosieniak, Grażyna; Sliwinska, Małgorzata A; Przybylska, Dorota; Grabowska, Wioleta; Sunderland, Piotr; Bielak-Zmijewska, Anna; Sikora, Ewa

2016-05-01

Cellular senescence is recognized as a potent anticancer mechanism that inhibits carcinogenesis. Cancer cells can also undergo senescence upon chemo- or radiotherapy. Curcumin, a natural polyphenol derived from the rhizome of Curcuma longa, shows anticancer properties both in vitro and in vivo. Previously, we have shown that treatment with curcumin leads to senescence of human cancer cells. Now we identified the molecular mechanism underlying this phenomenon. We observed a time-dependent accumulation of mitotic cells upon curcumin treatment. The time-lapse analysis proved that those cells progressed through mitosis for a significantly longer period of time. A fraction of cells managed to divide or undergo mitotic slippage and then enter the next phase of the cell cycle. Cells arrested in mitosis had an improperly formed mitotic spindle and were positive for γH2AX, which shows that they acquired DNA damage during prolonged mitosis. Moreover, the DNA damage response pathway was activated upon curcumin treatment and the components of this pathway remained upregulated while cells were undergoing senescence. Inhibition of the DNA damage response decreased the number of senescent cells. Thus, our studies revealed that the induction of cell senescence upon curcumin treatment resulted from aberrant progression through the cell cycle. Moreover, the DNA damage acquired by cancer cells, due to mitotic disturbances, activates an important molecular mechanism that determines the potential anticancer activity of curcumin. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Yeast Polo Kinase Cdc5 Regulates the Shape of the Mitotic Nucleus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walters, Alison D.; May, Christopher K.; Dauster, Emma S.

Abnormal nuclear size and shape are hallmarks of aging and cancer. However, the mechanisms regulating nuclear morphology and nuclear envelope (NE) expansion are poorly understood. In metazoans, the NE disassembles prior to chromosome segregation and reassembles at the end of mitosis. In budding yeast, the NE remains intact. The nucleus elongates as chromosomes segregate and then divides at the end of mitosis to form two daughter nuclei without NE disassembly. The budding yeast nucleus also undergoes remodeling during a mitotic arrest; the NE continues to expand despite the pause in chromosome segregation, forming a nuclear extension, or "flare," that encompassesmore » the nucleolus. The distinct nucleolar localization of the mitotic flare indicates that the NE is compartmentalized and that there is a mechanism by which NE expansion is confined to the region adjacent to the nucleolus. Here we show that mitotic flare formation is dependent on the yeast polo kinase Cdc5. This function of Cdc5 is independent of its known mitotic roles, including rDNA condensation. High-resolution imaging revealed that following Cdc5 inactivation, nuclei expand isometrically rather than forming a flare, indicating that Cdc5 is needed for NE compartmentalization. Lastly, even in an uninterrupted cell cycle, a small NE expansion occurs adjacent to the nucleolus prior to anaphase in a Cdc5-dependent manner. Our data provide the first evidence that polo kinase, a key regulator of mitosis, plays a role in regulating nuclear morphology and NE expansion.« less

The Yeast Polo Kinase Cdc5 Regulates the Shape of the Mitotic Nucleus

DOE PAGES

Walters, Alison D.; May, Christopher K.; Dauster, Emma S.; ...

2014-11-20

Abnormal nuclear size and shape are hallmarks of aging and cancer. However, the mechanisms regulating nuclear morphology and nuclear envelope (NE) expansion are poorly understood. In metazoans, the NE disassembles prior to chromosome segregation and reassembles at the end of mitosis. In budding yeast, the NE remains intact. The nucleus elongates as chromosomes segregate and then divides at the end of mitosis to form two daughter nuclei without NE disassembly. The budding yeast nucleus also undergoes remodeling during a mitotic arrest; the NE continues to expand despite the pause in chromosome segregation, forming a nuclear extension, or "flare," that encompassesmore » the nucleolus. The distinct nucleolar localization of the mitotic flare indicates that the NE is compartmentalized and that there is a mechanism by which NE expansion is confined to the region adjacent to the nucleolus. Here we show that mitotic flare formation is dependent on the yeast polo kinase Cdc5. This function of Cdc5 is independent of its known mitotic roles, including rDNA condensation. High-resolution imaging revealed that following Cdc5 inactivation, nuclei expand isometrically rather than forming a flare, indicating that Cdc5 is needed for NE compartmentalization. Lastly, even in an uninterrupted cell cycle, a small NE expansion occurs adjacent to the nucleolus prior to anaphase in a Cdc5-dependent manner. Our data provide the first evidence that polo kinase, a key regulator of mitosis, plays a role in regulating nuclear morphology and NE expansion.« less

The GPER agonist G-1 induces mitotic arrest and apoptosis in human vascular smooth muscle cells independent of GPER.

PubMed

Gui, Yu; Shi, Zhan; Wang, ZengYong; Li, Jing-Jing; Xu, Can; Tian, RuiJuan; Song, XinXing; Walsh, Michael P; Li, Dong; Gao, Jie; Zheng, Xi-Long

2015-04-01

The G protein-coupled estrogen receptor (GPER) has been implicated in the regulation of smooth muscle cell (SMC) proliferation. The GPER selective agonist G-1 has been a useful tool for exploring the biological roles of GPER in a variety of experimental settings, including SMC proliferation. The present study, originally designed to investigate cellular and signaling mechanisms underlying the regulatory role of GPER in vascular SMC proliferation using G-1, unexpectedly revealed off-target effects of G-1. G-1(1-10 μM) inhibited bromodeoxyuridine (BrdU) incorporation of human SMCs and caused G2/M cell accumulation. G-1 treatment also increased mitotic index concurrent with a decrease in phosphorylation of Cdk1 (Tyr 15) and an increase in phosphorylation of the mitotic checkpoint protein BuBR1. Furthermore, G-1 caused microtubule disruption, mitotic spindle damage, and tubulin depolymerization. G-1 induced cell apoptosis as indicated by the appearance of TUNEL-positive and annexin V-positive cells with enhanced cleavage of caspases 3 and 9. However, neither the GPER antagonist G-15 nor the MAPK kinase inhibitor PD98059 prevented these G-1 effects. Down-regulation of GPER or p44/42 MAPK with siRNA transfection also did not affect the G-1-induced apoptosis. We conclude that G-1 inhibits proliferation of SMCs through mechanisms involving mitotic arrest and apoptosis, independent of GPER and the MAPK pathway. © 2014 Wiley Periodicals, Inc.

VDAC3 regulates centriole assembly by targeting Mps1 to centrosomes.

PubMed

Majumder, Shubhra; Slabodnick, Mark; Pike, Amanda; Marquardt, Joseph; Fisk, Harold A

2012-10-01

Centrioles are duplicated during S-phase to generate the two centrosomes that serve as mitotic spindle poles during mitosis. The centrosomal pool of the Mps1 kinase is important for centriole assembly, but how Mps1 is delivered to centrosomes is unknown. Here we have identified a centrosome localization domain within Mps1 and identified the mitochondrial porin VDAC3 as a protein that binds to this region of Mps1. Moreover, we show that VDAC3 is present at the mother centriole and modulates centriole assembly by recruiting Mps1 to centrosomes.

VDAC3 regulates centriole assembly by targeting Mps1 to centrosomes

PubMed Central

Majumder, Shubhra; Slabodnick, Mark; Pike, Amanda; Marquardt, Joseph; Fisk, Harold A.

2012-01-01

Centrioles are duplicated during S-phase to generate the two centrosomes that serve as mitotic spindle poles during mitosis. The centrosomal pool of the Mps1 kinase is important for centriole assembly, but how Mps1 is delivered to centrosomes is unknown. Here we have identified a centrosome localization domain within Mps1 and identified the mitochondrial porin VDAC3 as a protein that binds to this region of Mps1. Moreover, we show that VDAC3 is present at the mother centriole and modulates centriole assembly by recruiting Mps1 to centrosomes. PMID:22935710

Saccharomyces cerevisiae Mob1p Is Required for Cytokinesis and Mitotic Exit

PubMed Central

Luca, Francis C.; Mody, Manali; Kurischko, Cornelia; Roof, David M.; Giddings, Thomas H.; Winey, Mark

2001-01-01

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G1 by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants. These results suggest that the MEN functions during the mitosis-to-G1 transition to control cyclin-CDK inactivation and cytokinesis. PMID:11564880

Functional Characterization of G12, a Gene Required for Mitotic Progression during Gastrulation in Zebrafish

NASA Technical Reports Server (NTRS)

Reinsch, Sigrid; Conway, Gregory; Dalton, Bonnie P. (Technical Monitor)

2002-01-01

In a differential RNA display screen we have isolated a zebrafish gene, G12, for which homologs can only be found in DNA databases for vertebrates, but not invertebrates. This suggests that this is a gene required specifically in vertebrates. G12 expression is upregulated at mid-blastula transition (MBT). Morpholino inactivation of this gene by injection into 1-cell embryos results in mitotic defects and apoptosis shortly after MBT. Nuclei in morpholino treated embryos also display segregation defects. We have characterized the localization of this gene as a GFP fusion in live and fixed embryos. Overexpression of G12-GFP is non-toxic. Animals retain GFP expression for at least 7 days with no developmental defects, Interestingly in these animals G12-GFP is never detectable in blood cells though blood is present. In the deep cells of early embryos, G 12GFP is localized to nuclei and cytoskeletal elements in interphase and to the centrosome and spindle apparatus during mitosis. In the EVL, G12-GFP shows additional localization to the cell periphery, especially in mitosis. In the yolk syncytium, G12-GFP again localizes to nuclei and strongly to cytoplasmic microtubules of migrating nuclei at the YSL margin. Morpholinc, injection specifically into the YSL after cellularization blocks epiboly and nuclei of the YSL show mitotic defects while deep cells show no mitotic defects and continue to divide. Rescue experiments in which morpholino and G12-GFP RNA are co-injected indicate partial rescue by the G12-GFP. The rescue is cell autonomous; that is, regions of the embryo with higher G12-GFP expression show fewer mitotic defects. Spot 14, the human bomolog of G12, has been shown to be amplified in aggressive breast tumors. This finding, along with our functional and morphological data suggest that G12 and spot 14 are vertebrate-specific and may function either as mitotic checkpoints or as structural components of the spindle apparatus.

Primary mesenchymal (nonangiomatous/nonlymphomatous) neoplasms occurring in the canine spleen: anatomic classification, immunohistochemistry, and mitotic activity correlated with patient survival.

PubMed

Spangler, W L; Culbertson, M R; Kass, P H

1994-01-01

Surgical submissions from canine splenectomy cases spanning a 3-year period (1988-1990) were evaluated. Eighty seven neoplasms of the spleen considered to be of nonangiomatous and nonlymphomatous origin were selected for morphologic classification, mitotic index determination, immunohistochemical analysis, and patient survival determination. In 76/87 cases, patient survival information was available, and the mitotic index was determined in 83/87 cases. Immunohistochemistry for selected antigens (vimentin, desmin, smooth muscle actin, myosin, and factor VIII-related antigen) was performed in 58/87 of the cases. Morphologic classification of these lesions in standard HE preparations yielded the following neoplastic groups: fibrosarcoma (19/87), undifferentiated sarcoma (19/87), leiomyosarcoma (14/87), osteosarcoma (8/87), mesenchymoma (7/87), myxosarcoma (6/87), histiocytic sarcoma (6/87), leiomyoma (3/87), lipoma-myelolipoma (2/87), liposarcoma (2/87), and malignant fibrous histiocytoma (1/87). A lack of distinct morphologic characteristics among many of the neoplasms that were classified as either fibrosarcoma, leiomyosarcoma, or undifferentiated sarcoma contrasted these groups with the relatively unambiguous features that distinguished the other sarcoma groups. Using immunohistochemical staining for muscle-specific antigens (desmin, smooth muscle actin, and myosin), specific staining often overlapped extensively within the neoplastic groups of fibrosarcomas, leiomyosarcomas, and undifferentiated sarcomas, suggesting either ambiguous morphologic findings or the possibility of a common histogenesis from smooth muscle trabeculae or a distinct population of splenic myofibroblasts. The biological behavior of all tumors examined could be placed into three categories of patient survival: (1) benign, noninvasive tumors (leiomyoma, lipoma) with prolonged survival intervals; (2) malignant tumors (fibrosarcoma, undifferentiated sarcoma, leiomyosarcoma, osteosarcoma, myxosarcoma, histiocytic sarcoma, and liposarcoma), showing severely truncated survival (median 4 months with 80-100% mortality after 12 months; and (3) intermediate survival periods (median 12 months with 50% 1 year survival) attributed to a single group of neoplasm, the mesenchymomas. The biological behavior of primary splenic nonangiomatous, nonlymphomatous sarcomas was most closely correlated with observed mitotic index. Splenic neoplasms of this type with a mitotic index < 9 showed significantly (P < 0.0001) longer survival intervals than those with an index > 9. With the exception of osteosarcoma, all anatomically defined tumor groups contained one or more specimens with a mitotic index < 9. The clinical prognosis given for splenic sarcomas should be modified according to the mitotic index as a predictive value for patient survival.

Role of apoptosis and mitosis during human eye development.

PubMed

Bozanić, Darka; Tafra, Robert; Saraga-Babić, Mirna

2003-08-01

The spatial and temporal distribution as well as ultrastructural and biochemical characteristics of apoptotic and mitotic cells during human eye development were investigated in 14 human conceptuses of 4-9 postovulatory weeks, using electron and light microscopy. In the 5th developmental week, apoptotic and mitotic cells were found in the neuroepithelium of the optic cup and stalk, being the most numerous at the borderline between the two layers of the optic cup, and at the place of transition of the optic cup into stalk. They were also found at the region of detachment of the lens pit from the surface ectoderm. In the later developmental stages (the 6th-the 9th week), apoptotic and mitotic cells were observed in the neural retina and the anterior lens epithelium. Throughout all stages examined, mitotic cells were found exclusively adjacent to the lumen either of the intraretinal space or the optic stalk ventricle, or were restricted to the superficial epithelial layer of the lens primordium. Unlike mitotic cells, apoptotic cells occurred throughout the whole width both of the neuroepithelium and the surface epithelium. Ultrastructurally, apoptotic cells were characterised by round- or crescent-shaped condensations of chromatin near the nuclear membrane, while in the more advanced stages of apoptosis by apoptotic bodies. The distribution of caspase-3-positive cells coincided with the location of apoptotic cells described by morphological techniques indicating that the caspase-3-dependent apoptotic pathway operates during the all stages of human eye development. The location of cells positive for anti-apoptotic bcl-2 protein was in accordance with the regions of eye with high mitotic activity, confirming the role of bcl-2 in protecting cells from apoptosis. In the earliest stage of eye development, apoptosis and mitosis might be associated with the sculpturing of the walls of optic cup and stalk, while high mitotic activity along the intraretinal space and optic stalk ventricle indicates its role in the gradual luminal closure. These processes also participate in the detachment of the lens pit epithelium from the surface ectoderm as well as in further closure of the lens vesicle. Later on, both processes seem to be involved in the neural retina differentiation, lens morphogenesis and secondary lens fibre differentiation.

Directional Bleb Formation in Spherical Cells under Temperature Gradient

PubMed Central

Oyama, Kotaro; Arai, Tomomi; Isaka, Akira; Sekiguchi, Taku; Itoh, Hideki; Seto, Yusuke; Miyazaki, Makito; Itabashi, Takeshi; Ohki, Takashi; Suzuki, Madoka; Ishiwata, Shin'ichi

2015-01-01

Living cells sense absolute temperature and temporal changes in temperature using biological thermosensors such as ion channels. Here, we reveal, to our knowledge, a novel mechanism of sensing spatial temperature gradients within single cells. Spherical mitotic cells form directional membrane extensions (polar blebs) under sharp temperature gradients (≥∼0.065°C μm−1; 1.3°C temperature difference within a cell), which are created by local heating with a focused 1455-nm laser beam under an optical microscope. On the other hand, multiple nondirectional blebs are formed under gradual temperature gradients or uniform heating. During heating, the distribution of actomyosin complexes becomes inhomogeneous due to a break in the symmetry of its contractile force, highlighting the role of the actomyosin complex as a sensor of local temperature gradients. PMID:26200871

Mechanical design principles of a mitotic spindle

PubMed Central

Ward, Jonathan J; Roque, Hélio; Antony, Claude; Nédélec, François

2014-01-01

An organised spindle is crucial to the fidelity of chromosome segregation, but the relationship between spindle structure and function is not well understood in any cell type. The anaphase B spindle in fission yeast has a slender morphology and must elongate against compressive forces. This ‘pushing’ mode of chromosome transport renders the spindle susceptible to breakage, as observed in cells with a variety of defects. Here we perform electron tomographic analyses of the spindle, which suggest that it organises a limited supply of structural components to increase its compressive strength. Structural integrity is maintained throughout the spindle's fourfold elongation by organising microtubules into a rigid transverse array, preserving correct microtubule number and dynamically rescaling microtubule length. DOI: http://dx.doi.org/10.7554/eLife.03398.001 PMID:25521247

MELK expression correlates with tumor mitotic activity but is not required for cancer growth

PubMed Central

Smith, Joan C; Palladino, Ann C

2018-01-01

The Maternal Embryonic Leucine Zipper Kinase (MELK) has been identified as a promising therapeutic target in multiple cancer types. MELK over-expression is associated with aggressive disease, and MELK has been implicated in numerous cancer-related processes, including chemotherapy resistance, stem cell renewal, and tumor growth. Previously, we established that triple-negative breast cancer cell lines harboring CRISPR/Cas9-induced null mutations in MELK proliferate at wild-type levels in vitro (Lin et al., 2017). Here, we generate several additional knockout clones of MELK and demonstrate that across cancer types, cells lacking MELK exhibit wild-type growth in vitro, under environmental stress, in the presence of cytotoxic chemotherapies, and in vivo. By combining our MELK-knockout clones with a recently described, highly specific MELK inhibitor, we further demonstrate that the acute inhibition of MELK results in no specific anti-proliferative phenotype. Analysis of gene expression data from cohorts of cancer patients identifies MELK expression as a correlate of tumor mitotic activity, explaining its association with poor clinical prognosis. In total, our results demonstrate the power of CRISPR/Cas9-based genetic approaches to investigate cancer drug targets, and call into question the rationale for treating patients with anti-MELK monotherapies. PMID:29417930

DOE Office of Scientific and Technical Information (OSTI.GOV)

Erenpreisa, Jekaterina; Cragg, Mark S.; Salmina, Kristine

Escape from mitotic catastrophe and generation of endopolyploid tumour cells (ETCs) represents a potential survival strategy of tumour cells in response to genotoxic treatments. ETCs that resume the mitotic cell cycle have reduced ploidy and are often resistant to these treatments. In search for a mechanism for genome reduction, we previously observed that ETCs express meiotic proteins among which REC8 (a meiotic cohesin component) is of particular interest, since it favours reductional cell division in meiosis. In the present investigation, we induced endopolyploidy in p53-dysfunctional human tumour cell lines (Namalwa, WI-L2-NS, HeLa) by gamma irradiation, and analysed the sub-cellular localisationmore » of REC8 in the resulting ETCs. We observed by RT-PCR and Western blot that REC8 is constitutively expressed in these tumour cells, along with SGOL1 and SGOL2, and that REC8 becomes modified after irradiation. REC8 localised to paired sister centromeres in ETCs, the former co-segregating to opposite poles. Furthermore, REC8 localised to the centrosome of interphase ETCs and to the astral poles in anaphase cells where it colocalised with the microtubule-associated protein NuMA. Altogether, our observations indicate that radiation-induced ETCs express features of meiotic cell divisions and that these may facilitate chromosome segregation and genome reduction.« less

Aurora A regulates the activity of HURP by controlling the accessibility of its microtubule-binding domain.

PubMed

Wong, Jim; Lerrigo, Robert; Jang, Chang-Young; Fang, Guowei

2008-05-01

HURP is a spindle-associated protein that mediates Ran-GTP-dependent assembly of the bipolar spindle and promotes chromosome congression and interkinetochore tension during mitosis. We report here a biochemical mechanism of HURP regulation by Aurora A, a key mitotic kinase that controls the assembly and function of the spindle. We found that HURP binds to microtubules through its N-terminal domain that hyperstabilizes spindle microtubules. Ectopic expression of this domain generates defects in spindle morphology and function that reduce the level of tension across sister kinetochores and activate the spindle checkpoint. Interestingly, the microtubule binding activity of this N-terminal domain is regulated by the C-terminal region of HURP: in its hypophosphorylated state, C-terminal HURP associates with the microtubule-binding domain, abrogating its affinity for microtubules. However, when the C-terminal domain is phosphorylated by Aurora A, it no longer binds to N-terminal HURP, thereby releasing the inhibition on its microtubule binding and stabilizing activity. In fact, ectopic expression of this C-terminal domain depletes endogenous HURP from the mitotic spindle in HeLa cells in trans, suggesting the physiological importance for this mode of regulation. We concluded that phosphorylation of HURP by Aurora A provides a regulatory mechanism for the control of spindle assembly and function.

The Symbiosome: Legume and Rhizobia Co-evolution toward a Nitrogen-Fixing Organelle?

PubMed Central

Coba de la Peña, Teodoro; Fedorova, Elena; Pueyo, José J.; Lucas, M. Mercedes

2018-01-01

In legume nodules, symbiosomes containing endosymbiotic rhizobial bacteria act as temporary plant organelles that are responsible for nitrogen fixation, these bacteria develop mutual metabolic dependence with the host legume. In most legumes, the rhizobia infect post-mitotic cells that have lost their ability to divide, although in some nodules cells do maintain their mitotic capacity after infection. Here, we review what is currently known about legume symbiosomes from an evolutionary and developmental perspective, and in the context of the different interactions between diazotroph bacteria and eukaryotes. As a result, it can be concluded that the symbiosome possesses organelle-like characteristics due to its metabolic behavior, the composite origin and differentiation of its membrane, the retargeting of host cell proteins, the control of microsymbiont proliferation and differentiation by the host legume, and the cytoskeletal dynamics and symbiosome segregation during the division of rhizobia-infected cells. Different degrees of symbiosome evolution can be defined, specifically in relation to rhizobial infection and to the different types of nodule. Thus, our current understanding of the symbiosome suggests that it might be considered a nitrogen-fixing link in organelle evolution and that the distinct types of legume symbiosomes could represent different evolutionary stages toward the generation of a nitrogen-fixing organelle. PMID:29403508

Apomixis in hawkweed: Mendel's experimental nemesis.

PubMed

Koltunow, Anna M G; Johnson, Susan D; Okada, Takashi

2011-03-01

Mendel used hawkweeds and other plants to verify the laws of inheritance he discovered using Pisum. Trait segregation was not evident in hawkweeds because many form seeds asexually by apomixis. Meiosis does not occur during female gametophyte formation and the mitotically formed embryo sacs do not require fertilization for seed development. The resulting progeny retain a maternal genotype. Hawkweeds in Hieracium subgenus Pilosella form mitotic embryo sacs by apospory. The initiation of sexual reproduction is required to stimulate apospory in ovules and to promote the function of the dominant locus, LOSS OF APOMEIOSIS, which stimulates the differentiation of somatic aposporous initial (AI) cells near sexually programmed cells. As AI cells undergo nuclear mitosis the sexual pathway terminates. The function of the dominant locus LOSS OF PARTHENOGENESIS in aposporous embryo sacs enables fertilization-independent embryo and endosperm development. Deletion of either locus results in partial reversion to sexual reproduction, and loss of function in both loci results in reversion to sexual development. In these apomicts, sexual reproduction is therefore the default reproductive mode upon which apomixis is superimposed. These loci are unlikely to encode factors critical for sexual reproduction but might recruit the sexual pathway to enable apomixis. Incomplete functional penetrance of these dominant loci is likely to lead to the generation of rare sexual progeny also derived from these facultative apomicts.

The small molecule SI113 synergizes with mitotic spindle poisons in arresting the growth of human glioblastoma multiforme

PubMed Central

Abbruzzese, Claudia; Catalogna, Giada; Gallo, Enzo; di Martino, Simona; Mileo, Anna M.; Carosi, Mariantonia; Dattilo, Vincenzo; Schenone, Silvia; Musumeci, Francesca; Lavia, Patrizia; Perrotti, Nicola; Amato, Rosario; Paggi, Marco G.

2017-01-01

Glioblastoma multiforme (GBM) is the deadliest brain tumor. State-of-art GBM therapy often fails to ensure control of a disease characterized by high frequency of recurrences and progression. In search for novel therapeutic approaches, we assayed the effect of compounds from a cancer drug library on the ADF GBM cell line, establishing their elevated sensitivity to mitotic spindle poisons. Our previous work showed that the effectiveness of the spindle poison paclitaxel in inhibiting cancer cell growth was dependent on the expression of RANBP1, a regulatory target of the serine/threonine kinase SGK1. Recently, we developed the small molecule SI113 to inhibit SGK1 activity. Therefore, we explored the outcome of the association between SI113 and selected spindle poisons, finding that these drugs generated a synergistic cytotoxic effect in GBM cells, drastically reducing their viability and clonogenic capabilities in vitro, as well as inhibiting tumor growth in vivo. We also defined the molecular bases of such a synergistic effect. Because SI113 displays low systemic toxicity, yet strong activity in potentiating the effect of radiotherapy in GBM cells, we believe that this drug could be a strong candidate for clinical trials, with the aim to add it to the current GBM therapeutic approaches. PMID:29340013

Neocortical dendritic complexity is controlled during development by NOMA-GAP-dependent inhibition of Cdc42 and activation of cofilin.

PubMed

Rosário, Marta; Schuster, Steffen; Jüttner, René; Parthasarathy, Srinivas; Tarabykin, Victor; Birchmeier, Walter

2012-08-01

Neocortical neurons have highly branched dendritic trees that are essential for their function. Indeed, defects in dendritic arborization are associated with human neurodevelopmental disorders. The molecular mechanisms regulating dendritic arbor complexity, however, are still poorly understood. Here, we uncover the molecular basis for the regulation of dendritic branching during cortical development. We show that during development, dendritic branching requires post-mitotic suppression of the RhoGTPase Cdc42. By generating genetically modified mice, we demonstrate that this is catalyzed in vivo by the novel Cdc42-GAP NOMA-GAP. Loss of NOMA-GAP leads to decreased neocortical volume, associated specifically with profound oversimplification of cortical dendritic arborization and hyperactivation of Cdc42. Remarkably, dendritic complexity and cortical thickness can be partially restored by genetic reduction of post-mitotic Cdc42 levels. Furthermore, we identify the actin regulator cofilin as a key regulator of dendritic complexity in vivo. Cofilin activation during late cortical development depends on NOMA-GAP expression and subsequent inhibition of Cdc42. Strikingly, in utero expression of active cofilin is sufficient to restore postnatal dendritic complexity in NOMA-GAP-deficient animals. Our findings define a novel cell-intrinsic mechanism to regulate dendritic branching and thus neuronal complexity in the cerebral cortex.

Loss of BubR1 acetylation causes defects in spindle assembly checkpoint signaling and promotes tumor formation

PubMed Central

Park, Inai; Lee, Hae-ock; Choi, Eunhee; Lee, Yoo-Kyung; Kwon, Mi-Sun; Min, Jaewon; Park, Pil-Gu; Lee, Seonju; Kong, Young-Yun; Gong, Gyungyub

2013-01-01

BubR1 acetylation is essential in mitosis. Mice heterozygous for the acetylation-deficient BubR1 allele (K243R/+) spontaneously developed tumors with massive chromosome missegregations. K243R/+ mouse embryonic fibroblasts (MEFs) exhibited a weakened spindle assembly checkpoint (SAC) with shortened mitotic timing. The generation of the SAC signal was intact, as Mad2 localization to the unattached kinetochore (KT) was unaltered; however, because of the premature degradation of K243R-BubR1, the mitotic checkpoint complex disassociated prematurely in the nocodazole-treated condition, suggesting that maintenance of the SAC is compromised. BubR1 acetylation was also required to counteract excessive Aurora B activity at the KT for stable chromosome–spindle attachments. The association of acetylation-deficient BubR1 with PP2A-B56α phosphatase was reduced, and the phosphorylated Ndc80 at the KT was elevated in K243R/+ MEFs. In relation, there was a marked increase of micronuclei and p53 mutation was frequently detected in primary tumors of K243R/+ mice. Collectively, the combined effects of failure in chromosome–spindle attachment and weakened SAC cause genetic instability and cancer in K243R/+ mice. PMID:23878276

The small molecule SI113 synergizes with mitotic spindle poisons in arresting the growth of human glioblastoma multiforme.

PubMed

Abbruzzese, Claudia; Catalogna, Giada; Gallo, Enzo; di Martino, Simona; Mileo, Anna M; Carosi, Mariantonia; Dattilo, Vincenzo; Schenone, Silvia; Musumeci, Francesca; Lavia, Patrizia; Perrotti, Nicola; Amato, Rosario; Paggi, Marco G

2017-12-19

Glioblastoma multiforme (GBM) is the deadliest brain tumor. State-of-art GBM therapy often fails to ensure control of a disease characterized by high frequency of recurrences and progression. In search for novel therapeutic approaches, we assayed the effect of compounds from a cancer drug library on the ADF GBM cell line, establishing their elevated sensitivity to mitotic spindle poisons. Our previous work showed that the effectiveness of the spindle poison paclitaxel in inhibiting cancer cell growth was dependent on the expression of RANBP1, a regulatory target of the serine/threonine kinase SGK1. Recently, we developed the small molecule SI113 to inhibit SGK1 activity. Therefore, we explored the outcome of the association between SI113 and selected spindle poisons, finding that these drugs generated a synergistic cytotoxic effect in GBM cells, drastically reducing their viability and clonogenic capabilities in vitro , as well as inhibiting tumor growth in vivo . We also defined the molecular bases of such a synergistic effect. Because SI113 displays low systemic toxicity, yet strong activity in potentiating the effect of radiotherapy in GBM cells, we believe that this drug could be a strong candidate for clinical trials, with the aim to add it to the current GBM therapeutic approaches.

Human Artificial Chromosomes with Alpha Satellite-Based De Novo Centromeres Show Increased Frequency of Nondisjunction and Anaphase Lag

PubMed Central

Rudd, M. Katharine; Mays, Robert W.; Schwartz, Stuart; Willard, Huntington F.

2003-01-01

Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes. PMID:14560014

Human artificial chromosomes with alpha satellite-based de novo centromeres show increased frequency of nondisjunction and anaphase lag.

PubMed

Rudd, M Katharine; Mays, Robert W; Schwartz, Stuart; Willard, Huntington F

2003-11-01

Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes.

Synergistic inhibition of the APC/C by the removal of APC15 in HCT116 cells lacking UBE2C.

PubMed

Garvanska, Dimitriya H; Larsen, Marie Sofie Yoo; Nilsson, Jakob

2016-10-15

The spindle assembly checkpoint (SAC) inhibits the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores by generating a diffusible inhibitor termed the mitotic checkpoint complex (MCC). At metaphase, rapid activation of the APC/C requires removal of the MCC, a process that has been shown to depend on the APC/C E2 enzymes, UBE2C and UBE2S. Here we investigate the in vivo role of the APC/C E2 enzymes in SAC silencing using CRISPR/Cas9 genetically engineered HCT116 UBE2C or UBE2S null cell lines. Using live cell assays, we show that UBE2C and UBE2S make a minor contribution to SAC silencing in HCT116 cells. Strikingly, in cells specifically lacking UBE2C, we observe a strong synergistic inhibition of mitotic progression when we stabilize the MCC on the APC/C by depleting APC15, potentially reflecting increased competition between the MCC and the remaining initiating E2 enzyme UBE2D. In conclusion, we provide in vivo insight into the APC/C E2 module and its interplay with SAC silencing components. © 2016. Published by The Company of Biologists Ltd.

Molecular basis of APC/C regulation by the spindle assembly checkpoint

PubMed Central

Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

2016-01-01

In the dividing eukaryotic cell the spindle assembly checkpoint (SAC) ensures each daughter cell inherits an identical set of chromosomes. The SAC coordinates the correct attachment of sister chromatid kinetochores to the mitotic spindle with activation of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase that initiates chromosome separation. In response to unattached kinetochores, the SAC generates the mitotic checkpoint complex (MCC), a multimeric assembly that inhibits the APC/C, delaying chromosome segregation. Here, using cryo-electron microscopy we determined the near-atomic resolution structure of an APC/C-MCC complex (APC/CMCC). We reveal how degron-like sequences of the MCC subunit BubR1 block degron recognition sites on Cdc20, the APC/C coactivator subunit (Cdc20APC/C) responsible for substrate interactions. BubR1 also obstructs binding of UbcH10 (APC/C’s initiating E2) to repress APC/C ubiquitination activity. Conformational variability of the complex allows for UbcH10 association, and we show from a structure of APC/CMCC in complex with UbcH10 how the Cdc20 subunit intrinsic to the MCC (Cdc20MCC) is ubiquitinated, a process that results in APC/C reactivation when the SAC is silenced. PMID:27509861

The RanGTP Pathway: From Nucleo-Cytoplasmic Transport to Spindle Assembly and Beyond

PubMed Central

Cavazza, Tommaso; Vernos, Isabelle

2016-01-01

The small GTPase Ran regulates the interaction of transport receptors with a number of cellular cargo proteins. The high affinity binding of the GTP-bound form of Ran to import receptors promotes cargo release, whereas its binding to export receptors stabilizes their interaction with the cargo. This basic mechanism linked to the asymmetric distribution of the two nucleotide-bound forms of Ran between the nucleus and the cytoplasm generates a switch like mechanism controlling nucleo-cytoplasmic transport. Since 1999, we have known that after nuclear envelope breakdown (NEBD) Ran and the above transport receptors also provide a local control over the activity of factors driving spindle assembly and regulating other aspects of cell division. The identification and functional characterization of RanGTP mitotic targets is providing novel insights into mechanisms essential for cell division. Here we review our current knowledge on the RanGTP system and its regulation and we focus on the recent advances made through the characterization of its mitotic targets. We then briefly review the novel functions of the pathway that were recently described. Altogether, the RanGTP system has moonlighting functions exerting a spatial control over protein interactions that drive specific functions depending on the cellular context. PMID:26793706

Single Site α-Tubulin Mutation Affects Astral Microtubules and Nuclear Positioning during Anaphase in Saccharomyces cerevisiae: Possible Role for Palmitoylation of α-Tubulin

PubMed Central

Caron, Joan M.; Vega, Leticia R.; Fleming, James; Bishop, Robert; Solomon, Frank

2001-01-01

We generated a strain of Saccharomyces cerevisiae in which the sole source of α-tubulin protein has a cys-to-ser mutation at cys-377, and then we examined microtubule morphology and nuclear positioning through the cell cycle. During G1 of the cell cycle, microtubules in the C377S α-tubulin (C377S tub1) mutant were indistinguishable from those in the control (TUB1) strain. However, mitotic C377S tub1 cells displayed astral microtubules that often appeared excessive in number, abnormally long, and/or misoriented compared with TUB1 cells. Although mitotic spindles were always correctly aligned along the mother-bud axis, translocation of spindles through the bud neck was affected. In late anaphase, spindles were often not laterally centered but instead appeared to rest along the sides of cells. When the doubling time was increased by growing cells at a lower temperature (15°C), we often found abnormally long mitotic spindles. No increase in the number of anucleate or multinucleate C377S mutant cells was found at any temperature, suggesting that, despite the microtubule abnormalities, mitosis proceeded normally. Because cys-377 is a presumptive site of palmitoylation in α-tubulin in S. cerevisiae, we next compared in vivo palmitoylation of wild-type and C377S mutant forms of the protein. We detected palmitoylated α-tubulin in TUB1 cells, but the cys-377 mutation resulted in approximately a 60% decrease in the level of palmitoylated α-tubulin in C377S tub1 cells. Our results suggest that cys-377 of α-tubulin, and possibly palmitoylation of this amino acid, plays a role in a subset of astral microtubule functions during nuclear migration in M phase of the cell cycle. PMID:11553707

Use of DEAD-box polypeptide-4 (Ddx4) gene promoter-driven fluorescent reporter mice to identify mitotically active germ cells in post-natal mouse ovaries.

PubMed

Park, Eun-Sil; Tilly, Jonathan L

2015-01-01

Several laboratories have independently isolated mitotically active germ cells, termed female germline stem cells or oogonial stem cells (OSCs), from adult mouse ovaries. However, a recent study using Ddx4-Cre;Rosa26 reporter mice concluded that such germ cells do not exist. Given the disparity in conclusions drawn in this study compared with others, we felt it was important to re-assess the utility of Ddx4-Cre;Rosa26 reporter mice for identification of OSCs in adult mouse ovaries. Transgenic Ddx4-Cre mice were crossed with Rosa26(tdTm/tdTm) mice to drive restricted tomato red (tdTm) gene expression in cells in which the Ddx4 gene promoter has been activated. Crude dispersion of ovaries from recombined offspring generated cell fractions containing tdTm-positive immature oocytes, which are incapable of proliferation and thus probably represent the uncharacterized reporter-positive ovarian cells identified in the paper Zhang et al. (2012) as being mitotically inactive. Dispersed ovaries further subjected to fluorescence-activated cell sorting yielded a large population of non-germline tdTm-positive cells, indicative of promoter 'leakiness' in the Ddx4-Cre mouse line. Nonetheless, a small percentage of these tdTm-positive cells exhibited externalized (extracellular, ec) expression of Ddx4 protein (ecDdx4-positive), expressed markers of primitive germ cells but not of oocytes, and actively proliferated in culture, all of which are characteristic features of OSCs. Thus, crude dispersion of ovaries collected from Ddx4 gene promoter-driven reporter mice is not, by itself, a reliable approach to identify OSCs, whereas the same ovarian dispersates further subjected to cell sorting strategies yield purified OSCs that can be expanded in culture. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

BRIT1/MCPH1 is essential for mitotic and meiotic recombination DNA repair and maintaining genomic stability in mice.

PubMed

Liang, Yulong; Gao, Hong; Lin, Shiaw-Yih; Peng, Guang; Huang, Xingxu; Zhang, Pumin; Goss, John A; Brunicardi, Francis C; Multani, Asha S; Chang, Sandy; Li, Kaiyi

2010-01-22

BRIT1 protein (also known as MCPH1) contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1(-/-) mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1(-/-) mice and mouse embryonic fibroblasts (MEFs) were hypersensitive to gamma-irradiation. BRIT1(-/-) MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1(-/-) mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs) were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2's function and as a result leads to infertility and genomic instability in mice.

Local traction force in the proximal leading process triggers nuclear translocation during neuronal migration.

PubMed

Umeshima, Hiroki; Nomura, Ken-Ichi; Yoshikawa, Shuhei; Hörning, Marcel; Tanaka, Motomu; Sakuma, Shinya; Arai, Fumihito; Kaneko, Makoto; Kengaku, Mineko

2018-04-05

Somal translocation in long bipolar neurons is regulated by actomyosin contractile forces, yet the precise spatiotemporal sites of force generation are unknown. Here we investigate the force dynamics generated during somal translocation using traction force microscopy. Neurons with a short leading process generated a traction force in the growth cone and counteracting forces in the leading and trailing processes. In contrast, neurons with a long leading process generated a force dipole with opposing traction forces in the proximal leading process during nuclear translocation. Transient accumulation of actin filaments was observed at the dipole center of the two opposing forces, which was abolished by inhibition of myosin II activity. A swelling in the leading process emerged and generated a traction force that pulled the nucleus when nuclear translocation was physically hampered. The traction force in the leading process swelling was uncoupled from somal translocation in neurons expressing a dominant negative mutant of the KASH protein, which disrupts the interaction between cytoskeletal components and the nuclear envelope. Our results suggest that the leading process is the site of generation of actomyosin-dependent traction force in long bipolar neurons, and that the traction force is transmitted to the nucleus via KASH proteins. Copyright © 2018 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.

Dual mechanism controls asymmetric spindle position in ascidian germ cell precursors.

PubMed

Prodon, François; Chenevert, Janet; Hébras, Céline; Dumollard, Rémi; Faure, Emmanuel; Gonzalez-Garcia, Jose; Nishida, Hiroki; Sardet, Christian; McDougall, Alex

2010-06-01

Mitotic spindle orientation with respect to cortical polarity cues generates molecularly distinct daughter cells during asymmetric cell division (ACD). However, during ACD it remains unknown how the orientation of the mitotic spindle is regulated by cortical polarity cues until furrowing begins. In ascidians, the cortical centrosome-attracting body (CAB) generates three successive unequal cleavages and the asymmetric segregation of 40 localized postplasmic/PEM RNAs in germ cell precursors from the 8-64 cell stage. By combining fast 4D confocal fluorescence imaging with gene-silencing and classical blastomere isolation experiments, we show that spindle repositioning mechanisms are active from prometaphase until anaphase, when furrowing is initiated in B5.2 cells. We show that the vegetal-most spindle pole/centrosome is attracted towards the CAB during prometaphase, causing the spindle to position asymmetrically near the cortex. Next, during anaphase, the opposite spindle pole/centrosome is attracted towards the border with neighbouring B5.1 blastomeres, causing the spindle to rotate (10 degrees /minute) and migrate (3 microm/minute). Dynamic 4D fluorescence imaging of filamentous actin and plasma membrane shows that precise orientation of the cleavage furrow is determined by this second phase of rotational spindle displacement. Furthermore, in pairs of isolated B5.2 blastomeres, the second phase of rotational spindle displacement was lost. Finally, knockdown of PEM1, a protein localized in the CAB and required for unequal cleavage in B5.2 cells, completely randomizes spindle orientation. Together these data show that two separate mechanisms active during mitosis are responsible for spindle positioning, leading to precise orientation of the cleavage furrow during ACD in the cells that give rise to the germ lineage in ascidians.

Detection of mitotic figures and G2+ tumor nuclei with histone markers correlates with worse overall survival in patients with Merkel cell carcinoma.

PubMed

Henderson, Samuel A; Tetzlaff, Michael T; Pattanaprichakul, Penvadee; Fox, Patricia; Torres-Cabala, Carlos A; Bassett, Roland L; Prieto, Victor G; Richards, Hunter W; Curry, Jonathan L

2014-11-01

High mitotic figure count (MFC) correlates with low survival rate in Merkel cell carcinoma (MCC). However, the prognostic impact of histone biomarkers as surrogates of MFC in MCC is unknown. We evaluated the prognostic significance of the immunodetection of mitotic figures and of G2+ tumor nuclei with histone-associated mitotic markers H3K79me3T80ph (H3KT) and phosphohistone H3 (PHH3) in MCC. Immunohistochemical analyses of H3KT and PHH3 and proliferative marker Ki-67 were performed in a series of 21 cases of MCC. The significance of the pathologic data and immunoreactivity with these markers was evaluated with Pearson correlation and paired Student t-test. Univariate Cox proportional hazards regression models were performed to assess the relationships between these markers and survival. H3KT detected a higher number of mitotic figure (p<0.0001) and G2+ tumor nuclei (p<0.0052) than did PHH3. Furthermore, the MFC combined with G2+ tumor nuclei detected with H3KT compared to PHH3 and manual MFC was a significant predictor of impaired survival in patients with MCC (p=0.035; HR=1.0172), corresponding to a 1.72% increased risk of death for each unit increase in H3KT. Biomarker analysis of proliferative rates with histone markers may have relevance in stratifying risk in patients with MCC. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Synergistic Blockade of Mitotic Exit by Two Chemical Inhibitors of the APC/C

PubMed Central

Sackton, Katharine L.; Dimova, Nevena; Zeng, Xing; Tian, Wei; Zhang, Mengmeng; Sackton, Timothy B.; Meaders, Johnathan; Pfaff, Kathleen L.; Sigoillot, Frederic; Yu, Hongtao; Luo, Xuelian; King, Randall W.

2014-01-01

Summary Protein machines are multi-subunit protein complexes that orchestrate highly regulated biochemical tasks. An example is the Anaphase-Promoting Complex/Cyclosome (APC/C), a thirteen-subunit ubiquitin ligase that initiates the metaphase-anaphase transition and mitotic exit by targeting proteins such as securin and cyclin B1 for ubiquitin-dependent destruction by the proteasome1,2. Because blocking mitotic exit is an effective approach for inducing tumor cell death3,4, the APC/C represents a potential novel target for cancer therapy. APC/C activation in mitosis requires binding of Cdc205, which forms a co-receptor with the APC/C to recognize substrates containing a Destruction box (D-box)6-14. Here we demonstrate that we can synergistically inhibit APC/C-dependent proteolysis and mitotic exit by simultaneously disrupting two protein-protein interactions within the APC/C-Cdc20-substrate ternary complex. We identified a small molecule, called apcin (APC inhibitor), which binds to Cdc20 and competitively inhibits the ubiquitylation of D-box-containing substrates. Analysis of the crystal structure of the apcin-Cdc20 complex suggests that apcin occupies the D-box-binding pocket on the side face of the WD40-domain. The ability of apcin to block mitotic exit is synergistically amplified by co-addition of tosyl-L-arginine methyl ester (TAME), a small molecule that blocks the APC/C-Cdc20 interaction15,16. This work suggests that simultaneous disruption of multiple, weak protein-protein interactions is an effective approach for inactivating a protein machine. PMID:25156254

The Echinoid Mitotic Gradient: Effect of Cell Size on the Micromere Cleavage Cycle

PubMed Central

Langelan Duncan, Rosalie E.; Whiteley, Arthur H.

2012-01-01

SUMMARY Like other euechinoids, the fertilized eggs of the sand dollar Dendraster excentricus proceed through cleavages that produce a pattern of macromeres, mesomeres, and micromeres at the 4th division. The 8 cells of the macro-mesomere lineage proceed through 6 additional cleavages before hatching. At the fifth overall division, the 4 micromeres produce a lineage of large micromeres that will divide 3 additional times, and a lineage of small micromeres that will divide once more before hatching. Irrespective of lineage, the length of the cell cycles is closely related to the size of the blastomere; cells of the same size have the same cell cycle time. A consequence is that at the fourth cleavage, there is a gradient of mitotic activity from the fastest dividers at the animal pole and the slowest cleacing micromeres at the vegetal pole. By the time of hatching, which is the 10th division of meso-macromeres, all cells are the same small size, the metachronic pattern of division gives way to asynchrony, and the mitotic gradient along the polar axis is lost. Experimental pre-exposure to sodium dodecyl sulfate (SDS), however, blocks the appearance of the gradients in cell size, the mitotic gradient, and the differential in cell cycle times. It is proposed that the mitotic gradients, cell cycle times, and attainment of a state of asynchrony are functions of cell size. Developmental consequences of the transition are large, and include coordinated activation of transcriptions, synthesis of new patterns of proteins, alterations of metabolism, and onset of morphogenesis. PMID:22006441

Cleavage in conical sand dollar eggs.

PubMed

Rappaport, R; Rappaport, B N

1994-07-01

Previous experiments have shown that the mitotic apparatus and the surface can interact and produce functional furrows in various unusual geometrical circumstances. The consistent development of the furrow in the plane equidistant from the aster centers has led to conjecture about the need for a special structural configuration of the subsurface in the future cleavage plane. In most experiments involving altered cell geometry, the relation between each aster and nearby surface was symmetrical, and the effect of that symmetry upon the position and orientation of the cleavage mechanism in the cortex has not been systematically analyzed. The normal symmetry of sand dollar eggs can be changed by reshaping them into cones. When the cone and mitotic axes are parallel, the aster center closer to the vertex is also closer to the nearby surface, and the cleavage plane develops on the vertex side of the midpoint between the asters. A mitotic apparatus oriented perpendicular to the cone axis produces in the base of the cone a normal unilateral furrow that advances toward the vertex, and a second contractile band that isolates the vertex region. This event only occurs when the surface is conical and the mitotic apparatus is perpendicular to the cone axis. Furrow formation is not restricted to the plane of the metaphase plate or the midpoint between the aster centers. The orientation of mitotic apparatus-produced contractile bands is not limited to the circumstances in normal cytokinesis, but may vary according to surface contour. These results confirm predictions of the Harris and Gewalt model of contractile ring induction.

NUP98 fusion oncoproteins interact with the APC/C(Cdc20) as a pseudosubstrate and prevent mitotic checkpoint complex binding.

PubMed

Salsi, Valentina; Fantini, Sebastian; Zappavigna, Vincenzo

2016-09-01

NUP98 is a recurrent partner gene in translocations causing acute myeloid leukemias and myelodisplastic syndrome. The expression of NUP98 fusion oncoproteins has been shown to induce mitotic spindle defects and chromosome missegregation, which correlate with the capability of NUP98 fusions to cause mitotic checkpoint attenuation. We show that NUP98 oncoproteins physically interact with the APC/C(Cdc20) in the absence of the NUP98 partner protein RAE1, and prevent the binding of the mitotic checkpoint complex to the APC/C(Cdc20). NUP98 oncoproteins require the GLEBS-like domain present in their NUP98 moiety to bind the APC/C(Cdc20). We found that NUP98 wild-type is a substrate of APC/C(Cdc20) prior to mitotic entry, and that its binding to APC/C(Cdc20) is controlled via phosphorylation of a PEST sequence located within its C-terminal portion. We identify S606, within the PEST sequence, as a key target site, whose phosphorylation modulates the capability of NUP98 to interact with APC/C(Cdc20). We finally provide evidence for an involvement of the peptidyl-prolyl isomerase PIN1 in modulating the possible conformational changes within NUP98 that lead to its dissociation from the APC/C(Cdc20) during mitosis. Our results provide novel insight into the mechanisms underlying the aberrant capability of NUP98 oncoproteins to interact with APC/C(Cdc20) and to interfere with its function.

Determining local and contextual features describing appearance of difficult to identify mitotic figures

NASA Astrophysics Data System (ADS)

Gandomkar, Ziba; Brennan, Patrick C.; Mello-Thoms, Claudia

2017-03-01

Mitotic count is helpful in determining the aggressiveness of breast cancer. In previous studies, it was shown that the agreement among pathologists for grading mitotic index is fairly modest, as mitoses have a large variety of appearances and they could be mistaken for other similar objects. In this study, we determined local and contextual features that differ significantly between easily identifiable mitoses and challenging ones. The images were obtained from the Mitosis-Atypia 2014 challenge. In total, the dataset contained 453 mitotic figures. Two pathologists annotated each mitotic figure. In case of disagreement, an opinion from a third pathologist was requested. The mitoses were grouped into three categories, those recognized as "a true mitosis" by both pathologists ,those labelled as "a true mitosis" by only one of the first two readers and also the third pathologist, and those annotated as "probably a mitosis" by all readers or the majority of them. After color unmixing, the mitoses were segmented from H channel. Shape-based features along with intensity-based and textural features were extracted from H-channel, blue ratio channel and five different color spaces. Holistic features describing each image were also considered. The Kruskal-Wallis H test was used to identify significantly different features. Multiple comparisons were done using the rank-based version of Tukey-Kramer test. The results indicated that there are local and global features which differ significantly among different groups. In addition, variations between mitoses in different groups were captured in the features from HSL and LCH color space more than other ones.

Condensins under the microscope.

PubMed

Maeshima, Kazuhiro; Hibino, Kayo; Hudson, Damien F

2018-04-30

Condensins are key players in mitotic chromosome condensation. Using an elegant combination of state-of-the-art imaging techniques, Walther et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201801048) counted the number of Condensins, examined their behaviors on human mitotic chromosomes, and integrated the quantitative data to propose a new mechanistic model for chromosome condensation. © 2018 Maeshima et al.

DREAMs make plant cells to cycle or to become quiescent.

PubMed

Magyar, Zoltán; Bögre, László; Ito, Masaki

2016-12-01

Cell cycle phase specific oscillation of gene transcription has long been recognized as an underlying principle for ordered processes during cell proliferation. The G1/S-specific and G2/M-specific cohorts of genes in plants are regulated by the E2F and the MYB3R transcription factors. Mutant analysis suggests that activator E2F functions might not be fully required for cell cycle entry. In contrast, the two activator-type MYB3Rs are part of positive feedback loops to drive the burst of mitotic gene expression, which is necessary at least to accomplish cytokinesis. Repressor MYB3Rs act outside the mitotic time window during cell cycle progression, and are important for the shutdown of mitotic genes to impose quiescence in mature organs. The two distinct classes of E2Fs and MYB3Rs together with the RETINOBLATOMA RELATED are part of multiprotein complexes that may be evolutionary related to what is known as DREAM complex in animals. In plants, there are multiple such complexes with distinct compositions and functions that may be involved in the coordinated cell cycle and developmental regulation of E2F targets and mitotic genes. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Fanconi Anemia C Protein Binds to and Regulates Stathmin-1 Phosphorylation

PubMed Central

Magron, Audrey; Elowe, Sabine; Carreau, Madeleine

2015-01-01

The Fanconi anemia (FA) proteins are involved in a signaling network that assures the safeguard of chromosomes. To understand the function of FA proteins in cellular division events, we investigated the interaction between Stathmin-1 (STMN1) and the FA group C (FANCC) protein. STMN1 is a ubiquitous cytosolic protein that regulates microtubule dynamics. STMN1 activities are regulated through phosphorylation-dephosphorylation mechanisms that control assembly of the mitotic spindle, and dysregulation of STMN1 phosphorylation is associated with mitotic aberrancies leading to chromosome instability and cancer progression. Using different biochemical approaches, we showed that FANCC interacts and co-localizes with STMN1 at centrosomes during mitosis. We also showed that FANCC is required for STMN1 phosphorylation, as mutations in FANCC reduced serine 16- and 38-phosphorylated forms of STMN1. Phosphorylation of STMN1 at serine 16 is likely an event dependent on a functional FA pathway, as it is reduced in FANCA- and FANCD2-mutant cells. Furthermore, FA-mutant cells exhibited mitotic spindle anomalies such as supernumerary centrosomes and shorter mitotic spindles. These results suggest that FA proteins participate in the regulation of cellular division via the microtubule-associated protein STMN1. PMID:26466335

Influence of acute promyelocytic leukemia therapeutic drugs on nuclear pore complex density and integrity.

PubMed

Lång, Anna; Øye, Alexander; Eriksson, Jens; Rowe, Alexander D; Lång, Emma; Bøe, Stig Ove

2018-05-15

During cell division, a large number of nuclear proteins are released into the cytoplasm due to nuclear envelope breakdown. Timely nuclear import of these proteins following exit from mitosis is critical for establishment of the G1 nuclear environment. Dysregulation of post-mitotic nuclear import may affect the fate of newly divided stem or progenitor cells and may lead to cancer. Acute promyelocytic leukemia (APL) is a malignant disorder that involves a defect in blood cell differentiation at the promyelocytic stage. Recent studies suggest that pharmacological concentrations of the APL therapeutic drugs, all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), affect post-mitotic nuclear import of the APL-associated oncoprotein PML/RARA. In the present study, we have investigated the possibility that ATRA and ATO affect post-mitotic nuclear import through interference with components of the nuclear import machinery. We observe reduced density and impaired integrity of nuclear pore complexes after ATRA and/or ATO exposure. Using a post-mitotic nuclear import assay, we demonstrate distinct import kinetics among different nuclear import pathways while nuclear import rates were similar in the presence or absence of APL therapeutic drugs. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Effect of 60-Hz magnetic fields on ultraviolet light-induced mutation and mitotic recombination in Saccharomyces cerevisiae.

PubMed

Ager, D D; Radul, J A

1992-12-01

The purpose of this study was to examine the effect of extremely low frequency (ELF) magnetic fields on the induction of genetic damage. In general, mutational studies involving ELF magnetic fields have proven negative. However, studies examining sister-chromatid exchange and chromosome aberrations have yielded conflicting results. In this study, we have examined whether 60-Hz magnetic fields are capable of inducing mutation or mitotic recombination in the yeast Saccharomyces cerevisiae. In addition we determined whether magnetic fields were capable of altering the genetic response of S. cerevisiae to UV (254 nm). We measured the frequencies of induced mutation, gene conversion and reciprocal mitotic crossing-over for exposures to magnetic fields alone (1 mT) or in combination with various UV exposures (2-50 J/m2). These experiments were performed using a repair-proficient strain (RAD+), as well as a strain of yeast (rad3) which is incapable of excising UV-induced thymine dimers. Magnetic field exposures did not induce mutation, gene conversion or reciprocal mitotic crossing-over in either of these strains, nor did the fields influence the frequencies of UV-induced genetic events.

Prognosis after surgical excision of canine fibrous connective tissue sarcomas.

PubMed

Bostock, D E; Dye, M T

1980-09-01

One hundred eighty seven dogs from which fibrous connective tissue sarcomas had been excised were studied until death or for at least 3 years after surgery. Dogs with a skin fibrosarcoma had a median survival time of 80 weeks, compared with 140 weeks for animals with haemangiopericytoma in similar sites, this difference being statistically significant. However, the difference in survival time between the two histologic types disappeared when tumours with a similar mitotic index were compared. Dogs with a tumour of mitotic index 9 or more had a median survival time of 49 weeks, compared with 118 weeks for those with a tumour of mitotic index less than 9, regardless of tumour morphology. Tumour recurrence rates of 62% and 25% respectively for the two groups were also significantly different.

Endocycles: a recurrent evolutionary innovation for post-mitotic cell growth.

PubMed

Edgar, Bruce A; Zielke, Norman; Gutierrez, Crisanto

2014-03-01

In endoreplication cell cycles, known as endocycles, cells successively replicate their genomes without segregating chromosomes during mitosis and thereby become polyploid. Such cycles, for which there are many variants, are widespread in protozoa, plants and animals. Endocycling cells can achieve ploidies of >200,000 C (chromatin-value); this increase in genomic DNA content allows a higher genomic output, which can facilitate the construction of very large cells or enhance macromolecular secretion. These cells execute normal S phases, using a G1-S regulatory apparatus similar to the one used by mitotic cells, but their capability to segregate chromosomes has been suppressed, typically by downregulation of mitotic cyclin-dependent kinase activity. Endocycles probably evolved many times, and the various endocycle mechanisms found in nature highlight the versatility of the cell cycle control machinery.

Recent findings and future directions for interpolar mitotic kinesin inhibitors in cancer therapy

PubMed Central

Myers, Stephanie M.; Collins, Ian

2016-01-01

The kinesin class of microtubule-associated motor proteins present attractive anti-cancer targets owing to their roles in key functions in dividing cells. Two interpolar mitotic kinesins Eg5 and HSET have opposing motor functions in mitotic spindle assembly with respect to microtubule movement, but both offer opportunities to develop cancer selective therapeutic agents. Here, we summarize the progress to date in developing inhibitors of Eg5 and HSET, with an emphasis on structural biology insights into the binding modes of allosteric inhibitors, compound selectivity and mechanisms of action of different chemical scaffolds. We discuss translation of preclinical studies to clinical experience with Eg5 inhibitors, recent findings on potential resistance mechanisms, and explore the implications for future anticancer drug development against these targets. PMID:26976726

Recent findings and future directions for interpolar mitotic kinesin inhibitors in cancer therapy.

PubMed

Myers, Stephanie M; Collins, Ian

2016-01-01

The kinesin class of microtubule-associated motor proteins present attractive anticancer targets owing to their roles in key functions in dividing cells. Two interpolar mitotic kinesins Eg5 and HSET have opposing motor functions in mitotic spindle assembly with respect to microtubule movement, but both offer opportunities to develop cancer selective therapeutic agents. Here, we summarize the progress to date in developing inhibitors of Eg5 and HSET, with an emphasis on structural biology insights into the binding modes of allosteric inhibitors, compound selectivity and mechanisms of action of different chemical scaffolds. We discuss translation of preclinical studies to clinical experience with Eg5 inhibitors, recent findings on potential resistance mechanisms and explore the implications for future anticancer drug development against these targets.

Elusive inheritance: Transgenerational effects and epigenetic inheritance in human environmental disease.

PubMed

Martos, Suzanne N; Tang, Wan-Yee; Wang, Zhibin

2015-07-01

Epigenetic mechanisms involving DNA methylation, histone modification, histone variants and nucleosome positioning, and noncoding RNAs regulate cell-, tissue-, and developmental stage-specific gene expression by influencing chromatin structure and modulating interactions between proteins and DNA. Epigenetic marks are mitotically inherited in somatic cells and may be altered in response to internal and external stimuli. The idea that environment-induced epigenetic changes in mammals could be inherited through the germline, independent of genetic mechanisms, has stimulated much debate. Many experimental models have been designed to interrogate the possibility of transgenerational epigenetic inheritance and provide insight into how environmental exposures influence phenotypes over multiple generations in the absence of any apparent genetic mutation. Unexpected molecular evidence has forced us to reevaluate not only our understanding of the plasticity and heritability of epigenetic factors, but of the stability of the genome as well. Recent reviews have described the difference between transgenerational and intergenerational effects; the two major epigenetic reprogramming events in the mammalian lifecycle; these two events making transgenerational epigenetic inheritance of environment-induced perturbations rare, if at all possible, in mammals; and mechanisms of transgenerational epigenetic inheritance in non-mammalian eukaryotic organisms. This paper briefly introduces these topics and mainly focuses on (1) transgenerational phenotypes and epigenetic effects in mammals, (2) environment-induced intergenerational epigenetic effects, and (3) the inherent difficulties in establishing a role for epigenetic inheritance in human environmental disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

Targeted interactomics reveals a complex core cell cycle machinery in Arabidopsis thaliana

PubMed Central

Van Leene, Jelle; Hollunder, Jens; Eeckhout, Dominique; Persiau, Geert; Van De Slijke, Eveline; Stals, Hilde; Van Isterdael, Gert; Verkest, Aurine; Neirynck, Sandy; Buffel, Yelle; De Bodt, Stefanie; Maere, Steven; Laukens, Kris; Pharazyn, Anne; Ferreira, Paulo C G; Eloy, Nubia; Renne, Charlotte; Meyer, Christian; Faure, Jean-Denis; Steinbrenner, Jens; Beynon, Jim; Larkin, John C; Van de Peer, Yves; Hilson, Pierre; Kuiper, Martin; De Veylder, Lieven; Van Onckelen, Harry; Inzé, Dirk; Witters, Erwin; De Jaeger, Geert

2010-01-01

Cell proliferation is the main driving force for plant growth. Although genome sequence analysis revealed a high number of cell cycle genes in plants, little is known about the molecular complexes steering cell division. In a targeted proteomics approach, we mapped the core complex machinery at the heart of the Arabidopsis thaliana cell cycle control. Besides a central regulatory network of core complexes, we distinguished a peripheral network that links the core machinery to up- and downstream pathways. Over 100 new candidate cell cycle proteins were predicted and an in-depth biological interpretation demonstrated the hypothesis-generating power of the interaction data. The data set provided a comprehensive view on heterodimeric cyclin-dependent kinase (CDK)–cyclin complexes in plants. For the first time, inhibitory proteins of plant-specific B-type CDKs were discovered and the anaphase-promoting complex was characterized and extended. Important conclusions were that mitotic A- and B-type cyclins form complexes with the plant-specific B-type CDKs and not with CDKA;1, and that D-type cyclins and S-phase-specific A-type cyclins seem to be associated exclusively with CDKA;1. Furthermore, we could show that plants have evolved a combinatorial toolkit consisting of at least 92 different CDK–cyclin complex variants, which strongly underscores the functional diversification among the large family of cyclins and reflects the pivotal role of cell cycle regulation in the developmental plasticity of plants. PMID:20706207

Ancient Evolutionary Trade-Offs between Yeast Ploidy States

PubMed Central

Zörgö, Enikö; Chwialkowska, Karolina; Gjuvsland, Arne B.; Garré, Elena; Sunnerhagen, Per; Liti, Gianni; Blomberg, Anders; Omholt, Stig W.; Warringer, Jonas

2013-01-01

The number of chromosome sets contained within the nucleus of eukaryotic organisms is a fundamental yet evolutionarily poorly characterized genetic variable of life. Here, we mapped the impact of ploidy on the mitotic fitness of baker's yeast and its never domesticated relative Saccharomyces paradoxus across wide swaths of their natural genotypic and phenotypic space. Surprisingly, environment-specific influences of ploidy on reproduction were found to be the rule rather than the exception. These ploidy–environment interactions were well conserved across the 2 billion generations separating the two species, suggesting that they are the products of strong selection. Previous hypotheses of generalizable advantages of haploidy or diploidy in ecological contexts imposing nutrient restriction, toxin exposure, and elevated mutational loads were rejected in favor of more fine-grained models of the interplay between ecology and ploidy. On a molecular level, cell size and mating type locus composition had equal, but limited, explanatory power, each explaining 12.5%–17% of ploidy–environment interactions. The mechanism of the cell size–based superior reproductive efficiency of haploids during Li+ exposure was traced to the Li+ exporter ENA. Removal of the Ena transporters, forcing dependence on the Nha1 extrusion system, completely altered the effects of ploidy on Li+ tolerance and evoked a strong diploid superiority, demonstrating how genetic variation at a single locus can completely reverse the relative merits of haploidy and diploidy. Taken together, our findings unmasked a dynamic interplay between ploidy and ecology that was of unpredicted evolutionary importance and had multiple molecular roots. PMID:23555297

Manufacture of Third-Generation Lentivirus for Preclinical Use, with Process Development Considerations for Translation to Good Manufacturing Practice.

PubMed

Gándara, Carolina; Affleck, Valerie; Stoll, Elizabeth Ann

2018-02-01

Lentiviral vectors are used in laboratories around the world for in vivo and ex vivo delivery of gene therapies, and increasingly clinical investigation as well as preclinical applications. The third-generation lentiviral vector system has many advantages, including high packaging capacity, stable gene expression in both dividing and post-mitotic cells, and low immunogenicity in the recipient organism. Yet, the manufacture of these vectors is challenging, especially at high titers required for direct use in vivo, and further challenges are presented by the process of translating preclinical gene therapies toward manufacture of products for clinical investigation. The goals of this paper are to report the protocol for manufacturing high-titer third-generation lentivirus for preclinical testing and to provide detailed information on considerations for translating preclinical viral vector manufacture toward scaled-up platforms and processes in order to make gene therapies under Good Manufacturing Practice that are suitable for clinical trials.

Manufacture of Third-Generation Lentivirus for Preclinical Use, with Process Development Considerations for Translation to Good Manufacturing Practice

PubMed Central

Gándara, Carolina; Affleck, Valerie; Stoll, Elizabeth Ann

2018-01-01

Lentiviral vectors are used in laboratories around the world for in vivo and ex vivo delivery of gene therapies, and increasingly clinical investigation as well as preclinical applications. The third-generation lentiviral vector system has many advantages, including high packaging capacity, stable gene expression in both dividing and post-mitotic cells, and low immunogenicity in the recipient organism. Yet, the manufacture of these vectors is challenging, especially at high titers required for direct use in vivo, and further challenges are presented by the process of translating preclinical gene therapies toward manufacture of products for clinical investigation. The goals of this paper are to report the protocol for manufacturing high-titer third-generation lentivirus for preclinical testing and to provide detailed information on considerations for translating preclinical viral vector manufacture toward scaled-up platforms and processes in order to make gene therapies under Good Manufacturing Practice that are suitable for clinical trials. PMID:29212357

How muscle fiber lengths and velocities affect muscle force generation as humans walk and run at different speeds

PubMed Central

Arnold, Edith M.; Hamner, Samuel R.; Seth, Ajay; Millard, Matthew; Delp, Scott L.

2013-01-01

SUMMARY The lengths and velocities of muscle fibers have a dramatic effect on muscle force generation. It is unknown, however, whether the lengths and velocities of lower limb muscle fibers substantially affect the ability of muscles to generate force during walking and running. We examined this issue by developing simulations of muscle–tendon dynamics to calculate the lengths and velocities of muscle fibers from electromyographic recordings of 11 lower limb muscles and kinematic measurements of the hip, knee and ankle made as five subjects walked at speeds of 1.0–1.75 m s−1 and ran at speeds of 2.0–5.0 m s−1. We analyzed the simulated fiber lengths, fiber velocities and forces to evaluate the influence of force–length and force–velocity properties on force generation at different walking and running speeds. The simulations revealed that force generation ability (i.e. the force generated per unit of activation) of eight of the 11 muscles was significantly affected by walking or running speed. Soleus force generation ability decreased with increasing walking speed, but the transition from walking to running increased the force generation ability by reducing fiber velocities. Our results demonstrate the influence of soleus muscle architecture on the walk-to-run transition and the effects of muscle–tendon compliance on the plantarflexors' ability to generate ankle moment and power. The study presents data that permit lower limb muscles to be studied in unprecedented detail by relating muscle fiber dynamics and force generation to the mechanical demands of walking and running. PMID:23470656

Loop L5 Assumes Three Distinct Orientations during the ATPase Cycle of the Mitotic Kinesin Eg5

PubMed Central

Muretta, Joseph M.; Behnke-Parks, William M.; Major, Jennifer; Petersen, Karl J.; Goulet, Adeline; Moores, Carolyn A.; Thomas, David D.; Rosenfeld, Steven S.

2013-01-01

Members of the kinesin superfamily of molecular motors differ in several key structural domains, which probably allows these molecular motors to serve the different physiologies required of them. One of the most variable of these is a stem-loop motif referred to as L5. This loop is longest in the mitotic kinesin Eg5, and previous structural studies have shown that it can assume different conformations in different nucleotide states. However, enzymatic domains often consist of a mixture of conformations whose distribution shifts in response to substrate binding or product release, and this information is not available from the “static” images that structural studies provide. We have addressed this issue in the case of Eg5 by attaching a fluorescent probe to L5 and examining its fluorescence, using both steady state and time-resolved methods. This reveals that L5 assumes an equilibrium mixture of three orientations that differ in their local environment and segmental mobility. Combining these studies with transient state kinetics demonstrates that there is a major shift in this distribution during transitions that interconvert weak and strong microtubule binding states. Finally, in conjunction with previous cryo-EM reconstructions of Eg5·microtubule complexes, these fluorescence studies suggest a model in which L5 regulates both nucleotide and microtubule binding through a set of reversible interactions with helix α3. We propose that these features facilitate the production of sustained opposing force by Eg5, which underlies its role in supporting formation of a bipolar spindle in mitosis. PMID:24145034

Mitotic Spindle Positioning in the EMS Cell of Caenorhabditis elegans Requires LET-99 and LIN-5/NuMA.

PubMed

Liro, Małgorzata J; Rose, Lesilee S

2016-11-01

Asymmetric divisions produce daughter cells with different fates, and thus are critical for animal development. During asymmetric divisions, the mitotic spindle must be positioned on a polarized axis to ensure the differential segregation of cell fate determinants into the daughter cells. In many cell types, a cortically localized complex consisting of Gα, GPR-1/2, and LIN-5 (Gαi/Pins/Mud, Gαi/LGN/NuMA) mediates the recruitment of dynactin/dynein, which exerts pulling forces on astral microtubules to physically position the spindle. The conserved PAR polarity proteins are known to regulate both cytoplasmic asymmetry and spindle positioning in many cases. However, spindle positioning also occurs in response to cell signaling cues that appear to be PAR-independent. In the four-cell Caenorhabditis elegans embryo, Wnt and Mes-1/Src-1 signaling pathways act partially redundantly to align the spindle on the anterior/posterior axis of the endomesodermal (EMS) precursor cell. It is unclear how those extrinsic signals individually contribute to spindle positioning and whether either pathway acts via conserved spindle positioning regulators. Here, we genetically test the involvement of Gα, LIN-5, and their negative regulator LET-99, in transducing EMS spindle positioning polarity cues. We also examined whether the C. elegans ortholog of another spindle positioning regulator, DLG-1, is required. We show that LET-99 acts in the Mes-1/Src-1 pathway for spindle positioning. LIN-5 is also required for EMS spindle positioning, possibly through a Gα- and DLG-1-independent mechanism. Copyright © 2016 by the Genetics Society of America.

Mitotic Spindle Positioning in the EMS Cell of Caenorhabditis elegans Requires LET-99 and LIN-5/NuMA

PubMed Central

Liro, Małgorzata J.; Rose, Lesilee S.

2016-01-01

Asymmetric divisions produce daughter cells with different fates, and thus are critical for animal development. During asymmetric divisions, the mitotic spindle must be positioned on a polarized axis to ensure the differential segregation of cell fate determinants into the daughter cells. In many cell types, a cortically localized complex consisting of Gα, GPR-1/2, and LIN-5 (Gαi/Pins/Mud, Gαi/LGN/NuMA) mediates the recruitment of dynactin/dynein, which exerts pulling forces on astral microtubules to physically position the spindle. The conserved PAR polarity proteins are known to regulate both cytoplasmic asymmetry and spindle positioning in many cases. However, spindle positioning also occurs in response to cell signaling cues that appear to be PAR-independent. In the four-cell Caenorhabditis elegans embryo, Wnt and Mes-1/Src-1 signaling pathways act partially redundantly to align the spindle on the anterior/posterior axis of the endomesodermal (EMS) precursor cell. It is unclear how those extrinsic signals individually contribute to spindle positioning and whether either pathway acts via conserved spindle positioning regulators. Here, we genetically test the involvement of Gα, LIN-5, and their negative regulator LET-99, in transducing EMS spindle positioning polarity cues. We also examined whether the C. elegans ortholog of another spindle positioning regulator, DLG-1, is required. We show that LET-99 acts in the Mes-1/Src-1 pathway for spindle positioning. LIN-5 is also required for EMS spindle positioning, possibly through a Gα- and DLG-1-independent mechanism. PMID:27672093

Slip slidin’ away of mitosis with CRL2Zyg11

PubMed Central

2016-01-01

The spindle assembly checkpoint arrests mitotic cells by preventing degradation of cyclin B1 by the anaphase-promoting complex/cyclosome, but some cells evade this checkpoint and slip out of mitosis. Balachandran et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201601083) show that the E3 ligase CRL2ZYG11 degrades cyclin B1, allowing mitotic slippage. PMID:27810907

Mitotic activity in dorsal epidermis of Rana pipiens.

NASA Technical Reports Server (NTRS)

Garcia-Arce, H.; Mizell, S.

1972-01-01

Study of statistically significant rhythms of mitotic division in dorsal epidermis of frogs, Rana pipiens, exposed to a 12:12 light:dark environment for 14 days. The results include the findings that (1) male animals have a primary period of 22 hr in summer and 18 hr in winter, (2) female animals have an 18 hr period, and (3) parapinealectomy and blinding abolish the rhythm.

Effects of Taxol plus radiation on the apoptotic and mitotic indices of mouse intestinal crypt cells.

PubMed

Ozkan, L; Ozuysal, S; Egeli, U; Adim, S B; Tunca, B; Aydemir, N; Ceçener, G; Ergül, E; Akpinar, G; Cimen, C; Engin, K; Ahmed, M M

2001-07-01

In this study we investigated the effect of Taxol, radiation, or Taxol plus radiation on highly proliferative normal tissue--the intestinal crypt cells of Swiss albino mice. Swiss-albino mice, 3-4 months old, were used in this study. Taxol was administered by bolus intravenously through the tail vein. Radiation was given using a linear accelerator. There were four treatment categories, which comprised a total of 34 groups. Each group consisted of five animals. The first category was a control category which comprised one group (n = 5). The second treatment category was Taxol alone which comprised three groups (n = 15). The third treatment category was radiation alone which comprised three groups (n = 15). The fourth treatment category was Taxol plus radiation which comprised 27 groups (n = 135). Mice were killed 24 h after Taxol or radiation or combined administration using ether anesthesia. Using a light microscope, apoptotic and mitotic indices were counted on jejunal crypt cells of mice that were stained with hematoxylin-eosin. Differences between groups were statistically evaluated with Student's t-test. Taxol caused a dose-dependent increase in apoptosis (P = 0.045) and decreased the mitotic index (P = 0.006) at high doses. Similarly, radiation caused a dose-dependent increase in apoptosis (P = 0.046) and decreased the mitotic index (P = 0.299) at higher radiation doses. Compared to radiation alone, Taxol caused a significant induction of apoptosis (P = 0.010). In combination, no significant radiosensitizing effect of Taxol was observed (enhancement ratio < 1), when compared to radiation alone. However, an increase in apoptosis was observed after 24 h of Taxol exposure when compared to 12 or 48 h of Taxol exposure (P = 0.0001 and P = 0.0001). These findings suggest that Taxol did not cause a radiosensitizing effect in intestinal crypt cells. However, a 24-hour pretreatment of Taxol exposure followed by radiation caused significant induction of apoptosis and reduction of the mitotic index when compared to other Taxol timing sequences. Thus, the lack of a radiosensitizing effect of Taxol in these proliferative cells may be due to enhanced mitotic death rather than apoptotic death.

Motor unit firing rate patterns during voluntary muscle force generation: a simulation study

NASA Astrophysics Data System (ADS)

Hu, Xiaogang; Rymer, William Z.; Suresh, Nina L.

2014-04-01

Objective. Muscle force is generated by a combination of motor unit (MU) recruitment and changes in the discharge rate of active MUs. There have been two basic MU recruitment and firing rate paradigms reported in the literature, which describe the control of the MUs during force generation. The first (termed the reverse ‘onion skin’ profile), exhibits lower firing rates for lower threshold units, with higher firing rates occurring in higher threshold units. The second (termed the ‘onion skin’ profile), exhibits an inverse arrangement, with lower threshold units reaching higher firing rates. Approach. Using a simulation of the MU activity in a hand muscle, this study examined the force generation capacity and the variability of the muscle force magnitude at different excitation levels of the MU pool under these two different MU control paradigms. We sought to determine which rate/recruitment scheme was more efficient for force generation, and which scheme gave rise to the lowest force variability. Main results. We found that the force output of both firing patterns leads to graded force output at low excitation levels, and that the force generation capacity of the two different paradigms diverged around 50% excitation. In the reverse ‘onion skin’ pattern, at 100% excitation, the force output reached up to 88% of maximum force, whereas for the ‘onion skin’ pattern, the force output only reached up to 54% of maximum force at 100% excitation. The force variability was lower at the low to moderate force levels under the ‘onion skin’ paradigm than with the reverse ‘onion skin’ firing patterns, but this effect was reversed at high force levels. Significance. This study captures the influence of MU recruitment and firing rate organization on muscle force properties, and our results suggest that the different firing organizations can be beneficial at different levels of voluntary muscle force generation and perhaps for different tasks.

Racial disparities in molecular subtypes of endometrial cancer.

PubMed

Dubil, Elizabeth A; Tian, Chunqiao; Wang, Guisong; Tarney, Christopher M; Bateman, Nicholas W; Levine, Douglas A; Conrads, Thomas P; Hamilton, Chad A; Maxwell, George Larry; Darcy, Kathleen M

2018-04-01

Racial differences in the molecular subtypes of endometrial cancer and associations with progression-free survival (PFS) were evaluated. Molecular, clinical and PFS data were acquired from the Cancer Genome Atlas (TCGA) including classification into the integrative, somatic copy number alteration and transcript-based subtypes. The prevalence and prognostic value of the aggressive molecular subtypes (copy number variant [CNV]-high, cluster 4 or mitotic) were evaluated in Black and White patients. There were 337 patients including 14% self-designated as Black, 27% with advanced stage, and 82% with endometrioid histology. The CNV-high subtype was more common in Black than White patients (61.9% vs. 23.5%, P=0.0005) and suggested worse PFS in Black patients (hazard ratio [HR]=3.4, P=0.189). The cluster 4 subtype was more prevalent in Black patients (56.8% vs. 20.9%, P<0.0001) and associated with worse PFS in Black patients (HR=3.4, P=0.049). The mitotic subtype was more abundant in Black patients (64.1% vs. 33.7%, P=0.002), indicated worse PFS in Black patients (HR=4.1, P=0.044) including the endometrioid histology (HR=6.1, P=0.024) and exhibited race-associated enrichment in cell cycle signaling and pathways in cancer including PLK1 and BIRC7. All of these aggressive molecular subtypes also indicated worse PFS in White patients, with unique enrichments in mitotic signaling different from Black patients. The aggressive molecular subtypes from TCGA were more common in Black endometrial cancer patients and indicated worse PFS in both Black and White patients. The mitotic subtypes also indicated worse PFS in Black patients with endometrioid histology. Enrichment patterns in mitotic signaling may represent therapeutic opportunities. Copyright © 2017. Published by Elsevier Inc.

Microtubule-dependent path to the cell cortex for cytoplasmic dynein in mitotic spindle orientation

PubMed Central

Markus, Steven M.; Lee, Wei-Lih

2011-01-01

During animal development, microtubules (MTs) play a major role in directing cellular and subcellular patterning, impacting cell polarization and subcellular organization, thereby affecting cell fate determination and tissue architecture. In particular, when progenitor cells divide asymmetrically along an anterior-posterior or apical-basal axis, MTs must coordinate the position of the mitotic spindle with the site of cell division to ensure normal distribution of cell fate determinants and equal sequestration of genetic material into the two daughter cells. Emerging data from diverse model systems have led to the prevailing view that, during mitotic spindle positioning, polarity cues at the cell cortex signal for the recruitment of NuMA and the minus-end directed MT motor cytoplasmic dynein.1 The NuMA/dynein complex is believed to connect, in turn, to the mitotic spindle via astral MTs, thus aligning and tethering the spindle, but how this connection is achieved faithfully is unclear. Do astral MTs need to search for and then capture cortical NuMA/dynein? How does dynein capture the astral MTs emanating from the correct spindle pole? Recently, using the classical model of asymmetric cell division—budding yeast S. cerevisiae—we successfully demonstrated that astral MTs assume an active role in cortical dynein targeting, in that astral MTs utilize their distal plus ends to deliver dynein to the daughter cell cortex, the site where dynein activity is needed to perform its spindle alignment function. This observation introduced the novel idea that, during mitotic spindle orientation processes, polarity cues at the cell cortex may actually signal to prime the cortical receptors for MT-dependent dynein delivery. This model is consistent with the observation that dynein/dynactin accumulate prominently at the astral MT plus ends during metaphase in a wide range of cultured mammalian cells. PMID:22754610

Redistribution of fluorescently labeled tubulin in the mitotic apparatus of sand dollar eggs and the effects of taxol.

PubMed

Hamaguchi, Y; Toriyama, M; Sakai, H; Hiramoto, Y

1987-02-01

Fluorescently labeled tubulin was quickly incorporated into the mitotic apparatus when injected into a live sand dollar egg. After a rectangular area (1.6 X 16 microns) of the mitotic spindle was photobleached at metaphase or anaphase by the irradiation of a laser microbeam, redistribution of fluorescence was almost complete within 30 sec. The photobleached area did not change in shape during the redistribution. During the period of redistribution, the bleached area moved slightly toward the near pole at metaphase and anaphase (means: 1.6 and 1.8 micron/min, respectively). These results indicate that redistribution was not due to the exchange of tubulin subunits only at the ends of microtubules but to their rapid exchange at sites along the microtubules in the bleached region. Furthermore, treadmilling of tubulin molecules along with the spindle microtubules possibly occurred at the rate of 1.6 micron/min at metaphase. Birefringence of the mitotic apparatus increased with a large increase in both the number and length of astral rays shortly after taxol was injected. However, the microtubules did not all seem to elongate at the same rate but appeared to become equalized in length. Chromosome movement stopped within 60 sec after the injection. Centrospheres became large and the labeled tubulin already incorporated into the centrospheres was excluded from the enlarged centrospheres. Shortly after the labeled tubulin was injected following the injection of taxol, it accumulated in the peripheral region of the centrospheres, suggesting that microtubules first assembled at this region. Fluorescently labeled tubulin in the mitotic apparatus in the egg after injection of taxol was redistributed much more slowly after photobleaching than in uninjected eggs.

Cytologic anaplasia is a prognostic factor in osteosarcoma biopsies, but mitotic rate or extent of spontaneous tumor necrosis are not: a critique of the College of American Pathologists Bone Biopsy template.

PubMed

Cates, Justin Mm; Dupont, William D

2017-01-01

The current College of American Pathologists cancer template for reporting biopsies of bone tumors recommends including information that is of unproven prognostic significance for osteosarcoma, such as the presence of spontaneous tumor necrosis and mitotic rate. Conversely, the degree of cytologic anaplasia (degree of differentiation) is not reported in this template. This retrospective cohort study of 125 patients with high-grade osteosarcoma was performed to evaluate the prognostic impact of these factors in diagnostic biopsy specimens in predicting the clinical outcome and response to neoadjuvant chemotherapy. Multivariate Cox regression was performed to adjust survival analyses for well-established prognostic factors. Multivariate logistic regression was used to determine odds ratios for good chemotherapy response (≥90% tumor necrosis). Osteosarcomas with severe anaplasia were independently associated with increased overall and disease-free survival, but mitotic rate and spontaneous necrosis had no prognostic impact after controlling for other confounding factors. Mitotic rate showed a trend towards increased odds of a good histologic response, but this effect was diminished after controlling for other predictive factors. Neither spontaneous necrosis nor the degree of cytologic anaplasia observed in biopsy specimens was predictive of a good response to chemotherapy. Mitotic rate and spontaneous tumor necrosis observed in pretreatment biopsy specimens of high-grade osteosarcoma are not strong independent prognostic factors for clinical outcome or predictors of response to neoadjuvant chemotherapy. Therefore, reporting these parameters for osteosarcoma, as recommended in the College of American Pathologists Bone Biopsy template, does not appear to have clinical utility. In contrast, histologic grading schemes for osteosarcoma based on the degree of cytologic anaplasia may have independent prognostic value and should continue to be evaluated.

Effective killing of the human pathogen Candida albicans by a specific inhibitor of non-essential mitotic kinesin Kip1p

PubMed Central

Chua, Penelope R; Roof, David M; Lee, Yan; Sakowicz, Roman; Clarke, David; Pierce, Dan; Stephens, Thoryn; Hamilton, Matthew; Morgan, Brad; Morgans, David; Nakai, Takashi; Tomasi, Adam; Maxon, Mary E

2007-01-01

Kinesins from the bipolar (Kinesin-5) family are conserved in eukaryotic organisms and play critical roles during the earliest stages of mitosis to mediate spindle pole body separation and formation of a bipolar mitotic spindle. To date, genes encoding bipolar kinesins have been reported to be essential in all organisms studied. We report the characterization of CaKip1p, the sole member of this family in the human pathogenic yeast Candida albicans. C. albicans Kip1p appears to localize to the mitotic spindle and loss of CaKip1p function interferes with normal progression through mitosis. Inducible excision of CaKIP1 revealed phenotypes unique to C. albicans, including viable homozygous Cakip1 mutants and an aberrant spindle morphology in which multiple spindle poles accumulate in close proximity to each other. Expression of the C. albicans Kip1 motor domain in Escherichia coli produced a protein with microtubule-stimulated ATPase activity that was inhibited by an aminobenzothiazole (ABT) compound in an ATP-competitive fashion. This inhibition results in ‘rigor-like’, tight association with microtubules in vitro. Upon treatment of C. albicans cells with the ABT compound, cells were killed, and terminal phenotype analysis revealed an aberrant spindle morphology similar to that induced by loss of the CaKIP1 gene. The ABT compound discovered is the first example of a fungal spindle inhibitor targeted to a mitotic kinesin. Our results also show that the non-essential nature and implementation of the bipolar motor in C. albicans differs from that seen in other organisms, and suggest that inhibitors of a non-essential mitotic kinesin may offer promise as cidal agents for antifungal drug discovery. PMID:17573815

Induction of Mitotic Cell Death by Overriding G2/M Checkpoint in Endometrial Cancer Cells with Non-functional p53

PubMed Central

Meng, Xiangbing; Laidler, Laura L.; Kosmacek, Elizabeth A.; Yang, Shujie; Xiong, Zhi; Zhu, Danlin; Wang, Xinjun; Dai, Donghai; Zhang, Yuping; Wang, Xiaofang; Brachova, Pavla; Albitar, Lina; Liu, Dawei; Ianzini, Fiorenza; Mackey, Michael A.; Leslie, Kimberly K.

2012-01-01

Objective Endometrial tumors with non-functional p53, such as serous uterine endometrial carcinomas, are aggressive malignancies with a poor outcome, yet they have an Achilles’ heel: due to loss of p53 function, these tumors may be sensitive to treatments which abrogate the G2/M checkpoint. Our objective was to exploit this weakness to induce mitotic cell death using two strategies: (1) EGFR inhibitor gefitinib combined with paclitaxel to arrest cells at mitosis, or (2) BI2536, an inhibitor of polo-like kinase 1 (PLK1), to block PLK1 activity. Methods We examined the impact of combining gefitinib and paclitaxel or PLK1 inhibitor on expression of G2/M checkpoint controllers, cell viability, and cell cycle progression in endometrial cancer cells with mutant p53. Results In cells lacking normal p53 activity, each treatment activated CDC25C and inactivated Wee1, which in turn activated cdc2 and sent cells rapidly through the G2/M checkpoint and into mitosis. Live cell imaging demonstrated irreversible mitotic arrest and eventual cell death. Combinatorial therapy with paclitaxel and gefitinib was highly synergistic and resulted in a 10-fold reduction in the IC50 for paclitaxel, from 14 nM as a single agent to 1.3 nM in the presence of gefitinib. However, BI2536 alone at low concentrations (5 nM) was the most effective treatment and resulted in massive mitotic cell death. In a xenograft mouse model with p53-deficient cells, low dose BI2536 significantly inhibited tumor growth. Conclusions These findings reveal induction of mitotic cell death as a therapeutic strategy for endometrial tumors lacking functional p53. PMID:23146687

Misexpression of cyclin D1 in embryonic germ cells promotes testicular teratoma initiation

PubMed Central

Lanza, Denise G.; Dawson, Emily P.; Rao, Priya; Heaney, Jason D.

2016-01-01

ABSTRACT Testicular teratomas result from anomalies in embryonic germ cell development. In the 129 family of inbred mouse strains, teratomas arise during the same developmental period that male germ cells normally enter G1/G0 mitotic arrest and female germ cells initiate meiosis (the mitotic:meiotic switch). Dysregulation of this switch associates with teratoma susceptibility and involves three germ cell developmental abnormalities seemingly critical for tumor initiation: delayed G1/G0 mitotic arrest, retention of pluripotency, and misexpression of genes normally restricted to embryonic female and adult male germ cells. One misexpressed gene, cyclin D1 (Ccnd1), is a known regulator of cell cycle progression and an oncogene in many tissues. Here, we investigated whether Ccnd1 misexpression in embryonic germ cells is a determinant of teratoma susceptibility in mice. We found that CCND1 localizes to teratoma-susceptible germ cells that fail to enter G1/G0 arrest during the mitotic:meiotic switch and is the only D-type cyclin misexpressed during this critical developmental time frame. We discovered that Ccnd1 deficiency in teratoma-susceptible mice significantly reduced teratoma incidence and suppressed the germ cell proliferation and pluripotency abnormalities associated with tumor initiation. Importantly, Ccnd1 expression was dispensable for somatic cell development and male germ cell specification and maturation in tumor-susceptible mice, implying that the mechanisms by which Ccnd1 deficiency reduced teratoma incidence were germ cell autonomous and specific to tumorigenesis. We conclude that misexpression of Ccnd1 in male germ cells is a key component of a larger pro-proliferative program that disrupts the mitotic:meiotic switch and predisposes 129 inbred mice to testicular teratocarcinogenesis. PMID:26901436

Measuring mitotic spindle dynamics in budding yeast

NASA Astrophysics Data System (ADS)

Plumb, Kemp

In order to carry out its life cycle and produce viable progeny through cell division, a cell must successfully coordinate and execute a number of complex processes with high fidelity, in an environment dominated by thermal noise. One important example of such a process is the assembly and positioning of the mitotic spindle prior to chromosome segregation. The mitotic spindle is a modular structure composed of two spindle pole bodies, separated in space and spanned by filamentous proteins called microtubules, along which the genetic material of the cell is held. The spindle is responsible for alignment and subsequent segregation of chromosomes into two equal parts; proper spindle positioning and timing ensure that genetic material is appropriately divided amongst mother and daughter cells. In this thesis, I describe fluorescence confocal microscopy and automated image analysis algorithms, which I have used to observe and analyze the real space dynamics of the mitotic spindle in budding yeast. The software can locate structures in three spatial dimensions and track their movement in time. By selecting fluorescent proteins which specifically label the spindle poles and cell periphery, mitotic spindle dynamics have been measured in a coordinate system relevant to the cell division. I describe how I have characterised the accuracy and precision of the algorithms by simulating fluorescence data for both spindle poles and the budding yeast cell surface. In this thesis I also describe the construction of a microfluidic apparatus that allows for the measurement of long time-scale dynamics of individual cells and the development of a cell population. The tools developed in this thesis work will facilitate in-depth quantitative analysis of the non-equilibrium processes in living cells.

PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis.

PubMed

Terzaghi, L; Tessaro, I; Raucci, F; Merico, V; Mazzini, G; Garagna, S; Zuccotti, M; Franciosi, F; Lodde, V

2016-08-02

Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC1's localization at the mid-zone and mid-body of the mitotic and meiotic spindle.

The Set1/COMPASS histone H3 methyltransferase helps regulate mitosis with the CDK1 and NIMA mitotic kinases in Aspergillus nidulans.

PubMed

Govindaraghavan, Meera; Anglin, Sarah Lea; Osmani, Aysha H; Osmani, Stephen A

2014-08-01

Mitosis is promoted and regulated by reversible protein phosphorylation catalyzed by the essential NIMA and CDK1 kinases in the model filamentous fungus Aspergillus nidulans. Protein methylation mediated by the Set1/COMPASS methyltransferase complex has also been shown to regulate mitosis in budding yeast with the Aurora mitotic kinase. We uncover a genetic interaction between An-swd1, which encodes a subunit of the Set1 protein methyltransferase complex, with NIMA as partial inactivation of nimA is poorly tolerated in the absence of swd1. This genetic interaction is additionally seen without the Set1 methyltransferase catalytic subunit. Importantly partial inactivation of NIMT, a mitotic activator of the CDK1 kinase, also causes lethality in the absence of Set1 function, revealing a functional relationship between the Set1 complex and two pivotal mitotic kinases. The main target for Set1-mediated methylation is histone H3K4. Mutational analysis of histone H3 revealed that modifying the H3K4 target residue of Set1 methyltransferase activity phenocopied the lethality seen when either NIMA or CDK1 are partially functional. We probed the mechanistic basis of these genetic interactions and find that the Set1 complex performs functions with CDK1 for initiating mitosis and with NIMA during progression through mitosis. The studies uncover a joint requirement for the Set1 methyltransferase complex with the CDK1 and NIMA kinases for successful mitosis. The findings extend the roles of the Set1 complex to include the initiation of mitosis with CDK1 and mitotic progression with NIMA in addition to its previously identified interactions with Aurora and type 1 phosphatase in budding yeast. Copyright © 2014 by the Genetics Society of America.

Mitotic Defects Lead to Pervasive Aneuploidy and Accompany Loss of RB1 Activity in Mouse LmnaDhe Dermal Fibroblasts

PubMed Central

Pratt, C. Herbert; Curtain, Michelle; Donahue, Leah Rae; Shopland, Lindsay S.

2011-01-01

Background Lamin A (LMNA) is a component of the nuclear lamina and is mutated in several human diseases, including Emery-Dreifuss muscular dystrophy (EDMD; OMIM ID# 181350) and the premature aging syndrome Hutchinson-Gilford progeria syndrome (HGPS; OMIM ID# 176670). Cells from progeria patients exhibit cell cycle defects in both interphase and mitosis. Mouse models with loss of LMNA function have reduced Retinoblastoma protein (RB1) activity, leading to aberrant cell cycle control in interphase, but how mitosis is affected by LMNA is not well understood. Results We examined the cell cycle and structural phenotypes of cells from mice with the Lmna allele, Disheveled hair and ears (LmnaDhe). We found that dermal fibroblasts from heterozygous LmnaDhe (LmnaDhe/+) mice exhibit many phenotypes of human laminopathy cells. These include severe perturbations to the nuclear shape and lamina, increased DNA damage, and slow growth rates due to mitotic delay. Interestingly, LmnaDhe/+ fibroblasts also had reduced levels of hypophosphorylated RB1 and the non-SMC condensin II-subunit D3 (NCAP-D3), a mitosis specific centromere condensin subunit that depends on RB1 activity. Mitotic check point control by mitotic arrest deficient-like 1 (MAD2L1) also was perturbed in LmnaDhe /+ cells. LmnaDhe /+ fibroblasts were consistently aneuploid and had higher levels of micronuclei and anaphase bridges than normal fibroblasts, consistent with chromosome segregation defects. Conclusions These data indicate that RB1 may be a key regulator of cellular phenotype in laminopathy-related cells, and suggest that the effects of LMNA on RB1 include both interphase and mitotic cell cycle control. PMID:21464947

The kinetochore proteins CENP-E and CENP-F directly and specifically interact with distinct BUB mitotic checkpoint Ser/Thr kinases.

PubMed

Ciossani, Giuseppe; Overlack, Katharina; Petrovic, Arsen; Huis In 't Veld, Pim J; Koerner, Carolin; Wohlgemuth, Sabine; Maffini, Stefano; Musacchio, Andrea

2018-05-10

The segregation of chromosomes during cell division relies on the function of the kinetochores, protein complexes that physically connect chromosomes with microtubules of the spindle. The metazoan proteins, centromere protein E (CENP-E) and CENP-F, are components of a fibrous layer of mitotic kinetochores named the corona. Several of their features suggest that CENP-E and CENP-F are paralogs: they are very large (comprising approximately 2700 and 3200 residues, respectively), contain abundant predicted coiled-coil structures, are C-terminally prenylated, and are endowed with microtubule-binding sites at their termini. Moreover, CENP-E contains an ATP-hydrolyzing motor domain that promotes microtubule plus end-directed motion. Here, we show that both CENP-E and CENP-F are recruited to mitotic kinetochores independently of the main corona constituent, the Rod-Zwilch-ZW10 (RZZ) complex. We identified specific interactions of CENP-F and CENP-E with budding uninhibited by benzimidazole 1 (BUB1) and BUB1-related (BUBR1) mitotic checkpoint Ser/Thr kinases, respectively, paralogous proteins involved in mitotic checkpoint control and chromosome alignment. Whereas BUBR1 was dispensable for kinetochore localization of CENP-E, BUB1 was stringently required for CENP-F localization. Through biochemical reconstitution, we demonstrated that the CENP-E-BUBR1 and CENP-F-BUB1 interactions are direct and require similar determinants, a dimeric coiled-coil in CENP-E or CENP-F and a kinase domain in BUBR1 or BUB1. Our findings are consistent with the existence of structurally similar BUB1-CENP-F and BUBR1-CENP-E complexes, supporting the notion that CENP-E and CENP-F are evolutionarily related. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Nuclear Migration During Retinal Development

PubMed Central

Baye, Lisa M.; Link, Brian A.

2009-01-01

In this review we focus on the mechanisms, regulation, and cellular consequences of nuclear migration in the developing retina. In the nervous system, nuclear migration is prominent during both proliferative and post-mitotic phases of development. Interkinetic nuclear migration is the process where the nucleus oscillates from the apical to basal surfaces in proliferative neuroepithelia. Proliferative nuclear movement occurs in step with the cell cycle, with M-phase being confined to the apical surface and G1-, S-, and G2-phases occurring at more basal locations. Later, following cell cycle exit, some neuron precursors migrate by nuclear translocation. In this mode of cellular migration, nuclear movement is the driving force for motility. Following discussion of the key components and important regulators for each of these processes, we present an emerging model where interkinetic nuclear migration functions to distinguish cell fates among retinal neuroepithelia. PMID:17560964

Differentiation by colchicine and vincristine sulphate of regenerative hepatocellular mitosis and tumour-associated hepatocellular mitosis.

PubMed

Parry, E W

1977-10-01

Colchicine and vincristine sulphate differentiate sharply between hepatocyte mitosis associated with the Ehrlich ascites tumour, and regenerative (post-CCl4 necrosis) hepatocyte mitosis. In the former case there is a gross depression of prophase index and a marked lowering of the overall mitotic index. In the latter, although a degree of prophase depression is evident, this is insufficient to prevent an overall substantial elevation of mitotic index due to accumulation at metaphase. Very low doses of colchicine claimed to be effective in stathmokinesis of hepatocytes under influence of hepatomitogenic tumour brei and tumour extracts, produced no discernible effect on the mitotic pattern of hepatocytes in Ehrlich ascites tumour-bearing mice nor in post-CCl4 regenerating livers. The results are discussed in the context of known features of tumour-associated hepatocellular mitosis.

Detection of mitotic nuclei in breast histopathology images using localized ACM and Random Kitchen Sink based classifier.

PubMed

Beevi, K Sabeena; Nair, Madhu S; Bindu, G R

2016-08-01

The exact measure of mitotic nuclei is a crucial parameter in breast cancer grading and prognosis. This can be achieved by improving the mitotic detection accuracy by careful design of segmentation and classification techniques. In this paper, segmentation of nuclei from breast histopathology images are carried out by Localized Active Contour Model (LACM) utilizing bio-inspired optimization techniques in the detection stage, in order to handle diffused intensities present along object boundaries. Further, the application of a new optimal machine learning algorithm capable of classifying strong non-linear data such as Random Kitchen Sink (RKS), shows improved classification performance. The proposed method has been tested on Mitosis detection in breast cancer histological images (MITOS) dataset provided for MITOS-ATYPIA CONTEST 2014. The proposed framework achieved 95% recall, 98% precision and 96% F-score.

Dynamics of the Establishment of Multinucleate Compartments in Fusarium oxysporum

PubMed Central

Shahi, Shermineh; Beerens, Bas; Manders, Erik M. M.

2014-01-01

Nuclear dynamics can vary widely between fungal species and between stages of development of fungal colonies. Here we compared nuclear dynamics and mitotic patterns between germlings and mature hyphae in Fusarium oxysporum. Using fluorescently labeled nuclei and live-cell imaging, we show that F. oxysporum is subject to a developmental transition from a uninucleate to a multinucleate state after completion of colony initiation. We observed a special type of hypha that exhibits a higher growth rate, possibly acting as a nutrient scout. The higher growth rate is associated with a higher nuclear count and mitotic waves involving 2 to 6 nuclei in the apical compartment. Further, we found that dormant nuclei of intercalary compartments can reenter the mitotic cycle, resulting in multinucleate compartments with up to 18 nuclei in a single compartment. PMID:25398376

Motility of catalytic nanoparticles through self-generated forces.

PubMed

Paxton, Walter F; Sen, Ayusman; Mallouk, Thomas E

2005-11-04

Small-scale synthetic motors capable of generating their own motive forces by exploiting the chemical free energy of their environment represent an important step in developing practical nanomachines. Catalytic particles are capable of generating concentration and other gradients that can be used to self-propel small objects. However, the autonomous movement of catalytic nanoparticles by self-generated forces is a relatively unexplored area in colloid and interfacial chemistry. This paper explores the potential of catalytically self-generated forces for propulsion of small objects through fluids.

Studies on Axonal Transport in an Animal Model for Gulf War Syndrome

DTIC Science & Technology

2008-07-01

designated by other documentation. REPORT DOCUMENTATION PAGE Form Approved OMB No. 0704-0188 Public reporting burden for this collection of...therapeutic strategies. With regard to kinesin-5, a homotetrameric motor protein that interacts with adjacent microtubules in the mitotic spindle , we...sets of antiparallel motor domains (Kashina et al., 1996). In the mitotic spindle , the primary func- tion of kinesin-5 is to maintain spindle bipolarity

Role of senescence and mitotic catastrophe in cancer therapy

PubMed Central

2010-01-01

Senescence and mitotic catastrophe (MC) are two distinct crucial non-apoptotic mechanisms, often triggered in cancer cells and tissues in response to anti-cancer drugs. Chemotherapeuticals and myriad other factors induce cell eradication via these routes. While senescence drives the cells to a state of quiescence, MC drives the cells towards death during the course of mitosis. The senescent phenotype distinguishes tumor cells that survived drug exposure but lost the ability to form colonies from those that recover and proliferate after treatment. Although senescent cells do not proliferate, they are metabolically active and may secrete proteins with potential tumor-promoting activities. The other anti-proliferative response of tumor cells is MC that is a form of cell death that results from abnormal mitosis and leads to the formation of interphase cells with multiple micronuclei. Different classes of cytotoxic agents induce MC, but the pathways of abnormal mitosis differ depending on the nature of the inducer and the status of cell-cycle checkpoints. In this review, we compare the two pathways and mention that they are activated to curb the growth of tumors. Altogether, we have highlighted the possibilities of the use of senescence targeting drugs, mitotic kinases and anti-mitotic agents in fabricating novel strategies in cancer control. PMID:20205872

Allyl Isothiocyanate Arrests Cancer Cells in Mitosis, and Mitotic Arrest in Turn Leads to Apoptosis via Bcl-2 Protein Phosphorylation*

PubMed Central

Geng, Feng; Tang, Li; Li, Yun; Yang, Lu; Choi, Kyoung-Soo; Kazim, A. Latif; Zhang, Yuesheng

2011-01-01

Allyl isothiocyanate (AITC) occurs in many commonly consumed cruciferous vegetables and exhibits significant anti-cancer activities. Available data suggest that it is particularly promising for bladder cancer prevention and/or treatment. Here, we show that AITC arrests human bladder cancer cells in mitosis and also induces apoptosis. Mitotic arrest by AITC was associated with increased ubiquitination and degradation of α- and β-tubulin. AITC directly binds to multiple cysteine residues of the tubulins. AITC induced mitochondrion-mediated apoptosis, as shown by cytochrome c release from mitochondria to cytoplasm, activation of caspase-9 and caspase-3, and formation of TUNEL-positive cells. Inhibition of caspase-9 blocked AITC-induced apoptosis. Moreover, we found that apoptosis induction by AITC depended entirely on mitotic arrest and was mediated via Bcl-2 phosphorylation at Ser-70. Pre-arresting cells in G1 phase by hydroxyurea abrogated both AITC-induced mitotic arrest and Bcl-2 phosphorylation. Overexpression of a Bcl-2 mutant prevented AITC from inducing apoptosis. We further showed that AITC-induced Bcl-2 phosphorylation was caused by c-Jun N-terminal kinase (JNK), and AITC activates JNK. Taken together, this study has revealed a novel anticancer mechanism of a phytochemical that is commonly present in human diet. PMID:21778226

Mitotic trafficking of silicon microparticles†

PubMed Central

Serda, Rita E.; Ferrati, Silvia; Godin, Biana; Tasciotti, Ennio; Liu, XueWu

2010-01-01

Multistage carriers were recently introduced by our laboratory, with the concurrent objectives of co-localized delivery of multiple therapeutic agents, the “theranostic” integration of bioactive moieties with imaging contrast, and the selective, potentially personalized bypassing of the multiplicity of biological barriers that adversely impact biodistribution of vascularly injected particulates. Mesoporous (“nanoporous”) silicon microparticles were selected as primary carriers in multi-stage devices, with targets including vascular endothelia at pathological lesions. The objective of this study was to evaluate biocompatibility of mesoporous silicon microparticles with endothelial cells using in vitro assays with an emphasis on microparticle compatibility with mitotic events. We observed that vascular endothelial cells, following internalization of silicon microparticles, maintain cellular integrity, as demonstrated by cellular morphology, viability and intact mitotic trafficking of vesicles bearing silicon microparticles. The presence of gold or iron oxide nanoparticles within the porous matrix did not alter the cellular uptake of particles or the viability of endothelial cells subsequent to engulfment of microparticles. Endothelial cells maintained basal levels of IL-6 and IL-8 release in the presence of silicon microparticles. This is the first study that demonstrates polarized, ordered partitioning of endosomes based on tracking microparticles. The finding that mitotic sorting of endosomes is unencumbered by the presence of nanoporous silicon microparticles advocates the use of silicon microparticles for biomedical applications. PMID:20644846

Adrenergic factors involved in the control of crypt cell proliferation in jejunum and descending colon of mouse.

PubMed

Kennedy, M F; Tutton, P J; Barkla, D H

1983-01-01

The mitotic rates in the crypts of Lieberkühn of the proximal jejunum and descending colon of mouse, following different treatments, were measured using a stathmokinetic technique. Regression coefficients, representing mitotic rates, were then calculated by the method of least squares. Treatment with adrenaline, isoprenaline, phenylephrine, phentolamine, and yohimbine all resulted in decreased mitotic rate of jejunal and colonic crypt cells. Chemical sympathectomy and cryosympathectomy had a similar effect, and chemical sympathectomy was followed by a supersensitivity to clonidine. Intraperitoneal injection of metaraminol, clonidine, propranolol, prazosin, labetolol and simultaneous injection of propranolol and adrenaline all resulted in an increased rate of crypt cell proliferation in both jejunum and colon. A significant increase in mitotic rate was observed in both tissues at night. The amplitude of this diurnal variation was decreased in both jejunum and colon following chemical sympathectomy. In addition, the amplitude of this variation in jejunum was decreased after treatment with yohimbine or phentolamine. The results of the study suggest that the sympathetic nervous system stimulates epithelial cell proliferation in both the small and large intestine and that this effect is mediated by an alpha 2-adrenoceptor. By contrast, stimulation of alpha 1- and beta-adrenoceptors is inhibitory to cell proliferation in these tissues.

Analysis and Modeling of Chromosome Congression During Mitosis in the Chemotherapy Drug Cisplatin.

PubMed

Chacón, Jeremy M; Gardner, Melissa K

2013-12-01

The chemotherapy drug Cisplatin (cis-diamminedichloroplatinum(II)) induces crosslinks within and between DNA strands, and between DNA and nearby proteins. Therefore, Cisplatin-treated cells which progress into cell division may do so with altered chromosome mechanical properties. This could have important consequences for the successful completion of mitosis. Using Total Internal Reflection Fluorescence (TIRF) microscopy of live Cisplatin-treated Saccharomyces cerevisiae cells, we found that metaphase mitotic spindles have disorganized kinetochores relative to untreated cells, and also that there is increased variability in the chromosome stretching distance between sister centromeres. This suggests that chromosome stiffness may become more variable after Cisplatin treatment. We explored the effect of variable chromosome stiffness during mitosis using a stochastic model in which kinetochore microtubule dynamics were regulated by tension imparted by stretched sister chromosomes. Consistent with experimental results, increased variability of chromosome stiffness in the model led to disorganization of kinetochores in simulated metaphase mitotic spindles. Furthermore, the variability in simulated chromosome stretching tension was increased as chromosome stiffness became more variable. Because proper chromosome stretching tension may serve as a signal that is required for proper progression through mitosis, tension variability could act to impair this signal and thus prevent proper mitotic progression. Our results suggest a possible mitotic mode of action for the anti-cancer drug Cisplatin.

Mitotic Vulnerability in Triple-Negative Breast Cancer Associated with LIN9 Is Targetable with BET Inhibitors.

PubMed

Sahni, Jennifer M; Gayle, Sylvia S; Webb, Bryan M; Weber-Bonk, Kristen L; Seachrist, Darcie D; Singh, Salendra; Sizemore, Steven T; Restrepo, Nicole A; Bebek, Gurkan; Scacheri, Peter C; Varadan, Vinay; Summers, Matthew K; Keri, Ruth A

2017-10-01

Triple-negative breast cancers (TNBC) are highly aggressive, lack FDA-approved targeted therapies, and frequently recur, making the discovery of novel therapeutic targets for this disease imperative. Our previous analysis of the molecular mechanisms of action of bromodomain and extraterminal protein inhibitors (BETi) in TNBC revealed these drugs cause multinucleation, indicating BET proteins are essential for efficient mitosis and cytokinesis. Here, using live cell imaging, we show that BET inhibition prolonged mitotic progression and induced mitotic cell death, both of which are indicative of mitotic catastrophe. Mechanistically, the mitosis regulator LIN9 was a direct target of BET proteins that mediated the effects of BET proteins on mitosis in TNBC. Although BETi have been proposed to function by dismantling super-enhancers (SE), the LIN9 gene lacks an SE but was amplified or overexpressed in the majority of TNBCs. In addition, its mRNA expression predicted poor outcome across breast cancer subtypes. Together, these results provide a mechanism for cancer selectivity of BETi that extends beyond modulation of SE-associated genes and suggest that cancers dependent upon LIN9 overexpression may be particularly vulnerable to BETi. Cancer Res; 77(19); 5395-408. ©2017 AACR . ©2017 American Association for Cancer Research.

The Light Intermediate Chain 2 Subpopulation of Dynein Regulates Mitotic Spindle Orientation.

PubMed

Mahale, Sagar; Kumar, Megha; Sharma, Amit; Babu, Aswini; Ranjan, Shashi; Sachidanandan, Chetana; Mylavarapu, Sivaram V S

2016-12-23

Cytoplasmic dynein 1 is a multi-protein intracellular motor essential for mediating several mitotic functions, including the establishment of proper spindle orientation. The functional relevance and mechanistic distinctions between two discrete dynein subpopulations distinguished only by Light Intermediate Chain (LIC) homologues, LIC1 and LIC2 is unknown during mitosis. Here, we identify LIC2-dynein as the major mediator of proper spindle orientation and uncover its underlying molecular mechanism. Cortically localized dynein, essential for maintaining correct spindle orientation, consists majorly of LIC2-dynein, which interacts with cortical 14-3-3 ε- ζ and Par3, conserved proteins required for orienting the spindle. LIC2-dynein is also responsible for the majority of dynein-mediated asymmetric poleward transport of NuMA, helping focus microtubule minus ends. In addition, LIC2-dynein dominates in equatorially aligning chromosomes at metaphase and in regulating mitotic spindle length. Key mitotic functions of LIC2 were remarkably conserved in and essential for early embryonic divisions and development in zebrafish. Thus LIC2-dynein exclusively engages with two major cortical pathways to govern spindle orientation. Overall, we identify a novel selectivity of molecular interactions between the two LICs in mitosis as the underlying basis for their uneven distribution of labour in ensuring proper spindle orientation.

Cdk1 and okadaic acid-sensitive phosphatases control assembly of nuclear pore complexes in Drosophila embryos.

PubMed

Onischenko, Evgeny A; Gubanova, Natalia V; Kiseleva, Elena V; Hallberg, Einar

2005-11-01

Disassembly and reassembly of the nuclear pore complexes (NPCs) is one of the major events during open mitosis in higher eukaryotes. However, how this process is controlled by the mitotic machinery is not clear. To investigate this we developed a novel in vivo model system based on syncytial Drosophila embryos. We microinjected different mitotic effectors into the embryonic cytoplasm and monitored the dynamics of disassembly/reassembly of NPCs in live embryos using fluorescently labeled wheat germ agglutinin (WGA) or in fixed embryos using electron microscopy and immunostaining techniques. We found that in live embryos Cdk1 activity was necessary and sufficient to induce disassembly of NPCs as well as their cytoplasmic mimics: annulate lamellae pore complexes (ALPCs). Cdk1 activity was also required for keeping NPCs and ALPCs disassembled during mitosis. In agreement recombinant Cdk1/cyclin B was able to induce phosphorylation and dissociation of nucleoporins from the NPCs in vitro. Conversely, reassembly of NPCs and ALPCs was dependent on the activity of protein phosphatases, sensitive to okadaic acid (OA). Our findings suggest a model where mitotic disassembly/reassembly of the NPCs is regulated by a dynamic equilibrium of Cdk1 and OA-sensitive phosphatase activities and provide evidence that mitotic phosphorylation mediates disassembly of the NPC.

Aerodynamic effects of flexibility in flapping wings.

PubMed

Zhao, Liang; Huang, Qingfeng; Deng, Xinyan; Sane, Sanjay P

2010-03-06

Recent work on the aerodynamics of flapping flight reveals fundamental differences in the mechanisms of aerodynamic force generation between fixed and flapping wings. When fixed wings translate at high angles of attack, they periodically generate and shed leading and trailing edge vortices as reflected in their fluctuating aerodynamic force traces and associated flow visualization. In contrast, wings flapping at high angles of attack generate stable leading edge vorticity, which persists throughout the duration of the stroke and enhances mean aerodynamic forces. Here, we show that aerodynamic forces can be controlled by altering the trailing edge flexibility of a flapping wing. We used a dynamically scaled mechanical model of flapping flight (Re approximately 2000) to measure the aerodynamic forces on flapping wings of variable flexural stiffness (EI). For low to medium angles of attack, as flexibility of the wing increases, its ability to generate aerodynamic forces decreases monotonically but its lift-to-drag ratios remain approximately constant. The instantaneous force traces reveal no major differences in the underlying modes of force generation for flexible and rigid wings, but the magnitude of force, the angle of net force vector and centre of pressure all vary systematically with wing flexibility. Even a rudimentary framework of wing veins is sufficient to restore the ability of flexible wings to generate forces at near-rigid values. Thus, the magnitude of force generation can be controlled by modulating the trailing edge flexibility and thereby controlling the magnitude of the leading edge vorticity. To characterize this, we have generated a detailed database of aerodynamic forces as a function of several variables including material properties, kinematics, aerodynamic forces and centre of pressure, which can also be used to help validate computational models of aeroelastic flapping wings. These experiments will also be useful for wing design for small robotic insects and, to a limited extent, in understanding the aerodynamics of flapping insect wings.

Aerodynamic effects of flexibility in flapping wings

PubMed Central

Zhao, Liang; Huang, Qingfeng; Deng, Xinyan; Sane, Sanjay P.

2010-01-01

Recent work on the aerodynamics of flapping flight reveals fundamental differences in the mechanisms of aerodynamic force generation between fixed and flapping wings. When fixed wings translate at high angles of attack, they periodically generate and shed leading and trailing edge vortices as reflected in their fluctuating aerodynamic force traces and associated flow visualization. In contrast, wings flapping at high angles of attack generate stable leading edge vorticity, which persists throughout the duration of the stroke and enhances mean aerodynamic forces. Here, we show that aerodynamic forces can be controlled by altering the trailing edge flexibility of a flapping wing. We used a dynamically scaled mechanical model of flapping flight (Re ≈ 2000) to measure the aerodynamic forces on flapping wings of variable flexural stiffness (EI). For low to medium angles of attack, as flexibility of the wing increases, its ability to generate aerodynamic forces decreases monotonically but its lift-to-drag ratios remain approximately constant. The instantaneous force traces reveal no major differences in the underlying modes of force generation for flexible and rigid wings, but the magnitude of force, the angle of net force vector and centre of pressure all vary systematically with wing flexibility. Even a rudimentary framework of wing veins is sufficient to restore the ability of flexible wings to generate forces at near-rigid values. Thus, the magnitude of force generation can be controlled by modulating the trailing edge flexibility and thereby controlling the magnitude of the leading edge vorticity. To characterize this, we have generated a detailed database of aerodynamic forces as a function of several variables including material properties, kinematics, aerodynamic forces and centre of pressure, which can also be used to help validate computational models of aeroelastic flapping wings. These experiments will also be useful for wing design for small robotic insects and, to a limited extent, in understanding the aerodynamics of flapping insect wings. PMID:19692394

Integrity of the Pericentriolar Material Is Essential for Maintaining Centriole Association during M Phase

PubMed Central

Rhee, Kunsoo

2015-01-01

A procentriole is assembled next to the mother centriole during S phase and remains associated until M phase. After functioning as a spindle pole during mitosis, the mother centriole and procentriole are separated at the end of mitosis. A close association of the centriole pair is regarded as an intrinsic block to the centriole reduplication. Therefore, deregulation of this process may cause a problem in the centriole number control, resulting in increased genomic instability. Despite its importance for faithful centriole duplication, the mechanism of centriole separation is not fully understood yet. Here, we report that centriole pairs are prematurely separated in cells whose cell cycle is arrested at M phase by STLC. Dispersal of the pericentriolar material (PCM) was accompanied. This phenomenon was independent of the separase activity but needed the PLK1 activity. Nocodazole effectively inhibited centriole scattering in STLC-treated cells, possibly by reducing the microtubule pulling force around centrosomes. Inhibition of PLK1 also reduced the premature separation of centrioles and the PCM dispersal as well. These results revealed the importance of PCM integrity in centriole association. Therefore, we propose that PCM disassembly is one of the driving forces for centriole separation during mitotic exit. PMID:26407333

Generation of β-glucuronidase reporter-tagged strain to monitor Ustilaginoidea virens infection in rice.

PubMed

Andargie, Mebeaselassie; Yang, Chao; Li, Jianxiong

2016-12-01

An Agrobacterium-mediated genetic transformation system for the rice false smut fungus Ustilaginoidea virens was developed using conidia as recipients. A binary vector, pCAMBIA1301-P gpdA -GUS-T trpC , was constructed. The gpdA promoter (P gpdA ) from Aspergillus nidulans was used to drive the expression of the β-glucuronidase (GUS) gene which enabled GUS activity visualization. The conidia transformation efficiency reached approximately 110 to 250 transformants per 1×10 5 conidia. Based on the analysis made on five successive generations of subcultures and PCR, the pCAMBIA1301-GUS cassette had integrated into the genomes of all transformants and clearly showed mitotic stability. The novel reporter vector constructed will promote the functional characterization of genes and the construction of genetically engineered strains of this important fungus. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of a new stem cell population that generates Drosophila flight muscles.

PubMed

Gunage, Rajesh D; Reichert, Heinrich; VijayRaghavan, K

2014-08-18

How myoblast populations are regulated for the formation of muscles of different sizes is an essentially unanswered question. The large flight muscles of Drosophila develop from adult muscle progenitor (AMP) cells set-aside embryonically. The thoracic segments are all allotted the same small AMP number, while those associated with the wing-disc proliferate extensively to give rise to over 2500 myoblasts. An initial amplification occurs through symmetric divisions and is followed by a switch to asymmetric divisions in which the AMPs self-renew and generate post-mitotic myoblasts. Notch signaling controls the initial amplification of AMPs, while the switch to asymmetric division additionally requires Wingless, which regulates Numb expression in the AMP lineage. In both cases, the epidermal tissue of the wing imaginal disc acts as a niche expressing the ligands Serrate and Wingless. The disc-associated AMPs are a novel muscle stem cell population that orchestrates the early phases of adult flight muscle development.

Neural Stem Cells: Historical Perspective and Future Prospects

PubMed Central

Breunig, Joshua J.; Haydar, Tarik F.; Rakic, Pasko

2011-01-01

How a single fertilized cell generates diverse neuronal populations has been a fundamental biological problem since the 19th century. Classical histological methods revealed that post-mitotic neurons are produced in a precise temporal and spatial order from germinal cells lining the cerebral ventricles. In the 20th century DNA labeling and histo- and immuno-histochemistry helped to distinguish the subtypes of dividing cells and delineate their locations in the ventricular and subventricular zones. Recently, genetic and cell biological methods have provided insights into sequential gene expression and molecular and cellular interactions that generate heterogeneous populations of NSCs leading to specific neuronal classes. This precisely regulated developmental process does not tolerate significant in vivo deviation, making replacement of adult neurons by NSCs during pathology a colossal challenge. In contrast, utilizing the trophic factors emanating from the NSC or their derivatives to slow down deterioration or prevent death of degenerating neurons may be a more feasible strategy. PMID:21609820

Ectopic expression of the Drosophila Cdk1 inhibitory kinases, Wee1 and Myt1, interferes with the second mitotic wave and disrupts pattern formation during eye development.

PubMed Central

Price, Donald M; Jin, Zhigang; Rabinovitch, Simon; Campbell, Shelagh D

2002-01-01

Wee1 kinases catalyze inhibitory phosphorylation of the mitotic regulator Cdk1, preventing mitosis during S phase and delaying it in response to DNA damage or developmental signals during G2. Unlike yeast, metazoans have two distinct Wee1-like kinases, a nuclear protein (Wee1) and a cytoplasmic protein (Myt1). We have isolated the genes encoding Drosophila Wee1 and Myt1 and are using genetic approaches to dissect their functions during normal development. Overexpression of Dwee1 or Dmyt1 during eye development generates a rough adult eye phenotype. The phenotype can be modified by altering the gene dosage of known regulators of the G2/M transition, suggesting that we could use these transgenic strains in modifier screens to identify potential regulators of Wee1 and Myt1. To confirm this idea, we tested a collection of deletions for loci that can modify the eye overexpression phenotypes and identified several loci as dominant modifiers. Mutations affecting the Delta/Notch signaling pathway strongly enhance a GMR-Dmyt1 eye phenotype but do not affect a GMR-Dwee1 eye phenotype, suggesting that Myt1 is potentially a downstream target for Notch activity during eye development. We also observed interactions with p53, which suggest that Wee1 and Myt1 activity can block apoptosis. PMID:12072468

The role of meiotic cohesin REC8 in chromosome segregation in gamma irradiation-induced endopolyploid tumour cells.

PubMed

Erenpreisa, Jekaterina; Cragg, Mark S; Salmina, Kristine; Hausmann, Michael; Scherthan, Harry

2009-09-10

Escape from mitotic catastrophe and generation of endopolyploid tumour cells (ETCs) represents a potential survival strategy of tumour cells in response to genotoxic treatments. ETCs that resume the mitotic cell cycle have reduced ploidy and are often resistant to these treatments. In search for a mechanism for genome reduction, we previously observed that ETCs express meiotic proteins among which REC8 (a meiotic cohesin component) is of particular interest, since it favours reductional cell division in meiosis. In the present investigation, we induced endopolyploidy in p53-dysfunctional human tumour cell lines (Namalwa, WI-L2-NS, HeLa) by gamma irradiation, and analysed the sub-cellular localisation of REC8 in the resulting ETCs. We observed by RT-PCR and Western blot that REC8 is constitutively expressed in these tumour cells, along with SGOL1 and SGOL2, and that REC8 becomes modified after irradiation. REC8 localised to paired sister centromeres in ETCs, the former co-segregating to opposite poles. Furthermore, REC8 localised to the centrosome of interphase ETCs and to the astral poles in anaphase cells where it colocalised with the microtubule-associated protein NuMA. Altogether, our observations indicate that radiation-induced ETCs express features of meiotic cell divisions and that these may facilitate chromosome segregation and genome reduction.

Biophysical modelling of early and delayed radiation damage at chromosome level

NASA Astrophysics Data System (ADS)

Andreev, S.; Eidelman, Y.

Exposure by ionising radiation increases cancer risk in human population Cancer is thought to originate from an altered expression of certain number of specific genes It is now widely recognised that chromosome aberrations CA are involved in stable change in expression of genes by gain or loss of their functions Thus CA can contribute to initiation or progression of cancer Therefore understanding mechanisms of CA formation in the course of cancer development might be valuable tool for quantification and prognosis of different stages of radiation carcinogenesis Early CA are defined as aberrations induced in first post-irradiation mitotic cycle The present work describes the original biophysical technique for early CA modelling It includes the following simulation steps the ionising particle track structure the structural organisation of all chromosomes in G 0 G 1 cell nucleus spatial distribution of radiation induced DNA double-strand breaks dsb within chromosomes dsb rejoining and misrejoining modelling cell cycle taking into account mitotic delay which results in complex time dependence of aberrant cells in first mitosis The results on prediction of dose-response curves for simple and complex CA measured in cells undergoing first division cycle are presented in comparison with recent experimental data There is increasing evidence that CA are also observed in descendents of irradiated cells many generations after direct DNA damage These delayed CA or chromosome instability CI are thought to be a manifestation of genome

Cerebrospinal fluid from subarachnoid haemorrhage patients causes excessive oxidative metabolism compared to vascular smooth muscle force generation.

PubMed

Pyne, G J; Cadoux-Hudson, T A; Clark, J F

2001-01-01

Cerebrospinal fluid (CSF) from subarachnoid haemorrhage (SAH) patients can stimulate vascular smooth muscle to generate force in vitro. CSF from SAH patients suffering from delayed ischaemic neurological deficits due to cerebral vasospasm can generate near maximal force in vitro and previous experiments have ascribed this generation of force to be a calcium mediated event. The intracellular calcium concentration has been demonstrated to rise during the vasospastic process. Calcium also stimulates oxidative metabolism as does adenosine diphosphate (ADP), the product of adenosine triphosphate (ATP) hydrolysis. Significant alteration in high energy metabolites such as ATP, ADP and phosphocreatine have also been demonstrated in various models of SAH mediated vasospasm. Vascular smooth muscle predominantly uses oxidative metabolism for force generation and reserves glycolytic metabolism for ion homeostasis. A decrease in oxidative metabolism during force generation would imply failing mitochondria and increased glycolytic high-energy phosphate supply. Increased oxidative metabolism would imply a decreased efficiency of the contractile apparatus or mitochondria. The aim of this study was to see if SAH CSF stimulation of porcine carotid artery oxidative metabolism was altered during force generation when compared with incremental calcium stimulation with potassium chloride depolarisation. CSF from patients (n = 10) who had subarachnoid haemorrhage stimulated force generation but with a significant 'right shift' in oxygen consumption. This 'right shift' is indicative of an increased energy cost for contractile work. These results suggest that vascular smooth muscle contractile apparatus, when stimulated by subarachnoid cerebrospinal fluid, is consuming excess adenosine triphosphate during force generation.

Chromatin fibers: from classical descriptions to modern interpretation.

PubMed

Kuznetsova, Maria A; Sheval, Eugene V

2016-11-01

The first description of intrachromosomal fibers was made by Baranetzky in 1880. Since that time, a plethora of fibrillar substructures have been described inside the mitotic chromosomes, and published data indicate that chromosomes may be formed as a result of the hierarchical folding of chromatin fibers. In this review, we examine the evolution and the current state of research on the morphological organization of mitotic chromosomes. © 2016 International Federation for Cell Biology.

Centrosome Amplification: A Potential Marker of Breast Cancer Agressiveness

DTIC Science & Technology

2006-07-01

centrosome amplification. Introduction of DNA damage in the MCF-7 cell line by treatment with hydroxyurea (HU) or daunorubicin (DR) resulted in the...cycles of DNA synthesis and mitotic division in hydroxyurea - arrested Chinese hamster ovary cells. J Cell Biol, 130: 105-115, 1995. 23. D’Assoro, A. B...from cycles of DNA synthesis and mitotic division in hydroxyurea -arrested Chinese hamster ovary cells. J Cell Biol, 1995. 130(1): p. 105-15. 22

Effects of Extremely Low Frequency Electric and Magnetic Fields on Roots of ’Vicia faba’.

DTIC Science & Technology

those near the Sanguine transmitter: growth rate, mitotic index , chromosomal abnormalities in dividing meristematic cells. The choice of Vicia faba ...Roots of Vicia faba were exposed to electric and magnetic fields comparable to but at levels higher than those associated with Project Sanguine...There were no differences among control and exposed roots for growth or mitotic index . Also, there were no chromosomal anomalies. Three indices are

Extremely Low Frequency (ELF) Communications Program: Non-Ionizing Electromagnetic Radiation Literature Evaluation and Assessment, 1986-1987 Literature Review.

DTIC Science & Technology

1987-11-01

and Carsteen E L (1985). Growth Rate and Mitotic Index Analysis of Vicia Faba L. Roots Exposed to 60-Hz Electric Fields. Bioelectromagnetics, Vol. 6...the observed effects. The mitotic index was also influenced. 5) In monkeys, central nervous system excitability was influenced by applying fields...Literature Search and Retrieval ...................... 16 2.2 Literature Culling and Indexing ...................... 18 2.3 Foreign Literature

EFHC1, a protein mutated in juvenile myoclonic epilepsy, associates with the mitotic spindle through its N-terminus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nijs, Laurence de; Lakaye, Bernard; Coumans, Bernard

2006-09-10

A novel gene, EFHC1, mutated in juvenile myoclonic epilepsy (JME) encodes a protein with three DM10 domains of unknown function and one putative EF-hand motif. To study the properties of EFHC1, we expressed EGFP-tagged protein in various cell lines. In interphase cells, the fusion protein was present in the cytoplasm and in the nucleus with specific accumulation at the centrosome. During mitosis EGFP-EFHC1 colocalized with the mitotic spindle, especially at spindle poles and with the midbody during cytokinesis. Using a specific antibody, we demonstrated the same distribution of the endogenous protein. Deletion analyses revealed that the N-terminal region of EFHC1more » is crucial for the association with the mitotic spindle and the midbody. Our results suggest that EFHC1 could play an important role during cell division.« less

Microtubule-dependent regulation of mitotic protein degradation

PubMed Central

Song, Ling; Craney, Allison; Rape, Michael

2014-01-01

Accurate cell division depends on tightly regulated ubiquitylation events catalyzed by the anaphase-promoting complex. Among its many substrates, the APC/C triggers the degradation of proteins that stabilize the mitotic spindle, and loss or accumulation of such spindle assembly factors can result in aneuploidy and cancer. Although critical for cell division, it has remained poorly understood how the timing of spindle assembly factor degradation is established during mitosis. Here, we report that active spindle assembly factors are protected from APC/C-dependent degradation by microtubules. In contrast, those molecules that are not bound to microtubules are highly susceptible to proteolysis and turned over immediately after APC/C-activation. The correct timing of spindle assembly factor degradation, as achieved by this regulatory circuit, is required for accurate spindle structure and function. We propose that the localized stabilization of APC/C-substrates provides a mechanism for the selective disposal of cell cycle regulators that have fulfilled their mitotic roles. PMID:24462202

[Cytogenetic characteristics of seed offspring of leafy tree plants from one-kilometer zone of Novovoronezh nuclear power station].

PubMed

Artiukhov, V G; Kalaev, V N; Sen'kevich, E V; Vakhtel', V M; Savko, A D

2004-01-01

Cytogenetic characteristics (mitotic activity, level and spectrum of pathological mitoses, nucleoly characteristics) of seed offspring of Quercus robur L. and Betula pendula Roth from Novovoronezh nuclear power station's 1-kilometer zone have been studied. It has been shown the change of time of passing though mitotic stages by cells, the increasing of bridges frequency occur in spectrum of mitotic aberrations (that shows activation of reparation systems), the change in nucleoly characteristics (the part of polynucleolaris cells increase in case of oak and decrease in case of birch, the rase of surface square of single nucleolies). The phenomena, mean above, probably, induced by synergic effects of Novovoronezh nuclear power station and environment pollutants. The most contaminated territories of 1-kilometer zone of Novovoronezh nuclear power station have been discovered by means of methods of cluster analysis of total cytogenetic characteristics of tree plants seed offspring.

[Cytogenetic characteristics of seed progeny of trees under condition of antropogenic contamination in Voronezh town].

PubMed

Butorina, A K; Kalaev, V N; Vostrikova, T V; Miagkova, O E

2000-01-01

It has been shown that in seed progeny of Quercus robur L., Pinus sylvestris L. and Betula pendula Roth. some cytogenetical characteristics vary under conditions of contamination. Such changes may be common or specific type. Thus, the frequency of pathological mitosis increases under such conditions in all the investigated species of trees. Inhibition of mitosis was found in the progeny of the pine, and variability in the number of nucleoli was detected in the pine and oak. However, in some cases the level of pathological mitosis in the oak progeny did not differ from the control, but the mitotic activity was higher due to the presence of much more cells being at the prophase stage. In the birch progeny under conditions of contamination the mitotic index increased, with a simultaneous shifts in the peaks of mitotic activity. The possibility of using these cytological characteristics for the aims of cytogenetical monitoring is considered.

CELL POPULATION KINETICS OF EXCISED ROOTS OF PISUM SATIVUM

PubMed Central

Van't Hof, Jack

1965-01-01

The cell population kinetics of excised, cultured pea roots was studied with the use of tritiated thymidine and colchicine to determine (1) the influence of excision, (2) the influence of sucrose concentration, (3) the average mitotic cycle duration, and (4) the duration of mitosis and the G 1, S, and G 2 periods of interphase.1 The results indicate that the process of excision causes a drop in the frequency of mitotic figures when performed either at the beginning of the culture period or after 100 hours in culture. This initial decrease in frequency of cell division is independent of sucrose concentration, but the subsequent rise in frequency of division, after 12 hours in culture, is dependent upon sucrose concentration. Two per cent sucrose maintains the shortest mitotic cycle duration. The use of colchicine indicated an average cycle duration of 20 hours, whereas the use of tritiated thymidine produced an average cycle duration of 17 hours. PMID:5857253

SON is a spliceosome-associated factor required for mitotic progression.

PubMed

Huen, Michael S Y; Sy, Shirley M H; Leung, Ka Man; Ching, Yick-Pang; Tipoe, George L; Man, Cornelia; Dong, Shuo; Chen, Junjie

2010-07-01

The eukaryotic RNA splicing machinery is dedicated to the daunting task of excising intronic sequences on the many nascent RNA transcripts in a cell, and in doing so facilitates proper translation of its transcriptome. Notably, emerging evidence suggests that RNA splicing may also play direct roles in maintaining genome stability. Here we report the identification of the RNA/DNA-binding protein SON as a component of spliceosome that plays pleiotropic roles during mitotic progression. We found that SON is essential for cell proliferation, and that its inactivation triggers a MAD2-dependent mitotic delay. Moreover, SON deficiency is accompanied by defective chromosome congression, compromised chromosome segregation and cytokinesis, which in turn contributes to cellular aneuploidy and cell death. In summary, our study uncovers a specific link between SON and mitosis, and highlights the potential of RNA processing as additional regulatory mechanisms that govern cell proliferation and division. © 2010 Landes Bioscience

SON is a spliceosome-associated factor required for mitotic progression

PubMed Central

Sy, Shirley MH; Leung, Ka Man; Ching, Yick-Pang; Tipoe, George L; Man, Cornelia; Dong, Shuo

2010-01-01

The eukaryotic RNA splicing machinery is dedicated to the daunting task of excising intronic sequences on the many nascent RNA transcripts in a cell, and in doing so facilitates proper translation of its transcriptome. Notably, emerging evidence suggests that RNA splicing may also play direct roles in maintaining genome stability. Here we report the identification of the RNA/DNA-binding protein SON as a component of spliceosome that plays pleiotropic roles during mitotic progression. We found that SON is essential for cell proliferation, and that its inactivation triggers a MAD2-dependent mitotic delay. Moreover, SON deficiency is accompanied by defective chromosome congression, compromised chromosome segregation and cytokinesis, which in turn contributes to cellular aneuploidy and cell death. In summary, our study uncovers a specific link between SON and mitosis, and highlights the potential of RNA processing as additional regulatory mechanisms that govern cell proliferation and division. PMID:20581448

Imaging Mitosis in the Moss Physcomitrella patens.

PubMed

Yamada, Moé; Miki, Tomohiro; Goshima, Gohta

2016-01-01

At first glance, mitosis in plants looks quite different from that in animals. In fact, terrestrial plants have lost the centrosome during evolution, and the mitotic spindle is assembled independently of a strong microtubule organizing center. The phragmoplast is a plant-specific mitotic apparatus formed after anaphase, which expands centrifugally towards the cell cortex. However, the extent to which plant mitosis differs from that of animals at the level of the protein repertoire is uncertain, largely because of the difficulty in the identification and in vivo characterization of mitotic genes of plants. Here, we discuss protocols for mitosis imaging that can be combined with endogenous green fluorescent protein (GFP) tagging or conditional RNA interference (RNAi) in the moss Physcomitrella patens, which is an emergent model plant for cell and developmental biology. This system has potential for use in the high-throughput study of mitosis and other intracellular processes, as is being done with various animal cell lines.

Oncogenic transformation through the cell cycle and the LET dependent inverse dose rate effect

NASA Technical Reports Server (NTRS)

Geard, C. R.; Miller, R. C.; Brenner, D. J.; Hall, E. J.; Wachholz, B. W. (Principal Investigator)

1994-01-01

Synchronised populations of mouse C3H/10T-1/2 cells were obtained by a stringent mitotic dislodgment procedure. Mitotic cells rapidly attach and progress sequentially through the cell cycle. Irradiation (3 Gy of X rays) was carried out at intervals from 0 to 18 h after initiating cell cycle progression of the mitotic cells. Oncogenic transformation was enhanced 10-fold over cells irradiated soon after replating (G1 and S phases) for cells in a near 2 h period corresponding to cells in G2 phase but not in mitosis. The cell surviving fraction had a 2-1/2-fold variation with resistant peaks corresponding to the late G1 and late S phases. These findings provide experimental support for the hypothesis initiated by Rossi and Kellerer and developed by Brenner and Hall to explain the LET dependent inverse dose rate effect for oncogenic transformation.

Prenatal Organophosphates Exposure Alternates the Cleavage Plane Orientation of Apical Neural Progenitor in Developing Neocortex

PubMed Central

Chen, Xiao-Ping; Chen, Wei-Feng; Wang, Da-Wei

2014-01-01

Prenatal organophosphate exposure elicits long-term brain cytoarchitecture and cognitive function impairments, but the mechanism underlying the onset and development of neural progenitors remain largely unclear. Using precise positioned brain slices, we observed an alternated cleavage plane bias that emerged in the mitotic neural progenitors of embryonal neocortex with diazinion (DZN) and chlorpyrifos (CPF) pretreatment. In comparison with the control, DZN and CPF treatment induced decrease of vertical orientation, increase of oblique orientation, and increase of horizontal orientation. That is, the cleavage plane orientation bias had been rotated from vertical to horizontal after DZN and CPF treatment. Meanwhile, general morphology and mitotic index of the progenitors were unchanged. Acephate (ACP), another common organophosphate, had no significant effects on the cleavage plane orientation, cell morphology and mitotic index. These results represent direct evidence for the toxicity mechanism in onset multiplication of neural progenitors. PMID:24740262

Mitotic Cortical Waves Predict Future Division Sites by Encoding Positional and Size Information.

PubMed

Xiao, Shengping; Tong, Cheesan; Yang, Yang; Wu, Min

2017-11-20

Dynamic spatial patterns such as traveling waves could theoretically encode spatial information, but little is known about whether or how they are employed by biological systems, especially higher eukaryotes. Here, we show that concentric target or spiral waves of active Cdc42 and the F-BAR protein FBP17 are invoked in adherent cells at the onset of mitosis. These waves predict the future sites of cell divisions and represent the earliest known spatial cues for furrow assembly. Unlike interphase waves, the frequencies and wavelengths of the mitotic waves display size-dependent scaling properties. While the positioning role of the metaphase waves requires microtubule dynamics, spindle and microtubule-independent inhibitory signals are propagated by the mitotic waves to ensure the singularity of furrow formation. Taken together, we propose that metaphase cortical waves integrate positional and cell size information for division-plane specification in adhesion-dependent cytokinesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Growth rate and mitotic index analysis of Vicia faba L. roots exposed to 60-Hz electric fields.

PubMed

Inoue, M; Miller, M W; Cox, C; Carstesen, E L

1985-01-01

Growth, mitotic index, and growth rate recovery were determined for Vicia faba L. roots exposed to 60-Hz electric fields of 200, 290, and 360 V/m in an aqueous inorganic nutrient medium (conductivity 0.07-0.09 S/m). Root growth rate decreased in proportion to the increasing strength; the electric field threshold for a growth rate effect was about 230 V/m. The induced transmembrane potential at the threshold exposure was about 4-7 mV. The mitotic index was not affected by an electric field exposure sufficient to reduce root growth rate to about 35% of control. Root growth rate recovery from 31-96% of control occurred in 4 days after cessation of the 360 V/m exposure. The results support the postulate that the site of action of the applied electric fields is the cell membrane.