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Sample records for cell nucleus mitosis

  1. Origin of the cell nucleus, mitosis and sex: roles of intracellular coevolution

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    Cavalier-Smith Thomas

    2010-02-01

    Full Text Available Abstract Background The transition from prokaryotes to eukaryotes was the most radical change in cell organisation since life began, with the largest ever burst of gene duplication and novelty. According to the coevolutionary theory of eukaryote origins, the fundamental innovations were the concerted origins of the endomembrane system and cytoskeleton, subsequently recruited to form the cell nucleus and coevolving mitotic apparatus, with numerous genetic eukaryotic novelties inevitable consequences of this compartmentation and novel DNA segregation mechanism. Physical and mutational mechanisms of origin of the nucleus are seldom considered beyond the long-standing assumption that it involved wrapping pre-existing endomembranes around chromatin. Discussions on the origin of sex typically overlook its association with protozoan entry into dormant walled cysts and the likely simultaneous coevolutionary, not sequential, origin of mitosis and meiosis. Results I elucidate nuclear and mitotic coevolution, explaining the origins of dicer and small centromeric RNAs for positionally controlling centromeric heterochromatin, and how 27 major features of the cell nucleus evolved in four logical stages, making both mechanisms and selective advantages explicit: two initial stages (origin of 30 nm chromatin fibres, enabling DNA compaction; and firmer attachment of endomembranes to heterochromatin protected DNA and nascent RNA from shearing by novel molecular motors mediating vesicle transport, division, and cytoplasmic motility. Then octagonal nuclear pore complexes (NPCs arguably evolved from COPII coated vesicle proteins trapped in clumps by Ran GTPase-mediated cisternal fusion that generated the fenestrated nuclear envelope, preventing lethal complete cisternal fusion, and allowing passive protein and RNA exchange. Finally, plugging NPC lumens by an FG-nucleoporin meshwork and adopting karyopherins for nucleocytoplasmic exchange conferred compartmentation

  2. Origin of the cell nucleus, mitosis and sex: roles of intracellular coevolution

    OpenAIRE

    Cavalier-Smith Thomas

    2010-01-01

    Abstract Background The transition from prokaryotes to eukaryotes was the most radical change in cell organisation since life began, with the largest ever burst of gene duplication and novelty. According to the coevolutionary theory of eukaryote origins, the fundamental innovations were the concerted origins of the endomembrane system and cytoskeleton, subsequently recruited to form the cell nucleus and coevolving mitotic apparatus, with numerous genetic eukaryotic novelties inevitable conseq...

  3. Killing cells by targeting mitosis.

    Science.gov (United States)

    Manchado, E; Guillamot, M; Malumbres, M

    2012-03-01

    Cell cycle deregulation is a common feature of human cancer. Tumor cells accumulate mutations that result in unscheduled proliferation, genomic instability and chromosomal instability. Several therapeutic strategies have been proposed for targeting the cell division cycle in cancer. Whereas inhibiting the initial phases of the cell cycle is likely to generate viable quiescent cells, targeting mitosis offers several possibilities for killing cancer cells. Microtubule poisons have proved efficacy in the clinic against a broad range of malignancies, and novel targeted strategies are now evaluating the inhibition of critical activities, such as cyclin-dependent kinase 1, Aurora or Polo kinases or spindle kinesins. Abrogation of the mitotic checkpoint or targeting the energetic or proteotoxic stress of aneuploid or chromosomally instable cells may also provide further benefits by inducing lethal levels of instability. Although cancer cells may display different responses to these treatments, recent data suggest that targeting mitotic exit by inhibiting the anaphase-promoting complex generates metaphase cells that invariably die in mitosis. As the efficacy of cell-cycle targeting approaches has been limited so far, further understanding of the molecular pathways modulating mitotic cell death will be required to move forward these new proposals to the clinic.

  4. Mitosis.

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    Henderson, Paula

    Cytology is the subject that is dealt with in this autoinstructional program. The process to be understood by secondary school students who are taking biology is mitosis. The material is presented to be adequate for achievers at the middle level. Knowledge of the structure of the DNA molecule and of the parts of the cell are considered as…

  5. Chlorpromazine inhibits mitosis of mammalian cells.

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    Boder, G B; Paul, D C; Williams, D C

    1983-09-01

    Chlorpromazine (CPZ) at minimally effective concentrations accumulates mammalian cells in mitosis without lethal effects on the cells. Star-metaphase morphology similar to effects seen with classical antimitotic compounds probably results from the preferential action of CPZ on a specific class of microtubules--the pole-to-pole microtubules of the mitotic spindle. At CPZ concentrations of 8 X 10(-6) M, flow cytometry indicates no effect of CPZ on the progress of cells through phases of the cell cycle other than mitosis (M). These results suggest a possible mechanism for toxic side effects of CPZ in man such as granulocytopenia and light sensitization.

  6. Strasburger's legacy to mitosis and cytokinesis and its relevance for the Cell Theory.

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    Baluška, František; Volkmann, Dieter; Menzel, Diedrik; Barlow, Peter

    2012-10-01

    Eduard Strasburger was one of the most prominent biologists contributing to the development of the Cell Theory during the nineteenth century. His major contribution related to the characterization of mitosis and cytokinesis and especially to the discovery of the discrete stages of mitosis, which he termed prophase, metaphase and anaphase. Besides his observations on uninucleate plant and animal cells, he also investigated division processes in multinucleate cells. Here, he emphasised the independent nature of mitosis and cytokinesis. We discuss these issues from the perspective of new discoveries in the field of cell division and conclude that Strasburger's legacy will in the future lead to a reformulation of the Cell Theory and that this will accommodate the independent and primary nature of the nucleus, together with its complement of perinuclear microtubules, for the organisation of the eukaryotic cell.

  7. Role of liver functions on liver cell mitosis

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    Takata,Tameyuki

    1974-06-01

    Full Text Available The control mechanism of mitosis in the regenerating rat liver was studied in relation to the cell functions. Partial hepatec· tomy induces a series of changes prior to the initiation of mitosis, i. e. decrease in serum glucose and albumin levels, loss of glycogen from liver cells, and increased lipid mobilization to liver cells. Massive supplies of glucose and fructose suppressed significantly hepatocellu. lar mitosis with suppression of lipid accumulation and preservation of glycogen in the liver cells and of blood sugar level. Homologous serum administration also suppressed the rate of liver cell mitosis after hepatectomy preventing the decrease in serum albumin level, but did not suppress the lipid accumulation in the liver. Starvation, which would relieve the liver cell from the work of detoxication of intesti. nal toxic products, did not show any suppressive effect on the mitotic rate of liver cells after partial hepatectomy in single animals. But starvation induced severe hypoglycemia, moderate hypoalbuminemia and loss of glycogen content in the liver. These changes in metabo. lism by starvation and partial hepatectomy were suppressed by con· jugating the animals with nonhepatectomized fed.partners by aortic anastomosis, and mitosis was suppressed in the residual liver of the fasting animals in this parabiosis. The results indicate that all the major functions of parenchymal live cells tested, sugar metabolism, serum albumin production, and detoxication, are closely related to the control of liver cell mitosis. Accumulation of lipids in the liver remnant after partial hepatectomy is thought to be for the compensa. tion of reduced glycogen storage and not concerned directly with the liver cell mitosis. Discussion was made briefly on the humoral factor and portal blood factor in relation to excess load of functions on resi. dual liver cells.

  8. Live-cell imaging of mitosis in Caenorhabditis elegans embryos.

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    Powers, James A

    2010-06-01

    Caenorhabditis elegans is a wonderful model system for live imaging studies of mitosis. A huge collection of research tools is readily available to facilitate experimentation. For imaging, C. elegans embryos provide large clear cells, an invariant pattern of cell division, only six chromosomes, a very short cell cycle, and remain healthy and happy at room temperature. Mitosis is a complicated process and the types of research questions being asked about the mechanisms involved are continuously expanding. For each experiment, the details of imaging methods need to be tailored to the question. Specific imaging methods will depend on the microscopy hardware and software available to each researcher. This article presents points to consider when choosing a microscope, designing an imaging experiment, or selecting appropriate worm strains for imaging. A method for mounting C. elegans embryos and guidelines for fluorescence and differential interference contrast imaging of mitosis in live embryos are presented.

  9. Circadian-independent cell mitosis in immortalized fibroblasts.

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    Yeom, Mijung; Pendergast, Julie S; Ohmiya, Yoshihiro; Yamazaki, Shin

    2010-05-25

    Two prominent timekeeping systems, the cell cycle, which controls cell division, and the circadian system, which controls 24-h rhythms of physiology and behavior, are found in nearly all living organisms. A distinct feature of circadian rhythms is that they are temperature-compensated such that the period of the rhythm remains constant (approximately 24 h) at different ambient temperatures. Even though the speed of cell division, or growth rate, is highly temperature-dependent, the cell-mitosis rhythm is temperature-compensated. Twenty-four-hour fluctuations in cell division have also been observed in numerous species, suggesting that the circadian system is regulating the timing of cell division. We tested whether the cell-cycle rhythm was coupled to the circadian system in immortalized rat-1 fibroblasts by monitoring cell-cycle gene promoter-driven luciferase activity. We found that there was no consistent phase relationship between the circadian and cell cycles, and that the cell-cycle rhythm was not temperature-compensated in rat-1 fibroblasts. These data suggest that the circadian system does not regulate the cell-mitosis rhythm in rat-1 fibroblasts. These findings are inconsistent with numerous studies that suggest that cell mitosis is regulated by the circadian system in mammalian tissues in vivo. To account for this discrepancy, we propose two possibilities: (i) There is no direct coupling between the circadian rhythm and cell cycle but the timing of cell mitosis is synchronized with the rhythmic host environment, or (ii) coupling between the circadian rhythm and cell cycle exists in normal cells but it is disconnected in immortalized cells.

  10. Circadian-independent cell mitosis in immortalized fibroblasts

    OpenAIRE

    Yeom, Mijung; Pendergast, Julie S.; Ohmiya, Yoshihiro; Yamazaki, Shin

    2010-01-01

    Two prominent timekeeping systems, the cell cycle, which controls cell division, and the circadian system, which controls 24-h rhythms of physiology and behavior, are found in nearly all living organisms. A distinct feature of circadian rhythms is that they are temperature-compensated such that the period of the rhythm remains constant (~24 h) at different ambient temperatures. Even though the speed of cell division, or growth rate, is highly temperature-dependent, the cell-mitosis rhythm is ...

  11. Mitosis orientation in prostate epithelial cells changed by endocrine effect

    Institute of Scientific and Technical Information of China (English)

    Xiang-yun LIU; Dong-mei Li; Xiao-fang ZHANG; Jian-hui WU; Zu-yue SUN

    2008-01-01

    Aim: The aim of the present study was to investigate the effect of androgen and estrogen on mitosis orientation in the prostate epithelial cells of male rats. Methods: Castrated rats were treated with a single injection of testosterone propionate (TP) or benzogynestry (E2). There were 8 rats in the control group and TP-treated or E2-treated group. Prostate, liver, a specimen of skin, and a segment of the jejunum and colon were removed after the corresponding treatment. The results were observed through immunohistochemistry and iron hematoxylin-eosin staining.Results: All mitoses found in the prostate epithelial cells of castrated rats with TP were oriented parallel to the basement membrane; however, mitoses found in the prostate epithelial cells of castrated rats in E2 and the control group were oriented perpendicular to the basement membrane. TP treatment resulted in marked changes in mitosis orientation in the prostate epithelial cells. Bromodeoxyuridine-labeled positive cells could be seen throughout the stroma and prostate epithelial cells with an injection of TP; however, the positive cells could only be seen in the stroma of prostate with an injection of E2, and the positive cells could hardly be seen in the control group. Conclusion: We found a novel effect of TP in the prostate as a marked change of mitosis orientation in prostate epithelial cells.

  12. Autophagic flux is highly active in early mitosis and differentially regulated throughout the cell cycle

    OpenAIRE

    Li, Zhiyuan; Ji, Xinmiao; Wang, Dongmei; Liu, Juanjuan; Zhang, Xin

    2016-01-01

    Mitosis is a fast process that involves dramatic cellular remodeling and has a high energy demand. Whether autophagy is active or inactive during the early stages of mitosis in a naturally dividing cell is still debated. Here we aimed to use multiple assays to resolve this apparent discrepancy. Although the LC3 puncta number was reduced in mitosis, the four different cell lines we tested all have active autophagic flux in both interphase and mitosis. In addition, the autophagic flux was highl...

  13. Investigation of MEK activity in COS7 cells entering mitosis.

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    Shi, Huaiping; Zhang, Tianying; Yi, Yongqing; Luo, Jun

    2014-12-01

    Although the mitogen-activated protein kinase (MAPK) pathway has been extensively investigated, numerous events remain unclear. In the present study, we examined mitogen-activated protein kinase kinase (MEK) expression from interphase to mitosis. Following nocodazole treatment, COS7 cells gradually became round as early as 4 h after treatment. Cyclin B1 expression gradually increased from 4 to 24 h in the presence of nocodazole. When cells were treated with nocodazole for 4 h, the level of epidermal growth factor (EGF)-mediated MEK phosphorylation did not significantly change between nocodazole-untreated and -treated (4 h) cells (P>0.05). However, EGF-mediated MEK phosphorylation was significantly inhibited upon treatment with nocodazole for 8 and 24 h compared to nocodazole-untreated cells (P0.05). The results showed that MEK expression is gradually inhibited from cell interphase to mitosis, and that MEK downstream signaling is affected by this inhibition, which probably reflects the requirements of cell physiology during mitosis.

  14. Dissecting mitosis by RNAi in Drosophila tissue culture cells

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    Maiato Helder

    2003-01-01

    Full Text Available Here we describe a detailed methodology to study the function of genes whose products function during mitosis by dsRNA-mediated interference (RNAi in cultured cells of Drosophila melanogaster. This procedure is particularly useful for the analysis of genes for which genetic mutations are not available or for the dissection of complicated phenotypes derived from the analysis of such mutants. With the advent of whole genome sequencing it is expected that RNAi-based screenings will be one method of choice for the identification and study of novel genes involved in particular cellular processes. In this paper we focused particularly on the procedures for the proper phenotypic analysis of cells after RNAi-mediated depletion of proteins required for mitosis, the process by which the genetic information is segregated equally between daughter cells. We use RNAi of the microtubule-associated protein MAST/Orbit as an example for the usefulness of the technique.

  15. Controlling the switches: Rho GTPase regulation during animal cell mitosis.

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    Zuo, Yan; Oh, Wonkyung; Frost, Jeffrey A

    2014-12-01

    Animal cell division is a fundamental process that requires complex changes in cytoskeletal organization and function. Aberrant cell division often has disastrous consequences for the cell and can lead to cell senescence, neoplastic transformation or death. As important regulators of the actin cytoskeleton, Rho GTPases play major roles in regulating many aspects of mitosis and cytokinesis. These include centrosome duplication and separation, generation of cortical rigidity, microtubule-kinetochore stabilization, cleavage furrow formation, contractile ring formation and constriction, and abscission. The ability of Rho proteins to function as regulators of cell division depends on their ability to cycle between their active, GTP-bound and inactive, GDP-bound states. However, Rho proteins are inherently inefficient at fulfilling this cycle and require the actions of regulatory proteins that enhance GTP binding (RhoGEFs), stimulate GTPase activity (RhoGAPs), and sequester inactive Rho proteins in the cytosol (RhoGDIs). The roles of these regulatory proteins in controlling cell division are an area of active investigation. In this review we will delineate the current state of knowledge of how specific RhoGEFs, RhoGAPs and RhoGDIs control mitosis and cytokinesis, and highlight the mechanisms by which their functions are controlled.

  16. Monitoring the elasticity changes of HeLa cells during mitosis by atomic force microscopy

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    Jiang, Ningcheng; Wang, Yuhua; Zeng, Jinshu; Ding, Xuemei; Xie, Shusen; Yang, Hongqin

    2016-10-01

    Cell mitosis plays a crucial role in cell life activity, which is one of the important phases in cell division cycle. During the mitosis, the cytoskeleton micro-structure of the cell changed and the biomechanical properties of the cell may vary depending upon different mitosis stages. In this study, the elasticity property of HeLa cells during mitosis was monitored by atomic force microscopy. Also, the actin filaments in different mitosis stages of the cells were observed by confocal imaging. Our results show that the cell in anaphase is stiffer than that in metaphase and telophase. Furthermore, lots of actin filaments gathered in cells' center area in anaphase, which contributes to the rigidity of the cell in this phase. Our findings demonstrate that the nano-biomechanics of living cells could provide a new index for characterizing cell physiological states.

  17. Presenting Mitosis

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    Roche, Stephanie; Sterling, Donna R.

    2005-01-01

    When the topic of cell division is introduced in the classroom, students can showcase their interpretations of the stages of mitosis by creating a slide show illustrating prophase, metaphase, anaphase, and telophase (see samples in Figure 1). With the help of a computer, they can create a model of mitosis that will help them distinguish the…

  18. The positional control of mitosis and cytokinesis in higher-plant cells.

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    Giménez-Abián, M I; Utrilla, L; Cánovas, J L; Giménez-Martín, G; Navarrete, M H; De la Torre, C

    1998-01-01

    The present work establishes a correlation between cell length and patterns of mitotic microtubular assemblies in Allium cepa L. root meristems. Binucleate cells were formed by a short caffeine treatment which aborted the formation of the phragmoplast during telophase. The largest binucleate cells (about 50 microns in length) behaved as two contiguous mononucleate cells in their next mitosis: they developed two preprophase bands (PPBs), one around each nucleus, where two spindles and two phragmoplasts were subsequently formed. On the other hand, the shortest binucleate cells (about 36 microns in length) formed a single PPB at the site of the aborted phragmoplast and, in the medium-sized cells (about 44 microns) in which the single PPB formed around the nucleus possessing the largest cytoplasmic environment, the two mitotic spindles and the new phragmoplasts moved to, or were assembled in the position of the phragmoplast that had been aborted one cycle earlier. Some rules derive from these observations. First of all, the aborted phragmoplast left a signal for microtubule positioning which was still operative one cycle later, in two-thirds of the bimitoses. Also, that formation of the PPB is dispensable. Moreover, its development does not always predict the future division plane, because of the presence of competing old signals which are stronger than those shed by the PPB in the same mitosis, but which fade away with distance. Finally, the positional signals were reinforced when the ratio of monomeric to fibrillar actin was increased by cytochalasin D during their shedding. When this drug was given simultaneously with caffeine, the frequency of bimitoses which, one cycle later, developed spindles and phragmoplasts in the positions of the old phragmoplast increased. On the other hand, those frequencies dropped in relation to control when the cytochalasin D treatment took place during bimitosis, indicating that at this time the treatment reinforced the signals

  19. Abnormal mitosis triggers p53-dependent cell cycle arrest in human tetraploid cells.

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    Kuffer, Christian; Kuznetsova, Anastasia Yurievna; Storchová, Zuzana

    2013-08-01

    Erroneously arising tetraploid mammalian cells are chromosomally instable and may facilitate cell transformation. An increasing body of evidence shows that the propagation of mammalian tetraploid cells is limited by a p53-dependent arrest. The trigger of this arrest has not been identified so far. Here we show by live cell imaging of tetraploid cells generated by an induced cytokinesis failure that most tetraploids arrest and die in a p53-dependent manner after the first tetraploid mitosis. Furthermore, we found that the main trigger is a mitotic defect, in particular, chromosome missegregation during bipolar mitosis or spindle multipolarity. Both a transient multipolar spindle followed by efficient clustering in anaphase as well as a multipolar spindle followed by multipolar mitosis inhibited subsequent proliferation to a similar degree. We found that the tetraploid cells did not accumulate double-strand breaks that could cause the cell cycle arrest after tetraploid mitosis. In contrast, tetraploid cells showed increased levels of oxidative DNA damage coinciding with the p53 activation. To further elucidate the pathways involved in the proliferation control of tetraploid cells, we knocked down specific kinases that had been previously linked to the cell cycle arrest and p53 phosphorylation. Our results suggest that the checkpoint kinase ATM phosphorylates p53 in tetraploid cells after abnormal mitosis and thus contributes to proliferation control of human aberrantly arising tetraploids.

  20. The effect of oleander glycosides on the germination of pollen grains and the mitosis of the generative nucleus in Tradescantia bracteata Small and Allium cepa L.

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    J. A. Tarkowska

    2015-01-01

    The effect of water solution of a mixture of glycosides from oleander (Nerium oleander L.) on the germination of pollen grains and on the mitosis of the generative nucleus in Tradescantia bracteata Small and Allium cepa L. has been studied. An inhibition of the germination and of the growth of pollen tubes was observed, proportionally to the concentration of glycosides. The pollen grains of A. cepa are more sensitive. The disturbances in mitosis lead to the formation of two or more uneven-siz...

  1. Immunolocalization of phospho-S6 kinases: a new way to detect mitosis in tissue sections and in cell culture.

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    Schmidt, Thomas; Wahl, Patricia; Wüthrich, Rudolf P; Vogetseder, Alexander; Picard, Nicolas; Kaissling, Brigitte; Le Hir, Michel

    2007-02-01

    During a study on the mTor pathway in the rat kidney we observed a striking increase of the phosphorylation of the S6 kinase in mitosis. In cryostat sections of perfusion-fixed tissue mitotic cells appeared as bright spots in immunofluorescence using an antibody specific for the phosphorylation site Thr421/Ser424. They were easily spotted in overviews with the objective 4x and 10x. Immunofluorescence was weak during the interphase. During the prophase it increased in both the nucleus and the cytoplasm and it remained bright during the subsequent phases of mitosis. All mitotic cells which were found in tubules and in the interstitium of the kidney using a chromatin stain displayed the bright immunofluorescence for phospho-S6 kinase. The same phenomenon was observed in rat liver and in mouse kidney as well as in a human cell line. Provided a rapid fixation, mitotic cells could be identified with the immunoperoxidase technique in paraffin sections of immersion-fixed tissue. This is the first report of phosphorylation of S6 kinase during mitosis in vivo. Thus, immunohistochemistry with anti-phospho-S6 kinase (Thr421/Ser424) appears to provide a convenient way to detect mitotic cells at low magnification.

  2. Prolonged Mitosis of Neural Progenitors Alters Cell Fate in the Developing Brain.

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    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.

  3. Hyperphosphorylation by cyclin B/CDK1 in mitosis resets CUX1 DNA binding clock at each cell cycle.

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    Sansregret, Laurent; Gallo, David; Santaguida, Marianne; Leduy, Lam; Harada, Ryoko; Nepveu, Alain

    2010-10-22

    The p110 CUX1 homeodomain protein participates in the activation of DNA replication genes in part by increasing the affinity of E2F factors for the promoters of these genes. CUX1 expression is very weak in quiescent cells and increases during G(1). Biochemical activities associated with transcriptional activation by CUX1 are potentiated by post-translational modifications in late G(1), notably a proteolytic processing event that generates p110 CUX1. Constitutive expression of p110 CUX1, as observed in some transformed cells, leads to accelerated entry into the S phase. In this study, we investigated the post-translation regulation of CUX1 during mitosis and the early G(1) phases of proliferating cells. We observed a major electrophoretic mobility shift and a complete inhibition of DNA binding during mitosis. We show that cyclin B/CDK1 interacts with CUX1 and phosphorylates it at multiple sites. Serine to alanine replacement mutations at 10 SP dipeptide sites were required to restore DNA binding in mitosis. Passage into G(1) was associated with the degradation of some p110 CUX1 proteins, and the remaining proteins were gradually dephosphorylated. Indirect immunofluorescence and subfractionation assays using a phospho-specific antibody showed that most of the phosphorylated protein remained in the cytoplasm, whereas the dephosphorylated protein was preferentially located in the nucleus. Globally, our results indicate that the hyperphosphorylation of CUX1 by cyclin B/CDK1 inhibits its DNA binding activity in mitosis and interferes with its nuclear localization following cell division and formation of the nuclear membrane, whereas dephosphorylation and de novo synthesis contribute to gradually restore CUX1 expression and activity in G(1).

  4. Kinesin-II is not essential for mitosis and cell growth in Chlamydomonas.

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    Matsuura, Kumi; Lefebvre, Paul A; Kamiya, Ritsu; Hirono, Masafumi

    2002-08-01

    The FLA10 gene product (Fla10p) in Chlamydomonas, a heterotrimeric kinesin-II, plays a crucial role in flagellar assembly as a motor protein driving intraflagellar transport. This protein has also been suggested to play a role in mitosis based on its localization to mitotic spindle. A role for Fla10p in mitosis has been difficult to test because to date only conditional (temperature-sensitive) mutant alleles were available, and it is not known whether these retain residual function for mitosis at the non-permissive temperature. In this report, we describe a null allele of fla10 produced by insertional mutagenesis. This mutant does not assemble flagella, but proliferates at a rate identical to that of wild type cells. Observation of microtubule organization in the cell body revealed that normal mitotic spindles are formed in dividing mutant cells. Thus, we conclude that FLA10 kinesin plays no significant roles in mitosis.

  5. Positive Feedback Keeps Duration of Mitosis Temporally Insulated from Upstream Cell-Cycle Events

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    Araujo, Ana Rita; Gelens, Lendert; Sheriff, Rahuman; Santos, Silvia D.M.

    2016-01-01

    Cell division is characterized by a sequence of events by which a cell gives rise to two daughter cells. Quan- titative measurements of cell-cycle dynamics in sin- gle cells showed that despite variability in G1-, S-, and G2 phases, duration of mitosis is short and remarkably constant. Surprisingly, there is no corre- lation between cell-cycle length and mitotic duration, suggesting that mitosis is temporally insulated from variability in earlier cell-cycle phases. By combining live cell imag...

  6. Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis.

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    Özlü, Nurhan; Qureshi, Mohammad H; Toyoda, Yusuke; Renard, Bernhard Y; Mollaoglu, Gürkan; Özkan, Nazlı E; Bulbul, Selda; Poser, Ina; Timm, Wiebke; Hyman, Anthony A; Mitchison, Timothy J; Steen, Judith A

    2015-01-13

    The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy.

  7. Cell death associated with abnormal mitosis observed by confocal imaging in live cancer cells.

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    Castiel, Asher; Visochek, Leonid; Mittelman, Leonid; Zilberstein, Yael; Dantzer, Francoise; Izraeli, Shai; Cohen-Armon, Malka

    2013-08-21

    Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent γ-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered γ-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

  8. Sex and the eukaryotic cell cycle is consistent with a viral ancestry for the eukaryotic nucleus.

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    Bell, Philip John Livingstone

    2006-11-07

    The origin of the eukaryotic cell cycle, including mitosis, meiosis, and sex are as yet unresolved aspects of the evolution of the eukaryotes. The wide phylogenetic distribution of both mitosis and meiosis suggest that these processes are integrally related to the origin of the earliest eukaryotic cells. According to the viral eukaryogenesis (VE) hypothesis, the eukaryotes are a composite of three phylogenetically unrelated organisms: a viral lysogen that evolved into the nucleus, an archaeal cell that evolved into the eukaryotic cytoplasm, and an alpha-proteobacterium that evolved into the mitochondria. In the extended VE hypothesis presented here, the eukaryotic cell cycle arises as a consequence of the derivation of the nucleus from a lysogenic DNA virus.

  9. The effect of oleander glycosides on the germination of pollen grains and the mitosis of the generative nucleus in Tradescantia bracteata Small and Allium cepa L.

    Directory of Open Access Journals (Sweden)

    J. A. Tarkowska

    2015-01-01

    Full Text Available The effect of water solution of a mixture of glycosides from oleander (Nerium oleander L. on the germination of pollen grains and on the mitosis of the generative nucleus in Tradescantia bracteata Small and Allium cepa L. has been studied. An inhibition of the germination and of the growth of pollen tubes was observed, proportionally to the concentration of glycosides. The pollen grains of A. cepa are more sensitive. The disturbances in mitosis lead to the formation of two or more uneven-sized doughter nuclei, or to the formation of restitution nuclei. These anomalies are more numerous in T. bracteata. From these results d t appears that pollen grains of A. cepa are characterized by a generally high physiological sensitivity and a small mitotic sensitivity, wheras for T. bracteata the opposite is true.

  10. Skp2 is required for Aurora B activation in cell mitosis and spindle checkpoint.

    Science.gov (United States)

    Wu, Juan; Huang, Yu-Fan; Zhou, Xin-Ke; Zhang, Wei; Lian, Yi-Fan; Lv, Xiao-Bin; Gao, Xiu-Rong; Lin, Hui-Kuan; Zeng, Yi-Xin; Huang, Jian-Qing

    2015-01-01

    The Aurora B kinase plays a critical role in cell mitosis and spindle checkpoint. Here, we showed that the ubiquitin E3-ligase protein Skp2, also as a cell-cycle regulatory protein, was required for the activation of Aurora B and its downstream protein. When we restored Skp2 knockdown Hela cells with Skp2 and Skp2-LRR E3 ligase dead mutant we found that Skp2 could rescue the defect in the activation of Aurora B, but the mutant failed to do so. Furthermore, we discovered that Skp2 could interact with Aurora B and trigger Aurora B Lysine (K) 63-linked ubiquitination. Finally, we demonstrated the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which was Aurora B dependent. Our results identified a novel ubiquitinated substrate of Skp2, and also indicated that Aurora B ubiquitination might serve as an important event for Aurora B activation in cell mitosis and spindle checkpoint.

  11. [Flow cytometric analysis of ICRF-193 influence on cell passage through mitosis].

    Science.gov (United States)

    Shatrova, A N; Aksenov, N D; Zenin, V V

    2002-01-01

    Studying the effect of topoisomerase II (topo II) inhibitors on cell passage through mitosis seems to be important for understanding the role of this enzyme during chromosome condensation and segregation. A flow cytometric assay (Zenin et al., 2001) allowed to determine the mitotic index, and to discriminate between not only cells in G2 and M phases (including metaphase and anaphase cells), but also cells in pseudo-G1 with 4c DNA content. It is shown that topo II catalytic inhibitor ICRF-193 blocks G2-M transition in a lymphoblastoid cell line GM-130. Addition of caffeine to cells abrogated a block of their entering mitosis but not the inhibitor action. Cells entered mitosis, which was proven by the presence of chromosomes in the examined specimen, and, bypassing anaphase, appeared in pseudo-G1 with 4c DNA content. We have found that in the presence of ICRF-193 cells, GM-130 and Hep-2 lines, previously blocked by nocodazole when in mitosis and then washed, pass through metaphase, enter anaphase and leave it to pass to pseudo-G1 with the 4c DNA content. Thus, by inhibiting topo II activity ICRF-193 causes abnormal mitotic transition.

  12. Expression of HSF2 decreases in mitosis to enable stress-inducible transcription and cell survival.

    Science.gov (United States)

    Elsing, Alexandra N; Aspelin, Camilla; Björk, Johanna K; Bergman, Heidi A; Himanen, Samu V; Kallio, Marko J; Roos-Mattjus, Pia; Sistonen, Lea

    2014-09-15

    Unless mitigated, external and physiological stresses are detrimental for cells, especially in mitosis, resulting in chromosomal missegregation, aneuploidy, or apoptosis. Heat shock proteins (Hsps) maintain protein homeostasis and promote cell survival. Hsps are transcriptionally regulated by heat shock factors (HSFs). Of these, HSF1 is the master regulator and HSF2 modulates Hsp expression by interacting with HSF1. Due to global inhibition of transcription in mitosis, including HSF1-mediated expression of Hsps, mitotic cells are highly vulnerable to stress. Here, we show that cells can counteract transcriptional silencing and protect themselves against proteotoxicity in mitosis. We found that the condensed chromatin of HSF2-deficient cells is accessible for HSF1 and RNA polymerase II, allowing stress-inducible Hsp expression. Consequently, HSF2-deficient cells exposed to acute stress display diminished mitotic errors and have a survival advantage. We also show that HSF2 expression declines during mitosis in several but not all human cell lines, which corresponds to the Hsp70 induction and protection against stress-induced mitotic abnormalities and apoptosis.

  13. Inhibitory effect of Polo-like kinase 1 depletion on mitosis and apoptosis of gastric cancer cells

    OpenAIRE

    Chen, Xue-Hua; Lan, Bin; Qu, Ying; ZHANG, XIAO-QING; Cai, Qu; Liu, Bing-Ya; Zhu, Zheng-Gang

    2006-01-01

    AIM: Polo-like kinase 1 (PLK1) serine/threonine kinase plays a vital role in multiple phases of mitosis in gastric cancer cells. To investigate the effect of PLK1 depletion on mitosis and apoptosis of gastric cancer cells.

  14. Histone deacetylation is required for progression through mitosis in tobacco cells.

    Science.gov (United States)

    Li, Yan; Butenko, Yana; Grafi, Gideon

    2005-02-01

    Post-translational modifications of core histone proteins play a key role in chromatin structure and function. Here, we study histone post-translational modifications during reentry of protoplasts derived from tobacco mesophyll cells into the cell cycle and evaluate their significance for progression through mitosis. Methylation of histone H3 at lysine residues 4 and 9 persisted in chromosomes during all phases of the cell cycle. However, acetylation of H4 and H3 was dramatically reduced during mitosis in a stage-specific manner; while deacetylation of histone H4 commenced at prophase and persisted up to telophase, histone H3 remained acetylated up to metaphase but was deacetylated at anaphase and telophase. Phosphorylation of histone H3 at serine 10 was initiated at prophase, concomitantly with deacetylation of histone H4, and persisted up to telophase. Preventing histone deacetylation by the histone deacetylase inhibitor trichostatin A (TSA) led to accumulation of protoplasts at metaphase-anaphase, and reduced S10 phosphorylation during anaphase and telophase; in cultured tobacco cells, TSA significantly reduced the frequency of mitotic figures. Our results indicate that deacetylation of histone H4 and H3 in tobacco protoplasts occurs during mitosis in a phase-specific manner, and is important for progression through mitosis.

  15. Cell degeneration and mitosis in the buccopharyngeal and branchial membranes in the mouse embryo.

    Science.gov (United States)

    Poelmann, R E; Dubois, S V; Hermsen, C; Smits-van Prooije, A E; Vermeij-Keers, C

    1985-01-01

    The frequencies of cell degeneration and mitosis were investigated in the rupturing buccopharyngeal membrane (BPM) and in the persistent first branchial membrane (BM). In the BPM, cell degeneration starts many hours before rupture is visible, but mitotic figures are absent. In the BM this situation is reversed: mitotic figures are regularly observed, but a degenerating cell only occasionally. It is concluded that the ratio between the numbers of degenerating and dividing cells regulates the fate of both the BPM and the BM.

  16. [Effect of temperature on the duration of mitosis in mammalian cells cultivated outside the body].

    Science.gov (United States)

    Sushkov, F V; Smirnova, T M; Savik, Z F

    1978-02-01

    Nine cell strains of different origin were cultivated at 28--36 degrees with the interval of 2 degrees. During the phase of logarithmic culture growth, the duration of mitosis (Tm) was determined by means of colchicine method. A strict temperatural dependence Tm, obeyed to Arrenius' law was revealed. Temperature range within which Arreinius' law is valid in different cell strains is not alike. Cultivation of L cells and connective tissue cells from Chinese hamster to 39, 41and 42 degrees demonstrated their upper critical point Tm to be for L cells 39 degrees, for connective tissue cells from the Chinese hamster--41 degrees. Electron microscopic investigations demonstrated that cell cultivation within physiological (mitosis destroying) range of temperatures does not notably effect their ultrastructural organization.

  17. Monitoring impedance changes associated with motility and mitosis of a single cell.

    Science.gov (United States)

    Ghenim, Lamya; Kaji, Hirokazu; Hoshino, Yu; Ishibashi, Takeshi; Haguet, Vincent; Gidrol, Xavier; Nishizawa, Matsuhiko

    2010-10-07

    We present a device enabling impedance measurements that probe the motility and mitosis of a single adherent cell in a controlled way. The micrometre-sized electrodes are designed for adhesion of an isolated cell and enhanced sensitivity to cell motion. The electrode surface is switched electro-chemically to favour cell adhesion, and single cells are attracted to the electrode using positive dielectrophoresis. Periods of linear variation in impedance with time correspond to the motility of a single cell adherent to the surface estimated at 0.6 μm h(-1). In the course of our study we observed the impedance changes associated with mitosis of a single cell. Electrical measurements, carried out concomitantly with optical observations, revealed three phases, prophase, metaphase and anaphase in the time variation of the impedance during cell division. Maximal impedance was observed at metaphase with a 20% increase of the impedance. We argue that at mitosis, the changes detected were due to the charge density distribution at the cell surface. Our data demonstrate subtle electrical changes associated with cell motility and for the first time with division at the single-cell level. We speculate that this could open up new avenues for characterizing healthy and pathological cells.

  18. Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells.

    Science.gov (United States)

    Sheremet, Ya A; Yemets, A I; Azmi, A; Vissenberg, K; Verbelen, J P; Blume, Ya B

    2012-01-01

    To test whether reversible tubulin phosphorylation plays any role in the process of plant mitosis the effects of inhibitors of tyrosine kinases, herbimycin A, genistein and tyrphostin AG 18, and of an inhibitor of tyrosine phosphatases, sodium orthovanadate, on microtubule organization and mitosis progression in a synchronized BY-2 culture has been investigated. It was found that treatment with inhibitors of tyrosine kinases of BY-2 cells at the G2/M transition did not lead to visible disturbances of mitotic microtubule structures, while it did reduce the frequency of their appearance. We assume that a decreased tyrosine phosphorylation level could alter the microtubule dynamic instability parameters during interphase/prophase transition. All types of tyrosine kinase inhibitors used caused a prophase delay: herbimycin A and genistein for 2 h, and tyrphostin AG18 for 1 h. Thereafter the peak of mitosis was displaced for 1 h by herbimycin A or genistein exposure, but after tyrphostin AG18 treatment the timing of the mitosis-peak was comparable to that in control cells. Enhancement of tyrosine phosphorylation induced by the tyrosine phosphatase inhibitor resulted in the opposite effect on BY-2 mitosis transition. Culture treatment with sodium orthovanadate during 1 h resulted in an accelerated start of the prophase and did not lead to the alteration in time of the mitotic index peak formation, as compared to control cells. We suppose that the reversible tyrosine phosphorylation can be involved in the regulation of interphase to M phase transition possibly through regulation of microtubule dynamics in plant cells.

  19. Resonant microchannel volume and mass measurements show that suspended cells swell during mitosis.

    Science.gov (United States)

    Son, Sungmin; Kang, Joon Ho; Oh, Seungeun; Kirschner, Marc W; Mitchison, T J; Manalis, Scott

    2015-11-23

    Osmotic regulation of intracellular water during mitosis is poorly understood because methods for monitoring relevant cellular physical properties with sufficient precision have been limited. Here we use a suspended microchannel resonator to monitor the volume and density of single cells in suspension with a precision of 1% and 0.03%, respectively. We find that for transformed murine lymphocytic leukemia and mouse pro-B cell lymphoid cell lines, mitotic cells reversibly increase their volume by more than 10% and decrease their density by 0.4% over a 20-min period. This response is correlated with the mitotic cell cycle but is not coupled to nuclear osmolytes released by nuclear envelope breakdown, chromatin condensation, or cytokinesis and does not result from endocytosis of the surrounding fluid. Inhibiting Na-H exchange eliminates the response. Although mitotic rounding of adherent cells is necessary for proper cell division, our observations that suspended cells undergo reversible swelling during mitosis suggest that regulation of intracellular water may be a more general component of mitosis than previously appreciated.

  20. Systematic Analysis of the Crosstalk between Mitosis and DNA Damage by a Live Cell siRNA Screen

    DEFF Research Database (Denmark)

    Pedersen, Ronni Sølvhøi

    Recent research has shown, that the biological processes of DNA replication, DNA damage, cell cycle and mitosis cannot be considered as isolated cellular functions but are mechanistically linked in many ways. For instance, when cells are exposed to replication stress and enter mitosis...... propose that this strong p53 response, which often occurs without detectable increase in DNA damage, is caused by the acute increase in chromosomal aneuploidy. Finally, our systematic approach to the DNA damage-mitosis crosstalk reveals widespread cell death in response to mitotic pertubations, showing...

  1. Tau excess impairs mitosis and kinesin-5 function, leading to aneuploidy and cell death

    Directory of Open Access Journals (Sweden)

    Anne-Laure Bougé

    2016-03-01

    Full Text Available In neurodegenerative diseases such as Alzheimer's disease (AD, cell cycle defects and associated aneuploidy have been described. However, the importance of these defects in the physiopathology of AD and the underlying mechanistic processes are largely unknown, in particular with respect to the microtubule (MT-binding protein Tau, which is found in excess in the brain and cerebrospinal fluid of affected individuals. Although it has long been known that Tau is phosphorylated during mitosis to generate a lower affinity for MTs, there is, to our knowledge, no indication that an excess of this protein could affect mitosis. Here, we studied the effect of an excess of human Tau (hTau protein on cell mitosis in vivo. Using the Drosophila developing wing disc epithelium as a model, we show that an excess of hTau induces a mitotic arrest, with the presence of monopolar spindles. This mitotic defect leads to aneuploidy and apoptotic cell death. We studied the mechanism of action of hTau and found that the MT-binding domain of hTau is responsible for these defects. We also demonstrate that the effects of hTau occur via the inhibition of the function of the kinesin Klp61F, the Drosophila homologue of kinesin-5 (also called Eg5 or KIF11. We finally show that this deleterious effect of hTau is also found in other Drosophila cell types (neuroblasts and tissues (the developing eye disc, as well as in human HeLa cells. By demonstrating that MT-bound Tau inhibits the Eg5 kinesin and cell mitosis, our work provides a new framework to consider the role of Tau in neurodegenerative diseases.

  2. Tau excess impairs mitosis and kinesin-5 function, leading to aneuploidy and cell death.

    Science.gov (United States)

    Bougé, Anne-Laure; Parmentier, Marie-Laure

    2016-03-01

    In neurodegenerative diseases such as Alzheimer's disease (AD), cell cycle defects and associated aneuploidy have been described. However, the importance of these defects in the physiopathology of AD and the underlying mechanistic processes are largely unknown, in particular with respect to the microtubule (MT)-binding protein Tau, which is found in excess in the brain and cerebrospinal fluid of affected individuals. Although it has long been known that Tau is phosphorylated during mitosis to generate a lower affinity for MTs, there is, to our knowledge, no indication that an excess of this protein could affect mitosis. Here, we studied the effect of an excess of human Tau (hTau) protein on cell mitosis in vivo. Using the Drosophila developing wing disc epithelium as a model, we show that an excess of hTau induces a mitotic arrest, with the presence of monopolar spindles. This mitotic defect leads to aneuploidy and apoptotic cell death. We studied the mechanism of action of hTau and found that the MT-binding domain of hTau is responsible for these defects. We also demonstrate that the effects of hTau occur via the inhibition of the function of the kinesin Klp61F, the Drosophila homologue of kinesin-5 (also called Eg5 or KIF11). We finally show that this deleterious effect of hTau is also found in other Drosophila cell types (neuroblasts) and tissues (the developing eye disc), as well as in human HeLa cells. By demonstrating that MT-bound Tau inhibits the Eg5 kinesin and cell mitosis, our work provides a new framework to consider the role of Tau in neurodegenerative diseases.

  3. The mitosis and immunocytochemistry of olfactory ensheathing cells from nasal olfactory mucosa

    Institute of Scientific and Technical Information of China (English)

    LIU Jin-bo; TANG Tian-si; GONG Ai-hua; SHENG Wei-hua; YANG Ji-cheng

    2005-01-01

    Objective: To culture olfactory ensheathing cells (OECs) of rats in vitro and to investigate its morphology, mitosis and immunocytochemistry, and to explore if the OECs could be a new donation for transplantation. Methods: OECs were harvested from olfactory mucosa of Sprague Dawleys rats based on the differing rates of attachment of the various cell types, followed by glial fibrillary acidic protein (GFAP), nerve growth factor (NGF), anti-low affinity receptor for NGF (NGFRp75), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and S-100 immunocytochemistry. The morphological changes and mitosis were observed under a phase contrast microscope at different culture time.Results: Three morphologically distinct types of cells, bipolar,multipolar and flat morphology were present in the primary culture of adult rat olfactory mucosa. Mitosis was characterized by a retraction of all processes, forming a sphere that divided into spherical daughter cells, the daughter cells sent out their processes. The OECs were immunoreactive for GFAP, NGFRp75, S-100, NGF, BDNF and NT-3. Conclusions: The OECs from nasal olfactory mucosa cultivated in the medium with fetal bovine serum could survive, divide, differentiate, and express the neurotrophin. It may become an accessible source for autologous grafting in spinal cord injury.

  4. [Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells].

    Science.gov (United States)

    Sheremet, Ia A; Emets, A I; Azmi, A; Vissenberg, K; Verbelen, J-P; Blium, Ia B

    2012-01-01

    In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

  5. Effect of hypothermia (20-25 degrees C) on mitosis in PtK1 cells.

    Science.gov (United States)

    Rieder, C L

    1981-06-01

    PtK1 cells enter prophase and complete mitosis at 24-25 degrees C but are inhibited from entering prophase at 20-21 degrees C. Cells which have progressed up to midprophase at 24-37 degrees C return to interphase when cooled to 20-21 degrees C, but those in late prophase complete a normal, although prolonged mitosis. If prophase cells which have reverted to interphase at 20-21 degrees C are incubated at 24-37 degrees C they reenter prophase and complete mitosis. This temperature-induced prophase-interphase-prophase transition can be repeated several times on the same cell. At 24-25 degrees C the process of spindle formation (i.e. prometaphase to the initiation of anaphase) encompasses approximately 75% of the total mitotic interval, with a duration of 8-12 h, compared to about 50% of the mitotic interval and a duration of 0.5 to 1.0 h at 37 degrees C.

  6. Deciphering the evolutionary history of open and closed mitosis.

    Science.gov (United States)

    Sazer, Shelley; Lynch, Michael; Needleman, Daniel

    2014-11-17

    The origin of the nucleus at the prokaryote-to-eukaryote transition represents one of the most important events in the evolution of cellular organization. The nuclear envelope encircles the chromosomes in interphase and is a selectively permeable barrier between the nucleoplasm and cytoplasm and an organizational scaffold for the nucleus. It remains intact in the 'closed' mitosis of some yeasts, but loses its integrity in the 'open' mitosis of mammals. Instances of both types of mitosis within two evolutionary clades indicate multiple evolutionary transitions between open and closed mitosis, although the underlying genetic changes that influenced these transitions remain unknown. A survey of the diversity of mitotic nuclei that fall between these extremes is the starting point from which to determine the physiologically relevant characteristics distinguishing open from closed mitosis and to understand how they evolved and why they are retained in present-day organisms. The field is now poised to begin addressing these issues by defining and documenting patterns of mitotic nuclear variation within and among species and mapping them onto a phylogenic tree. Deciphering the evolutionary history of open and closed mitosis will complement cell biological and genetic approaches aimed at deciphering the fundamental organizational principles of the nucleus.

  7. Unreplicated DNA remaining from unperturbed S phases passes through mitosis for resolution in daughter cells

    Science.gov (United States)

    Moreno, Alberto; Carrington, Jamie T.; Al Mamun, Mohammed; Haagensen, Emma J.; Komseli, Eirini-Stavroula; Gorgoulis, Vassilis G.; Newman, Timothy J.; Blow, J. Julian

    2016-01-01

    To prevent rereplication of genomic segments, the eukaryotic cell cycle is divided into two nonoverlapping phases. During late mitosis and G1 replication origins are “licensed” by loading MCM2-7 double hexamers and during S phase licensed replication origins activate to initiate bidirectional replication forks. Replication forks can stall irreversibly, and if two converging forks stall with no intervening licensed origin—a “double fork stall” (DFS)—replication cannot be completed by conventional means. We previously showed how the distribution of replication origins in yeasts promotes complete genome replication even in the presence of irreversible fork stalling. This analysis predicts that DFSs are rare in yeasts but highly likely in large mammalian genomes. Here we show that complementary strand synthesis in early mitosis, ultrafine anaphase bridges, and G1-specific p53-binding protein 1 (53BP1) nuclear bodies provide a mechanism for resolving unreplicated DNA at DFSs in human cells. When origin number was experimentally altered, the number of these structures closely agreed with theoretical predictions of DFSs. The 53BP1 is preferentially bound to larger replicons, where the probability of DFSs is higher. Loss of 53BP1 caused hypersensitivity to licensing inhibition when replication origins were removed. These results provide a striking convergence of experimental and theoretical evidence that unreplicated DNA can pass through mitosis for resolution in the following cell cycle. PMID:27516545

  8. Unreplicated DNA remaining from unperturbed S phases passes through mitosis for resolution in daughter cells.

    Science.gov (United States)

    Moreno, Alberto; Carrington, Jamie T; Albergante, Luca; Al Mamun, Mohammed; Haagensen, Emma J; Komseli, Eirini-Stavroula; Gorgoulis, Vassilis G; Newman, Timothy J; Blow, J Julian

    2016-09-27

    To prevent rereplication of genomic segments, the eukaryotic cell cycle is divided into two nonoverlapping phases. During late mitosis and G1 replication origins are "licensed" by loading MCM2-7 double hexamers and during S phase licensed replication origins activate to initiate bidirectional replication forks. Replication forks can stall irreversibly, and if two converging forks stall with no intervening licensed origin-a "double fork stall" (DFS)-replication cannot be completed by conventional means. We previously showed how the distribution of replication origins in yeasts promotes complete genome replication even in the presence of irreversible fork stalling. This analysis predicts that DFSs are rare in yeasts but highly likely in large mammalian genomes. Here we show that complementary strand synthesis in early mitosis, ultrafine anaphase bridges, and G1-specific p53-binding protein 1 (53BP1) nuclear bodies provide a mechanism for resolving unreplicated DNA at DFSs in human cells. When origin number was experimentally altered, the number of these structures closely agreed with theoretical predictions of DFSs. The 53BP1 is preferentially bound to larger replicons, where the probability of DFSs is higher. Loss of 53BP1 caused hypersensitivity to licensing inhibition when replication origins were removed. These results provide a striking convergence of experimental and theoretical evidence that unreplicated DNA can pass through mitosis for resolution in the following cell cycle.

  9. Meeting at mitosis: cell cycle-specific regulation of c-Src by RPTPalpha.

    Science.gov (United States)

    Mustelin, Tomas; Hunter, Tony

    2002-01-15

    Exquisite regulation is required for cells to properly enter and exit the phases of the cell cycle. The transmembrane receptor-like protein tyrosine phosphatase RPTPalpha, an important protein that participates in the transition of the cell cycle from G2 to mitosis activates the protein tyrosine kinase c-Src in vivo. Mustelin and Hunter discuss new findings that describe the highly regulated activation of RPTPalpha and c-Src that occurs just before entry into the mitotic phase. These findings also raise several questions that pertain to redistribution of RPTPalpha in the cell, and the role of phosphorylation and dimerization in regulating RPTPalpha activity.

  10. An imaging flow cytometric method for measuring cell division history and molecular symmetry during mitosis.

    Science.gov (United States)

    Filby, Andrew; Perucha, Esperanza; Summers, Huw; Rees, Paul; Chana, Prabhjoat; Heck, Susanne; Lord, Graham M; Davies, Derek

    2011-07-01

    Asymmetric cell division is an important mechanism for generating cellular diversity, however, techniques for measuring the distribution of fate-regulating molecules during mitosis have been hampered by a lack of objectivity, quantitation, and statistical robustness. Here we describe a novel imaging flow cytometric approach that is able to report a cells proliferative history and cell cycle position using dye dilution, pH3, and PI staining to then measure the spatial distribution of fluorescent signals during mitosis using CCD-derived imagery. Using Jurkat cells, resolution of the fluorescently labeled populations was comparable to traditional PMT based cytometers thus eliminating the need to sort cells with specific division histories for microscopy. Subdividing mitotic stages by morphology allowed us to determine the time spent in each cell cycle phase using mathematical modeling approaches. Furthermore high sample throughput allowed us to collect statistically relevant numbers of cells without the need to use blocking agents that artificially enrich for mitotic events. The fluorescent imagery was used to measure PKCζ protein and EEA-1+ endosome distribution during different mitotic phases in Jurkat cells. While telophase cells represented the favorable population for measuring asymmetry, asynchronously dividing cells spent approximately 43 seconds in this stage, explaining why they were present at such low frequencies. This necessitated the acquisition of large cell numbers. Interestingly we found that PKCζ was inherited asymmetrically in 2.5% of all telophasic events whereas endosome inheritance was significantly more symmetrical. Furthermore, molecular polarity at early mitotic phases was a poor indicator of asymmetry during telophase highlighting that, though rare, telophasic events represented the best candidates for asymmetry studies. In summary, this technique combines the spatial information afforded by fluorescence microscopy with the statistical

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

    Science.gov (United States)

    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

    2016-02-25

    The orientation of cell division along the long axis of the interphase cell--the century-old Hertwig's rule--has profound roles in tissue proliferation, morphogenesis, architecture and mechanics. In epithelial tissues, the shape of the interphase cell is influenced by cell adhesion, mechanical stress, neighbour topology, and planar polarity pathways. At mitosis, epithelial cells usually adopt a rounded shape to ensure faithful chromosome segregation and to promote morphogenesis. The mechanisms underlying interphase cell shape sensing in tissues are therefore unknown. Here we show that in Drosophila epithelia, tricellular junctions (TCJs) localize force generators, pulling on astral microtubules and 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 TCJs 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.

  12. Hepatitis C virus infection causes cell cycle arrest at the level of initiation of mitosis.

    Science.gov (United States)

    Kannan, Rathi P; Hensley, Lucinda L; Evers, Lauren E; Lemon, Stanley M; McGivern, David R

    2011-08-01

    Chronic infection with the hepatitis C virus (HCV) is associated with increased risk for hepatocellular carcinoma (HCC). Chronic immune-mediated inflammation is likely to be an important factor in the development of HCV-associated HCC, but direct effects of HCV infection on the host cell cycle may also play a role. Although overexpression studies have revealed multiple interactions between HCV-encoded proteins and host cell cycle regulators and tumor suppressor proteins, the relevance of these observations to HCV-associated liver disease is not clear. We determined the net effect of these interactions on regulation of the cell cycle in the context of virus infection. Flow cytometry of HCV-infected carboxyfluorescein succinimidyl ester-labeled hepatoma cells indicated a slowdown in proliferation that correlated with abundance of viral antigen. A decrease in the proportions of infected cells in G(1) and S phases with an accumulation of cells in G(2)/M phase was observed, compared to mock-infected controls. Dramatic decreases in markers of mitosis, such as phospho-histone H3, in infected cells suggested a block to mitotic entry. In common with findings described in the published literature, we observed caspase 3 activation, suggesting that cell cycle arrest is associated with apoptosis. Differences were observed in patterns of cell cycle disturbance and levels of apoptosis with different strains of HCV. However, the data suggest that cell cycle arrest at the interface of G(2) and mitosis is a common feature of HCV infection.

  13. Role-Playing Mitosis.

    Science.gov (United States)

    Wyn, Mark A.; Stegink, Steven J.

    2000-01-01

    Introduces a role playing activity that actively engages students in the learning process of mitosis. Students play either chromosomes carrying information, or cells in the cell membrane. (Contains 11 references.) (Author/YDS)

  14. Arrangement of nuclear structures is not transmitted through mitosis but is identical in sister cells.

    Science.gov (United States)

    Orlova, Darya Yu; Stixová, Lenka; Kozubek, Stanislav; Gierman, Hinco J; Šustáčková, Gabriela; Chernyshev, Andrei V; Medvedev, Ruslan N; Legartová, Soňa; Versteeg, Rogier; Matula, Pavel; Stoklasa, Roman; Bártová, Eva

    2012-11-01

    Although it is well known that chromosomes are non-randomly organized during interphase, it is not completely clear whether higher-order chromatin structure is transmitted from mother to daughter cells. Therefore, we addressed the question of how chromatin is rearranged during interphase and whether heterochromatin pattern is transmitted after mitosis. We additionally tested the similarity of chromatin arrangement in sister interphase nuclei. We noticed a very active cell rotation during interphase, especially when histone hyperacetylation was induced or transcription was inhibited. This natural phenomenon can influence the analysis of nuclear arrangement. Using photoconversion of Dendra2-tagged core histone H4 we showed that the distribution of chromatin in daughter interphase nuclei differed from that in mother cells. Similarly, the nuclear distribution of heterochromatin protein 1β (HP1β) was not completely identical in mother and daughter cells. However, identity between mother and daughter cells was in many cases evidenced by nucleolar composition. Moreover, morphology of nucleoli, HP1β protein, Cajal bodies, chromosome territories, and gene transcripts were identical in sister cell nuclei. We conclude that the arrangement of interphase chromatin is not transmitted through mitosis, but the nuclear pattern is identical in naturally synchronized sister cells. It is also necessary to take into account the possibility that cell rotation and the degree of chromatin condensation during functionally specific cell cycle phases might influence our view of nuclear architecture.

  15. Quantitative characterization of mitosis-blocked tetraploid cells using high content analysis.

    Science.gov (United States)

    Grove, Linnette E; Ghosh, Richik N

    2006-08-01

    A range of cellular evidence supporting a G1 tetraploidy checkpoint was obtained from different assay methods including flow cytometry, immunoblotting, and microscopy. Cancer research would benefit if these cellular properties could instead be measured by a single, quantitative, automated assay method, such as high content analysis (HCA). Thus, nocodazole-treated cells were fluorescently labeled for different cell cycle-associated properties, including DNA content, retinoblastoma (Rb) and histone H3 phosphorylation, p53 and p21(WAF1) expression, nuclear and cell sizes, and cell morphology, and automatically imaged, analyzed, and correlated using HCA. HCA verified that nocodazole-induced mitosis block resulted in tetraploid cells. Rb and histone H3 were maximally hyperphosphorylated by 24 h of nocodazole treatment, accompanied by cell and nuclear size decreases and cellular rounding. Cells remained tetraploid and mononucleated with longer treatments, but other targets reverted to G1 levels, including Rb and histone H3 dephosphorylation accompanied by cellular respreading. This was accompanied by increased p53 and p21(WAF1) expression levels. The range of effects accompanying nocodazole-induced block of mitosis and the resulting tetraploid cells' reversal to a pseudo-G1 state can be quantitatively measured by HCA in an automated manner, recommending this assay method for the large-scale biology challenges of modern cancer drug discovery.

  16. Planar Cell Polarity Breaks the Symmetry of PAR Protein Distribution prior to Mitosis in Drosophila Sensory Organ Precursor Cells.

    Science.gov (United States)

    Besson, Charlotte; Bernard, Fred; Corson, Francis; Rouault, Hervé; Reynaud, Elodie; Keder, Alyona; Mazouni, Khalil; Schweisguth, François

    2015-04-20

    During development, cell-fate diversity can result from the unequal segregation of fate determinants at mitosis. Polarization of the mother cell is essential for asymmetric cell division (ACD). It often involves the formation of a cortical domain containing the PAR complex proteins Par3, Par6, and atypical protein kinase C (aPKC). In the fly notum, sensory organ precursor cells (SOPs) divide asymmetrically within the plane of the epithelium and along the body axis to generate two distinct cells. Fate asymmetry depends on the asymmetric localization of the PAR complex. In the absence of planar cell polarity (PCP), SOPs divide with a random planar orientation but still asymmetrically, showing that PCP is dispensable for PAR asymmetry at mitosis. To study when and how the PAR complex localizes asymmetrically, we have used a quantitative imaging approach to measure the planar polarization of the proteins Bazooka (Baz, fly Par3), Par6, and aPKC in living pupae. By using imaging of functional GFP-tagged proteins with image processing and computational modeling, we find that Baz, Par6, and aPKC become planar polarized prior to mitosis in a manner independent of the AuroraA kinase and that PCP is required for the planar polarization of Baz, Par6, and aPKC during interphase. This indicates that a "mitosis rescue" mechanism establishes asymmetry at mitosis in PCP mutants. This study therefore identifies PCP as the initial symmetry-breaking signal for the planar polarization of PAR proteins in asymmetrically dividing SOPs.

  17. PCTAIRE1 phosphorylates p27 and regulates mitosis in cancer cells.

    Science.gov (United States)

    Yanagi, Teruki; Krajewska, Maryla; Matsuzawa, Shu-ichi; Reed, John C

    2014-10-15

    PCTAIRE1 is distant relative of the cyclin-dependent kinase family that has been implicated in spermatogenesis and neuronal development, but it has not been studied in cancer. Here, we report that PCTAIRE1 is expressed in prostate, breast, and cervical cancer cells, where its RNAi-mediated silencing causes growth inhibition with aberrant mitosis due to defects in centrosome dynamics. PCTAIRE1 was not similarly involved in proliferation of nontransformed cells, including diploid human IMR-90 fibroblasts. Through yeast two-hybrid screening, we identified tumor suppressor p27 as a PCTAIRE1 interactor. In vitro kinase assays showed PCTAIRE1 phosphorylates p27 at Ser10. PCTAIRE1 silencing modulated Ser10 phosphorylation on p27 and led to its accumulation in cancer cells but not in nontransformed cells. In a mouse xenograft model of PPC1 prostate cancer, conditional silencing of PCTAIRE1 restored p27 protein expression and suppressed tumor growth. Mechanistic studies in HeLa cells showed that PCTAIRE1 phosphorylates p27 during the S and M phases of the cell cycle. Notably, p27 silencing was sufficient to rescue cells from mitotic arrest caused by PCTAIRE1 silencing. Clinically, PCTAIRE1 was highly expressed in primary breast and prostate tumors compared with adjacent normal epithelial tissues. Together our findings reveal an unexpected role for PCTAIRE1 in regulating p27 stability, mitosis, and tumor growth, suggesting PCTAIRE1 as a candidate cancer therapeutic target.

  18. Cholesterol is essential for mitosis progression and its deficiency induces polyploid cell formation.

    Science.gov (United States)

    Fernández, Carlos; Lobo Md, María del Val T; Gómez-Coronado, Diego; Lasunción, Miguel A

    2004-10-15

    As an essential component of mammalian cell membranes, cells require cholesterol for proliferation, which is either obtained from plasma lipoproteins or synthesized intracellularly from acetyl-CoA. In addition to cholesterol, other non-sterol mevalonate derivatives are necessary for DNA synthesis, such as the phosphorylated forms of isopentane, farnesol, geranylgeraniol, and dolichol. The aim of the present study was to elucidate the role of cholesterol in mitosis. For this, human leukemia cells (HL-60) were incubated in a cholesterol-free medium and treated with SKF 104976, which inhibits cholesterol biosynthesis by blocking sterol 14alpha-demethylase, and the expression of relevant cyclins in the different phases of the cell cycle was analyzed by flow cytometry. Prolonged cholesterol starvation induced the inhibition of cytokinesis and the formation of polyploid cells, which were multinucleated and had mitotic aberrations. Supplementing the medium with cholesterol completely abolished these effects, demonstrating they were specifically due to cholesterol deficiency. This is the first evidence that cholesterol is essential for mitosis completion and that, in the absence of cholesterol, the cells fail to undergo cytokinesis, entered G1 phase at higher DNA ploidy (tetraploidy), and then progressed through S (rereplication) into G2, generating polyploid cells.

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

    Science.gov (United States)

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

    2015-11-23

    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.

  20. Comparative analysis of mitosis-specific antibodies for bulk purification of mitotic populations by fluorescence-activated cell sorting.

    Science.gov (United States)

    Campbell, Amy E; Hsiung, Chris C-S; Blobel, Gerd A

    2014-01-01

    Mitosis entails complex chromatin changes that have garnered increasing interest from biologists who study genome structure and regulation-fields that are being advanced by high-throughput sequencing (Seq) technologies. The application of these technologies to study the mitotic genome requires large numbers of highly pure mitotic cells, with minimal contamination from interphase cells, to ensure accurate measurement of phenomena specific to mitosis. Here, we optimized a fluorescence-activated cell sorting (FACS)-based method for isolating formaldehyde-fixed mitotic cells--at virtually 100% mitotic purity and in quantities sufficient for high-throughput genomic studies. We compared several commercially available antibodies that react with mitosis-specific epitopes over a range of concentrations and cell numbers, finding antibody MPM2 to be the most robust and cost-effective.

  1. Chk1 prevents abnormal mitosis of S-phase HeLa cells containing DNA damage

    Institute of Scientific and Technical Information of China (English)

    LI XiaoFang; WARD Tarsha; YAO XueBiao; WU JiaRui

    2009-01-01

    To explore effects of DNA damage on cell-cycle progression in p53-deficient tumor cells,synchronized HeLa cells at G1,S and G2/M phases were treated with methyl methanesulfnate (MMS).The results showed that the MMS treatment resulted in the cell-cycle arrest or delay in all 3 phases,while the S-phase cells were the most sensitive to MMS.Further studies demonstrated that ATM-Chk2 and p38 MAPK signaling pathways were activated in all 3 phases when the cells were treated with MMS;whereas Chk1 was activated only in S phase under the drug treatment,indicating that Chk1 specifically participated in S-phase checkpoints.To analyze the role of Chk1 in S-phase checkpoints,we administered a specific Chk1 inhibitor,UCN-01,to the S-phase cells.The results showed that the S-phase cells treated with MMS+UCN-01 could enter aberrant mitosis without finishing DNA replication,indicating that Chk1 mainly functions in the DNA damage checkpoint rather than in the replication checkpoint.In addition,MMS treatment alone inhibited the accumulation of cyclin B1,a key component of M-phase CDK-cyclin complex,in the S-phase cells,whereas the inhibition of Chk1 activation resulted in the accumulation of cyclin B1 in the MMS-treated S-phase cells.This observation further supports the view that DNA-damaged S-phase cells enter abnormal mitosis when Chk1 activation is inhibited.Our results demonstrate that Chk1 is a specific kinase that plays an important role in the MMS-induced S-phase DNA damage checkpoint.As p53 is not involved in this process,Chk1 may be a potential target for p53-deficient tumor therapy.

  2. Movie Mitosis

    Science.gov (United States)

    Bogiages, Christopher; Hitt, Austin M.

    2008-01-01

    Mitosis and meiosis are essential for the growth, development, and reproduction of organisms. Because these processes are essential to life, both are emphasized in biology texts, state standards, and the National Science Education Standards. In this article, the authors present their methodology for teaching mitosis by having students produce…

  3. The MAP kinase pathway is required for entry into mitosis and cell survival.

    Science.gov (United States)

    Liu, Xiaoqi; Yan, Shi; Zhou, Tianhua; Terada, Yasuhiko; Erikson, Raymond L

    2004-01-22

    In this communication, we examined the role of the MAP kinase pathway in the G2/M phase of the cell cycle. Activation of the Plk1 and MAP kinase pathways was initially evaluated in FT210 cells, which arrest at G2 phase at the restrictive temperature (39 degrees C), due to a mutation in the cdc2 gene. Previous studies had shown that these cells enter mitosis at the nonpermissive temperature upon incubation with okadaic acid, a protein phosphatase 1 and 2A inhibitor. We show that treatment of FT210 cells at 39 degrees C with okadaic acid activated Plk1, as shown by hyperphosphorylation and elevated protein kinase activity, and also induced activation of the MAP kinase pathway. The specific Mek inhibitor PD98059 antagonized the okadaic acid-induced activation of both Plk1 and MAP kinases. This suggests that activation of the MAP kinase pathway may contribute to the okadaic acid-induced activation of Plk1 in FT210 cells at 39 degrees C. We also found that PD98059 strongly attenuated progression of HeLa cells through mitosis, and active Mek colocalizes with Plk1 at mitotic structures. To study the potential function of the MAP kinase pathway during mitosis, RNAi was used to specifically deplete five members of this pathway (Raf1, Mek1/2, Erk1/2). Each of these five protein kinases is required for cell proliferation and survival, and depletion of any of these proteins eventually leads to apoptosis. Treatment with Mek inhibitors also inhibited cell proliferation and caused apoptosis. A dramatic increase of Plk1 activities and a moderate increase of Cdc2 activities in Raf1-depleted cells indicate that Raf1-depleted cells arrest in the late G2 or M phase. Mek1 and Erk1 depletion also caused cell cycle arrest at G2, suggesting that these enzymes are required for the G2/M transition, whereas the loss of Mek2 or Erk2 caused arrest at G1.

  4. The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis

    DEFF Research Database (Denmark)

    Yde, C W; Olsen, B B; Meek, D

    2008-01-01

    Cell-cycle transition from the G(2) phase into mitosis is regulated by the cyclin-dependent protein kinase 1 (CDK1) in complex with cyclin B. CDK1 activity is controlled by both inhibitory phosphorylation, catalysed by the Myt1 and Wee1 kinases, and activating dephosphorylation, mediated by the CDC...... interference results in delayed cell-cycle progression at the onset of mitosis. Knockdown of CK2beta causes stabilization of Wee1 and increased phosphorylation of CDK1 at the inhibitory Tyr15. PLK1-Wee1 association is an essential event in the degradation of Wee1 in unperturbed cell cycle. We have found...... regulatory subunit, identifying it as a new component of signaling pathways that regulate cell-cycle progression at the entry of mitosis.Oncogene advance online publication, 12 May 2008; doi:10.1038/onc.2008.146....

  5. Mitosis and its regulation

    Directory of Open Access Journals (Sweden)

    Frías Vázquez Sara

    2014-07-01

    Full Text Available Cell division by mitosis is essential for the development of organisms and their reproduction; it is also neces- sary that each new cell is genetically identical to that from which it comes. In eukaryotes this is achieved by the presence of complex mechanisms that ensure the integrity of genomic material and their proper segregation during mitosis. The traditional view of mitosis has been divided into different stages that were characterized by morphological studies in dividing cells; advances in molecular biology have led beyond this characterization, so that we now know a range of participant molecules. This article will discuss the process of mitosis, both at the cellular and molecular level and a brief summary of the molecular players that regulate this process.

  6. CDy6, a photostable probe for long-term real-time visualization of mitosis and proliferating cells.

    Science.gov (United States)

    Jeong, Yun-Mi; Duanting, Zhai; Hennig, Holger; Samanta, Animesh; Agrawalla, Bikram Keshari; Bray, Mark-Anthony; Carpenter, Anne E; Chang, Young-Tae

    2015-02-19

    Long-term real-time visualization of lysosomal dynamics has been challenging at the onset of mitosis due to the lack of fluorescent probes enabling convenient imaging of dividing cells. We developed a long-term real-time photostable mitotic or proliferating marker, CDy6, a BODIPY-derived compound of designation yellow 6, which labels lysosome. In long-term real-time, CDy6 displayed a sharp increase in intensity and change in localization in mitosis, improved photostability, and decreased toxicity compared with other widely used lysosomal and DNA markers, and the ability to label cells in mouse xenograft models. Therefore, CDy6 may open new possibilities to target and trace lysosomal contents during mitosis and to monitor cell proliferation, which can further our knowledge of the basic underlying biological mechanisms in the management of cancer.

  7. TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells

    DEFF Research Database (Denmark)

    Pedersen, Rune Troelsgaard; Kruse, Thomas; Nilsson, Jakob

    2015-01-01

    mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise...... temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin...

  8. Unraveling the biomolecular snapshots of mitosis in healthy and cancer cells using plasmonically-enhanced Raman spectroscopy.

    Science.gov (United States)

    Panikkanvalappil, Sajanlal R; Hira, Steven M; Mahmoud, Mahmoud A; El-Sayed, Mostafa A

    2014-11-12

    Owing to the dynamic and complex nature of mitosis, precise and timely executions of biomolecular events are critical for high fidelity cell division. In this context, visualization of such complex events at the molecular level can provide vital information on the biomolecular processes in abnormal cells. Here, we explored the plasmonically enhanced light scattering properties of functionalized gold nanocubes (AuNCs) together with surface-enhanced Raman spectroscopy (SERS) to unravel the complex and dynamic biological processes involved in mitosis of healthy and cancerous cells from its molecular perspectives. By monitoring various stages of mitosis using SERS, we noticed that relatively high rate of conversion of mitotic proteins from their α-helix structure to β-sheet conformation is likely in the cancer cells during meta-, ana-, and telophases. Unique biochemical modifications to the lipid and amino acid moieties, associated with the observed protein conformational modifications, were also identified. However, in healthy cells, the existence of proteins in their β conformation was momentary and was largely in the α-helix form. The role of abnormal conformational modifications of mitotic proteins on the development of anomalous mitotic activities was further confirmed by looking at plasmonic nanoparticle-induced cytokinesis failure in cancer cells. Our findings illustrate the vast possibilities of SERS in real-time tracking of complex, subtle, and momentary modifications of biomolecules in live cells, which could provide new insights to the role of protein conformation dynamics during mitosis on the development of cancer and many other diseases.

  9. Cdc42 is not essential for filopodium formation, directed migration, cell polarization, and mitosis in fibroblastoid cells

    DEFF Research Database (Denmark)

    Czuchra, Aleksandra; Wu, Xunwei; Meyer, Hannelore

    2005-01-01

    apparatus into the direction of migration was decreased. However, expression of dominant negative Cdc42 in Cdc42-null cells resulted in strongly reduced directed migration, severely reduced single cell directionality, and complete loss of Golgi polarization and of directionality of protrusion formation...... of Cdc42 did not affect filopodium or lamellipodium formation and had no significant influence on the speed of directed migration nor on mitosis. Cdc42-deficient cells displayed a more elongated cell shape and had a reduced area. Furthermore, directionality during migration and reorientation of the Golgi...

  10. Control of Outer Radial Glial Stem Cell Mitosis in the Human Brain

    Directory of Open Access Journals (Sweden)

    Bridget E.L. Ostrem

    2014-08-01

    Full Text Available Evolutionary expansion of the human neocortex is partially attributed to a relative abundance of neural stem cells in the fetal brain called outer radial glia (oRG. oRG cells display a characteristic division mode, mitotic somal translocation (MST, in which the soma rapidly translocates toward the cortical plate immediately prior to cytokinesis. MST may be essential for progenitor zone expansion, but the mechanism of MST is unknown, hindering exploration of its function in development and disease. Here, we show that MST requires activation of the Rho effector ROCK and nonmuscle myosin II, but not intact microtubules, centrosomal translocation into the leading process, or calcium influx. MST is independent of mitosis and distinct from interkinetic nuclear migration and saltatory migration. Our findings suggest that disrupted MST may underlie neurodevelopmental diseases affecting the Rho-ROCK-myosin pathway and provide a foundation for future exploration of the role of MST in neocortical development, evolution, and disease.

  11. Automatic detection of cell divisions (mitosis) in live-imaging microscopy images using Convolutional Neural Networks.

    Science.gov (United States)

    Shkolyar, Anat; Gefen, Amit; Benayahu, Dafna; Greenspan, Hayit

    2015-08-01

    We propose a semi-automated pipeline for the detection of possible cell divisions in live-imaging microscopy and the classification of these mitosis candidates using a Convolutional Neural Network (CNN). We use time-lapse images of NIH3T3 scratch assay cultures, extract patches around bright candidate regions that then undergo segmentation and binarization, followed by a classification of the binary patches into either containing or not containing cell division. The classification is performed by training a Convolutional Neural Network on a specially constructed database. We show strong results of AUC = 0.91 and F-score = 0.89, competitive with state-of-the-art methods in this field.

  12. Mitosis and microtubule organizational changes in rice root-tip cells

    Institute of Scientific and Technical Information of China (English)

    XUSHIXIONG(SYZEE); CHUNGUILI; CHENGZHU

    1993-01-01

    The pattern of change of the microtubule cytoskeleton of the root-tip cells of rice during mitosis was studied using immunofluorescence technic and confocal laser scanning microscopy. All the major stages of ceil division including preprophase, prophase, metaphase, anaphase and telophase were observed. The most significant finding was that in the preprophase cells microtubules radiating from the nuclear surface to the cortex were frequently seen. During development these microtubules became closely associated with the preprophase band and prophase spindie indicating that the microtubules radiating from the nuclear surface, the preprophase band and the prophazc spindle were structurally and functionally closely related to each other. Granule-like anchorage sites for the radiating microtubules at the muclear surface were often seen and the possibility that these gramle-like anchorage sites might represent the microtubule organizing centres was discussed.

  13. Developmental Control of Cell-Cycle Compensation Provides a Switch for Patterned Mitosis at the Onset of Chordate Neurulation.

    Science.gov (United States)

    Ogura, Yosuke; Sasakura, Yasunori

    2016-04-18

    During neurulation of chordate ascidians, the 11th mitotic division within the epidermal layer shows a posterior-to-anterior wave that is precisely coordinated with the unidirectional progression of the morphogenetic movement. Here we show that the first sign of this patterned mitosis is an asynchronous anterior-to-posterior S-phase length and that mitotic synchrony is reestablished by a compensatory asynchronous G2-phase length. Live imaging combined with genetic experiments demonstrated that compensatory G2-phase regulation requires transcriptional activation of the G2/M regulator cdc25 by the patterning genes GATA and AP-2. The downregulation of GATA and AP-2 at the onset of neurulation leads to loss of compensatory G2-phase regulation and promotes the transition to patterned mitosis. We propose that such developmentally regulated cell-cycle compensation provides an abrupt switch to spatially patterned mitosis in order to achieve the coordination between mitotic timing and morphogenesis.

  14. Trichomonas vaginalis: chromatin and mitotic spindle during mitosis.

    Science.gov (United States)

    Gómez-Conde, E; Mena-López, R; Hernández-Jaúregui, P; González-Camacho, M; Arroyo, R

    2000-11-01

    The mitotic phases and the changes that the chromatin and mitotic microtubules undergo during mitosis in the sexually transmitted parasite Trichomonas vaginalis are described. Parasites arrested in the gap 2 phase of the cell cycle by nutrient starvation were induced to mitosis by addition of fresh whole medium. [(3)H] Thymidine labeling of trichomonad parasites for 24 h showed that parasites have at least four synchronic duplications after mitosis induction. Fixed or live and acridine orange (AO)-stained trichomonads analyzed at different times during mitosis by epifluorescence microscopy showed that mitosis took about 45 min and is divided into five stages: prophase, metaphase, early and late anaphase, early and late telophase, and cytokinesis. The AO-stained nucleus of live trichomonads showed green (DNA) and orange (RNA) fluorescence, and the nucleic acid nature was confirmed by DNase and RNase treatment, respectively. The chromatin appeared partially condensed during interphase. At metaphase, it appeared as six condensed chromosomes, as recently reported, which decondensed at anaphase and migrated to the nuclear poles at telophase. In addition, small bundles of microtubules (as hemispindles) were detected only in metaphase with the polyclonal antibody anti-Entamoeba histolytica alpha-tubulin. This antibody showed that the hemispindle and an atractophore-like structure seem to duplicate and polarize during metaphase. In conclusion, T. vaginalis mitosis involves five mitotic phases in which the chromatin undergoes different degrees of condensation, from chromosomes to decondensed chromatin, and two hemispindles that are observed only in the metaphase stage.

  15. Association of Rictor with chromosomes during mitosis in MCF7 cells

    Directory of Open Access Journals (Sweden)

    Malanchuk O. M.

    2015-12-01

    Full Text Available The mTOR kinase forms two multicomponent protein complexes (mTORC1 and mTORC2, where protein Rictor is a unique component of mTORC2. These two complexes differ both structurally and functionally and transmit signals to distinct downstream substrates. In contrast to mTORC1, comparatively little is known about functioning and regulation of mTORC2 and its components. Aim. To analyze of the particularity of Rictor subcellular localization in MCF7 cells. Methods. Immunofluorescence assay using confocal microscopy was applied. Results. Analysis of Rictor localization with anti N-terminal mAb revealed a different pattern of immunostaining in cells that was depended on cell cycle phase. For the first time it was shown that Rictor is at the exposed edge of condensed chromatin pool in MCF7 cells. Furthermore Z-stacking of cells revealed that character of association depends on mitosis phases. Conclusions. Our novel findings add to our understanding of functioning of both Rictor and mTORC2 in cell cycle progression.

  16. A semi-Markov model for mitosis segmentation in time-lapse phase contrast microscopy image sequences of stem cell populations.

    Science.gov (United States)

    Liu, An-An; Li, Kang; Kanade, Takeo

    2012-02-01

    We propose a semi-Markov model trained in a max-margin learning framework for mitosis event segmentation in large-scale time-lapse phase contrast microscopy image sequences of stem cell populations. Our method consists of three steps. First, we apply a constrained optimization based microscopy image segmentation method that exploits phase contrast optics to extract candidate subsequences in the input image sequence that contains mitosis events. Then, we apply a max-margin hidden conditional random field (MM-HCRF) classifier learned from human-annotated mitotic and nonmitotic sequences to classify each candidate subsequence as a mitosis or not. Finally, a max-margin semi-Markov model (MM-SMM) trained on manually-segmented mitotic sequences is utilized to reinforce the mitosis classification results, and to further segment each mitosis into four predefined temporal stages. The proposed method outperforms the event-detection CRF model recently reported by Huh as well as several other competing methods in very challenging image sequences of multipolar-shaped C3H10T1/2 mesenchymal stem cells. For mitosis detection, an overall precision of 95.8% and a recall of 88.1% were achieved. For mitosis segmentation, the mean and standard deviation for the localization errors of the start and end points of all mitosis stages were well below 1 and 2 frames, respectively. In particular, an overall temporal location error of 0.73 ± 1.29 frames was achieved for locating daughter cell birth events.

  17. Dyskerin localizes to the mitotic apparatus and is required for orderly mitosis in human cells.

    Directory of Open Access Journals (Sweden)

    Faizan Alawi

    Full Text Available Dyskerin is a highly conserved, nucleolar RNA-binding protein with established roles in small nuclear ribonucleoprotein biogenesis, telomerase and telomere maintenance and precursor rRNA processing. Telomerase is functional during S phase and the bulk of rRNA maturation occurs during G1 and S phases; both processes are inactivated during mitosis. Yet, we show that during the course of cell cycle progression, human dyskerin expression peaks during G2/M in parallel with the upregulation of pro-mitotic factors. Dyskerin redistributed from the nucleolus in interphase cells to the perichromosomal region during prometaphase, metaphase and anaphase. With continued anaphase progression, dyskerin also localized to the cytoplasm within the mid-pole region. Loss of dyskerin function via siRNA-mediated depletion promoted G2/M accumulation and this was accompanied by an increased mitotic index and activation of the spindle assembly checkpoint. Live cell imaging further revealed an array of mitotic defects including delayed prometaphase progression, a significantly increased incidence of multi-polar spindles, and anaphase bridges culminating in micronucleus formation. Together, these findings suggest that dyskerin is a highly dynamic protein throughout the cell cycle and increases the repertoire of fundamental cellular processes that are disrupted by absence of its normal function.

  18. Dyskerin localizes to the mitotic apparatus and is required for orderly mitosis in human cells.

    Science.gov (United States)

    Alawi, Faizan; Lin, Ping

    2013-01-01

    Dyskerin is a highly conserved, nucleolar RNA-binding protein with established roles in small nuclear ribonucleoprotein biogenesis, telomerase and telomere maintenance and precursor rRNA processing. Telomerase is functional during S phase and the bulk of rRNA maturation occurs during G1 and S phases; both processes are inactivated during mitosis. Yet, we show that during the course of cell cycle progression, human dyskerin expression peaks during G2/M in parallel with the upregulation of pro-mitotic factors. Dyskerin redistributed from the nucleolus in interphase cells to the perichromosomal region during prometaphase, metaphase and anaphase. With continued anaphase progression, dyskerin also localized to the cytoplasm within the mid-pole region. Loss of dyskerin function via siRNA-mediated depletion promoted G2/M accumulation and this was accompanied by an increased mitotic index and activation of the spindle assembly checkpoint. Live cell imaging further revealed an array of mitotic defects including delayed prometaphase progression, a significantly increased incidence of multi-polar spindles, and anaphase bridges culminating in micronucleus formation. Together, these findings suggest that dyskerin is a highly dynamic protein throughout the cell cycle and increases the repertoire of fundamental cellular processes that are disrupted by absence of its normal function.

  19. Inhibitory effect of Polo-like kinase 1 depletion on mitosis and apoptosis of gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Xue-Hua Chen; Bin Lan; Ying Qu; Xiao-Qing Zhang; Qu Cai; Bing-Ya Liu; Zheng-Gang Zhu

    2006-01-01

    AIM: Polo-like kinase 1 (PLK1) serine/threonine kinase plays a vital role in multiple phases of mitosis in gastric cancer cells. To investigate the effect of PLK1 depletion on mitosis and apoptosis of gastric cancer cells.METHODS: PLK1 expression was blocked by small RNA interference(siRNA). The expression levels of PLK1, cdc2, cyclin B and caspase 3 were detected by Western blotting. Then, PLK1 depletion, cdc2 activity,cell proliferation, cell cycle phase distribution, mitotic spindle structure, and the rate of apoptosis of the PLK1knockdown cells were observed.RESULTS: PLK1 gene knockdown was associated with increased cyclin B expression, increased cdc2 activity (but not with the expression levels), accumulation of gastric cancer cells at G2/M, improper mitotic spindle formation,delayed chromosome separation and delayed or arrested cytokinesis. Moreover, PLK1 depletion in gastric cancer cells was associated with decreased proliferation,attenuated pro-caspase 3 levels and increased apoptosis.CONCLUSION: Blockage of PLK1 expression may lead to decreased mitosis or even apoptosis in gastric cancer cells, indicating that PLK1 may be a valuable therapeutic target for gastric cancer.

  20. Cancer: Mitosis Run Amok

    Science.gov (United States)

    Science Scope, 2005

    2005-01-01

    Virtually every student knows someone who has battled cancer. It is a topic that is of great interest to many students because of their personal connection to the subject. Mitosis is an important topic in a middle school unit on cells and cell processes (National Science Standards, Grades 5?8: Life Sciences: Content Standard C). Studying cancer…

  1. Mitosis in neurons: Roughex and APC/C maintain cell cycle exit to prevent cytokinetic and axonal defects in Drosophila photoreceptor neurons.

    Directory of Open Access Journals (Sweden)

    Robert Ruggiero

    Full Text Available The mechanisms of cell cycle exit by neurons remain poorly understood. Through genetic and developmental analysis of Drosophila eye development, we found that the cyclin-dependent kinase-inhibitor Roughex maintains G1 cell cycle exit during differentiation of the R8 class of photoreceptor neurons. The roughex mutant neurons re-enter the mitotic cell cycle and progress without executing cytokinesis, unlike non-neuronal cells in the roughex mutant that perform complete cell divisions. After mitosis, the binucleated R8 neurons usually transport one daughter nucleus away from the cell body into the developing axon towards the brain in a kinesin-dependent manner resembling anterograde axonal trafficking. Similar cell cycle and photoreceptor neuron defects occurred in mutants for components of the Anaphase Promoting Complex/Cyclosome. These findings indicate a neuron-specific defect in cytokinesis and demonstrate a critical role for mitotic cyclin downregulation both to maintain cell cycle exit during neuronal differentiation and to prevent axonal defects following failed cytokinesis.

  2. Mitosis in neurons: Roughex and APC/C maintain cell cycle exit to prevent cytokinetic and axonal defects in Drosophila photoreceptor neurons.

    Science.gov (United States)

    Ruggiero, Robert; Kale, Abhijit; Thomas, Barbara; Baker, Nicholas E

    2012-01-01

    The mechanisms of cell cycle exit by neurons remain poorly understood. Through genetic and developmental analysis of Drosophila eye development, we found that the cyclin-dependent kinase-inhibitor Roughex maintains G1 cell cycle exit during differentiation of the R8 class of photoreceptor neurons. The roughex mutant neurons re-enter the mitotic cell cycle and progress without executing cytokinesis, unlike non-neuronal cells in the roughex mutant that perform complete cell divisions. After mitosis, the binucleated R8 neurons usually transport one daughter nucleus away from the cell body into the developing axon towards the brain in a kinesin-dependent manner resembling anterograde axonal trafficking. Similar cell cycle and photoreceptor neuron defects occurred in mutants for components of the Anaphase Promoting Complex/Cyclosome. These findings indicate a neuron-specific defect in cytokinesis and demonstrate a critical role for mitotic cyclin downregulation both to maintain cell cycle exit during neuronal differentiation and to prevent axonal defects following failed cytokinesis.

  3. Mitosis is swell.

    Science.gov (United States)

    Zatulovskiy, Evgeny; Skotheim, Jan M

    2015-11-23

    Cell volume and dry mass are typically correlated. However, in this issue, Zlotek-Zlotkiewicz et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201505056) and Son et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201505058) use new live-cell techniques to show that entry to mitosis coincides with rapid cell swelling, which is reversed before division.

  4. TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells.

    Science.gov (United States)

    Pedersen, Rune Troelsgaard; Kruse, Thomas; Nilsson, Jakob; Oestergaard, Vibe H; Lisby, Michael

    2015-08-17

    Genome integrity is critically dependent on timely DNA replication and accurate chromosome segregation. Replication stress delays replication into G2/M, which in turn impairs proper chromosome segregation and inflicts DNA damage on the daughter cells. Here we show that TopBP1 forms foci upon mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin recruitment of SLX4 and by facilitating unscheduled DNA synthesis.

  5. Microtubule remodeling mediates the inhibition of store-operated calcium entry (SOCE) during mitosis in COS-7 cells.

    Science.gov (United States)

    Russa, Afadhali Denis; Ishikita, Naoyuki; Masu, Kazuki; Akutsu, Hitomi; Saino, Tomoyuki; Satoh, Yoh-ichi

    2008-12-01

    Regulation of the intracellular calcium ion concentration ([Ca(2+)](i)) is critical, because calcium signaling controls diverse and vital cellular processes such as secretion, proliferation, division, gene transcription, and apoptosis. Store-operated calcium entry (SOCE) is the main mechanism through which non-excitable cells replenish and thus maintain this delicate balance. There is limited evidence which indicates that SOCE may be inhibited during mitosis, and the mechanisms leading to the presumed inhibition has not been elucidated. In the present study, we examined and compared the [Ca(2+)](i) dynamics of COS-7 cells in mitotic and non-mitotic phases with special reference paid to SOCE. Laser scanning confocal microscopy to monitor [Ca(2+)](i) dynamics revealed that SOCE was progressively inhibited in mitosis and became virtually absent during the metaphase. We used various cytoskeletal modifying drugs and immunofluorescence to assess the contribution of microtubule and actin filaments in SOCE signaling. Nocodazole treatment caused microtubule reorganization and retraction from the cell periphery that mimicked the natural mitotic microtubule remodeling that was also accompanied by SOCE inhibition. Short exposure to paclitaxel, a microtubule-stabilizing drug, bolstered SOCE, whereas long exposure resulted in microtubule disruption and SOCE inhibition. Actin-modifying drugs did not affect SOCE. These findings indicate that mitotic microtubule remodeling plays a significant role in the inhibition of SOCE during mitosis.

  6. EFFECTS OF p53 OVEREXPRESSION ON NEOPLASTIC CELL MITOSIS AND APOPTOSIS IN NASOPHARYNGEAL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To investigate the p53 overexpression and its correlation withneoplastic cell mitosis and apoptosis in 43 nasopharyngeal carcinomas (NPCs). Methods: Forty-three pretreated NPC biopsy samples were randomly collected in the year 1997 for this study. p53 overexpression was detected by LSAB immunohistochemistry using DO-7 primary antibody. Mitotic figures were counted on H&E stained slides, and apoptotic cells on TUNEL-stained slides by use of in-situ cell death detection kit. Both of mitotic and apoptotic cells were quantitated by cell numbers per one high power field (5′ 40) averagely in terms of mitotic index (MI) and TUNEL index (TI), respectively. To compare the mean MIs of two groups categorized by different percentages of positive p53 positive cells found in NPC specimens was taken for the purpose of designating the criterion of p53 overexpression. And then, the correlation of p53 overexpression with MI and TI was made by statistical analysis. Results: Because statistically significant difference appeared at the criterion of 20%, the p53 overexpression of NPC was defined as≥20% of positive cells found. The p53 overexpression thus could be detected in 37 out of 43 NPCs, reaching 86.05% (37/43). The mean MI (1.87± 1.78/HPF) of 37 NPCs with p53 overexpression was significantly higher than that (0.76± 0.63/HPF) of 6 NPCs without p53 overexpression, the P value being <0.05. However, there was no statistical difference between the mean TI (24.50± 26.66HPF) of 37 NPCs with p53 overexpression and TI (23.17± 25.30/HPF) of 6 NPCs without p53 overexpression. Conclusions: p53 overexpression of NPC could be designated by ≥20% of positive neoplastic cells found in pretreated NPC specimens, and the rate of which reached 86.05% (37/43). The overexpressed p53 could enhance cell proliferative activity in pretreated NPCs represented by increasing of MI, but showed no effect on neoplastic cell apoptosis.

  7. [The molecular organizational characteristics of the cell nucleus components at different phases of the mitotic cycle and in the resting state].

    Science.gov (United States)

    S'iakste, N I

    1992-01-01

    Data about the changes of the cell nucleus structure at different levels of its organization are summarized in the review. The data about the change of the DNA break number during the cycle and in resting state are presented and the role of the changes of the repair efficiency in this process is discussed. The changes of the chromatin protein spectrum, the chromatin structure at nucleosomal and supranucleosomal levels, the DNA superhelicity, topoisomerase activity, nuclear matrix composition and structure are discussed as well. The nucleus structure during the S-phase and mitosis and the cycle-related changes of the chromatin structure in lower eukaryotes are reviewed separately.

  8. Genotoxic Anti-Cancer Agents and Their Relationship to DNA Damage, Mitosis, and Checkpoint Adaptation in Proliferating Cancer Cells

    Directory of Open Access Journals (Sweden)

    Lucy H. Swift

    2014-02-01

    Full Text Available When a human cell detects damaged DNA, it initiates the DNA damage response (DDR that permits it to repair the damage and avoid transmitting it to daughter cells. Despite this response, changes to the genome occur and some cells, such as proliferating cancer cells, are prone to genome instability. The cellular processes that lead to genomic changes after a genotoxic event are not well understood. Our research focuses on the relationship between genotoxic cancer drugs and checkpoint adaptation, which is the process of mitosis with damaged DNA. We examine the types of DNA damage induced by widely used cancer drugs and describe their effects upon proliferating cancer cells. There is evidence that cell death caused by genotoxic cancer drugs in some cases includes exiting a DNA damage cell cycle arrest and entry into mitosis. Furthermore, some cells are able to survive this process at a time when the genome is most susceptible to change or rearrangement. Checkpoint adaptation is poorly characterised in human cells; we predict that increasing our understanding of this pathway may help to understand genomic instability in cancer cells and provide insight into methods to improve the efficacy of current cancer therapies.

  9. Sp1 phosphorylation by cyclin-dependent kinase 1/cyclin B1 represses its DNA-binding activity during mitosis in cancer cells.

    Science.gov (United States)

    Chuang, J-Y; Wang, S-A; Yang, W-B; Yang, H-C; Hung, C-Y; Su, T-P; Chang, W-C; Hung, J-J

    2012-11-22

    Sp1 is important for the transcription of many genes. Our previous studies have shown that Sp1 is degraded in normal cell, but it is preserved in cancer cells during mitosis and exists a priori in the daughter cells, ready to engage in gene transcription and thereby contributes to the proliferation and survival of cancer cells. The mechanism by which Sp1 is preserved in cancer cells during mitosis remains unknown. In this study, we observed that Sp1 strongly colocalized with cyclin-dependent kinase 1 (CDK1)/cyclin B1 during mitosis. Moreover, we showed that Sp1 is a novel mitotic substrate of CDK1/cyclin B1 and is phosphorylated by it at Thr 739 before the onset of mitosis. Phospho-Sp1 reduced its DNA-binding ability and facilitated the chromatin condensation process during mitosis. Mutation of Thr739 to alanine resulted in Sp1 remaining in the chromosomes, delayed cell-cycle progression, and eventually led to apoptosis. Screening of Sp1-associated proteins during mitosis by using liquid chromatography/mass spectrometry indicated the tethering of Sp1 to myosin/F-actin. Furthermore, phospho-Sp1 and myosin/F-actin appeared to exist as a congregated ring at the periphery of the chromosome. However, at the end of mitosis and the beginning of interphase, Sp1 was dephosphorylated by PP2A and returned to the chromatin. These results indicate that cancer cells use CDK1 and PP2A to regulate the movement of Sp1 in and out of the chromosomes during cell-cycle progression, which may benefit cancer-cell proliferation.

  10. Nonthermal Fluctuations and Mechanics of the Active Cell Nucleus

    CERN Document Server

    Smith, K; Byrd, H; MacKintosh, F C; Kilfoil, M L

    2013-01-01

    We present direct measurements of fluctuations in the nucleus of yeast cells. While prior work has shown these fluctuations to be active and non-thermal in character, their origin and time dependence are not understood. We show that nuclear fluctuations can be quantitatively understood by uncorrelated, active force fluctuations driving a nuclear medium that is dominated by an uncondensed DNA solution, for which we perform rheological measurements on an in vitro model system under similar conditions to what is expected in the nucleus. We conclude that the eukaryotic nucleus of living cells is a nonequilibrium soft material whose fluctuations are actively driven, and are far from thermal in their time dependence.

  11. NO-sensitive guanylyl cyclase beta1 subunit is peripherally associated to chromosomes during mitosis. Novel role in chromatin condensation and cell cycle progression.

    Science.gov (United States)

    Pifarré, Paula; Baltrons, María Antonia; Földi, Istvan; García, Agustina

    2009-01-01

    NO-sensitive guanylyl cyclase (GC(NO)), the major NO target, is involved in important regulatory functions in the cardiovascular, gastrointestinal and central nervous systems. GC(NO) exists as heterodimers of alpha(1/2) and beta1 subunits. Deletion of the obligate beta1 dimerizing partner abrogates NO/cGMP signaling and shortens the life span of KO mice. Localization studies in the CNS have shown that beta1 is more widespread than alpha subunits and in some areas is the only GC(NO) subunit expressed, suggesting that beta1 may have GC(NO)-independent functions. GC(NO) is predominantly cytosolic, but association to membranes and other intracellular structures has been described. Here, we show localization of beta1 in cytoplasm and nucleus of cells expressing alpha subunits and GC(NO) activity (astrocytes, C6 cells), as well as in cells devoid of alpha subunits and GC(NO) activity (microglia). In both cell types beta1 associates peripherally to chromosomes in all phases of mitosis. Immunodepletion of beta1 in C6 cells enhances chromatin condensation in an in vitro assay. Moreover, silencing beta1 by siRNA induces cell cycle re-entry as determined by flow cytometry, and increases proliferation rate in a MTT-assay, whereas infection with beta1-containing adenovirus has the opposite effect. These actions are independent of cGMP formation. We postulate that beta1 is a multifunctional protein that regulates chromatin condensation and cell cycle progression, in addition to being an obligate monomer in functional GC(NO) heterodimers.

  12. Synthesis of microtubule-interfering halogenated noscapine analogs that perturb mitosis in cancer cells followed by cell death.

    Science.gov (United States)

    Aneja, Ritu; Vangapandu, Surya N; Lopus, Manu; Viswesarappa, Vijaya G; Dhiman, Neerupma; Verma, Akhilesh; Chandra, Ramesh; Panda, Dulal; Joshi, Harish C

    2006-08-14

    We have previously identified the naturally occurring non-toxic antitussive phthalideisoquinoline alkaloid, noscapine as a tubulin-binding agent that arrests mitosis and induces apoptosis. Here we present high-yield efficient synthetic methods and an evaluation of anticancer activity of halogenated noscapine analogs. Our results show that all analogs display higher tubulin-binding activity than noscapine and inhibit proliferation of human cancer cells (MCF-7, MDA-MB-231 and CEM). Surprisingly, the bromo-analog is approximately 40-fold more potent than noscapine in inhibiting cellular proliferation of MCF-7 cells. The ability of these analogs to inhibit cellular proliferation is mediated by cell cycle arrest at the G2/M phase, in that all analogs except 9-iodonoscapine, caused selective mitotic arrest with a higher efficiency than noscapine followed by apoptotic cell death as shown by immunofluorescence and quantitative FACS analyses. Furthermore, our results reveal the appearance of numerous fragmented nuclei as evidenced by DAPI staining. Thus, our data indicate a great potential of these compounds for studying microtubule-mediated processes and as chemotherapeutic agents for the management of human cancers.

  13. A FIBER APPARATUS IN THE NUCLEUS OF THE YEAST CELL

    Science.gov (United States)

    Robinow, C. F.; Marak, J.

    1966-01-01

    The structure and mode of division of the nucleus of budding yeast cells have been studied by phase-contrast microscopy during life and by ordinary microscopy after Helly fixation. The components of the nucleus were differentially stained by the Feulgen procedure, with Giemsa solution after hydrolysis, and with iron alum haematoxylin. New information was obtained in cells fixed in Helly's by directly staining them with 0.005% acid fuchsin in 1% acetic acid in water. Electron micrographs have been made of sections of cells that were first fixed with 3% glutaraldehyde, then divested of their walls with snail juice, and postfixed with osmium tetroxide. Light and electron microscopy have given concordant information about the organization of the yeast nucleus. A peripheral segment of the nucleus is occupied by relatively dense matter (the "peripheral cluster" of Mundkur) which is Feulgen negative. The greater part of the nucleus is filled with fine-grained Feulgen-positive matter of low density in which chromosomes could not be identified. Chromosomes become visible in this region under the light microscope at meiosis. In the chromatin lies a short fiber with strong affinity for acid fuchsin. The nucleus divides by elongation and constriction, and during this process the fiber becomes long and thin. Electron microscopy has resolved it into a bundle of dark-edged 150 to 180 A filaments which extends between "centriolar plaques" that are attached to the nuclear envelope. PMID:5331666

  14. Conversion from mitosis to meiosis: morphology and expression of proliferating cell nuclear antigen (PCNA) and Dmc1 during newt spermatogenesis.

    Science.gov (United States)

    Yazawa, T; Yamamoto, T; Nakayama, Y; Hamada, S; Abé, S

    2000-12-01

    The conversion from mitosis to meiosis is a phenomenon specific to the cellular progenitors of gametes; however, the mechanism or mechanisms responsible for this conversion are poorly understood. To this end, some morphological and molecular changes that occur during the initiation of meiosis in newt spermatogenesis are reported in the present paper. In situ morphologic studies revealed that spermatogonial stages comprise two phases: early mitotic generations (G1-G4) and late mitotic generations (G5-G8). Morphologic conversion from secondary spermatogonia to primary spermatocytes occurred during the intermediate stage of premeiotic DNA replication. The expression of proliferating cell nuclear antigen (PCNA), a DNA polymerase-delta auxiliary protein, in spermatogonia was weak in G1, highest during DNA synthesis (S), decreased in G2 and was not detectable in dividing cells. Complementary DNA for newt homologs of DMC1 (disrupted meiotic cDNA), which is an Escherichia coli RecA-like protein specifically active during meiosis, were isolated. The newt Dmc1 mRNA was first expressed significantly during the preleptotene stage and this continued into the spermatid stage. These observations present a basis for investigating the mechanism(s) controlling the conversion of newt spermatogonial cells from mitosis to meiosis.

  15. A vertebrate N-end rule degron reveals that Orc6 is required in mitosis for daughter cell abscission.

    Science.gov (United States)

    Bernal, Juan A; Venkitaraman, Ashok R

    2011-03-21

    Orc6, an evolutionarily conserved component of the origin recognition complex, is essential for deoxyribonucleic acid (DNA) replication initiation from yeast to humans. Whether vertebrate Orc6 has a mitotic function remains unresolved. In vertebrates, but not yeast, its depletion causes centrosome amplification and multinucleate division, but replication stress indirectly causes similar abnormalities. In this paper, we exploit Varshavsky's N-end rule to create a temperature-sensitive degron form of avian Orc6. Orc6 depletion during the S phase triggers centrosome amplification suppressed by G2 checkpoint inhibition, reflecting an indirect consequence of aberrant DNA replication. However, Orc6 depletion during mitosis suffices to cause asymmetric division and failure in cytokinesis, with a delay in daughter cell abscission revealed by a fluorescence-bleaching assay. A mutant lacking the C-terminal 25 residues cannot rescue these defects. Thus, vertebrate Orc6 is necessary during mitosis for the abscission stage of cytokinesis. Our findings exemplify N-end rule degrons as tools to unravel functions of a single protein during different phases of the vertebrate cell cycle.

  16. Chemical genetic inhibition of Mps1 in stable human cell lines reveals novel aspects of Mps1 function in mitosis.

    Directory of Open Access Journals (Sweden)

    Tale Sliedrecht

    Full Text Available BACKGROUND: Proper execution of chromosome segregation relies on tight control of attachment of chromosomes to spindle microtubules. This is monitored by the mitotic checkpoint that allows chromosome segregation only when all chromosomes are stably attached. Proper functioning of the attachment and checkpoint processes is thus important to prevent chromosomal instability. Both processes rely on the mitotic kinase Mps1. PRINCIPAL FINDING: We present here two cell lines in which endogenous Mps1 has been stably replaced with a mutant kinase (Mps1-as that is specifically inhibited by bulky PP1 analogs. Mps1 inhibition in these cell lines is highly penetrant and reversible. Timed inhibition during bipolar spindle assembly shows that Mps1 is critical for attachment error-correction and confirms its role in Aurora B regulation. We furthermore show that Mps1 has multiple controls over mitotic checkpoint activity. Mps1 inhibition precludes Mad1 localization to unattached kinetochores but also accelerates mitosis. This acceleration correlates with absence of detectable mitotic checkpoint complex after Mps1 inhibition. Finally, we show that short-term inhibition of Mps1 catalytic activity is sufficient to kill cells. CONCLUSIONS/SIGNIFICANCE: Mps1 is involved in the regulation of multiple key processes that ensure correct chromosome segregation and is a promising target for inhibition in anti-cancer strategies. We report here two cell lines that allow specific and highly penetrant inhibition of Mps1 in a reproducible manner through the use of chemical genetics. Using these cell lines we confirm previously suggested roles for Mps1 activity in mitosis, present evidence for novel functions and examine cell viability after short and prolonged Mps1 inhibition. These cell lines present the best cellular model system to date for investigations into Mps1 biology and the effects of penetrance and duration of Mps1 inhibition on cell viability.

  17. Immunogold electron microscopy and confocal analyses reveal distinctive patterns of histone H3 phosphorylation during mitosis in MCF-7 cells.

    Science.gov (United States)

    Yan, Yitang; Cummings, Connie A; Sutton, Deloris; Yu, Linda; Castro, Lysandra; Moore, Alicia B; Gao, Xiaohua; Dixon, Darlene

    2016-04-01

    Histone phosphorylation has a profound impact on epigenetic regulation of gene expression, chromosome condensation and segregation, and maintenance of genome integrity. Histone H3 Serine 10 is evolutionally conserved and heavily phosphorylated during mitosis. To examine Histone H3 Serine 10 phosphorylation (H3S10ph) dynamics in mitosis, we applied immunogold labeling and confocal microscopy to visualize H3S10ph expression in MCF-7 cells. Confocal observations showed that MCF-7 cells had abundant H3S10ph expression in prophase and metaphase. In anaphase, the H3S10ph expression was significantly decreased and displayed only sparsely localized staining that mainly associated with the chromatid tips. We showed that immunogold bead density distribution followed the H3S10ph expression patterns observed in confocal analysis. At a higher magnification in metaphase, the immunogold beads were readily visible and the bead distribution along the condensed chromosomes was distinctive, indicating the specificity and reliability of the immunogold staining procedure. In anaphase, the beads were found to distribute focally in specific regions of chromatids, reinforcing the confocal observations of differential H3 phosphorylation. To our knowledge, this is the first report to show the specific H3S10ph expression with an immunogold technique and transmission electron microscopy. Additionally, with confocal microscopy, we analyzed H3S10ph expression in an immortalized cell line derived from benign uterine smooth muscle tumor cells. H3S10ph epitope was expressed more abundantly during anaphase in the benign tumor cells, and there was no dramatic differential expression within the condensed chromatid clusters as observed in MCF-7 cells. The differences in H3S10ph expression pattern and dynamics may contribute to the differential proliferative potential between benign tumor cells and MCF-7 cells.

  18. The terminal basal mitosis of chicken retinal Lim1 horizontal cells is not sensitive to cisplatin-induced cell cycle arrest.

    Science.gov (United States)

    Shirazi Fard, Shahrzad; Thyselius, Malin; All-Ericsson, Charlotta; Hallböök, Finn

    2014-01-01

    For proper development, cells need to coordinate proliferation and cell cycle-exit. This is mediated by a cascade of proteins making sure that each phase of the cell cycle is controlled before the initiation of the next. Retinal progenitor cells divide during the process of interkinetic nuclear migration, where they undergo S-phase on the basal side, followed by mitoses on the apical side of the neuroepithelium. The final cell cycle of chicken retinal horizontal cells (HCs) is an exception to this general cell cycle behavior. Lim1 expressing (+) horizontal progenitor cells (HPCs) have a heterogenic final cell cycle, with some cells undergoing a terminal mitosis on the basal side of the retina. The results in this study show that this terminal basal mitosis of Lim1+ HPCs is not dependent on Chk1/2 for its regulation compared to retinal cells undergoing interkinetic nuclear migration. Neither activating nor blocking Chk1 had an effect on the basal mitosis of Lim1+ HPCs. Furthermore, the Lim1+ HPCs were not sensitive to cisplatin-induced DNA damage and were able to continue into mitosis in the presence of γ-H2AX without activation of caspase-3. However, Nutlin3a-induced expression of p21 did reduce the mitoses, suggesting the presence of a functional p53/p21 response in HPCs. In contrast, the apical mitoses were blocked upon activation of either Chk1/2 or p21, indicating the importance of these proteins during the process of interkinetic nuclear migration. Inhibiting Cdk1 blocked M-phase transition both for apical and basal mitoses. This confirmed that the cyclin B1-Cdk1 complex was active and functional during the basal mitosis of Lim1+ HPCs. The regulation of the final cell cycle of Lim1+ HPCs is of particular interest since it has been shown that the HCs are able to sustain persistent DNA damage, remain in the cell cycle for an extended period of time and, consequently, survive for months.

  19. Biosynthesis of copper nanoparticles and its effect on actively dividing cells of mitosis in Allium cepa.

    Science.gov (United States)

    Nagaonkar, Dipali; Shende, Sudhir; Rai, Mahendra

    2015-01-01

    Nanobiotechnological application of copper nanoparticles has paved the way for advancement in agriculture owing to its bactericidal and fungicidal activities. Recently, researchers have focussed on bioinspired synthesis of copper nanoparticles as a viable alternative to existing physicochemical techniques. For the commercialization of nanocopper, the toxicity evaluation is a major issue. In this context, Citrus medica (L.) fruit extract-mediated copper nanoparticles were synthesized and its different concentrations (10, 20, 40, 60, 80, and 100 µg mL(-1) ) were evaluated for its effect on actively dividing cells of Allium cepa. The study clearly revealed that copper nanoparticles increased mitotic index up to the concentration of 20 µg mL(-1) . In addition, a gradual decline in mitotic index and increase in abnormality index was observed as the concentration of copper nanoparticles and treatment duration were increased. Aberrations in chromosomal behavior such as sticky and disturbed chromosomes in metaphase and anaphase, c-metaphase, bridges, laggard, disturbed telophase, and vacuolated nucleus were also observed.

  20. Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial Glia

    Directory of Open Access Journals (Sweden)

    Marco Onorati

    2016-09-01

    Full Text Available The mechanisms underlying Zika virus (ZIKV-related microcephaly and other neurodevelopment defects remain poorly understood. Here, we describe the derivation and characterization, including single-cell RNA-seq, of neocortical and spinal cord neuroepithelial stem (NES cells to model early human neurodevelopment and ZIKV-related neuropathogenesis. By analyzing human NES cells, organotypic fetal brain slices, and a ZIKV-infected micrencephalic brain, we show that ZIKV infects both neocortical and spinal NES cells as well as their fetal homolog, radial glial cells (RGCs, causing disrupted mitoses, supernumerary centrosomes, structural disorganization, and cell death. ZIKV infection of NES cells and RGCs causes centrosomal depletion and mitochondrial sequestration of phospho-TBK1 during mitosis. We also found that nucleoside analogs inhibit ZIKV replication in NES cells, protecting them from ZIKV-induced pTBK1 relocalization and cell death. We established a model system of human neural stem cells to reveal cellular and molecular mechanisms underlying neurodevelopmental defects associated with ZIKV infection and its potential treatment.

  1. Effect of apelin on mitosis, apoptosis and DNA repair enzyme OGG 1/2 expression in intestinal cell lines IEC-6 and Caco-2.

    Science.gov (United States)

    Antushevich, Hanna; Krawczynska, Agata; Kapica, Malgorzata; Herman, Andrzej Przemyslaw; Zabielski, Romuald

    2014-01-01

    Apelin is a regulatory peptide, identified as an endogenous ligand of the Apelin receptor (APJ). Both the apelin and the APJ were detected in brain, lung, heart, mammary gland, kidney, placenta, adipose tissues and the gastrointestinal tract. Apelin is considered an important regulatory gut peptide with a potential physiological role in gastrointestinal cytoprotection, regulation of food intake and drinking behaviour. The aim of the present study was to assess the effect of the apelin on mitosis, apoptosis and the expression of DNA repair enzyme (OGG 1/2), and APJ receptor in intestinal cell lines: rat crypt (IEC-6) and human enterocyte model (Caco-2). The cell cultures were incubated with the apelin-12 (10-8 M) for 4, 6, 12, 24 and 48 h and the apoptosis (caspase 3), mitosis (Ki-67) and DNA repair enzyme (OGG1/2) markers were studied by Real-Time qRT-PCR and immunofluorescent methods. The results of Real-Time qRT-PCR and immunocytochemical analysis showed that the levels of mRNAs were inversely related to the expression level of corresponding proteins. Immunofluorescent studies revealed inhibitory effect of apelin-12 on apoptosis, mitosis and the expression of OGG1/2 in the intestinal crypt cell line IEC-6. However, in the enterocyte model Caco-2 cells apelin stimulated apoptosis and mitosis, and reduced OGG1/2 expression. These findings suggest that apelin may be involved in the control of epithelial cell turnover in the gastrointestinal tract.

  2. C. elegans RNA-binding proteins PUF-8 and MEX-3 function redundantly to promote germline stem cell mitosis.

    Science.gov (United States)

    Ariz, Mohd; Mainpal, Rana; Subramaniam, Kuppuswamy

    2009-02-15

    Maintenance of mitotically cycling germline stem cells (GSCs) is vital for continuous production of gametes. In worms and insects, signaling from surrounding somatic cells play an essential role in the maintenance of GSCs by preventing premature differentiation. In addition, germ cell proteins such as the Drosophila Pumilio and Caenorhabditis elegans FBF, both members of the PUF family translational regulators, contribute to GSC maintenance. FBF functions by suppressing GLD-1, which promotes meiotic entry. However, factors that directly promote GSC proliferation, rather than prevent differentiation, are not known. Here we show that PUF-8, another C. elegans member of the PUF family and MEX-3, a KH domain translational regulator, function redundantly to promote GSC mitosis. We find that PUF-8 protein is highly enriched in mitotic germ cells, which is similar to the expression pattern of MEX-3 described earlier. The puf-8(-) mex-3(-) double mutant gonads contain far fewer germ cells than both single mutants and wild-type. While these cells lack mitotic, meiotic and sperm markers, they retain the germ cell-specific P granules, and are capable of gametogenesis if GLP-1, which normally blocks meiotic entry, is removed. Significantly, we find that at least one of these two proteins is essential for germ cell proliferation even in meiotic entry-defective mutants, which otherwise produce germ cell tumors. We conclude PUF-8 and MEX-3 contribute to GSC maintenance by promoting mitotic proliferation rather than by blocking meiotic entry.

  3. The Biochemistry of Mitosis

    Science.gov (United States)

    Wieser, Samuel; Pines, Jonathon

    2015-01-01

    In this article, we will discuss the biochemistry of mitosis in eukaryotic cells. We will focus on conserved principles that, importantly, are adapted to the biology of the organism. It is vital to bear in mind that the structural requirements for division in a rapidly dividing syncytial Drosophila embryo, for example, are markedly different from those in a unicellular yeast cell. Nevertheless, division in both systems is driven by conserved modules of antagonistic protein kinases and phosphatases, underpinned by ubiquitin-mediated proteolysis, which create molecular switches to drive each stage of division forward. These conserved control modules combine with the self-organizing properties of the subcellular architecture to meet the specific needs of the cell. Our discussion will draw on discoveries in several model systems that have been important in the long history of research on mitosis, and we will try to point out those principles that appear to apply to all cells, compared with those in which the biochemistry has been specifically adapted in a particular organism. PMID:25663668

  4. Regulation of mitosis onset and thymidine kinase activity during the cell cycle of Physarum polycephalum plasmodia: effect of fluorodeoxyuridine.

    Science.gov (United States)

    Wright, M; Tollon, Y

    1989-05-01

    The effects of fluorodeoxyuridine were investigated during three events of the cell cycle: S-phase, mitosis, and the cyclic synthesis of thymidine kinase in the synchronous plasmodium of the myxomycete Physarum. DNA synthesis was inhibited, and there was limited action on other macromolecular syntheses. When DNA synthesis was slowed down, onset of the following increase of thymidine kinase synthesis occurred at approximately the same time as in the control, but mitosis was blocked in a very early prophase stage and metaphase was never observed. These effects were suppressed when the action of fluorodeoxyuridine was prevented by the addition of thymidine to the medium. In agreement with the action of aphidicolin and hydroxyurea, these observations show that: 1) perturbation of the S-phase does not prevent the nuclei from entering a very early prophase stage, but it does prevent them from proceeding through metaphase; 2) blockage of DNA synthesis does not perturb the normal timing of the triggering of thymidine kinase synthesis; and 3) the signal that triggers the arrest of thymidine kinase synthesis is postmitotic and does not require extensive DNA synthesis. In contrast with hydroxyurea and aphidicolin, in the presence of fluorodeoxyuridine metaphase was not observed. Thus, the triggering of thymidine kinase synthesis is unambiguously dissociated from metaphase and postmitotic events. Because synthesis of thymidine kinase remains under the control of temperature shifts from 22 to 32 degrees C, a simple model of the cell cycle involving two regulatory pathways could account for the triggering of thymidine kinase synthesis, early prophase stage, and metaphase.

  5. Long Term Aggresome Accumulation Leads to DNA Damage, p53-dependent Cell Cycle Arrest, and Steric Interference in Mitosis.

    Science.gov (United States)

    Lu, Meng; Boschetti, Chiara; Tunnacliffe, Alan

    2015-11-13

    Juxtanuclear aggresomes form in cells when levels of aggregation-prone proteins exceed the capacity of the proteasome to degrade them. It is widely believed that aggresomes have a protective function, sequestering potentially damaging aggregates until these can be removed by autophagy. However, most in-cell studies have been carried out over a few days at most, and there is little information on the long term effects of aggresomes. To examine these long term effects, we created inducible, single-copy cell lines that expressed aggregation-prone polyglutamine proteins over several months. We present evidence that, as perinuclear aggresomes accumulate, they are associated with abnormal nuclear morphology and DNA double-strand breaks, resulting in cell cycle arrest via the phosphorylated p53 (Ser-15)-dependent pathway. Further analysis reveals that aggresomes can have a detrimental effect on mitosis by steric interference with chromosome alignment, centrosome positioning, and spindle formation. The incidence of apoptosis also increased in aggresome-containing cells. These severe defects developed gradually after juxtanuclear aggresome formation and were not associated with small cytoplasmic aggregates alone. Thus, our findings demonstrate that, in dividing cells, aggresomes are detrimental over the long term, rather than protective. This suggests a novel mechanism for polyglutamine-associated developmental and cell biological abnormalities, particularly those with early onset and non-neuronal pathologies.

  6. Incorporation of mammalian actin into microfilaments in plant cell nucleus

    Directory of Open Access Journals (Sweden)

    Paves Heiti

    2004-04-01

    Full Text Available Abstract Background Actin is an ancient molecule that shows more than 90% amino acid homology between mammalian and plant actins. The regions of the actin molecule that are involved in F-actin assembly are largely conserved, and it is likely that mammalian actin is able to incorporate into microfilaments in plant cells but there is no experimental evidence until now. Results Visualization of microfilaments in onion bulb scale epidermis cells by different techniques revealed that rhodamine-phalloidin stained F-actin besides cytoplasm also in the nuclei whereas GFP-mouse talin hybrid protein did not enter the nuclei. Microinjection of fluorescently labeled actin was applied to study the presence of nuclear microfilaments in plant cells. Ratio imaging of injected fluorescent rabbit skeletal muscle actin and phalloidin staining of the microinjected cells showed that mammalian actin was able to incorporate into plant F-actin. The incorporation occurred preferentially in the nucleus and in the perinuclear region of plant cells whereas part of plant microfilaments, mostly in the periphery of cytoplasm, did not incorporate mammalian actin. Conclusions Microinjected mammalian actin is able to enter plant cell's nucleus, whereas incorporation of mammalian actin into plant F-actin occurs preferentially in the nucleus and perinuclear area.

  7. Omcg1 is critically required for mitosis in rapidly dividing mouse intestinal progenitors and embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Teddy Léguillier

    2012-05-01

    Recent studies have shown that factors involved in transcription-coupled mRNA processing are important for the maintenance of genome integrity. How these processes are linked and regulated in vivo remains largely unknown. In this study, we addressed in the mouse model the function of Omcg1, which has been shown to participate in co-transcriptional processes, including splicing and transcription-coupled repair. Using inducible mouse models, we found that Omcg1 is most critically required in intestinal progenitors. In absence of OMCG1, proliferating intestinal epithelial cells underwent abnormal mitosis followed by apoptotic cell death. As a consequence, the crypt proliferative compartment of the small intestine was quickly and totally abrogated leading to the rapid death of the mice. Lack of OMCG1 in embryonic stem cells led to a similar cellular phenotype, with multiple mitotic defects and rapid cell death. We showed that mutant intestinal progenitors and embryonic stem cells exhibited a reduced cell cycle arrest following irradiation, suggesting that mitotic defects may be consecutive to M phase entry with unrepaired DNA damages. These findings unravel a crucial role for pre-mRNA processing in the homeostasis of the small intestine and point to a major role of OMCG1 in the maintenance of genome integrity.

  8. Silencing of E2F3 suppresses tumor growth of Her2+ breast cancer cells by restricting mitosis.

    Science.gov (United States)

    Lee, Miyoung; Oprea-Ilies, Gabriela; Saavedra, Harold I

    2015-11-10

    The E2F transcriptional activators E2F1, E2F2 and E2F3a regulate many important cellular processes, including DNA replication, apoptosis and centrosome duplication. Previously, we demonstrated that silencing E2F1 or E2F3 suppresses centrosome amplification (CA) and chromosome instability (CIN) in Her2+ breast cancer cells without markedly altering proliferation. However, it is unknown whether and how silencing a single E2F activator, E2F3, affects malignancy of human breast cancer cells. Thus, we injected HCC1954 Her2+ breast cancer cells silenced for E2F3 into mammary fat pads of immunodeficient mice and demonstrated that loss of E2F3 retards tumor growth. Surprisingly, silencing of E2F3 led to significant reductions in mitotic indices relative to vector controls, while the percentage of cells undergoing S phase were not affected. Nek2 is a mitotic kinase commonly upregulated in breast cancers and a critical regulator of Cdk4- or E2F-mediated CA. In this report, we found that Nek2 overexpression rescued back the CA caused by silencing of shE2F3. However, the effects of Nek2 overexpression in affecting tumor growth rates of shE2F3 and shE2F3; GFP cells were inconclusive. Taken together, our results indicate that E2F3 silencing decreases mammary tumor growth by reducing percentage of cells undergoing mitosis.

  9. Dynamic distribution of Ser-10 phosphorylated histone H3 in cytoplasm of MCF-7 and CHO cells during mitosis

    Institute of Scientific and Technical Information of China (English)

    Deng Wen LI; Qin YANG; Jia Tong CHEN; Hao ZHOU; Ru Ming LIU; Xi Tai HUANG

    2005-01-01

    The dynamic distribution of phosphorylated Histone H3 on Ser10 (phospho-H3) in cells was investigated to determine its function during mitosis. Human breast adenocarcinoma cells MCF-7, and Chinese hamster cells CHO were analyzed by indirect immunofluorescence staining with an antibody against phospho-H3. We found that the phosphorylation begins at early prophase, and spreads throughout the chromosomes at late prophase. At metaphase, most of the phosphoH3 aggregates at the end of the condensed entity of chromosomes at equatorial plate. During anaphase and telophase,the fluorescent signal of phospho-H3 is detached from chromosomes into cytoplasm. At early anaphase, phospho-H3shows ladder bands between two sets of separated chromosome, and forms "sandwich-like structure" when the chromosomes condensed. With the cleavage progressing, the "ladders" of the histone contract into a bigger bright dot. Then the histone aggregates and some of compacted microtubules in the midbody region are composed into a "bar-like"complex to separate daughter cells. The daughter cells seal their plasma membrane along with the ends of the "bar",inside which locates microtubules and modified histones, to finish the cytokinesis and keep the "bar complex" out of the cells. The specific distribution and kinetics of phospho-H3 in cytoplasm suggest that the modified histones may take part in the formation of midbody and play a crucial role in cytokinesis.

  10. Expression of progesterone receptor membrane component-2 within the immature rat ovary and its role in regulating mitosis and apoptosis of spontaneously immortalized granulosa cells.

    Science.gov (United States)

    Griffin, Daniel; Liu, Xiufang; Pru, Cindy; Pru, James K; Peluso, John J

    2014-08-01

    Progesterone receptor membrane component 2 (Pgrmc2) mRNA was detected in the immature rat ovary. By 48 h after eCG, Pgrmc2 mRNA levels decreased by 40% and were maintained at 48 h post-hCG. Immunohistochemical studies detected PGRMC2 in oocytes and ovarian surface epithelial, interstitial, thecal, granulosa, and luteal cells. PGRMC2 was also present in spontaneously immortalized granulosa cells, localizing to the cytoplasm of interphase cells and apparently to the mitotic spindle of cells in metaphase. Interestingly, PGRMC2 levels appeared to decrease during the G1 stage of the cell cycle. Moreover, overexpression of PGRMC2 suppressed entry into the cell cycle, possibly by binding the p58 form of cyclin dependent kinase 11b. Conversely, Pgrmc2 small interfering RNA (siRNA) treatment increased the percentage of cells in G1 and M stage but did not increase the number of cells, which was likely due to an increase in apoptosis. Depleting PGRMC2 did not inhibit cellular (3)H-progesterone binding, but attenuated the ability of progesterone to suppress mitosis and apoptosis. Taken together these studies suggest that PGRMC2 affects granulosa cell mitosis by acting at two specific stages of the cell cycle. First, PGRMC2 regulates the progression from the G0 into the G1 stage of the cell cycle. Second, PGRMC2 appears to localize to the mitotic spindle, where it likely promotes the final stages of mitosis. Finally, siRNA knockdown studies indicate that PGRMC2 is required for progesterone to slow the rate of granulosa cell mitosis and apoptosis. These findings support a role for PGRMC2 in ovarian follicle development.

  11. How nuclei of Giardia pass through cell differentiation: semi-open mitosis followed by nuclear interconnection.

    Science.gov (United States)

    Jiráková, Klára; Kulda, Jaroslav; Nohýnková, Eva

    2012-05-01

    Differentiation into infectious cysts (encystation) and multiplication of pathogenic trophozoites after hatching from the cyst (excystation) are fundamental processes in the life cycle of the human intestinal parasite Giardia intestinalis. During encystation, a bi-nucleated trophozoite transforms to a dormant tetra-nucleated cyst enveloped by a protective cyst wall. Nuclear division during encystation is not followed by cytokinesis. In contrast to the well-studied mechanism of cyst wall formation, information on nuclei behavior is incomplete and basic cytological data are lacking. Here we present evidence that (1) the nuclei divide by semi-open mitosis during early encystment; (2) the daughter nuclei coming from different parent nuclei are always arranged in pairs; (3) in both pairs, the nuclei are interconnected via bridges formed by fusion of their nuclear envelopes; (4) each interconnected nuclear pair is associated with one basal body tetrad of the undivided diplomonad mastigont; and (5) the interconnection between nuclei persists through the cyst stage being a characteristic feature of encysted Giardia. Based on the presented results, a model of nuclei behavior during Giardia differentiation is proposed.

  12. Appearance of actin microfilament 'twin peaks' in mitosis and their function in cell plate formation, as visualized in tobacco BY-2 cells expressing GFP-fimbrin.

    Science.gov (United States)

    Sano, Toshio; Higaki, Takumi; Oda, Yoshihisa; Hayashi, Tomomi; Hasezawa, Seiichiro

    2005-11-01

    The actin cytoskeleton of higher plants plays an essential role in plant morphogenesis and in maintaining various cellular activities. In this study we established a tobacco BY-2 cell line, stably transformed with a GFP-fimbrin actin-binding domain (ABD) 2 construct, that allows visualization of actin microfilaments (MFs) in living cells. Using this cell line, designated BY-GF11, we performed time-sequential observations of MF dynamics during cell-cycle progression. Detailed analyses revealed the appearance of a broad MF band in the late G2 phase that separated to form a structure corresponding to the so-called actin-depleted zone (ADZ) in mitosis. In BY-GF11, the MF structure at the cell cortex in mitosis appeared to form two bands rather than the ADZ. Measurements of fluorescent intensities of the cell cortex indicated an MF distribution that resembled two peaks, and we therefore named the structure MF 'twin peaks' (MFTP). The cell plate formed exactly within the valley between the MFTP at cytokinesis, and this cell-plate guidance was distorted by disruption of the MFTP by an inhibitor of actin polymerization. These results suggest that the MFTP originates from the broad MF band in the G2 phase and functions as a marker of cytokinesis.

  13. Nucleus- and cell-specific gene expression in monkey thalamus.

    Science.gov (United States)

    Murray, Karl D; Choudary, Prabhakara V; Jones, Edward G

    2007-02-06

    Nuclei of the mammalian thalamus are aggregations of neurons with unique architectures and input-output connections, yet the molecular determinants of their organizational specificity remain unknown. By comparing expression profiles of thalamus and cerebral cortex in adult rhesus monkeys, we identified transcripts that are unique to dorsal thalamus or to individual nuclei within it. Real-time quantitative PCR and in situ hybridization analyses confirmed the findings. Expression profiling of individual nuclei microdissected from the dorsal thalamus revealed additional subsets of nucleus-specific genes. Functional annotation using Gene Ontology (GO) vocabulary and Ingenuity Pathways Analysis revealed overrepresentation of GO categories related to development, morphogenesis, cell-cell interactions, and extracellular matrix within the thalamus- and nucleus-specific genes, many involved in the Wnt signaling pathway. Examples included the transcription factor TCF7L2, localized exclusively to excitatory neurons; a calmodulin-binding protein PCP4; the bone extracellular matrix molecules SPP1 and SPARC; and other genes involved in axon outgrowth and cell matrix interactions. Other nucleus-specific genes such as CBLN1 are involved in synaptogenesis. The genes identified likely underlie nuclear specification, cell phenotype, and connectivity during development and their maintenance in the adult thalamus.

  14. The phosphorylation-dependent regulation of nuclear SREBP1 during mitosis links lipid metabolism and cell growth

    Science.gov (United States)

    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

  15. Model-based investigation of the circadian clock and cell cycle coupling in mouse embryonic fibroblasts: Prediction of RevErb-α up-regulation during mitosis.

    Science.gov (United States)

    Traynard, Pauline; Feillet, Céline; Soliman, Sylvain; Delaunay, Franck; Fages, François

    2016-11-01

    Experimental observations have put in evidence autonomous self-sustained circadian oscillators in most mammalian cells, and proved the existence of molecular links between the circadian clock and the cell cycle. Some mathematical models have also been built to assess conditions of control of the cell cycle by the circadian clock. However, recent studies in individual NIH3T3 fibroblasts have shown an unexpected acceleration of the circadian clock together with the cell cycle when the culture medium is enriched with growth factors, and the absence of such acceleration in confluent cells. In order to explain these observations, we study a possible entrainment of the circadian clock by the cell cycle through a regulation of clock genes around the mitosis phase. We develop a computational model and a formal specification of the observed behavior to investigate the conditions of entrainment in period and phase. We show that either the selective activation of RevErb-α or the selective inhibition of Bmal1 transcription during the mitosis phase, allow us to fit the experimental data on both period and phase, while a uniform inhibition of transcription during mitosis seems incompatible with the phase data. We conclude on the arguments favoring the RevErb-α up-regulation hypothesis and on some further predictions of the model.

  16. Novel subcellular localization of the DNA helicase Twinkle at the kinetochore complex during mitosis in neuronal-like progenitor cells.

    Science.gov (United States)

    Uittenbogaard, Martine; Chiaramello, Anne

    2016-03-01

    During mitosis, the kinetochore, a multi-protein structure located on the centromeric DNA, is responsible for proper segregation of the replicated genome. More specifically, the outer kinetochore complex component Ndc80/Hec1 plays a critical role in regulating microtubule attachment to the spindle for accurate sister chromatid segregation. In addition, DNA helicases play a key contribution for precise and complete disjunction of sister chromatids held together through double-stranded DNA catenations until anaphase. In this study, we focused our attention on the nuclear-encoded DNA helicase Twinkle, which functions as an essential helicase for replication of mitochondrial DNA. It regulates the copy number of the mitochondrial genome, while maintaining its integrity, two processes essential for mitochondrial biogenesis and bioenergetic functions. Although the majority of the Twinkle protein is imported into mitochondria, a small fraction remains cytosolic with an unknown function. In this study, we report a novel expression pattern of Twinkle during chromosomal segregation at distinct mitotic phases. By immunofluorescence microscopy, we found that Twinkle protein colocalizes with the outer kinetochore protein HEC1 as early as prophase until late anaphase in neuronal-like progenitor cells. Thus, our collective results have revealed an unexpected cell cycle-regulated expression pattern of the DNA helicase Twinkle, known for its role in mtDNA replication. Therefore, its recruitment to the kinetochore suggests an evolutionary conserved function for both mitochondrial and nuclear genomic inheritance.

  17. Calcium and Mitosis

    Science.gov (United States)

    Hepler, P.

    1983-01-01

    Although the mechanism of calcium regulation is not understood, there is evidence that calcium plays a role in mitosis. Experiments conducted show that: (1) the spindle apparatus contains a highly developed membrane system that has many characteristics of sarcoplasmic reticulum of muscle; (2) this membrane system contains calcium; and (3) there are ionic fluxes occurring during mitosis which can be seen by a variety of fluorescence probes. Whether the process of mitosis can be modulated by experimentally modulating calcium is discussed.

  18. Romancing mitosis and the mitotic apparatus.

    Science.gov (United States)

    Brinkley, William B R

    2014-11-01

    One of the earliest lessons students learn in biology is the process of mitosis and how cells divide to produce daughter cells. Although first described more than a century ago by early investigators such as E. B. Wilson, many aspects of mitosis and cell division remain the subject of considerable research today. My personal investigations and research contributions to the study of mitosis were made possible by recent developments in the field when I began my career, including access to novel mammalian cell culture models and electron and fluorescence microscopy. Building upon those innovations, my laboratory and other contemporary investigators first charted the ultrastructure and molecular organization of mitosis and chromosome movement and the assembly and function of the cytoskeleton. This field of research remains a significant challenge for future investigators in cell biology and medicine.

  19. Src activity increases and Yes activity decreases during mitosis of human colon carcinoma cells.

    OpenAIRE

    Park, J.; Cartwright, C A

    1995-01-01

    Src and Yes protein-tyrosine kinase activities are elevated in malignant and premalignant tumors of the colon. To determine whether Src activity is elevated throughout the human colon carcinoma cell cycle as it is in polyomavirus middle T antigen- or F527 Src-transformed cells, and whether Yes activity, which is lower than that of Src in the carcinoma cells, is regulated differently, we measured their activities in cycling cells. We observed that the activities of both kinases were higher thr...

  20. The mitotic spindle and associated membranes in the closed mitosis of trichomonads.

    Science.gov (United States)

    Ribeiro, Karla Consort; Pereira-Neves, Antonio; Benchimol, Marlene

    2002-06-01

    In the present work, we followed the several phases of Tritrichomonas foetus and Trichomonas vaginalis cell cycles using immunofluorescence, serial thin sections, three-dimensional (3D) reconstruction, and transmission electron microscopy. In motile trichomonad cells or in pseudocyst forms, the nuclear envelope persists throughout mitosis, and the spindle is extranuclear. We found three types of spindle microtubules: pole-to-nucleus microtubules which are attached to the nuclear envelope, pole-to-pole microtubules forming a cylindrical, cytoplasmic groove on the nuclear compartment in pseudocysts of T. foetus cells, and pole-to-cytosol microtubules which extend freely into the cytoplasm. We demonstrated that: (1) in T. foetus, the spindle is assembled from an MTOC located at the base of the costa, underneath one of the basal bodies; (2) the spindle presents an unusual arc shape during the initial phases of mitosis in motile trophozoites; (3) the spindle microtubules are glutamylated, but not acetylated; (4) several membranes similar to those of the endoplasmic reticulum follow the spindle microtubules; (5) finger-like projections extend from the nucleus towards the cell poles in pseudocysts and multinucleated cells; and (6) vesicles formed in between the two nuclear membranes are seen in the course of mitosis in both trophozoite and pseudocyst forms.

  1. Understanding mitosis in secondary school in Slovenia

    OpenAIRE

    Frank, Aleksandra

    2012-01-01

    Teaching experience show that students have trouble understands thematic of the cell and genetics. The purpose of our research was to evaluate students' knowledge of the cell and genetics. Next to evaluation of students’ knowledge we also tested a successfulness of a method of teaching the mitosis by letting students to sort the pictures with 15 different phases of mitosis. 99 first¬-year high school students were included in the research. First the students answered a questionnaire containin...

  2. Nucleus pulposus cell-matrix interactions with laminins.

    Science.gov (United States)

    Gilchrist, C L; Francisco, A T; Plopper, G E; Chen, J; Setton, L A

    2011-06-20

    The cells of the nucleus pulposus (NP) region of the intervertebral disc play a critical role in this tissue's generation and maintenance, and alterations in NP cell viability, metabolism, and phenotype with aging may be key contributors to progressive disc degeneration. Relatively little is understood about the phenotype of NP cells, including their cell-matrix interactions which may modulate phenotype and survival. Our previous work has identified strong and region-specific expression of laminins and laminin cell-surface receptors in immature NP tissues, suggesting laminin cell-matrix interactions are uniquely important to the biology of NP cells. Whether these observed tissue-level laminin expression patterns reflect functional adhesion behaviors for these cells is not known. In this study, we examined NP cell-matrix interactions with specific matrix ligands, including various laminin isoforms, using quantitative assays of cell attachment, spreading, and adhesion strength. NP cells were found to attach in higher numbers and exhibited rapid cell spreading and higher resistance to detachment force on two laminin isoforms (LM-511,LM-332) identified to be uniquely expressed in the NP region, as compared to another laminin isoform (LM-111) and several other matrix ligands (collagen, fibronectin). Additionally, NP cells were found to attach in higher numbers to laminins as compared to cells isolated from the disc's annulus fibrosus region. These findings confirm that laminin and laminin receptor expression documented in NP tissues translates into unique functional NP cell adhesion behaviors that may be useful tools for in vitro cell culture and biomaterials that support NP cells.

  3. Nucleus pulposus cell-matrix interactions with laminins

    Directory of Open Access Journals (Sweden)

    CL Gilchrist

    2011-06-01

    Full Text Available The cells of the nucleus pulposus (NP region of the intervertebral disc play a critical role in this tissue’s generation and maintenance, and alterations in NP cell viability, metabolism, and phenotype with aging may be key contributors to progressive disc degeneration. Relatively little is understood about the phenotype of NP cells, including their cell-matrix interactions which may modulate phenotype and survival. Our previous work has identified strong and region-specific expression of laminins and laminin cell-surface receptors in immature NP tissues, suggesting laminin cell-matrix interactions are uniquely important to the biology of NP cells. Whether these observed tissue-level laminin expression patterns reflect functional adhesion behaviors for these cells is not known. In this study, we examined NP cell-matrix interactions with specific matrix ligands, including various laminin isoforms, using quantitative assays of cell attachment, spreading, and adhesion strength. NP cells were found to attach in higher numbers and exhibited rapid cell spreading and higher resistance to detachment force on two laminin isoforms (LM-511,LM-332 identified to be uniquely expressed in the NP region, as compared to another laminin isoform (LM-111 and several other matrix ligands (collagen, fibronectin. Additionally, NP cells were found to attach in higher numbers to laminins as compared to cells isolated from the disc’s annulus fibrosus region. These findings confirm that laminin and laminin receptor expression documented in NP tissues translates into unique functional NP cell adhesion behaviors that may be useful tools for in vitro cell culture and biomaterials that support NP cells.

  4. Pancreatic cancer cells express CD44 variant 9 and multidrug resistance protein 1 during mitosis.

    Science.gov (United States)

    Kiuchi, Shizuka; Ikeshita, Shunji; Miyatake, Yukiko; Kasahara, Masanori

    2015-02-01

    Pancreatic cancer is one of the most lethal cancers with high metastatic potential and strong chemoresistance. Its intractable natures are attributed to high robustness in tumor cells for their survival. We demonstrate here that pancreatic cancer cells (PCCs) with an epithelial phenotype upregulate cell surface expression of CD44 variant 9 (CD44v9), an important cancer stem cell marker, during the mitotic phases of the cell cycle. Of five human CD44(+) PCC lines examined, three cell lines, PCI-24, PCI-43 and PCI-55, expressed E-cadherin and CD44 variants, suggesting that they have an epithelial phenotype. By contrast, PANC-1 and MIA PaCa-2 cells expressed vimentin and ZEB1, suggesting that they have a mesenchymal phenotype. PCCs with an epithelial phenotype upregulated cell surface expression of CD44v9 in prophase, metaphase, anaphase and telophase and downregulated CD44v9 expression in late-telophase, cytokinesis and interphase. Sorted CD44v9-negative PCI-55 cells resumed CD44v9 expression when they re-entered the mitotic stage. Interestingly, CD44v9(bright) mitotic cells expressed multidrug resistance protein 1 (MDR1) intracellularly. Upregulated expression of CD44v9 and MDR1 might contribute to the intractable nature of PCCs with high proliferative activity.

  5. Phospholipase C-beta 2 promotes mitosis and migration of human breast cancer-derived cells.

    Science.gov (United States)

    Bertagnolo, Valeria; Benedusi, Mascia; Brugnoli, Federica; Lanuti, Paola; Marchisio, Marco; Querzoli, Patrizia; Capitani, Silvano

    2007-08-01

    Like most human neoplasm, breast cancer has aberrations in signal transduction elements that can lead to increased proliferative potential, apoptosis inhibition, tissue invasion and metastasis. Due to the high heterogeneity of this tumor, currently, no markers are clearly associated with the insurgence of breast cancer, as well as with its progression from in situ lesion to invasive carcinoma. We have recently demonstrated an altered expression of the beta2 isoform of the phosphoinositide-dependent phospholipase C (PLC) in invasive breast tumors with different histopathological features. In primary breast tumor cells, elevated amounts of this protein are closely correlated with a poor prognosis of patients with mammary carcinoma, suggesting that PLC-beta2 may be involved in the development and worsening of the malignant phenotype. Here we demonstrate that PLC-beta2 may improve some malignant characteristics of tumor cells, like motility and invasion capability, but it fails to induce tumorigenesis in non-transformed breast-derived cells. We also report that, compared with the G(0)/G(1) phases of the cell cycle, the cells in S/G(2)/M phases show high PLC-beta2 expressions that reach the greatest levels during the late mitotic stages. In addition, even if unable to modify the proliferation rate and the expression of cell cycle-related enzymes of malignant cells, PLC-beta2 may promote the G(2)/M progression, a critical event in cancer evolution. Since phosphoinositides, substrates of PLC, are involved in regulating cytoskeleton architecture, PLC-beta2 in breast tumor cells may mediate the modification of cell shape that characterizes cell division, motility and invasion. On the basis of these data, PLC-beta2 may constitute a molecular marker of breast tumor cells able to monitor the progression to invasive cancers and a target for novel therapeutic breast cancer strategies.

  6. A force-generating machinery maintains the spindle at the cell center during mitosis.

    Science.gov (United States)

    Garzon-Coral, Carlos; Fantana, Horatiu A; Howard, Jonathon

    2016-05-27

    The position and orientation of the mitotic spindle is precisely regulated to ensure the accurate partition of the cytoplasm between daughter cells and the correct localization of the daughters within growing tissue. Using magnetic tweezers to perturb the position of the spindle in intact cells, we discovered a force-generating machinery that maintains the spindle at the cell center during metaphase and anaphase in one- and two-cell Caenorhabditis elegans embryos. The forces increase with the number of microtubules and are larger in smaller cells. The machinery is rigid enough to suppress thermal fluctuations to ensure precise localization of the mitotic spindle, yet compliant enough to allow molecular force generators to fine-tune the position of the mitotic spindle to facilitate asymmetric division.

  7. Multipolar mitosis of tetraploid cells: inhibition by p53 and dependency on Mos.

    Science.gov (United States)

    Vitale, Ilio; Senovilla, Laura; Jemaà, Mohamed; Michaud, Mickaël; Galluzzi, Lorenzo; Kepp, Oliver; Nanty, Lisa; Criollo, Alfredo; Rello-Varona, Santiago; Manic, Gwenola; Métivier, Didier; Vivet, Sonia; Tajeddine, Nicolas; Joza, Nicholas; Valent, Alexander; Castedo, Maria; Kroemer, Guido

    2010-04-07

    Tetraploidy can constitute a metastable intermediate between normal diploidy and oncogenic aneuploidy. Here, we show that the absence of p53 is not only permissive for the survival but also for multipolar asymmetric divisions of tetraploid cells, which lead to the generation of aneuploid cells with a near-to-diploid chromosome content. Multipolar mitoses (which reduce the tetraploid genome to a sub-tetraploid state) are more frequent when p53 is downregulated and the product of the Mos oncogene is upregulated. Mos inhibits the coalescence of supernumerary centrosomes that allow for normal bipolar mitoses of tetraploid cells. In the absence of p53, Mos knockdown prevents multipolar mitoses and exerts genome-stabilizing effects. These results elucidate the mechanisms through which asymmetric cell division drives chromosomal instability in tetraploid cells.

  8. Influence of the bn gene on mitosis of immature red blood cells in turkeys.

    Science.gov (United States)

    Searle, B M; Bloom, S E

    1979-01-01

    The binucleated and large mononucleated red blood cells found in the blood of bnbn mutant turkeys result from nondisjunction of chromosomes in bone marrow polychromatic erythrocytes. The major ultrastructural abnormality that is observed in these mutant cells is malpositioning of the centrioles in the cell. This involves failure to assume a normal pole-to-pole position in the center of the cell, and, often, centrioles are seen close together near the cell membrane. In addition to the abnormalities in centrioles, incomplete spindles are formed with large masses of chromatin unattached to microtubules. Cytokinesis is blocked in many instances because large amounts of chromatin remain at the region of the metaphase plate. None of the aforementioned abnormalities were seen in bone marrow cells from genetically normal turkeys. The results of this study suggest that malorientation of the centrioles has adverse effects on chromosome movement in animal cells. The concept that the spatial orientation of the centrioles is fundamental in achieving normal separation of chromosomes during anaphase movement is supported by our work. Finally, the close ultrastructural parallels with the human blood disease congenital dyserythropoietic anemia type I are discussed.

  9. Fresh WNT into the regulation of mitosis.

    Science.gov (United States)

    Stolz, Ailine; Bastians, Holger

    2015-01-01

    Canonical Wnt signaling triggering β-catenin-dependent gene expression contributes to cell cycle progression, in particular at the G1/S transition. Recently, however, it became clear that the cell cycle can also feed back on Wnt signaling at the G2/M transition. This is illustrated by the fact that mitosis-specific cyclin-dependent kinases can phosphorylate the Wnt co-receptor LRP6 to prime the pathway for incoming Wnt signals when cells enter mitosis. In addition, there is accumulating evidence that various Wnt pathway components might exert additional, Wnt-independent functions that are important for proper regulation of mitosis. The importance of Wnt pathways during mitosis was most recently enforced by the discovery of Wnt signaling contributing to the stabilization of proteins other than β-catenin, specifically at G2/M and during mitosis. This Wnt-mediated stabilization of proteins, now referred to as Wnt/STOP, might on one hand contribute to maintaining a critical cell size required for cell division and, on the other hand, for the faithful execution of mitosis itself. In fact, most recently we have shown that Wnt/STOP is required for ensuring proper microtubule dynamics within mitotic spindles, which is pivotal for accurate chromosome segregation and for the maintenance of euploidy.

  10. Atomic force microscope tracking observation of Chinese hamster ovary cell mitosis.

    Science.gov (United States)

    Wu, Yangzhe; Cai, Jiye; Cheng, Longqiu; Xu, Yanfang; Lin, Zhiyan; Wang, Chenxi; Chen, Yong

    2006-01-01

    CHO cells possess easily identifiable karyotypes, and CHO cell chromosomes are large and few in number, making these cells ideal for mutational and drug toxicity studies and suitable for investigations of animal chromosome structure. Here, we used atomic force microscopy (AFM) in the tapping mode for detailed visualizations of Chinese hamster ovary (CHO) cell chromosomes during various mitotic phases, including typical prophase, prometaphase, metaphase, anaphase and telophase. Based on our detailed observations, we were able to divide metaphase and anaphase into sub-phases: metaphase I, II and III, and anaphase I and II. Furthermore, we used the AFM error-signal mode to visualize chromosomal ultrastructures and cytokinesis. While these visualizations were all successful, we found that the image quality was affected by cellular debris, contamination. Collectively, our results show that the AFM technique has great potential for the detailed study of chromosomes and chromosomal ultrastructures during all phases of the cell cycle, but that careful standards of sample preparation must be maintained.

  11. Microtubules contribute to maintain nucleus shape in epithelial cell monolayer

    Science.gov (United States)

    Tremblay, Dominique; Andrzejewski, Lukasz; Pelling, Andrew

    2013-03-01

    INTRODUCTION: Tissue strains can result in significant nuclear deformations and may regulate gene expression. However, the precise role of the cytoskeleton in regulating nuclear mechanics remains poorly understood. Here, we investigate the nuclear deformability of Madin-Darky canine kidney cells (MDCK) under various stretching conditions to clarify the role of the microtubules and actin network on the mechanical behavior of the nucleus. METHODS: A custom-built cell-stretching device allowing for real time imaging of MDCK nuclei was used. Cells were seeded on a silicone membrane coated with rat-tail collagen I. A nuclear stain, Hoechst-33342, was used to image nuclei during stretching. We exposed cells to a compressive and non-compressive stretching strain field of 25%. Nocodazole and cytochalasin-D were used to depolymerize the microtubules and actin network. RESULTS: Nuclei in control cells stretched more along their minor axis than major axis with a deformation of 5% and 2% respectively. This anisotropy vanished completely in microtubule-deprived cells and these cells showed a very high nuclear deformability along the minor axis when exposed to a compressive stretching strain field. CONCLUSIONS: The microtubules drive the anisotropic deformability of MDCK nuclei in a monolayer and maintain nuclear shape when exposed to compressive strain. Such intrinsic mechanical behavior indicates that microtubules are essential to maintain nuclear shape and may prevent down regulation of gene expression.

  12. Superficial stellate cells of the dorsal cochlear nucleus

    Directory of Open Access Journals (Sweden)

    Pierre F. Apostolides

    2014-06-01

    Full Text Available The dorsal cochlear nucleus (DCN integrates auditory and multisensory signals at the earliest levels of auditory processing. Proposed roles for this region include sound localization in the vertical plane, head orientation to sounds of interest, and suppression of sensitivity to expected sounds. Auditory and non-auditory information streams to the DCN are refined by a remarkably complex array of inhibitory and excitatory interneurons, and the role of each cell type is gaining increasing attention. One inhibitory neuron that has been poorly appreciated to date is the superficial stellate cell. Here we review previous studies and describe new results that reveal the surprisingly rich interactions that this tiny interneuron has with its neighbors, interactions which enable it to respond to both multisensory and auditory afferents.

  13. Symmetric pollen mitosis I and suppression of pollen mitosis II prevent pollen development in Brachiaria jubata (Gramineae

    Directory of Open Access Journals (Sweden)

    C. Risso-Pascotto

    2005-11-01

    Full Text Available Microsporogenesis and pollen development were analyzed in a tetraploid (2n = 4x = 36 accession of the forage grass Brachiaria jubata (BRA 007820 from the Embrapa Beef Cattle Brachiaria collection that showed partial male sterility. Microsporocytes and pollen grains were prepared by squashing and staining with 0.5% propionic carmine. The meiotic process was typical of polyploids, with precocious chromosome migration to the poles and laggards in both meiosis I and II, resulting in tetrads with micronuclei in some microspores. After callose dissolution, microspores were released into the anther locule and appeared to be normal. Although each microspore initiated its differentiation into a pollen grain, in 11.1% of them nucleus polarization was not observed, i.e., pollen mitosis I was symmetric and the typical hemispherical cell plate was not detected. After a central cytokinesis, two equal-sized cells showing equal chromatin condensation and the same nuclear shape and size were formed. Generative cells and vegetative cells could not be distinguished. These cells did not undergo the second pollen mitosis and after completion of pollen wall synthesis each gave rise to a sterile and uninucleate pollen grain. The frequency of abnormal pollen mitosis varied among flowers and also among inflorescences. All plants were equally affected. The absence of fertile sperm cells in a considerable amount of pollen grains in this accession of B. jubata may compromise its use in breeding and could explain, at least in part, why seed production is low when compared with the amount of flowers per raceme.

  14. The DNA damage response during mitosis.

    Science.gov (United States)

    Heijink, Anne Margriet; Krajewska, Małgorzata; van Vugt, Marcel A T M

    2013-10-01

    Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed.

  15. The DNA damage response during mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Heijink, Anne Margriet; Krajewska, Małgorzata; Vugt, Marcel A.T.M. van, E-mail: m.vugt@umcg.nl

    2013-10-15

    Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed.

  16. Mitosis and cell death in the tail of the chick embryo.

    Science.gov (United States)

    Mills, C L; Bellairs, R

    1989-01-01

    Although somites develop from the mesoderm in the tail of the chick embryo, they do not form to the tip of the tail. Previous work has shown that this terminal mesoderm possesses many of the characteristics of the segmental plate mesoderm which gives rise to the somites in the trunk. This investigation is aimed therefore at understanding why the terminal mesoderm fails to form somites. Mitotic and pyknotic rates have been obtained for the tail region of chick embryos between stages 13 and 27. Embryos were treated with colchicine, so that the mitoses were blocked in metaphase, and counts were made on serial sections. The overall mitotic rates were highest between stages 15 and 18. Regions of high mitotic rate, which are an indication of cell synchrony, were found in the tail bud mesoderm though not in a consistent location, and only infrequently near the anterior end of the tail segmental plate. In the trunk however (Stern and Bellairs 1984) a single peak of cell synchrony was routinely found near the cranial end of the segmental plate. It is concluded that the cells of the tail mesoderm are less synchronised in preparation for somitogenesis than are the corresponding mesoderm cells in the trunk. A further conclusion is that the tail bud is not per se a region of high proliferation, though there are patches of high mitotic rate. The overall pyknotic rate reached a maximum at stage 25; peaks of pyknosis corresponded initially with the mitotic peaks and were associated with the ventral ectodermal ridge and the tail gut. By stage 25 however, the high levels of cell death were restricted mainly to the tip of the tail.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Incoming human papillomavirus type 16 genome resides in a vesicular compartment throughout mitosis.

    Science.gov (United States)

    DiGiuseppe, Stephen; Luszczek, Wioleta; Keiffer, Timothy R; Bienkowska-Haba, Malgorzata; Guion, Lucile G M; Sapp, Martin J

    2016-05-31

    During the entry process, the human papillomavirus (HPV) capsid is trafficked to the trans-Golgi network (TGN), whereupon it enters the nucleus during mitosis. We previously demonstrated that the minor capsid protein L2 assumes a transmembranous conformation in the TGN. Here we provide evidence that the incoming viral genome dissociates from the TGN and associates with microtubules after the onset of mitosis. Deposition onto mitotic chromosomes is L2-mediated. Using differential staining of an incoming viral genome by small molecular dyes in selectively permeabilized cells, nuclease protection, and flotation assays, we found that HPV resides in a membrane-bound vesicle until mitosis is completed and the nuclear envelope has reformed. As a result, expression of the incoming viral genome is delayed. Taken together, these data provide evidence that HPV has evolved a unique strategy for delivering the viral genome to the nucleus of dividing cells. Furthermore, it is unlikely that nuclear vesicles are unique to HPV, and thus we may have uncovered a hitherto unrecognized cellular pathway that may be of interest for future cell biological studies.

  18. Calpastatin is regulated by protein never in mitosis gene A interacting-1 (PIN1) in endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Tongzheng, E-mail: liu.tongzheng@mayo.edu [Division of Oncology Research, Department of Oncology, Mayo Clinic, Rochester, MN 55905 (United States); Schneider, Ryan A., E-mail: schneiderr@findlay.edu [College of Pharmacy, The University of Findlay, Findlay, OH 45840 (United States); Hoyt, Dale G., E-mail: hoyt.27@osu.edu [The Dorothy M. Davis Heart and Lung Research Institute, and the Division of Pharmacology, College of Pharmacy, The Ohio State University, 500 West Twelfth Avenue, Columbus, OH 43210 (United States)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer Depletion of PIN1 increases inhibitory effect of calpastatin against calpain in endothelial cells. Black-Right-Pointing-Pointer PIN1 associates with calpastatin. Black-Right-Pointing-Pointer PIN1, but not mutants, reduces the inhibitory activity of calpastatin in vitro. Black-Right-Pointing-Pointer Depletion of calpastatin shows that it is required for PIN1 depletion to reduce calpain activity. -- Abstract: The peptidyl-proline isomerase, protein never in mitosis gene A interacting-1 (PIN1) binds and isomerizes proteins phosphorylated on serine/threonine before a proline. It was previously found that depletion of PIN1 greatly increased induction of cyclooxygenase-2 and inducible nitric oxide synthase by lowering calpain activity in murine aortic endothelial cells (MAEC). Here we investigated the effect of PIN1 on the endogenous inhibitor of heterodimeric {mu}- and m-calpains, calpastatin. MAEC were transduced with small hairpin (sh) RNA to knock down PIN1 (KD) or an inactive Control shRNA. Cells were also treated with non-targeted double stranded small inhibitory RNA (siRNA) or siRNA designed to deplete calpastatin. Despite reducing calpain activity, PIN1 KD did not significantly affect the expression of {mu}- and m-calpains, or calpastatin, compared to Control shRNA. Instead, depletion of PIN1 increased the inhibitory activity of calpastatin. Calpastatin co-immunoprecipitated with endogenous PIN1 and was pulled down with glutathione-S-transferase (GST)-PIN1 fusion protein. Adding GST-PIN1 to KD cell extracts lacking PIN1 reduced calpastatin inhibitory activity. Substrate binding and catalytic domain mutants of PIN1 failed to do so. These results suggest that protein interaction and the proline isomerase functions of PIN1 are required for it to inhibit calpastatin. Furthermore, depletion of calpastatin raised calpain activity and reduced calpain inhibitory activity to similar levels in KD and Control MAEC, indicating that

  19. Mitotic accumulation of dimethylated lysine 79 of histone H3 is important for maintaining genome integrity during mitosis in human cells.

    Science.gov (United States)

    Guppy, Brent J; McManus, Kirk J

    2015-02-01

    The loss of genome stability is an early event that drives the development and progression of virtually all tumor types. Recent studies have revealed that certain histone post-translational modifications exhibit dynamic and global increases in abundance that coincide with mitosis and exhibit essential roles in maintaining genomic stability. Histone H2B ubiquitination at lysine 120 (H2Bub1) is regulated by RNF20, an E3 ubiquitin ligase that is altered in many tumor types. Through an evolutionarily conserved trans-histone pathway, H2Bub1 is an essential prerequisite for subsequent downstream dimethylation events at lysines 4 (H3K4me2) and 79 (H3K79me2) of histone H3. Although the role that RNF20 plays in tumorigenesis has garnered much attention, the downstream components of the trans-histone pathway, H3K4me2 and H3K79me2, and their potential contributions to genome stability remain largely overlooked. In this study, we employ single-cell imaging and biochemical approaches to investigate the spatial and temporal patterning of RNF20, H2Bub1, H3K4me2, and H3K79me2 throughout the cell cycle, with a particular focus on mitosis. We show that H2Bub1, H3K4me2, and H3K79me2 exhibit distinct temporal progression patterns throughout the cell cycle. Most notably, we demonstrate that H3K79me2 is a highly dynamic histone post-translational modification that reaches maximal abundance during mitosis in an H2Bub1-independent manner. Using RNAi and chemical genetic approaches, we identify DOT1L as a histone methyltransferase required for the mitotic-associated increases in H3K79me2. We also demonstrate that the loss of mitotic H3K79me2 levels correlates with increases in chromosome numbers and increases in mitotic defects. Collectively, these data suggest that H3K79me2 dynamics during mitosis are normally required to maintain genome stability and further implicate the loss of H3K79me2 during mitosis as a pathogenic event that contributes to the development and progression of tumors.

  20. Okadaic acid (1 microM) accelerates S phase and mitosis but inhibits heterochromatin replication and metaphase anaphase transition in Vicia faba meristem cells.

    Science.gov (United States)

    Polit, Justyna Teresa; Kazmierczak, Andrzej

    2007-01-01

    Protein kinases and phosphatases are the foremost agents which take part in cell cycle regulation in both plants and other eukaryotes. Protein kinases are a very well examined group of proteins with respect to chemical structure and function. Nowadays protein phosphatases, including PP1 and PP2A belonging to the PSP family, are the focus of interest. Okadaic acid (OA) which is a specific inhibitor of protein phosphatase activity is widely used to study them. In the present research, the involvement of OA-sensitive phosphatases in the regulation of progression of the plant cell cycle was analysed (in planta) using Vicia faba root meristems synchronized with hydroxyurea and divided into five series. Each series was treated with 1 muM OA for 3 h for different time periods corresponding to the consecutive cell cycle phases. The results showed that in the OA-treated cells DNA replication and mitosis began earlier than in the control cells, since G(1) and G(2) phases were significantly shorter and the H1 histone kinases activity was higher. Moreover, autoradiography and morphological analyses of mitotic figures revealed that the OA-treated cells entered mitosis before the end of heterochromatin replication. An immunocytochemical search showed that earlier initiation of S phase in the OA-treated cells correlated with more abundant phosphorylation of Rb-like protein in comparison with the control cells. OA also induced significant condensation of metaphase chromosomes and blocked metaphase-anaphase transition.

  1. Golgi twins in late mitosis revealed by genetically encoded tags for live cell imaging and correlated electron microscopy

    NARCIS (Netherlands)

    Gaietta, Guido M; Giepmans, Ben N G; Deerinck, Thomas J; Smith, W Bryan; Ngan, Lucy; Llopis, Juan; Adams, Stephen R; Tsien, Roger Y; Ellisman, Mark H

    2006-01-01

    Combinations of molecular tags visible in light and electron microscopes become particularly advantageous in the analysis of dynamic cellular components like the Golgi apparatus. This organelle disassembles at the onset of mitosis and, after a sequence of poorly understood events, reassembles after

  2. Cell kinetic studies in the epidermis of mouse. III. The percent labelled mitosis (PLM) technique.

    Science.gov (United States)

    Potten, C S; Wichmann, H E; Dobek, K; Birch, J; Codd, T M; Horrocks, L; Pedrick, M; Tickle, S P

    1985-01-01

    Full PLM curves have been obtained for four sites in the mouse. The first peaks have been analysed by computer and the duration of the G2 + M and S phases determined together with their standard deviations. The full curves showed a general similarity for all four sites with no clear second peak. The data are compared with the published data for mouse and human epidermis using the in vivo PLM technique. The timing and shape of the first peak can vary considerably even for one site in mice. Hence, both G2 + M and S can vary in their durations. Cells labelled at one time of day exhibit different kinetic properties to those labelled at another time of day. The duration of G2 + M is shortest in dorsum labelled at 03.00 hours (3 X 2 hr) and longest in tail (up to 7 X 5 hr). The S-phase is shortest in dorsum (6 X 3-7 X 2 hr) and longest in tail or ear (13 X 3-14 X 1 hr). There is also a very large standard deviation in tail and foot. There is little general variability when the psoriatic human data are considered, which is surprising. The general variability amongst the data from experimental mice might also be expected amongst humans which might make comparisons between the cell kinetics of normal and diseased skin difficult.

  3. The Stimulatory Effect of Notochordal Cell-Conditioned Medium in a Nucleus Pulposus Explant Culture

    NARCIS (Netherlands)

    de Vries, Stefan A H; van Doeselaar, Marina; Meij, Björn P; Tryfonidou, Marianna A; Ito, K

    2016-01-01

    Objectives: Notochordal cell-conditioned medium (NCCM) has previously shown to have a stimulatory effect on nucleus pulposus cells (NPCs) and bone marrow stromal cells (BMSCs) in alginate and pellet cultures. These culture methods provide a different environment than the nucleus pulposus (NP) tissue

  4. The Stimulatory Effect of Notochordal-Cell Conditioned Medium in a Nucleus Pulposus Explant Culture

    NARCIS (Netherlands)

    de Vries, Stefan; Doeselaar, Marina van; Meij, Björn; Tryfonidou, M; Ito, Keita

    2015-01-01

    OBJECTIVES: Notochordal cell-conditioned medium (NCCM) has previously shown to have a stimulatory effect on nucleus pulposus cells (NPCs) and bone marrow stromal cells (BMSCs) in alginate and pellet cultures. These culture methods provide a different environment than the nucleus pulposus (NP) tissue

  5. Differentiation of adipose stem cells by nucleus pulposus cells: Configuration effect

    NARCIS (Netherlands)

    Lu, Z.F.; Zandieh Doulabi, B.; Wuisman, P.I.; Bank, R.A.; Helder, M.N.

    2007-01-01

    Degenerative disc disease (DDD) is a major cause of chronic low back pain. For mild/intermediate DDD, regeneration by injecting adipose stem cells (ASCs) into the nucleus pulposus (NP) may be considered. The goal of this study is to investigate whether NP cells can direct ASCs towards the NP phenoty

  6. Meiosis: an overview of key differences from mitosis.

    Science.gov (United States)

    Ohkura, Hiroyuki

    2015-01-20

    Meiosis is the specialized cell division that generates gametes. In contrast to mitosis, molecular mechanisms and regulation of meiosis are much less understood. Meiosis shares mechanisms and regulation with mitosis in many aspects, but also has critical differences from mitosis. This review highlights these differences between meiosis and mitosis. Recent studies using various model systems revealed differences in a surprisingly wide range of aspects, including cell-cycle regulation, recombination, postrecombination events, spindle assembly, chromosome-spindle interaction, and chromosome segregation. Although a great degree of diversity can be found among organisms, meiosis-specific processes, and regulation are generally conserved.

  7. Dynamics of the mitochondrial network during mitosis.

    Science.gov (United States)

    Kanfer, Gil; Kornmann, Benoît

    2016-04-15

    During mitosis, cells undergo massive deformation and reorganization, impacting on all cellular structures. Mitochondria, in particular, are highly dynamic organelles, which constantly undergo events of fission, fusion and cytoskeleton-based transport. This plasticity ensures the proper distribution of the metabolism, and the proper inheritance of functional organelles. During cell cycle, mitochondria undergo dramatic changes in distribution. In this review, we focus on the dynamic events that target mitochondria during mitosis. We describe how the cell-cycle-dependent microtubule-associated protein centromeric protein F (Cenp-F) is recruited to mitochondria by the mitochondrial Rho GTPase (Miro) to promote mitochondrial transport and re-distribution following cell division.

  8. Spatial and dynamic organization of molecular structures in the cell nucleus

    NARCIS (Netherlands)

    Brouwer, Anne-Kee

    2010-01-01

    In this thesis we attempt to provide a better understanding of the principles that underlie the spatial dynamic organization of the cell nucleus. Chapter 1 reviews the current status of knowledge about the structural and functional organization of the cell nucleus. In chapter 2, the development of a

  9. OsRAN2, essential for mitosis, enhances cold tolerance in rice by promoting export of intranuclear tubulin and maintaining cell division under cold stress.

    Science.gov (United States)

    Chen, Na; Xu, Yunyuan; Wang, Xin; DU, Cheng; DU, Jizhou; Yuan, Ming; Xu, Zhihong; Chong, Kang

    2011-01-01

    With global climate change, abnormally low temperatures have affected the world's rice production. Many genes have been shown to be essential for molecular improvement of rice cold-tolerance traits. However, less is known about the molecular cellular mechanism of their response to cold stress. Here, we investigated OsRAN2 involved in regulation of cell division during cold stress in rice. Expression of OsRAN2 was increased under cold treatment, but not during salt and drought stress. The mean root mitotic index was closely related to the expression level of OsRAN2. Knockdown transgenic rice lines showed an aberrant organization of spindles during mitosis and stunted growth during development. Overexpression of OsRAN2 enhanced cold tolerance in rice. The transgenic rice overexpressing OsRAN2 showed maintained cell division, decreased proportion of cells with intranuclear tubulin and formation of a normal nuclear envelope under the cold condition. Our study suggests a mechanism for OsRAN2 in regulating cold resistance in rice by maintaining cell division through promoting the normal export of intranuclear tubulin at the end of mitosis. This insight could help improve the cold-tolerance trait in rice.

  10. Stress induced by premature chromatin condensation triggers chromosome shattering and chromothripsis at DNA sites still replicating in micronuclei or multinucleate cells when primary nuclei enter mitosis.

    Science.gov (United States)

    Terzoudi, Georgia I; Karakosta, Maria; Pantelias, Antonio; Hatzi, Vasiliki I; Karachristou, Ioanna; Pantelias, Gabriel

    2015-11-01

    Combination of next-generation DNA sequencing, single nucleotide polymorphism array analyses and bioinformatics has revealed the striking phenomenon of chromothripsis, described as complex genomic rearrangements acquired in a single catastrophic event affecting one or a few chromosomes. Via an unproven mechanism, it is postulated that mechanical stress causes chromosome shattering into small lengths of DNA, which are then randomly reassembled by DNA repair machinery. Chromothripsis is currently examined as an alternative mechanism of oncogenesis, in contrast to the present paradigm that considers a stepwise development of cancer. While evidence for the mechanism(s) underlying chromosome shattering during cancer development remains elusive, a number of hypotheses have been proposed to explain chromothripsis, including ionizing radiation, DNA replication stress, breakage-fusion-bridge cycles, micronuclei formation and premature chromosome compaction. In the present work, we provide experimental evidence on the mechanistic basis of chromothripsis and on how chromosomes can get locally shattered in a single catastrophic event. Considering the dynamic nature of chromatin nucleoprotein complex, capable of rapid unfolding, disassembling, assembling and refolding, we first show that chromatin condensation at repairing or replicating DNA sites induces the mechanical stress needed for chromosome shattering to ensue. Premature chromosome condensation is then used to visualize the dynamic nature of interphase chromatin and demonstrate that such mechanical stress and chromosome shattering can also occur in chromosomes within micronuclei or asynchronous multinucleate cells when primary nuclei enter mitosis. Following an aberrant mitosis, chromosomes could find themselves in the wrong place at the wrong time so that they may undergo massive DNA breakage and rearrangement in a single catastrophic event. Specifically, our results support the hypothesis that premature chromosome

  11. TopBP1-mediated DNA processing during mitosis.

    Science.gov (United States)

    Gallina, Irene; Christiansen, Signe Korbo; Pedersen, Rune Troelsgaard; Lisby, Michael; Oestergaard, Vibe H

    2016-01-01

    Maintenance of genome integrity is crucial to avoid cancer and other genetic diseases. Thus faced with DNA damage, cells mount a DNA damage response to avoid genome instability. The DNA damage response is partially inhibited during mitosis presumably to avoid erroneous processing of the segregating chromosomes. Yet our recent study shows that TopBP1-mediated DNA processing during mitosis is highly important to reduce transmission of DNA damage to daughter cells. (1) Here we provide an overview of the DNA damage response and DNA repair during mitosis. One role of TopBP1 during mitosis is to stimulate unscheduled DNA synthesis at underreplicated regions. We speculated that such genomic regions are likely to hold stalled replication forks or post-replicative gaps, which become the substrate for DNA synthesis upon entry into mitosis. Thus, we addressed whether the translesion pathways for fork restart or post-replicative gap filling are required for unscheduled DNA synthesis in mitosis. Using genetics in the avian DT40 cell line, we provide evidence that unscheduled DNA synthesis in mitosis does not require the translesion synthesis scaffold factor Rev1 or PCNA ubiquitylation at K164, which serve to recruit translesion polymerases to stalled forks. In line with this finding, translesion polymerase η foci do not colocalize with TopBP1 or FANCD2 in mitosis. Taken together, we conclude that TopBP1 promotes unscheduled DNA synthesis in mitosis independently of the examined translesion polymerases.

  12. [An involvement of polokinases in control of progress of the cell-cycle--the mechanism of transient translocation and formation of an activated protein-protein complexes during mitosis].

    Science.gov (United States)

    Kaczanowska, Janina; Piwońska, Dominika; Kaczanowski, Andrzej

    2006-01-01

    Polokinases are a subfamily of the mitotic serine/threonine kinases involved in coordination of a run of mitosis of eukaryotic cells. The main polo-like-kinase 1p (PLK1) is a passenger protein transiently localized to centrosomes, kinetochores and central spindle during mitosis and is required for bi-orientation of the normal metaphase spindle. Its activity is regulated at the level of protein stability and by action of upstream kinases, so that it peaks in metaphase and drops as cells exit mitosis. Regulation of location and activity of Plk1p is bi-phasic: the COOH terminal polo box domain binds to an array of mitotic phosphoproteins and followed by an allosteric conformation is activated to phosphorylate many its substrates. These mode of action involves polokinases into critical transitions of the cell cycle phases, and in control at some checkpoints of this cycle.

  13. The Cell Nucleus in Physiological and Experimentally Induced Hypometabolism

    Science.gov (United States)

    Malatesta, M.

    The main problem for manned space mission is, at present, represented by the mass penalty associated to the human presence. An efficient approach could be the induction of a hypometabolic stasis in the astronauts, thus drastically reducing the physical and psychological requirements of the crew. On the other hand, in the wild, a reduction in resource consumptions physiologi- cally occurs in certain animals which periodically enter hibernation, a hypometabolic state in which both the energy need and energy offer are kept at a minimum. During the last twelve years, we have been studying different tissues of hibernating dormice, with the aim of analyzing their features during the euthermia -hibernation-arousal cycle as well as getting insight into the mechanisms allowing adaptation to hypometabolism. We paid particular attention to the cell nucleus, as it is the site of chief metabolic functions, such as DNA replication and RNA transcription. Our observations revealed no significant modification in the basic features of cell nuclei during hibernation; however, the cell nuclei of hibernating dormice showed unusual nuclear bodies containing molecules involved in RNA pathways. Therefore, we supposed that they could represent storage/assembly sites of several factors for processing some RNA which could be slowly synthesised during hibernation and rapidly and abundantly released in early arousal in order to meet the increased metabolic needs of the cell. The nucleolus also underwent structural and molecular modifications during hibernation, maybe to continue important nucleolar functions, or, alternatively, permit a most efficient reactivation upon arousal. On the basis of the observations made in vivo , we recently tried to experimentally induce a reversible hypometabolic state in in vitro models, using cell lines derived from hibernating and non-hibernating species. By administering the synthetic opioid DADLE, we could significantly reduce both RNA transcrip- tion and

  14. The cell cycle timing of centromeric chromatin assembly in Drosophila meiosis is distinct from mitosis yet requires CAL1 and CENP-C.

    Science.gov (United States)

    Dunleavy, Elaine M; Beier, Nicole L; Gorgescu, Walter; Tang, Jonathan; Costes, Sylvain V; Karpen, Gary H

    2012-01-01

    CENP-A (CID in flies) is the histone H3 variant essential for centromere specification, kinetochore formation, and chromosome segregation during cell division. Recent studies have elucidated major cell cycle mechanisms and factors critical for CENP-A incorporation in mitosis, predominantly in cultured cells. However, we do not understand the roles, regulation, and cell cycle timing of CENP-A assembly in somatic tissues in multicellular organisms and in meiosis, the specialized cell division cycle that gives rise to haploid gametes. Here we investigate the timing and requirements for CID assembly in mitotic tissues and male and female meiosis in Drosophila melanogaster, using fixed and live imaging combined with genetic approaches. We find that CID assembly initiates at late telophase and continues during G1 phase in somatic tissues in the organism, later than the metaphase assembly observed in cultured cells. Furthermore, CID assembly occurs at two distinct cell cycle phases during male meiosis: prophase of meiosis I and after exit from meiosis II, in spermatids. CID assembly in prophase I is also conserved in female meiosis. Interestingly, we observe a novel decrease in CID levels after the end of meiosis I and before meiosis II, which correlates temporally with changes in kinetochore organization and orientation. We also demonstrate that CID is retained on mature sperm despite the gross chromatin remodeling that occurs during protamine exchange. Finally, we show that the centromere proteins CAL1 and CENP-C are both required for CID assembly in meiosis and normal progression through spermatogenesis. We conclude that the cell cycle timing of CID assembly in meiosis is different from mitosis and that the efficient propagation of CID through meiotic divisions and on sperm is likely to be important for centromere specification in the developing zygote.

  15. The cell cycle timing of centromeric chromatin assembly in Drosophila meiosis is distinct from mitosis yet requires CAL1 and CENP-C.

    Directory of Open Access Journals (Sweden)

    Elaine M Dunleavy

    Full Text Available CENP-A (CID in flies is the histone H3 variant essential for centromere specification, kinetochore formation, and chromosome segregation during cell division. Recent studies have elucidated major cell cycle mechanisms and factors critical for CENP-A incorporation in mitosis, predominantly in cultured cells. However, we do not understand the roles, regulation, and cell cycle timing of CENP-A assembly in somatic tissues in multicellular organisms and in meiosis, the specialized cell division cycle that gives rise to haploid gametes. Here we investigate the timing and requirements for CID assembly in mitotic tissues and male and female meiosis in Drosophila melanogaster, using fixed and live imaging combined with genetic approaches. We find that CID assembly initiates at late telophase and continues during G1 phase in somatic tissues in the organism, later than the metaphase assembly observed in cultured cells. Furthermore, CID assembly occurs at two distinct cell cycle phases during male meiosis: prophase of meiosis I and after exit from meiosis II, in spermatids. CID assembly in prophase I is also conserved in female meiosis. Interestingly, we observe a novel decrease in CID levels after the end of meiosis I and before meiosis II, which correlates temporally with changes in kinetochore organization and orientation. We also demonstrate that CID is retained on mature sperm despite the gross chromatin remodeling that occurs during protamine exchange. Finally, we show that the centromere proteins CAL1 and CENP-C are both required for CID assembly in meiosis and normal progression through spermatogenesis. We conclude that the cell cycle timing of CID assembly in meiosis is different from mitosis and that the efficient propagation of CID through meiotic divisions and on sperm is likely to be important for centromere specification in the developing zygote.

  16. Cell nucleus - physical limitation of a quasar-galaxy association. Cell, DNA and cosmological background

    Energy Technology Data Exchange (ETDEWEB)

    Muheim, J.T. (Eidgenoessische Technische Hochschule, Zurich (Switzerland). Lab. fuer Festkoerperphysik)

    1985-03-15

    The author extends his analogy of atomic structure to cosmological structures to include the cell nucleus and DNA replication. From this the author believes that there is extraterrestrial life within 34 light years of us and that telepathy is possible within the solar system.

  17. Planar multipolar cells in the cochlear nucleus project to medial olivocochlear neurons in mouse.

    Science.gov (United States)

    Darrow, Keith N; Benson, Thane E; Brown, M Christian

    2012-05-01

    Medial olivocochlear (MOC) neurons originate in the superior olivary complex and project to the cochlea, where they act to reduce the effects of noise masking and protect the cochlea from damage. MOC neurons respond to sound via a reflex pathway; however, in this pathway the cochlear nucleus cell type that provides input to MOC neurons is not known. We investigated whether multipolar cells of the ventral cochlear nucleus have projections to MOC neurons by labeling them with injections into the dorsal cochlear nucleus. The projections of one type of labeled multipolar cell, planar neurons, were traced into the ventral nucleus of the trapezoid body, where they were observed terminating on MOC neurons (labeled in some cases by a second cochlear injection of FluoroGold). These terminations formed what appear to be excitatory synapses, i.e., containing small, round vesicles and prominent postsynaptic densities. These data suggest that cochlear nucleus planar multipolar neurons drive the MOC neuron's response to sound.

  18. PKR is activated by cellular dsRNAs during mitosis and acts as a mitotic regulator.

    Science.gov (United States)

    Kim, Yoosik; Lee, Jung Hyun; Park, Jong-Eun; Cho, Jun; Yi, Hyerim; Kim, V Narry

    2014-06-15

    dsRNA-dependent protein kinase R (PKR) is a ubiquitously expressed enzyme well known for its roles in immune response. Upon binding to viral dsRNA, PKR undergoes autophosphorylation, and the phosphorylated PKR (pPKR) regulates translation and multiple signaling pathways in infected cells. Here, we found that PKR is activated in uninfected cells, specifically during mitosis, by binding to dsRNAs formed by inverted Alu repeats (IRAlus). While PKR and IRAlu-containing RNAs are segregated in the cytosol and nucleus of interphase cells, respectively, they interact during mitosis when nuclear structure is disrupted. Once phosphorylated, PKR suppresses global translation by phosphorylating the α subunit of eukaryotic initiation factor 2 (eIF2α). In addition, pPKR acts as an upstream kinase for c-Jun N-terminal kinase and regulates the levels of multiple mitotic factors such as cyclins A and B and Polo-like kinase 1 and phosphorylation of histone H3. Disruption of PKR activation via RNAi or expression of a transdominant-negative mutant leads to misregulation of the mitotic factors, delay in mitotic progression, and defects in cytokinesis. Our study unveils a novel function of PKR and endogenous dsRNAs as signaling molecules during the mitosis of uninfected cells.

  19. Promoters active in interphase are bookmarked during mitosis by ubiquitination.

    Science.gov (United States)

    Arora, Mansi; Zhang, Jie; Heine, George F; Ozer, Gulcin; Liu, Hui-wen; Huang, Kun; Parvin, Jeffrey D

    2012-11-01

    We analyzed modification of chromatin by ubiquitination in human cells and whether this mark changes through the cell cycle. HeLa cells were synchronized at different stages and regions of the genome with ubiquitinated chromatin were identified by affinity purification coupled with next-generation sequencing. During interphase, ubiquitin marked the chromatin on the transcribed regions of ∼70% of highly active genes and deposition of this mark was sensitive to transcriptional inhibition. Promoters of nearly half of the active genes were highly ubiquitinated specifically during mitosis. The ubiquitination at the coding regions in interphase but not at promoters during mitosis was enriched for ubH2B and dependent on the presence of RNF20. Ubiquitin labeling of both promoters during mitosis and transcribed regions during interphase, correlated with active histone marks H3K4me3 and H3K36me3 but not a repressive histone modification, H3K27me3. The high level of ubiquitination at the promoter chromatin during mitosis was transient and was removed within 2 h after the cells exited mitosis and entered the next cell cycle. These results reveal that the ubiquitination of promoter chromatin during mitosis is a bookmark identifying active genes during chromosomal condensation in mitosis, and we suggest that this process facilitates transcriptional reactivation post-mitosis.

  20. Using a Case-Study Article to Effectively Introduce Mitosis

    Science.gov (United States)

    Van Hoewyk, Doug

    2007-01-01

    Community college students in a nonmajors biology class are introduced to mitosis by reading a case-study article that allows them to gauge how many times various parts of their bodies have been regenerated. The case-study article allows students to develop a conceptual framework of the cell cycle prior to a lecture on mitosis. (Contains 1 figure.)

  1. The tumor suppressor CDKN3 controls mitosis.

    Science.gov (United States)

    Nalepa, Grzegorz; Barnholtz-Sloan, Jill; Enzor, Rikki; Dey, Dilip; He, Ying; Gehlhausen, Jeff R; Lehmann, Amalia S; Park, Su-Jung; Yang, Yanzhu; Yang, Xianlin; Chen, Shi; Guan, Xiaowei; Chen, Yanwen; Renbarger, Jamie; Yang, Feng-Chun; Parada, Luis F; Clapp, Wade

    2013-06-24

    Mitosis is controlled by a network of kinases and phosphatases. We screened a library of small interfering RNAs against a genome-wide set of phosphatases to comprehensively evaluate the role of human phosphatases in mitosis. We found four candidate spindle checkpoint phosphatases, including the tumor suppressor CDKN3. We show that CDKN3 is essential for normal mitosis and G1/S transition. We demonstrate that subcellular localization of CDKN3 changes throughout the cell cycle. We show that CDKN3 dephosphorylates threonine-161 of CDC2 during mitotic exit and we visualize CDC2(pThr-161) at kinetochores and centrosomes in early mitosis. We performed a phosphokinome-wide mass spectrometry screen to find effectors of the CDKN3-CDC2 signaling axis. We found that one of the identified downstream phosphotargets, CKβ phosphorylated at serine 209, localizes to mitotic centrosomes and controls the spindle checkpoint. Finally, we show that CDKN3 protein is down-regulated in brain tumors. Our findings indicate that CDKN3 controls mitosis through the CDC2 signaling axis. These results have implications for targeted anticancer therapeutics.

  2. Chromosome Bridges Maintain Kinetochore-Microtubule Attachment throughout Mitosis and Rarely Break during Anaphase.

    Directory of Open Access Journals (Sweden)

    Judit Pampalona

    Full Text Available Accurate chromosome segregation during cell division is essential to maintain genome stability, and chromosome segregation errors are causally linked to genetic disorders and cancer. An anaphase chromosome bridge is a particular chromosome segregation error observed in cells that enter mitosis with fused chromosomes/sister chromatids. The widely accepted Breakage/Fusion/Bridge cycle model proposes that anaphase chromosome bridges break during mitosis to generate chromosome ends that will fuse during the following cell cycle, thus forming new bridges that will break, and so on. However, various studies have also shown a link between chromosome bridges and aneuploidy and/or polyploidy. In this study, we investigated the behavior and properties of chromosome bridges during mitosis, with the idea to gain insight into the potential mechanism underlying chromosome bridge-induced aneuploidy. We find that only a small number of chromosome bridges break during anaphase, whereas the rest persist through mitosis into the subsequent cell cycle. We also find that the microtubule bundles (k-fibers bound to bridge kinetochores are not prone to breakage/detachment, thus supporting the conclusion that k-fiber detachment is not the cause of chromosome bridge-induced aneuploidy. Instead, our data suggest that while the microtubules bound to the kinetochores of normally segregating chromosomes shorten substantially during anaphase, the k-fibers bound to bridge kinetochores shorten only slightly, and may even lengthen, during anaphase. This causes some of the bridge kinetochores/chromosomes to lag behind in a position that is proximal to the cell/spindle equator and may cause the bridged chromosomes to be segregated into the same daughter nucleus or to form a micronucleus.

  3. Chromosome Bridges Maintain Kinetochore-Microtubule Attachment throughout Mitosis and Rarely Break during Anaphase.

    Science.gov (United States)

    Pampalona, Judit; Roscioli, Emanuele; Silkworth, William T; Bowden, Brent; Genescà, Anna; Tusell, Laura; Cimini, Daniela

    2016-01-01

    Accurate chromosome segregation during cell division is essential to maintain genome stability, and chromosome segregation errors are causally linked to genetic disorders and cancer. An anaphase chromosome bridge is a particular chromosome segregation error observed in cells that enter mitosis with fused chromosomes/sister chromatids. The widely accepted Breakage/Fusion/Bridge cycle model proposes that anaphase chromosome bridges break during mitosis to generate chromosome ends that will fuse during the following cell cycle, thus forming new bridges that will break, and so on. However, various studies have also shown a link between chromosome bridges and aneuploidy and/or polyploidy. In this study, we investigated the behavior and properties of chromosome bridges during mitosis, with the idea to gain insight into the potential mechanism underlying chromosome bridge-induced aneuploidy. We find that only a small number of chromosome bridges break during anaphase, whereas the rest persist through mitosis into the subsequent cell cycle. We also find that the microtubule bundles (k-fibers) bound to bridge kinetochores are not prone to breakage/detachment, thus supporting the conclusion that k-fiber detachment is not the cause of chromosome bridge-induced aneuploidy. Instead, our data suggest that while the microtubules bound to the kinetochores of normally segregating chromosomes shorten substantially during anaphase, the k-fibers bound to bridge kinetochores shorten only slightly, and may even lengthen, during anaphase. This causes some of the bridge kinetochores/chromosomes to lag behind in a position that is proximal to the cell/spindle equator and may cause the bridged chromosomes to be segregated into the same daughter nucleus or to form a micronucleus.

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

    Science.gov (United States)

    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.

  5. Action of mercury in plant mitosis II

    Energy Technology Data Exchange (ETDEWEB)

    Lorente, R.

    1972-01-01

    The cytological abnormalities induced by mercurochrome on mitosis and meiosis of Allium cepa are studied and the capacity of the chemical agent to induce c-mitosis is shown. Inhibition of the cytokinetic process as well as alterations of the nucleoli and pollen-mother cells (from pachytene to division II) have also been observed. These cytological effects may be ascribed to the affinity of the mercurial compounds for the thyolic groups existing in the nucleoproteins and protoplasmic proteins, with the subsequent inhibitory effect on the enzymatic mechanisms.

  6. Shuttling of the autoantigen La between nucleus and cell surface after uv irradiation of human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bachmann, M.; Chang, S.; Slor, H.; Kukulies, J.; Mueller, W.E. (Universitaet, Mainz (Germany, F.R.))

    1990-12-01

    During the past years we have established that the nuclear autoantigen La shuttles between the nucleus and the cytoplasm in tumor cells after inhibition of transcription or virus infection. We reinvestigated this shuttling using primary human keratinocytes from both healthy donors and patients with xeroderma pigmentosum. Ultraviolet irradiation resulted in both an inhibition of transcription and a translocation of La protein from the nucleus to the cytoplasm. After a prolonged inhibition of transcription La protein relocated into the nucleus and assembled with nuclear storage regions. The uv-induced shuttling included a translocation to the cell surface, where La protein colocalized with epidermal growth factor receptors.

  7. Mitosis Methods & Protocols

    Directory of Open Access Journals (Sweden)

    CarloAlberto Redi

    2010-06-01

    Full Text Available Mitosis Methods & Protocols Andrew D. McAinsh (Edt Humana press, Totowa, New Jersey (USA Series: Springer Protocols Methods in Molecular Biology, Volume 545, 2009 ISBN: 978-1-60327-992-5   It is quite clear from the contents of this book that the remarkably fascinating phenomenon of mitosis (that captured, and still is capturing, the attention of entire generations of scientists is still open to research. This is mainly due to our lack of knowledge of so many multifaced events of this extraordinarly complex process. The reader giving a glace through the Contents and Contributors sections is speechless: All of the first-class models (i.e., budding yeast, Caenorabditis, Drosophila, Xenopus and Human are presented..... 

  8. Clathrin-mediated endocytosis is inhibited during mitosis.

    Science.gov (United States)

    Fielding, Andrew B; Willox, Anna K; Okeke, Emmanuel; Royle, Stephen J

    2012-04-24

    A long-standing paradigm in cell biology is the shutdown of endocytosis during mitosis. There is consensus that transferrin uptake is inhibited after entry into prophase and that it resumes in telophase. A recent study proposed that endocytosis is continuous throughout the cell cycle and that the observed inhibition of transferrin uptake is due to a decrease in available transferrin receptor at the cell surface, and not to a shutdown of endocytosis. This challenge to the established view is gradually becoming accepted. Because of this controversy, we revisited the question of endocytic activity during mitosis. Using an antibody uptake assay and controlling for potential changes in surface receptor density, we demonstrate the strong inhibition of endocytosis in mitosis of CD8 chimeras containing any of the three major internalization motifs for clathrin-mediated endocytosis (YXXΦ, [DE]XXXL[LI], or FXNPXY) or a CD8 protein with the cytoplasmic tail of the cation-independent mannose 6-phosphate receptor. The shutdown is not gradual: We describe a binary switch from endocytosis being "on" in interphase to "off" in mitosis as cells traverse the G(2)/M checkpoint. In addition, we show that the inhibition of transferrin uptake in mitosis occurs despite abundant transferrin receptor at the surface of HeLa cells. Our study finds no support for the recent idea that endocytosis continues during mitosis, and we conclude that endocytosis is temporarily shutdown during early mitosis.

  9. In situ label-free cell viability assessment of nucleus pulposus tissue.

    Science.gov (United States)

    Dittmar, Roman; van Dijk, Bart G M; van Zandvoort, Marc A M J; Ito, Keita

    2014-04-01

    Regenerative medicine approaches aiming at treating degenerating intervertebral discs, a major cause of back pain, are increasingly tested in ex-vivo disc explant models mimicking in-vivo conditions. For assessing the efficacy of regenerative therapies, cell viability is commonly measured requiring specific labels to stain cells. Here, we demonstrate and evaluate how cellular auto-fluorescence can be utilized to non-invasively assess viability in disc tissue in-situ using label-free two-photon microscopy. Live and dead bovine disc cells (0% and 100% cell viability) from the nucleus pulposus were seeded into collagen gels and auto-fluorescence was characterized. Subsequently, nucleus pulposus explants were cultured for 6 days in media with different glucose supplementation (0, 0.25, 0.5, and 1 g/L) to induce different degrees of cell death. Then, samples were split and viability was assessed using label-free two-photon microscopy and conventional staining. Results show that live and dead nucleus pulposus cells systematically emit auto-fluorescent light with distinct characteristics. Cell viability values obtained with label-free microscopy did not significantly differ from those acquired with staining. In summary, monitoring auto-fluorescence facilitates accurate cell viability assessment in nucleus tissue requiring no additional dyes. Thus, this technique may be suitable for pre-clinical testing of regenerative therapies in nucleus pulposus cultures. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:545-550, 2014.

  10. Action of mercury in plant mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Lorente, R.

    1969-01-01

    The p-chloromercuriphenylsulfonate (PMPS) acts on mitosis of Allium cepa as a substance typically c-mitotic. Metaphases with the classic c-pair as well as polyploid cells, multipolar anaphases, polynucleate and aneuploid cells were observed. These effects were observed from the first 24 hr post-treatment and after 12 and 15 days of recuperation in some cases. These effects by PMPS are immediate and persist in root meristematic cells of Allium cepa.

  11. Mitosis and growth in biological tissues

    OpenAIRE

    Mombach, Jose Carlos Merino; Almeida, Rita Maria Cunha de; Iglesias, Jose Roberto

    1993-01-01

    We present a simulation of the growth of a two-dimensional biological cellular system in which the cells experience mitosis whenever the (area)/(perimeter) ratio reaches a critical value. The model also includes the effect of interfacial energy and temperature. A stationary state with a constant average area is attained. We calculate the distribution of cells as a function of area, perimeter, and number of sides and also the two-cell correlation function. The results depend on temperature and...

  12. Parvoviruses cause nuclear envelope breakdown by activating key enzymes of mitosis.

    Science.gov (United States)

    Porwal, Manvi; Cohen, Sarah; Snoussi, Kenza; Popa-Wagner, Ruth; Anderson, Fenja; Dugot-Senant, Nathalie; Wodrich, Harald; Dinsart, Christiane; Kleinschmidt, Jürgen A; Panté, Nelly; Kann, Michael

    2013-10-01

    Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca⁺⁺ efflux from the lumen between inner and outer nuclear membrane we found that Ca⁺⁺ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis.

  13. Parvoviruses cause nuclear envelope breakdown by activating key enzymes of mitosis.

    Directory of Open Access Journals (Sweden)

    Manvi Porwal

    2013-10-01

    Full Text Available Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca⁺⁺ efflux from the lumen between inner and outer nuclear membrane we found that Ca⁺⁺ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis.

  14. Unlike in Drosophila Meroistic Ovaries, hippo represses notch in Blattella germanica Panoistic ovaries, triggering the mitosis-endocycle switch in the follicular cells.

    Directory of Open Access Journals (Sweden)

    Paula Irles

    Full Text Available During insect oogenesis, the follicular epithelium undergoes both cell proliferation and apoptosis, thus modulating ovarian follicle growth. The Hippo pathway is key in these processes, and has been thoroughly studied in the meroistic ovaries of Drosophila melanogaster. However, nothing is known about the role of the Hippo pathway in primitive panoistic ovaries. This work examines the mRNA expression levels of the main components of the Hippo pathway in the panoistic ovary of the basal insect species Blattella germanica, and demonstrates the function of Hippo through RNAi. In Hippo-depleted specimens, the follicular cells of the basal ovarian follicles proliferate without arresting cytokinesis; the epithelium therefore becomes bilayered, impairing ovarian follicle growth. This phenotype is accompanied by long stalks between the ovarian follicles. In D. melanogaster loss of function of Notch determines that the stalk is not developed. With this in mind, we tested whether Hippo and Notch pathways are related in B. germanica. In Notch (only-depleted females, no stalks were formed between the ovarian follicles. Simultaneous depletion of Hippo and Notch rescued partially the stalk to wild-type. Unlike in the meroistic ovaries of D. melanogaster, in panoistic ovaries the Hippo pathway appears to regulate follicular cell proliferation by acting as a repressor of Notch, triggering the switch from mitosis to the endocycle in the follicular cells. The phylogenetically basal position of B. germanica suggests that this might be the ancestral function of Hippo in insect ovaries.

  15. The Change of Microtubule Cytoskeleton in the Stem-Tip Cells of Sugarcane during Mitosis%甘蔗茎尖细胞有丝分裂过程中微管骨架的变化

    Institute of Scientific and Technical Information of China (English)

    李志刚; 赵洪波; 李素丽; 杨丽涛; 李杨瑞

    2008-01-01

    In order to understand the microtubule change of monoeotyls stem-tip during mitosis,the arrangement,transformation of microtubule array and its relation with chromosome movement during mitosis were studied with freezing microtome,indirect immunofluorescence,DAPI staining and fluorescence microscopy.The results showed that nueleolus was intact when the cortical miemtubules formed;cortical mierotubulos were changed into phramoplast microtubule bands at mitosis prophase.When phramoplast microtubule came into being,nuclear membrane was ruptured and chromosome was arranged at the position of cell plate;subsequently,phramoplast microtubules were changed into phragmoplast mierotubules,phramoplast mierotubules were shortening and microtubules on the sides of cell plate were increasing gradually,during this course sister chromatid was separated by microtubules at cell plate and tract to the two poles,forming phragmoplast microtubules.Then the nucleolus of two daughter cells formed and separated in the end with the increase of cells numbers.Therefore,cell division orientation could be judged from the arrangement of cell microtubules in different periods in order to understand its growth status.

  16. On the robustness of SAC silencing in closed mitosis

    Science.gov (United States)

    Ruth, Donovan; Liu, Jian

    Mitosis equally partitions sister chromatids to two daughter cells. This is achieved by properly attaching these chromatids via their kinetochores to microtubules that emanate from the spindle poles. Once the last kinetochore is properly attached, the spindle microtubules pull the sister chromatids apart. Due to the dynamic nature of microtubules, however, kinetochore-microtubule attachment often goes wrong. When this erroneous attachment occurs, it locally activates an ensemble of proteins, called the spindle assembly checkpoint proteins (SAC), which halts the mitotic progression until all the kinetochores are properly attached by spindle microtubules. The timing of SAC silencing thus determines the fidelity of chromosome segregation. We previously established a spatiotemporal model that addresses the robustness of SAC silencing in open mitosis for the first time. Here, we focus on closed mitosis by examining yeast mitosis as a model system. Though much experimental work has been done to study the SAC in cells undergoing closed mitosis, the processes responsible are not well understood. We leverage and extend our previous model to study SAC silencing mechanism in closed mitosis. We show that a robust signal of the SAC protein accumulation at the spindle pole body can be achieved. This signal is a nonlinear increasing function of number of kinetochore-microtubule attachments, and can thus serve as a robust trigger to time the SAC silencing. Together, our mechanism provides a unified framework across species that ensures robust SAC silencing and fidelity of chromosome segregation in mitosis. Intramural research program in NHLBI at NIH.

  17. ANA detected by ELISA using nucleus of egg cell as antigen.

    Science.gov (United States)

    Hui, Liu; Shijun, Li; Yue, Ma

    2008-01-01

    Antinuclear antibodies, ANA, were usually detected with antigen of somatic cell nucleus. It has not been reported to detect ANA with egg cell nucleus as antigen. Enzyme linked immuosorbent assay, ELISA, coated with yolk was developed to detect ANA in our laboratory. A quality control test, cross absorption test, and cross antibody-induced test with yolk were performed. Results showed a good agreement between our method and IFA through measurement of the same samples from patients suspected of having rheumatic connective tissue diseases (Kappa=0.668, P=0.000). The results were not influenced by the RF and different sources of egg. CVs of inter-assay, were less than 10%. The cross absorption test was negative, as well; the ANA to somatic cell nucleus could be induced with egg cell nucleus. It is implied that there were both cross as well as overlapped Egg-ANA and Somatic-ANA. As egg nucleus, its volume was large, its purification was simple, so the better method might be established.

  18. Cloning and characterization of a Dim1-like mitosis gene of Spodoptera frugiperda cells (Sf9) induced by Autographa californica multiple nucleopolyhedrovirus.

    Science.gov (United States)

    Mehrabadi, Mohammad; Hussain, Mazhar; Asgari, Sassan

    2013-06-01

    Dim1 proteins are evolutionarily highly conserved throughout the eukaryotes and are present in numerous species. These proteins are essential for mitosis and pre-mRNA splicing. In this study, the full-length cDNA of Dim1-like gene from Spodoptera frugiperda cells (Sf9) was obtained. S. frugiperda Dim1 (SfDim1) cDNA is comprised of 975 bp including a 429 bp open reading frame (ORF), 225 bp 5' untranslated region (5' UTR), and 321 bp 3' untranslated region (3' UTR) with a poly A tail. The predicted polypeptide encoded by this gene is 142 aa with a molecular weight of 16.76 kDa and a PI of 5.53. Sequence alignment and phylogenetic analysis showed high similarities with Dim1 of other species. The evolutionary conserved site of Dim1 proteins ((35)Asp-Pro-Thr-Cys(38)) is also present in SfDim1. Silencing of SfDim1 gene decreased cell proliferation at 72 h post-treatment in comparison to mock and control transfected cells. Using RT-PCR, we found relatively higher SfDim1 transcript levels following Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infection compared to mock-infected cells from 4h post-infection (hpi) up until 24 hpi. The expression level diminished dramatically at 36 hpi up to 120 hpi with no expression detected at 144 hpi. Silencing of SfDim1 resulted in lower levels of virus DNA production in comparison to mock-infected cells, which suggested that SfDim1 might benefit the virus and facilitate viral replication. Overall, the results showed that SfDim1 protein is involved in cell proliferation as well as cell-virus interaction.

  19. Regulatory Genes Controlling Mitosis in the Fission Yeast SCHIZOSACCHAROMYCES POMBE

    OpenAIRE

    Nurse, Paul; Thuriaux, Pierre

    1980-01-01

    Fifty-two wee mutants that undergo mitosis and cell division at a reduced size compared with wild type have been genetically analyzed. The mutants define two genes, wee1 and cdc2, which control the timing of mitosis. Fifty-one of the mutants map at the wee1 locus, which is unlinked to any known cdc gene. One of the wee1 alleles has been shown to be nonsense suppressible. The 52nd wee mutant maps within cdc2. Previously, only temperature-sensitive mutants that become blocked at mitosis have be...

  20. Histone H1 of Trypanosoma cruzi is concentrated in the nucleolus region and disperses upon phosphorylation during progression to mitosis.

    Science.gov (United States)

    Gutiyama, Luciana M; da Cunha, Julia P Chagas; Schenkman, Sergio

    2008-04-01

    Phosphorylation of histone H1 is intimately related to the cell cycle progression in higher eukaryotes, reaching maximum levels during mitosis. We have previously shown that in the flagellated protozoan Trypanosoma cruzi, which does not condense chromatin during mitosis, histone H1 is phosphorylated at a single cyclin-dependent kinase site. By using an antibody that recognizes specifically the phosphorylated T. cruzi histone H1 site, we have now confirmed that T. cruzi histone H1 is also phosphorylated in a cell cycle-dependent manner. Differently from core histones, the bulk of nonphosphorylated histone H1 in G(1) and S phases of the cell cycle is concentrated in the central regions of the nucleus, which contains the nucleolus and less densely packed chromatin. When cells pass G(2), histone H1 becomes phosphorylated and starts to diffuse. At the onset of mitosis, histone H1 phosphorylation is maximal and found in the entire nuclear space. As permeabilized parasites preferentially lose phosphorylated histone H1, we conclude that this modification promotes its release from less condensed and nucleolar chromatin after G(2).

  1. Temperature-induced labelling of Fluo-3 AM selectively yields brighter nucleus in adherent cells

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Guixian [The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, School of Physics and TEDA Applied Physics Institute, Nankai University, Tianjin (China); Pan, Leiting, E-mail: plt@nankai.edu.cn [The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, School of Physics and TEDA Applied Physics Institute, Nankai University, Tianjin (China); Li, Cunbo; Hu, Fen; Shi, Xuechen; Lee, Imshik [The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, School of Physics and TEDA Applied Physics Institute, Nankai University, Tianjin (China); Drevenšek-Olenik, Irena [Faculty of Mathematics and Physics, University of Ljubljana, and J. Stefan Institute, Ljubljana (Slovenia); Zhang, Xinzheng; Xu, Jingjun [The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, School of Physics and TEDA Applied Physics Institute, Nankai University, Tianjin (China)

    2014-01-17

    Highlights: •We detailedly examine temperature effects of Fluo-3 AM labelling in adherent cells. •4 °C Loading and 20 °C de-esterification of Fluo-3 AM yields brighter nuclei. •Brighter nuclei labelling by Fluo-3 AM also depends on cell adhesion quality. •A qualitative model of the brighter nucleus is proposed. -- Abstract: Fluo-3 is widely used to study cell calcium. Two traditional approaches: (1) direct injection and (2) Fluo-3 acetoxymethyl ester (AM) loading, often bring conflicting results in cytoplasmic calcium ([Ca{sup 2+}]{sub c}) and nuclear calcium ([Ca{sup 2+}]{sub n}) imaging. AM loading usually yields a darker nucleus than in cytoplasm, while direct injection always induces a brighter nucleus which is more responsive to [Ca{sup 2+}]{sub n} detection. In this work, we detailedly investigated the effects of loading and de-esterification temperatures on the fluorescence intensity of Fluo-3 in response to [Ca{sup 2+}]{sub n} and [Ca{sup 2+}]{sub c} in adherent cells, including osteoblast, HeLa and BV2 cells. Interestingly, it showed that fluorescence intensity of nucleus in osteoblast cells was about two times larger than that of cytoplasm when cells were loaded with Fluo-3 AM at 4 °C and allowed a subsequent step for de-esterification at 20 °C. Brighter nuclei were also acquired in HeLa and BV2 cells using the same experimental condition. Furthermore, loading time and adhesion quality of cells had effect on fluorescence intensity. Taken together, cold loading and room temperature de-esterification treatment of Fluo-3 AM selectively yielded brighter nucleus in adherent cells.

  2. Inactivation of Cdk1/Cyclin B in metaphase-arrested mouse FT210 cells induces exit from mitosis without chromosome segregation or cytokinesis and allows passage through another cell cycle.

    Science.gov (United States)

    Paulson, James R

    2007-04-01

    It is well known that inactivation of Cdk1/Cyclin B is required for cells to exit mitosis. The work reported here tests the hypothesis that Cdk1/Cyclin B inactivation is not only necessary but also sufficient to induce mitotic exit and reestablishment of the interphase state. This hypothesis predicts that inactivation of Cdk1 in metaphase-arrested cells will induce the M to G1-phase transition. It is shown that when mouse FT210 cells (in which Cdk1 is temperature-sensitive) are arrested in metaphase and then shifted to their non-permissive temperature, they rapidly exit mitosis as evidenced by reassembly of interphase nuclei, decondensation of chromosomes, and dephosphorylation of histones H1 and H3. The resulting interphase cells are functionally normal as judged by their ability to progress through another cell cycle. However, they have double the normal number of chromosomes because they previously bypassed anaphase, chromosome segregation, and cytokinesis. These results, taken together with other observations in the literature, strongly suggest that in mammalian cells, inactivation of Cdk1/cyclin B is the trigger for mitotic exit and reestablishment of the interphase state.

  3. Ultrastructure and immunocytochemical characteristics of cells in the octopus cell area of the rat cochlear nucleus: comparison with multipolar cells.

    Science.gov (United States)

    Alibardi, Lorenzo

    2003-01-01

    Cells in the octopus cell area of the rat ventral cochlear nucleus have been connected to the monaural interpretation of spectral patterns of sound such as those derived from speech. This is possible by their fast onset of firing after each octopus cell and its dendrites have been contacted by many auditory fibres carrying different frequencies. The cytological characteristics that make these large cells able to perform such a function have been studied with ultrastructural immunocytochemistry for glycine, GABA and glutamate, and compared to that of other multipolar neurons of other regions of the ventral cochlear nucleus. Cells in the octopus cell area have an ultrastructure similar to large-giant D-multipolar neurons present in other areas of the cochlear nucleus, from which they differ by the presence of a larger excitatory axo-somatic synaptic input and larger mitochondria. Octopus cells are glycine and GABA negative, and glutamate positive with different degree. Large octopus cells receive more axo-somatic boutons than smaller octopus cells. Fusiform octopus cells are found sparsely within the intermediate acoustic striae. These cells are large to giant excitatory neurons (23-35 microm) with 62-85% of their irregular perimeter covered with large axo-somatic synaptic boutons. Most boutons contain round vesicles and are glycine and GABA negative but glutamate positive. The latter excitatory boutons represent about 70% of the input to octopus cells. Glycine positive boutons with flat and pleomorphic vesicles account for 9-10% of the input while GABA-ergic boutons with pleomorphic vesicles represent about 20% of the synaptic input. Other few, multipolar cells within the rat octopus cell area are surrounded by more inhibitory than excitatory terminals which contain flat and pleomorphic vesicles, a feature distinctive from that of true octopus cells. The latter resemble multipolar cells seen outside the octopus cell area that project to the contralateral inferior

  4. Genome accessibility is widely preserved and locally modulated during mitosis.

    Science.gov (United States)

    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.

  5. Synchronizing Progression of Schizosaccharomyces pombe Cells from Prophase through Mitosis and into S Phase with nda3-KM311 Arrest Release.

    Science.gov (United States)

    Hagan, Iain M; Grallert, Agnes; Simanis, Viesturs

    2016-08-01

    Here, we describe how the rapid reversibility of the nda3-KM311 cold-sensitive β-tubulin mutation was optimized by Mitsuhiro Yanagida's laboratory to synchronize mitotic progression in an entire cell population. The inability to form microtubules following the loss of β-tubulin function at 20°C triggers the spindle assembly checkpoint, which arrests mitotic progression. Restoration of β-tubulin function by rewarming to 30°C (or higher) releases the arrest, generating a highly synchronous progression through mitosis. The viability of nda3-KM311 strains at 30°C makes it feasible to generate double mutants between nda3-KM311 and any temperature-sensitive mutant that can also grow at 30°C. These double mutants can be used in reciprocal shift analyses, in which cold-induced early mitotic arrest is relieved by a shift to 36°C, which then inactivates the product of the second mutant gene. The addition of microtubule depolymerizing drugs before the return to 36°C will maintain checkpoint signaling at 36°C transiently, permitting analysis of the impact of temperature-sensitive mutations on checkpoint function. Silencing the checkpoint of nda3-KM311-arrested cells at 20°C through chemical inhibition of aurora kinase is a powerful way to study checkpoint recovery pathways and mitotic exit without anaphase.

  6. The three-dimensional organization of telomeres in the nucleus of mammalian cells

    Directory of Open Access Journals (Sweden)

    Perrin Mathilde

    2004-06-01

    Full Text Available Abstract Background The observation of multiple genetic markers in situ by optical microscopy and their relevance to the study of three-dimensional (3D chromosomal organization in the nucleus have been greatly developed in the last decade. These methods are important in cancer research because cancer is characterized by multiple alterations that affect the modulation of gene expression and the stability of the genome. It is, therefore, essential to analyze the 3D genome organization of the interphase nucleus in both normal and cancer cells. Results We describe a novel approach to study the distribution of all telomeres inside the nucleus of mammalian cells throughout the cell cycle. It is based on 3D telomere fluorescence in situ hybridization followed by quantitative analysis that determines the telomeres' distribution in the nucleus throughout the cell cycle. This method enables us to determine, for the first time, that telomere organization is cell-cycle dependent, with assembly of telomeres into a telomeric disk in the G2 phase. In tumor cells, the 3D telomere organization is distorted and aggregates are formed. Conclusions The results emphasize a non-random and dynamic 3D nuclear telomeric organization and its importance to genomic stability. Based on our findings, it appears possible to examine telomeric aggregates suggestive of genomic instability in individual interphase nuclei and tissues without the need to examine metaphases. Such new avenues of monitoring genomic instability could potentially impact on cancer biology, genetics, diagnostic innovations and surveillance of treatment response in medicine.

  7. Nuclear Reprogramming and Mitosis--how does mitosis enhance changes in gene expression?

    Science.gov (United States)

    Halley-Stott, Richard P

    2015-01-01

    Nuclear reprogramming changes the identity of cells by changing gene expression programmes. Two recent pieces of work have highlighted the role that mitosis plays in enhancing the success of nuclear reprogramming. This Point of View article examines this work in the context of nuclear reprogramming.

  8. Mitosis in Oedogonium: spindle microfilaments and the origin of the kinetochore fiber.

    Science.gov (United States)

    Schibler, M J; Pickett-Heaps, J D

    1980-10-01

    New ultrastructural observations of mitosis in the closed spindle of Oedogonium cardiacum have been made using cells fixed with glutaraldehyde and tannic acid. Fine filaments 5 to 8 nm in diameter are attached to kinetochores from prophase through anaphase. Some are free in the early division nucleus while others emanate from forming kinetochores at prophase when few if any microtubules (MTs) are inside the nucleus. During prometaphase, MTs invade the nucleus from the poles and appear to interact with the microfilaments. Early in prometaphase, numerous MTs are laterally associated with kinetochores, and the kinetochore fiber is often formed first at one kinetochore of a pair. During metaphase and anaphase, the microfilaments are interspersed among the MTs of these kinetochore fibers. There also is an ill-defined matrix concentrated in the kinetochore fiber, and MTs are often coated irregularly with osmiophilic material. Live mitotic cells of Oedogonium were studied using time lapse cinematography, and we correlate these observations with the above results. We conclude that these microfilaments may constitute one structural component of the traction apparatus that moves chromosomes during metakinesis and anaphase, and that at least some (and possibly many) of the MTs of the kinetochore fiber are derived from those entering the nucleus at prometaphase.

  9. The Role of BRCA1/BARD1 Heterodimers in the Mitosis-Interphase Transition

    Science.gov (United States)

    2007-05-01

    Littlepage, L., Wu, H., and Ruderman, J. V. (2003). Aurora A, meiosis and mitosis. Biol Cell 96, 215-229. Deng, C. X., and Wang, R. H. (2003...mitosis. J. Cell Biol. 120, 947–957. Crane, R., Gadea, B., Littlepage, L., Wu, H., and Ruderman, J.V. (2003). Aurora A, meiosis and mitosis. Biol. Cell...and the mitotic state. Extract naturally arrested in metaphase of meiosis II (CSF-arrested extract) was prepared by crushing unfertilized eggs in

  10. Turning meiosis into mitosis.

    Directory of Open Access Journals (Sweden)

    Isabelle d'Erfurth

    2009-06-01

    Full Text Available Apomixis, or asexual clonal reproduction through seeds, is of immense interest due to its potential application in agriculture. One key element of apomixis is apomeiosis, a deregulation of meiosis that results in a mitotic-like division. We isolated and characterised a novel gene that is directly involved in controlling entry into the second meiotic division. By combining a mutation in this gene with two others that affect key meiotic processes, we created a genotype called MiMe in which meiosis is totally replaced by mitosis. The obtained plants produce functional diploid gametes that are genetically identical to their mother. The creation of the MiMe genotype and apomeiosis phenotype is an important step towards understanding and engineering apomixis.

  11. Differentiation of Mesenchymal Stem Cells into Nucleus Pulposus Cells In Vitro

    Institute of Scientific and Technical Information of China (English)

    Fenghua TAO; Feng LI; Guanghui LI; Feng PAN

    2008-01-01

    To find a new source of seed cells for constructing tissue-engineered intervertebral disc, nucleus pulposus (NP) cells and mesenchymal stem cells (MSCs) were isolated from New Zealand white rabbits. The nuclcus pulposus cells population was fluorescence-laelled and co-cultured with MSCs with or without direct contact. Morphological changes were observed every 12 h. Semi-quantitaive reverse transcriptase-polymerase chain reaction was performed to assess the expression levels of Sox-9, aggreacan and type Ⅱ collagen every 24 h after the co-culture. MSCs treated with direct contact rounded up and presented a ring-like appearance. The expression of marker genes was significantly increased when cells were co-cultured with direct contact for 24 h. No significant change was found after coculture without direct contact. Co-culture of NP cells and MSCs with direct contact is a reliable method for generating large amount of NP cells used for cell-based tissue engineering therapy.

  12. Promyelocytic leukemia bodies tether to early endosomes during mitosis.

    Science.gov (United States)

    Palibrk, Vuk; Lång, Emma; Lång, Anna; Schink, Kay Oliver; Rowe, Alexander D; Bøe, Stig Ove

    2014-01-01

    During mitosis the nuclear envelope breaks down, leading to potential interactions between cytoplasmic and nuclear components. PML bodies are nuclear structures with tumor suppressor and antiviral functions. Early endosomes, on the other hand, are cytoplasmic vesicles involved in transport and growth factor signaling. Here we demonstrate that PML bodies form stable interactions with early endosomes immediately following entry into mitosis. The 2 compartments remain stably associated throughout mitosis and dissociate in the cytoplasm of newly divided daughter cells. We also show that a minor subset of PML bodies becomes anchored to the mitotic spindle poles during cell division. The study demonstrates a stable mitosis-specific interaction between a cytoplasmic and a nuclear compartment.

  13. Extracellular Matrix Ligand and Stiffness Modulate Immature Nucleus Pulposus Cell-Cell Interactions

    Science.gov (United States)

    Gilchrist, Christopher L.; Darling, Eric M.; Chen, Jun; Setton, Lori A.

    2011-01-01

    The nucleus pulposus (NP) of the intervertebral disc functions to provide compressive load support in the spine, and contains cells that play a critical role in the generation and maintenance of this tissue. The NP cell population undergoes significant morphological and phenotypic changes during maturation and aging, transitioning from large, vacuolated immature cells arranged in cell clusters to a sparse population of smaller, isolated chondrocyte-like cells. These morphological and organizational changes appear to correlate with the first signs of degenerative changes within the intervertebral disc. The extracellular matrix of the immature NP is a soft, gelatinous material containing multiple laminin isoforms, features that are unique to the NP relative to other regions of the disc and that change with aging and degeneration. Based on this knowledge, we hypothesized that a soft, laminin-rich extracellular matrix environment would promote NP cell-cell interactions and phenotypes similar to those found in immature NP tissues. NP cells were isolated from porcine intervertebral discs and cultured in matrix environments of varying mechanical stiffness that were functionalized with various matrix ligands; cellular responses to periods of culture were assessed using quantitative measures of cell organization and phenotype. Results show that soft (<720 Pa), laminin-containing extracellular matrix substrates promote NP cell morphologies, cell-cell interactions, and proteoglycan production in vitro, and that this behavior is dependent upon both extracellular matrix ligand and substrate mechanical properties. These findings indicate that NP cell organization and phenotype may be highly sensitive to their surrounding extracellular matrix environment. PMID:22087260

  14. Extracellular matrix ligand and stiffness modulate immature nucleus pulposus cell-cell interactions.

    Directory of Open Access Journals (Sweden)

    Christopher L Gilchrist

    Full Text Available The nucleus pulposus (NP of the intervertebral disc functions to provide compressive load support in the spine, and contains cells that play a critical role in the generation and maintenance of this tissue. The NP cell population undergoes significant morphological and phenotypic changes during maturation and aging, transitioning from large, vacuolated immature cells arranged in cell clusters to a sparse population of smaller, isolated chondrocyte-like cells. These morphological and organizational changes appear to correlate with the first signs of degenerative changes within the intervertebral disc. The extracellular matrix of the immature NP is a soft, gelatinous material containing multiple laminin isoforms, features that are unique to the NP relative to other regions of the disc and that change with aging and degeneration. Based on this knowledge, we hypothesized that a soft, laminin-rich extracellular matrix environment would promote NP cell-cell interactions and phenotypes similar to those found in immature NP tissues. NP cells were isolated from porcine intervertebral discs and cultured in matrix environments of varying mechanical stiffness that were functionalized with various matrix ligands; cellular responses to periods of culture were assessed using quantitative measures of cell organization and phenotype. Results show that soft (<720 Pa, laminin-containing extracellular matrix substrates promote NP cell morphologies, cell-cell interactions, and proteoglycan production in vitro, and that this behavior is dependent upon both extracellular matrix ligand and substrate mechanical properties. These findings indicate that NP cell organization and phenotype may be highly sensitive to their surrounding extracellular matrix environment.

  15. The plant cell nucleus: a true arena for the fight between plants and pathogens.

    Science.gov (United States)

    Deslandes, Laurent; Rivas, Susana

    2011-01-01

    Communication between the cytoplasm and the nucleus is a fundamental feature shared by both plant and animal cells. Cellular factors involved in the transport of macromolecules through the nuclear envelope, including nucleoporins, importins and Ran-GTP related components, are conserved among a variety of eukaryotic systems. Interestingly, mutations in these nuclear components compromise resistance signalling, illustrating the importance of nucleocytoplasmic trafficking in plant innate immunity. Indeed, spatial restriction of defence regulators by the nuclear envelope and stimulus-induced nuclear translocation constitute an important level of defence-associated gene regulation in plants. A significant number of effectors from different microbial pathogens are targeted to the plant cell nucleus. In addition, key host factors, including resistance proteins, immunity components, transcription factors and transcriptional regulators shuttle between the cytoplasm and the nucleus, and their level of nuclear accumulation determines the output of the defence response, further confirming the crucial role played by the nucleus during the interaction between plants and pathogens. Here, we discuss recent findings that situate the nucleus at the frontline of the mutual recognition between plants and invading microbes.

  16. Rhodococcus fascians infection accelerates progression of tobacco BY-2 cells into mitosis through rapid changes in plant gene expression.

    Science.gov (United States)

    Vandeputte, Olivier; Vereecke, Danny; Mol, Adeline; Lenjou, Marc; Van Bockstaele, Dirk; El Jaziri, Mondher; Baucher, Marie

    2007-01-01

    * To characterize plant cell cycle activation following Rhodococcus fascians infection, bacterial impact on cell cycle progression of tobacco BY-2 cells was investigated. * S-phase-synchronized BY-2 cells were cocultivated with R. fascians and cell cycle progression was monitored by measuring mitotic index, cell cycle gene expression and flow cytometry parameters. Cell cycle alteration was further investigated by cDNA-AFLP (amplified fragment length polymorphism). * It was shown that cell cycle progression of BY-2 cells was accelerated only upon infection with bacteria whose virulence gene expression was induced by a leafy gall extract. Thirty-eight BY-2 genes showed a differential expression within 6 h post-infection. Among these, seven were previously associated with specific plant cell cycle phases (in particular S and G2/M phases). Several genes also showed a differential expression during leafy gall formation. * R. fascians-infected BY-2 cells provide a simple model to identify plant genes related to leafy gall development. R. fascians can also be regarded as a useful biotic agent to alter cell cycle progression and, thereby, gain a better understanding of cell cycle regulation in plants.

  17. Distinct chromatin environment associated with phosphorylated H3S10 histone during pollen mitosis I in orchids.

    Science.gov (United States)

    Sharma, Santosh Kumar; Yamamoto, Maki; Mukai, Yasuhiko

    2017-01-01

    Pollen developmental pathway in plants involving synchronized transferal of cellular divisions from meiosis (microsporogenesis) to mitosis (pollen mitosis I/II) eventually offers a unique "meiosis-mitosis shift" at pollen mitosis I. Since the cell type (haploid microspore) and fate of pollen mitosis I differ from typical mitosis (in meristem cells), it is immensely important to analyze the chromosomal distribution of phosphorylated H3S10 histone during atypical pollen mitosis I to comprehend the role of histone phosphorylation in pollen development. We investigated the chromosomal phosphorylation of H3S10 histone during pollen mitosis I in orchids using immunostaining technique. The chromosomal distribution of H3S10ph during pollen mitosis I revealed differential pattern than that of typical mitosis in plants, however, eventually following the similar trends of mitosis in animals where H3S10 phosphorylation begins in the pericentromeric regions first, later extending to the whole chromosomes, and finally declining at anaphase/early cytokinesis (differentiation of vegetative and generative cells). The study suggests that the chromosomal distribution of H3S10ph during cell division is not universal and can be altered between different cell types encoded for diverse cellular processes. During pollen development, phosphorylation of histone might play a critical role in chromosome condensation events throughout pollen mitosis I in plants.

  18. Three-Dimensional Organization of Chromosome Territories and the Human Interphase Cell Nucleus

    NARCIS (Netherlands)

    T.A. Knoch (Tobias); C. Münkel (Christian); J. Langowski (Jörg)

    1998-01-01

    textabstractTo study the three-dimensional organization of chromosome territories and the human interphase cell nucleus we developed models which could be compared to experiments. Despite the successful linear sequencing of the human genome its 3D-organization is widely unknown. Using Monte Carl

  19. Three-dimensional organization of chromosome territories in the human interphase cell nucleus

    NARCIS (Netherlands)

    T.A. Knoch (Tobias); C. Münkel (Christian); J. Langowski (Jörg)

    1999-01-01

    markdownabstractTo study the three-dimensional organization of chromosome territories and the human interphase cell nucleus we developed models which could be compared to experiments. Despite the successful linear sequencing of the human genome its 3D-organization is widely unknown. Using Monte Carl

  20. Three-Dimensional Organization of Chromosome Territories and the Human Cell Nucleus

    NARCIS (Netherlands)

    T.A. Knoch (Tobias)

    1999-01-01

    textabstractTo study the three-dimensional organization of chromosome territories and the human interphase cell nucleus we developed models, which could be compared to experiments. Despite the successful linear sequencing of the human genome its 3D-organization is widely unknown. Using Monte Car

  1. Nucleus Retroambiguus Projections To Lumbosacral Mononeuronal Cell Groups In The Male Cat.

    NARCIS (Netherlands)

    VanderHorst, Veronique G.J.M.; Holstege, Gert

    1997-01-01

    Recently, in the female cat, nucleus retroambiguus (NRA) projections have been described as distinct motoneuronal cell groups in the lumbar enlargement, possibly involved in lordosis behavior. The present study deals with the NRA-lumbosacral pathway in the male cat. Lumbosacral injections of wheat g

  2. Nucleus retroambiguous projections to lumbosacral motoneuronal cell groups in the male cat

    NARCIS (Netherlands)

    Vanderhorst, VGJM; Holstege, G

    1997-01-01

    Recently, in the female cat, nucleus retroambiguus (NRA) projections have been described as distinct motoneuronal cell groups in the lumbar enlargement, possibly involved in lordosis behavior. The present study deals with the NRA-lumbosacral pathway in the male cat, Lumbosacral injections of wheat g

  3. Concentration Sensing by the Moving Nucleus in Cell Fate Determination: A Computational Analysis.

    Directory of Open Access Journals (Sweden)

    Varun Aggarwal

    Full Text Available During development of the vertebrate neuroepithelium, the nucleus in neural progenitor cells (NPCs moves from the apex toward the base and returns to the apex (called interkinetic nuclear migration at which point the cell divides. The fate of the resulting daughter cells is thought to depend on the sampling by the moving nucleus of a spatial concentration profile of the cytoplasmic Notch intracellular domain (NICD. However, the nucleus executes complex stochastic motions including random waiting and back and forth motions, which can expose the nucleus to randomly varying levels of cytoplasmic NICD. How nuclear position can determine daughter cell fate despite the stochastic nature of nuclear migration is not clear. Here we derived a mathematical model for reaction, diffusion, and nuclear accumulation of NICD in NPCs during interkinetic nuclear migration (INM. Using experimentally measured trajectory-dependent probabilities of nuclear turning, nuclear waiting times and average nuclear speeds in NPCs in the developing zebrafish retina, we performed stochastic simulations to compute the nuclear trajectory-dependent probabilities of NPC differentiation. Comparison with experimentally measured nuclear NICD concentrations and trajectory-dependent probabilities of differentiation allowed estimation of the NICD cytoplasmic gradient. Spatially polarized production of NICD, rapid NICD cytoplasmic consumption and the time-averaging effect of nuclear import/export kinetics are sufficient to explain the experimentally observed differentiation probabilities. Our computational studies lend quantitative support to the feasibility of the nuclear concentration-sensing mechanism for NPC fate determination in zebrafish retina.

  4. THE COMPLEX ORGANIZATION OF EUKARYOTIC CELL NUCLEUS: THE NUCLEAR BODIES (I

    Directory of Open Access Journals (Sweden)

    Cristian Campeanu

    2012-10-01

    Full Text Available Identified short time after the discovery of cells, over 300 years ago, the cell nucleus of eukaryotes continuously focused the interest of scientists, which used increasingly sophisticated research tools to clarify its complex structure and functions. The results of all these studies, especially those carried out in the second half of the past century, proved and confirmed that the eukaryotic cell nucleus is the control center of all cellular activities and also ensures the continuity of genetic information along successive generations of cells. These vital functions are the result of selective expression of genes contained in the nuclear chromatin, which is a high ordered and dynamic structure, in permanent and bilateral relations with other nuclear components. Based on these considerations, the present review aims to synthetize the latest researches and concepts about the cell nuclear territory in three distinctive parts, according to the complexity of the topic

  5. Entry into mitosis without Cdc2 kinase activation.

    Science.gov (United States)

    Gowdy, P M; Anderson, H J; Roberge, M

    1998-11-01

    Mouse FT210 cells at 39 degreesC cannot enter mitosis but arrest in G2 phase, because they lack Cdc2 kinase activity as a result of a temperature-sensitive lesion in the cdc2 gene. Incubation of arrested cells with the protein phosphatase 1 and 2A inhibitor okadaic acid induces morphologically normal chromosome condensation. We now show that okadaic acid also induces two other landmark events of early mitosis, nuclear lamina depolymerization and centrosome separation, in the absence of Cdc2 kinase activity. Okadaic acid-induced entry into mitosis is accompanied by partial activation of Cdc25C and may be prevented by tyrosine phosphatase inhibitors and by the protein kinase inhibitor staurosporine, suggesting that Cdc25C and kinases distinct from Cdc2 are required for these mitotic events. Using in-gel assays, we show that a 45-kDa protein kinase normally activated at mitosis is also activated by okadaic acid independently of Cdc2 kinase. The 45-kDa kinase can utilize GTP, is stimulated by spermine and is inhibited by heparin. These properties are characteristic of the kinase CK2, but immunoprecipitation studies indicate that it is not CK2. The data underline the importance of a tyrosine phosphatase, possibly Cdc25C, and of kinases other than Cdc2 in the structural changes the cell undergoes at mitosis, and indicate that entry into mitosis involves the activation of multiple kinases working in concert with Cdc2 kinase.

  6. Releasing dentate nucleus cells from Purkinje cell inhibition generates output from the cerebrocerebellum.

    Directory of Open Access Journals (Sweden)

    Takahiro Ishikawa

    Full Text Available The cerebellum generates its vast amount of output to the cerebral cortex through the dentate nucleus (DN that is essential for precise limb movements in primates. Nuclear cells in DN generate burst activity prior to limb movement, and inactivation of DN results in cerebellar ataxia. The question is how DN cells become active under intensive inhibitory drive from Purkinje cells (PCs. There are two excitatory inputs to DN, mossy fiber and climbing fiber collaterals, but neither of them appears to have sufficient strength for generation of burst activity in DN. Therefore, we can assume two possible mechanisms: post-inhibitory rebound excitation and disinhibition. If rebound excitation works, phasic excitation of PCs and a concomitant inhibition of DN cells should precede the excitation of DN cells. On the other hand, if disinhibition plays a primary role, phasic suppression of PCs and activation of DN cells should be observed at the same timing. To examine these two hypotheses, we compared the activity patterns of PCs in the cerebrocerebellum and DN cells during step-tracking wrist movements in three Japanese monkeys. As a result, we found that the majority of wrist-movement-related PCs were suppressed prior to movement onset and the majority of wrist-movement-related DN cells showed concurrent burst activity without prior suppression. In a minority of PCs and DN cells, movement-related increases and decreases in activity, respectively, developed later. These activity patterns suggest that the initial burst activity in DN cells is generated by reduced inhibition from PCs, i.e., by disinhibition. Our results indicate that suppression of PCs, which has been considered secondary to facilitation, plays the primary role in generating outputs from DN. Our findings provide a new perspective on the mechanisms used by PCs to influence limb motor control and on the plastic changes that underlie motor learning in the cerebrocerebellum.

  7. Effect of Cinnamomum longepaniculatum allelopathy on the mitosis of Vicia faba root tip cells%宜宾油樟的化感作用对蚕豆根尖细胞有丝分裂的影响

    Institute of Scientific and Technical Information of China (English)

    邓骛远; 罗通; 郑田喜

    2011-01-01

    利用油樟水浸提液作用蚕豆根尖研究了油樟化感作用对蚕豆根尖细胞有丝分裂的影响.结果表明:油樟对蚕豆根尖细胞有丝分裂的化感作用和浸提液浓度、作用时间有密切关系.在实验设计的范围内,随油樟水浸提液浓度的加大,蚕豆根尖细胞有丝分裂指数显著减小,而细胞微核率显著提高;油樟浸提液作用48 h时,对蚕豆根尖细胞有丝分裂的化感作用最强;油樟水浸提液诱导蚕豆根尖细胞产生染色体断片和滞后染色体等多种畸变染色体,并使有丝分裂各时期的分裂相减少.说明油樟化感物质抑制了细胞的有丝分裂,促进微核的产生,从而影响蚕豆根尖的生长和细胞的遗传稳定性.%The effect of Youzhang [Cinnamomum longepaniculatum (Gamble), N.Chan]allelopathy on the mitosis of Vicia faba root tip cells was studied with Youzhang water extracts acting Vicia faba root tips.The result shows the allelopathy has a close relation to the extract concentration and treating time.Within designed range of the experiment, the mitosis index of Vicia faba root tip cells decreases obviously, and the micronucleus frequency rises remarkably along with the increase of Youzhang extract concentrations.When acting time comes to 48 hours, the allelopathy of Youzhang extracts on the mitosis of Vicia faba root tip cells is the strongest.Youzhang water extracts induce a variety of aberrant chromosomes, such as chromosome fregments and delaying chromosomes, and inhibit mitosis different phases.It is implied that Youzhang's allelopthy matter inhibits cell mitosis, and promotes micronucleus generation of cells, and finally affects the growth of Vicia faba root tips and the genetic stability of cells.

  8. The multiple functions of T stellate/multipolar/chopper cells in the ventral cochlear nucleus.

    Science.gov (United States)

    Oertel, Donata; Wright, Samantha; Cao, Xiao-Jie; Ferragamo, Michael; Bal, Ramazan

    2011-06-01

    Acoustic information is brought to the brain by auditory nerve fibers, all of which terminate in the cochlear nuclei, and is passed up the auditory pathway through the principal cells of the cochlear nuclei. A population of neurons variously known as T stellate, type I multipolar, planar multipolar, or chopper cells forms one of the major ascending auditory pathways through the brainstem. T Stellate cells are sharply tuned; as a population they encode the spectrum of sounds. In these neurons, phasic excitation from the auditory nerve is made more tonic by feedforward excitation, coactivation of inhibitory with excitatory inputs, relatively large excitatory currents through NMDA receptors, and relatively little synaptic depression. The mechanisms that make firing tonic also obscure the fine structure of sounds that is represented in the excitatory inputs from the auditory nerve and account for the characteristic chopping response patterns with which T stellate cells respond to tones. In contrast with other principal cells of the ventral cochlear nucleus (VCN), T stellate cells lack a low-voltage-activated potassium conductance and are therefore sensitive to small, steady, neuromodulating currents. The presence of cholinergic, serotonergic and noradrenergic receptors allows the excitability of these cells to be modulated by medial olivocochlear efferent neurons and by neuronal circuits associated with arousal. T Stellate cells deliver acoustic information to the ipsilateral dorsal cochlear nucleus (DCN), ventral nucleus of the trapezoid body (VNTB), periolivary regions around the lateral superior olivary nucleus (LSO), and to the contralateral ventral lemniscal nuclei (VNLL) and inferior colliculus (IC). It is likely that T stellate cells participate in feedback loops through both medial and lateral olivocochlear efferent neurons and they may be a source of ipsilateral excitation of the LSO.

  9. Nonlinear optical imaging and Raman microspectrometry of the cell nucleus throughout the cell cycle.

    Science.gov (United States)

    Pliss, Artem; Kuzmin, Andrey N; Kachynski, Aliaksandr V; Prasad, Paras N

    2010-11-17

    Fundamental understanding of cellular processes at molecular level is of considerable importance in cell biology as well as in biomedical disciplines for early diagnosis of infection and cancer diseases, and for developing new molecular medicine-based therapies. Modern biophotonics offers exclusive capabilities to obtain information on molecular composition, organization, and dynamics in a cell by utilizing a combination of optical spectroscopy and optical imaging. We introduce here a combination of Raman microspectrometry, together with coherent anti-Stokes Raman scattering (CARS) and two-photon excited fluorescence (TPEF) nonlinear optical microscopy, to study macromolecular organization of the nucleus throughout the cell cycle. Site-specific concentrations of proteins, DNA, RNA, and lipids were determined in nucleoli, nucleoplasmic transcription sites, nuclear speckles, constitutive heterochromatin domains, mitotic chromosomes, and extrachromosomal regions of mitotic cells by quantitative confocal Raman microspectrometry. A surprising finding, obtained in our study, is that the local concentration of proteins does not increase during DNA compaction. We also demonstrate that postmitotic DNA decondensation is a gradual process, continuing for several hours. The quantitative Raman spectroscopic analysis was corroborated with CARS/TPEF multimodal imaging to visualize the distribution of protein, DNA, RNA, and lipid macromolecules throughout the cell cycle.

  10. Communication Between the Cell Membrane and the Nucleus: Role of Protein Compartmentalization

    Energy Technology Data Exchange (ETDEWEB)

    Lelievre, Sophie A; Bissell, Mina J

    1998-10-21

    Understanding how the information is conveyed from outside to inside the cell is a critical challenge for all biologists involved in signal transduction. The flow of information initiated by cell-cell and cell-extracellular matrix contacts is mediated by the formation of adhesion complexes involving multiple proteins. Inside adhesion complexes, connective membrane skeleton (CMS) proteins are signal transducers that bind to adhesion molecules, organize the cytoskeleton, and initiate biochemical cascades. Adhesion complex-mediated signal transduction ultimately directs the formation of supramolecular structures in the cell nucleus, as illustrated by the establishment of multi complexes of DNA-bound transcription factors, and the redistribution of nuclear structural proteins to form nuclear subdomains. Recently, several CMS proteins have been observed to travel to the cell nucleus, suggesting a distinctive role for these proteins in signal transduction. This review focuses on the nuclear translocation of structural signal transducers of the membrane skeleton and also extends our analysis to possible translocation of resident nuclear proteins to the membrane skeleton. This leads us to envision the communication between spatially distant cellular compartments (i.e., membrane skeleton and cell nucleus) as a bidirectional flow of information (a dynamic reciprocity) based on subtle multilevel structural and biochemical equilibria. At one level, it is mediated by the interaction between structural signal transducers and their binding partners, at another level it may be mediated by the balance and integration of signal transducers in different cellular compartments.

  11. Regulatory genes controlling mitosis in the fission yeast Schizosaccharomyces pombe.

    Science.gov (United States)

    Nurse, P; Thuriaux, P

    1980-11-01

    Fifty-two wee mutants that undergo mitosis and cell division at a reduced size compared with wild type have been genetically analyzed. The mutants define two genes, wee1 and cdc2, which control the timing of mitosis. Fifty-one of the mutants map at the wee1 locus, which is unlinked to any known cdc gene. One of the wee1 alleles has been shown to be nonsense suppressible. The 52nd were mutant maps within cdc2. Previously, only temperature-sensitive mutants that become blocked at mitosis have been found at the cdc2 locus. The simplest interpretation of these observations is that wee1+ codes for a negative element or inhibitor, and cdc2+ codes for a positive element or activator in the mitotic control. The gene dosage of wee1+ plays some role in determining the timing of mitosis, but the gene dosage of cdc2+ has little effect. However, some aspect of the cdc2 gene product activity is important for determining when mitosis takes place. The possible roles of wee1 and cdc2 in the mitotic control are discussed, with particular reference to the part they may play in the monitoring of cell growth rate, both of which influence the timing of mitosis.

  12. Perturbation of nucleo-cytoplasmic transport affects size of nucleus and nucleolus in human cells.

    Science.gov (United States)

    Ganguly, Abira; Bhattacharjee, Chumki; Bhave, Madhura; Kailaje, Vaishali; Jain, Bhawik K; Sengupta, Isha; Rangarajan, Annapoorni; Bhattacharyya, Dibyendu

    2016-03-01

    Size regulation of human cell nucleus and nucleolus are poorly understood subjects. 3D reconstruction of live image shows that the karyoplasmic ratio (KR) increases by 30-80% in transformed cell lines compared to their immortalized counterpart. The attenuation of nucleo-cytoplasmic transport causes the KR value to increase by 30-50% in immortalized cell lines. Nucleolus volumes are significantly increased in transformed cell lines and the attenuation of nucleo-cytoplasmic transport causes a significant increase in the nucleolus volume of immortalized cell lines. A cytosol and nuclear fraction swapping experiment emphasizes the potential role of unknown cytosolic factors in nuclear and nucleolar size regulation.

  13. HEK293 cells express dystrophin Dp71 with nucleus-specific localization of Dp71ab.

    Science.gov (United States)

    Nishida, Atsushi; Yasuno, Sato; Takeuchi, Atsuko; Awano, Hiroyuki; Lee, Tomoko; Niba, Emma Tabe Eko; Fujimoto, Takahiro; Itoh, Kyoko; Takeshima, Yasuhiro; Nishio, Hisahide; Matsuo, Masafumi

    2016-09-01

    The dystrophin gene consists of 79 exons and encodes tissue-specific isoforms. Mutations in the dystrophin gene cause Duchenne muscular dystrophy, of which a substantial proportion of cases are complicated by non-progressive mental retardation. Abnormalities of Dp71, an isoform transcribed from a promoter in intron 62, are a suspected cause of mental retardation. However, the roles of Dp71 in human brain have not been fully elucidated. Here, we characterized dystrophin in human HEK293 cells with the neuronal lineage. Reverse transcription-PCR amplification of the full-length dystrophin transcript revealed the absence of fragments covering the 5' part of the dystrophin cDNA. In contrast, fragments covering exons 64-79 were present. The Dp71 promoter-specific exon G1 was shown spliced to exon 63. We demonstrated that the Dp71 transcript comprised two subisoforms: one lacking exon 78 (Dp71b) and the other lacking both exons 71 and 78 (Dp71ab). Western blotting of cell lysates using an antibody against the dystrophin C-terminal region revealed two bands, corresponding to Dp71b and Dp71ab. Immunohistochemical examination with the dystrophin antibody revealed scattered punctate signals in the cytoplasm and the nucleus. Western blotting revealed one band corresponding to Dp71b in the cytoplasm and two bands corresponding to Dp71b and Dp71ab in the nucleus, with Dp71b being predominant. These results indicated that Dp71ab is a nucleus-specific subisoform. We concluded that Dp71, comprising Dp71b and Dp71ab, was expressed exclusively in HEK293 cells and that Dp71ab was specifically localized to the nucleus. Our findings suggest that Dp71ab in the nucleus contributes to the diverse functions of HEK293 cells.

  14. Mitosis and growth in biological tissues

    Science.gov (United States)

    Mombach, J. C. M.; de Almeida, Rita M. C.; Iglesias, J. R.

    1993-07-01

    We present a simulation of the growth of a two-dimensional biological cellular system in which the cells experience mitosis whenever the area-to-perimeter ratio reaches a critical value. The model also includes the effect of interfacial energy and temperature. A stationary state with a constant average area is attained. We calculate the distribution of cells as a function of area, perimeter, and number of sides and also the two-cell correlation function. The results depend on temperature and are in agreement with experimental data, simulations, and theoretical models.

  15. A hyperactive transcriptional state marks genome reactivation at the mitosis-G1 transition.

    Science.gov (United States)

    Hsiung, Chris C-S; Bartman, Caroline R; Huang, Peng; Ginart, Paul; Stonestrom, Aaron J; Keller, Cheryl A; Face, Carolyne; Jahn, Kristen S; Evans, Perry; Sankaranarayanan, Laavanya; Giardine, Belinda; Hardison, Ross C; Raj, Arjun; Blobel, Gerd A

    2016-06-15

    During mitosis, RNA polymerase II (Pol II) and many transcription factors dissociate from chromatin, and transcription ceases globally. Transcription is known to restart in bulk by telophase, but whether de novo transcription at the mitosis-G1 transition is in any way distinct from later in interphase remains unknown. We tracked Pol II occupancy genome-wide in mammalian cells progressing from mitosis through late G1. Unexpectedly, during the earliest rounds of transcription at the mitosis-G1 transition, ∼50% of active genes and distal enhancers exhibit a spike in transcription, exceeding levels observed later in G1 phase. Enhancer-promoter chromatin contacts are depleted during mitosis and restored rapidly upon G1 entry but do not spike. Of the chromatin-associated features examined, histone H3 Lys27 acetylation levels at individual loci in mitosis best predict the mitosis-G1 transcriptional spike. Single-molecule RNA imaging supports that the mitosis-G1 transcriptional spike can constitute the maximum transcriptional activity per DNA copy throughout the cell division cycle. The transcriptional spike occurs heterogeneously and propagates to cell-to-cell differences in mature mRNA expression. Our results raise the possibility that passage through the mitosis-G1 transition might predispose cells to diverge in gene expression states.

  16. DNA damage-induced translocation of S100A11 into the nucleus regulates cell proliferation

    Directory of Open Access Journals (Sweden)

    Ulbricht Tobias

    2010-12-01

    Full Text Available Abstract Background Proteins are able to react in response to distinct stress stimuli by alteration of their subcellular distribution. The stress-responsive protein S100A11 belongs to the family of multifunctional S100 proteins which have been implicated in several key biological processes. Previously, we have shown that S100A11 is directly involved in DNA repair processes at damaged chromatin in the nucleus. To gain further insight into the underlying mechanism subcellular trafficking of S100A11 in response to DNA damage was analyzed. Results We show that DNA damage induces a nucleolin-mediated translocation of S100A11 from the cytoplasm into the nucleus. This translocation is impeded by inhibition of the phosphorylation activity of PKCα. Translocation of S100A11 into the nucleus correlates with an increased cellular p21 protein level. Depletion of nucleolin by siRNA severely impairs translocation of S100A11 into the nucleus resulting in a decreased p21 protein level. Additionally, cells lacking nucleolin showed a reduced colony forming capacity. Conclusions These observations suggest that regulation of the subcellular distribution of S100A11 plays an important role in the DNA damage response and p21-mediated cell cycle control.

  17. Characterization of the role of the tumor marker Nup88 in mitosis

    Directory of Open Access Journals (Sweden)

    Yoshida Kimihisa

    2010-05-01

    Full Text Available Abstract Nuclear pore complexes are massive multiprotein channels responsible for traffic between the nucleus and cytoplasm, and are composed of approximately 30 proteins, termed nucleoporins (Nup. Our recent studies indicated that the nucleoporins Rae1 and Tpr play critical roles in maintaining the spindle bipolarity during cell division. In the present study, we found that another nucleoporin, Nup88, was localized on the spindles together with Nup214 during mitosis. Nup88 expression is linked to the progression of carcinogenesis, Nup88 has been proposed as a tumor marker. Overexpression of Nup88 enhanced multinucleated cell formation. RNAi-mediated knockdown of Nup88 disrupted Nup214 expression and localization and caused multipolar spindle phenotypes. Our data indicate that proper expression of Nup88 is critical for preventing aneuploidy formation and tumorigenesis.

  18. Formation of tRNA granules in the nucleus of heat-induced human cells

    Energy Technology Data Exchange (ETDEWEB)

    Miyagawa, Ryu [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Department of Biological Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654 (Japan); Mizuno, Rie [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Watanabe, Kazunori, E-mail: watanabe@ric.u-tokyo.ac.jp [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Ijiri, Kenichi [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Department of Biological Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654 (Japan)

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer tRNAs are tranlocated into the nucleus in heat-induced HeLa cells. Black-Right-Pointing-Pointer tRNAs form the unique granules in the nucleus. Black-Right-Pointing-Pointer tRNA ganules overlap with nuclear stress granules. -- Abstract: The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA{sup Met} (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA{sup Met} was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules.

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

    NARCIS (Netherlands)

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

    2011-01-01

    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

  20. Bacterial mitosis: partitioning protein ParA oscillates in spiral-shaped structures and positions plasmids at mid-cell

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Gerdes, Kenn; Charbon, Gitte Ebersbach

    2004-01-01

    The par2 locus of Escherichia coli plasmid pB171 encodes oscillating ATPase ParA, DNA binding protein ParB and two cis-acting DNA regions to which ParB binds (parC1 and parC2). Three independent techniques were used to investigate the subcellular localization of plasmids carrying par2. In cells w...

  1. Temporal properties of pattern adaptation of relay cells in the lateral geniculate nucleus of cats

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The temporal properties of pattern adaptation of relay cells induced by repeated sinusoidal drifting grating were investigated in the dorsal lateral geniculate nucleus (dLGN) of cats. The results showed that the response amplitude declined and the response latency prolonged when relay cells were pattern-adapted in dLGN, like the similar fmdings in visual cortex. However, in contrast to the result in cortex,the response phase of relay cells advanced. This implies that an inhibition with relatively long latency may participate in the pattern adaptation of dLGN cells and the adaptation in dLGN may be via a mechanism different from that of visual cortex.``

  2. Acentrosomal Microtubule Assembly in Mitosis: The Where, When, and How.

    Science.gov (United States)

    Meunier, Sylvain; Vernos, Isabelle

    2016-02-01

    In mitosis the cell assembles the bipolar spindle, a microtubule (MT)-based apparatus that segregates the duplicated chromosomes into two daughter cells. Most animal cells enter mitosis with duplicated centrosomes that provide an active source of dynamic MTs. However, it is now established that spindle assembly relies on the nucleation of acentrosomal MTs occurring around the chromosomes after nuclear envelope breakdown, and on pre-existing microtubules. Where chromosome-dependent MT nucleation occurs, when MT amplification takes place and how the two pathways function are still key questions that generate some controversies. We reconcile the data and present an integrated model accounting for acentrosomal microtubule assembly in the dividing cell.

  3. Spatial reorganization of the endoplasmic reticulum during mitosis relies on mitotic kinase cyclin A in the early Drosophila embryo.

    Science.gov (United States)

    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.

  4. An autoantibody is modified for use as a delivery system to target the cell nucleus: therapeutic implications.

    Science.gov (United States)

    Weisbart, R H; Stempniak, M; Harris, S; Zack, D J; Ferreri, K

    1998-10-01

    A murine monoclonal anti-dsDNA antibody was found to penetrate living cells and localize in the nucleus without pathologic effects. A single mutation in VH markedly enhanced cellular penetration. The mutant antibody was produced as recombinant Fab and single chain antibody fragments to investigate its use as a delivery system to target the cell nucleus. Complexes were made containing Fab fragments and alkaline phosphatase conjugated goat antibodies to mouse |gk chains. Fab fragments transported 305 kDa goat antibody-enzyme complexes into the nucleus in COS-7 and CHO cells. A single chain antibody cDNA was constructed by splice overlap extension PCR and expressed in COS-7 cells. Binding of the single chain antibody to dsDNA was shown by ELISA, and cellular penetration and nuclear localization were demonstrated in COS-7 and CHO cells. The single chain antibody cDNA was ligated into the expression vector, pEGFP, to produce a fusion protein with green fluorescent protein. The fusion protein penetrated COS-7 cells and localized in the cell nucleus. The single chain antibody produced during sustained expression in CHO cells re-entered antibody-producing cells and localized in the nucleus without affecting cell viability. Our results demonstrate the potential use of a modified autoantibody as a delivery system to target the cell nucleus.

  5. Role of the first mitosis in the remodeling of the parental genomes in mouse embryos

    Institute of Scientific and Technical Information of China (English)

    Hong Lin LIU; Kentaro T.HARA; Fugaku AOKI

    2005-01-01

    Although male and female pronuclei reside in the same zygotic cytoplasm, they differ in many respects, such as volume and transcriptional activity. The aim of this study is to investigate whether these differences are lost during the first mitosis. For this purpose, a new method was developed to inhibit the mixing of two parental chromosomes during mitosis, thus to induce the formation of two nuclei after they exit from the mitotic phase. In this method, one-cell embryos are arrested at metaphase by treatment with nocodazole, and whn exitting from the mitotic phase, two nuclei were formed in a single karyocyte following treatment with 6-dimethylaminopurine (6-DMAP). These embryos were designated as post-mitotic embryos (PM-embryos), in which the two nuclei were derived from the male and female genomes. We found that in the control one-cell embryos that had not been treated with the reagents, the volume of the male pronucleus was about 1.65-fold greater than that of the female pronucleus, whereas the volumes of the two nuclei in the PM-embryos were similar (volume ratio of 1.01). Although a two-fold difference in transcriptional activity was detected between the male and female pronuclei in the control embryos, no difference in transcriptional activity was detected between the two nuclei of PM-embryos. The ratio of transcriptional activity in the nucleus derived from the paternal genome to that from the maternal genome was 1.02, for which no significant difference was detected by the x2fitness test. Therefore, the volumes and transcriptional activities of the male and female nuclei were approximately equal in PM-embryos, which suggests that the asymmetries of pronuclear volume and transcriptional activity between male and female genomes are somehow losted during the first mitosis.

  6. Segmentation of White Blood Cells through Nucleus Mark Watershed Operations and Mean Shift Clustering

    Directory of Open Access Journals (Sweden)

    Zhi Liu

    2015-09-01

    Full Text Available This paper presents a novel method for segmentation of white blood cells (WBCs in peripheral blood and bone marrow images under different lights through mean shift clustering, color space conversion and nucleus mark watershed operation (NMWO. The proposed method focuses on obtaining seed points. First, color space transformation and image enhancement techniques are used to obtain nucleus groups as inside seeds. Second, mean shift clustering, selection of the C channel component in the CMYK model, and illumination intensity adjustment are employed to acquire WBCs as outside seeds. Third, the seeds and NMWO are employed to precisely determine WBCs and solve the cell adhesion problem. Morphological operations are further used to improve segmentation accuracy. Experimental results demonstrate that the algorithm exhibits higher segmentation accuracy and robustness compared with traditional methods.

  7. Nucleocytoplasmic protein translocation during mitosis in the social amoebozoan Dictyostelium discoideum.

    Science.gov (United States)

    O'Day, Danton H; Budniak, Aldona

    2015-02-01

    Mitosis is a fundamental and essential life process. It underlies the duplication and survival of all cells and, as a result, all eukaryotic organisms. Since uncontrolled mitosis is a dreaded component of many cancers, a full understanding of the process is critical. Evolution has led to the existence of three types of mitosis: closed, open, and semi-open. The significance of these different mitotic species, how they can lead to a full understanding of the critical events that underlie the asexual duplication of all cells, and how they may generate new insights into controlling unregulated cell division remains to be determined. The eukaryotic microbe Dictyostelium discoideum has proved to be a valuable biomedical model organism. While it appears to utilize closed mitosis, a review of the literature suggests that it possesses a form of mitosis that lies in the middle between truly open and fully closed mitosis-it utilizes a form of semi-open mitosis. Here, the nucleocytoplasmic translocation patterns of the proteins that have been studied during mitosis in the social amoebozoan D. discoideum are detailed followed by a discussion of how some of them provide support for the hypothesis of semi-open mitosis.

  8. Polyimidazole conjugated oligonucleotides reach the nucleus of HeLa cells.

    Science.gov (United States)

    Morvan, F; Castex, C; Vivès, E; Imbach, J L

    2001-01-01

    Oligonucleotide models bearing 6, 12 or 18 histamine residues were synthesized on solid support and labeled with fluorescein. Only the oligo with 6 histamine residues showed a high uptake in HeLa cells with a nuclear localization. Experiment a 4 degrees C or with bafilomicyn A1 suggest that uptake proceeded by an endocytosis mechanism followed by a destabilization of the membrane. Once in the cytoplasm the oligo reached rapidly the nucleus.

  9. Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei, E-mail: biehzw@nus.edu.sg [Optical Bioimaging Laboratory, Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore 117576 (Singapore)

    2014-09-08

    We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

  10. The Functional Role of TopBP1 in DNA Maintenance at Mitosis

    DEFF Research Database (Denmark)

    Pedersen, Rune Troelsgaard

    When cells traverse mitosis, genome integrity of the emerging daughter cells is dependent on replication of the entire genome during the preceding S-phase and accurate chromosome segregation in mitosis. Replication stress may cause cells to enter mitosis with underreplicated loci, consisting...... can lead to anaphase bridges that impair accurate chromosome segregation. The recent decade featured many advances in our understanding of how cells cope with underreplicated loci in mitosis. A major advance was the description of ultra-fine anaphase bridges (UFBs), a class of anaphase bridges...... established Saccharomyces cerevisiae as a model organism to study anaphase bridges, and we identified Dpb11/TopBP1 as a novel UFB-associated protein in yeast and avian DT40 cells, respectively. TopBP1 localized to confined areas on replication-stress induced UFBs. Upon onset of mitosis we observed a burst...

  11. The role of model organisms in the history of mitosis research.

    Science.gov (United States)

    Yanagida, Mitsuhiro

    2014-09-02

    Mitosis is a cell-cycle stage during which condensed chromosomes migrate to the middle of the cell and segregate into two daughter nuclei before cytokinesis (cell division) with the aid of a dynamic mitotic spindle. The history of mitosis research is quite long, commencing well before the discovery of DNA as the repository of genetic information. However, great and rapid progress has been made since the introduction of recombinant DNA technology and discovery of universal cell-cycle control. A large number of conserved eukaryotic genes required for the progression from early to late mitotic stages have been discovered, confirming that DNA replication and mitosis are the two main events in the cell-division cycle. In this article, a historical overview of mitosis is given, emphasizing the importance of diverse model organisms that have been used to solve fundamental questions about mitosis.

  12. Leishmania amazonensis Promastigotes Present Two Distinct Modes of Nucleus and Kinetoplast Segregation during Cell Cycle

    Science.gov (United States)

    da Silva, Marcelo Santos; Monteiro, Jomar Patrício; Nunes, Vinícius Santana; Vasconcelos, Elton José; Perez, Arina Marina; Freitas-Júnior, Lúcio de Holanda; Elias, Maria Carolina; Cano, Maria Isabel Nogueira

    2013-01-01

    Here, we show the morphological events associated with organelle segregation and their timing in the cell cycle of a reference strain of Leishmania (L.) amazonensis promastigotes, the main causative agent of Tegumentary leishmaniasis in the Americas. We show evidences that during the cell cycle, L. amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation, which occur in different temporal order in different proportions of cells. We used DAPI-staining and EdU-labeling to monitor the segregation of DNA-containing organelles and DNA replication in wild-type parasites. The emergence of a new flagellum was observed using a specific monoclonal antibody. The results show that L. amazonensis cell cycle division is peculiar, with 65% of the dividing cells duplicating the kinetoplast before the nucleus, and the remaining 35% doing the opposite or duplicating both organelles concomitantly. In both cases, the new flagellum appeared during S to G2 phase in 1N1K cells and thus before the segregation of both DNA-containing organelles; however, we could not determine the exact timing of flagellar synthesis. Most of these results were confirmed by the synchronization of parasites using hydroxyurea. Altogether, our data show that during the cell cycle of L. amazonensis promastigotes, similarly to L. donovani, the segregation of nucleus and kinetoplast do not follow a specific order, especially when compared to other trypanosomatids, reinforcing the idea that this characteristic seems to be species-specific and may represent differences in cellular biology among members of the Leishmania genus. PMID:24278433

  13. Transplantation of gene-modified nucleus pulposus cells reverses rabbit intervertebral disc degeneration

    Institute of Scientific and Technical Information of China (English)

    LIU Yong; LI Jian-min; HU You-gu

    2011-01-01

    Background Intervertebral disc degeneration is the main cause of low back pain. The purpose of this study was to explore potential methods for reversing the degeneration of lumbar intervertebral discs by transplantation of gene-modified nucleus pulposus cells into rabbit degenerative lumbar intervertebral discs after transfecting rabbit nucleus pulposus cells with adeno-associated virus 2 (AAV2)-mediated connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinases 1 (TIMP1) genes in vitro.Methods Computer tomography (CT)-guided percutaneous annulus fibrosus injury was performed to build degenerative lumbar intervertebral disc models in 60 New Zealand white rabbits. rAAV2-CTGF-IRES-TIMP1-transfected rabbit nucleus pulposus cells were transplanted into degenerative lumbar intervertebral discs (transplantation group),phosphate-buffered saline (PBS) was injected into degenerative lumbar intervertebral discs (degeneration control group)and normal lumbar intervertebral discs served as a blank control group. After 6, 10 and 14 weeks, the disc height index (DHI) and signal intensity in intervertebral discs were observed by X-ray and magnetic resonance imaging (MRI) analysis.The expression of CTGF and TIMP1 in nucleus pulposus tissue was determined by Western blotting analysis, the synthesis efficiency of proteoglycan was determined by a 35S-sulfate incorporation assay, and the mRNA expression of type Ⅱ collagen and proteoglycan was detected by RT-PCR.Results MRI confirmed that degenerative intervertebral discs appeared two weeks after percutaneous puncture.Transgenic nucleus pulposus cell transplantation could retard the rapid deterioration of the DHI. MRI indicated that degenerative intervertebral discs were relieved in the transplantation group compared with the degeneration control group. The expression of collagen Ⅱ mRNA and proteoglycan mRNA was significantly higher in the transplantation group and the blank control group compared with the

  14. On the move: organelle dynamics during mitosis.

    Science.gov (United States)

    Jongsma, Marlieke L M; Berlin, Ilana; Neefjes, Jacques

    2015-03-01

    A cell constitutes the minimal self-replicating unit of all organisms, programmed to propagate its genome as it proceeds through mitotic cell division. The molecular processes entrusted with ensuring high fidelity of DNA replication and subsequent segregation of chromosomes between daughter cells have therefore been studied extensively. However, to process the information encoded in its genome a cell must also pass on its non-genomic identity to future generations. To achieve productive sharing of intracellular organelles, cells have evolved complex mechanisms of organelle inheritance. Many membranous compartments undergo vast spatiotemporal rearrangements throughout mitosis. These controlled organizational changes are crucial to enabling completion of the division cycle and ensuring successful progeny. Herein we review current understanding of intracellular organelle segregation during mitotic division in mammalian cells, with a focus on compartment organization and integrity throughout the inheritance process.

  15. Identifying mitosis deep in tissue using dynamic light scattering fluctuation spectroscopy

    Science.gov (United States)

    An, Ran; Jeong, Kwan; Turek, John; Nolte, David

    2012-03-01

    In the cell cycle, mitosis is the most dramatic phase, especially in Telophase and Cytokinesis. For single cells and cell monolayer, there are precise microscopic studies of mitosis, while for 3-D tissue such as tumor spheroids the light signal is obscured by the high background of diffusely scattered light. Therefore, the mitosis phase cannot be detected deep inside 3-D tissue using conventional microscopic techniques. In this work, we detect mitosis in living tissue using Tissue Dynamic Imaging (TDI). We trace depth-gated dynamic speckles from a tumor spheroid (up to 1mm in diameter) using coherence-gated digital holography imaging. Frequency-versus-time spectrograms depend on specific types of perturbation such as cell shape change, membrane undulation and cell organelles movements. By using these spectral responses as functional finger prints, we can identify mitosis events from different voxels at a specified depth inside tumor spheroids. By performing B-scans of the tumor spheroid, we generate 3-D mitosis maps (or movies) for the entire tumor spheroids. We show that for healthy tumor spheroids, the mitosis events only happen within the proliferating shell. We also compare results when anti-cancer drugs are applied to arrest, release and synchronize mitosis. This shows the application of TDI for drug screening. The technique can identify and monitor complex motilities inside 3-D tissue with a strong potential for drug diagnosis and developmental biology studies.

  16. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Brunak, Søren; Olsen, JV

    2010-01-01

    ) or CDK2 were almost fully phosphorylated in mitotic cells. In particular, nuclear proteins and proteins involved in regulating metabolic processes have high phosphorylation site occupancy in mitosis. This suggests that these proteins may be inactivated by phosphorylation in mitotic cells....

  17. Replication stress activates DNA repair synthesis in mitosis

    DEFF Research Database (Denmark)

    Minocherhomji, Sheroy; Ying, Songmin; Bjerregaard, Victoria A

    2015-01-01

    mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest...

  18. Structural and functional classes of multipolar cells in the ventral cochlear nucleus.

    Science.gov (United States)

    Doucet, John R; Ryugo, David K

    2006-04-01

    Multipolar cells in the ventral cochlear nucleus (VCN) are a structurally and functionally diverse group of projection neurons. Understanding their role in the ascending pathway involves partitioning multipolar cells into distinct populations and determining where in the brain each sends its coded messages. In this study, we used retrograde labeling techniques in rats to identify multipolar neurons that project their axons to the ipsilateral dorsal cochlear nucleus (DCN), the contralateral CN, or both structures. Three rats received injections of biotinylated dextran amine in the ipsilateral DCN and diamidino yellow in the contralateral CN. Several radiate multipolar neurons (defined by their axonal projections to the ipsilateral DCN and their dendrites that traverse VCN isofrequency sheets) were double-labeled but over 70% were not. This result suggests two distinct populations: (1) radiate-commissural (RC) multipolar cells that project to the ipsilateral DCN and the contralateral CN, and (2) radiate multipolar cells that project exclusively (in this context) to the ipsilateral DCN. In a different group of animals, we retrogradely labeled multipolar neurons that project their axons to the contralateral CN and measured the size of their cell bodies. The mean size of this population (266 +/- 156 microm2) was significantly smaller than those of RC-multipolar cells (418 +/- 140 microm2). We conclude that the CN commissural pathway is composed of at least two components: (1) RC multipolar cells and (2) commissural multipolar cells that are small- and medium-sized neurons that project exclusively (in this context) to the contralateral CN. These results identify separate structural groups of multipolar cells that may correspond to physiological unit types described in the literature. They also provide protocols for isolating and studying different populations of multipolar cells to determine the neural mechanisms that govern their responses to sound.

  19. Polo样激酶1在细胞有丝分裂期作用的研究进展%Research progress of the roles of Polo-like kinase 1 in cell mitosis

    Institute of Scientific and Technical Information of China (English)

    李伟

    2014-01-01

    Polo样激酶1(Plk1)是一类广泛存在于真核细胞中的丝氨酸/苏氨酸激酶,是细胞有丝分裂期的关键调控因子.在有丝分裂的不同时期,Plk1在细胞中的定位不同,并与不同的底物相互作用,从而具有多种生物学功能.文章对Plk1在细胞有丝分裂各个时期的功能作一综述.%Polo-like kinase 1 (Plk1) is a serine/threonine kinase that is widely expressed in eukaryotic cells and a key regulator of the cell mitosis.During different stages of mitosis,the locations of Plk1 in cells are different,and can interaction with different substrates,thus it has many biological functions.This article focuses on the functions of Plk1 in different mitotic stages.

  20. Ion channels generating complex spikes in cartwheel cells of the dorsal cochlear nucleus.

    Science.gov (United States)

    Kim, Yuil; Trussell, Laurence O

    2007-02-01

    Cartwheel cells are glycinergic interneurons that modify somatosensory input to the dorsal cochlear nucleus. They are characterized by firing of mixtures of both simple and complex action potentials. To understand what ion channels determine the generation of these two types of spike waveforms, we recorded from cartwheel cells using the gramicidin perforated-patch technique in brain slices of mouse dorsal cochlear nucleus and applied channel-selective blockers. Complex spikes were distinguished by whether they arose directly from a negative membrane potential or later during a long depolarization. Ca(2+) channels and Ca(2+)-dependent K(+) channels were major determinants of complex spikes. Onset complex spikes required T-type and possibly R-type Ca(2+) channels and were shaped by BK and SK K(+) channels. Complex spikes arising later in a depolarization were dependent on P/Q- and L-type Ca(2+) channels as well as BK and SK channels. BK channels also contributed to fast repolarization of simple spikes. Simple spikes featured an afterdepolarization that is probably the trigger for complex spiking and is shaped by T/R-type Ca(2+) and SK channels. Fast spikes were dependent on Na(+) channels; a large persistent Na(+) current may provide a depolarizing drive for spontaneous activity in cartwheel cells. Thus the diverse electrical behavior of cartwheel cells is determined by the interaction of a wide variety of ion channels with a prominent role played by Ca(2+).

  1. Thymosin Beta-4 Recombinant Adeno-associated Virus Enhances Human Nucleus Pulposus Cell Proliferation and Reduces Cell Apoptosis and Senescence

    Institute of Scientific and Technical Information of China (English)

    Yuan-Yi Wang; Qing-San Zhu; Yi-Wei Wang; Ruo-Feng Yin

    2015-01-01

    Background:Thymosin beta-4 (TB-4) is considered key roles in tissue development,maintenance and pathological processes.The study aimed to prove TB-4 positive biological function on nucleus pulposus (NP) cell apoptosis and slowing the process of cell aging while increasing the cell proliferation.Methods:TB-4 recombinant adeno-associated virus (AAV) was constructed and induced to human NP cells.Cell of same group were cultured without gene modification as controlled group.Proliferation capacity and cell apoptosis were observed during 6 passages of the cells.Morphology and expression of the TB-4 gene were documented as parameter of cell activity during cell passage.Results:NP cells with TB-4 transfection has normal TB-4 expression and exocytosis.NP cells with TB-4 transfection performed significantly higher cell activity than that at the control group in each generation.TB-4 recombinant AAV-transfected human NP cells also show slower cell aging,lower cell apoptosis and higher cell proliferation than control group.Conclusions:TB-4 can prevent NP cell apoptosis,slow NP cell aging and promote NP cell proliferation.AAV transfection technique was able to highly and stably express TB-4 in human NP cells,which may provide a new pathway for innovation in the treatment of intervertebral disc degenerative diseases.

  2. Folded genome as a platform for the functional compartmentalization of the eukaryotic cell nucleus

    Directory of Open Access Journals (Sweden)

    Ioudinkova E. S.

    2014-03-01

    Full Text Available In a number of recent studies a tight interconnection between the spatial organization of the eukaryotic genome and its functioning has been demonstrated. Moreover, it is becoming evident that the folded DNA by itself consti- tutes an important, if not the key, factor supporting the internal nuclear organization. In this review, we will discuss the current state of chromatin research with the special attention focused on chromosome territories, chromatin folding and dynamics, chromatin domains, transcription and replication factories. Based on this analysis we will show how interphase chromosomes define the assembly of different nuclear compartments and underlie the spatial compartmentalization of the cell nucleus.

  3. Novel functions of endocytic player clathrin in mitosis

    Institute of Scientific and Technical Information of China (English)

    Wenxiang Fu; Qing Jiang; Chuanmao Zhang

    2011-01-01

    Clathrin has been widely recognized as a pivotal player in endocytosis,in which several adaptors and accessory proteins are involved.Recent studies suggested that clathrin is also essential for cell division.Here this review mainly focuses on the clathrin-dependent mechanisms involved in spindle assembly and chromosome alignment.In mitosis,clathrin forms a complex with phosphorylated TACC3 to ensure spindle stability and proper chromosome alignment.The clathrin-regulated mechanism in mitosis requires the crosstalk among clathrin,spindle assembly factors (SAFs),Ran-GTP and mitotic kinases.Meanwhile,a coordinated mechanism is required for role transitions of clathrin during endocytosis and mitosis.Taken together,the findings of the multiple functions of clathrin besides endocytosis have expanded our understanding of the basic cellular activities.

  4. Cocaine exposure reorganizes cell type- and input-specific connectivity in the nucleus accumbens.

    Science.gov (United States)

    MacAskill, Andrew F; Cassel, John M; Carter, Adam G

    2014-09-01

    Repeated exposure to cocaine alters the structural and functional properties of medium spiny neurons (MSNs) in the nucleus accumbens (NAc). These changes suggest a rewiring of the NAc circuit, with an enhancement of excitatory synaptic connections onto MSNs. However, it is unknown how drug exposure alters the balance of long-range afferents onto different cell types in the NAc. Here we used whole-cell recordings, two-photon microscopy, optogenetics and pharmacogenetics to show how repeated cocaine exposure alters connectivity in the mouse NAc medial shell. Cocaine selectively enhanced amygdala innervation of MSNs expressing D1 dopamine receptors (D1-MSNs) relative to D2-MSNs. We also found that amygdala activity was required for cocaine-induced changes to behavior and connectivity. Finally, we established how heightened amygdala innervation can explain the structural and functional changes evoked by cocaine. Our findings reveal how exposure to drugs of abuse fundamentally reorganizes cell type- and input-specific connectivity in the NAc.

  5. Primary immune system responders to nucleus pulposus cells: evidence for immune response in disc herniation

    Directory of Open Access Journals (Sweden)

    K Murai

    2010-01-01

    Full Text Available Although intervertebral disc herniation and associated sciatica is a common disease, its molecular pathogenesis is not well understood. Immune responses are thought to be involved. This study provides direct evidence that even non-degenerated nucleus pulposus (NP cells elicit immune responses. An in vitro colony forming inhibition assay demonstrated the suppressive effects of autologous spleen cells on NP cells and an in vitro cytotoxicity assay showed the positive cytotoxic effects of natural killer (NK cells and macrophages on NP cells. Non-degenerated rat NP tissues transplanted into wild type rats and immune-deficient mice demonstrated a significantly higher NP cell survival rate in immune-deficient mice. Immunohistochemical staining showed the presence of macrophages and NK cells in the transplanted NP tissues. These results suggest that even non-degenerated autologous NP cells are recognized by macrophages and NK cells, which may have an immunological function in the early phase of disc herniation. These findings contribute to understanding resorption and the inflammatory reaction to disc herniation.

  6. Modulating notochordal differentiation of human induced pluripotent stem cells using natural nucleus pulposus tissue matrix.

    Directory of Open Access Journals (Sweden)

    Yongxing Liu

    Full Text Available Human induced pluripotent stem cells (hiPSCs can differentiate into notochordal cell (NC-like cells when cultured in the presence of natural porcine nucleus pulposus (NP tissue matrix. The method promises massive production of high-quality, functional cells to treat degenerative intervertebral discs (IVDs. Based on our previous work, we further examined the effect of cell-NP matrix contact and culture medium on the differentiation, and further assessed the functional differentiation ability of the generated NC-like. The study showed that direct contact between hiPSCs and NP matrix can promote the differentiation yield, whilst both the contact and non-contact cultures can generate functional NC-like cells. The generated NC-like cells are highly homogenous regarding the expression of notochordal marker genes. A culture medium containing a cocktail of growth factors (FGF, EGF, VEGF and IGF-1 also supported the notochordal differentiation in the presence of NP matrix. The NC-like cells showed excellent functional differentiation ability to generate NP-like tissue which was rich in aggrecan and collagen type II; and particularly, the proteoglycan to collagen content ratio was as high as 12.5-17.5 which represents a phenotype close to NP rather than hyaline cartilage. Collectively, the present study confirmed the effectiveness and flexibility of using natural NP tissue matrix to direct notochordal differentiation of hiPSCs, and the potential of using the generated NC-like cells for treating IVD degeneration.

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

    Science.gov (United States)

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

    2016-09-01

    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.

  8. Chromosome oscillations in mitosis

    Science.gov (United States)

    Campas, Otger

    2008-03-01

    Successful cell division necessitates a tight regulation of chromosome movement via the activity of molecular motors. Many of the key players at the origin of the forces generating the motion have been identified, but their spatial and temporal organization remains elusive. In animal cells, chromosomes periodically switch between phases of movement towards and away from the pole. This characteristic oscillatory behaviour cannot be explained by the current models of chromosome positioning and congression. We perform a self-contained theoretical analysis in which the motion of mono-oriented chromosomes results from the competition between the activity of the kinetochore and chromokinesin motors on the chromosome arms. Our analysis, consistent with the available experimental data, proposes that the interplay between the aster-like morphology of the spindle and the collective kinetics of molecular motors is at the origin of chromosome oscillations, positioning and congression. It provides a natural explanation for the so-called chromosome directional instability and for the mechanism by which chromosomes sense their position in space. In addition, we estimate the in vivo velocity of chromokinesins at vanishing load and propose new experiments to assess the mechanism at the origin of chromosome movement in cell division.

  9. Pathologic mitoses and pathology of mitosis in tumorigenesis

    Directory of Open Access Journals (Sweden)

    RG Steinbeck

    2009-12-01

    Full Text Available The gist of my hypothesis (.. is a certain abnormal chromatin constitution. Each process, which brings about this chromatin constitution, would result in the origin of a malignant tumour. Certainly, I consider irregularities with mitosis as the normal mode of the origin of an incorrectly assembled nucleus. This statement by Boveri (1914 has considered earlier observations of asymmetric divisions in human cancers (Hansemann, 1890. The hypothesis is based on the understanding of mitosis as an equational bipartition of the hereditary substance (Flemming, 1879; Roux, 1883. Latest since it was known that genes are located on chromosomes (Sturtevant, 1913, their balanced transport in anaphase appeared as a condition of correct somatic proliferation. True mitoses guarantee the constancy of terminally differentiated tissues. Politzer (1934 has performed X-ray experiments to investigate abnormal karyokinesis with regard to anomalous chromatin condensation, chromosome breakage, spindle malformation, and failure in cytokinesis. On the basis of light microscopy, further significant progress in understanding the pathology of mitosis was not possible. Tumour cases with reduced chromosome numbers seduced to the idea that mitotic activity is rather under cytoplasmic than under nuclear control (Koller, 1947.

  10. Visually guided whole cell patch clamp of mouse supraoptic nucleus neurons in cultured and acute conditions.

    Science.gov (United States)

    Stachniak, Tevye J E; Bourque, Charles W

    2006-07-01

    Recent advances in neuronal culturing techniques have supplied a new set of tools for studying neural tissue, providing effective means to study molecular aspects of regulatory elements in the supraoptic nucleus of the hypothalamus (SON). To combine molecular biology techniques with electrophysiological recording, we modified an organotypic culture protocol to permit transfection and whole cell patch-clamp recordings from SON cells. Neonatal mouse brain coronal sections containing the SON were dissected out, placed on a filter insert in culture medium, and incubated for at least 4 days to allow attachment to the insert. The SON was identifiable using gross anatomical landmarks, which remained intact throughout the culturing period. Immunohistochemical staining identified both vasopressinergic and oxytocinergic cells present in the cultures, typically appearing in well-defined clusters. Whole cell recordings from these cultures demonstrated that certain properties of the neonatal mouse SON were comparable to adult mouse magnocellular neurons. SON neurons in both neonatal cultures and acute adult slices showed similar sustained outward rectification above -60 mV and action potential broadening during evoked activity. Membrane potential, input resistance, and rapidly inactivating potassium current density (IA) were reduced in the cultures, whereas whole cell capacitance and spontaneous synaptic excitation were increased, perhaps reflecting developmental changes in cell physiology that warrant further study. The use of the outlined organotypic culturing procedures will allow the study of such electrophysiological properties of mouse SON using whole cell patch-clamp, in addition to various molecular, techniques that require longer incubation times.

  11. The development of fluorescence turn-on probe for Al(III) sensing and live cell nucleus-nucleoli staining

    Science.gov (United States)

    Saini, Anoop Kumar; Sharma, Vinay; Mathur, Pradeep; Shaikh, Mobin M.

    2016-01-01

    The morphology of nucleus and nucleolus is powerful indicator of physiological and pathological conditions. The specific staining of nucleolus recently gained much attention due to the limited and expensive availability of the only existing stain “SYTO RNA-Select”. Here, a new multifunctional salen type ligand (L1) and its Al3+ complex (1) are designed and synthesized. L1 acts as a chemosensor for Al3+ whereas 1 demonstrates specific staining of nucleus as well as nucleoli. The binding of 1 with nucleic acid is probed by DNase and RNase digestion in stained cells. 1 shows an excellent photostability, which is a limitation for existing nucleus stains during long term observations. 1 is assumed to be a potential candidate as an alternative to expensive commercial dyes for nucleus and nucleoli staining. PMID:27721431

  12. Mitosis-specific phosphorylation of PML at T409 regulates spindle checkpoint.

    Science.gov (United States)

    Jin, J; Liu, J

    2016-08-31

    During mitosis, Promyelocytic leukemia nuclear bodies (PML NBs) change dramatically in morphology and composition, but little is known about function of PML in mitosis. Here, we show that PML is phosphorylated at T409 (PML p409) in a mitosis-specific manner. More importantly, PML p409 contributes to maintain the duration of pro-metaphase and regulates spindle checkpoint. Deficient PML p409 caused a shortening of pro-metaphase and challenged the nocodazole-triggered mitotic arrest. T409A mutation led to a higher frequency of misaligned chromosomes on metaphase plate, and subsequently death in late mitosis. In addition, inhibition of PML p409 repressed growth of tumor cells, suggesting that PML p409 is a potential target for cancer therapy. Collectively, our study demonstrated an important phosphorylated site of PML, which contributed to explore the role of PML in mitosis.

  13. Disruption of microtubule integrity initiates mitosis during CNS repair.

    Science.gov (United States)

    Bossing, Torsten; Barros, Claudia S; Fischer, Bettina; Russell, Steven; Shepherd, David

    2012-08-14

    Mechanisms of CNS repair have vital medical implications. We show that traumatic injury to the ventral midline of the embryonic Drosophila CNS activates cell divisions to replace lost cells. A pilot screen analyzing transcriptomes of single cells during repair pointed to downregulation of the microtubule-stabilizing GTPase mitochondrial Rho (Miro) and upregulation of the Jun transcription factor Jun-related antigen (Jra). Ectopic Miro expression can prevent midline divisions after damage, whereas Miro depletion destabilizes cortical β-tubulin and increases divisions. Disruption of cortical microtubules, either by chemical depolymerization or by overexpression of monomeric tubulin, triggers ectopic mitosis in the midline and induces Jra expression. Conversely, loss of Jra renders midline cells unable to replace damaged siblings. Our data indicate that upon injury, the integrity of the microtubule cytoskeleton controls cell division in the CNS midline, triggering extra mitosis to replace lost cells. The conservation of the identified molecules suggests that similar mechanisms may operate in vertebrates.

  14. Matrix stiffness determines the fate of nucleus pulposus-derived stem cells.

    Science.gov (United States)

    Navaro, Yosi; Bleich-Kimelman, Nadav; Hazanov, Lena; Mironi-Harpaz, Iris; Shachaf, Yonatan; Garty, Shai; Smith, Yoav; Pelled, Gadi; Gazit, Dan; Seliktar, Dror; Gazit, Zulma

    2015-05-01

    Intervertebral disc (IVD) degeneration and consequent low-back pain present a major medical challenge. Nucleus pulposus-derived stem cells (NP-SCs) may lead to a novel therapy for this severe disease. It was recently shown that survival and function of mature NP cells are regulated in part by tissue stiffness. We hypothesized that modification of matrix stiffness will influence the ability of cultured NP-SCs to proliferate, survive, and differentiate into mature NP cells. NP-SCs were subcultured in three-dimensional matrices of varying degrees of stiffness as measured by the material's shear storage modulus. Cell survival, activity, and rate of differentiation toward the chondrogenic or osteogenic lineage were analyzed. NP-SCs were found to proliferate and differentiate in all matrices, irrespective of matrix stiffness. However, matrices with a low shear storage modulus (G' = 1 kPa) promoted significantly more proliferation and chondrogenic differentiation, whereas matrices with a high modulus (G' = 2 kPa) promoted osteogenic differentiation. Imaging performed via confocal and scanning electron microscopes validated cell survival and highlighted stiffness-dependent cell-matrix interactions. These results underscore the effect of the matrix modulus on the fate of NP-SCs. This research may facilitate elucidation of the complex cross-talk between NP-SCs and their surrounding matrix in healthy as well as pathological conditions.

  15. A nutrient dependant switch explains mutually exclusive existence of meiosis and mitosis initiation in budding yeast.

    Science.gov (United States)

    Wannige, C T; Kulasiri, D; Samarasinghe, S

    2014-01-21

    Nutrients from living environment are vital for the survival and growth of any organism. Budding yeast diploid cells decide to grow by mitosis type cell division or decide to create unique, stress resistant spores by meiosis type cell division depending on the available nutrient conditions. To gain a molecular systems level understanding of the nutrient dependant switching between meiosis and mitosis initiation in diploid cells of budding yeast, we develop a theoretical model based on ordinary differential equations (ODEs) including the mitosis initiator and its relations to budding yeast meiosis initiation network. Our model accurately and qualitatively predicts the experimentally revealed temporal variations of related proteins under different nutrient conditions as well as the diverse mutant studies related to meiosis and mitosis initiation. Using this model, we show how the meiosis and mitosis initiators form an all-or-none type bistable switch in response to available nutrient level (mainly nitrogen). The transitions to and from meiosis or mitosis initiation states occur via saddle node bifurcation. This bidirectional switch helps the optimal usage of available nutrients and explains the mutually exclusive existence of meiosis and mitosis pathways.

  16. ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Inesta-Vaquera, Francisco A. [Departamento de Inmunologia y Oncologia, Centro Nacional de Biotecnologia-CSIC, Campus de Cantoblanco-UAM, 28049 Madrid (Spain); Campbell, David G.; Arthur, J. Simon C. [MRC Protein Phosphorylation Unit, Sir James Black Building, School of Life Sciences, University of Dundee, Dundee DD1 5EH (United Kingdom); Cuenda, Ana, E-mail: acuenda@cnb.csic.es [Departamento de Inmunologia y Oncologia, Centro Nacional de Biotecnologia-CSIC, Campus de Cantoblanco-UAM, 28049 Madrid (Spain)

    2010-08-13

    Research highlights: {yields} hDlg is phosphorylated during mitosis in multiple residues. {yields} Prospho-hDlg is excluded from the midbody during mitosis. {yields} hDlg is not phosphorylated by p38{gamma} or JNK1/2 during mitosis. {yields} ERK5 pathway mediates hDlg phosphorylation in mitosis. -- Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

  17. Dynamic interaction between actin and nesprin2 maintain the cell nucleus in a prestressed state

    Science.gov (United States)

    Kumar, Abhishek; Shivashankar, G. V.

    2016-12-01

    Mechanical coupling between the nucleus and the cytoskeleton is indispensable for direct force transduction from the extra cellular matrix (ECM) to the chromatin. Although this physical coupling has been shown to be crucial for nuclear positioning and its function, the quantification of nuclear-cytoskeleton interaction has been lacking. In this paper, using various quantitative fluorescence spectroscopy techniques, we investigate the nature of this connection. High-resolution 3D imaging shows that nesprin2G forms short linear structures along actin stress fibers (ASFs) in the apical region of the nucleus. Fluorescence recovery after photobleaching (FRAP) revealed that the alignment of nesprin2G becomes heterogeneous when cell shape is engineered from elongated rectangular shape to square using micropatterned substrates. Further, fluorescence cross-correlation spectroscopy (FCCS) revealed that actin interacts transiently with outer nuclear membrane protein nesprin2G with a time scale of 12 ms. In addition, fluorescence resonance energy transfer (FRET) experiments show that the apical ASFs and nesprin2G are in close physical proximity. This interaction is spatially heterogeneous with high FRET along the ASFs. Lastly, we show that the disruption of actin to nuclear connection by over-expression of Dominant Negative Klarsicht, ANC-1, Syne Homology (DNKASH) leads to an increase in nuclear height. These results not only reveal the characteristics of actin-nesprin2G interaction and its significance in regulating nuclear morphology, but also validate the utility of quantitative fluorescence techniques in deciphering physical connections that are essential for mechanotransduction.

  18. Toxic effects of mercury on the cell nucleus of Dictyostelium discoideum.

    Science.gov (United States)

    Boatti, Lara; Rapallo, Fabio; Viarengo, Aldo; Marsano, Francesco

    2017-02-01

    Governmental agencies (www.epa.gov/mercury) and the scientific community have reported on the high toxicity due to mercury. Indeed, exposure to mercury can cause severe injury to the central nervous system and kidney in humans. Beyond its recognized toxicity, little is known regarding the molecular mechanisms involved in the actions of this heavy metal. Mercury has been also observed to form insoluble fibrous protein aggregates in the cell nucleus. We used D. discoideum to evaluate micronuclei formation and, since mercury is able to induce oxidative stress that could bring to protein aggregation, we assessed nuclear protein carbonylation by Western Blot. We observed a significant increase in micronuclei formation and 14 carbonylated proteins were identified. Moreover, we used isotope-coded protein label (ICPL) and mass spectrometry analysis of proteins obtained by lysis of purified nuclei, before of tryptic digestion to quantify nuclear proteins affected by mercury. In particular, we examined the effects of mercury that associate a classical genotoxic assay to proteomic effects into the nucleus. The data present direct evidences for mercury genotoxicity, nuclear protein carbonylation, quantitative change in core histones, and the involvement of pseudouridine synthase in mercury toxicity. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 417-425, 2017.

  19. Retinal ganglion cell projections to the hamster suprachiasmatic nucleus, intergeniculate leaflet, and visual midbrain: bifurcation and melanopsin immunoreactivity

    Science.gov (United States)

    Morin, Lawrence P.; Blanchard, Jane H.; Provencio, Ignacio

    2003-01-01

    The circadian clock in the suprachiasmatic nucleus (SCN) receives direct retinal input via the retinohypothalamic tract (RHT), and the retinal ganglion cells contributing to this projection may be specialized with respect to direct regulation of the circadian clock. However, some ganglion cells forming the RHT bifurcate, sending axon collaterals to the intergeniculate leaflet (IGL) through which light has secondary access to the circadian clock. The present studies provide a more extensive examination of ganglion cell bifurcation and evaluate whether ganglion cells projecting to several subcortical visual nuclei contain melanopsin, a putative ganglion cell photopigment. The results showed that retinal ganglion cells projecting to the SCN send collaterals to the IGL, olivary pretectal nucleus, and superior colliculus, among other places. Melanopsin-immunoreactive (IR) ganglion cells are present in the hamster retina, and some of these cells project to the SCN, IGL, olivary pretectal nucleus, or superior colliculus. Triple-label analysis showed that melanopsin-IR cells bifurcate and project bilaterally to each SCN, but not to the other visual nuclei evaluated. The melanopsin-IR cells have photoreceptive characteristics optimal for circadian rhythm regulation. However, the presence of moderately widespread bifurcation among ganglion cells projecting to the SCN, and projection by melanopsin-IR cells to locations distinct from the SCN and without known rhythm function, suggest that this ganglion cell type is generalized, rather than specialized, with respect to the conveyance of photic information to the brain. Copyright 2003 Wiley-Liss, Inc.

  20. EphB4 localises to the nucleus of prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Mertens-Walker, Inga, E-mail: inga.mertenswalker@qut.edu.au [Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD (Australia); Australian Prostate Cancer Research Centre—Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD (Australia); Lisle, Jessica E. [Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD (Australia); Australian Prostate Cancer Research Centre—Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD (Australia); Nyberg, William A. [Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD (Australia); Stephens, Carson R. [Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD (Australia); Australian Prostate Cancer Research Centre—Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD (Australia); Burke, Leslie [Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD (Australia); Rutkowski, Raphael; Herington, Adrian C.; Stephenson, Sally-Anne [Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD (Australia); Australian Prostate Cancer Research Centre—Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD (Australia)

    2015-04-10

    The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of both NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor. - Highlights: • The EphB4 protein can be found in the nucleus of prostate cancer cell lines. • EphB4 contains two functional nuclear localisation signals. • Chromatin immunoprecipitation has identified potential genome sequences to which EphB4 binds. • Lef1 is a confirmed target for EphB4-mediated gene regulation.

  1. Increased expression of stefin B in the nucleus of T98G astrocytoma cells delays caspase activation

    Directory of Open Access Journals (Sweden)

    Tao eSun

    2012-09-01

    Full Text Available Stefin B (cystatin B is an endogenous inhibitor of cysteine proteinases localized in the nucleus and the cytosol. Loss-of-function mutations in the stefin B gene (CSTB gene were reported in patients with Unverricht-Lundborg disease (EPM1. Our previous results showed that thymocytes isolated from stefin B-deficient mice are more sensitive to apoptosis induced by the protein kinase C inhibitor staurosporin (STS than the wild-type control cells. We have also shown that the increased expression of stefin B in the nucleus of T98G astrocytoma cells delayed cell cycle progression through the S phase. In the present study we examined if the nuclear or cytosolic functions of stefin B are responsible for the accelerated induction of apoptosis observed in the cells from stefin B-deficient mice. We have shown that the overexpression of stefin B in the nucleus, but not in the cytosol of astrocytoma T98G cells, delayed caspase-3 and-7 activation. Pretreatment of cells with the pan-caspase inhibitor z-Val-Ala-Asp(OMe-fluoromethylketone completely inhibited caspase activation, while treatment with the inhibitor of calpains- and papain-like cathepsins (2S,3S-trans-epoxysuccinyl-leucylamido-3-methyl-butane ethyl ester did not prevent caspase activation. We concluded that the delay of caspase activation in T98G cells overexpressing stefin B in the nucleus is independent of cathepsin inhibition.

  2. The polo-like kinase PLKA is required for initiation and progression through mitosis in the filamentous fungus Aspergillus nidulans.

    Science.gov (United States)

    Bachewich, Catherine; Masker, Kathryn; Osmani, Stephen

    2005-01-01

    Polo-like kinases (PLK) function during multiple stages of mitotic progression and in cytokinesis. We identified and cloned a PLK homologue in Aspergillus nidulans, plkA, which is the first PLK reported in a filamentous fungus and the largest member of the PLK family to date. As plkA was essential, the effects of overexpression and localization of protein in living cells were explored to determine PLKA function. Overexpression of PLKA permitted hyphal formation, but blocked nuclear division in interphase. In NIMA or NIMT temperature-sensitive backgrounds, overexpression of PLKA impaired normal entry into mitosis upon release from restrictive temperature, supporting a role for PLKA during G2/M. In the few mitotic cells present, spindles were monopolar or disorganized, and chromatin condensation and segregation were impaired, suggesting additional roles for PLKA in spindle formation and in chromosome dynamics. Consistent with this, green fluorescent protein (GFP)-tagged PLKA could localize to the spb during interphase, and to the spb and nucleus throughout mitosis. Intriguingly, PLKA remained on the spb during telophase and into G1, in contrast to other PLK. In addition, spb localization was independent of NIMA function, unlike that demonstrated in Schizosaccharomyces pombe where PLK localization to the spb required the NIMA homologue Fin1. PLKA was not detected at cortical, septation-associated sites, and overexpression did not drive septum formation, also in contrast to that observed with other PLK. Therefore, PLKA is important for multiple events during mitosis, similar to PLK in higher organisms, but exhibits differences in size, localization and influence on septation/cytokinesis, suggesting additional novel regulatory features.

  3. Transient, afferent input-dependent, postnatal niche for neural progenitor cells in the cochlear nucleus.

    Science.gov (United States)

    Volkenstein, Stefan; Oshima, Kazuo; Sinkkonen, Saku T; Corrales, C Eduardo; Most, Sam P; Chai, Renjie; Jan, Taha A; van Amerongen, Renée; Cheng, Alan G; Heller, Stefan

    2013-08-27

    In the cochlear nucleus (CN), the first central relay of the auditory pathway, the survival of neurons during the first weeks after birth depends on afferent innervation from the cochlea. Although input-dependent neuron survival has been extensively studied in the CN, neurogenesis has not been evaluated as a possible mechanism of postnatal plasticity. Here we show that new neurons are born in the CN during the critical period of postnatal plasticity. Coincidently, we found a population of neural progenitor cells that are controlled by a complex interplay of Wnt, Notch, and TGFβ/BMP signaling, in which low levels of TGFβ/BMP signaling are permissive for progenitor proliferation that is promoted by Wnt and Notch activation. We further show that cells with activated Wnt signaling reside in the CN and that these cells have high propensity for neurosphere formation. Cochlear ablation resulted in diminishment of progenitors and Wnt/β-catenin-active cells, suggesting that the neonatal CN maintains an afferent innervation-dependent population of progenitor cells that display active canonical Wnt signaling.

  4. Influence of recipient cytoplasm cell stage on transcription in bovine nucleus transfer embryos

    DEFF Research Database (Denmark)

    Smith, Steven D.; Soloy, Eva; Kanka, Jiri

    1996-01-01

    relies upon maternally derived RNA transcripts up to the 8-cell stage, at which time it begins to transcribe its own RNA. In this experiment, RNA synthesis was detected in nucleus transfer embryos (NTE) and control embryos by pulsing with 3H-uridine, fixation, and autoradiography on semithin sections...... of maturation. Control in-vitro-produced embryos were 3H-uridine-labelled and fixed at the 2-, 4-, early 8-, and late 8-cell stages. NTE were similarly prepared at 1, 3, and 20 hr postfusion and at the 2-, 4-, and 8-cell stages. In the control embryos, RNA synthesis was absent in the 2-, 4-, and early 8-cell...... stages, whereas in all late 8-cell stages, it was present. In NTE from nonactivated (MII phase) cytoplasts, there was a sharp decline in RNA synthesis at 1 hr and 3 hr after fusion and a total absence by 20 hr after fusion. In contrast, NTE from activated (S phase) cytoplasts exhibited continued high...

  5. Glucosamine protects nucleus pulposus cells and induces autophagy via the mTOR-dependent pathway.

    Science.gov (United States)

    Jiang, LiBo; Jin, YongLong; Wang, HuiRen; Jiang, YunQi; Dong, Jian

    2014-11-01

    Although glucosamine has been suggested to be effective in the treatment of osteoarthritis, its effect on disc degeneration remains unclear. We sought to explore whether glucosamine can activate autophagy in rat nucleus pulposus (NP) cells and protect cells treated with IL-1β or hydrogen peroxide (H2 O2 ). Autophagy in cells was examined by detecting for LC3, Beclin-1, m-TOR, and p70S6K, as well as by analyzing autophagosomes. To inhibit autophagy, 3-methyladenine (3-MA) was used. In the cells treated with IL-1β, the levels of Adamts-4, Mmp-13, aggrecan, and Col2a1 were analyzed by real-time PCR and immunofluorescence. Apoptosis was analyzed by TUNEL. Cell senescence under H2 O2 was revealed by SA-β-Gal staining. Glucosamine could activate autophagy in a dose-dependent manner within 24 h and inhibit the phosphorylation of m-TOR and p70S6K. Autophagy in IL-1β or H2 O2 -treated cells was increased by glucosamine. Glucosamine attenuated the decrease of aggrecan and prevented the apoptosis of the NP cells induced by IL-1β, whereas 3-MA partly reversed these effects. The percentage of SA-β-Gal-positive cells induced by H2 O2 treatment was decreased by glucosamine, accompanied by the decline of p70S6K phosphorylation. Glucosamine protects NP cells and up-regulates autophagy by inhibiting the m-TOR pathway, which might point a potential therapeutic agent for disc degeneration.

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

    Science.gov (United States)

    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.

  7. Location of chromosomes in the nucleus of human mesenchymal stem cells.

    Science.gov (United States)

    Lavrov, A V; Voldgorn, Y I

    2011-08-01

    For evaluation of the spatial structure of chromatin in nuclei of mesenchymal SC we determined the position of centromeres and individual chromosomes in interphase nucleus of mesenchymal SC. More than 300 nuclei in 7 cultures of mesenchymal SC were analyzed. Centromeres of chromosomes 6, 8, and 11 lie at a longer (0.68, 0.67, 0.7), while centromere of chromosome 18 at a shorter radial distance (0.49). Homologues of each chromosome had different radial distances. No differences in radial distances of centromeres were detected between mesenchymal SC from the adipose tissue and BM. After passage 8, distal displacement of chromosome 6 centromere (from 0.66 to 0.72) was observed, which probably indicates aging or spontaneous differentiation of cells.

  8. A Simulation-Based Study of Dorsal Cochlear Nucleus Pyramidal Cell Firing Patterns

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Daliri

    2012-02-01

    Full Text Available A two-variable integrate and fire model is presented to study the role of transient outward potassium currents in producing temporal aspects of dorsal cochlear nucleus (DCN pyramidal cells with different profiles namely the chopper, the pauser and the buildup. This conductance based model is a reduced version of KM-LIF model (Meng & Rinzel, 2010 which captures qualitative firing features of a detailed physiological model (Kanold & Manis, 2000.For our development we benefit from transient potassium currents properties i.e.fast activation and slow inactivation to generate long latency before start of firing.We compare our minimal model outputs in response to a hyperpolarizing stimulus fallowed by a depolarizing one with the data of KM-LIF model.The results conform well to the KM-LIF model with lower complexity.

  9. The function of the chicken p34CDC2 protein kinase in fission yeast is cold sensitive for cell cycle progression through the G1 phase and temperature sensitive for traversal of mitosis.

    Science.gov (United States)

    Schmitz, N

    1999-06-01

    The protein kinase p34cdc2 is required at the onset of DNA replication and for entry into mitosis. The catalytic subunit and its regulatory proteins, notably the cyclins, are conserved from yeast to man. This suggests that the control mechanisms necessary for progression through the cell cycle in fission yeast are conserved throughout evolution. This work describes the characterization of a fission yeast strain that is dependent for cell cycle progression on the activity of the p34CDC2 protein kinase from chicken. The response of the chicken p34CDC2 protein kinase to cell cycle components of fission yeast was examined. Cells expressing the chicken p34CDC2 protein divide at reduced size at 31 degrees C. Cells are temperature sensitive at 35.5 degrees C and die as a result of mitotic catastrophe. This phenotype can be rescued by delaying cell cycle progression at the G1-S transition by adding low concentrations of hydroxyurea. Schizosaccharomyces pombe cells that are dependent on chicken p34CDC2 are cold sensitive. At 19 degrees C to 25 degrees C cells arrest in the G1 phase, while traversal of the G2-M transition is not blocked at low temperature. Expression of chicken p34CDC2 in the cold-sensitive G2-M mutant cdc2A21 suppresses the G1 arrest.

  10. Regenerative and immunogenic characteristics of cultured nucleus pulposus cells from human cervical intervertebral discs.

    Directory of Open Access Journals (Sweden)

    Stefan Stich

    Full Text Available Cell-based regenerative approaches have been suggested as primary or adjuvant procedures for the treatment of degenerated intervertebral disc (IVD diseases. Our aim was to evaluate the regenerative and immunogenic properties of mildly and severely degenerated cervical nucleus pulposus (NP cells with regard to cell isolation, proliferation and differentiation, as well as to cell surface markers and co-cultures with autologous or allogeneic peripheral blood mononuclear cells (PBMC including changes in their immunogenic properties after 3-dimensional (3D-culture. Tissue from the NP compartment of 10 patients with mild or severe grades of IVD degeneration was collected. Cells were isolated, expanded with and without basic fibroblast growth factor and cultured in 3D fibrin/poly (lactic-co-glycolic acid transplants for 21 days. Real-time reverse-transcription polymerase chain reaction (RT-PCR showed the expression of characteristic NP markers ACAN, COL1A1 and COL2A1 in 2D- and 3D-culture with degeneration- and culture-dependent differences. In a 5,6-carboxyfluorescein diacetate N-succinimidyl ester-based proliferation assay, NP cells in monolayer, regardless of their grade of degeneration, did not provoke a significant proliferation response in T cells, natural killer (NK cells or B cells, not only with donor PBMC, but also with allogeneic PBMC. In conjunction with low inflammatory cytokine expression, analyzed by Cytometric Bead Array and fluorescence-activated cell sorting (FACS, a low immunogenicity can be assumed, facilitating possible therapeutic approaches. In 3D-culture, however, we found elevated immune cell proliferation levels, and there was a general trend to higher responses for NP cells from severely degenerated IVD tissue. This emphasizes the importance of considering the specific immunological alterations when including biomaterials in a therapeutic concept. The overall expression of Fas receptor, found on cultured NP cells, could have

  11. The planar cell polarity (PCP) protein Diversin translocates to the nucleus to interact with the transcription factor AF9

    Energy Technology Data Exchange (ETDEWEB)

    Haribaskar, Ramachandran; Puetz, Michael; Schupp, Birte; Skouloudaki, Kassiani; Bietenbeck, Andreas; Walz, Gerd [Renal Division, University Hospital Freiburg, Hugstetter Strasse 55, D-79106 Freiburg (Germany); Schaefer, Tobias, E-mail: tobias.schaefer@uniklinik-freiburg.de [Renal Division, University Hospital Freiburg, Hugstetter Strasse 55, D-79106 Freiburg (Germany)

    2009-09-11

    The planar cell polarity (PCP) pathway, a {beta}-catenin-independent branch of the Wnt signaling pathway, orients cells and their appendages with respect to the body axes. Diversin, the mammalian homolog of the Drosophila PCP protein Diego, acts as a molecular switch that blocks {beta}-catenin-dependent and promotes {beta}-catenin-independent Wnt signaling. We report now that Diversin, containing several nuclear localization signals, translocates to the nucleus, where it interacts with the transcription factor AF9. Both Diversin and AF9 block canonical Wnt signaling; however, this occurs independently of each other, and does not require nuclear Diversin. In contrast, AF9 strongly augments the Diversin-driven activation of c-Jun N-terminal kinase (JNK)-dependent gene expression in the nucleus, and this augmentation largely depends on the presence of nuclear Diversin. Thus, our findings reveal that components of the PCP cascade translocate to the nucleus to participate in transcriptional regulation and PCP signaling.

  12. Identification of mayaro virus nucleocapsid protein in nucleus of Aedes albopictus cells.

    Science.gov (United States)

    Mitchell, C; de Andrade-Rozental, A F; Souto-Padrón, T; Carvalho, M G

    1997-01-01

    The modifications in the pattern of nuclear proteins of Aedes albopictus cells in response to Mayaro virus infection were analysed early and late after infection. The viral capsid (C) protein of 34 kDa (p34) could be detected in the nuclear compartment 4 h after infection, soon after its synthesis in the cytoplasm. In addition to p34, a group of high molecular weight proteins was also present in this compartment late after infection. The exposition of infected cells to supra optimal temperature of growth modifies significantly the pattern of nuclear proteins. However, the stress condition does not inhibit the transport of p34 to the nucleus. The transport of proteins into nuclei was also followed under "in vitro' conditions by incubating radiolabeled post-mitochondrial extract of infected cells with unlabeled nuclei. Under these conditions, as observed "in vivo', a specific transport of viral C protein and of a group of proteins of high molecular weight to the nuclei was also detected. These results indicate that Mayaro virus infection modifies the nuclear protein pattern in invertebrate cells.

  13. Dysfunction of nucleus accumbens-1 activates cellular senescence and inhibits tumor cell proliferation and oncogenesis.

    Science.gov (United States)

    Zhang, Yi; Cheng, Yan; Ren, Xingcong; Hori, Tsukasa; Huber-Keener, Kathryn J; Zhang, Li; Yap, Kai Lee; Liu, David; Shantz, Lisa; Qin, Zheng-Hong; Zhang, Suping; Wang, Jianrong; Wang, Hong-Gang; Shih, Ie-Ming; Yang, Jin-Ming

    2012-08-15

    Nucleus accumbens-1 (NAC1), a nuclear factor belonging to the BTB/POZ gene family, has emerging roles in cancer. We report here that NAC1 acts as a negative regulator of cellular senescence in transformed and nontransformed cells, and dysfunction of NAC1 induces senescence and inhibits its oncogenic potential. We show that NAC1 deficiency markedly activates senescence and inhibits proliferation in tumor cells treated with sublethal doses of γ-irradiation. In mouse embryonic fibroblasts from NAC1 knockout mice, following infection with a Ras virus, NAC1-/- cells undergo significantly more senescence and are either nontransformed or less transformed in vitro and less tumorigenic in vivo when compared with NAC1+/+ cells. Furthermore, we show that the NAC1-caused senescence blunting is mediated by ΔNp63, which exerts its effect on senescence through p21, and that NAC1 activates transcription of ΔNp63 under stressful conditions. Our results not only reveal a previously unrecognized function of NAC1, the molecular pathway involved and its impact on pathogenesis of tumor initiation and development, but also identify a novel senescence regulator that may be exploited as a potential target for cancer prevention and treatment.

  14. 植物细胞有丝分裂实验中盐酸浓度对实验效果影响的研究%Effective research on different concentrations of hydrochloric acid in plant cell mitosis experiment

    Institute of Scientific and Technical Information of China (English)

    归改霞

    2015-01-01

    目的:探讨有丝分裂实验中盐酸浓度对实验效果的影响。方法:通过使用不同浓度的盐酸做为解离液进行实验,从中选取最适合的盐酸浓度,从而达到最理想的实验效果。结果:通过实验比较,解离液中盐酸的浓度为25%比较合适。结论:植物细胞有丝分裂实验,在选材、培养、取材均为最优条件下,解离液中盐酸的浓度是实验成功的关键。当盐酸的浓度为25%时,实验成功率最高,能达到最佳效果。%Objective: To investigate effects of different concentrations of hydrochloric acid in plant cell mitosis experiment. Methods: In order to achieve the best experimental results, the most suitable concentration of hydrochloric acid was selected in experiments. Results: Based on experimental comparison, concentration of hydrochloric acid of 25% in dissociation solution is suitable. Conclusion: When other conditions are the best, concentration of hydrochloric acid is the key in plant cell mitosis experiment. When concentration of hydrochloric acid was 25%, there was highest efficiency and the best result.

  15. Characterization of novel photocrosslinked carboxymethylcellulose hydrogels for encapsulation of nucleus pulposus cells.

    Science.gov (United States)

    Reza, Anna T; Nicoll, Steven B

    2010-01-01

    Back pain is a significant clinical concern often associated with degeneration of the intervertebral disc (IVD). Tissue engineering strategies may provide a viable IVD replacement therapy; however, an ideal biomaterial scaffold has yet to be identified. One candidate material is carboxymethylcellulose (CMC), a water-soluble derivative of cellulose. In this study, 90 and 250 kDa CMC polymers were modified with functional methacrylate groups and photocrosslinked to produce hydrogels at different macromer concentrations. At 7 days, bovine nucleus pulposus (NP) cells encapsulated in these hydrogels were viable, with values for the elastic modulus ranging from 1.07 + or - 0.06 to 4.29 + or - 1.25 kPa. Three specific formulations were chosen for further study based on cell viability and mechanical integrity assessments: 4% 90 kDa, 2% 250 kDa and 3% 250 kDa CMC. The equilibrium weight swelling ratio of these formulations remained steady throughout the 2 week study (46.45 + or - 3.14, 48.55 + or - 2.91 and 42.41 + or - 3.06, respectively). The equilibrium Young's modulus of all cell-laden and cell-free control samples decreased over time, with the exception of cell-laden 3% 250 kDa CMC constructs, indicating an interplay between limited hydrolysis of interchain crosslinks and the elaboration of a functional matrix. Histological analyses of 3% 250 kDa CMC hydrogels confirmed the presence of rounded cells in lacunae and the pericellular deposition of chondroitin sulfate proteoglycan, a phenotypic NP marker. Taken together, these studies support the use of photocrosslinked CMC hydrogels as tunable biomaterials for NP cell encapsulation.

  16. Herbicide effects on freshwater benthic diatoms: Induction of nucleus alterations and silica cell wall abnormalities

    Energy Technology Data Exchange (ETDEWEB)

    Debenest, T. [Ecolab UMR 5245 (INP ENSAT, CNRS, UPS), Equipe ECOGEN, Avenue de l' Agrobiopole - BP 32607 Auzeville Tolosane, 31326 Castanet Tolosan Cedex (France); Cemagref, 50 avenue de Verdun, 33612 Cestas Cedex (France); Silvestre, J. [Ecolab UMR 5245 (INP ENSAT, CNRS, UPS), Equipe ECOGEN, Avenue de l' Agrobiopole - BP 32607 Auzeville Tolosane, 31326 Castanet Tolosan Cedex (France); Coste, M.; Delmas, F. [Cemagref, 50 avenue de Verdun, 33612 Cestas Cedex (France); Pinelli, E. [Ecolab UMR 5245 (INP ENSAT, CNRS, UPS), Equipe ECOGEN, Avenue de l' Agrobiopole - BP 32607 Auzeville Tolosane, 31326 Castanet Tolosan Cedex (France)], E-mail: pinelli@ensat.fr

    2008-06-02

    Benthic diatoms are well known bio-indicators of river pollution by nutrients (nitrogen and phosphorus). Biological indexes, based on diatom sensitivity for non-toxic pollution, have been developed to assess the water quality. Nevertheless, they are not reliable tools to detect pollution by pesticides. Many authors have suggested that toxic agents, like pesticides, induce abnormalities of the diatom cell wall (frustule). High abnormal frustule abundances have been reported in natural diatom communities sampled in streams contaminated by pesticides. However, no direct link was found between the abundances of abnormal frustules in these communities and the pesticide concentrations in stream water. In the present study, a freshwater benthic diatom community, isolated from natural biofilm and cultured under controlled conditions, was treated with a known genotoxic herbicide, maleic hydrazide (MH). Cells were exposed to three concentrations of MH (5 x 10{sup -6}, 10{sup -6}, 10{sup -7} M) for 6 h followed by a 24 h-recovery time. After MH treatments, nucleus alterations were observed: abnormal nucleus location, micronucleus, multinuclear cell or disruption of the nuclear membrane. A dose-dependent increase of nuclear alterations was observed. The difference between the control (9.65 nuclear alterations per 1000 cells observed (9.65 per mille), S.D. = 4.23) and the highest concentrations (29.40 per mille, S.D. = 8.49 for 10{sup -6} M and 35.96 per mille , S.D. = 3.71 for 5 x 10{sup -6} M) was statistically significant (Tukey test, P < 0.05). Diatoms also exhibited frustules with deformed morphology and abnormal ornamentation. Significantly increased abundances of abnormal frustules were observed for the highest concentrations (10{sup -6} and 5 x 10{sup -6} M; Tukey test, P < 0.05). These two parameters tended to increase together (Pearson correlation = 0.702, P < 0.05). The results suggest that the induction of abnormal frustules could be associated with the genotoxic

  17. Population control of resident and immigrant microglia by mitosis and apoptosis

    DEFF Research Database (Denmark)

    Wirenfeldt, Martin; Dissing-Olesen, Lasse; Babcock, Alicia

    2007-01-01

    Microglial population expansion occurs in response to neural damage via processes that involve mitosis and immigration of bone marrow-derived cells. However, little is known of the mechanisms that regulate clearance of reactive microglia, when microgliosis diminishes days to weeks later. We have...... in bone marrow chimeric mice. These results broaden our understanding of the microglial response to neural damage by demonstrating that simultaneously occurring mitosis and apoptosis regulate expansion and reduction of both resident and immigrant microglial cell populations....

  18. PICH promotes sister chromatid disjunction and co-operates with topoisomerase II in mitosis

    DEFF Research Database (Denmark)

    Nielsen, Christian Thomas Friberg; Huttner, Diana; Bizard, Anna H

    2015-01-01

    PICH is a SNF2 family DNA translocase that binds to ultra-fine DNA bridges (UFBs) in mitosis. Numerous roles for PICH have been proposed from protein depletion experiments, but a consensus has failed to emerge. Here, we report that deletion of PICH in avian cells causes chromosome structural......-193-treated cells. We propose that PICH and Topo II cooperate to prevent chromosome missegregation events in mitosis....

  19. Immobility, inheritance and plasticity of shape of the yeast nucleus

    Directory of Open Access Journals (Sweden)

    Andrulis Erik D

    2007-11-01

    Full Text Available Abstract Background Since S. cerevisiae undergoes closed mitosis, the nuclear envelope of the daughter nucleus is continuous with that of the maternal nucleus at anaphase. Nevertheless, several constitutents of the maternal nucleus are not present in the daughter nucleus. The present study aims to identify proteins which impact the shape of the yeast nucleus and to learn whether modifications of shape are passed on to the next mitotic generation. The Esc1p protein of S. cerevisiae localizes to the periphery of the nucleoplasm, can anchor chromatin, and has been implicated in targeted silencing both at telomeres and at HMR. Results Upon increased Esc1p expression, cell division continues and dramatic elaborations of the nuclear envelope extend into the cytoplasm. These "escapades" include nuclear pores and associate with the nucleolus, but exclude chromatin. Escapades are not inherited by daughter nuclei. This exclusion reflects their relative immobility, which we document in studies of prezygotes. Moreover, excess Esc1p affects the levels of multiple transcripts, not all of which originate at telomere-proximal loci. Unlike Esc1p and the colocalizing protein, Mlp1p, overexpression of selected proteins of the inner nuclear membrane is toxic. Conclusion Esc1p is the first non-membrane protein of the nuclear periphery which – like proteins of the nuclear lamina of higher eukaryotes – can modify the shape of the yeast nucleus. The elaborations of the nuclear envelope ("escapades" which appear upon induction of excess Esc1p are not inherited during mitotic growth. The lack of inheritance of such components could help sustain cell growth when parental nuclei have acquired potentially deleterious characteristics.

  20. Insights into centromeric transcription in mitosis.

    Science.gov (United States)

    Liu, Hong

    2016-01-01

    The major role of RNA polymerase II (RNAP II) is to generate mRNAs. I recently uncovered a novel function of RNAP II in chromosome segregation in mitosis, installing the cohesin protector, Shugoshin, at centromeres. Here I will discuss the current understanding of RNAP II-dependent centromeric transcription in mitosis.

  1. Gastrointestinal stromal tumor and mitosis, pay attention

    Institute of Scientific and Technical Information of China (English)

    Federico Coccolini; Fausto Catena; Luca Ansaloni; Antonio Daniele Pinna

    2012-01-01

    The difference between stages I and III of gastric gastrointestinal stromal tumor depends principally on the number of mitosis. According with TNM classification, the presence in the tumor of high mitotic rate determines the upgrading. Many studies exposed different count techniques in evaluating the number of mitosis. An international standardized method to assess mitotic rate is needed.

  2. The dynamic nature of the nuclear envelope: lessons from closed mitosis.

    Science.gov (United States)

    Arnone, James T; Walters, Alison D; Cohen-Fix, Orna

    2013-01-01

    In eukaryotes, chromosomes are encased by a dynamic nuclear envelope. In contrast to metazoans, where the nuclear envelope disassembles during mitosis, many fungi including budding yeast undergo "closed mitosis," where the nuclear envelope remains intact throughout the cell cycle. Consequently, during closed mitosis the nuclear envelope must expand to accommodate chromosome segregation to the two daughter cells. A recent study by Witkin et al. in budding yeast showed that if progression through mitosis is delayed, for example due to checkpoint activation, the nuclear envelope continues to expand despite the block to chromosome segregation. Moreover, this expansion occurs at a specific region of the nuclear envelope- adjacent to the nucleolus- forming an extension referred to as a "flare." These observations raise questions regarding the regulation of nuclear envelope expansion both in budding yeast and in higher eukaryotes, the mechanisms confining mitotic nuclear envelope expansion to a particular region and the possible consequences of failing to regulate nuclear envelope expansion during the cell cycle.

  3. HHV-8 encoded LANA-1 alters the higher organization of the cell nucleus

    Directory of Open Access Journals (Sweden)

    Klein George

    2007-04-01

    Full Text Available Abstract The latency-associated nuclear antigen (LANA-1 of Human Herpes Virus 8 (HHV-8, alternatively called Kaposi Sarcoma Herpes Virus (KSHV is constitutively expressed in all HHV-8 infected cells. LANA-1 accumulates in well-defined foci that co-localize with the viral episomes. We have previously shown that these foci are tightly associated with the borders of heterochromatin 1. We have also shown that exogenously expressed LANA-1 causes an extensive re-organization of Hoechst 33248 DNA staining patterns of the nuclei in non-HHV-8 infected cells 2. Here we show that this effect includes the release of the bulk of DNA from heterochromatic areas, in both human and mouse cells, without affecting the overall levels of heterochromatin associated histone H3 lysine 9 tri-methylation (3MK9H3. The release of DNA from the heterochromatic chromocenters in LANA-1 transfected mouse cells co-incides with the dispersion of the chromocenter associated methylcytosin binding protein 2 (MECP2. The localization of 3MK9H3 to the remnants of the chromocenters remains unaltered. Moreover, exogeneously expressed LANA-1 leads to the relocation of the chromocenters to the nuclear periphery, indicating extensive changes in the positioning of the chromosomal domains in the LANA-1 harboring interphase nucleus. Using a series of deletion mutants we have shown that the chromatin rearranging effects of LANA-1 require the presence of a short (57 amino acid region that is located immediately upstream of the internal acidic repeats. This sequence lies within the previously mapped binding site to histone methyltransferase SUV39H1. We suggest that the highly concentrated LANA-1, anchored to the host genome in the nuclear foci of latently infected cells and replicated through each cell generation, may function as "epigenetic modifier". The induction of histone modification in adjacent host genes may lead to altered gene expression, thereby contributing to the viral oncogenesis.

  4. Periodic mechanical stress activates EGFR-dependent Rac1 mitogenic signals in rat nucleus pulpous cells via ERK1/2.

    Science.gov (United States)

    Gao, Gongming; Shen, Nan; Jiang, Xuefeng; Sun, Huiqing; Xu, Nanwei; Zhou, Dong; Nong, Luming; Ren, Kewei

    2016-01-15

    The mitogenic effects of periodic mechanical stress on nucleus pulpous cells have been studied extensively but the mechanisms whereby nucleus pulpous cells sense and respond to mechanical stimulation remain a matter of debate. We explored this question by performing cell culture experiments in our self-developed periodic stress field and perfusion culture system. Under periodic mechanical stress, rat nucleus pulpous cell proliferation was significantly increased (p mechanical stress-induced nucleus pulpous cell proliferation (p mechanical stress (p mechanical stress (p mechanical stress promotes nucleus pulpous cell proliferation in part through the EGFR-Rac1-ERK1/2 signaling pathway, which links these three important signaling molecules into a mitogenic cascade.

  5. Frailty of two cell cycle checkpoints which prevent entry into mitosis and progression through early mitotic stages in higher plant cells.

    Science.gov (United States)

    del Campo, A; Giménez-Martín, G; López-Sáez, J F; de la Torre, C

    1997-11-01

    Allium cepa L. root meristems were given two short caffeine treatments spaced by 15 hours, the time which roughly corresponds to the duration of one cell cycle. In this way two subsequent cytokineses were prevented, and multinucleate cells with their in complement distributed into two, three or four nuclei were formed. Though all nuclei started to replicate synchronously in these cells, some of them (fast nuclei) completed their replication earlier than others (slow nuclei). The present report shows that two successive checkpoints operate before prometaphase in these cells. The first one prevents the entry of the fast nuclei into prophase until the slow ones have completed their replication. The second checkpoint ensures the synchronous entry into prometaphase after all nuclei have reached and finished prophase. By treating the multinucleate cells with an inhibitor of DNA synthesis at that time when fast but not slow nuclei had finished their replication, it was observed that both checkpoint mechanisms became leaky with time. Under these conditions the fast nuclei entered prophase in the presence of nuclei which were prevented from finishing the replication of their DNA. Subsequently, even prometaphase was triggered after a prolonged prophase. Finally, as expected from the presence of mitotic stages in these cells, nuclei with incompletely replicated DNA endured premature chromosome condensation. The prematurely condensed chromosomes either remained in a prometaphase-like stage until reconstitution nuclei formed or they followed the progression of the fast nuclei into metaphase and anaphase leading to the appearance of acentric chromosomal segments which after reconstitution gave rise to aneuploid nuclei containing unstable and broken DNA.

  6. Computational prediction of Mycoplasma hominis proteins targeting in nucleus of host cell and their implication in prostate cancer etiology.

    Science.gov (United States)

    Khan, Shahanavaj; Zakariah, Mohammed; Palaniappan, Sellappan

    2016-08-01

    Cancer has long been assumed to be a genetic disease. However, recent evidence supports the enigmatic connection of bacterial infection with the growth and development of various types of cancers. The cause and mechanism of the growth and development of prostate cancer due to Mycoplasma hominis remain unclear. Prostate cancer cells are infected and colonized by enteroinvasive M. hominis, which controls several factors that can affect prostate cancer growth in susceptible persons. We investigated M. hominis proteins targeting the nucleus of host cells and their implications in prostate cancer etiology. Many vital processes are controlled in the nucleus, where the proteins targeting M. hominis may have various potential implications. A total of 29/563 M. hominis proteins were predicted to target the nucleus of host cells. These include numerous proteins with the capability to alter normal growth activities. In conclusion, our results emphasize that various proteins of M. hominis targeted the nucleus of host cells and were involved in prostate cancer etiology through different mechanisms and strategies.

  7. Cycling with BRCA2 from DNA repair to mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hyunsook, E-mail: HL212@snu.ac.kr

    2014-11-15

    Genetic integrity in proliferating cells is guaranteed by the harmony of DNA replication, appropriate DNA repair, and segregation of the duplicated genome. Breast cancer susceptibility gene BRCA2 is a unique tumor suppressor that is involved in all three processes. Hence, it is critical in genome maintenance. The functions of BRCA2 in DNA repair and homology-directed recombination (HDR) have been reviewed numerous times. Here, I will briefly go through the functions of BRCA2 in HDR and focus on the emerging roles of BRCA2 in telomere homeostasis and mitosis, then discuss how BRCA2 exerts distinct functions in a cell-cycle specific manner in the maintenance of genomic integrity. - Highlights: • BRCA2 is a multifaceted tumor suppressor and is crucial in genetic integrity. • BRCA2 exerts distinct functions in cell cycle-specific manner. • Mitotic kinases regulate diverse functions of BRCA2 in mitosis and cytokinesis.

  8. The nucleus of endothelial cell as a sensor of blood flow direction

    Directory of Open Access Journals (Sweden)

    Eugene Tkachenko

    2013-08-01

    Hemodynamic shear stresses cause endothelial cells (ECs to polarize in the plane of the flow. Paradoxically, under strong shear flows, ECs disassemble their primary cilia, common sensors of shear, and thus must use an alternative mechanism of sensing the strength and direction of flow. In our experiments in microfluidic perfusion chambers, confluent ECs developed planar cell polarity at a rate proportional to the shear stress. The location of Golgi apparatus and microtubule organizing center was biased to the upstream side of the nucleus, i.e. the ECs polarized against the flow. These in vitro results agreed with observations in murine blood vessels, where EC polarization against the flow was stronger in high flow arteries than in veins. Once established, flow-induced polarization persisted over long time intervals without external shear. Transient destabilization of acto-myosin cytoskeleton by inhibition of myosin II or depolymerization of actin promoted polarization of EC against the flow, indicating that an intact acto-myosin cytoskeleton resists flow-induced polarization. These results suggested that polarization was induced by mechanical displacement of EC nuclei downstream under the hydrodynamic drag. This hypothesis was confirmed by the observation that acute application of a large hydrodynamic force to ECs resulted in an immediate downstream displacement of nuclei and was sufficient to induce persistent polarization. Taken together, our data indicate that ECs can sense the direction and strength of blood flow through the hydrodynamic drag applied to their nuclei.

  9. DGCR8 Localizes to the Nucleus as well as Cytoplasmic Structures in Mammalian Spermatogenic Cells and Epididymal Sperm

    Directory of Open Access Journals (Sweden)

    Akane Nakano

    2013-01-01

    Full Text Available The localization of DGCR8 in spermatogenic cells and sperm from rat and mouse was studied by immunofluorescence and immunoelectron microscopy. Spermatogenic cells from these species yielded similar DGCR8 localization pattern. Immunofluorescence microscopy results showed that DGCR8 localized to both the cytoplasm and nucleus. In the cytoplasm, diffuse cytosolic and discrete granular staining was observed. Dual staining showed that DGCR8 colocalized to the granules with MAEL (a nuage marker. In the nucleus of spermatocytes, both the nucleoli and nucleoplasm were stained, whereas in the nucleus of early spermatids small spots were stained. In late spermatids, DGCR8 localized to the tip of their head and to small granules (neck granules of the neck cytoplasm. The neck granules were also observed in the neck of epididymal sperm. Immunoelectron microscopy results showed that DGCR8 localized to nuage structures. Moreover, DGCR8 localized to nonnuage structures in late spermatids. DGCR8 also localized to the nucleolus and euchromatin in spermatocytes and round spermatids and to small granules in the nucleus of late spermatids. The results suggest that in spermatogenic cells DGCR8 localizes not only to the nuclei but also to the cytoplasmic structures such as nuage and nonnuage structures. Furthermore, DGCR8 seems to be imported into the egg with neck granules in sperm during fertilization.

  10. Activity of cells in the lateral vestibular nucleus as a function of head position

    Science.gov (United States)

    Fujita, Y.; Rosenberg, Jay; Segundo, J. P.

    1968-01-01

    1. The spike activity of cells in the lateral vestibular nucleus was recorded in cats anaesthetized with pentobarbital sodium. Natural labyrinthine stimulation was applied by fixing the animal at different positions reached through roations about a longitudinal or transverse axis. 2. The majority of cells responded to rotations only about the longitudinal axis. Two types of response were found. The first was characterized by a transient change in activity which occurred only during the movement. The second type had an initial transient component and a subsequent steady component that persisted as long as the head remained fixed. 3. The interspike interval means, standard deviations, histograms and autocorrelograms of the steady response components of cells sensitive to lateral tilt were calculated. In every cell the relation between the head position with respect to gravity and the mean interspike interval of the steady discharge showed two main features. (a) `Directional sensitivity': the mean interval increased following rotation in one sense, and decreased following rotation in the other. In twenty-two out of thirty-three cells, the mean increased when the recording side was raised. The remaining cells showed the opposite relation. (b) `Multivaluedness': each particular position is associated with several different values of mean interval and these values had a relatively wide scatter. The curve that resulted from joining points in the order in which they occurred during the experiment was either closed, open, or combined closed and open portions. 4. The standard deviations, histograms and autocorrelograms also showed directional sensitivity and multivaluedness with respect to position. Several types of interspike interval histograms and autocorrelograms characterized lateral vestibular activity. The forms of the histogram and the autocorrelogram of the discharge from each cell usually remained unchanged during stimulation. 5. The extensive spread of the

  11. Uncoupling of S phase and mitosis in cardiomyocytes and hepatocytes lacking the winged-helix transcription factor Trident

    NARCIS (Netherlands)

    Korver, W.; Schilham, M.W.; Moerer, P.; Hoff, M.J. van den; Dam, K.; Lamers, W.H.; Medema, R.H.; Clevers, H.C.

    1998-01-01

    In order to maintain a stable karyotype, the eukaryotic cell cycle is coordinated such that only one round of S phase precedes each mitosis, and mitosis is not initiated until DNA replication is completed. Several checkpoints and regulatory proteins have been defined in lower eukaryotes that govern

  12. An epigenetic regulator emerges as microtubule minus-end binding and stabilizing factor in mitosis.

    Science.gov (United States)

    Meunier, Sylvain; Shvedunova, Maria; Van Nguyen, Nhuong; Avila, Leonor; Vernos, Isabelle; Akhtar, Asifa

    2015-08-05

    The evolutionary conserved NSL complex is a prominent epigenetic regulator controlling expression of thousands of genes. Here we uncover a novel function of the NSL complex members in mitosis. As the cell enters mitosis, KANSL1 and KANSL3 undergo a marked relocalisation from the chromatin to the mitotic spindle. By stabilizing microtubule minus ends in a RanGTP-dependent manner, they are essential for spindle assembly and chromosome segregation. Moreover, we identify KANSL3 as a microtubule minus-end-binding protein, revealing a new class of mitosis-specific microtubule minus-end regulators. By adopting distinct functions in interphase and mitosis, KANSL proteins provide a link to coordinate the tasks of faithful expression and inheritance of the genome during different phases of the cell cycle.

  13. Checkpoint for DNA integrity at the first mitosis after oocyte activation.

    Science.gov (United States)

    Liu, Lin; Trimarchi, James R; Smith, Peter J S; Keefe, David L

    2002-06-01

    Activation of oocytes, arrested at the meiosis II (MII) in mammals, initiates meiotic release, mitotic divisions, and development. Unlike most somatic cell types, MII arrested female germ cells lack an efficient DNA integrity checkpoint control. Here we present evidence showing a unique checkpoint for DNA integrity at first mitosis after oocyte activation. Mouse oocytes carrying intact DNA cleaved normally after meiotic release, whereas 50% of oocytes harboring damaged DNA manifested cytofragmentation, a morphological hallmark of apoptosis. If not activated, DNA-damaged MII oocytes did not show apoptotic fragmentation. Further, activated, enucleated oocytes or enucleated fertilized oocytes also underwent cytofragmentation, implicating cytoplasmic coordination of the fragmentation process, independent of the nucleus. Depolymerization of either actin filaments or microtubules induced no cytofragmentation, but inhibited fragmentation upon oocyte activation. During the process of fragmentation, microtubule networks formed, then microtubule asters congregated at discrete locations, around which fragmented cellular bodies formed. Mitotic spindles, however, were not formed inactivated oocytes with damaged or absent DNA; in contrast, normal mitotic spindles were formed in activated oocytes with intact DNA. These results demonstrate that damaged DNA or absence of DNA leads to cytofragmentation after oocyte activation. Further, we found a mechanism of cytoskeletal involvement in the process of cytofragmentation. In addition, possible implication of the present findings in somatic cell cloning and human clinical embryology is discussed.

  14. CDK1-mediated phosphorylation of the RIIalpha regulatory subunit of PKA works as a molecular switch that promotes dissociation of RIIalpha from centrosomes at mitosis.

    Science.gov (United States)

    Carlson, C R; Witczak, O; Vossebein, L; Labbé, J C; Skålhegg, B S; Keryer, G; Herberg, F W; Collas, P; Taskén, K

    2001-09-01

    Protein kinase A regulatory subunit RIIalpha is tightly bound to centrosomal structures during interphase through interaction with the A-kinase anchoring protein AKAP450, but dissociates and redistributes from centrosomes at mitosis. The cyclin B-p34(cdc2) kinase (CDK1) has been shown to phosphorylate RIIalpha on T54 and this has been proposed to alter the subcellular localization of RIIalpha. We have made stable transfectants from an RIIalpha-deficient leukemia cell line (Reh) that expresses either wild-type or mutant RIIalpha (RIIalpha(T54E)). When expressed, RIIalpha detaches from centrosomes at mitosis and dissociates from its centrosomal location in purified nucleus-centrosome complexes by incubation with CDK1 in vitro. By contrast, centrosomal RIIalpha(T54E) is not redistributed at mitosis, remains mostly associated with centrosomes during all phases of the cell cycle and cannot be solubilized by CDK1 in vitro. Furthermore, RIIalpha is solubilized from particular cell fractions and changes affinity for AKAP450 in the presence of CDK1. D and V mutations of T54 also reduce affinity for the N-terminal RII-binding domain of AKAP450, whereas small neutral residues do not change affinity detected by surface plasmon resonance. In addition, only RIIalpha(T54E) interacts with AKAP450 in a RIPA-soluble extract from mitotic cells. Finally, microtubule repolymerization from mitotic centrosomes of the RIIalpha(T54E) transfectant is poorer and occurs at a lower frequency than that of RIIalpha transfectants. Our results suggest that T54 phosphorylation of RIIalpha by CDK1 might serve to regulate the centrosomal association of PKA during the cell cycle.

  15. An epigenetic regulator emerges as microtubule minus-end binding and stabilizing factor in mitosis

    OpenAIRE

    Meunier, Sylvain; Shvedunova, Maria; Van Nguyen, Nhuong; Ávila, Leonor; Vernos, Isabelle; Akhtar, Asifa

    2015-01-01

    The evolutionary conserved NSL complex is a prominent epigenetic regulator controlling expression of thousands of genes. Here we uncover a novel function of the NSL complex members in mitosis. As the cell enters mitosis, KANSL1 and KANSL3 undergo a marked relocalisation from the chromatin to the mitotic spindle. By stabilizing microtubule minus ends in a RanGTP-dependent manner, they are essential for spindle assembly and chromosome segregation. Moreover, we identify KANSL3 as a microtubule m...

  16. Meeting report--Getting Into and Out of Mitosis.

    Science.gov (United States)

    Mchedlishvili, Nunu; Jonak, Katarzyna; Saurin, Adrian T

    2015-11-15

    The Company of Biologists Workshop 'Getting Into and Out of Mitosis' was held 10-13 May 2015 at Wiston House in West Sussex, UK. The workshop brought together researchers from wide-ranging disciplines and provided a forum to discuss their latest work on the control of cell division from mitotic entry to exit. This report highlights the main topics and summarises the discussion around the key themes and questions that emerged from the meeting.

  17. Chordoma-derived cell line U-CH1-N recapitulates the biological properties of notochordal nucleus pulposus cells.

    Science.gov (United States)

    Fujita, Nobuyuki; Suzuki, Satoshi; Watanabe, Kota; Ishii, Ken; Watanabe, Ryuichi; Shimoda, Masayuki; Takubo, Keiyo; Tsuji, Takashi; Toyama, Yoshiaki; Miyamoto, Takeshi; Horiuchi, Keisuke; Nakamura, Masaya; Matsumoto, Morio

    2016-08-01

    Intervertebral disc degeneration proceeds with age and is one of the major causes of lumbar pain and degenerative lumbar spine diseases. However, studies in the field of intervertebral disc biology have been hampered by the lack of reliable cell lines that can be used for in vitro assays. In this study, we show that a chordoma-derived cell line U-CH1-N cells highly express the nucleus pulposus (NP) marker genes, including T (encodes T brachyury transcription factor), KRT19, and CD24. These observations were further confirmed by immunocytochemistry and flow cytometry. Reporter analyses showed that transcriptional activity of T was enhanced in U-CH1-N cells. Chondrogenic capacity of U-CH1-N cells was verified by evaluating the expression of extracellular matrix (ECM) genes and Alcian blue staining. Of note, we found that proliferation and synthesis of chondrogenic ECM proteins were largely dependent on T in U-CH1-N cells. In accordance, knockdown of the T transcripts suppressed the expression of PCNA, a gene essential for DNA replication, and SOX5 and SOX6, the master regulators of chondrogenesis. On the other hand, the CD24-silenced cells showed no reduction in the mRNA expression level of the chondrogenic ECM genes. These results suggest that U-CH1-N shares important biological properties with notochordal NP cells and that T plays crucial roles in maintaining the notochordal NP cell-like phenotype in this cell line. Taken together, our data indicate that U-CH1-N may serve as a useful tool in studying the biology of intervertebral disc. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:1341-1350, 2016.

  18. The three-dimensional architecture of the notochordal nucleus pulposus: novel observations on cell structures in the canine intervertebral disc.

    Science.gov (United States)

    Hunter, Christopher J; Matyas, John R; Duncan, Neil A

    2003-03-01

    Cells from the nucleus pulposus of young ( 5 years) non-chondrodystrophoid dogs were studied using routine histology, confocal laser scanning microscopy and transmission electron microscopy. The architecture of cell structures--from the tissue scale down to subcellular scale--was reported. Clusters of notochordal cells were observed in young nuclei pulposi, ranging from 10 to 426 cells each. These clusters resisted mechanical disruption and showed evidence of cell-cell signalling via gap junctions. Cells (30-40 microm in diameter) within the clusters had a physaliferous appearance, containing numerous large inclusions which ranged from 1 to 20 microm in diameter. The inclusions were surrounded by a dense actin cortex but were not contained by a lipid bilayer. The contents of the inclusions were determined not to be predominantly carbohydrate or neutral lipid as assessed by histochemical staining, but the exact composition of the contents remained uncertain. There were striking differences in the cell architecture of young vs. old nuclei pulposi, with a loss of both cell clusters and physaliferous cells during ageing. These observations demonstrate unique cell structures, which may influence our understanding of the differences between notochordal and chondrocytic cells in the nucleus pulposus. Such differences could have substantial impact upon how we think about development, degeneration and repair of the intervertebral disc.

  19. Identification of a Novel Nucleus Protein Involved in the Regulation of Urokinase in 95D Cells

    Institute of Scientific and Technical Information of China (English)

    Chang TONG; Li TAN; Ping LI; Yun-Song ZHU

    2005-01-01

    The urokinase-type plasminogen activator (uPA) plays an important role in cellular invasion.By using the downstream part of a 74 bp DNA region called the cooperation mediator (COM) of the uPA promoter as a bait sequence in the yeast one-hybrid screen, a gene called PBK1 was previously cloned from the cDNA library of the 95D lung cancer cell strain. In this study, the intracellular distribution of PBK1 was studied by using the transient transfection of pEGFP-C3-PBK1, and PBK1 was found to be localized in the nucleus. Co-transfection of pEGFP-C3-PBK1 and the deletion mutants of the pGL3-uPA promoter indicated that PBK1 can increase the uPA promoter activity by about 25% and this effect is uPA enhancer-dependent.Western blotting and Enzyme-linked immunoadsordent assay further confirmed that PBK1 can upregulate the expression of uPA. Our results suggest that PBK1 is involved in the regulation of uPA expression, which might provide a new clue to further understanding the regulation mechanism of uPA expression.

  20. Inflammatory Kinetics and Efficacy of Anti-inflammatory Treatments on Human Nucleus Pulposus Cells

    Science.gov (United States)

    Walter, Benjamin A; Purmessur, Devina; Likhitpanichkul, Morakot; Weinberg, Alan; Cho, Samuel K.; Qureshi, Sheeraz A.; Hecht, Andrew C.; Iatridis, James C.

    2015-01-01

    Study Design Human nucleus pulposus (NP) cell culture study investigating response to tumor necrosis factor-α (TNFα), effectiveness of clinically available anti-inflammatory drugs, and interactions between pro-inflammatory cytokines. Objective To characterize the kinetic response of pro-inflammatory cytokines released by human NP cells to TNFα stimulation and the effectiveness of multiple anti-inflammatories with 3 sub-studies: Timecourse, Same-time blocking, Delayed blocking. Summary of Background Data Chronic inflammation is a key component of painful intervertebral disc (IVD) degeneration. Improved efficacy of anti-inflammatories requires better understanding of how quickly NP cells produce pro-inflammatory cytokines and which pro-inflammatory mediators are most therapeutically advantageous to target. Methods Degenerated human NP cells (n=10) were cultured in alginate with or without TNFα (10ng/mL). Cells were incubated with one of four anti-inflammatories (anti-IL-6 receptor/atlizumab, IL-1 receptor anatagonist, anti-TNFα/infliximab and sodium pentosan polysulfate/PPS) in two blocking-studies designed to determine how intervention timing influences drug efficacy. Cell viability, protein and gene expression for IL-1β, IL-6 & IL-8 were assessed. Results Timecourse: TNFα substantially increased the amount of IL-6, IL-8 & IL-1β, with IL-1β and IL-8 reaching equilibrium within ~72 hours (IL-1β: 111±40pg/mL, IL-8: 8478±957pg/mL), and IL-6 not reaching steady state after 144 hours (1570±435 pg/mL). Anti-TNFα treatment was most effective at reducing the expression of all cytokines measured when added at the same time as TNFα stimulation. Similar trends were observed when drugs were added 72 hours after TNFα stimulation, however, no anti-inflammatories significantly reduced cytokine levels compared to TNF control. Conclusion IL-1β, IL-6 and IL-8 were expressed at different rates and magnitudes suggesting different roles for these cytokines in disease

  1. Fission yeast Mog1p homologue, which interacts with the small GTPase Ran, is required for mitosis-to-interphase transition and poly(A)(+) RNA metabolism.

    Science.gov (United States)

    Tatebayashi, K; Tani, T; Ikeda, H

    2001-04-01

    We have cloned and characterized the Schizosaccharomyces pombe gene mog1(+), which encodes a protein with homology to the Saccharomyces cerevisiae Mog1p participating in the Ran-GTPase system. The S. pombe Mog1p is predominantly localized in the nucleus. In contrast to the S. cerevisiae MOG1 gene, the S. pombe mog1(+) gene is essential for cell viability. mog1(+) is required for the mitosis-to-interphase transition, as the mog1-1 mutant arrests at restrictive temperatures as septated, binucleated cells with highly condensed chromosomes and an aberrant nuclear envelope. FACS analysis showed that these cells do not undergo a subsequent round of DNA replication. Surprisingly, also unlike the Delta mog1 mutation in S. cerevisiae, the mog1-1 mutation causes nucleolar accumulation of poly(A)(+) RNA at the restrictive temperature in S. pombe, but the signals do not overlap with the fibrillarin-rich region of the nucleolus. Thus, we found that mog1(+) is required for the mitosis-to-interphase transition and a class of RNA metabolism. In our attempt to identify suppressors of mog1-1, we isolated the spi1(+) gene, which encodes the fission yeast homologue of Ran. We found that overexpression of Spi1p rescues the S. pombe Delta mog1 cells from death. On the basis of these results, we conclude that mog1(+) is involved in the Ran-GTPase system.

  2. Requirement of cell nucleus for Sindbis virus replication in cultured Aedes albopictus cells.

    Science.gov (United States)

    Erwin, C; Brown, D T

    1983-02-01

    The ability of Sindbis virus to grow in enucleated BHK-21 (vertebrate) and Aedes albopictus (invertebrate) cells was tested to determine the dependence of this virus upon nuclear function in these two phylogenetically unrelated hosts. Although both cell types could be demonstrated to produce viable cytoplasts (enucleated cells) which produced virus-specific antigen subsequent to infection. BHK cytoplasts produced a significant number of progeny virions, whereas mosquito cytoplasts did not. The production of vesicular stomatitis virus in mosquito cells was not significantly reduced by enucleation. That such a host function was not essential for vesicular stomatitis virus growth in insect cells is supported by the observation that the production of this virus by mosquito cells is not actinomycin D sensitive. This result agrees with a previously published report in which it was shown that Sindbis virus maturation in invertebrate cells is inhibited by actinomycin D, indicating a possible requirement for host cell nuclear function (Scheefers-Borchel et al., Virology, 110:292-301, 1981).

  3. Neuronal cell death in the arcuate nucleus of the medulla oblongata in stillbirth.

    Science.gov (United States)

    Folkerth, Rebecca D; Zanoni, Sallie; Andiman, Sarah E; Billiards, Saraid S

    2008-02-01

    The hypothesis that unexplained stillbirth arises in a similar manner as the sudden infant death syndrome (SIDS) is based in part on shared neuropathologic features between the two entities, including hypoxic-ischemic lesions such as white matter and brainstem gliosis, as well as aplasia or hypoplasia of the arcuate nucleus on the ventral surface of the medulla. The arcuate nucleus is the putative homologue of the respiratory chemosensory region at the ventral medullary surface in animals that is involved in central chemosensitivity. To determine arcuate nucleus pathology in stillbirth, and its co-occurrence with evidence of hypoxia-ischemia, we reviewed brain specimens from the archives of our hospitals from 22 consecutive stillbirths from 22 to 41 gestational weeks. Explained causes of death (n=17) included nuchal cord, acute chorioamnionitis, placental abruption, and fetal glomerulosclerosis; 5 cases were unexplained. In 12 brains, we observed nuclear karyorrhexis and/or pyknosis with cytoplasmic hypereosinophilia in neurons in the arcuate nucleus in both explained (n=8) and unexplained (n=4) cases (54.5% of total cases). Three additional cases had arcuate aplasia (n=1) or hypoplasia (n=2) (13.6% of total cases); one of the latter cases also had neuronal necrosis in the hypoplastic arcuate. The degree of gliosis in the region of the arcuate nucleus was variable across all cases, without statistically significant differences between groups with and without arcuate nucleus necrosis. Other lesions in association with (n=14) and without (n=8) arcuate nucleus abnormalities were diffuse cerebral white matter gliosis, periventricular leukomalacia (PVL), and neuronal necrosis in the hippocampus, basal ganglia, thalamus, basis pontis, and brainstem tegmentum. In 16/20 (80.0%) cases (with or without histologic necrosis of the arcuate), immunostaining with caspase-3 demonstrated positive neurons. Our findings suggest that neuronal pathology in the arcuate nucleus may be

  4. Mitotic Transcriptional Activation: Clearance of Actively Engaged Pol II via Transcriptional Elongation Control in Mitosis.

    Science.gov (United States)

    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.

  5. 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.

    Science.gov (United States)

    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.

  6. Bora and Aurora-A continue to activate Plk1 in mitosis.

    Science.gov (United States)

    Bruinsma, Wytse; Macurek, Libor; Freire, Raimundo; Lindqvist, Arne; Medema, René H

    2014-02-15

    Polo-like kinase-1 (Plk1) is required for proper cell division. Activation of Plk1 requires phosphorylation on a conserved threonine in the T-loop of the kinase domain (T210). Plk1 is first phosphorylated on T210 in G2 phase by the kinase Aurora-A, in concert with its cofactor Bora. However, Bora was shown to be degraded prior to entry into mitosis, and it is currently unclear how Plk1 activity is sustained in mitosis. Here we show that the Bora-Aurora-A complex remains the major activator of Plk1 in mitosis. We show that a small amount of Aurora-A activity is sufficient to phosphorylate and activate Plk1 in mitosis. In addition, a fraction of Bora is retained in mitosis, which is essential for continued Aurora-A-dependent T210 phosphorylation of Plk1. We find that once Plk1 is activated, minimal amounts of the Bora-Aurora-A complex are sufficient to sustain Plk1 activity. Thus, the activation of Plk1 by Aurora-A may function as a bistable switch; highly sensitive to inhibition of Aurora-A in its initial activation, but refractory to fluctuations in Aurora-A activity once Plk1 is fully activated. This provides a cell with robust Plk1 activity once it has committed to mitosis.

  7. Endosomal recycling controls plasma membrane area during mitosis.

    Science.gov (United States)

    Boucrot, Emmanuel; Kirchhausen, Tomas

    2007-05-08

    The shape and total surface of a cell and its daughters change during mitosis. Many cells round up during prophase and metaphase and reacquire their extended and flattened shape during cytokinesis. How does the total area of plasma membrane change to accommodate these morphological changes and by what mechanism is control of total membrane area achieved? Using single-cell imaging methods, we have found that the amount of plasma membrane in attached cells in culture decreases at the beginning of mitosis and recovers rapidly by the end. Clathrin-based endocytosis is normal throughout all phases of cell division, whereas recycling of internalized membranes back to the cell surface slows considerably during the rounding up period and resumes at the time at which recovery of cell membrane begins. Interference with either one of these processes by genetic or chemical means impairs cell division. The total cell-membrane area recovers even in the absence of a functional Golgi apparatus, which would be needed for export of newly synthesized membrane lipids and proteins. We propose a mechanism by which modulation of endosomal recycling controls cell area and surface expression of membrane-bound proteins during cell division.

  8. Inducement of mitosis delay by cucurbitacin E, a novel tetracyclic triterpene from climbing stem of Cucumis melo L., through GADD45γ in human brain malignant glioma (GBM) 8401 cells.

    Science.gov (United States)

    Hsu, Y-C; Chen, M-J; Huang, T-Y

    2014-02-27

    Cucurbitacin E (CuE) is a natural compound previously shown to have anti-feedant, antioxidant and antitumor activities as well as a potent chemo-preventive action against cancer. The present study investigates its anti-proliferative property using MTT assay; CuE demonstrated cytotoxic activity against malignant glioma GBM 8401 cells and induced cell cycle G2/M arrest in these cells. CuE-treated cells accumulated in metaphase (CuE 2.5-10 μM) as determined using MPM-2 by flow cytometry. We attempted to characterize the molecular pathways responsible for cytotoxic effects of CuE in GBM 8401 cells. We studied the genome-wide gene expression profile on microarrays and molecular networks by using pathway analysis tools of bioinformatics. The CuE reduced the expression of 558 genes and elevated the levels of 1354 genes, suggesting an existence of the common pathways involved in induction of G2/M arrest. We identified the RB (GADD45β and GADD45γ) and the p53 (GADD45α) signaling pathways as the common pathways, serving as key molecules that regulate cell cycle. Results indicate that CuE produced G2/M arrest as well as the upregulation of GADD45 γ and binding with CDC2. Both effects increased proportionally with the dose of CuE, suggesting that the CuE-induced mitosis delay is regulated by GADD45γ overexpression. Our findings suggest that, in addition to the known effects on cancer prevention, CuE may have antitumor activity in glioma therapy.

  9. Mitosis-specific promoter of the alfalfa cyclin-dependent kinase gene (Medsa;CDKB2;1) is activated by wounding and ethylene in a non-cell division-dependent manner.

    Science.gov (United States)

    Zhiponova, Miroslava K; Pettkó-Szandtner, Aladár; Stelkovics, Eva; Neer, Zsuzsanna; Bottka, Sándor; Krenács, Tibor; Dudits, Dénes; Fehér, Attila; Szilák, László

    2006-02-01

    Cyclin-dependent serine/threonine kinases (CDKs) have pivotal roles in regulating the eukaryotic cell cycle. Plants possess a unique class of CDKs (B-type CDKs) with preferential protein accumulation at G2/M-phases; however, their exact functions are still enigmatic. Here we describe the functional characterization of a 360-bp promoter region of the alfalfa (Medicago sativa) CDKB2;1 gene in transgenic plants and cell lines. It is shown that the activity of the analyzed promoter was characteristic for proliferating meristematic regions in planta and specific for cells in the G2/M-phases in synchronized cell cultures. Immunohistochemical analysis of transgenic root sections further confirmed the correlation of the expression of the CDKB2;1 promoter-linked reporter genes with the accumulation of the correspondent kinase. It was found that, in addition to auxin (2,4-dichlorophenoxyacetic acid) treatment, wounding could also induce both the reporter and endogenous genes in transgenic leaf explants. Furthermore, ethylene, known as a wound-response mediator, had a similar effect. The gene activation in response to wounding or ethephon was faster and occurred without the induction of cell cycle progression in contrast to the control auxin treatment. In silico analysis of this promoter indeed revealed the presence of a set of cis-elements, indicating not only cell cycle- but wound- and ethylene-dependent regulation of this CDK gene. Based on the presented data, we discuss the functional significance of the complex regulation of mitosis-specific CDK genes in plants.

  10. Mesenchymal stem cells regulate mechanical properties of human degenerated nucleus pulposus cells through SDF-1/CXCR4/AKT axis.

    Science.gov (United States)

    Liu, Ming-Han; Bian, Bai-Shi-Jiao; Cui, Xiang; Liu, Lan-Tao; Liu, Huan; Huang, Bo; Cui, You-Hong; Bian, Xiu-Wu; Zhou, Yue

    2016-08-01

    Transplantation of mesenchymal stem cells (MSCs) into the degenerated intervertebral disc (IVD) has shown promise for decelerating or arresting IVD degeneration. Cellular mechanical properties play crucial roles in regulating cell-matrix interactions, potentially reflecting specific changes that occur based on cellular phenotype and behavior. However, the effect of co-culturing of MSCs with nucleus pulposus cells (NPCs) on the mechanical properties of NPCs remains unknown. In our study, we demonstrated that co-culture of degenerated NPCs with MSCs resulted in significantly decreased mechanical moduli (elastic modulus, relaxed modulus, and instantaneous modulus) and increased biological activity (proliferation and expression of matrix genes) in degenerated NPCs, but not normal NPCs. SDF-1, CXCR4 ligand, was highly expressed in MSCs when co-cultured with degenerated NPCs. Inhibition of SDF-1 using CXCR4 antagonist AMD3100 or knocking-down CXCR4 in degenerated NPCs abolished the MSCs-induced decrease in the mechanical moduli and increased biological activity of degenerated NPCs, suggesting a crucial role for SDF-1/CXCR4 signaling. AKT and FAK inhibition attenuated the MSCs- or SDF-1-induced decrease in the mechanical moduli of degenerated NPCs. In conclusion, it was demonstrated in vitro that MSCs regulate the mechanical properties of degenerated NPCs through SDF-1/CXCR4/AKT signaling. These findings highlight a possible mechanical mechanism for MSCs-induced modulation with degenerated NPCs, which may be applicable to MSCs-based therapy for disc degeneration.

  11. Can notochordal cells promote bone marrow stromal cell potential for nucleus pulposus enrichment? A simplified in vitro system.

    Science.gov (United States)

    Potier, Esther; Ito, Keita

    2014-12-01

    Bone marrow stromal cells (BMSCs) have shown promising potential to stop intervertebral disc degeneration in several animal models. In order to restore a healthy state, though, this potential should be further stimulated. Notochordal cells (NCs), influential in disc development, have been shown to stimulate BMSC differentiation, but it is unclear how this effect will translate in an environment where resident disc cells (nucleus pulposus cells [NPCs]) could also influence BMSCs. The goal of this study was, therefore, to evaluate the effects of NCs on BMSCs when cocultured with NPCs, in a simplified 3D in vitro system. Bovine BMSCs and NPCs were mixed (Mix) and seeded into alginate beads. Using culture inserts, the Mix was then cocultured with porcine NCs (alginate beads) and compared to coculture with empty beads or porcine skin fibroblasts (SFs, alginate beads). NPCs alone were also cocultured with NCs, and BMSCs alone cultured under chondrogenic conditions. The effects of coculture conditions on cell viability, matrix production (proteoglycan and collagen), and gene expression of disc markers (aggrecan, type II collagen, and SOX9) were assessed after 4 weeks of culture. The NC phenotype and gene expression profile were also analyzed. Coculture with NCs did not significantly influence cell viability, proteoglycan production, or disc marker gene expression of the Mix. When compared to NPCs, the Mix produced the same amount of proteoglycan and displayed a higher expression of disc marker, indicating a stimulation of the BMSCs (and/or NPCs) in the Mix. Additionally, during the 4 weeks of culture, the NC phenotype changed drastically (morphology, gene expression profile). These results show that NCs might not be as stimulatory for BMSCs in an NPC-rich environment, as believed from individual cultures. This absence of effects could be explained by a mild stimulation provided by (de)differentiating NCs and the costimulation of BMSCs and NPCs by each other.

  12. κ-Opioid receptor in the nucleus is a novel prognostic factor of esophageal squamous cell carcinoma.

    Science.gov (United States)

    Zhang, Yong-Fa; Xu, Qing-Xia; Liao, Lian-Di; Xu, Xiu-E; Wu, Jian-Yi; Shen, Jian; Wu, Zhi-Yong; Shen, Jin-Hui; Li, En-Min; Xu, Li-Yan

    2013-09-01

    Opioid receptors, members of the G-protein-coupled receptor superfamily, appear to be involved in cancer progression. However, the expression and significance of opioid receptors in esophageal squamous cell carcinoma (ESCC) remain unclear. In this study, we demonstrated by flow cytometry that μ, δ, and κ-opioid receptors (MOR, DOR, and KOR) are expressed to various degrees in ESCC cell lines. The KOR protein was further examined by several methods in ESCC cell lines and tissues. Immunocytochemical staining localized KOR to the cell membrane in KYSE180 cells and the nucleus in EC109 cells, whereas no signal or weak staining of the cytoplasm was observed in KYSE150 cells. The expression of KOR was confirmed in ESCC cells by Western blotting. Furthermore, immunohistochemistry staining showed that KOR was up-regulated in ESCC tissues compared with nontumorous esophageal epithelium (P = .004, χ(2) test). Moreover, high nuclear KOR expression was significantly correlated with lymph node metastasis in 256 ESCC cases (R = 0.144; P = .030, Kendall τB test). Patients with high nuclear KOR expression in ESCC had a significantly poorer prognosis (P = .001, log-rank test). Multivariate Cox analysis revealed that KOR in the nucleus was an independent prognostic factor (hazard ratio, 1.789; 95% confidence interval, 1.177-2.720; P = .006). Our results suggest that KOR is involved in the carcinogenesis or progression of ESCC and that nuclear KOR may be indicative of prognosis.

  13. EGF stimulates the activation of EGF receptors and the selective activation of major signaling pathways during mitosis.

    Science.gov (United States)

    Wee, Ping; Shi, Huaiping; Jiang, Jennifer; Wang, Yuluan; Wang, Zhixiang

    2015-03-01

    Mitosis and epidermal growth factor (EGF) receptor (EGFR) are both targets for cancer therapy. The role of EGFR signaling in mitosis has been rarely studied and poorly understood. The limited studies indicate that the activation of EGFR and downstream signaling pathways is mostly inhibited during mitosis. However, we recently showed that EGFR is phosphorylated in response to EGF stimulation in mitosis. Here we studied EGF-induced EGFR activation and the activation of major signaling pathways downstream of EGFR during mitosis. We showed that EGFR was strongly activated by EGF during mitosis as all the five major tyrosine residues including Y992, Y1045, Y1068, Y1086, and Y1173 were phosphorylated to a level similar to that in the interphase. We further showed that the activated EGFR is able to selectively activate some downstream signaling pathways while avoiding others. Activated EGFR is able to activate PI3K and AKT2, but not AKT1, which may be responsible for the observed effects of EGF against nocodazole-induced cell death. Activated EGFR is also able to activate c-Src, c-Cbl and PLC-γ1 during mitosis. However, activated EGFR is unable to activate ERK1/2 and their downstream substrates RSK and Elk-1. While it activated Ras, EGFR failed to fully activate Raf-1 in mitosis due to the lack of phosphorylation at Y341 and the lack of dephosphorylation at pS259. We conclude that contrary to the dogma, EGFR is activated by EGF during mitosis. Moreover, EGFR-mediated cell signaling is regulated differently from the interphase to specifically serve the needs of the cell in mitosis.

  14. From equator to pole: splitting chromosomes in mitosis and meiosis.

    Science.gov (United States)

    Duro, Eris; Marston, Adèle L

    2015-01-15

    During eukaryotic cell division, chromosomes must be precisely partitioned to daughter cells. This relies on a mechanism to move chromosomes in defined directions within the parental cell. While sister chromatids are segregated from one another in mitosis and meiosis II, specific adaptations enable the segregation of homologous chromosomes during meiosis I to reduce ploidy for gamete production. Many of the factors that drive these directed chromosome movements are known, and their molecular mechanism has started to be uncovered. Here we review the mechanisms of eukaryotic chromosome segregation, with a particular emphasis on the modifications that ensure the segregation of homologous chromosomes during meiosis I.

  15. Integrin-mediated interactions with extracellular matrix proteins for nucleus pulposus cells of the human intervertebral disc.

    Science.gov (United States)

    Bridgen, D T; Gilchrist, C L; Richardson, W J; Isaacs, R E; Brown, C R; Yang, K L; Chen, J; Setton, L A

    2013-10-01

    The extracellular matrix (ECM) of the human intervertebral disc is rich in molecules that interact with cells through integrin-mediated attachments. Porcine nucleus pulposus (NP) cells have been shown to interact with laminin (LM) isoforms LM-111 and LM-511 through select integrins that regulate biosynthesis and cell attachment. Since human NP cells lose many phenotypic characteristics with age, attachment and interaction with the ECM may be altered. Expression of LM-binding integrins was quantified for human NP cells using flow cytometry. The cell-ECM attachment mechanism was determined by quantifying cell attachment to LM-111, LM-511, or type II collagen after functionally blocking specific integrin subunits. Human NP cells express integrins β1, α3, and α5, with over 70% of cells positive for each subunit. Blocking subunit β1 inhibited NP cell attachment to all substrates. Blocking subunits α1, α2, α3, and α5 simultaneously, but not individually, inhibits NP cell attachment to laminins. While integrin α6β1 mediated porcine NP cell attachment to LM-111, we found integrins α3, α5, and β1 instead contributed to human NP cell attachment. These findings identify integrin subunits that may mediate interactions with the ECM for human NP cells and could be used to promote cell attachment, survival, and biosynthesis in cell-based therapeutics.

  16. Comprehensive identification of SUMO2/3 targets and their dynamics during mitosis.

    Science.gov (United States)

    Schou, Julie; Kelstrup, Christian D; Hayward, Daniel G; Olsen, Jesper V; Nilsson, Jakob

    2014-01-01

    During mitosis large alterations in cellular structures occur rapidly, which to a large extent is regulated by post-translational modification of proteins. Modification of proteins with the small ubiquitin-related protein SUMO2/3 regulates mitotic progression, but few mitotic targets have been identified so far. To deepen our understanding of SUMO2/3 during this window of the cell cycle, we undertook a comprehensive proteomic characterization of SUMO2/3 modified proteins in mitosis and upon mitotic exit. We developed an efficient tandem affinity purification strategy of SUMO2/3 modified proteins from mitotic cells. Combining this purification strategy with cell synchronization procedures and quantitative mass spectrometry allowed for the mapping of numerous novel targets and their dynamics as cells progressed out of mitosis. This identified RhoGDIα as a major SUMO2/3 modified protein, specifically during mitosis, mediated by the SUMO ligases PIAS2 and PIAS3. Our data provide a rich resource for further exploring the role of SUMO2/3 modifications in mitosis and cell cycle regulation.

  17. Akt is transferred to the nucleus of cells treated with apoptin, and it participates in apoptin-induced cell death

    DEFF Research Database (Denmark)

    Bay, GH; Kroczak, TJ; Ande, SR;

    2007-01-01

    . Downstream of PI3-K, Akt is activated and translocated to the nucleus together with apoptin. Direct interaction between apoptin and Akt is documented. Co-expression of nuclear Akt significantly potentiates cell death induced by apoptin. Thus, apoptin-facilitated nuclear Akt, in contrast to when in its...

  18. Comprehensive Identification of SUMO2/3 Targets and Their Dynamics during Mitosis

    DEFF Research Database (Denmark)

    Schou, Julie; Kelstrup, Christian D; Hayward, Daniel G

    2014-01-01

    During mitosis large alterations in cellular structures occur rapidly, which to a large extent is regulated by post-translational modification of proteins. Modification of proteins with the small ubiquitin-related protein SUMO2/3 regulates mitotic progression, but few mitotic targets have been...... identified so far. To deepen our understanding of SUMO2/3 during this window of the cell cycle, we undertook a comprehensive proteomic characterization of SUMO2/3 modified proteins in mitosis and upon mitotic exit. We developed an efficient tandem affinity purification strategy of SUMO2/3 modified proteins...... from mitotic cells. Combining this purification strategy with cell synchronization procedures and quantitative mass spectrometry allowed for the mapping of numerous novel targets and their dynamics as cells progressed out of mitosis. This identified RhoGDIα as a major SUMO2/3 modified protein...

  19. Restarting life: fertilization and the transition from meiosis to mitosis.

    Science.gov (United States)

    Clift, Dean; Schuh, Melina

    2013-09-01

    Fertilization triggers a complex cellular programme that transforms two highly specialized meiotic germ cells, the oocyte and the sperm, into a totipotent mitotic embryo. Linkages between sister chromatids are remodelled to support the switch from reductional meiotic to equational mitotic divisions; the centrosome, which is absent from the egg, is reintroduced; cell division shifts from being extremely asymmetric to symmetric; genomic imprinting is selectively erased and re-established; and protein expression shifts from translational control to transcriptional control. Recent work has started to reveal how this remarkable transition from meiosis to mitosis is achieved.

  20. The Ran pathway in Drosophila melanogaster mitosis

    Directory of Open Access Journals (Sweden)

    James G Wakefield

    2015-11-01

    Full Text Available Over the last two decades, the small GTPase Ran has emerged as a central regulator of both mitosis and meiosis, particularly in the generation, maintenance and regulation of the microtubule (MT-based bipolar spindle. Ran-regulated pathways in mitosis bear many similarities to the well-characterized functions of Ran in nuclear transport and, as with transport, the majority of these mitotic effects are mediated through affecting the physical interaction between karyopherins and Spindle Assembly Factors (SAFs - a loose term describing proteins or protein complexes involved in spindle assembly through promoting nucleation, stabilization, and/or depolymerization of MTs, through anchoring MTs to specific structures such as centrosomes, chromatin or kinetochores, or through sliding MTs along each other to generate the force required to achieve bipolarity. As such, the Ran-mediated pathway represents a crucial functional module within the wider spindle assembly landscape. Research into mitosis using the model organism Drosophila melanogaster has contributed substantially to our understanding of centrosome and spindle function. However, in comparison to mammalian systems, very little is known about the contribution of Ran-mediated pathways in Drosophila mitosis. This article sets out to summarize our understanding of the roles of the Ran pathway components in Drosophila mitosis, focusing on the syncytial blastoderm embryo, arguing that, far from being superfluous, it can provide important insights into the conserved functions on Ran during spindle formation.

  1. A new theory of the origin of cancer: quantum coherent entanglement, centrioles, mitosis, and differentiation.

    Science.gov (United States)

    Hameroff, Stuart R

    2004-11-01

    Malignant cells are characterized by abnormal segregation of chromosomes during mitosis ("aneuploidy"), generally considered a result of malignancy originating in genetic mutations. However, recent evidence supports a century-old concept that maldistribution of chromosomes (and resultant genomic instability) due to abnormalities in mitosis itself is the primary cause of malignancy rather than a mere byproduct. In normal mitosis chromosomes replicate into sister chromatids which are then precisely separated and transported into mirror-like sets by structural protein assemblies called mitotic spindles and centrioles, both composed of microtubules. The elegant yet poorly understood ballet-like movements and geometric organization occurring in mitosis have suggested guidance by some type of organizing field, however neither electromagnetic nor chemical gradient fields have been demonstrated or shown to be sufficient. It is proposed here that normal mirror-like mitosis is organized by quantum coherence and quantum entanglement among microtubule-based centrioles and mitotic spindles which ensure precise, complementary duplication of daughter cell genomes and recognition of daughter cell boundaries. Evidence and theory supporting organized quantum states in cytoplasm/nucleoplasm (and quantum optical properties of centrioles in particular) at physiological temperature are presented. Impairment of quantum coherence and/or entanglement among microtubule-based mitotic spindles and centrioles can result in abnormal distribution of chromosomes, abnormal differentiation and uncontrolled growth, and account for all aspects of malignancy. New approaches to cancer therapy and stem cell production are suggested via non-thermal laser-mediated effects aimed at quantum optical states of centrioles.

  2. Toxoplasma exports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression.

    Science.gov (United States)

    Bougdour, Alexandre; Tardieux, Isabelle; Hakimi, Mohamed-Ali

    2014-03-01

    Toxoplasma gondii is the most widespread apicomplexan parasite and occupies a large spectrum of niches by infecting virtually any warm-blooded animals. As an obligate intracellular parasite, Toxoplasma has evolved a repertoire of strategies to fine-tune the cellular environment in an optimal way to promote growth and persistence in host tissues hence increasing the chance to be transmitted to new hosts. Short and long-term intracellular survival is associated with Toxoplasma ability to both evade the host deleterious immune defences and to stimulate a beneficial immune balance by governing host cell gene expression. It is only recently that parasite proteins responsible for driving these transcriptional changes have been identified. While proteins contained in the apical secretory Rhoptry organelle have already been identified as bona fide secreted effectors that divert host signalling pathways, recent findings revealed that dense granule proteins should be added to the growing list of effectors as they reach the host cell cytoplasm and nucleus and target various host cell pathways in the course of cell infection. Herein, we emphasize on a novel subfamily of dense granule residentproteins, exemplified with the GRA16 and GRA24 members we recently discovered as both are exported beyond the vacuole-containing parasites and reach the host cell nucleus to reshape the host genome expression.

  3. Chemical Topography of Efferent Projections from the Median Preoptic Nucleus to Pontine Monoaminergic Cell Groups in the Rat

    Science.gov (United States)

    Zardetto-Smith, Andrea M.; Johnson, Alan Kim

    1995-01-01

    This study examined efferent output from the median preoptic nucleus (MNPO) to pontine noradrenergic and serotonergic cell groups using an anterograde tracing technique (Phaseolus vulgaris leucoagglutinin, PHA-L) combined with glucose oxidase immunocytochemistry to serotonin (5-HT) or to dopamine-beta-hydroxylase (DBH). Injections of PHA-L into the ventral MNPO resulted in moderate axonal labeling within the region of the B7 and B8 serotonergic groups in the dorsal raphe. PHA-L labeled fibers and punctate processes were observed in close apposition to many of the 5-HT immunoreactive neurons in these regions. In contrast, sparse terminal labeling was found within the B5 group in the raphe pontis nucleus, and only trace fiber labeling observed in the B3 and B6 groups. Efferents from the MNPO also provided moderate innervation to the A6 and A7 noradrenergic groups. PHA-L labeled punctate processes were found most frequently in close apposition to DBH-immunoreactive neurons at mid- to caudal levels of the locus coeruleus. Some labeled axons were also present within the A7 and A5 groups. Additionally, a close apposition between labeled MNPO efferents and 5-HT fibers within the lateral parabrachial nucleus was observed. The results indicate the MNPO provides a topographic innervation of monoaminergic groups in the upper brainstem.

  4. Nuclear transport factors: global regulation of mitosis.

    Science.gov (United States)

    Forbes, Douglass J; Travesa, Anna; Nord, Matthew S; Bernis, Cyril

    2015-08-01

    The unexpected repurposing of nuclear transport proteins from their function in interphase to an equally vital and very different set of functions in mitosis was very surprising. The multi-talented cast when first revealed included the import receptors, importin alpha and beta, the small regulatory GTPase RanGTP, and a subset of nuclear pore proteins. In this review, we report that recent years have revealed new discoveries in each area of this expanding story in vertebrates: (a) The cast of nuclear import receptors playing a role in mitotic spindle regulation has expanded: both transportin, a nuclear import receptor, and Crm1/Xpo1, an export receptor, are involved in different aspects of spindle assembly. Importin beta and transportin also regulate nuclear envelope and pore assembly. (b) The role of nucleoporins has grown to include recruiting the key microtubule nucleator - the γ-TuRC complex - and the exportin Crm1 to the mitotic kinetochores of humans. Together they nucleate microtubule formation from the kinetochores toward the centrosomes. (c) New research finds that the original importin beta/RanGTP team have been further co-opted by evolution to help regulate other cellular and organismal activities, ranging from the actual positioning of the spindle within the cell perimeter, to regulation of a newly discovered spindle microtubule branching activity, to regulation of the interaction of microtubule structures with specific actin structures. (d) Lastly, because of the multitudinous roles of karyopherins throughout the cell cycle, a recent large push toward testing their potential as chemotherapeutic targets has begun to yield burgeoning progress in the clinic.

  5. Deciphering the spatio-temporal regulation of entry and progression through mitosis.

    Science.gov (United States)

    Gheghiani, Lilia; Gavet, Olivier

    2014-02-01

    Mitosis has been studied since the early 1880s as a key event of the cell division cycle where remarkable changes in cellular architecture take place and ultimately lead to an equal segregation of duplicated chromosomes into two daughter cells. A detailed description of the complex and highly ordered cellular events taking place is now available. Many regulators involved in key steps including entry into mitosis, nuclear envelope breakdown, microtubule (MT) spindle formation, and chromosome attachment, as well as mitotic exit and cytokinesis, have also been identified. However, understanding the precise spatio-temporal contribution of each regulator in the cell reorganization process has been technically challenging. This review will focus on a number of recent advances in our understanding of the spatial distribution of protein activities and the temporal regulation of their activation and inactivation during entry and progression through mitosis by the use of intramolecular Förster resonance energy transfer (FRET)-based biosensors.

  6. Phosphorylation of SAF-A/hnRNP-U Serine 59 by Polo-Like Kinase 1 Is Required for Mitosis.

    Science.gov (United States)

    Douglas, Pauline; Ye, Ruiqiong; Morrice, Nicholas; Britton, Sébastien; Trinkle-Mulcahy, Laura; Lees-Miller, Susan P

    2015-08-01

    Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis.

  7. Stereological analysis of the mediodorsal thalamic nucleus in schizophrenia: volume, neuron number, and cell types

    DEFF Research Database (Denmark)

    Dorph-Petersen, Karl-Anton; Pierri, Joseph N; Sun, Zhuoxin

    2004-01-01

    The mediodorsal thalamic nucleus (MD) is the principal relay nucleus for the prefrontal cortex, a brain region thought to be dysfunctional in schizophrenia. Several, but not all, postmortem studies of the MD in schizophrenia have reported decreased volume and total neuronal number. However......, it is not clear whether the findings are specific for schizophrenia nor is it known which subtypes of thalamic neurons are affected. We studied the left MD in 11 subjects with schizophrenia, 9 control subjects, and 12 subjects with mood disorders. Based on morphological criteria, we divided the neurons into two...... subclasses, presumably corresponding to projection neurons and local circuit neurons. We estimated MD volume and the neuron number of each subclass using methods based on modern unbiased stereological principles. We also estimated the somal volumes of each subclass using a robust, but biased, approach...

  8. Mitosis Detection for Invasive Breast Cancer Grading in Histopathological Images.

    Science.gov (United States)

    Paul, Angshuman; Mukherjee, Dipti Prasad

    2015-11-01

    Histopathological grading of cancer not only offers an insight to the patients' prognosis but also helps in making individual treatment plans. Mitosis counts in histopathological slides play a crucial role for invasive breast cancer grading using the Nottingham grading system. Pathologists perform this grading by manual examinations of a few thousand images for each patient. Hence, finding the mitotic figures from these images is a tedious job and also prone to observer variability due to variations in the appearances of the mitotic cells. We propose a fast and accurate approach for automatic mitosis detection from histopathological images. We employ area morphological scale space for cell segmentation. The scale space is constructed in a novel manner by restricting the scales with the maximization of relative-entropy between the cells and the background. This results in precise cell segmentation. The segmented cells are classified in mitotic and non-mitotic category using the random forest classifier. Experiments show at least 12% improvement in F1 score on more than 450 histopathological images at 40× magnification.

  9. FANCA safeguards interphase and mitosis during hematopoiesis in vivo.

    Science.gov (United States)

    Abdul-Sater, Zahi; Cerabona, Donna; Potchanant, Elizabeth Sierra; Sun, Zejin; Enzor, Rikki; He, Ying; Robertson, Kent; Goebel, W Scott; Nalepa, Grzegorz

    2015-12-01

    The Fanconi anemia (FA/BRCA) signaling network controls multiple genome-housekeeping checkpoints, from interphase DNA repair to mitosis. The in vivo role of abnormal cell division in FA remains unknown. Here, we quantified the origins of genomic instability in FA patients and mice in vivo and ex vivo. We found that both mitotic errors and interphase DNA damage significantly contribute to genomic instability during FA-deficient hematopoiesis and in nonhematopoietic human and murine FA primary cells. Super-resolution microscopy coupled with functional assays revealed that FANCA shuttles to the pericentriolar material to regulate spindle assembly at mitotic entry. Loss of FA signaling rendered cells hypersensitive to spindle chemotherapeutics and allowed escape from the chemotherapy-induced spindle assembly checkpoint. In support of these findings, direct comparison of DNA crosslinking and anti-mitotic chemotherapeutics in primary FANCA-/- cells revealed genomic instability originating through divergent cell cycle checkpoint aberrations. Our data indicate that FA/BRCA signaling functions as an in vivo gatekeeper of genomic integrity throughout interphase and mitosis, which may have implications for future targeted therapies in FA and FA-deficient cancers.

  10. Cdc25B and Cdc25C differ markedly in their properties as initiators of mitosis.

    Science.gov (United States)

    Karlsson, C; Katich, S; Hagting, A; Hoffmann, I; Pines, J

    1999-08-09

    We have used time-lapse fluorescence microscopy to study the properties of the Cdc25B and Cdc25C phosphatases that have both been implicated as initiators of mitosis in human cells. To differentiate between the functions of the two proteins, we have microinjected expression constructs encoding Cdc25B or Cdc25C or their GFP-chimeras into synchronized tissue culture cells. This assay allows us to express the proteins at defined points in the cell cycle. We have followed the microinjected cells by time-lapse microscopy, in the presence or absence of DNA synthesis inhibitors, and assayed whether they enter mitosis prematurely or at the correct time. We find that overexpressing Cdc25B alone rapidly causes S phase and G2 phase cells to enter mitosis, whether or not DNA replication is complete, whereas overexpressing Cdc25C does not cause premature mitosis. Overexpressing Cdc25C together with cyclin B1 does shorten the G2 phase and can override the unreplicated DNA checkpoint, but much less efficiently than overexpressing Cdc25B. These results suggest that Cdc25B and Cdc25C do not respond identically to the same cell cycle checkpoints. This difference may be related to the differential localization of the proteins; Cdc25C is nuclear throughout interphase, whereas Cdc25B is nuclear in the G1 phase and cytoplasmic in the S and G2 phases. We have found that the change in subcellular localization of Cdc25B is due to nuclear export and that this is dependent on cyclin B1. Our data suggest that although both Cdc25B and Cdc25C can promote mitosis, they are likely to have distinct roles in the controlling the initiation of mitosis.

  11. Analysis of the G2/M Checkpoint in fanconi anemia cells via examinating chromosomal instability during G2-phase and mitosis

    OpenAIRE

    Sauer, Rica

    2013-01-01

    Fanconi anemia is a genetical and phenotypical heterogenous disease, characterized through loss of one of the 15 identified genes of the Fanconi anemia pathway what causes congenital anomalies, bone marrow failure and solid tumors. In this work the G2/M checkpoint is analysed by use of the phosphatase inhibitor Calyculin A to examine the chromosomal instability, which is typical for fanconi anemia cells, not only in mitoses but also in the G2 phase of the cell cycle. It is proved that the che...

  12. SU-E-T-494: Influence of Proton Track-Cell Nucleus Incidence Angle On Relative Biological Effectiveness

    Energy Technology Data Exchange (ETDEWEB)

    Pater, P; Backstrom, G; Enger, S; Seuntjens, J; El Naqa, I [McGill University, Montreal, Quebec (Canada); Villegas, F; Ahnesjo, A [Uppsala University, Uppsala (Sweden)

    2015-06-15

    Purpose: To explain a Monte Carlo (MC) simulation artifact whereby differences in relative biological effectiveness (RBE) in the induction of initial double strand breaks are observed as a function of the proton track incidence angles in a geometric cell nucleus model. Secondly, to offer an alternative isotropic irradiation procedure to mitigate this effect. Methods: MC tracks of 1 MeV protons were generated in an event-by-event mode. They were overlaid on a cylindrical model of a cell nucleus containing 6×109 nucleotide base pairs. The tracks incidence angle θ with respect to the cell nucleus’s axis was varied in 10 degrees intervals, each time generating one hundred fractions of ∼2 Gy. Strand breaks were scored in the modeled DNA sugar-phosphate groups and further sub-classified into single or double strand breaks (ssbs or dsbs). For each angle, an RBE for the induction of initial dsbs with reference to Co-60 was calculated. Results: Our results show significant angular dependencies of RBE, with maximum values for incidence angles parallel to the nucleus central axis. Further examination shows that the higher cross-sections for the creation of dsbs is due to the preferential alignment of tracks with geometrical sub-parts of the cell nucleus model, especially the nucleosomes containing the sugar-phosphate groups. To alleviate the impact of this simulation artifact, an average RBE was calculated with a procedure based on a weighted sampling of the angular data. Conclusion: This work demonstrates a possible numerical artifact in estimated RBE if the influence of the particle incidence angle is not correctly taken into account. A correction procedure is presented to better conform the simulations to real-life experimental conditions. We would like to acknowledge support from the Fonds de recherche du Quebec Sante (FRQS), from the CREATE Medical Physics Research Training Network grant (number 432290) of NSERC, support from NSERC under grants RGPIN 397711-11 and

  13. The effect of extracellular pH on matrix turnover by cells of the bovine nucleus pulposus

    OpenAIRE

    Razaq, Sajjad; Wilkins, Robert J.; Urban, Jill P. G.

    2003-01-01

    It has long been known that very acidic conditions can be found in degenerate discs. The effect of these acid conditions on matrix turnover are, however, unknown. This study aimed to examine the effect of acidity on production of matrix components and on agents which break down the matrix in order to gain insight into the effect of pathological values of pH on matrix turnover. Cells were isolated from the nucleus of bovine discs and from bovine articular cartilage, embedded in alginate beads ...

  14. Nonchromosomal cytogenetic analysis of mammal somatic cells

    Directory of Open Access Journals (Sweden)

    Kovalova O. А.

    2013-01-01

    Full Text Available The mutational events that take place in mammalian somatic cells influenced with different endogenous and exogenous factors are presented in this review. The nonchromosomal method of research allows taking into account the complex cell characteristics without time-consuming analysis of the chromosomes as such. As a result, the information can be obtained about the mitotic (phases of mitosis, the number of nuclei per cell, micronuclei, pathology of mitosis and vital (mitotic index, apoptosis cell statuses, as well as about the state of chromosomal integrity (the presence of nucleoplasmic bridges, nucleus protrusions, chromosome fragmentation, micronuclei. Depending on the material studied (erythrocytes and lymphocytes of peripheral blood, buccal cells, permanent cell lines etc., a complex of cytogenetic characteristics can be selected for each case which is the most informative for determination of the mutational spectra in mammalian somatic cells.

  15. Cloning of genes whose expression is correlated with mitosis and localized in dividing cells in root caps of Pisum sativum L.

    Science.gov (United States)

    Woo, H H; Hawes, M C

    1997-12-01

    Removal of border cells from pea roots synchronizes and induces root cap cell division, wall biogenesis and differentiation. Three messages which are expressed differentially in such induced root caps have been cloned. Sequence analyses showed that the PsHRGP1-encoded protein has high homology with a homology with a hydroxyproline-rich glycoprotein. The PsCaP23-encoded protein has high homology with an alfalfa callus protein or translationally controlled human or mouse tumor protein P23. The PsRbL41-encoded protein has high homology with a highly basic 60S ribosomal protein L41. In situ hybridization showed that PsHRGP1. PsCaP23 and PsRbL41 messages are localized within dividing cells of the root cap. PsHRGP1 is highly expressed in uninduced root caps, but its message is repressed by 10-11 times as soon as cell division and differentiation begin. Expression of PsHRGP1 recovers to higher than (180%) its initial level in 30 min. PsHRGP1 is root-specific. PsCaP23 and PsRbL41 messages increase ca. 3-fold within 15 min after root cap induction. All three genes represent small families of 3-5 closely related genes in the pea genome.

  16. Synchronizing Progression of Schizosaccharomyces pombe Cells from G2 through Repeated Rounds of Mitosis and S Phase with cdc25-22 Arrest Release.

    Science.gov (United States)

    Hagan, Iain M; Grallert, Agnes; Simanis, Viesturs

    2016-08-01

    Transient inactivation of the cdc25(+) gene product by manipulation of the culture temperature for cdc25-22 cells is the most commonly exploited approach to mitotic synchronization in fission yeast. Because Cdc25 removes the inhibitory phosphate placed on Cdk1 by Wee1, inactivation of Cdc25 arrests cells at the G2/M boundary. Incubation at the restrictive temperature of 36°C for just over one generation time forces all cells in the culture to accumulate at the G2/M boundary. Restoration of Cdc25 function via a return to the permissive temperature or chemical inhibition of Wee1 activity at 36°C can then promote a highly synchronous wave of cell division throughout the culture. These approaches can be performed on any scale and thus support simultaneous assessment of numerous events within a single culture. After describing this simple and widely applicable procedure, we discuss frequently overlooked issues that can have a considerable impact on the interpretation of data from cdc25-22 induction-synchronized cultures.

  17. A Vivens Ex Vivo Study on the Synergistic Effect of Electrolysis and Freezing on the Cell Nucleus.

    Science.gov (United States)

    Lugnani, Franco; Zanconati, Fabrizio; Marcuzzo, Thomas; Bottin, Cristina; Mikus, Paul; Guenther, Enric; Klein, Nina; Rubinsky, Liel; Stehling, Michael K; Rubinsky, Boris

    2015-01-01

    Freezing-cryosurgery, and electrolysis-electrochemical therapy (EChT), are two important minimally invasive surgery tissue ablation technologies. Despite major advantages they also have some disadvantages. Cryosurgery cannot induce cell death at high subzero freezing temperatures and requires multiple freeze thaw cycles, while EChT requires high concentrations of electrolytic products-which makes it a lengthy procedure. Based on the observation that freezing increases the concentration of solutes (including products of electrolysis) in the frozen region and permeabilizes the cell membrane to these products, this study examines the hypothesis that there could be a synergistic effect between freezing and electrolysis in their use together for tissue ablation. Using an animal model we refer to as vivens ex vivo, which may be of value in reducing the use of animals for experiments, combined with a Hematoxylin stain of the nucleus, we show that there are clinically relevant protocols in which the cell nucleus appears intact when electrolysis and freezing are used separately but is affected by certain combinations of electrolysis and freezing.

  18. A Vivens Ex Vivo Study on the Synergistic Effect of Electrolysis and Freezing on the Cell Nucleus

    Science.gov (United States)

    Lugnani, Franco; Zanconati, Fabrizio; Marcuzzo, Thomas; Bottin, Cristina; Mikus, Paul; Guenther, Enric; Klein, Nina; Rubinsky, Liel; Stehling, Michael K.; Rubinsky, Boris

    2015-01-01

    Freezing—cryosurgery, and electrolysis—electrochemical therapy (EChT), are two important minimally invasive surgery tissue ablation technologies. Despite major advantages they also have some disadvantages. Cryosurgery cannot induce cell death at high subzero freezing temperatures and requires multiple freeze thaw cycles, while EChT requires high concentrations of electrolytic products—which makes it a lengthy procedure. Based on the observation that freezing increases the concentration of solutes (including products of electrolysis) in the frozen region and permeabilizes the cell membrane to these products, this study examines the hypothesis that there could be a synergistic effect between freezing and electrolysis in their use together for tissue ablation. Using an animal model we refer to as vivens ex vivo, which may be of value in reducing the use of animals for experiments, combined with a Hematoxylin stain of the nucleus, we show that there are clinically relevant protocols in which the cell nucleus appears intact when electrolysis and freezing are used separately but is affected by certain combinations of electrolysis and freezing. PMID:26695185

  19. Fission Yeast dim1 + Encodes a Functionally Conserved Polypeptide Essential for Mitosis

    OpenAIRE

    Berry, Lynne D.; Gould, Kathleen L.

    1997-01-01

    In a screen for second site mutations capable of reducing the restrictive temperature of the fission yeast mutant cdc2-D217N, we have isolated a novel temperature-sensitive mutant, dim1-35. When shifted to restrictive temperature, dim1-35 mutant cells arrest before entry into mitosis or proceed through mitosis in the absence of nuclear division, demonstrating an uncoupling of proper DNA segregation from other cell cycle events. Deletion of dim1 from the Schizosaccharomyces pombe genome produc...

  20. Human geminin promotes pre-RC formation and DNA replication by stabilizing CDT1 in mitosis

    DEFF Research Database (Denmark)

    Ballabeni, Andrea; Melixetian, Marina; Zamponi, Raffaella

    2004-01-01

    -mediated degradation by inhibiting its ubiquitination. In particular, Geminin ensures basal levels of CDT1 during S phase and its accumulation during mitosis. Consistently, inhibition of Geminin synthesis during M phase leads to impairment of pre-RC formation and DNA replication during the following cell cycle....... Moreover, we show that inhibition of CDK1 during mitosis, and not Geminin depletion, is sufficient for premature formation of pre-RCs, indicating that CDK activity is the major mitotic inhibitor of licensing in human cells. Taken together with recent data from our laboratory, our results demonstrate...

  1. Correlative Analysis on the Relationship between PMI and DNA Degradation of Cell Nucleus in Human Different Tissues

    Institute of Scientific and Technical Information of China (English)

    SHU Xiji; LIU Yaling; REN Liang; HE Fanggang; ZHOU Hongyan; LIU Lijiang; LIU Liang

    2005-01-01

    To determining the postmortem interval (PMI) through quantitative analysis of the DNA degradation of cell nucleus in human brain and spleen by using image analysis technique (IAT). The brain and spleen tissues from 32 cadavers with known PMI were collected, subjected to cell smear every 1 h within the first 5-36 h after death, stained by Feulgen-Van's staining, Three indices reflecting DNA in brain cells (astrocytes) and splenic lymphocytes, including integral optical density (IOD), average optical density (AOD), average gray (AG) were measured by employing the mage analysis instrument. The results showed that IOD and AOD declined and AG increased with the prolongation of dead time within 5-36 h. A correlation between the PMI and gray parameters (IOD,AOD and AG) was identified and the corresponding regression equation was obtained. The parameters (IOD,AOD and AG) were proved to be effective quantitative indicators for accurate estimation of PMI within 5-36 h after death.

  2. Kinetics of low threshold calcium channels of relay cells in cat lateral geniculate nucleus

    Institute of Scientific and Technical Information of China (English)

    罗茀荪; S.M.Sherman

    1996-01-01

    Kinetics of the low threshold T-type Ca2+ channel is studied with single electrode voltage damp technique on brain slices of the cat lateral geniculate nucleus (LGN). Space damp is dramatically improved by blocking various K+ and Na+ channels, decreasing Ca2+ current and selecting proper holding potentials. Results from this study are similar to those obtained from acutely dissociated LGN neurons of the rat, indicating that the kinetics of T-Ca2+ channels of the cat LGN neurons is the same as that of the rat LGN. The result reported previously on the cat LGN may result from a defect in space damp.

  3. Mitosis and cell death in the optic lobes of workers, queens and drones of the honey bee (Apis mellifera) during metamorphosis

    Indian Academy of Sciences (India)

    Thaisa Cristina Roat; Carminda Da Cruz Landim

    2010-09-01

    Colonies of the honey bee, Apis mellifera, consist of males and two female castes: workers and queens. The castes and males from A. mellifera have a distinct morphology, physiology and behaviour that correlate with their roles in the society and are characterized by some brain polymorphisms. Compound eyes are one of the characteristics that differ among the castes and sexes. A. mellifera is a holometabolous insect; therefore, the development of adult organs during metamorphosis, which will produce these differences, requires the precise coordination of three main programmed cellular processes: proliferation, differentiation and death. These processes take place simultaneously during pupation. Our purpose was to investigate cell division and death in the optic lobes (OL) of workers, queens and males during pupation to identify how the differences in the compound eyes in adults of these classes are achieved. The results showed that OL differentiation follows a similar pattern in the three classes of individuals studied, without structural differences in their development. The main non-structural differences involve cell division, mortality rates and timing. The results suggest a modelling of the brain during differentiation, which contributes to the specific functions of each individual class.

  4. Antioxidants in aqueous extract of Myristica fragrans (Houtt.) suppress mitosis and cyclophosphamide-induced chromosomal aberrations in Allium cepa L. cells.

    Science.gov (United States)

    Akinboro, Akeem; Mohamed, Kamaruzaman Bin; Asmawi, Mohd Zaini; Sulaiman, Shaida Fariza; Sofiman, Othman Ahmad

    2011-11-01

    In this study, freeze-dried water extract from the leaves of Myristica fragrans (Houtt.) was tested for mutagenic and antimutagenic potentials using the Allium cepa assay. Freeze-dried water extract alone and its combination with cyclophosphamide (CP) (50 mg/kg) were separately dissolved in tap water at 500, 1000, 2000, and 4000 mg/kg. Onions (A. cepa) were suspended in the solutions and controls for 48 h in the dark. Root tips were prepared for microscopic evaluation. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radicals' scavenging power of the extract was tested using butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) as standards. Water extract of Myristica fragrans scavenged free radicals better than BHA, but worse than BHT. The extract alone, as well as in combination with CP suppressed cell division, and induced chromosomal aberrations that were insignificantly different from the negative control (P ≤ 0.05). However, cytotoxic and mutagenic actions of CP were considerably suppressed. The observed effects on cell division and chromosomes of A. cepa may be principally connected to the antioxidant properties of the extract. The obtained results suggest mitodepressive and antimutagenic potentials of water extract of the leaves of M. fragrans as desirable properties of a promising anticancer agent.

  5. Antioxidants in aqueous extract of Myristicafragrans (Houtt.) suppress mitosis and cyclophosphamide-induced chromosomal aberrations in Allium cepa L.cells

    Institute of Scientific and Technical Information of China (English)

    Akeem AKINBORO; Kamaruzaman Bin MOHAMED; Mohd Zaini ASMAWI; Shaida Fariza SULAIMAN; Othman Ahmad SOFIMAN

    2011-01-01

    In this study,freeze-dried water extract from the leaves of Myristica fragrans (Houtt.) was tested for mutagenic and antimutagenic potentials using the Allium cepa assay.Freeze-dried water extract alone and its combination with cyclophosphamide (CP) (50 mg/kg) were separately dissolved in tap water at 500,1000,2000,and 4000 mg/kg.Onions (A.cepa) were suspended in the solutions and controls for 48 h in the dark.Root tips were prepared for microscopic evaluation.2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radicals' scavenging power of the extract was tested using butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) as standards.Water extract of Myristica fragrans scavenged free radicals better than BHA,but worse than BHT.The extract alone,as well as in combination with CP suppressed cell division,and induced chromosomal aberrations that were insignificantly different from the negative control (P≤0.05).However,cytotoxic and mutagenic actions of CP were considerably suppressed.The observed effects on cell division and chromosomes of A.cepa may be principally connected to the antioxidant properties of the extract.The obtained results suggest mitodepressive and antimutagenic potentials of water extract of the leaves of M.fragrans as desirable properties of a promising anticancer agent.

  6. Mitosis and cell death in the optic lobes of workers, queens and drones of the honey bee (Apis mellifera) during metamorphosis.

    Science.gov (United States)

    Roat, Thaisa Cristina; Landim, Carminda da Cruz

    2010-09-01

    Colonies of the honey bee, Apis mellifera, consist of males and two female castes: workers and queens. The castes and males from A. mellifera have a distinct morphology, physiology and behaviour that correlate with their roles in the society and are characterized by some brain polymorphisms. Compound eyes are one of the characteristics that differ among the castes and sexes. A. mellifera is a holometabolous insect; therefore, the development of adult organs during metamorphosis, which will produce these differences, requires the precise coordination of three main programmed cellular processes: proliferation, differentiation and death. These processes take place simultaneously during pupation. Our purpose was to investigate cell division and death in the optic lobes (OL) of workers, queens and males during pupation to identify how the differences in the compound eyes in adults of these classes are achieved. The results showed that OL differentiation follows a similar pattern in the three classes of individuals studied, without structural differences in their development. The main non-structural differences involve cell division, mortality rates and timing. The results suggest a modelling of the brain during differentiation, which contributes to the specific functions of each individual class.

  7. Clathrin is spindle-associated but not essential for mitosis.

    Directory of Open Access Journals (Sweden)

    Joana Borlido

    Full Text Available Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis.Previously a chicken pre-B lymphoma cell line (DKO-R was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression.We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the

  8. Characterization of the tight junction protein ZO-2 localized at the nucleus of epithelial cells.

    Science.gov (United States)

    Jaramillo, Blanca Estela; Ponce, Arturo; Moreno, Jacqueline; Betanzos, Abigail; Huerta, Miriam; Lopez-Bayghen, Esther; Gonzalez-Mariscal, Lorenza

    2004-07-01

    ZO-2 is a MAGUK protein that in confluent epithelial sheets localizes at tight junctions (TJ) whereas in sparse cultures accumulates in clusters at the nucleus. Here, we have characterized several nuclear properties of ZO-2. We observe that ZO-2 is present in the nuclear matrix and co-immunoprecipitates with lamin B(1) and actin from the nuclei of sparse cultures. We show that ZO-2 presents several NLS at its amino region, that when deleted, diminish the nuclear import of the ZO-2 amino segment and impair the ability of the region to regulate the transcriptional activity of promoters controlled by AP-1. Several RS repeats are detected in the ZO-2 amino segment, however, their deletion does not preclude the display of a speckled nuclear pattern. ZO-2 displays two putative NES. However, only the second one appears to be functional, as when conjugated to ovalbumin (OV), it is able to translocate this protein from the nucleus to the cytoplasm in a leptomycin B-sensitive way.

  9. Extracellular matrix production by nucleus pulposus and bone marrow stem cells in response to altered oxygen and glucose microenvironments.

    Science.gov (United States)

    Naqvi, Syeda M; Buckley, Conor T

    2015-12-01

    Bone marrow (BM) stem cells may be an ideal source of cells for intervertebral disc (IVD) regeneration. However, the harsh biochemical microenvironment of the IVD may significantly influence the biological and metabolic vitality of injected stem cells and impair their repair potential. This study investigated the viability and production of key matrix proteins by nucleus pulposus (NP) and BM stem cells cultured in the typical biochemical microenvironment of the IVD consisting of altered oxygen and glucose concentrations. Culture-expanded NP cells and BM stem cells were encapsulated in 1.5% alginate and ionically crosslinked to form cylindrical hydrogel constructs. Hydrogel constructs were maintained under different glucose concentrations (1, 5 and 25 mM) and external oxygen concentrations (5 and 20%). Cell viability was measured using the Live/Dead® assay and the production of sulphated glycosaminoglycans (sGAG), and collagen was quantified biochemically and histologically. For BM stem cells, IVD-like micro-environmental conditions (5 mM glucose and 5% oxygen) increased the accumulation of sGAG and collagen. In contrast, low glucose conditions (1 mM glucose) combined with 5% external oxygen concentration promoted cell death, inhibiting proliferation and the accumulation of sGAG and collagen. NP-encapsulated alginate constructs were relatively insensitive to oxygen concentration or glucose condition in that they accumulated similar amounts of sGAG under all conditions. Under IVD-like microenvironmental conditions, NP cells were found to have a lower glucose consumption rate compared with BM cells and may in fact be more suitable to adapt and sustain the harsh microenvironmental conditions. Considering the highly specialised microenvironment of the central NP, these results indicate that IVD-like concentrations of low glucose and low oxygen are critical and influential for the survival and biological behaviour of stem cells. Such findings may promote and accelerate

  10. Block copolymer micelles target Auger electron radiotherapy to the nucleus of HER2-positive breast cancer cells.

    Science.gov (United States)

    Hoang, Bryan; Reilly, Raymond M; Allen, Christine

    2012-02-13

    Intracellular trafficking of Auger electron emitting radionuclides to perinuclear and nuclear regions of cells is critical to realizing their full therapeutic potential. In the present study, block copolymer micelles (BCMs) were labeled with the Auger electron emitter indium-111 ((111)In) and loaded with the radiosensitizer methotrexate. HER2 specific antibodies (trastuzumab fab) and nuclear localization signal (NLS; CGYGPKKKRKVGG) peptides were conjugated to the surface of the BCMs to direct uptake in HER2 expressing cells and subsequent localization in the cell nucleus. Cell uptake and intracellular distribution of the multifunctional BCMs were evaluated in a panel of breast cancer cell lines with different levels of HER2 expression. Indeed cell uptake was found to be HER2 density dependent, confirming receptor-mediated internalization of the BCMs. Importantly, conjugation of NLS peptides to the surface of BCMs was found to result in a significant increase in nuclear uptake of the radionuclide (111)In. Successful nuclear targeting was shown to improve the antipoliferative effect of the Auger electrons as measured by clonogenic assays. In addition, a significant radiation enhancement effect was observed by concurrent delivery of low-dose MTX and (111)In in all breast cancer cell lines evaluated.

  11. Properties of fiber cell plasma membranes isolated from the cortex and nucleus of the porcine eye lens.

    Science.gov (United States)

    Mainali, Laxman; Raguz, Marija; O'Brien, William J; Subczynski, Witold K

    2012-04-01

    The organization and physical properties of the lipid bilayer portion of intact cortical and nuclear fiber cell plasma membranes isolated from the eye lenses of two-year-old pigs were studied using electron paramagnetic resonance (EPR) spin-labeling. Membrane fluidity, hydrophobicity, and the oxygen transport parameter (OTP) were assessed from the EPR spectra of precisely positioned spin labels. Intact cortical and nuclear membranes, which include membrane proteins, were found to contain three distinct lipid environments. These lipid environments were termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain (lipids in protein aggregates). The amount of boundary and trapped lipids was greater in intact nuclear membranes than in cortical membranes. The properties of intact membranes were compared with the organization and properties of lens lipid membranes made of the total lipid extracts from the lens cortex or nucleus. In cortical lens lipid membranes, only one homogenous environment was detected, which was designated as a bulk lipid domain (phospholipid bilayer saturated with cholesterol). Lens lipid membranes prepared from the lens nucleus possessed two domains, assigned as a bulk lipid domain and a cholesterol bilayer domain (CBD). In intact nuclear membranes, it was difficult to discriminate the CBD, which was clearly detected in nuclear lens lipid membranes, because the OTP measured in the CBD is the same as in the domain formed by trapped lipids. The two domains unique to intact membranes-namely, the domain formed by boundary lipids and the domain formed by trapped lipids-were most likely formed due to the presence of membrane proteins. It is concluded that formation of rigid and practically impermeable domains is enhanced in the lens nucleus, indicating changes in membrane composition that may help to maintain low oxygen concentration in this lens region.

  12. Effects of Mg{sup 2+}, Co{sup 2+}, and Hg{sup 2+} on the nucleus and nucleolus in root tip cells of allium cepa

    Energy Technology Data Exchange (ETDEWEB)

    Liu, D.; Jiang, W.; Wang, W. [Tianjin Normal Univ. (China); Zhai, L. [Xinhua High School, Tianjin (China)

    1995-11-01

    Metal toxicity in plants has been known for a long time. Much importance has increasingly been attached to the problems of metal pollution with the development of modern industry and agriculture. If metals in plants are accumulated to a large extent, it might seriously affect them. The cytological effects of cobalt and mercury have been studied in Allium cepa by documentation of c-mitosis. Also, the quantification of chromosome aberration in Vicia faba root-tip cells treated by magnesium sulphate and in Allium cepa by metyl mercury chloride and mercuric chloride has been reported. Cytological research on the poisoning effects of Mg, Co and Hg on the nuclei and nucleoli in root-tip cells of plants has hardly been reported. The aim of this study was to determine the effects of different concentrations of magnesium, cobalt and mercury ions on root growth, and on the nuclei and nucleoli of root tip cells of Allium-cepa. 20 refs., 3 figs.

  13. Mechanisms of Chromosome Congression during Mitosis

    Science.gov (United States)

    Maiato, Helder; Gomes, Ana Margarida; Sousa, Filipe; Barisic, Marin

    2017-01-01

    Chromosome congression during prometaphase culminates with the establishment of a metaphase plate, a hallmark of mitosis in metazoans. Classical views resulting from more than 100 years of research on this topic have attempted to explain chromosome congression based on the balance between opposing pulling and/or pushing forces that reach an equilibrium near the spindle equator. However, in mammalian cells, chromosome bi-orientation and force balance at kinetochores are not required for chromosome congression, whereas the mechanisms of chromosome congression are not necessarily involved in the maintenance of chromosome alignment after congression. Thus, chromosome congression and maintenance of alignment are determined by different principles. Moreover, it is now clear that not all chromosomes use the same mechanism for congressing to the spindle equator. Those chromosomes that are favorably positioned between both poles when the nuclear envelope breaks down use the so-called “direct congression” pathway in which chromosomes align after bi-orientation and the establishment of end-on kinetochore-microtubule attachments. This favors the balanced action of kinetochore pulling forces and polar ejection forces along chromosome arms that drive chromosome oscillatory movements during and after congression. The other pathway, which we call “peripheral congression”, is independent of end-on kinetochore microtubule-attachments and relies on the dominant and coordinated action of the kinetochore motors Dynein and Centromere Protein E (CENP-E) that mediate the lateral transport of peripheral chromosomes along microtubules, first towards the poles and subsequently towards the equator. How the opposite polarities of kinetochore motors are regulated in space and time to drive congression of peripheral chromosomes only now starts to be understood. This appears to be regulated by position-dependent phosphorylation of both Dynein and CENP-E and by spindle microtubule

  14. Mechanisms of Chromosome Congression during Mitosis

    Directory of Open Access Journals (Sweden)

    Helder Maiato

    2017-02-01

    Full Text Available Chromosome congression during prometaphase culminates with the establishment of a metaphase plate, a hallmark of mitosis in metazoans. Classical views resulting from more than 100 years of research on this topic have attempted to explain chromosome congression based on the balance between opposing pulling and/or pushing forces that reach an equilibrium near the spindle equator. However, in mammalian cells, chromosome bi-orientation and force balance at kinetochores are not required for chromosome congression, whereas the mechanisms of chromosome congression are not necessarily involved in the maintenance of chromosome alignment after congression. Thus, chromosome congression and maintenance of alignment are determined by different principles. Moreover, it is now clear that not all chromosomes use the same mechanism for congressing to the spindle equator. Those chromosomes that are favorably positioned between both poles when the nuclear envelope breaks down use the so-called “direct congression” pathway in which chromosomes align after bi-orientation and the establishment of end-on kinetochore-microtubule attachments. This favors the balanced action of kinetochore pulling forces and polar ejection forces along chromosome arms that drive chromosome oscillatory movements during and after congression. The other pathway, which we call “peripheral congression”, is independent of end-on kinetochore microtubule-attachments and relies on the dominant and coordinated action of the kinetochore motors Dynein and Centromere Protein E (CENP-E that mediate the lateral transport of peripheral chromosomes along microtubules, first towards the poles and subsequently towards the equator. How the opposite polarities of kinetochore motors are regulated in space and time to drive congression of peripheral chromosomes only now starts to be understood. This appears to be regulated by position-dependent phosphorylation of both Dynein and CENP-E and by spindle

  15. The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation.

    Science.gov (United States)

    Chiu, Shao-Chih; Chen, Jo-Mei Maureen; Wei, Tong-You Wade; Cheng, Tai-Shan; Wang, Ya-Hui Candice; Ku, Chia-Feng; Lian, Chiao-Hsuan; Liu, Chun-Chih Jared; Kuo, Yi-Chun; Yu, Chang-Tze Ricky

    2014-09-01

    Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser(115). The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis.

  16. On the regulation of protein phosphatase 2A and its role in controlling entry into and exit from mitosis.

    Science.gov (United States)

    Hunt, Tim

    2013-05-01

    The process of mitosis involves a comprehensive reorganization of the cell: chromosomes condense, the nuclear envelope breaks down, the mitotic spindle is assembled, cells round up and release their ties to the substrate and so on and so forth. This reorganization is triggered by the activation of the protein kinase, Cyclin-Dependent Kinase 1 (CDK1). The end of mitosis is marked by the proteolysis of the cyclin subunit of CDK1, which terminates kinase activity. At this point, the phosphate moieties that altered the properties of hundreds of proteins to bring about the cellular reorganization are removed by protein phosphatases. At least one protein phosphatase, PP2A-B55, is completely shut off in mitosis. Depletion of this particular form of PP2A accelerates entry into mitosis, and blocks exit from mitosis. Control of this phosphatase is achieved by an inhibitor protein (α-endosulfine or ARPP-19) that becomes inhibitory when phosphorylated by a protein kinase called Greatwall, which is itself a substrate of CDK1. Failure to inhibit PP2A-B55 causes arrest of the cell cycle in G2 phase. I will discuss the role of this control mechanism in the control of mitosis.

  17. Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis.

    Science.gov (United States)

    Pillai, Smitha; Nguyen, Jonathan; Johnson, Joseph; Haura, Eric; Coppola, Domenico; Chellappan, Srikumar

    2015-12-10

    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.

  18. Microtubules as key coordinators of nuclear envelope and endoplasmic reticulum dynamics during mitosis.

    Science.gov (United States)

    Schlaitz, Anne-Lore

    2014-07-01

    During mitosis, cells comprehensively restructure their interior to promote the faithful inheritance of DNA and cytoplasmic contents. In metazoans, this restructuring entails disassembly of the nuclear envelope, redistribution of its components into the endoplasmic reticulum (ER) and eventually nuclear envelope reassembly around the segregated chromosomes. The microtubule cytoskeleton has recently emerged as a critical regulator of mitotic nuclear envelope and ER dynamics. Microtubules and associated molecular motors tear open the nuclear envelope in prophase and remove nuclear envelope remnants from chromatin. Additionally, two distinct mechanisms of microtubule-based regulation of ER dynamics operate later in mitosis. First, association of the ER with microtubules is reduced, preventing invasion of ER into the spindle area, and second, organelle membrane is actively cleared from metaphase chromosomes. However, we are only beginning to understand the role of microtubules in shaping and distributing ER and other organelles during mitosis.

  19. How unfinished business from S-phase affects mitosis and beyond

    DEFF Research Database (Denmark)

    Mankouri, H.W.; Huttner, D.; Hickson, I.D.

    2013-01-01

    The eukaryotic cell cycle is conventionally viewed as comprising several discrete steps, each of which must be completed before the next one is initiated. However, emerging evidence suggests that incompletely replicated, or unresolved, chromosomes from S-phase can persist into mitosis, where...... they present a potential threat to the faithful segregation of sister chromatids. In this review, we provide an overview of the different classes of loci where this 'unfinished S-phase business' can lead to a variety of cytogenetically distinct DNA structures throughout the various steps of mitosis...

  20. Novel functions for the endocytic regulatory proteins MICAL-L1 and EHD1 in mitosis.

    Science.gov (United States)

    Reinecke, James B; Katafiasz, Dawn; Naslavsky, Naava; Caplan, Steve

    2015-01-01

    During interphase, recycling endosomes mediate the transport of internalized cargo back to the plasma membrane. However, in mitotic cells, recycling endosomes are essential for the completion of cytokinesis, the last phase of mitosis that promotes the physical separation the two daughter cells. Despite recent advances, our understanding of the molecular determinants that regulate recycling endosome dynamics during cytokinesis remains incomplete. We have previously demonstrated that Molecule Interacting with CasL Like-1 (MICAL-L1) and C-terminal Eps15 Homology Domain protein 1 (EHD1) coordinately regulate receptor transport from tubular recycling endosomes during interphase. However, their potential roles in controlling cytokinesis had not been addressed. In this study, we show that MICAL-L1 and EHD1 regulate mitosis. Depletion of either protein resulted in increased numbers of bi-nucleated cells. We provide evidence that bi-nucleation in MICAL-L1- and EHD1-depleted cells is a consequence of impaired recycling endosome transport during late cytokinesis. However, depletion of MICAL-L1, but not EHD1, resulted in aberrant chromosome alignment and lagging chromosomes, suggesting an EHD1-independent function for MICAL-L1 earlier in mitosis. Moreover, we provide evidence that MICAL-L1 and EHD1 differentially influence microtubule dynamics during early and late mitosis. Collectively, our new data suggest several unanticipated roles for MICAL-L1 and EHD1 during the cell cycle.

  1. CDK-1 Inhibition in G2 Stabilizes Kinetochore-Microtubules in the following Mitosis.

    Science.gov (United States)

    Gayek, A Sophia; Ohi, Ryoma

    2016-01-01

    Cell proliferation is driven by cyclical activation of cyclin-dependent kinases (CDKs), which produce distinct biochemical cell cycle phases. Mitosis (M phase) is orchestrated by CDK-1, complexed with mitotic cyclins. During M phase, chromosomes are segregated by a bipolar array of microtubules called the mitotic spindle. The essential bipolarity of the mitotic spindle is established by the kinesin-5 Eg5, but factors influencing the maintenance of spindle bipolarity are not fully understood. Here, we describe an unexpected link between inhibiting CDK-1 before mitosis and bipolar spindle maintenance. Spindles in human RPE-1 cells normally collapse to monopolar structures when Eg5 is inhibited at metaphase. However, we found that inhibition of CDK-1 in the G2 phase of the cell cycle improved the ability of RPE-1 cells to maintain spindle bipolarity without Eg5 activity in the mitosis immediately after release from CDK-1 inhibition. This improved bipolarity maintenance correlated with an increase in the stability of kinetochore-microtubules, the subset of microtubules that link chromosomes to the spindle. The improvement in bipolarity maintenance after CDK-1 inhibition in G2 required both the kinesin-12 Kif15 and increased stability of kinetochore-microtubules. Consistent with increased kinetochore-microtubule stability, we find that inhibition of CDK-1 in G2 impairs mitotic fidelity by increasing the incidence of lagging chromosomes in anaphase. These results suggest that inhibition of CDK-1 in G2 causes unpredicted effects in mitosis, even after CDK-1 inhibition is relieved.

  2. One pot synthesis of highly luminescent polyethylene glycol anchored carbon dots functionalized with a nuclear localization signal peptide for cell nucleus imaging.

    Science.gov (United States)

    Yang, Lei; Jiang, Weihua; Qiu, Lipeng; Jiang, Xuewei; Zuo, Daiying; Wang, Dongkai; Yang, Li

    2015-04-14

    Strong blue fluorescent polyethylene glycol (PEG) anchored carbon nitride dots (CDs@PEG) with a high quantum yield (QY) of 75.8% have been synthesized by a one step hydrothermal treatment. CDs with a diameter of ca. 6 nm are well dispersed in water and present a graphite-like structure. Photoluminescence (PL) studies reveal that CDs display excitation-dependent behavior and are stable under various test conditions. Based on the as-prepared CDs, we designed novel cell nucleus targeting imaging carbon dots functionalized with a nuclear localization signal (NLS) peptide. The favourable biocompatibilities of CDs and NLS modified CDs (NLS-CDs) are confirmed by in vitro cytotoxicity assays. Importantly, intracellular localization experiments in MCF7 and A549 cells demonstrate that NLS-CDs could be internalized in the nucleus and show blue light, which indicates that CDs may serve as cell nucleus imaging probes.

  3. Intervertebral disc repair with activated nucleus pulposus cell transplantation: a three-year, prospective clinical study of its safety

    Directory of Open Access Journals (Sweden)

    J Mochida

    2015-03-01

    Full Text Available Degeneration of the lumbar intervertebral discs is irreversible, with no treatment currently available. Building upon experimental studies that demonstrated the importance of the nucleus pulposus (NP in preserving disc structure, we demonstrated that reinsertion of NP cells slowed further disc degeneration and that direct cell-to-cell contact co-culture with mesenchymal stromal cells (MSCs significantly upregulated the viability of NP cells in basic and pre-clinical studies in vitro and in vivo using animal models and human cells. Here, we report a 3-year result of a prospective clinical study, aimed to assess the safety and efficacy of activated NP cell transplantation in the degenerate lumbar intervertebral disc. Candidates were 9 patients aged 20-29 years who had Pfirrmann’s grade III disc degeneration at the level adjacent to the level scheduled for posterior lumbar intervertebral fusion. Viable NP cells from the fused disc were co-cultured in direct contact with autologous bone marrow-derived MSCs. One million activated NP cells were transplanted into the degenerated disc adjacent to the fused level at 7 d after the first fusion surgery. No adverse effects were observed during the 3-year follow-up period. Magnetic resonance imaging did not show any detrimental effects to the transplanted discs and revealed a mild improvement in 1 case. No cases reported any low back pain. Our clinical study confirmed the safety of activated NP cell transplantation, and the findings suggest the minimal efficacy of this treatment to slow the further degeneration of human intervertebral discs.

  4. Spindle mechanics and dynamics during mitosis in Drosophila.

    Science.gov (United States)

    Kwon, Mijung; Scholey, Jonathan M

    2004-04-01

    Drosophila melanogaster is an excellent model for studying mitosis. Syncytial embryos are amenable to time-lapse imaging of hundreds of synchronously dividing spindles, allowing the quantitation of spindle and chromosome dynamics with unprecedented fidelity. Other Drosophila cell types, including neuroblasts, cultured cells, spermatocytes and oocytes, contain spindles that differ in their design, providing cells amenable to different types of experiments and allowing identification of common core mechanisms. The function of mitotic proteins can be studied using mutants, inhibitor microinjection and RNA interference (RNAi) to identify the full inventory of mitotic proteins encoded by the genome. Here, we review recent advances in understanding how ensembles of mitotic proteins coordinate spindle assembly and chromosome motion in this system.

  5. Inhibitory action of oestrogen on calcium-induced mitosis in rat bone marrow and thymus.

    Science.gov (United States)

    Smith, G R; Gurson, M L; Riddell, A J; Perris, A D

    1975-04-01

    In the male rat injections of CaCl-2 and MgCl-2 stimulated mitosis in bone marrow and thymus tissue. The magnesium salt was also mitogenic in the normal female, but calcium only exerted its mitogenic effect after ovariectomy. Oestradiol, but not progesterone replacement therpy abolished calcium-induced mitosis in the ovariectomized rat. The inability of calcium to stimulate cell division was also apparent in the thyroparathyroidectomized female rat, suggesting the oestradiol blockage did not operate via some indirect action on the calcium homeostatic hormones calcitonin or parathyroid hormone. When thymic lymphocytes derived from male or female rats were isolated and maintained in suspension, increased calcium or magnesium concentrations in the culture medium stimulated the entry of cells into mitosis. Addition of oestradiol to the culture medium abolished the mitogenic effect of increased calcium levels, but had no effect on magnesium-induced proliferation. These experiments suggested that oestradiol might act at the cell surface to prevent the influx of calcium but not magnesium ions into the interior of the cell and thus to block the sequence of biochemical events which led to the initiation of DNA synthesis and culminate in mitosis.

  6. Centrosomes split in the presence of impaired DNA integrity during mitosis

    NARCIS (Netherlands)

    Hut, HMJ; Lemstra, W; Blaauw, EH; van Cappellen, GWA; Kampinga, HH; Sibon, OCM

    2003-01-01

    A well-established function of centrosomes is their role in accomplishing a successful mitosis that gives rise to a pair of identical daughter cells. We recently showed that DNA replication defects and DNA damage in Drosophila embryos trigger centrosomal changes, but it remained unclear whether comp

  7. Observation of Mitosis and Meiosis in Rice Cells by Simple Squash Method%简易压片法观察水稻有丝分裂和减数分裂行为

    Institute of Scientific and Technical Information of China (English)

    杨旭; 代西梅

    2009-01-01

    色体复制,减数分裂II 过程中仅发生了细胞分裂.[结论]改进后的水稻染色体压片技术能够获得准确的染色体资料,可为育种和遗传操作提供依据.%[Objective] The aim of this study was to establish a feasible squashing technique for chromosome and obtain data of rice chromosome. [Method] With the materials of rice root tips and anther, the specimen was prepared by the modified squash method, and microscopic observation of mitosis and meiosis in rice cells was also carried out. [Result] Mitosis in rice cells included interphase, prophase, metaphase, anaphase and telophase. Chromosome in metaphase shortened to the minmum, which was a good time for observing and investigating chromosome. However, meiosis in rice cells included meiosis I and meiosis II. Chromosome replication appeared in meiosis I, while cell division only appeared in meiosis II. [Conclusion] The modified squashing technique for rice chromosome can obtain accurate data of rice chromosome, which provides evidence for genetic breeding.

  8. Transient calcium-dependent potassium current in magnocellular neurosecretory cells of the rat supraoptic nucleus.

    Science.gov (United States)

    Bourque, C W

    1988-03-01

    1. Magnocellular neurosecretory neurones were impaled in the supraoptic nucleus of perfused explants of rat hypothalamus. Membrane currents were studied at 35 degrees C using the single-microelectrode voltage-clamp technique. 2. Depolarizing voltage steps applied from -100 mV evoked a transient outward current (TOC) from a threshold of -75 mV. From this potential, the amplitude of the current increased non-linearly with voltage. 3. Following its activation TOC reached a peak within 7 ms and subsequently decayed monotonically with a time constant of 30 ms. This time constant did not vary significantly with voltage between -75 and -55 mV. 4. The TOC showed complete steady-state inactivation at potentials positive to -55 mV. Inactivation was removed by hyperpolarization, with a mid-point near -80 mV. The removal of inactivation followed a complex time course with distinct fast (tens of milliseconds) and slow (hundreds of milliseconds) components. 5. Tail current measurements revealed that the TOC equilibrium potential (ETOC) lies near -97 mV in the presence of 3 mM [K+]o. Increasing [K+]o caused a decrease of TOC amplitude and a shift in ETOC of 57 mV/log [K+]o. The TOC is therefore predominantly a K+ current. 6. The TOC was unaffected by tetraethylammonium (up to 12 mM) but was reversibly reduced by 4-aminopyridine (ca. 50% block at 1.0 mM) and dendrotoxin (ca. 50% block at 4 nM). 7. The TOC was strongly inhibited (greater than 70%) by adding Co2+ or Mn2+ (1-3 mM) or Cd2+ (50-400 microM) to Ca-containing solutions, or by removal of Ca2+ from the perfusate. These effects were not accompanied by detectable changes in threshold voltage. The amplitude of TOC was also depressed by the organic Ca2+ channel blocker methoxyverapamil (D600). Finally replacement of Ca2+ by Ba2+ in the perfusate completely and reversibly abolished the TOC. 8. These findings suggest that neurosecretory neurones of the rat supraoptic nucleus display a transient K+ current which is strongly

  9. Mitosis gives a brief window of opportunity for a change in gene transcription.

    Directory of Open Access Journals (Sweden)

    Richard P Halley-Stott

    2014-07-01

    Full Text Available Cell differentiation is remarkably stable but can be reversed by somatic cell nuclear transfer, cell fusion, and iPS. Nuclear transfer to amphibian oocytes provides a special opportunity to test transcriptional reprogramming without cell division. We show here that, after nuclear transfer to amphibian oocytes, mitotic chromatin is reprogrammed up to 100 times faster than interphase nuclei. We find that, as cells traverse mitosis, their genes pass through a temporary phase of unusually high responsiveness to oocyte reprogramming factors (mitotic advantage. Mitotic advantage is not explained by nuclear penetration, DNA modifications, histone acetylation, phosphorylation, methylation, nor by salt soluble chromosomal proteins. Our results suggest that histone H2A deubiquitination may account, at least in part, for the acquisition of mitotic advantage. They support the general principle that a temporary access of cytoplasmic factors to genes during mitosis may facilitate somatic cell nuclear reprogramming and the acquisition of new cell fates in normal development.

  10. Negative regulation of the endocytic adaptor disabled-2 (Dab2) in mitosis.

    Science.gov (United States)

    Chetrit, David; Barzilay, Lior; Horn, Galit; Bielik, Tom; Smorodinsky, Nechama I; Ehrlich, Marcelo

    2011-02-18

    Mitotic cells undergo extensive changes in shape and size through the altered regulation and function of their membrane trafficking machinery. Disabled 2 (Dab2), a multidomain cargo-specific endocytic adaptor and a mediator of signal transduction, is a potential integrator of trafficking and signaling. Dab2 binds effectors of signaling and trafficking that localize to different intracellular compartments. Thus, differential localization is a putative regulatory mechanism of Dab2 function. Furthermore, Dab2 is phosphorylated in mitosis and is thus regulated in the cell cycle. However, a detailed description of the intracellular localization of Dab2 in the different phases of mitosis and an understanding of the functional consequences of its phosphorylation are lacking. Here, we show that Dab2 is progressively displaced from the membrane in mitosis. This phenomenon is paralleled by a loss of co-localization with clathrin. Both phenomena culminate in metaphase/anaphase and undergo partial recovery in cytokinesis. Treatment with 2-methoxyestradiol, which arrests cells at the spindle assembly checkpoint, induces the same effects observed in metaphase cells. Moreover, 2-methoxyestradiol also induced Dab2 phosphorylation and reduced Dab2/clathrin interactions, endocytic vesicle motility, clathrin exchange dynamics, and the internalization of a receptor endowed with an NPXY endocytic signal. Serine/threonine to alanine mutations, of residues localized to the central region of Dab2, attenuated its phosphorylation, reduced its membrane displacement, and maintained its endocytic abilities in mitosis. We propose that the negative regulation of Dab2 is part of an accommodation of the cell to the altered physicochemical conditions prevalent in mitosis, aimed at allowing endocytic activity throughout the cell cycle.

  11. Double-strand break repair-adox: Restoration of suppressed double-strand break repair during mitosis induces genomic instability.

    Science.gov (United States)

    Terasawa, Masahiro; Shinohara, Akira; Shinohara, Miki

    2014-12-01

    Double-strand breaks (DSBs) are one of the severest types of DNA damage. Unrepaired DSBs easily induce cell death and chromosome aberrations. To maintain genomic stability, cells have checkpoint and DSB repair systems to respond to DNA damage throughout most of the cell cycle. The failure of this process often results in apoptosis or genomic instability, such as aneuploidy, deletion, or translocation. Therefore, DSB repair is essential for maintenance of genomic stability. During mitosis, however, cells seem to suppress the DNA damage response and proceed to the next G1 phase, even if there are unrepaired DSBs. The biological significance of this suppression is not known. In this review, we summarize recent studies of mitotic DSB repair and discuss the mechanisms of suppression of DSB repair during mitosis. DSB repair, which maintains genomic integrity in other phases of the cell cycle, is rather toxic to cells during mitosis, often resulting in chromosome missegregation and aberration. Cells have multiple safeguards to prevent genomic instability during mitosis: inhibition of 53BP1 or BRCA1 localization to DSB sites, which is important to promote non-homologous end joining or homologous recombination, respectively, and also modulation of the non-homologous end joining core complex to inhibit DSB repair. We discuss how DSBs during mitosis are toxic and the multiple safeguard systems that suppress genomic instability.

  12. Tetraploid induction by inhibiting mitosis I in scallop Chlamys farreri

    Science.gov (United States)

    Yang, Hui-Ping; Guo, Xi-Ming; Chen, Zai-Zhong; Wang, Yong-Ping

    1999-12-01

    Tetraploid induction was carried out by inhibiting mitosis I in fertilized eggs of Chlamys farreri. Mitosis I was blocked with cold shock (5 7°C), Cytochalasin B (0.75 mg/L) and 6-dimethylaminopurine (6-DMAP) (60 75 mg/L) when 60% fertilized eggs released polar body II at 20°C. At 4-cells embryo stage, the ploidy was determined by counting chromosome number. In control groups, most embryos were diploids (72.22%) and aneuploids (24.78%). In Cytochalasin B, cold shock and 6-DMAP treated groups, tetraploids were respectively 10.51%, 4.08%, and 13.34%; aneuploids were 43.10%, 35.93% and 29.16%, and triploids were 7.84%, 8.52% and 18.33%. At D-larva stage, ploidy was determined by flow cytometry (FCM). The ploidy analysis of day 2 larvae showed diploids in control group and also in three treated groups. Juvenile scallops (0.2 0.3cm) which were harvested in two control groups and two CB treated groups were all diploids through checking ploidy individually by FCM.

  13. Aminopropyltransferases involved in polyamine biosynthesis localize preferentially in the nucleus of plant cells.

    Directory of Open Access Journals (Sweden)

    Borja Belda-Palazón

    Full Text Available Plant aminopropyltransferases consist of a group of enzymes that transfer aminopropyl groups derived from decarboxylated S-adenosyl-methionine (dcAdoMet or dcSAM to propylamine acceptors to produce polyamines, ubiquitous metabolites with positive charge at physiological pH. Spermidine synthase (SPDS uses putrescine as amino acceptor to form spermidine, whereas spermine synthase (SPMS and thermospermine synthase (TSPMS use spermidine as acceptor to synthesize the isomers spermine and thermospermine respectively. In previous work it was shown that both SPDS1 and SPDS2 can physically interact with SPMS although no data concerning the subcellular localization was reported. Here we study the subcellular localization of these enzymes and their protein dimer complexes with gateway-based Bimolecular Fluorescence Complementation (BiFC binary vectors. In addition, we have characterized the molecular weight of the enzyme complexes by gel filtration chromatography with in vitro assembled recombinant enzymes and with endogenous plant protein extracts. Our data suggest that aminopropyltransferases display a dual subcellular localization both in the cytosol and nuclear enriched fractions, and they assemble preferably as dimers. The BiFC transient expression data suggest that aminopropyltransferase heterodimer complexes take place preferentially inside the nucleus.

  14. The nucleoporin Mlp2 is involved in chromosomal distribution during mitosis in trypanosomatids.

    Science.gov (United States)

    Morelle, Christelle; Sterkers, Yvon; Crobu, Lucien; MBang-Benet, Diane-Ethna; Kuk, Nada; Portalès, Pierre; Bastien, Patrick; Pagès, Michel; Lachaud, Laurence

    2015-04-30

    Nucleoporins are evolutionary conserved proteins mainly involved in the constitution of the nuclear pores and trafficking between the nucleus and cytoplasm, but are also increasingly viewed as main actors in chromatin dynamics and intra-nuclear mitotic events. Here, we determined the cellular localization of the nucleoporin Mlp2 in the 'divergent' eukaryotes Leishmania major and Trypanosoma brucei. In both protozoa, Mlp2 displayed an atypical localization for a nucleoporin, essentially intranuclear, and preferentially in the periphery of the nucleolus during interphase; moreover, it relocated at the mitotic spindle poles during mitosis. In T. brucei, where most centromeres have been identified, TbMlp2 was found adjacent to the centromeric sequences, as well as to a recently described unconventional kinetochore protein, in the periphery of the nucleolus, during interphase and from the end of anaphase onwards. TbMlp2 and the centromeres/kinetochores exhibited a differential migration towards the poles during mitosis. RNAi knockdown of TbMlp2 disrupted the mitotic distribution of chromosomes, leading to a surprisingly well-tolerated aneuploidy. In addition, diploidy was restored in a complementation assay where LmMlp2, the orthologue of TbMlp2 in Leishmania, was expressed in TbMlp2-RNAi-knockdown parasites. Taken together, our results demonstrate that Mlp2 is involved in the distribution of chromosomes during mitosis in trypanosomatids.

  15. An examination of nucleus accumbens cell firing during extinction and reinstatement of water reinforcement behavior in rats.

    Science.gov (United States)

    Hollander, Jonathan A; Ijames, Stephanie G; Roop, Richard G; Carelli, Regina M

    2002-03-08

    Electrophysiological recording procedures were used to examine nucleus accumbens (Acb) cell firing in rats (n = 13) during water reinforcement sessions consisting of three phases. During phase one (maintenance), a lever press resulted in water reinforcement (fixed ratio 1; 0.05 ml/press) paired with an auditory stimulus (0.5 s). Of 128 Acb neurons recorded during maintenance, 40 cells (31%) exhibited one of three types of neuronal firing patterns described previously [J. Neurosci. 14 (12) (1994) 7735-7746; J. Neurosci. 20 (11) (2000) 4255-4266]. Briefly, Acb neurons exhibited increases in firing rate within seconds preceding the reinforced response (type PR) or increases (type RFe) or decreases (type RFi) in activity seconds following response completion. In phase two (extinction), subsequent lever pressing had no programmed consequences (i.e., water reinforcement and the auditory stimulus were not presented). After 30 min of no responding, animals were given water reinforcement/auditory stimulus 'primes' to reestablish lever pressing behavior during the third phase (reinstatement). Results indicated that all types of phasic neurons (PR, RFe and RFi) exhibited an attenuated firing rate during extinction, and in some cases recovery of patterned discharges were observed during reinstatement. No significant changes in cell firing were observed for any cell type during presentation of the stimulus prime used to reestablish operant responding following extinction. These findings are discussed in terms of how Acb neurons process information related to 'natural' reinforcers versus drugs of abuse.

  16. Variations in gene and protein expression in canine chondrodystrophic nucleus pulposus cells following long-term three-dimensional culture.

    Directory of Open Access Journals (Sweden)

    Munetaka Iwata

    Full Text Available Intervertebral disc (IVD degeneration greatly affects quality of life. The nucleus pulposus (NP of chondrodystrophic dog breeds (CDBs is similar to the human NP, because the cells disappear with age and are replaced by fibrochondrocyte-like cells. However, because IVD develops as early as within the first year of life, we used canines as a model to investigate in vitro the mechanisms underlying IVD degeneration. Specifically, we evaluated the potential of a three-dimensional (3D culture of healthy NP as an in vitro model system to investigate the mechanisms of IVD degeneration. Agarose hydrogels were populated with healthy NP cells from beagles after performing magnetic resonance imaging, and mRNA expression profiles and pericellular extracellular matrix (ECM protein distribution were determined. After 25 days of 3D culture, there was a tendency for redifferentiation into the native NP phenotype, and mRNA levels of Col2A1, COMP, and CK18 were not significantly different from those of freshly isolated cells. Our findings suggest that long-term 3D culture promoted chondrodystrophic NP redifferentiation through reconstruction of the pericellular microenvironment. Further, lipopolysaccharide (LPS induced expression of TNF-α, MMP3, MMP13, VEGF, and PGES mRNA in the 3D cultures, creating a molecular milieu that mimics that of degenerated NP. These results suggest that this in vitro model represents a reliable and cost-effective tool for evaluating new therapies for disc degeneration.

  17. Transfer of a eubacteria-type cell division site-determining factor CrMinD gene to the nucleus from the chloroplast genome in Chlamydomonas reinhardtii

    Institute of Scientific and Technical Information of China (English)

    LIU WeiZhong; HU Yong; ZHANG RunJie; ZHOU WeiWei; ZHU JiaYing; LIU XiangLin; HE YiKun

    2007-01-01

    MinD is a ubiquitous ATPase that plays a crucial role in selection of the division site in eubacteria, chloroplasts, and probably Archaea. In four green algae, Mesostigma viride, Nephroselmis olivacea, Chlorella vulgaris and Prototheca wickerhamii, MinD homologues are encoded in the plastid genome. However, in Arabidopsis, MinD is a nucleus-encoded, chloroplast-targeted protein involved in chloroplast division, which suggests that MinD has been transferred to the nucleus in higher land plants. Yet the lateral gene transfer (LGT) of MinD from plastid to nucleus during plastid evolution remains poorly understood. Here, we identified a nucleus-encoded MinD homologue from unicellular green alga Chlamydomonas reinhardtii, a basal species in the green plant lineage. Overexpression of CrMinD in wild type E. coli inhibited cell division and resulted in the filamentous cell formation, clearly demonstrated the conservation of the MinD protein during the evolution of photosynthetic eukaryotes. The transient expression of CrMinD-egfp confirmed the role of CrMinD protein in the regulation of plastid division. Searching all the published plastid genomic sequences of land plants, no MinD homologues were found, which suggests that the transfer of MinD from plastid to nucleus might have occurred before the evolution of land plants.

  18. Electron-microscope observations of mitosis and cytokinesis in multinucleate protoplasts of soybean.

    Science.gov (United States)

    Fowke, L C; Bech-Hansen, C W; Gamborg, O L; Constabel, F

    1975-08-01

    Multinucleate soybean protoplasts produced by spontaneous fusion during enzyme digestion of the cell wall initiated cell division after approximately 40 h in culture. The structure of these protoplasts during mitosis and cytokinesis was studied with both light and electron microscopes. Most nuclei did not fuse but divided synchronously. Interphase nuclei was commonly connected by short narrow nuclear bridges. At prophase and metaphase the nuclei appeared typical of those in most higher plants; technical difficulties prevented an adequate examination of protoplasts at anaphase. Telophase was characterized by cytokinesis involving phragmoplast and cell plate formation; however, complete partitioning of the cytoplasm by cell plants was not observed. Numerous coated vesicles were present near to or continuous with the cell plate and plasmalemma. The presence of a few dividing protoplasts with at least double the normal chromosome number suggests that some nuclear fusion occurred prior to mitosis. Very little cell wall material was detected at the margin of the dividing protoplasts.

  19. Regulation of nuclear envelope dynamics via APC/C is necessary for the progression of semi-open mitosis in Schizosaccharomyces japonicus.

    Science.gov (United States)

    Aoki, Keita; Shiwa, Yuh; Takada, Hiraku; Yoshikawa, Hirofumi; Niki, Hironori

    2013-09-01

    Three types of mitosis, which are open, closed or semi-open mitosis, function in eukaryotic cells, respectively. The open mitosis involves breakage of the nuclear envelope before nuclear division, whereas the closed mitosis proceeds with an intact nuclear envelope. To understand the mechanism and significance of three types of mitotic division in eukaryotes, we investigated the process of semi-open mitosis, in which the nuclear envelope is only partially broken, in the fission yeast Schizosaccharomyces japonicus. In anaphase-promoting complex/cyclosome (APC/C) mutants of Sz. japonicus, the nuclear envelope remained relatively intact during anaphase, resulting in impaired semi-open mitosis. As a suppressor of apc2 mutant, a mutation of Oar2, which was a 3-oxoacyl-[acyl carrier protein] reductase, was obtained. The level of the Oar2, which had two destruction-box motifs recognized by APC/C, was increased in APC/C mutants. Furthermore, the defective semi-open mitosis observed in an apc2 mutant was restored by mutated oar2+. Based on these findings, we propose that APC/C regulates the dynamics of the nuclear envelope through degradation of Oar2 dependent on APC/C during the metaphase-to-anaphase transition of semi-open mitosis in Sz. japonicus.

  20. The nucleus of differentiated root plant cells: modifications induced by arbuscular mycorrhizal fungi

    Directory of Open Access Journals (Sweden)

    G Lingua

    2009-12-01

    Full Text Available The nuclei of plant cells show marked differences in chromatin organisation, related to their DNA content, which ranges from the type with large strands of condensed chromatin (reticulate or chromonematic nuclei to one with mostly decondensed chromatin (chromocentric or diffuse nuclei. A loosening of the chromatin structure generally occurs in actively metabolising cells, such as differentiating and secretory cells, in relation to their high transcriptional activity. Endoreduplication may occur, especially in plants with a small genome, which increases the availability of nuclear templates, the synthesis of DNA, and probably regulates gene expression. Here we describe structural and quantitative changes of the chromatin and their relationship with transcription that occur in differentiated cells following an increase of their metabolism. The nuclei of root cortical cells of three plants with different 2C DNA content (Allium porrum, Pisum sativum and Lycopersicon esculentm and their modifications induced by arbuscular mycorrhization, which strongly increase the metabolic activity of colonised cells, are taken as examples.

  1. The RNA-binding protein TIAR is translocated from the nucleus to the cytoplasm during Fas-mediated apoptotic cell death.

    OpenAIRE

    Taupin, J L; Tian, Q.; Kedersha, N; Robertson, M.; Anderson, P

    1995-01-01

    We have determined the structure, intracellular localization, and tissue distribution of TIAR, a TIA-1-related RNA-binding protein. Two related isoforms of TIAR, migrating at 42 and 50 kDa, are expressed in primate cells. Unlike TIA-1, which is found in the granules of cytotoxic lymphocytes, TIAR is concentrated in the nucleus of hematopoietic and nonhematopoietic cells. Because TIAR can trigger DNA fragmentation in permeabilized thymocytes, it is a candidate effector of apoptotic cell death....

  2. Bicarbonate-sensing soluble adenylyl cyclase is present in the cell cytoplasm and nucleus of multiple shark tissues.

    Science.gov (United States)

    Roa, Jinae N; Tresguerres, Martin

    2017-01-01

    The enzyme soluble adenylyl cyclase (sAC) is directly stimulated by bicarbonate (HCO3(-)) to produce the signaling molecule cyclic adenosine monophosphate (cAMP). Because sAC and sAC-related enzymes are found throughout phyla from cyanobacteria to mammals and they regulate cell physiology in response to internal and external changes in pH, CO2, and HCO3(-), sAC is deemed an evolutionarily conserved acid-base sensor. Previously, sAC has been reported in dogfish shark and round ray gill cells, where they sense and counteract blood alkalosis by regulating the activity of V-type H(+)- ATPase. Here, we report the presence of sAC protein in gill, rectal gland, cornea, intestine, white muscle, and heart of leopard shark Triakis semifasciata Co-expression of sAC with transmembrane adenylyl cyclases supports the presence of cAMP signaling microdomains. Furthermore, immunohistochemistry on tissue sections, and western blots and cAMP-activity assays on nucleus-enriched fractions demonstrate the presence of sAC protein in and around nuclei. These results suggest that sAC modulates multiple physiological processes in shark cells, including nuclear functions.

  3. Qualitative and quantitative characteristics of the extracellular DNA delivered to the nucleus of a living cell

    Directory of Open Access Journals (Sweden)

    Bogachev Sergei S

    2006-10-01

    Full Text Available Abstract Background The blood plasma and other intertissue fluids usually contain a certain amount of DNA, getting there due to a natural cell death in the organism. Cells of this organism can capture the extracellular DNA, whereupon it is delivered to various cell compartments. It is hypothesized that the extracellular DNA is involved in the transfer of genetic information and its fixation in the genome of recipient cell. Results The existence of an active flow of extracellular DNA into the cell is demonstrated using human breast adenocarcinoma (MCF-7 cells as a recipient culture. The qualitative state of the DNA fragments delivered to the main cell compartments (cytoplasm and interchromosomal fraction was assessed. The extracellular DNA delivered to the cell is characterized quantitatively. Conclusion It is demonstrated that the extracellular DNA fragments in several minutes reach the nuclear space, where they are processed so that their linear size increases from about 500 bp to 10,000 bp. The amount of free extracellular DNA fragments simultaneously present in the nuclear space may reach up to 2% of the haploid genome. Using individual DNA fragments with a known molecular weight and sequence as an extracellular DNA, it is found that these fragments degrade instantly in the culture liquid in the absence of a competitor DNA and are delivered into the cell as degradants. When adding a sufficient amount of competitor DNA, the initial undegraded molecules of the DNA fragments with the known molecular weight and sequence are detectable both in the cytoplasm and nuclear space only at the zero point of experiments. The labeled precursor α-dNTP*, added to culture medium, was undetectable inside the cell in all the experiments.

  4. Differential expression of extracellular-signal-regulated kinase 5 (ERK5) in normal and degenerated human nucleus pulposus tissues and cells

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Weiguo, E-mail: liangweiguo@tom.com [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); Fang, Dejian [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); Ye, Dongping [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); School of Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia 6009 (Australia); Zou, Longqiang; Shen, Yan; Dai, Libing [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); Xu, Jiake, E-mail: jiake.xu@uwa.edu.au [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); School of Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia 6009 (Australia)

    2014-07-11

    Highlights: • ERK5 involved in NP cells. • ERK5 involved in NP tissue. • It was important modulator. - Abstract: Extracellular-signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and regulates a wide variety of cellular processes such as proliferation, differentiation, necrosis, apoptosis and degeneration. However, the expression of ERK5 and its role in degenerated human nucleus pulposus (NP) is hitherto unknown. In this study, we observed the differential expression of ERK5 in normal and degenerated human nucleus pulposus tissues by using immunohistochemical staining and Western blot. Treatment of NP cells with Pro-inflammatory cytokine, TNF-α decreased ERK5 gene expression as well as NP marker gene expression; including the type II collagen and aggrecan. Suppression of ERK5 gene expression in NP cells by ERK5 siRNA resulted in decreased gene expression of type II collagen and aggrecan. Furthermore, inhibition of ERK5 activation by BIX02188 (5 μM) decreased the gene expression of type II collagen and aggrecan in NP cells. Our results document the expression of ERK5 in degenerated nucleus pulposus tissues, and suggest a potential involvement of ERK5 in human degenerated nucleus pulposus.

  5. A polycomb group protein is retained at specific sites on chromatin in mitosis.

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    Nicole E Follmer

    Full Text Available Epigenetic regulation of gene expression, including by Polycomb Group (PcG proteins, may depend on heritable chromatin states, but how these states can be propagated through mitosis is unclear. Using immunofluorescence and biochemical fractionation, we find PcG proteins associated with mitotic chromosomes in Drosophila S2 cells. Genome-wide sequencing of chromatin immunoprecipitations (ChIP-SEQ from mitotic cells indicates that Posterior Sex Combs (PSC is not present at well-characterized PcG targets including Hox genes in mitosis, but does remain at a subset of interphase sites. Many of these persistent sites overlap with chromatin domain borders described by Sexton et al. (2012, which are genomic regions characterized by low levels of long range contacts. Persistent PSC binding sites flank both Hox gene clusters. We hypothesize that disruption of long-range chromatin contacts in mitosis contributes to PcG protein release from most sites, while persistent binding at sites with minimal long-range contacts may nucleate re-establishment of PcG binding and chromosome organization after mitosis.

  6. Translocation of histone H1 subtypes between chromatin and cytoplasm during mitosis in normal human fibroblasts.

    Science.gov (United States)

    Gréen, Anna; Lönn, Anita; Peterson, Kajsa Holmgren; Ollinger, Karin; Rundquist, Ingemar

    2010-05-01

    Histone H1 is an important constituent of chromatin, which undergoes major structural rearrangements during mitosis. However, the role of H1, multiple H1 subtypes, and H1 phosphorylation is still unclear. In normal human fibroblasts, phosphorylated H1 was found located in nuclei during prophase and in both cytoplasm and condensed chromosomes during metaphase, anaphase, and telophase as detected by immunocytochemistry. Moreover, we detected remarkable differences in the distribution of the histone H1 subtypes H1.2, H1.3, and H1.5 during mitosis. H1.2 was found in chromatin during prophase and almost solely in the cytoplasm of metaphase and early anaphase cells. In late anaphase, it appeared in both chromatin and cytoplasm and again in chromatin during telophase. H1.5 distribution pattern resembled that of H1.2, but H1.5 was partitioned between chromatin and cytoplasm during metaphase and early anaphase. H1.3 was detected in chromatin in all cell cycle phases. We propose therefore, that H1 subtype translocation during mitosis is controlled by phosphorylation, in combination with H1 subtype inherent affinity. We conclude that H1 subtypes, or theirphosphorylated forms, may leave chromatin in a regulated way to give access for chromatin condensing factors or transcriptional regulators during mitosis.

  7. Evidence that adrenergic ventrolateral medullary cells are activated whereas precerebellar lateral reticular nucleus neurons are suppressed during REM sleep.

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    Georg M Stettner

    Full Text Available Rapid eye movement sleep (REMS is generated in the brainstem by a distributed network of neurochemically distinct neurons. In the pons, the main subtypes are cholinergic and glutamatergic REMS-on cells and aminergic REMS-off cells. Pontine REMS-on cells send axons to the ventrolateral medulla (VLM, but little is known about REMS-related activity of VLM cells. In urethane-anesthetized rats, dorsomedial pontine injections of carbachol trigger REMS-like episodes that include cortical and hippocampal activation and suppression of motoneuronal activity; the episodes last 4-8 min and can be elicited repeatedly. We used this model to determine whether VLM catecholaminergic cells are silenced during REMS, as is typical of most aminergic neurons studied to date, and to investigate other REMS-related cells in this region. In 18 anesthetized, paralyzed and artificially ventilated rats, we obtained extracellular recordings from VLM cells when REMS-like episodes were elicited by pontine carbachol injections (10 mM, 10 nl. One major group were the cells that were activated during the episodes (n = 10. Their baseline firing rate of 3.7±2.1 (SD Hz increased to 9.7±2.1 Hz. Most were found in the adrenergic C1 region and at sites located less than 50 µm from dopamine β-hydroxylase-positive (DBH(+ neurons. Another major group were the silenced or suppressed cells (n = 35. Most were localized in the lateral reticular nucleus (LRN and distantly from any DBH(+ cells. Their baseline firing rates were 6.8±4.4 Hz and 15.8±7.1 Hz, respectively, with the activity of the latter reduced to 7.4±3.8 Hz. We conclude that, in contrast to the pontine noradrenergic cells that are silenced during REMS, medullary adrenergic C1 neurons, many of which drive the sympathetic output, are activated. Our data also show that afferent input transmitted to the cerebellum through the LRN is attenuated during REMS. This may distort the spatial representation of body position

  8. Live imaging of mitosis in the developing mouse embryonic cortex.

    Science.gov (United States)

    Pilaz, Louis-Jan; Silver, Debra L

    2014-06-04

    Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-α-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

  9. Phospho-Bcl-xL(Ser62) influences spindle assembly and chromosome segregation during mitosis.

    Science.gov (United States)

    Wang, Jianfang; Beauchemin, Myriam; Bertrand, Richard

    2014-01-01

    Functional analysis of a series of phosphorylation mutants reveals that Bcl-xL(Ser62Ala) influences cell entry into anaphase and mitotic exit in taxol-exposed cells compared with cells expressing wild-type Bcl-xL or a series of other phosphorylation mutants, an effect that appears to be independent of its anti-apoptotic activity. During normal mitosis progression, Bcl-xL(Ser62) is strongly phosphorylated by PLK1 and MAPK14/SAPKp38α at the prometaphase, metaphase, and the anaphase boundaries, while it is de-phosphorylated at telophase and cytokinesis. Phospho-Bcl-xL(Ser62) localizes in centrosomes with γ-tubulin and in the mitotic cytosol with some spindle-assembly checkpoint signaling components, including PLK1, BubR1, and Mad2. In taxol- and nocodazole-exposed cells, phospho-Bcl-xL(Ser62) also binds to Cdc20- Mad2-, BubR1-, and Bub3-bound complexes, while Bcl-xL(Ser62Ala) does not. Silencing Bcl-xL expression and expressing the phosphorylation mutant Bcl-xL(Ser62Ala) lead to an increased number of cells harboring mitotic spindle defects including multipolar spindle, chromosome lagging and bridging, aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h. Together, the data indicate that during mitosis, Bcl-xL(Ser62) phosphorylation impacts on spindle assembly and chromosome segregation, influencing chromosome stability. Observations of mitotic cells harboring aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h were also made with cells expressing the phosphorylation mutant Bcl-xL(Ser49Ala) and dual mutant Bcl-xL(Ser49/62Ala).

  10. Moxonidine into the lateral parabrachial nucleus reduces renal and hormonal responses to cell dehydration.

    Science.gov (United States)

    Andrade, C A F; Margatho, L O; Andrade-Franzé, G M F; De Luca, L A; Antunes-Rodrigues, J; Menani, J V

    2012-04-19

    The deactivation of the inhibitory mechanisms with injections of moxonidine (α2-adrenoceptor/imidazoline receptor agonist) into the lateral parabrachial nucleus (LPBN) increases hypertonic NaCl intake by intra- or extracellular dehydrated rats. In the present study, we investigated the changes in the urinary sodium and volume, sodium balance, and plasma vasopressin and oxytocin in rats treated with intragastric (i.g.) 2 M NaCl load (2 ml/rat) combined with injections of moxonidine into the LPBN. Male Holtzman rats (n=5-12/group) with stainless steel cannulas implanted bilaterally into LPBN were used. Bilateral injections of moxonidine (0.5 nmol/0.2 μl) into the LPBN decreased i.g. 2 M NaCl-induced diuresis (4.6±0.7 vs. vehicle: 7.4±0.6 ml/120 min) and natriuresis (1.65±0.29 vs. vehicle: 2.53±0.17 mEq/120 min), whereas the previous injection of the α2-adrenoceptor antagonist RX 821002 (10 nmol/0.2 μl) into the LPBN abolished the effects of moxonidine. Moxonidine injected into the LPBN reduced i.g. 2 M NaCl-induced increase in plasma oxytocin and vasopressin (14.6±2.8 and 2.2±0.3 vs. vehicle: 25.7±7 and 4.3±0.7 pg/ml, respectively). Moxonidine injected into the LPBN combined with i.g. 2 M NaCl also increased 0.3 M NaCl intake (7.5±1.7 vs. vehicle: 0.5±0.2 mEq/2 h) and produced positive sodium balance (2.3±1.4 vs. vehicle: -1.2±0.4 mEq/2 h) in rats that had access to water and NaCl. The present results show that LPBN α2-adrenoceptor activation reduces renal and hormonal responses to intracellular dehydration and increases sodium and water intake, which facilitates sodium retention and body fluid volume expansion.

  11. Cell-poor septa separate representations of digits in the ventroposterior nucleus of the thalamus in monkeys and prosimian galagos.

    Science.gov (United States)

    Qi, Hui-Xin; Gharbawie, Omar A; Wong, Peiyan; Kaas, Jon H

    2011-03-01

    The architectonic features of the ventroposterior nucleus (VP) were visualized in coronal brain sections from two macaque monkeys, two owl monkeys, two squirrel monkeys, and three galagos that were processed for cytochrome oxidase, Nissl bodies, or the vesicular glutamate transporter 2 (vGluT2). The traditional ventroposterior medial (VPM) and ventroposterior lateral (VPL) subnuclei were easily identified, as well as the forelimb and hindlimb compartments of VPL, as they were separated by poorly staining, cell-poor septa. Septa also separated other cell groups within VPM and VPL, specifically in the medial compartment of VPL representing the hand (hand VPL). In one squirrel monkey and one galago we demonstrated that these five groups of cells represent digits 1-5 in a mediolateral sequence by injecting tracers into the cortical representation of single digits, defined by microelectrode recordings, and relating concentrations of labeled neurons to specific cell groups in hand VPL. The results establish the existence of septa that isolate the representation of the five digits in VPL of primates and demonstrate that the isolated cell groups represent digits 1-5 in a mediolateral sequence. The present results show that the septa are especially prominent in brain sections processed for vGluT2, which is expressed in the synaptic terminals of excitatory neurons in most nuclei of the brainstem and thalamus. As vGluT2 is expressed in the synaptic terminations from dorsal columns and trigeminal brainstem nuclei, the effectiveness of vGluT2 preparations in revealing septa in VP likely reflects a lack of synapses using glutamate in the septa.

  12. THE USE OF FLUORESCENCE CORRELATION SPECTROSCOPY TO PROBE CHROMATIN IN THE CELL NUCLEUS

    Energy Technology Data Exchange (ETDEWEB)

    Sorscher, Stanley M.; Bartholemew, James C.; Klein, Melvin P.

    1980-03-01

    All systems in thermodynamic equilibrium are subject to spontaneous fluctuations from equilibrium. For very small systems, the fluctuations can be made apparent, and can be used to study the behavior of the system without introducing any external perturbations. The mean squared amplitude of these fluctuations contains information about the absolute size of the system. The characteristic time of the fluctuation autocorrelation function contains kinetic information. In the experiments reported here, these concepts are applied to the binding equilibrium between ethidium bromide and DNA, a system where the fluorescence properties of the dye greatly enhance the effect of spontaneous fluctuations in the binding equilibrium. Preliminary experiments employ well characterized DNA preparations, including calf thymus DNA, SV40 DNA, and calf thymus nucleohistone particles. Additional measurements are described which have been made in small regions of individual nuclei, isolated from green monkey kidney cells, observing as few as 5000 dye molecules. The data indicate that the strength of dye binding increases in nuclei isolated from cells which have been stimulated to enter the cell growth cycle. The viscosity of nuclear material is inferred to be between one and two orders of magnitude greater than that of water, and decreases as the cells leave the resting state, and enter the cell growth cycle. Washing the nuclei also lowers the viscosity. These experiments demonstrate that fluorescence correlation spectroscopy can provide information at the subnuclear level that is otherwise unavailable.

  13. Citron kinase is a regulator of mitosis and neurogenic cytokinesis in the neocortical ventricular zone.

    Science.gov (United States)

    LoTurco, Joseph J; Sarkisian, Mathew R; Cosker, Laurie; Bai, Jilin

    2003-06-01

    Successful cell division in neural progenitors in the neocortical ventricular zone (VZ), as in all dividing cells, depends critically upon coordinating chromosome segregation during mitosis with cytokinesis. This coordination further suggests that common molecular regulators may link events in mitosis with those in cytokinesis. Recent genetic evidence indicates that cytokinesis in CNS neuronal progenitors, but not in most other cell types of the body, requires the function of citron kinase. In neocortex, citron kinase is most critical for neurogenic cytokinesis. In citron kinase null mutants, a large proportion of neuronal cells within neocortex are binucleate; however, very few glial cells are binucleate. In addition, confocal time-lapse imaging of mitoses at the VZ surface shows that citron kinase is also necessary for phases of the cell cycle just prior to cytokinesis. Deficits in mitosis seen in mutants indicate aberrant mitotic spindle function, and like deficits in cytokinesis, occur in some but not all cells at the VZ surface. Citron kinase is therefore an essential multifunctional regulator of cell divisions in the VZ, and may serve to coordinate chromosome segregation with cytokinesis in neuronal precursors.

  14. β-Adrenergic activation enhances NMDA-induced current in pyramidal cells of the basolateral nucleus of amygdala

    Institute of Scientific and Technical Information of China (English)

    LIU Xinqiu; CAO Xiaohua; LI Bao-ming

    2005-01-01

    NMDA receptor (NMDA-R) in the amygdala complex is critical for both long-term potentiation (LTP) and formation of conditioned fear memory. It is reported that activation of β-adrenoceptors (β-AR) in the amygdala facilitates LTP and enhances memory consolidation. The present study examined the regulatory effect of β-AR activation on NMDA-R mediated current in pyramidal cells of the basolateral nucleus of amygdala (BLA), using whole-cell recording technique. Bath application of the β-AR agonist isoproterenol enhanced NMDA-induced current, and this facilitatory effect was blocked by co-administered propranolol, a β-AR antagonist. The facilitatory effect of isoproterenol on NMDA-induced current could not be induced when the protein kinase A (PKA) inhibitor Rp-cAMPs was added in electrode internal solution.The present results suggest that β-AR activation in the BLA could modulate NMDA-R activity directly and positively, probably via PKA.

  15. Cell-type specific oxytocin gene expression from AAV delivered promoter deletion constructs into the rat supraoptic nucleus in vivo.

    Directory of Open Access Journals (Sweden)

    Raymond L Fields

    Full Text Available The magnocellular neurons (MCNs in the hypothalamus selectively express either oxytocin (OXT or vasopressin (AVP neuropeptide genes, a property that defines their phenotypes. Here we examine the molecular basis of this selectivity in the OXT MCNs by stereotaxic microinjections of adeno-associated virus (AAV vectors that contain various OXT gene promoter deletion constructs using EGFP as the reporter into the rat supraoptic nucleus (SON. Two weeks following injection of the AAVs, immunohistochemical assays of EGFP expression from these constructs were done to determine whether the EGFP reporter co-localizes with either the OXT- or AVP-immunoreactivity in the MCNs. The results show that the key elements in the OT gene promoter that regulate the cell-type specific expression the SON are located -216 to -100 bp upstream of the transcription start site. We hypothesize that within this 116 bp domain a repressor exists that inhibits expression specifically in AVP MCNs, thereby leading to the cell-type specific expression of the OXT gene only in the OXT MCNs.

  16. A fraction of neurofibromin interacts with PML bodies in the nucleus of the CCF astrocytoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Godin, Fabienne; Villette, Sandrine; Vallee, Beatrice; Doudeau, Michel; Morisset-Lopez, Severine [Centre de Biophysique Moleculaire, Centre National de la Recherche Scientifique (CNRS), UPR 4301, Universite d' Orleans et INSERM, rue Charles Sadron, 45071 Orleans Cedex 2 (France); Ardourel, Maryvonne; Hevor, Tobias [Laboratoire de Neurobiologie, Universite d' Orleans, BP 6759, 45067 Orleans Cedex 2 (France); Pichon, Chantal [Centre de Biophysique Moleculaire, Centre National de la Recherche Scientifique (CNRS), UPR 4301, Universite d' Orleans et INSERM, rue Charles Sadron, 45071 Orleans Cedex 2 (France); Benedetti, Helene, E-mail: helene.benedetti@cnrs-orleans.fr [Centre de Biophysique Moleculaire, Centre National de la Recherche Scientifique (CNRS), UPR 4301, Universite d' Orleans et INSERM, rue Charles Sadron, 45071 Orleans Cedex 2 (France)

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer We validate the use of specific anti-Nf1 antibodies for immunofluorescence studies. Black-Right-Pointing-Pointer We detect Nf1 in the cytoplasm and nucleus of CCF cells. Black-Right-Pointing-Pointer We demonstrate that Nf1 partially colocalizes with PML nuclear bodies. Black-Right-Pointing-Pointer We demonstrate that there is a direct interaction between a fraction of Nf1 and the PML bodies. -- Abstract: Neurofibromatosis type 1 is a common genetic disease that causes nervous system tumors, and cognitive deficits. It is due to mutations within the NF1 gene, which encodes the Nf1 protein. Nf1 has been shown to be involved in the regulation of Ras, cAMP and actin cytoskeleton dynamics. In this study, using immunofluorescence experiments, we have shown a partial nuclear localization of Nf1 in the astrocytoma cell line: CCF and we have demonstrated that Nf1 partially colocalizes with PML (promyelocytic leukemia) nuclear bodies. A direct interaction between Nf1 and the multiprotein complex has further been demonstrated using 'in situ' proximity ligation assay (PLA).

  17. Increased number of TH-immunoreactive cells in the ventral tegmental area after deep brain stimulation of the anterior nucleus of the thalamus.

    Science.gov (United States)

    Dela Cruz, J A D; Hescham, S; Adriaanse, B; Campos, F L; Steinbusch, H W M; Rutten, B P F; Temel, Y; Jahanshahi, A

    2015-09-01

    Dopamine (DA) has been long implicated with the processes of memory. In long-term memory, the hippocampus and ventral tegmental area (VTA) use DA to enhance long-term potentiation, while prefrontal DA D1 receptors are involved in working memory. Deep brain stimulation (DBS) of specific brain areas have been shown to affect memory impairments in animal models. Here, we tested the hypothesis that DBS could reverse memory impairments by increasing the number of dopaminergic cells in the VTA. Rats received DBS at the level of the mammillothalamic tract, the anterior nucleus of the thalamus, and entorhinal cortex before euthanasia. These regions are part of the so-called memory circuit. Brain sections were processed for c-Fos and tyrosine hydroxylase (TH) immunocytochemistry in the VTA and the substantia nigra pars compacta (SNc). c-Fos, TH and c-Fos/TH immunoreactive cells were analyzed by means of stereology and confocal microscopy. Our results showed that DBS of the anterior nucleus of the thalamus induced substantial higher numbers of TH-immunoreactive cells in the VTA, while there were no significant differences between the experimental groups in the number of TH immunoreactive cells in the SNc, c-Fos immunoreactive cells and c-Fos/TH double-labeled cells in both the SNc and VTA. Our findings suggest a phenotypic switch, or neurotransmitter respecification, of DAergic cells specifically in the VTA which may be induced by DBS in the anterior nucleus of the thalamus.

  18. Protein Phosphatases Involved in Regulating Mitosis: Facts and Hypotheses.

    Science.gov (United States)

    Kim, Hyun-Soo; Fernandes, Gary; Lee, Chang-Woo

    2016-09-01

    Almost all eukaryotic proteins are subject to post-translational modifications during mitosis and cell cycle, and in particular, reversible phosphorylation being a key event. The recent use of high-throughput experimental analyses has revealed that more than 70% of all eukaryotic proteins are regulated by phosphorylation; however, the mechanism of dephosphorylation, counteracting phosphorylation, is relatively unknown. Recent discoveries have shown that many of the protein phosphatases are involved in the temporal and spatial control of mitotic events, such as mitotic entry, mitotic spindle assembly, chromosome architecture changes and cohesion, and mitotic exit. This implies that certain phosphatases are tightly regulated for timely dephosphorylation of key mitotic phosphoproteins and are essential for control of various mitotic processes. This review describes the physiological and pathological roles of mitotic phosphatases, as well as the versatile role of various protein phosphatases in several mitotic events.

  19. p21 is Responsible for Ionizing Radiation-induced Bypass of Mitosis

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xu Rui; LIU Yong Ai; SUN Fang; LI He; LEI Su Wen; WANG Ju Fang

    2016-01-01

    Objective To explore the role of p21 in ionizing radiation-induced changes in protein levels during the G2/M transition and long-term G2 arrest. Methods Protein expression levels were assessed by western blot in the human uveal melanoma 92-1 cells after treatment with ionizing radiation. Depletion of p21 was carried out by employing the siRNA technique. Cell cycle distribution was determined by flow cytometry combined with histone H3 phosphorylation at Ser28, an M-phase marker. Senescence was assessed by senescence-associated-β-galactosidase (SA-β-gal) staining combined with Ki67 staining, a cell proliferation marker. Results Accompanying increased p21, the protein levels of G2/M transition genes declined significantly in 92-1 cells irradiated with 5 Gy of X-rays. Furthermore, these irradiated cells were blocked at the G2 phase followed by cellular senescence. Depletion of p21 rescued radiation-induced G2 arrest as demonstrated by the upregulation of G2/M transition kinases, as well as the high expression of histone H3 phosphorylated at Ser28. Knockdown of p21 resulted in entry into mitosis of irradiated 92-1 cells. However, cells with serious DNA damage failed to undergo cytokinesis, leading to the accumulation of multinucleated cells. Conclusion Our results indicated that p21 was responsible for the downregulation of G2/M transition regulatory proteins and the bypass of mitosis induced by irradiation. Downregulation of p21 by siRNA resulted in G2-arrested cells entering into mitosis with serious DNA damage. This is the first report on elucidating the role of p21 in the bypass of mitosis.

  20. The three-dimensional organization of a self replicating nano fabrication site: The human cell nucleus.

    NARCIS (Netherlands)

    T.A. Knoch (Tobias); C. Münkel (Christian); J. Langowski (Jörg)

    1997-01-01

    markdownabstractThe eukaryotic cell is a prime example of a functioning nano-machinery. The synthesis of proteins, maintenance of structure and duplication of the machinery itself are all fine-tuned biochemical processes that depend on the precise structural arrangement of the cellular components. E

  1. Three-dimensional organization of chromosome territories in the human interphase cell nucleus.

    NARCIS (Netherlands)

    T.A. Knoch (Tobias); C. Münkel (Christian); J. Langowski (Jörg)

    1998-01-01

    markdownabstractThe synthesis of proteins, maintenance of structure and duplication of the eukaryotic cell itself are all fine-tuned biochemical processes that depend on the precise structural arrangement of the cellular components. The regulation of genes – their transcription and replication - has

  2. PICH promotes sister chromatid disjunction and co-operates with topoisomerase II in mitosis.

    Science.gov (United States)

    Nielsen, Christian F; Huttner, Diana; Bizard, Anna H; Hirano, Seiki; Li, Tian-Neng; Palmai-Pallag, Timea; Bjerregaard, Victoria A; Liu, Ying; Nigg, Erich A; Wang, Lily Hui-Ching; Hickson, Ian D

    2015-01-01

    PICH is a SNF2 family DNA translocase that binds to ultra-fine DNA bridges (UFBs) in mitosis. Numerous roles for PICH have been proposed from protein depletion experiments, but a consensus has failed to emerge. Here, we report that deletion of PICH in avian cells causes chromosome structural abnormalities, and hypersensitivity to an inhibitor of Topoisomerase II (Topo II), ICRF-193. ICRF-193-treated PICH(-/-) cells undergo sister chromatid non-disjunction in anaphase, and frequently abort cytokinesis. PICH co-localizes with Topo IIα on UFBs and at the ribosomal DNA locus, and the timely resolution of both structures depends on the ATPase activity of PICH. Purified PICH protein strongly stimulates the catalytic activity of Topo II in vitro. Consistent with this, a human PICH(-/-) cell line exhibits chromosome instability and chromosome condensation and decatenation defects similar to those of ICRF-193-treated cells. We propose that PICH and Topo II cooperate to prevent chromosome missegregation events in mitosis.

  3. Hydrogen sulfide protects against endoplasmic reticulum stress and mitochondrial injury in nucleus pulposus cells and ameliorates intervertebral disc degeneration.

    Science.gov (United States)

    Xu, Daoliang; Jin, Haiming; Wen, Jianxia; Chen, Jiaoxiang; Chen, Deheng; Cai, Ningyu; Wang, Yongli; Wang, Jianle; Chen, Yu; Zhang, Xiaolei; Wang, Xiangyang

    2017-03-01

    It has been suggested that excessive apoptosis in intervertebral disc cells induced by inflammatory cytokines, such as interleukin (IL)-1β, is related to the process of intervertebral disc degeneration (IVDD). Hydrogen sulfide (H2S), a gaseous signaling molecule, has drawn attention for its anti-apoptosis role in various pathophysiological processes in degenerative diseases. To date, there has been no investigation of the correlation of H2S production and IVDD or of the effects of H2S on IL-1β-induced apoptosis in nucleus pulposus (NP) cells. Here, we found that the expression levels of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), two key enzymes in the generation of H2S, were significantly decreased in human degenerate NP tissues as well as in IL-1β-treated NP cells. NaHS (H2S donor) administration showed a protective effect by inhibiting the endoplasmic reticulum (ER) stress response and mitochondrial dysfunction induced by IL-1β stimulation in vitro, the effect was related to activation of the PI3K/Akt and ERK1/2 signaling pathways. Suppression of these pathways by specific inhibitors, LY294002 and PD98059, partially reduced the protective effect of NaHS. Moreover, in the percutaneous needle puncture disc degeneration rat tail model, disc degeneration was partially reversed by NaHS administration. Taken together, our results suggest that H2S plays a protective role in IVDD and the underlying mechanism involves PI3K/Akt and ERK1/2 signaling pathways-mediated suppression of ER stress and mitochondrial dysfunction in IL-1β-induced NP cells.

  4. SDF-1/CXCR4 axis induces apoptosis of human degenerative nucleus pulposus cells via the NF-κB pathway

    Science.gov (United States)

    LIU, ZONGCHAO; MA, CHUAN; SHEN, JIELIANG; WANG, DAWU; HAO, JIE; HU, ZHENMING

    2016-01-01

    Intervertebral disc degeneration (IVDD) is a major cause of lower back pain, and increased cell apoptosis is a key characteristic of IVDD. The present study aimed to investigate the effects and mechanism of the stromal cell-derived factor-1 (SDF-1)/C-X-C motif chemokine receptor 4 (CXCR4) axis on apoptosis in human degenerative nucleus pulposus cells (NPCs). The expression levels of SDF-1 and CXCR4 in human intervertebral discs (IVD) were determined using immunohistochemistry and western blot analysis. Apoptosis of primary cultured NPCs was quantified by Annexin V/propidium iodide staining following stimulation with SDF-1 and knockdown of CXCR4 using small interfering RNA (siRNA). The association with the nuclear factor-κB (NF-κB) signaling pathway was investigated using CXCR4-siRNA and NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), treatment. The results demonstrated that SDF-1 and its receptor, CXCR4, were upregulated in degenerative IVD samples compared with normal samples. Stimulation with SDF-1 increased the level of apoptosis in cultured NPCs, and conversely, the apoptosis level was suppressed post-transfection with CXCR4 siRNA compared with SDF-1 stimulation alone. Furthermore, SDF-1 treatment increased the level of phosphorylated NF-κB subunit P65, which was downregulated following CXCR4 siRNA and PDTC treatment. In addition, CXCR4 siRNA and PDTC inhibited the nuclear translocation of P65, which was induced by SDF-1. Taken together, SDF-1-mediated apoptosis was suppressed by NF-κB inhibition using PDTC. In conclusion, the SDF-1/CXCR4 axis promoted cell apoptosis in human degenerative NPCs via the NF-κB pathway, thus suggesting that SDF-1/CXCR signaling may be a therapeutic target for the treatment of degenerative IVD diseases. PMID:27220474

  5. Characterization of two thioredoxins h with predominant localization in the nucleus of aleurone and scutellum cells of germinating wheat seeds.

    Science.gov (United States)

    Serrato, A J; Crespo, J L; Florencio, F J; Cejudo, F J

    2001-06-01

    Two full-length cDNA clones, designated TrxhA and TrxhB, encoding different but very similar thioredoxin h polypeptides were isolated from wheat (Triticum aestivum cv. Chinese Spring) aleurone cells. The deduced proteins show a high similarity to each other and to thioredoxin h from other sources, in particular from T. aestivum and T. durum. One of them, TRXhA, was expressed in E. coli as a His-tagged polypeptide and used to raise polyclonal antibodies by immunization of rabbits. These antibodies identified a single band (ca. 13.5 kDa) in western blot analysis of protein extracts from all wheat organs analyzed. TRXhA and TRXhB when expressed in E. coli as intact polypetides showed indistinguishable electrophoretic mobility, which corresponded to the 13.5 kDa polypeptide detected in wheat protein extracts. The amount of thioredoxin h transcripts increased in scutellum and aleurone cells during germination but GA3 did not exert any stimulatory effect on thioredoxin h expression. Although northern blot analysis detected a single band, competitive RT-PCR showed that this band is due to the accumulation of both TrxhA and TrxhB mRNAs. These results suggest that the single band detected in western blots is due to the presence of at least two thioredoxin h polypeptides. Immunolocalization experiments confirmed the high content of thioredoxins h in scutellum and aleurone cells, and showed a low content in the starchy endosperm of germinating grains. Interestingly, though these proteins are evenly distributed in the cytosol, the highest levels of thioredoxins h were detected in the nucleus, both in aleurone and scutellum cells.

  6. A novel human gene spindlin1,encoding a protein localized in the cell nucleus and inducing NIH3T3 cell's transformation

    Institute of Scientific and Technical Information of China (English)

    GAO Yanhong; QIN Lipeng; ZHANG Peng; CHEN Lin; YUAN Hongfeng; BAI Cixian; YAN Fang; YUE Wen; PEI Xuetao

    2004-01-01

    A novel human gene, spindlin1, recently cloned in our laboratory, is highly expressed in the tissue of ovary cancer. To study its biological function, a vector expressing green fluorescent-spindlin1 fusion protein was constructed and transfected into COS-7 and NIH3T3 cells by lipofectamine methods. The results showed that the fusion protein pEGFP-N1-spindlin1 was localized in the nucleus of COS-7 and NIH3T3 cells. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the parental NIH3T3 cells displayed a complete morphological change, improved the cell growth and increased the percentage of cells in G2/M phase (12.6% vs control cells at 3.4%). Furthermore, overexpressed spindlin1 cells formed colonies in soft agar, more motile in migration assay in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may be a prooncogene which is associated with tumorigenesis.

  7. p63cdc13, a B-type cyclin, is associated with both the nucleolar and chromatin domains of the fission yeast nucleus.

    Science.gov (United States)

    Gallagher, I M; Alfa, C E; Hyams, J S

    1993-11-01

    The cellular distribution of the fission yeast mitotic cyclin B, p63cdc13, was investigated by a combination of indirect immunofluorescence light microscopy, immunogold electron microscopy, and nuclear isolation and fractionation. Immunofluorescence microscopy of wild-type cells and the cold-sensitive mutant dis2.11 with a monospecific anti-p63cdc13 antiserum was consistent with the association of a major subpopulation of fission yeast M-phase protein kinase with the nucleolus. Immunogold electron microscopy of freeze-substituted wild-type cells identified two nuclear populations of p63cdc13, one associated with the nucleolus, the other with the chromatin domain. To investigate the cell cycle regulation of nuclear labeling, the mutant cdc25.22 was synchronized through mitosis by temperature arrest and release. Immunogold labeling of cells arrested at G2M revealed gold particles present abundantly over the nucleolus and less densely over the chromatin region of the nucleus. Small vesicles around the nucleus were also labeled by anti-p63cdc13, but few gold particles were detected over the cytoplasm. Labeling of all cell compartments declined to zero through mitosis. Cell fractionation confirmed that p63cdc13 was substantially enriched in both isolated nuclei and in a fraction containing small vesicles and organelles. p63cdc13 was not extracted from nuclei by treatment with RNase A, Nonidet P40 (NP-40), Triton X-100, and 0.1 M NaCl, although partial solubilization was observed with DNase I and 1 M NaCl. A known nucleolar protein NOP1, partitioned in a similar manner to p63cdc13, as did p34cdc2, the other subunit of the M-phase protein kinase. We conclude that a major subpopulation of the fission yeast mitotic cyclin B is targeted to structural elements of the nucleus and nucleolus.

  8. Cdk1/cyclin B-mediated phosphorylation stabilizes SREBP1 during mitosis.

    Science.gov (United States)

    Bengoechea-Alonso, Maria T; Ericsson, Johan

    2006-08-01

    Members of the sterol regulatory element-binding protein (SREBP) family of transcription factors control the biosynthesis of cholesterol and other lipids, and lipid synthesis is critical for cell growth and proliferation. We recently found that the mature forms of SREBP1a and SREBP1c are hyperphosphorylated in mitotic cells, giving rise to a phosphoepitope recognized by the mitotic protein monoclonal-2 (MPM-2) antibody. In addition, we found that mature SREBP1 was stabilized in a phosphorylation-dependent manner during mitosis. We have now mapped the major MPM-2 epitope to a serine residue, S439, in the C terminus of mature SREBP1. Using phosphorylation-specific antibodies, we demonstrate that endogenous SREBP1 is phosphorylated on S439 specifically during mitosis. Mature SREBP1 interacts with the Cdk1/cyclin B complex in mitotic cells and we demonstrate that Cdk1 phosphorylates S439, both in vitro and in vivo. Our results suggest that Cdk1-mediated phosphorylation of S439 stabilizes mature SREBP1 during mitosis, thereby preserving a critical pool of active transcription factors to support lipid synthesis. Taken together with our previous work, the current study suggests that SREBP1 may provide a link between lipid synthesis, proliferation and cell growth. This hypothesis was supported by our observation that siRNA-mediated inactivation of SREBP1 arrested cells in the G(1) phase of the cell cycle, thereby attenuating cell growth.

  9. Antagonistic effect of polyamines on ABA-induced suppression of mitosis in Allium cepa L.

    Science.gov (United States)

    Mahajan, Arpana; Sharma, Shashi

    2009-02-01

    Effect of abscisic acid (ABA) and polyamines (PAs) [putrescine (Put), spermidine (Spd) and spermine (Spm)] on mitosis in root tips of A. cepa was studied. Treatment with ABA (0.1 to 100 microM) for 24 hr suppressed the mitosis, measured as mitotic index (MI), in a concentration-dependent manner with approx. 50% suppression at 10 microM of ABA. Treatment with different PAs (1 to 100 microM) had differential mitosis suppression effect. Spm was most inhibitory followed by Spd and Put, respectively. The higher concentrations of PAs (1 mM Put; 0.1 and 1 mM Spd or Spm) caused cell distortion. Remarkably, a 24 hr pretreatment of root tips with PAs prior to ABA (100 microM) treatment resulted in a general concentration-dependent reversal of ABA-induced suppression of MI. Catalase (CAT) activity in the root tips, an indicator of redox metabolism, increased due to ABA treatment in a concentration-dependent manner, remained unaltered in response to Put and declined due to Spd and Spm (> or = 0.1 mM). However, all PAs, irrespective of their individual effects, generally antagonized the ABA-dependent increase in CAT activity. Data indicate the possibility of ABA-PA interaction in the regulation of mitosis.

  10. A highly efficient method for generation of therapeutic quality human pluripotent stem cells by using naive induced pluripotent stem cells nucleus for nuclear transfer

    Directory of Open Access Journals (Sweden)

    Madhusudana Girija Sanal

    2014-09-01

    Full Text Available Even after several years since the discovery of human embryonic stem cells and induced pluripotent stem cells (iPSC, we are still unable to make any significant therapeutic benefits out of them such as cell therapy or generation of organs for transplantation. Recent success in somatic cell nuclear transfer (SCNT made it possible to generate diploid embryonic stem cells, which opens up the way to make high-quality pluripotent stem cells. However, the process is highly inefficient and hence expensive compared to the generation of iPSC. Even with the latest SCNT technology, we are not sure whether one can make therapeutic quality pluripotent stem cell from any patient’s somatic cells or by using oocytes from any donor. Combining iPSC technology with SCNT, that is, by using the nucleus of the candidate somatic cell which got reprogrammed to pluripotent state instead that of the unmodified nucleus of the candidate somatic cell, would boost the efficiency of the technique, and we would be able to generate therapeutic quality pluripotent stem cells. Induced pluripotent stem cell nuclear transfer (iPSCNT combines the efficiency of iPSC generation with the speed and natural reprogramming environment of SCNT. The new technique may be called iPSCNT. This technique could prove to have very revolutionary benefits for humankind. This could be useful in generating organs for transplantation for patients and for reproductive cloning, especially for childless men and women who cannot have children by any other techniques. When combined with advanced gene editing techniques (such as CRISPR-Cas system this technique might also prove useful to those who want to have healthy children but suffer from inherited diseases. The current code of ethics may be against reproductive cloning. However, this will change with time as it happened with most of the revolutionary scientific breakthroughs. After all, it is the right of every human to have healthy offspring and it is

  11. Transcriptional intermediary factor 1γ binds to the anaphase-promoting complex/cyclosome and promotes mitosis

    DEFF Research Database (Denmark)

    Sedgwick, G.G.; Townsend, K.; Martin, A.

    2013-01-01

    The anaphase-promoting complex/cyclosome (APC/C) is an ubiquitin ligase that functions during mitosis. Here we identify the transcriptional regulator, transcriptional intermediary factor 1γ, TIF1γ, as an APC/C-interacting protein that regulates APC/C function. TIF1γ is not a substrate for APC....../C-dependent ubiquitylation but instead, associates specifically with the APC/C holoenzyme and Cdc20 to affect APC/C activity and progression through mitosis. RNA interference studies indicate that TIF1γ knockdown results in a specific reduction in APC/C ubiquitin ligase activity, the stabilization of APC/C substrates......, and an increase in the time taken for cells to progress through mitosis from nuclear envelope breakdown to anaphase. TIF1γ knockdown cells are also characterized by the inappropriate presence of cyclin A at metaphase, and an increase in the number of cells that fail to undergo metaphase-to-anaphase transition...

  12. Electric fields generated by synchronized oscillations of microtubules, centrosomes and chromosomes regulate the dynamics of mitosis and meiosis

    Directory of Open Access Journals (Sweden)

    Zhao Yue

    2012-07-01

    Full Text Available Abstract Super-macromolecular complexes play many important roles in eukaryotic cells. Classical structural biological studies focus on their complicated molecular structures, physical interactions and biochemical modifications. Recent advances concerning intracellular electric fields generated by cell organelles and super-macromolecular complexes shed new light on the mechanisms that govern the dynamics of mitosis and meiosis. In this review we synthesize this knowledge to provide an integrated theoretical model of these cellular events. We suggest that the electric fields generated by synchronized oscillation of microtubules, centrosomes, and chromatin fibers facilitate several events during mitosis and meiosis, including centrosome trafficking, chromosome congression in mitosis and synapsis between homologous chromosomes in meiosis. These intracellular electric fields are generated under energy excitation through the synchronized electric oscillations of the dipolar structures of microtubules, centrosomes and chromosomes, three of the super-macromolecular complexes within an animal cell.

  13. Electric fields generated by synchronized oscillations of microtubules, centrosomes and chromosomes regulate the dynamics of mitosis and meiosis.

    Science.gov (United States)

    Zhao, Yue; Zhan, Qimin

    2012-07-02

    Super-macromolecular complexes play many important roles in eukaryotic cells. Classical structural biological studies focus on their complicated molecular structures, physical interactions and biochemical modifications. Recent advances concerning intracellular electric fields generated by cell organelles and super-macromolecular complexes shed new light on the mechanisms that govern the dynamics of mitosis and meiosis. In this review we synthesize this knowledge to provide an integrated theoretical model of these cellular events. We suggest that the electric fields generated by synchronized oscillation of microtubules, centrosomes, and chromatin fibers facilitate several events during mitosis and meiosis, including centrosome trafficking, chromosome congression in mitosis and synapsis between homologous chromosomes in meiosis. These intracellular electric fields are generated under energy excitation through the synchronized electric oscillations of the dipolar structures of microtubules, centrosomes and chromosomes, three of the super-macromolecular complexes within an animal cell.

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

    Science.gov (United States)

    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.

  15. Polo-like kinase 1 inhibits DNA damage response during mitosis.

    Science.gov (United States)

    Benada, Jan; Burdová, Kamila; Lidak, Tomáš; von Morgen, Patrick; Macurek, Libor

    2015-01-01

    In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.

  16. Subcellular localization of the APOBEC3 proteins during mitosis and implications for genomic DNA deamination.

    Science.gov (United States)

    Lackey, Lela; Law, Emily K; Brown, William L; Harris, Reuben S

    2013-03-01

    Humans have seven APOBEC3 DNA cytosine deaminases. The activity of these enzymes allows them to restrict a variety of retroviruses and retrotransposons, but may also cause pro-mutagenic genomic uracil lesions. During interphase the APOBEC3 proteins have different subcellular localizations: cell-wide, cytoplasmic or nuclear. This implies that only a subset of APOBEC3s have contact with nuclear DNA. However, during mitosis, the nuclear envelope breaks down and cytoplasmic proteins may enter what was formerly a privileged zone. To address the hypothesis that all APOBEC3 proteins have access to genomic DNA, we analyzed the localization of the APOBEC3 proteins during mitosis. We show that APOBEC3A, APOBEC3C and APOBEC3H are excluded from condensed chromosomes, but become cell-wide during telophase. However, APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3G are excluded from chromatin throughout mitosis. After mitosis, APOBEC3B becomes nuclear, and APOBEC3D, APOBEC3F and APOBEC3G become cytoplasmic. Both structural motifs as well as size may be factors in regulating chromatin exclusion. Deaminase activity was not dependent on cell cycle phase. We also analyzed APOBEC3-induced cell cycle perturbations as a measure of each enzyme's capacity to inflict genomic DNA damage. AID, APOBEC3A and APOBEC3B altered the cell cycle profile, and, unexpectedly, APOBEC3D also caused changes. We conclude that several APOBEC3 family members have access to the nuclear compartment and can impede the cell cycle, most likely through DNA deamination and the ensuing DNA damage response. Such genomic damage may contribute to carcinogenesis, as demonstrated by AID in B cell cancers and, recently, APOBEC3B in breast cancers.

  17. Automated morphological analysis of bone marrow cells in microscopic images for diagnosis of leukemia: nucleus-plasma separation and cell classification using a hierarchical tree model of hematopoesis

    Science.gov (United States)

    Krappe, Sebastian; Wittenberg, Thomas; Haferlach, Torsten; Münzenmayer, Christian

    2016-03-01

    The morphological differentiation of bone marrow is fundamental for the diagnosis of leukemia. Currently, the counting and classification of the different types of bone marrow cells is done manually under the use of bright field microscopy. This is a time-consuming, subjective, tedious and error-prone process. Furthermore, repeated examinations of a slide may yield intra- and inter-observer variances. For that reason a computer assisted diagnosis system for bone marrow differentiation is pursued. In this work we focus (a) on a new method for the separation of nucleus and plasma parts and (b) on a knowledge-based hierarchical tree classifier for the differentiation of bone marrow cells in 16 different classes. Classification trees are easily interpretable and understandable and provide a classification together with an explanation. Using classification trees, expert knowledge (i.e. knowledge about similar classes and cell lines in the tree model of hematopoiesis) is integrated in the structure of the tree. The proposed segmentation method is evaluated with more than 10,000 manually segmented cells. For the evaluation of the proposed hierarchical classifier more than 140,000 automatically segmented bone marrow cells are used. Future automated solutions for the morphological analysis of bone marrow smears could potentially apply such an approach for the pre-classification of bone marrow cells and thereby shortening the examination time.

  18. Cultivation and identification of human cervical nucleus pulposus cells%人颈椎间盘髓核细胞的体外培养和鉴定

    Institute of Scientific and Technical Information of China (English)

    杨勇; 梁伟; 张世磊; 陶慧人; 罗卓荆

    2011-01-01

    Objective: To establish the system for culturing the human cervical nucleus pulposus cells and to identify the phenotype. Methods The human cervical nucleus pulposus cells were isolated by collagenase digestion method and cultured in monolayer by culture solutions of DF12+20%FBS. Morphologic changes and growth of the cells were detected by microscope. The cell cycle and apoptosis rate were detected by Flow cytometric. The morphological structure and the phenotype were identified with the toluidine blue staining and immunocytochemistry means. Result The apoptosis rate of prirnary cells was 6.1± 1.4%. The Cells at S phase were 7.3±0.5%. The cells were polygonal or short wedged morphology. The second passage cells grew more fast than that of the primary cells. The cells displayed tense toluidine blue metachromasia. The cells expressed collagen type Ⅱ, but only a few elliptic gaint cells expressed CK8. Conclusion The human cervical nucleus pulposus cells were isolated and cultured in monolayer successfully. A few cells of the nucleus pulposus still maintain notochord cells phenotype in adult specimens.%目的:建立人颈椎间盘髓核细胞体外培养体系,并对其细胞表型进行鉴定.方法:采用酶消化法分离人颈椎间盘髓核细胞,进行单层培养,倒置相差显微镜观察细胞生长和形态,流式细胞仪测定细胞周期和凋亡率,并行甲苯胺蓝、Ⅱ型胶原及CK8免疫组化染色对其细胞表型进行鉴定.结果:原代髓核细胞凋亡率6.1±1.4%,S期细胞比例7.3±0.5%.贴壁后形态为多角形或短楔形,传代后生长加速.细胞呈甲苯胺蓝异染性;Ⅱ型胶原免疫组化染色阳性;只有少量椭圆形大细胞CK8免疫组化染色阳性.结论:成功建立人颈椎间盘髓核细胞体外培养模型,并证实成年后髓核内仍有少量细胞保持脊索细胞表型.

  19. Zebrafish Noxa promotes mitosis in early embryonic development and regulates apoptosis in subsequent embryogenesis.

    Science.gov (United States)

    Zhong, J-X; Zhou, L; Li, Z; Wang, Y; Gui, J-F

    2014-06-01

    Noxa functions in apoptosis and immune system of vertebrates, but its activities in embryo development remain unclear. In this study, we have studied the role of zebrafish Noxa (zNoxa) by using zNoxa-specifc morpholino knockdown and overexpression approaches in developing zebrafish embryos. Expression pattern analysis indicates that zNoxa transcript is of maternal origin, which displays a uniform distribution in early embryonic development until shield stage, and the zygote zNoxa transcription is initiated from this stage and mainly localized in YSL of the embryos. The zNoxa expression alterations result in strong embryonic development defects, demonstrating that zNoxa regulates apoptosis from 75% epiboly stage of development onward, in which zNoxa firstly induces the expression of zBik, and then cooperates with zBik to regulate apoptosis. Moreover, zNoxa knockdown also causes a reduction in number of mitotic cells before 8 h.p.f., suggesting that zNoxa also promotes mitosis before 75% epiboly stage. The effect of zNoxa on mitosis is mediated by zWnt4b in early embryos, whereas zMcl1a and zMcl1b suppress the ability of zNoxa to regulate mitosis and apoptosis at different developmental stages. In addition, mammalian mouse Noxa (mNoxa) mRNA was demonstrated to rescue the arrest of mitosis when zNoxa was knocked down, suggesting that mouse and zebrafish Noxa might have similar dual functions. Therefore, the current findings indicate that Noxa is a novel regulator of early mitosis before 75% epiboly stage when it translates into a key mediator of apoptosis in subsequent embryogenesis.

  20. Cell biological mechanisms of activity-dependent synapse to nucleus translocation of CRTC1 in neurons

    Science.gov (United States)

    Ch'ng, Toh Hean; DeSalvo, Martina; Lin, Peter; Vashisht, Ajay; Wohlschlegel, James A.; Martin, Kelsey C.

    2015-01-01

    Previous studies have revealed a critical role for CREB-regulated transcriptional coactivator (CRTC1) in regulating neuronal gene expression during learning and memory. CRTC1 localizes to synapses but undergoes activity-dependent nuclear translocation to regulate the transcription of CREB target genes. Here we investigate the long-distance retrograde transport of CRTC1 in hippocampal neurons. We show that local elevations in calcium, triggered by activation of glutamate receptors and L-type voltage-gated calcium channels, initiate active, dynein-mediated retrograde transport of CRTC1 along microtubules. We identify a nuclear localization signal within CRTC1, and characterize three conserved serine residues whose dephosphorylation is required for nuclear import. Domain analysis reveals that the amino-terminal third of CRTC1 contains all of the signals required for regulated nucleocytoplasmic trafficking. We fuse this region to Dendra2 to generate a reporter construct and perform live-cell imaging coupled with local uncaging of glutamate and photoconversion to characterize the dynamics of stimulus-induced retrograde transport and nuclear accumulation. PMID:26388727

  1. Cell Biological Mechanisms of Activity-Dependent Synapse to Nucleus Translocation of CRTC1 in Neurons

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    Toh Hean eCh'ng

    2015-09-01

    Full Text Available Previous studies have revealed a critical role for CREB-regulated transcriptional coactivator (CRTC1 in regulating neuronal gene expression during learning and memory. CRTC1 localizes to synapses but undergoes activity-dependent nuclear translocation to regulate the transcription of CREB target genes. Here we investigate the long-distance retrograde transport of CRTC1 in hippocampal neurons. We show that local elevations in calcium, triggered by activation of synaptic glutamate receptors and L-type voltage-gated calcium channels, initiate active, dynein-mediated retrograde transport of CRTC1 along microtubules. We identify a nuclear localization signal within CRTC1, and characterize three conserved serine residues whose dephosphorylation is required for nuclear import. Domain analysis reveals that the amino-terminal third of CRTC1 contains all of the signals required for regulated nucleocytoplasmic trafficking. We fuse this region to Dendra2 to generate a reporter construct and perform live-cell imaging coupled with local uncaging of glutamate and photoconversion to characterize the dynamics of stimulus-induced retrograde transport and nuclear accumulation.

  2. Adenovirus replication and transcription sites are spatially separated in the nucleus of infected cells.

    Science.gov (United States)

    Pombo, A; Ferreira, J; Bridge, E; Carmo-Fonseca, M

    1994-11-01

    We have visualized the intranuclear topography of adenovirus replication and transcription in infected HeLa cells. The results show that viral DNA replication occurs in multiple foci that are highly organized in the nucleoplasm. Pulse-chase experiments indicate that newly synthesized viral double-stranded DNA molecules are displaced from the replication foci and spread throughout the nucleoplasm, while the single-stranded DNA replication intermediates accumulate in adjacent sites. Double-labelling experiments and confocal microscopy show that replication occurs in foci localized at the periphery of the sites where single-stranded DNA accumulates. The simultaneous visualization of viral replication and transcription reveals that the sites of transcription are predominantly separated from the sites of replication. Transcription is detected adjacent to the replication foci and extends around the sites of single-stranded DNA accumulation. These data indicate that newly synthesized double-stranded DNA molecules are displaced from the replication foci and spread in the surrounding nucleoplasm, where they are used as templates for transcription. Splicing snRNPs are shown to co-localize with the sites of transcription and to be excluded from the sites of replication. This provides evidence that splicing of viral RNAs occurs co-transcriptionally and that the sites of viral DNA replication are spatially distinct from the sites of RNA transcription and processing.

  3. Vegetative Cell Division and Nuclear Translocation in Three Algae Species of Netrium (Zygnematales, Chlorophyta

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    DIAN HENDRAYANTI

    2006-03-01

    Full Text Available Three species of Netrium oblongum, N. digitus v. latum, and N. interruptum were studied for their mode in the vegetative cell division and nuclear translocation during mitosis using light and fluorescence microscopy. The process of cell division in the three species began with the prominent constriction at the chloroplast in both semicells about half way from the apex. The constriction of chloroplast was mostly visible in N. digitus v. latum. Soon after nucleus divided, septum was formed across the cell and cytokinesis occurred. Observation with fluorescence microscope showed that the movement of nucleus moved back into the center of daughter cells was not always synchronous. Division of chloroplast in N. oblongum and N. digitus v. latum were different with that of N. interruptum. Chloroplast division in two former species occured following the movement of the nucleus down semicell. However, in N. interruptum, chloroplast divided later after nucleus occupied the position at the center of the daughter cells. Cell restoration started after the completion of mitosis and cytokinesis.

  4. Contributions of the axostyle and flagella to closed mitosis in the protists Tritrichomonas foetus and Trichomonas vaginalis.

    Science.gov (United States)

    Ribeiro, K C; Monteiro-Leal, L H; Benchimol, M

    2000-01-01

    Tritrichomonas foetus and Trichomonas vaginalis are protists that undergo closed mitosis: the nuclear envelope remains intact and the spindle remains extranuclear. Here we show, in disagreement with previous studies, that the axostyle does not disappear during mitosis but rather actively participates in it. We document the main structural modifications of the cell during its cell cycle using video enhanced microscopy and computer animation, bright field light microscopy, confocal laser scanning microscopy, and scanning and transmission electron microscopy. We propose six phases in the trichomonad's cell cycle: an orthodox interphase, a pre-mitotic phase, and four stages during the cell division process. We report that in T. foetus and T. vaginalis: a) all skeletal structures such as the costa, pelta-axostyle system, basal bodies, flagella, and associated filaments of the mastigont system are duplicated in a pre-mitotic phase; b) the axostyle does not disappear during mitosis, otherwise playing a fundamental role in this process; c) axostyles participate in the changes in the cell shape, contortion of the anterior region of the cell, and karyokinesis; d) flagella are not under assembly during mitosis, as previously stated by others, but completely formed before it; and e) cytokinesis is powered in part by cell locomotion.

  5. Immunosuppressive Yersinia Effector YopM Binds DEAD Box Helicase DDX3 to Control Ribosomal S6 Kinase in the Nucleus of Host Cells.

    Science.gov (United States)

    Berneking, Laura; Schnapp, Marie; Rumm, Andreas; Trasak, Claudia; Ruckdeschel, Klaus; Alawi, Malik; Grundhoff, Adam; Kikhney, Alexey G; Koch-Nolte, Friedrich; Buck, Friedrich; Perbandt, Markus; Betzel, Christian; Svergun, Dmitri I; Hentschke, Moritz; Aepfelbacher, Martin

    2016-06-01

    Yersinia outer protein M (YopM) is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3) as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1) increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1) in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10) mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines.

  6. Immunosuppressive Yersinia Effector YopM Binds DEAD Box Helicase DDX3 to Control Ribosomal S6 Kinase in the Nucleus of Host Cells.

    Directory of Open Access Journals (Sweden)

    Laura Berneking

    2016-06-01

    Full Text Available Yersinia outer protein M (YopM is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3 as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1 increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1 in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10 mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines.

  7. A gibberellin-induced nuclease is localized in the nucleus of wheat aleurone cells undergoing programmed cell death

    OpenAIRE

    Domínguez, Fernándo; Moreno Onorato, Francisco Javier; Cejudo Fernández, Francisco Javier

    2003-01-01

    The aleurone layer of cereal grains undergoes a gibberellin-regulated process of programmed cell death (PCD) following germination. We have applied a combination of ultrastructural and biochemical approaches to analyze aleurone PCD in intact wheat grains. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay revealed that PCD was initiated in aleurone cells proximal to the embryo and then extended to distal cells. DNA fragmentation and terminal deoxynucleotidyl trans...

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

    Science.gov (United States)

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

    2015-04-01

    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.

  9. The acetyllysine reader BRD3R promotes human nuclear reprogramming and regulates mitosis.

    Science.gov (United States)

    Shao, Zhicheng; Zhang, Ruowen; Khodadadi-Jamayran, Alireza; Chen, Bo; Crowley, Michael R; Festok, Muhamad A; Crossman, David K; Townes, Tim M; Hu, Kejin

    2016-01-01

    It is well known that both recipient cells and donor nuclei demonstrate a mitotic advantage as observed in the traditional reprogramming with somatic cell nuclear transfer (SCNT). However, it is not known whether a specific mitotic factor plays a critical role in reprogramming. Here we identify an isoform of human bromodomain-containing 3 (BRD3), BRD3R (BRD3 with Reprogramming activity), as a reprogramming factor. BRD3R positively regulates mitosis during reprogramming, upregulates a large set of mitotic genes at early stages of reprogramming, and associates with mitotic chromatin. Interestingly, a set of the mitotic genes upregulated by BRD3R constitutes a pluripotent molecular signature. The two BRD3 isoforms display differential binding to acetylated histones. Our results suggest a molecular interpretation for the mitotic advantage in reprogramming and show that mitosis may be a driving force of reprogramming.

  10. Cytological evidence for assortment mitosis leading to loss of heterozygosity in rice.

    Science.gov (United States)

    Wang, Richard R-C; Li, Xiaomei; Chatterton, N Jerry

    2006-05-01

    In the root meristem cells of the rice line AMR, which causes loss of heterozygosity in its hybrids, both normal and assortment mitoses were observed. During normal mitosis, chromosomes did not form homologous pairs at metaphase; all chromosomes lined up at the equatorial plate and 2 chromatids of each chromosome disjoined at the centromere and moved toward opposite poles. During assortment mitosis, varying numbers of paired homologues were observed at mitotic metaphase. Two groups of 12 chromosomes separated and moved towards the opposite poles of daughter cells with few chromosomes having their chromatids separated at anaphase. These observations support the proposed mechanism that is responsible for early genotype fixation in rice hybrids involving AMR.

  11. pkl1(+)and klp2(+): Two kinesins of the Kar3 subfamily in fission yeast perform different functions in both mitosis and meiosis.

    Science.gov (United States)

    Troxell, C L; Sweezy, M A; West, R R; Reed, K D; Carson, B D; Pidoux, A L; Cande, W Z; McIntosh, J R

    2001-11-01

    We have identified Klp2p, a new kinesin-like protein (KLP) of the KAR3 subfamily in fission yeast. The motor domain of this protein is 61% identical and 71% similar to Pkl1p, another fission yeast KAR3 protein, yet the two enzymes are different in behavior and function. Pkl1p is nuclear throughout the cell cycle, whereas Klp2p is cytoplasmic during interphase. During mitosis Klp2p enters the nucleus where it forms about six chromatin-associated dots. In metaphase-arrested cells these migrate back and forth across the nucleus. During early anaphase they segregate with the chromosomes into two sets of about three, fade, and are replaced by other dots that form on the spindle interzone. Neither klp2(+) nor pkl1(+) is essential, and the double deletion is also wild type for both vegetative and sexual reproduction. Each deletion rescues different alleles of cut7(ts), a KLP that contributes to spindle formation and elongation. When either or both deletions are combined with a dynein deletion, vegetative growth is normal, but sexual reproduction fails: klp2 Delta,dhc1-d1 in karyogamy, pkl1 Delta,dhc1-d1 in multiple phases of meiosis, and the triple deletion in both. Deletion of Klp2p elongates a metaphase-arrested spindle, but pkl1 Delta shortens it. The anaphase spindle of klp2 Delta becomes longer than the cell, leading it to curl around the cell's ends. Apparently, Klp2p promotes spindle disassembly and contributes to the behavior of mitotic chromosomes.

  12. Construction of a tissue engineered intervertebral disc with high biological activity using an allogeneic intervertebral disc supplemented with transfected nucleus pulposus cells expressing exogenous dopamine beta-hydroxylase.

    Science.gov (United States)

    Bai, M; Wang, Y H; Yin, H P; Li, S W

    2015-09-09

    This study addressed the in vitro construction and biological activity of tissue engineered intervertebral discs with exogenous human dopamine beta-hydroxylase (DBH) nucleus pulposus cells. pSNAV2.0-DBH expression plasmids were utilized to enhance the survival rates of intervertebral disc tissue cells. Various concentrations of transfected nucleus pulposus cells were injected into the discs, and DBH mRNA expression was determined using polymerase chain reaction amplification. Polysaccharide content and total collagen protein content in the engineered disc nucleus pulposus tissue were determined. The visible fluorescence intensities of the 1 x 10(5) and 1 x 10(6) groups vs the 1 x 10(4) group were significantly increased (P 0.05) at 7 days after injection. DBH mRNA expression could be detected in the all but the EGFP control group at 14 days culture. No significant difference was observed in the protein content between the 1 x 10(4) and the control groups at various times, while the protein content was significantly higher in the 1 x 10(5) vs the control and the 1 x 10(4) groups at 7-, 14-, and 21-day cultures. These results demonstrate that a tissue engineered intervertebral disc with high biological activity can be constructed by utilizing allogeneic intervertebral discs stored in liquid nitrogen and a 1 x 10(5) transfected nucleus pulposus cell complex with in vitro culture for 14 days. This model can be used in animal experiments to study the biological activity of the engineered discs.

  13. Replication stress activates DNA repair synthesis in mitosis.

    Science.gov (United States)

    Minocherhomji, Sheroy; Ying, Songmin; Bjerregaard, Victoria A; Bursomanno, Sara; Aleliunaite, Aiste; Wu, Wei; Mankouri, Hocine W; Shen, Huahao; Liu, Ying; Hickson, Ian D

    2015-12-10

    Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis. Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs). CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS 'expression'), particularly when cells have been exposed to replicative stress. The MUS81-EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress. Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach.

  14. Neurons of human nucleus accumbens

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    Sazdanović Maja

    2011-01-01

    Full Text Available Background/Aim. Nucleus accumbens is a part of the ventral striatum also known as a drug active brain region, especially related with drug addiction. The aim of the study was to investigate the Golgi morphology of the nucleus accumbens neurons. Methods. The study was performed on the frontal and sagittal sections of 15 human brains by the Golgi Kopsch method. We classified neurons in the human nucleus accumbens according to their morphology and size into four types: type I - fusiform neurons; type II - fusiform neurons with lateral dendrite, arising from a part of the cell body; type III - pyramidal-like neuron; type IV - multipolar neuron. The medium spiny neurons, which are mostly noted regarding to the drug addictive conditions of the brain, correspond to the type IV - multipolar neurons. Results. Two regions of human nucleus accumbens could be clearly recognized on Nissl and Golgi preparations each containing different predominant neuronal types. Central part of nucleus accumbens, core region, has a low density of impregnated neurons with predominant type III, pyramidal-like neurons, with spines on secondary branches and rare type IV, multipolar neurons. Contrary to the core, peripheral region, shell of nucleus, has a high density of impregnated neurons predominantly contained of type I and type IV - multipolar neurons, which all are rich in spines on secondary and tertiary dendritic branches. Conclusion. Our results indicate great morphological variability of human nucleus accumbens neurons. This requires further investigations and clarifying clinical significance of this important brain region.

  15. Hyperphosphorylation regulates the activity of SREBP1 during mitosis.

    Science.gov (United States)

    Bengoechea-Alonso, Maria T; Punga, Tanel; Ericsson, Johan

    2005-08-16

    The sterol regulatory element-binding protein (SREBP) family of transcription factors controls the biosynthesis of cholesterol and other lipids, and lipid synthesis is critical for cell growth and proliferation. We were, therefore, interested in the expression and activity of SREBPs during the cell cycle. We found that the expression of a number of SREBP-responsive promoter-reporter genes were induced in a SREBP-dependent manner in cells arrested in G2/M. In addition, the mature forms of SREBP1a and SREBP1c were hyperphosphorylated in mitotic cells, giving rise to a phosphoepitope recognized by the mitotic protein monoclonal-2 (MPM-2) antibody. In contrast, SREBP2 was not hyperphosphorylated in mitotic cells and was not recognized by the MPM-2 antibody. The MPM-2 epitope was mapped to the C terminus of mature SREBP1, and the mitosis-specific hyperphosphorylation of SREBP1 depended on this domain of the protein. The transcriptional and DNA-binding activity of SREBP1 was enhanced in cells arrested in G2/M, and these effects depended on the C-terminal domain of the protein. In part, these effects could be explained by our observation that mature SREBP1 was stabilized in G2/M. In agreement with these observations, we found that the synthesis of cholesterol was increased in G2/M-arrested cells. Thus, our results demonstrate that the activity of mature SREBP1 is regulated by phosphorylation during the cell cycle, suggesting that SREBP1 may provide a link between lipid synthesis, proliferation, and cell growth.

  16. DNA replication and spindle checkpoints cooperate during S phase to delay mitosis and preserve genome integrity.

    Science.gov (United States)

    Magiera, Maria M; Gueydon, Elisabeth; Schwob, Etienne

    2014-01-20

    Deoxyribonucleic acid (DNA) replication and chromosome segregation must occur in ordered sequence to maintain genome integrity during cell proliferation. Checkpoint mechanisms delay mitosis when DNA is damaged or upon replication stress, but little is known on the coupling of S and M phases in unperturbed conditions. To address this issue, we postponed replication onset in budding yeast so that DNA synthesis is still underway when cells should enter mitosis. This delayed mitotic entry and progression by transient activation of the S phase, G2/M, and spindle assembly checkpoints. Disabling both Mec1/ATR- and Mad2-dependent controls caused lethality in cells with deferred S phase, accompanied by Rad52 foci and chromosome missegregation. Thus, in contrast to acute replication stress that triggers a sustained Mec1/ATR response, multiple pathways cooperate to restrain mitosis transiently when replication forks progress unhindered. We suggest that these surveillance mechanisms arose when both S and M phases were coincidently set into motion by a unique ancestral cyclin-Cdk1 complex.

  17. A conserved role for kinesin-5 in plant mitosis.

    Science.gov (United States)

    Bannigan, Alex; Scheible, Wolf-Rüdiger; Lukowitz, Wolfgang; Fagerstrom, Carey; Wadsworth, Patricia; Somerville, Chris; Baskin, Tobias I

    2007-08-15

    The mitotic spindle of vascular plants is assembled and maintained by processes that remain poorly explored at a molecular level. Here, we report that AtKRP125c, one of four kinesin-5 motor proteins in arabidopsis, decorates microtubules throughout the cell cycle and appears to function in both interphase and mitosis. In a temperature-sensitive mutant, interphase cortical microtubules are disorganized at the restrictive temperature and mitotic spindles are massively disrupted, consistent with a defect in the stabilization of anti-parallel microtubules in the spindle midzone, as previously described in kinesin-5 mutants from animals and yeast. AtKRP125c introduced into mammalian epithelial cells by transfection decorates microtubules throughout the cell cycle but is unable to complement the loss of the endogenous kinesin-5 motor (Eg5). These results are among the first reports of any motor with a major role in anastral spindle structure in plants and demonstrate that the conservation of kinesin-5 motor function throughout eukaryotes extends to vascular plants.

  18. Polo kinase regulates the localization and activity of the chromosomal passenger complex in meiosis and mitosis in Drosophila melanogaster.

    Science.gov (United States)

    Carmena, Mar; Lombardia, Miguel Ortiz; Ogawa, Hiromi; Earnshaw, William C

    2014-11-01

    Cell cycle progression is regulated by members of the cyclin-dependent kinase (CDK), Polo and Aurora families of protein kinases. The levels of expression and localization of the key regulatory kinases are themselves subject to very tight control. There is increasing evidence that crosstalk between the mitotic kinases provides for an additional level of regulation. We have previously shown that Aurora B activates Polo kinase at the centromere in mitosis, and that the interaction between Polo and the chromosomal passenger complex (CPC) component INCENP is essential in this activation. In this report, we show that Polo kinase is required for the correct localization and activity of the CPC in meiosis and mitosis. Study of the phenotype of different polo allele combinations compared to the effect of chemical inhibition revealed significant differences in the localization and activity of the CPC in diploid tissues. Our results shed new light on the mechanisms that control the activity of Aurora B in meiosis and mitosis.

  19. Phosphorylation of the centrosomal protein, Cep169, by Cdk1 promotes its dissociation from centrosomes in mitosis.

    Science.gov (United States)

    Mori, Yusuke; Inoue, Yoko; Taniyama, Yuki; Tanaka, Sayori; Terada, Yasuhiko

    2015-12-25

    Cep169 is a centrosomal protein conserved among vertebrates. In our previous reports, we showed that mammalian Cep169 interacts and collaborates with CDK5RAP2 to regulate microtubule (MT) dynamics and stabilization. Although Cep169 is required for MT regulation, its precise cellular function remains largely elusive. Here we show that Cep169 associates with centrosomes during interphase, but dissociates from these structures from the onset of mitosis, although CDK5RAP2 (Cep215) is continuously located at the centrosomes throughout cell cycle. Interestingly, treatment with purvalanol A, a Cdk1 inhibitor, nearly completely blocked the dissociation of Cep169 from centrosomes during mitosis. In addition, mass spectrometry analyses identified 7 phosphorylated residues of Cep169 corresponding to consensus phosphorylation sequence for Cdk1. These data suggest that the dissociation of Cep169 from centrosomes is controlled by Cdk1/Cyclin B during mitosis, and that Cep169 might regulate MT dynamics of mitotic spindle.

  20. Body sodium overload modulates the firing rate and fos immunoreactivity of serotonergic cells of dorsal raphe nucleus.

    Directory of Open Access Journals (Sweden)

    Andrea Godino

    Full Text Available In order to determine whether serotonergic (5HT dorsal raphe nucleus (DRN cells are involved in body sodium status regulation, the effect of a s.c. infusion of either 2 M or 0.15 M NaCl on 5HT DRN neuron firing was studied using single unit extracellular recordings. In separate groups of 2 M and 0.15 M NaCl-infused rats, water intake, oxytocin (OT plasma concentration, urine and plasma sodium and protein concentrations were also measured. Also, to determine the involvement of particular brain nuclei and neurochemical systems in body sodium overload (SO, animals from both groups were perfused for brain immunohistochemical detection of Fos, Fos-OT and Fos-5HT expression. SO produced a significant increase in serotonergic DRN neuron firing rate compared to baseline and 0.15 M NaCl-infused rats. As expected, 2 M NaCl s.c. infusion also induced a significant increase of water intake, diuresis and natriuresis, plasma sodium concentration and osmolality, even though plasma volume did not increase as indicated by changes in plasma protein concentration. The distribution of neurons along the forebrain and brainstem expressing Fos after SO showed the participation of the lamina terminalis, extended amygdala, supraoptic and paraventricular hypothalamic nuclei in the neural network that controls osmoregulatory responses. Both Fos-OT immunoreactive and plasma OT concentration increased after s.c. hypertonic sodium infusion. Finally, matching the "in vivo" electrophysiological study, SO doubled the number of Fos-5HT immunolabeled cells within the DRN. In summary, the results characterize the behavioral, renal and endocrine responses after body sodium overload without volume expansion and specify the cerebral nuclei that participate at different CNS levels in the control of these responses. The electrophysiological approach also allows us to determine in an "in vivo" model that DRN 5HT neurons increase their firing frequency during an increase in systemic

  1. Accumulation of cytoplasmic calcium, but not apamin-sensitive afterhyperpolarization current, during high frequency firing in rat subthalamic nucleus cells.

    Science.gov (United States)

    Teagarden, Mark; Atherton, Jeremy F; Bevan, Mark D; Wilson, Charles J

    2008-02-01

    The autonomous firing pattern of neurons in the rat subthalamic nucleus (STN) is shaped by action potential afterhyperpolarization currents. One of these is an apamin-sensitive calcium-dependent potassium current (SK). The duration of SK current is usually considered to be limited by the clearance of calcium from the vicinity of the channel. When the cell is driven to fire faster, calcium is expected to accumulate, and this is expected to result in accumulation of calcium-dependent AHP current. We measured the time course of calcium transients in the soma and proximal dendrites of STN neurons during spontaneous firing and their accumulation during driven firing. We compared these to the time course and accumulation of AHP currents using whole-cell and perforated patch recordings. During spontaneous firing, a rise in free cytoplasmic calcium was seen after each action potential, and decayed with a time constant of about 200 ms in the soma, and 80 ms in the dendrites. At rates higher than 10 Hz, calcium transients accumulated as predicted. In addition, there was a slow calcium transient not predicted by summation of action potentials that became more pronounced at high firing frequency. Spike AHP currents were measured in voltage clamp as tail currents after 2 ms voltage pulses that triggered action currents. Apamin-sensitive AHP (SK) current was measured by subtraction of tail currents obtained before and after treatment with apamin. SK current peaked between 10 and 15 ms after an action potential, had a decay time constant of about 30 ms, and showed no accumulation. At frequencies between 5 and 200 spikes s(-1), the maximal SK current remained the same as that evoked by a single action potential. AHP current did not have time to decay between action potentials, so at frequencies above 50 spikes s(-1) the apamin-sensitive current was effectively constant. These results are inconsistent with the view that the decay of SK current is governed by calcium dynamics. They

  2. Low-concentration uranium enters the HepG2 cell nucleus rapidly and induces cell stress response.

    Science.gov (United States)

    Guéguen, Yann; Suhard, David; Poisson, Clémentine; Manens, Line; Elie, Christelle; Landon, Géraldine; Bouvier-Capely, Céline; Rouas, Caroline; Benderitter, Marc; Tessier, Christine

    2015-12-25

    This study aimed to compare the cell stress effects of low and high uranium concentrations and relate them to its localization, precipitate formation, and exposure time. The time-course analysis shows that uranium appears in cell nuclei as a soluble form within 5 min of exposure, and quickly induces expression of antioxidant and DNA repair genes. On the other hand, precipitate formations began at the very beginning of exposure at the 300-μM concentration, but took longer to appear at lower concentrations. Adaptive response might occur at low concentrations but are overwhelmed at high concentrations, especially when uranium precipitates are abundant.

  3. Mitosis-meiosis and sperm-oocyte fate decisions are separable regulatory events.

    Science.gov (United States)

    Morgan, Clinton T; Noble, Daniel; Kimble, Judith

    2013-02-26

    Germ cell fate decisions are poorly understood, despite their central role in reproduction. One fundamental question has been whether germ cells are regulated to enter the meiotic cell cycle (i.e., mitosis-meiosis decision) and to be sperm or oocyte (i.e., sperm-oocyte decision) through one or two cell fate choices. If a single decision is used, a male-specific or female-specific meiotic entry would lead necessarily toward spermatogenesis or oogenesis, respectively. If two distinct decisions are used, meiotic entry should be separable from specification as sperm or oocyte. Here, we investigate the relationship of these two decisions with tools uniquely available in the nematode Caenorhabditis elegans. Specifically, we used a temperature-sensitive Notch allele to drive germ-line stem cells into the meiotic cell cycle, followed by chemical inhibition of the Ras/ERK pathway to reprogram the sperm-oocyte decision. We found that germ cells already in meiotic prophase can nonetheless be sexually transformed from a spermatogenic to an oogenic fate. This finding cleanly uncouples the mitosis-meiosis decision from the sperm-oocyte decision. In addition, we show that chemical reprogramming occurs in a germ-line region where germ cells normally transition from the mitotic to the meiotic cell cycle and that it dramatically changes the abundance of key sperm-oocyte fate regulators in meiotic germ cells. We conclude that the C. elegans mitosis-meiosis and sperm-oocyte decisions are separable regulatory events and suggest that this fundamental conclusion will hold true for germ cells throughout the animal kingdom.

  4. Interaction with DNA and different effect on the nucleus of cancer cells for copper(II) complexes of N-benzyl di(pyridylmethyl)amine.

    Science.gov (United States)

    Chen, Qiu-Yun; Fu, Hai-Jian; Zhu, Wei-Hua; Qi, Yan; Ma, Zheng-Ping; Zhao, Kai-Di; Gao, Jing

    2011-05-01

    Three new copper(II) complexes of N-benzyl di(pyridylmethyl)amine (phdpa) were synthesized and characterized by spectroscopic methods. The interaction between CT-DNA and the complexes was studied by UV and fluorescence titration methods. It was found that the complex [(phdpa)Cu(H(2)O)Ac)](Ac), with the non-planar aromatic heterocyclic ring ligand (phdpa), showed good anticancer properties and could cause the fragmentation of the nucleus, although its interaction with CT-DNA was weaker than that of 1,10-phenanthroline (phen)-based copper(II) complexes. The anticancer activities of copper(II) complexes with phdpa and phen based ligands are correlated to their binding constants with DNA, but phen-based copper(II) complexes did not cause the nucleus fragmentation of HeLa cells. [(phdpa)Cu(H(2)O)Ac)](Ac) can noticeably decrease the oxygen content of a culture solution and of HeLa cells, which make it a new nucleus and oxygen related anticancer copper(II) complex. Information obtained here would be helpful in the design of new antitumor complexes in oxidative therapy.

  5. Cytotoxic Effects of (5 Medicinal Plants on Mitosis in Allium cepa Root Tips

    Directory of Open Access Journals (Sweden)

    I.J. Udo

    2014-03-01

    Full Text Available The study was conducted to investigate the effects that plant extracts from 5 medicinal plants may have on mitosis in Allium cepa. Root of A .cepa were immersed in alcoholic extracts at the concentrations of 0, 25, 50, 75 and 100 mg/mL, respectively for each of the following plants: Gnetum africanum Welw., Lasianther aafricana P. Beauv, Ocimum gratissimum Linn., Telfairia occidentalis Hook F. and Vernonia amygdalina Del. Leafy vegetable which are commonly used in herbal medicine. Results obtained show that the various concentrations of the extracts from test plants had toxic effects on the cells, which caused significant reduction (p<0.05 in the mitotic index when compared with the control. Other effects were prophase inhibition, the delay of mitosis and nuclear lesion. The cytotoxic effect makes a case for a precaution in the use of the leafy extracts in herbal medicine practice.

  6. Dynamic Alterations to α-Actinin Accompanying Sarcomere Disassembly and Reassembly during Cardiomyocyte Mitosis.

    Science.gov (United States)

    Fan, Xiaohu; Hughes, Bryan G; Ali, Mohammad A M; Cho, Woo Jung; Lopez, Waleska; Schulz, Richard

    2015-01-01

    Although mammals are thought to lose their capacity to regenerate heart muscle shortly after birth, embryonic and neonatal cardiomyocytes in mammals are hyperplastic. During proliferation these cells need to selectively disassemble their myofibrils for successful cytokinesis. The mechanism of sarcomere disassembly is, however, not understood. To study this, we performed a series of immunofluorescence studies of multiple sarcomeric proteins in proliferating neonatal rat ventricular myocytes and correlated these observations with biochemical changes at different cell cycle stages. During myocyte mitosis, α-actinin and titin were disassembled as early as prometaphase. α-actinin (representing the sarcomeric Z-disk) disassembly precedes that of titin (M-line), suggesting that titin disassembly occurs secondary to the collapse of the Z-disk. Sarcomere disassembly was concurrent with the dissolution of the nuclear envelope. Inhibitors of several intracellular proteases could not block the disassembly of α-actinin or titin. There was a dramatic increase in both cytosolic (soluble) and sarcomeric α-actinin during mitosis, and cytosolic α-actinin exhibited decreased phosphorylation compared to sarcomeric α-actinin. Inhibition of cyclin-dependent kinase 1 (CDK1) induced the quick reassembly of the sarcomere. Sarcomere dis- and re-assembly in cardiomyocyte mitosis is CDK1-dependent and features dynamic differential post-translational modifications of sarcomeric and cytosolic α-actinin.

  7. ANKRD53 interacts with DDA3 and regulates chromosome integrity during mitosis.

    Science.gov (United States)

    Kim, Seul; Jang, Chang-Young

    2016-02-12

    Spindle dynamics drives chromosome movement and mitotic progression during mitosis. Microtubule (MT)-associated proteins (MAPs) regulate MT stabilization/destabilization and MT polymerization/depolymerization for congression of sister chromatids at the mitotic equator and subsequent segregation toward the spindle poles. Here, we identified ANKRD53 as a novel DDA3-interacting protein through proteomic analysis. Based on expression profiles, ANKRD53 is phosphorylated by mitotic kinases during mitosis. In ANKRD53-depleted HeLa cells, the progression of mitosis was delayed and the number of unaligned chromosomes increased substantially. In addition, spindle MT polymerization decreased and the spindle assembly checkpoint (SAC) was concomitantly activated by the decreased spindle dynamics in ANKRD53-depleted cells. Although ANKRD53 is recruited to the mitotic spindle by DDA3, it counteracts the activity of DDA3 for spindle MT polymerization. Furthermore, ANKRD53 depletion increased the number of bi-nuclei and polylobed nuclei. Thus, ANKRD53 is recruited to the mitotic spindle by DDA3 and acts as a regulator of spindle dynamics and cytokinesis.

  8. Three-Dimensional Organization of Chromosome Territories and the Human Cell Nucleus: Comparison between simulated Parameters and Experiments

    NARCIS (Netherlands)

    T.A. Knoch (Tobias)

    2000-01-01

    textabstractDespite the successful linear sequencing of the human genome its three-dimensional structure is widely unknown, although it is important for gene regulation and replication. For a long time the interphase nucleus has been viewed as a 'spaghetti soup' of DNA without much internal stru

  9. Cytotoxic effects of cylindrospermopsin in mitotic and non-mitotic Vicia faba cells.

    Science.gov (United States)

    Garda, Tamás; Riba, Milán; Vasas, Gábor; Beyer, Dániel; M-Hamvas, Márta; Hajdu, Gréta; Tándor, Ildikó; Máthé, Csaba

    2015-02-01

    Cylindrospermopsin (CYN) is a cyanobacterial toxin known as a eukaryotic protein synthesis inhibitor. We aimed to study its effects on growth, stress responses and mitosis of a eukaryotic model, Vicia faba (broad bean). Growth responses depended on exposure time (3 or 6d), cyanotoxin concentration, culture conditions (dark or continuous light) and V. faba cultivar ("Standard" or "ARC Egypt Cross"). At 6d of exposure, CYN had a transient stimulatory effect on root system growth, roots being possibly capable of detoxification. The toxin induced nucleus fragmentation, blebbing and chromosomal breaks indicating double stranded DNA breaks and programmed cell death. Root necrotic tissue was observed at 0.1-20 μg mL(-1) CYN that probably impeded toxin uptake into vascular tissue. Growth and cell death processes observed were general stress responses. In lateral root tip meristems, lower CYN concentrations (0.01-0.1 μg mL(-1)) induced the stimulation of mitosis and distinct mitotic phases, irrespective of culture conditions or the cultivar used. Higher cyanotoxin concentrations inhibited mitosis. Short-term exposure of hydroxylurea-synchronized roots to 5 μg mL(-1) CYN induced delay of mitosis that might have been related to a delay of de novo protein synthesis. CYN induced the formation of double, split and asymmetric preprophase bands (PPBs), in parallel with the alteration of cell division planes, related to the interference of cyanotoxin with protein synthesis, thus it was a plant- and CYN specific alteration.

  10. Nuclear localization of P-glycoprotein is responsible for protection of the nucleus from doxorubicin in the resistant LoVo cell line.

    Science.gov (United States)

    Szaflarski, Witold; Sujka-Kordowska, Patrycja; Januchowski, Radosław; Wojtowicz, Karolina; Andrzejewska, Małgorzata; Nowicki, Michał; Zabel, Maciej

    2013-07-01

    The high expression of P-glycoprotein (P-gp) belongs to one of the most important factors causing multidrug-resistant (MDR) of cancer cells. P-gp is primarily associated with plasma membrane; however, small fraction of that protein is present in the nuclear envelope. Such phenomenon is observed in cancer cells and may result in the selection of MDR cells as the secondary tumor and/or resistant metastasis that significantly shorten patient survival rate. Here, we confirmed nuclear localization of P-gp in resistant LoVo cells and demonstrated its impact on doxorubicin efflux from the nucleus to cytoplasm. Furthermore, we showed that P-gp located at the nuclear envelope might have a different glycoside chain when compared to the form located in the cytoplasm. It suggests that the glycoside chain plays a role in the intracellular trafficking of P-gp and may decide about the destination place in the cell.

  11. Using a GFP-gene fusion technique to study the cell cycle-dependent distribution of calmodulin in living cells

    Institute of Scientific and Technical Information of China (English)

    李朝军; 吕品; 张东才

    1999-01-01

    In this study, a green fluorescent protein (GFP)-calmodulin (CaM) fusion gene method was used to examine the distribution of calmodulin during various stages of cell cycle. First, it was found that the distribution of CaM in living cells changes with the cell cycle. CaM was found mainly in the cytoplasm during G1 phase. It began to move into the nucleus when the cell entered S phase. At G2 phase, CaM became more concentrated in the nucleus than in cytoplasm. Second, the accumulation of CaM in the nucleus during G2 phase appeared to be related to the onset of mitosis, since inhibiting the activation of CaM at this stage resulted in blocking the nuclear membrane breakdown and chromatin condensation. Finally, after the cell entered mitosis, a high concentration of CaM was found at the polar regions of the mitotic spindle. At this time, inhibiting the activity of CaM would cause a disruption of the spindle structure. The relationship between the stage-specific distribution of CaM and its function in regulat

  12. The G2/M DNA damage checkpoint inhibits mitosis through Tyr15 phosphorylation of p34cdc2 in Aspergillus nidulans.

    Science.gov (United States)

    Ye, X S; Fincher, R R; Tang, A; Osmani, S A

    1997-01-02

    It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non-dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature-sensitive mutations cause an arrest in G2 at 42 degrees C. Addition of MMS to nimT23 G2-arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 degrees C and this delay was correlated with a long delay in the dephosphorylation and activation of p34cdc2. Addition of MMS to nimA5 G2-arrested cells caused inactivation of the H1 kinase activity of p34cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 degrees C. However, if Tyr15 phosphorylation of p34cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G2-arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 degrees C. Thus, Tyr15 phosphorylation of p34cdc2 in G2 arrests initiation of mitosis after DNA damage in A. nidulans.

  13. Amaranth lunasin-like peptide internalizes into the cell nucleus and inhibits chemical carcinogen-induced transformation of NIH-3T3 cells.

    Science.gov (United States)

    Maldonado-Cervantes, Enrique; Jeong, Hyung Jin; León-Galván, Fabiola; Barrera-Pacheco, Alberto; De León-Rodríguez, Antonio; González de Mejia, Elvira; de Lumen, Ben O; Barba de la Rosa, Ana P

    2010-09-01

    Because an unbalanced diet is an important risk factor for several illnesses, interest has increased in finding novel health-promoting foods. Amaranth produces seeds that not only have substantial nutritional properties but that also contain phytochemical compounds as rutin and nicotiflorin and peptides with antihypertensive and anticarcinogenic activities. We report that a cancer-preventive peptide in amaranth has activities similar to those of soybean lunasin. The amaranth lunasin-like peptide, however, requires less time than the soybean lunasin to internalize into the nucleus of NIH-3T3 cells, and inhibits histone acetylation (H(3) and H(4) in a 70 and 77%, respectively). The amaranth lunasin-like peptide inhibited the transformation of NIH-3T3 cells to cancerous foci. The open reading frame (ORF) of amaranth lunasin corresponds to a bifunctional inhibitor/lipid-transfer protein (LTP). LTPs are a family of proteins that in plants are implicated in different functions, albeit all linked to developmental processes and biotic and abiotic stress resistance. Our results open new intriguing questions about the function of lunasin in plants and support that amaranth is a food alternative containing natural peptides with health-promoting benefits.

  14. Multipolar mitosis and aneuploidy after chrysotile treatment: a consequence of abscission failure and cytokinesis regression.

    Science.gov (United States)

    Cortez, Beatriz Araujo; Rezende-Teixeira, Paula; Redick, Sambra; Doxsey, Stephen; Machado-Santelli, Glaucia Maria

    2016-02-23

    Chrysotile, like other types of asbestos, has been associated with mesothelioma, lung cancer and asbestosis. However, the cellular abnormalities induced by these fibers involved in cancer development have not been elucidated yet. Previous works show that chrysotile fibers induce features of cancer cells, such as aneuploidy, multinucleation and multipolar mitosis. In the present study, normal and cancer derived human cell lines were treated with chrysotile and the cellular and molecular mechanisms related to generation of aneuploid cells was elucidated. The first alteration observed was cytokinesis regression, the main cause of multinucleated cells formation and centrosome amplification. The multinucleated cells formed after cytokinesis regression were able to progress through cell cycle and generated aneuploid cells after abnormal mitosis. To understand the process of cytokinesis regression, localization of cytokinetic proteins was investigated. It was observed mislocalization of Anillin, Aurora B, Septin 9 and Alix in the intercellular bridge, and no determination of secondary constriction and abscission sites. Fiber treatment also led to overexpression of genes related to cancer, cytokinesis and cell cycle. The results show that chrysotile fibers induce cellular and molecular alterations in normal and tumor cells that have been related to cancer initiation and progression, and that tetraploidization and aneuploid cell formation are striking events after fiber internalization, which could generate a favorable context to cancer development.

  15. Chromatids segregate without centrosomes during Caenorhabditis elegans mitosis in a Ran- and CLASP-dependent manner.

    Science.gov (United States)

    Nahaboo, Wallis; Zouak, Melissa; Askjaer, Peter; Delattre, Marie

    2015-06-01

    During mitosis, chromosomes are connected to a microtubule-based spindle. Current models propose that displacement of the spindle poles and/or the activity of kinetochore microtubules generate mechanical forces that segregate sister chromatids. Using laser destruction of the centrosomes during Caenorhabditis elegans mitosis, we show that neither of these mechanisms is necessary to achieve proper chromatid segregation. Our results strongly suggest that an outward force generated by the spindle midzone, independently of centrosomes, is sufficient to segregate chromosomes in mitotic cells. Using mutant and RNAi analysis, we show that the microtubule-bundling protein SPD-1/MAP-65 and BMK-1/kinesin-5 act as a brake opposing the force generated by the spindle midzone. Conversely, we identify a novel role for two microtubule-growth and nucleation agents, Ran and CLASP, in the establishment of the centrosome-independent force during anaphase. Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is continuously required to sustain chromosome segregation during mitosis.

  16. Phosphorylation of human Sgo1 by NEK2A is essential for chromosome congression in mitosis

    Institute of Scientific and Technical Information of China (English)

    Guosheng Fu; Xia Ding; Kai Yuan; Felix Aikhionbare; Jianhui Yao; Xin Cai; Kai Jiang; Xuebiao Yao

    2007-01-01

    Chromosome segregation in mitosis is orchestrated by the interaction of the kinetochore with spindle microtubules. Our recent study shows that NEK2A interacts with MAD1 at the kinetochore and possibly functions as a novel integrator of spindle checkpoint signaling. However, it is unclear how NEK2 A regulates kinetochore-microtubule attachment in mitosis. Here we show that NEK2A phosphorylates human Sgol and such phosphorylation is essential for faithful chromosome congression in mitosis. NEK2A binds directly to HsSgol in vitro and co-distributes with HsSgol to the kinetochore of mitotic cells. Our in vitro phosphorylation experiment demonstrated that HsSgol is a substrate of NEK2A and the phosphorylation sites were mapped to Ser14 and Ser507 as judged by the incorporation of 32P. Although such phosphorylation is not required for assembly of HsSgol to the kinetochore, expression of non-phosphorylatable mutant HsSgol perturbed chromosome congression and resulted in a dramatic increase in microtubule attachment errors, including syntelic and monotelic attachments. These findings reveal a key role for the NEK2A-mediated phosphorylation of HsSgol in orchestrating dynamic kinetochore-microtubule interaction. We propose that NEK2A-mediated phosphorylation of human Sgol provides a link between centromeric cohesion and spindle microtubule attachment at the kinetochores.

  17. Cytoskeleton, endoplasmic reticulum and nucleus alterations in CHO-K1 cell line after Crotalus durissus terrificus (South American rattlesnake venom treatment

    Directory of Open Access Journals (Sweden)

    B. P. Tamieti

    2007-01-01

    Full Text Available Snake venoms are toxic to a variety of cell types. However, the intracellular damages and the cell death fate induced by venom are unclear. In the present work, the action of the South American rattlesnake Crotalus durissus terrificus venom on CHO-K1 cell line was analyzed. The cells CHO-K1 were incubated with C. d. terrificus venom (10, 50 and 100g/ml for 1 and 24 hours, and structural alterations of actin filaments, endoplasmic reticulum and nucleus were assessed using specific fluorescent probes and agarose gel electrophoresis for DNA fragmentation. Significant structural changes were observed in all analyzed structures. DNA fragmentation was detected suggesting that, at the concentrations used, the venom induced apoptosis.

  18. Surface functions during mitosis. II. Quantitation of pinocytosis and kinetic characterization of the mitotic cycle with a new fluorescence technique

    OpenAIRE

    1980-01-01

    The profound depression of fluid pinocytosis observed in mitotic cells (Berlin, R. D., et al. 1978. Cell. 15:327--341) is documented by quantitative microspectrofluorimetry of fluorescein-labeled dextran uptake in single cells. In J774.2 macrophages, fluid pinocytosis is reduced 30-fold during mitosis. The depression develops within 30 s of entry into prophase and recovers with equal rapidity upon emergence from telophase into G1. This characteristic pattern of fluid pinocytosis forms the bas...

  19. Intracellular localization of GBF proteins and blue light-induced import of GBF2 fusion proteins into the nucleus of cultured Arabidopsis and soybean cells.

    Science.gov (United States)

    Terzaghi, W B; Bertekap, R L; Cashmore, A R

    1997-05-01

    The G-box is an important regulatory element found in the promoters of many different genes. Four members of an Arabidopsis gene family encoding basic leucine zipper proteins (GBFs) which bind the G-box have previously been cloned. To study GBFs, a polyclonal antibody was raised against GBF1 expressed in bacteria. This antibody also recognized GBF2 and GBF3. Immunoblot analysis of nuclear and cytoplasmic fractions from Arabidopsis and soybean (SB-M) cell cultures indicated that over 90% of proteins detected with anti-GBF1 were cytoplasmic. Electrophoretic mobility shift assays indicated that over 90% of G-box binding activity was cytoplasmic. DNA affinity chromatography demonstrated that each protein detected with anti-GBF1 specifically bound the G-box. To study individual GBFs, DNA constructs fusing GBF1, GBF2 and GBF4 to GUS were made and assayed by transient expression in SB-M protoplasts. Of GUS:GBF1 proteins, 50-62% were localized in the cytoplasm under all conditions tested, while 97% of GUS:GBF4 was localized in the nucleus. By contrast, whereas about 50% of GUS:GBF2 was found in the cytoplasm of dark-grown cells, over 80% of this protein was found in the nucleus in cells cultured under blue light. Deletion analysis of GBF1 identified a region between amino acids 112 and 164 apparently required for cytoplasmic retention. These results suggest the intriguing possibility that limitation of nuclear access may be an important control on GBF activity. In particular, GBF2 is apparently specifically imported into the nucleus in response to light.

  20. Penetration of short fluorescence-labeled peptides into the nucleus in HeLa cells and in vitro specific interaction of the peptides with deoxyribooligonucleotides and DNA.

    Science.gov (United States)

    Fedoreyeva, L I; Kireev, I I; Khavinson, V Kh; Vanyushin, B F

    2011-11-01

    Marked fluorescence in cytoplasm, nucleus, and nucleolus was observed in HeLa cells after incubation with each of several fluorescein isothiocyanate-labeled peptides (epithalon, Ala-Glu-Asp-Gly; pinealon, Glu-Asp-Arg; testagen, Lys-Glu-Asp-Gly). This means that short biologically active peptides are able to penetrate into an animal cell and its nucleus and, in principle they may interact with various components of cytoplasm and nucleus including DNA and RNA. It was established that various initial (intact) peptides differently affect the fluorescence of the 5,6-carboxyfluorescein-labeled deoxyribooligonucleotides and DNA-ethidium bromide complexes. The Stern-Volmer constants characterizing the degree of fluorescence quenching of various single- and double-stranded fluorescence-labeled deoxyribooligonucleotides with short peptides used were different depending on the peptide primary structures. This indicates the specific interaction between short biologically active peptides and nucleic acid structures. On binding to them, the peptides discriminate between different nucleotide sequences and recognize even their cytosine methylation status. Judging from corresponding constants of the fluorescence quenching, the epithalon, pinealon, and bronchogen (Ala-Glu-Asp-Leu) bind preferentially with deoxyribooligonucleotides containing CNG sequence (CNG sites are targets for cytosine DNA methylation in eukaryotes). Epithalon, testagen, and pinealon seem to preferentially bind with CAG- but bronchogen with CTG-containing sequences. The site-specific interactions of peptides with DNA can control epigenetically the cell genetic functions, and they seem to play an important role in regulation of gene activity even at the earliest stages of life origin and in evolution.

  1. CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast.

    Science.gov (United States)

    Převorovský, Martin; Oravcová, Martina; Zach, Róbert; Jordáková, Anna; Bähler, Jürg; Půta, František; Folk, Petr

    2016-11-16

    For every eukaryotic cell to grow and divide, intricately coordinated action of numerous proteins is required to ensure proper cell-cycle progression. The fission yeast Schizosaccharomyces pombe has been instrumental in elucidating the fundamental principles of cell-cycle control. Mutations in S. pombe 'cut' (cell untimely torn) genes cause failed coordination between cell and nuclear division, resulting in catastrophic mitosis. Deletion of cbf11, a fission yeast CSL transcription factor gene, triggers a 'cut' phenotype, but the precise role of Cbf11 in promoting mitotic fidelity is not known. We report that Cbf11 directly activates the transcription of the acetyl-coenzyme A carboxylase gene cut6, and the biotin uptake/biosynthesis genes vht1 and bio2, with the former 2 implicated in mitotic fidelity. Cbf11 binds to a canonical, metazoan-like CSL response element (GTGGGAA) in the cut6 promoter. Expression of Cbf11 target genes shows apparent oscillations during the cell cycle using temperature-sensitive cdc25-22 and cdc10-M17 block-release experiments, but not with other synchronization methods. The penetrance of catastrophic mitosis in cbf11 and cut6 mutants is nutrient-dependent. We also show that drastic decrease in biotin availability arrests cell proliferation but does not cause mitotic defects. Taken together, our results raise the possibility that CSL proteins play conserved roles in regulating cell-cycle progression, and they could guide experiments into mitotic CSL functions in mammals.

  2. Matefin/SUN-1 Phosphorylation on Serine 43 Is Mediated by CDK-1 and Required for Its Localization to Centrosomes and Normal Mitosis in C. elegans Embryos

    Directory of Open Access Journals (Sweden)

    Noam Zuela

    2016-02-01

    Full Text Available Matefin/SUN-1 is an evolutionary conserved C. elegans inner nuclear membrane SUN-domain protein. By creating a bridge with the KASH-domain protein ZYG-12, it connects the nucleus to cytoplasmic filaments and organelles. Matefin/SUN-1 is expressed in the germline where it undergoes specific phosphorylation at its N-terminal domain, which is required for germline development and homologous chromosome pairing. The maternally deposited matefin/SUN-1 is then essential for embryonic development. Here, we show that in embryos, serine 43 of matefin/SUN-1 (S43 is phosphorylated in a CDK-1 dependent manner and is localized throughout the cell cycle mostly to centrosomes. By generating animals expressing phosphodead S43A and phosphomimetic S43E mutations, we show that phosphorylation of S43 is required to maintain centrosome integrity and function, as well as for the localization of ZYG-12 and lamin. Expression of S43E in early embryos also leads to an increase in chromatin structural changes, decreased progeny and to almost complete embryonic lethality. Down regulation of emerin further increases the occurrence of chromatin organization abnormalities, indicating possible collaborative roles for these proteins that is regulated by S43 phosphorylation. Taken together, these results support a role for phosphorylation of serine 43 in matefin/SUN-1 in mitosis.

  3. 不结球白菜根尖体细胞染色体制片及其二倍体和四倍体有丝分裂过程观察%Chromosome preparation of somatic cell in root-tip of non-heading Chinese cabbage ( Brassica campestris ssp.chinensis ) and observation of mitosis process of its diploid and tetraploid

    Institute of Scientific and Technical Information of China (English)

    郑金双; 张蜀宁; 孙成振; 王雅美

    2011-01-01

    L-1 colchicine, 0. 2 g · L-1 colchicine, saturated p-dichlorobenezene, 20 or 40 mg · L-1 cycloheximide, equivalent volume mixed solution of 40 mg · L-1 cycloheximide and 2 mmol · L-1 8-hydroxyquinoline) and pretreatment time (1.0 -3. 5 h) in chromosome preparation process were compared and selected. And on this basis, mitosis process of somatic cell in root-lip of diploid and tetraploid of non-heading Chinese cabbage was observed. The results show that root length obviously influences on number of split phase. And there are relatively more split phases in 1.0-2.0 cm root with an account for 64. 75% of total cell number. Some split phases can be obtained by frozen pretreating for 22-23 h. Using different pretreatment agents andtimes, number of split phase and chromosome morphology are obviously different. Pretreating for 3. 5 h with 40 mg · L-1 cycloheximide, number of split phase is the most but chromosome doubling is easy to be caused. And pretreating for 3. 5 h with 20 mg · L-1 cycloheximide, chromosomes are long with clear kinetochore and satellite, and number of split phase accounts for 53. 65% of total cell number. Therefore, suitable length of root is 1. 0-2. 0 cm, optimal pretreatment method is using 20 mg · L-1 cycloheximide soaking for 2. 0-3. 0 h. The mitosis process of somatic cell in root-tip of diploid and tetraploid of non-heading Chinese cabbage is generally similar, and their chromosome behavior is basically identical in interphase, prophase, metaphase, anaphase and telophase of mitosis. But there are some abnormal phenomena in mitosis process of tetraploid, such as, multivalent, chromosome bridge, lagging chromosome, chromosome abnormal segregation and endogenous mitosis.

  4. Regulation of mRNA translation during mitosis.

    Science.gov (United States)

    Tanenbaum, Marvin E; Stern-Ginossar, Noam; Weissman, Jonathan S; Vale, Ronald D

    2015-08-25

    Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ~200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.

  5. Poly(ADP-ribosyl)ation is recognized by ECT2 during mitosis.

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    Li, Mo; Bian, Chunjing; Yu, Xiaochun

    2014-01-01

    Poly(ADP-ribosyl)ation is an unique posttranslational modification and required for spindle assembly and function during mitosis. However, the molecular mechanism of poly(ADP-ribose) (PAR) in mitosis remains elusive. Here, we show the evidence that PAR is recognized by ECT2, a key guanine nucleotide exchange factor in mitosis. The BRCT domain of ECT2 directly binds to PAR both in vitro and in vivo. We further found that α-tubulin is PARylated during mitosis. PARylation of α-tubulin is recognized by ECT2 and recruits ECT2 to mitotic spindle for completing mitosis. Taken together, our study reveals a novel mechanism by which PAR regulates mitosis.

  6. The M-current contributes to high threshold membrane potential oscillations in a cell type-specific way in the pedunculopontine nucleus of mice

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    Csilla eBordas

    2015-04-01

    Full Text Available The pedunculopontine nucleus is known as a cholinergic nucleus of the reticular activating system, participating in regulation of sleep and wakefulness. Besides cholinergic neurons, it consists of GABAergic and glutamatergic neurons as well. According to classical and recent studies, more subgroups of neurons were defined. Groups based on the neurotransmitter released by a neuron are not homogenous, but can be further subdivided.The PPN neurons do not only provide cholinergic and non-cholinergic inputs to several subcortical brain areas but they are also targets of cholinergic and other different neuromodulatory actions. Although cholinergic neuromodulation has been already investigated in the nucleus, one of its characteristic targets, the M-type potassium current has not been described yet.Using slice electrophysiology, we provide evidence in the present work that cholinergic neurons possess M-current, whereas GABAergic neurons lack it. The M-current contributes to certain functional differences of cholinergic and GABAergic neurons, as spike frequency adaptation, action potential firing frequency or the amplitude difference of medium afterhyperpolarizations. Furthermore, we showed that high threshold membrane potential oscillation with high power, around 20 Hz frequency is a functional property of almost all cholinergic cells, whereas GABAergic neurons have only low amplitude oscillations. Blockade of the M-current abolished the oscillatory activity at 20 Hz, and largely diminished it at other frequencies.Taken together, the M-current seems to be characteristic for PPN cholinergic neurons. It provides a possibility for modulating gamma band activity of these cells, thus contributing to neuromodulatory regulation of the reticular activating system.

  7. The spindle pole bodies facilitate nuclear envelope division during closed mitosis in fission yeast.

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    Zheng, Liling; Schwartz, Cindi; Magidson, Valentin; Khodjakov, Alexey; Oliferenko, Snezhana

    2007-07-01

    Many organisms divide chromosomes within the confines of the nuclear envelope (NE) in a process known as closed mitosis. Thus, they must ensure coordination between segregation of the genetic material and division of the NE itself. Although many years of work have led to a reasonably clear understanding of mitotic spindle function in chromosome segregation, the NE division mechanism remains obscure. Here, we show that fission yeast cells overexpressing the transforming acid coiled coil (TACC)-related protein, Mia1p/Alp7p, failed to separate the spindle pole bodies (SPBs) at the onset of mitosis, but could assemble acentrosomal bipolar and antiparallel spindle structures. Most of these cells arrested in anaphase with fully extended spindles and nonsegregated chromosomes. Spindle poles that lacked the SPBs did not lead the division of the NE during spindle elongation, but deformed it, trapping the chromosomes within. When the SPBs were severed by laser microsurgery in wild-type cells, we observed analogous deformations of the NE by elongating spindle remnants, resulting in NE division failure. Analysis of dis1Delta cells that elongate spindles despite unattached kinetochores indicated that the SPBs were required for maintaining nuclear shape at anaphase onset. Strikingly, when the NE was disassembled by utilizing a temperature-sensitive allele of the Ran GEF, Pim1p, the abnormal spindles induced by Mia1p overexpression were capable of segregating sister chromatids to daughter cells, suggesting that the failure to divide the NE prevents chromosome partitioning. Our results imply that the SPBs preclude deformation of the NE during spindle elongation and thus serve as specialized structures enabling nuclear division during closed mitosis in fission yeast.

  8. The spindle pole bodies facilitate nuclear envelope division during closed mitosis in fission yeast.

    Directory of Open Access Journals (Sweden)

    Liling Zheng

    2007-07-01

    Full Text Available Many organisms divide chromosomes within the confines of the nuclear envelope (NE in a process known as closed mitosis. Thus, they must ensure coordination between segregation of the genetic material and division of the NE itself. Although many years of work have led to a reasonably clear understanding of mitotic spindle function in chromosome segregation, the NE division mechanism remains obscure. Here, we show that fission yeast cells overexpressing the transforming acid coiled coil (TACC-related protein, Mia1p/Alp7p, failed to separate the spindle pole bodies (SPBs at the onset of mitosis, but could assemble acentrosomal bipolar and antiparallel spindle structures. Most of these cells arrested in anaphase with fully extended spindles and nonsegregated chromosomes. Spindle poles that lacked the SPBs did not lead the division of the NE during spindle elongation, but deformed it, trapping the chromosomes within. When the SPBs were severed by laser microsurgery in wild-type cells, we observed analogous deformations of the NE by elongating spindle remnants, resulting in NE division failure. Analysis of dis1Delta cells that elongate spindles despite unattached kinetochores indicated that the SPBs were required for maintaining nuclear shape at anaphase onset. Strikingly, when the NE was disassembled by utilizing a temperature-sensitive allele of the Ran GEF, Pim1p, the abnormal spindles induced by Mia1p overexpression were capable of segregating sister chromatids to daughter cells, suggesting that the failure to divide the NE prevents chromosome partitioning. Our results imply that the SPBs preclude deformation of the NE during spindle elongation and thus serve as specialized structures enabling nuclear division during closed mitosis in fission yeast.

  9. Nucleoporin MOS7/Nup88 is required for mitosis in gametogenesis and seed development in Arabidopsis.

    Science.gov (United States)

    Park, Guen Tae; Frost, Jennifer M; Park, Jin-Sup; Kim, Tae Ho; Lee, Jong Seob; Oh, Sung Aeong; Twell, David; Brooks, Janie Sue; Fischer, Robert L; Choi, Yeonhee

    2014-12-23

    Angiosperm reproduction is characterized by alternate diploid sporophytic and haploid gametophytic generations. Gametogenesis shares similarities with that of animals except for the formation of the gametophyte, whereby haploid cells undergo several rounds of postmeiotic mitosis to form gametes and the accessory cells required for successful reproduction. The mechanisms regulating gametophyte development in angiosperms are incompletely understood. Here, we show that the nucleoporin Nup88-homolog MOS7 (Modifier of Snc1,7) plays a crucial role in mitosis during both male and female gametophyte formation in Arabidopsis thaliana. Using a mutagenesis screen, we identify the mos7-5 mutant allele, which causes ovule and pollen abortion in MOS7/mos7-5 heterozygous plants, and preglobular stage embryonic lethality in homozygous mos7-5 seeds. During interphase, we show that MOS7 is localized to the nuclear membrane but, like many nucleoporins, is associated with the spindle apparatus during mitosis. We detect interactions between MOS7 and several nucleoporins known to control spindle dynamics, and find that in pollen from MOS7/mos7-5 heterozygotes, abortion is accompanied by a failure of spindle formation, cell fate specification, and phragmoplast activity. Most intriguingly, we show that following gamete formation by MOS7/mos7-5 heterozygous spores, inheritance of either the MOS7 or the mos7-5 allele by a given gamete does not correlate with its respective survival or abortion. Instead, we suggest a model whereby MOS7, which is highly expressed in the Pollen- and Megaspore Mother Cells, enacts a dosage-limiting effect on the gametes to enable their progression through subsequent mitoses.

  10. The multi zinc-finger protein Trps1 acts as a regulator of histone deacetylation during mitosis.

    Science.gov (United States)

    Wuelling, Manuela; Pasdziernik, Markus; Moll, Carina N; Thiesen, Andrea M; Schneider, Sabine; Johannes, Christian; Vortkamp, Andrea

    2013-07-15

    TRPS1, the gene mutated in human "Tricho-Rhino-Phalangeal syndrome," encodes a multi zinc-finger nuclear regulator of chondrocyte proliferation and differentiation. Here, we have identified a new function of Trps1 in controlling mitotic progression in chondrocytes. Loss of Trps1 in mice leads to an increased proportion of cells arrested in mitosis and, subsequently, to chromosome segregation defects. Searching for the molecular basis of the defect, we found that Trps1 acts as regulator of histone deacetylation. Trps1 interacts with two histone deacetylases, Hdac1 and Hdac4, thereby increasing their activity. Loss of Trps1 results in histone H3 hyperacetylation, which is maintained during mitosis. Consequently, chromatin condensation and binding of HP1 is impaired, and Trps1-deficient chondrocytes accumulate in prometaphase. Overexpression of Hdac4 rescues the mitotic defect of Trps1-deficient chondrocytes, identifying Trps1 as an important regulator of chromatin deacetylation during mitosis in chondrocytes. Our data provide the first evidence that the control of mitosis can be linked to the regulation of chondrocyte differentiation by epigenetic consequences of altered Hdac activity.

  11. 3D/4D architecture of chromosomal break point regions in the cell nucleus following irradiation of normal cells and tumor cells; 3D/4D Architektur von chromosomalen Bruchpunktregionen im Zellkern nach Bestrahlung von Normalzellen und Tumorzellen

    Energy Technology Data Exchange (ETDEWEB)

    Hausmann, M.; Cremer, C.; Friedl, A.; Dollinger, G.; Loebrich, M.; Friedland, W.

    2015-01-15

    The development of an effective analytical methodology for a correct description of oncogenic chromosomal aberrations is the challenge of medical radiobiology with respect to preventive therapeutic methods. Scope of the project was a better understanding of the behavior of break point regions dependent on the genome loci, the chromatin folding, the involved repair proteins and the beam quality with respect to an improvement and an efficient prognosis of the health consequences following radiation exposure. New microscopic insights in the normal cell nucleus are supposed to allow a better understanding of the spatial interactions on a molecular scale.

  12. Differentiation of adipose-derived stem cells toward nucleus pulposuslike cells induced by hypoxia and a three-dimensional chitosan-alginate gel scaffold in vitro

    Institute of Scientific and Technical Information of China (English)

    Zhang Zhicheng; Li Fang; Tian Haiquan; Guan Kai; Zhao Guangmin; Shan Jianlin; Ren Dajiang

    2014-01-01

    Background Injectable three-dimensional (3D) scaffolds have the advantages of fluidity and moldability to fill irregularshaped defects,simple incorporation of bioactive factors,and limited surgical invasiveness.Adipose-derived stem cells (ADSCs) are multipotent and can be differentiated toward nucleus pulposus (NP)-Iike cells.A hypoxic environment may be important for differentiation to NP-like cells because the intervertebral disc is an avascular tissue.Hence,we investigated the induction effects of hypoxia and an injectable 3D chitosan-alginate (C/A) gel scaffold on ADSCs.Methods The C/A gel scaffold consisted of medical-grade chitosan and alginate.Gel porosity was calculated by liquid displacement method.Pore microstructure was analyzed by light and scanning electron microscopy.ADSCs were isolated and cultured by conventional methods.Passage 2 BrdU-labeled ADSCs were co-cultured with the C/A gel.ADSCs were divided into three groups (control,normoxia-induced,and hypoxia-induced groups).In the control group,cells were cultured in 10% FBS/DMEM.Hypoxia-induced and normoxia-induced groups were induced by adding transforming grow