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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.

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    Cavalier-Smith, Thomas

    2010-02-04

    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. 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 advantages. These successive changes took place

  3. 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…

  4. Mitosis can drive cell cannibalism through entosis

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

    2017-01-01

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

  5. Remodeling of bovine oviductal epithelium by mitosis of secretory cells.

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    Ito, Sayaka; Kobayashi, Yoshihiko; Yamamoto, Yuki; Kimura, Koji; Okuda, Kiyoshi

    2016-11-01

    Two types of oviductal epithelial cells, secretory and ciliated, play crucial roles in the first days after fertilization in mammals. Secretory cells produce various molecules promoting embryo development, while ciliated cells facilitate transport of oocytes and zygotes by ciliary beating. The proportions of the two cell types change during the estrous cycle. The proportion of ciliated cells on the oviductal luminal surface is abundant at the follicular phase, whereas the proportion of secretory cells gradually increases with the formation of the corpus luteum. In the present study, we hypothesize that the proportions of ciliated and secretory epithelial cells are regulated by mitosis. The proportion of the cells being positive for FOXJ1 (a ciliated cell marker) or Ki67 (a mitosis marker) in epithelial cells during the estrous cycle were immunohistochemically examined. Ki67 and FOXJ1 or PAX8 (a secretory cell marker), were double-stained to clarify which types of epithelial cells undergo mitosis. In the ampulla, the percentage of FOXJ1-positive cells was highest at the day of ovulation (Day 0) and decreased by about 50 % by Days 8-12, while in the isthmus it did not change during the estrous cycle. The proportion of Ki67-positive cells was highest at around the time of ovulation in both the ampulla and isthmus. All the Ki67-positive cells were PAX8-positive and FOXJ1-negative in both the ampulla and isthmus. These findings suggest that epithelial remodeling, which is regulated by differentiation and/or proliferation of secretory cells of the oviduct, provides the optimal environment for gamete transport, fertilization and embryonic development.

  6. The radiation hypersensitivity of cells at mitosis.

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

    2002-12-01

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

  7. How protein kinases co-ordinate mitosis in animal cells.

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    Ma, Hoi Tang; Poon, Randy Y C

    2011-04-01

    Mitosis is associated with profound changes in cell physiology and a spectacular surge in protein phosphorylation. To accomplish these, a remarkably large portion of the kinome is involved in the process. In the present review, we will focus on classic mitotic kinases, such as cyclin-dependent kinases, Polo-like kinases and Aurora kinases, as well as more recently characterized players such as NIMA (never in mitosis in Aspergillus nidulans)-related kinases, Greatwall and Haspin. Together, these kinases co-ordinate the proper timing and fidelity of processes including centrosomal functions, spindle assembly and microtubule-kinetochore attachment, as well as sister chromatid separation and cytokinesis. A recurrent theme of the mitotic kinase network is the prevalence of elaborated feedback loops that ensure bistable conditions. Sequential phosphorylation and priming phosphorylation on substrates are also frequently employed. Another important concept is the role of scaffolds, such as centrosomes for protein kinases during mitosis. Elucidating the entire repertoire of mitotic kinases, their functions, regulation and interactions is critical for our understanding of normal cell growth and in diseases such as cancers.

  8. 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.

  9. The duration of mitosis and daughter cell size are modulated by nutrients in budding yeast.

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    Leitao, Ricardo M; Kellogg, Douglas R

    2017-11-06

    The size of nearly all cells is modulated by nutrients. Thus, cells growing in poor nutrients can be nearly half the size of cells in rich nutrients. In budding yeast, cell size is thought to be controlled almost entirely by a mechanism that delays cell cycle entry until sufficient growth has occurred in G1 phase. Here, we show that most growth of a new daughter cell occurs in mitosis. When the rate of growth is slowed by poor nutrients, the duration of mitosis is increased, which suggests that cells compensate for slow growth in mitosis by increasing the duration of growth. The amount of growth required to complete mitosis is reduced in poor nutrients, leading to a large reduction in cell size. Together, these observations suggest that mechanisms that control the extent of growth in mitosis play a major role in cell size control in budding yeast. © 2017 Leitao and Kellogg.

  10. 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…

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

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

    2016-06-28

    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 highly active in nocodazole-induced, double-thymidine synchronization released as well as naturally occurring mitosis in HeLa cells. Multiple autophagy proteins are upregulated in mitosis and the increased Beclin-1 level likely contributes to the active autophagic flux in early mitosis. It is interesting that although the autophagic flux is active throughout the cell cycle, early mitosis and S phase have relatively higher autophagic flux than G1 and late G2 phases, which might be helpful to degrade the damaged organelles and provide energy during S phase and mitosis.

  12. Centriole distribution during tripolar mitosis in Chinese hamster ovary cells

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    1984-01-01

    During bipolar mitosis a pair of centrioles is distributed to each cell but the activities of the two centrioles within the pair are not equivalent. The parent is normally surrounded by a cloud of pericentriolar material that serves as a microtubule-organizing center. The daughter does not become associated with pericentriolar material until it becomes a parent in the next cell cycle (Rieder, C.L., and G. G. Borisy , 1982, Biol. Cell., 44:117-132). We asked whether the microtubule-organizing activity associated with a centriole was dependent on its becoming a parent. We induced multipolar mitosis in Chinese hamster ovary cells by treatment with 0.04 micrograms/ml colcemid for 4 h. After recovery from this colcemid block, the majority of cells divided into two, but 40% divided into three and 2% divided into four. The tripolar mitotic cells were examined by antitubulin immunofluorescence and by high voltage electron microscopy of serial thick (0.25-micron) sections. The electron microscope analysis showed that centriole number was conserved and that the centrioles were distributed among the three spindle poles, generally in a 2:1:1 or 2:2:0 pattern. The first pattern shows that centriole parenting is not prerequisite for association with pole function; the second pattern indicates that centrioles per se are not required at all. However, the frequency of midbody formation and successful division was higher when centrioles were present in the 2:1:1 pattern. We suggest that the centrioles may help the proper distribution and organization of the pericentriolar cloud, which is needed for the formation of a functional spindle pole. PMID:6373793

  13. 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.

  14. Positioning of the NOR-bearing chromosomes in relation to nucleoli in daughter cells after mitosis.

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    Kalmárová, M; Smirnov, E; Kovácik, L; Popov, A; Raska, I

    2008-01-01

    It is known that chromosomes occupy non-random positions in the cell nucleus. However, it is not clear to what extent their nuclear positions, together with their neighborhood, are conserved in daughter cells. To address specific aspects of this problem, we used the model of the chromosomes carrying ribosomal genes that are organized in clusters termed Nucleolus Organizer Regions (NORs). We compared the association of chosen NOR-bearing chromosomes (NOR-chromosomes) with nucleoli, as well as the numbers of nucleoli, in the pairs of daughter cells, and established how frequently the daughter cells had equal numbers of the homologs of certain NOR-chromosomes associated with individual nucleoli. The daughter cells typically had different numbers of nucleoli. At the same time, using immuno-FISH with probes for chromosomes 14 and 15 in HeLa cells, we found that the cell pairs with identical combinations appeared significantly more frequently than predicted by the random model. Thus, although the total number of chromosomes associated with nucleoli is variable, our data indicate that the position of the NOR-bearing chromosomes in relation to nucleoli is partly conserved through mitosis.

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

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

    2016-10-20

    Cell division is characterized by a sequence of events by which a cell gives rise to two daughter cells. Quantitative measurements of cell-cycle dynamics in single cells showed that despite variability in G1-, S-, and G2 phases, duration of mitosis is short and remarkably constant. Surprisingly, there is no correlation between cell-cycle length and mitotic duration, suggesting that mitosis is temporally insulated from variability in earlier cell-cycle phases. By combining live cell imaging and computational modeling, we showed that positive feedback is the molecular mechanism underlying the temporal insulation of mitosis. Perturbing positive feedback gave rise to a sluggish, variable entry and progression through mitosis and uncoupled duration of mitosis from variability in cell cycle length. We show that positive feedback is important to keep mitosis short, constant, and temporally insulated and anticipate it might be a commonly used regulatory strategy to create modularity in other biological systems. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  16. 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. © 2014 The Authors.

  17. Mathematical imaging methods for mitosis analysis in live-cell phase contrast microscopy.

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    Grah, Joana Sarah; Harrington, Jennifer Alison; Koh, Siang Boon; Pike, Jeremy Andrew; Schreiner, Alexander; Burger, Martin; Schönlieb, Carola-Bibiane; Reichelt, Stefanie

    2017-02-15

    In this paper we propose a workflow to detect and track mitotic cells in time-lapse microscopy image sequences. In order to avoid the requirement for cell lines expressing fluorescent markers and the associated phototoxicity, phase contrast microscopy is often preferred over fluorescence microscopy in live-cell imaging. However, common specific image characteristics complicate image processing and impede use of standard methods. Nevertheless, automated analysis is desirable due to manual analysis being subjective, biased and extremely time-consuming for large data sets. Here, we present the following workflow based on mathematical imaging methods. In the first step, mitosis detection is performed by means of the circular Hough transform. The obtained circular contour subsequently serves as an initialisation for the tracking algorithm based on variational methods. It is sub-divided into two parts: in order to determine the beginning of the whole mitosis cycle, a backwards tracking procedure is performed. After that, the cell is tracked forwards in time until the end of mitosis. As a result, the average of mitosis duration and ratios of different cell fates (cell death, no division, division into two or more daughter cells) can be measured and statistics on cell morphologies can be obtained. All of the tools are featured in the user-friendly MATLAB®Graphical User Interface MitosisAnalyser. Copyright © 2017. Published by Elsevier Inc.

  18. Apoptosis and mitosis as prognostic factors in pathologically staged N1 nonsmall cell lung cancer

    International Nuclear Information System (INIS)

    Komaki, Ritsuko; Fujii, Takashi; Perkins, Penny; Ro, Jae Y.; Allen, Pamela K.; Mason, Kathryn A.; Mountain, Clifton F.; Milas, Luka

    1996-01-01

    Purpose: This study aimed to establish whether spontaneous apoptosis or mitosis has prognostic value among patients with pathologically staged N1 nonsmall cell lung carcinoma (NSCLC) treated with surgical resection with or without adjuvant therapy. Methods and Materials: Material from 173 patients who had resections between 1970 and 1988 was analyzed for apoptosis and mitosis. There were 128 men and 45 women, with a median age of 61 years. There were 86 squamous cell carcinomas (SQ), 73 adenocarcinomas (AC), 3 large-cell carcinomas (LC), 6 SQ-AC, and 5 unclassified. Patients were observed from 2 to 209 months (median 27). Actuarial methods were used to assess survival and freedom from distant metastasis. Results: In NSCLC, apoptosis was found to range from 0.2% to 2.8% (median 1.0%) and mitosis from 0 to 1.8% (median 0.4%). Tumors having higher levels of apoptosis also had higher levels of mitosis (p = 0.001). The values of neither apoptosis nor mitosis depended on size, location, differentiation of tumors, age, performance status, or weight loss of patients. However, the values of apoptosis depended on tumor histology in that high values (greater than or equal to the median) were more frequent in SQ (49%) than in AC/LC (29%) (p 0.01). The overall survival for NSCLC patients, which was 33% at 5 years, did not depend on the level of either apoptosis or mitosis. The 5-year survival of patients having SQ was higher (43%) than that of patients having AC/LC (21%) (p = 0.03). Patients with high apoptosis showed significantly better 5-year overall (p = 0.008) and DMF (p = 0.0012) survivals in the SQ group compared to the AC/LC group. High mitosis compared to low mitosis was a significantly better predictor for 5-year survival (62% vs. 29%, respectively) (p = 0.035) in the SQ. However, high mitosis was a significantly worse 5-year DMF survival predictor compared to low mitosis: 13% vs. 56%, respectively (p = 0.05) in AC/LC. In the multivariate models for AC/LC, mitosis

  19. p53 Dependent Centrosome Clustering Prevents Multipolar Mitosis in Tetraploid Cells

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    Yi, Qiyi; Zhao, Xiaoyu; Huang, Yun; Ma, Tieliang; Zhang, Yingyin; Hou, Heli; Cooke, Howard J.; Yang, Da-Qing; Wu, Mian; Shi, Qinghua

    2011-01-01

    Background p53 abnormality and aneuploidy often coexist in human tumors, and tetraploidy is considered as an intermediate between normal diploidy and aneuploidy. The purpose of this study was to investigate whether and how p53 influences the transformation from tetraploidy to aneuploidy. Principal Findings Live cell imaging was performed to determine the fates and mitotic behaviors of several human and mouse tetraploid cells with different p53 status, and centrosome and spindle immunostaining was used to investigate centrosome behaviors. We found that p53 dominant-negative mutation, point mutation, or knockout led to a 2∼ 33-fold increase of multipolar mitosis in N/TERT1, 3T3 and mouse embryonic fibroblasts (MEFs), while mitotic entry and cell death were not significantly affected. In p53-/- tetraploid MEFs, the ability of centrosome clustering was compromised, while centrosome inactivation was not affected. Suppression of RhoA/ROCK activity by specific inhibitors in p53-/- tetraploid MEFs enhanced centrosome clustering, decreased multipolar mitosis from 38% to 20% and 16% for RhoA and ROCK, respectively, while expression of constitutively active RhoA in p53+/+ tetraploid 3T3 cells increased the frequency of multipolar mitosis from 15% to 35%. Conclusions p53 could not prevent tetraploid cells entering mitosis or induce tetraploid cell death. However, p53 abnormality impaired centrosome clustering and lead to multipolar mitosis in tetraploid cells by modulating the RhoA/ROCK signaling pathway. PMID:22076149

  20. Tradescantia cytogenetic tests (root-tip mitosis, pollen mitosis, pollen mother-cell meiosis). A report of the US Environmental Protection Agency gene-tox program

    Energy Technology Data Exchange (ETDEWEB)

    Ma, T H

    1982-01-01

    3 kinds of cytogenetic tests for screening of environmental mutagens were established for Tradescantia, namely, root-tip mitosis, pollen mitosis, and pollen mother-cell meiosis (commonly referred to as the Tradescantia-micronucleus (Trad-MCN) test). All these tests are technically simple, inexpensive, and can yield reliable results in a relatively short time (36 to 72 h). The root-tip mitosis test is suitable only for liquid agents, while pollen mitosis is suitable for both liquid and gaseous agents. Pollen tube mitotic chromosomes are extremely sensitive to mutagens; therefore, they are good materials for detecting very low concentrations of mutagens. Both root-tip mitosis and pollen mitosis tests use chromosome and/or chromatid aberrations as end points for scoring. The Trad-MCN test is suitable for both liquid and gaseous agents. In addition, it is especially suitable for in situ monitoring of water and air pollutants. Of the 12 chemicals tested, 5-fluorouracil and 1,2-dibromoethane indicate that they are very potent mutagens based on the effective dosages used to produce a positive response. Sulfur dioxide, ethyl methanesulfonate, sodium azide, Phosdrin, and Bladex rank next in potency.

  1. 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.

  2. Maximized Inter-Class Weighted Mean for Fast and Accurate Mitosis Cells Detection in Breast Cancer Histopathology Images.

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    Nateghi, Ramin; Danyali, Habibollah; Helfroush, Mohammad Sadegh

    2017-08-14

    Based on the Nottingham criteria, the number of mitosis cells in histopathological slides is an important factor in diagnosis and grading of breast cancer. For manual grading of mitosis cells, histopathology slides of the tissue are examined by pathologists at 40× magnification for each patient. This task is very difficult and time-consuming even for experts. In this paper, a fully automated method is presented for accurate detection of mitosis cells in histopathology slide images. First a method based on maximum-likelihood is employed for segmentation and extraction of mitosis cell. Then a novel Maximized Inter-class Weighted Mean (MIWM) method is proposed that aims at reducing the number of extracted non-mitosis candidates that results in reducing the false positive mitosis detection rate. Finally, segmented candidates are classified into mitosis and non-mitosis classes by using a support vector machine (SVM) classifier. Experimental results demonstrate a significant improvement in accuracy of mitosis cells detection in different grades of breast cancer histopathological images.

  3. Do migrating cells need a nucleus?

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    Hawkins, Rhoda J

    2018-03-05

    How the nucleus affects cell polarity and migration is unclear. In this issue, Graham et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201706097) show that enucleated cells polarize and migrate in two but not three dimensions and propose that the nucleus is a necessary component of the molecular clutch regulating normal mechanical responses. © 2018 Hawkins.

  4. Dll1 maintains quiescence of adult neural stem cells and segregates asymmetrically during mitosis.

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    Kawaguchi, Daichi; Furutachi, Shohei; Kawai, Hiroki; Hozumi, Katsuto; Gotoh, Yukiko

    2013-01-01

    Stem cells often divide asymmetrically to produce one stem cell and one differentiating cell, thus maintaining the stem cell pool. Although neural stem cells (NSCs) in the adult mouse subventricular zone have been suggested to divide asymmetrically, intrinsic cell fate determinants for asymmetric NSC division are largely unknown. Stem cell niches are important for stem cell maintenance, but the niche for the maintenance of adult quiescent NSCs has remained obscure. Here we show that the Notch ligand Delta-like 1 (Dll1) is required to maintain quiescent NSCs in the adult mouse subventricular zone. Dll1 protein is induced in activated NSCs and segregates to one daughter cell during mitosis. Dll1-expressing cells reside in close proximity to quiescent NSCs, suggesting a feedback signal for NSC maintenance by their sister cells and progeny. Our data suggest a model in which NSCs produce their own niche cells for their maintenance through asymmetric Dll1 inheritance at mitosis.

  5. Tripolar mitosis in human cells and embryos: occurrence, pathophysiology and medical implications.

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    Kalatova, Beata; Jesenska, Renata; Hlinka, Daniel; Dudas, Marek

    2015-01-01

    Tripolar mitosis is a specific case of cell division driven by typical molecular mechanisms of mitosis, but resulting in three daughter cells instead of the usual count of two. Other variants of multipolar mitosis show even more mitotic poles and are relatively rare. In nature, this phenomenon was frequently observed or suspected in multiple common cancers, infected cells, the placenta, and in early human embryos with impaired pregnancy-yielding potential. Artificial causes include radiation and various toxins. Here we combine several pieces of the most recent evidence for the existence of different types of multipolar mitosis in preimplantation embryos together with a detailed review of the literature. The related molecular and cellular mechanisms are discussed, including the regulation of centriole duplication, mitotic spindle biology, centromere functions, cell cycle checkpoints, mitotic autocorrection mechanisms, and the related complicating factors in healthy and affected cells, including post-mitotic cell-cell fusion often associated with multipolar cell division. Clinical relevance for oncology and embryo selection in assisted reproduction is also briefly discussed in this context. Copyright © 2014 Elsevier GmbH. All rights reserved.

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

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

    2014-01-01

    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. PMID:25202032

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

    Science.gov (United States)

    Pilaz, Louis-Jan; McMahon, John J; Miller, Emily E; Lennox, Ashley L; Suzuki, Aussie; Salmon, Edward; Silver, Debra L

    2016-01-06

    Embryonic neocortical development depends on balanced production of progenitors and neurons. Genetic mutations disrupting progenitor mitosis frequently impair neurogenesis; however, the link between altered mitosis and cell fate remains poorly understood. Here we demonstrate that prolonged mitosis of radial glial progenitors directly alters neuronal fate specification and progeny viability. Live imaging of progenitors from a neurogenesis mutant, Magoh(+/-), reveals that mitotic delay significantly correlates with preferential production of neurons instead of progenitors, as well as apoptotic progeny. Independently, two pharmacological approaches reveal a causal relationship between mitotic delay and progeny fate. As mitotic duration increases, progenitors produce substantially more apoptotic progeny or neurons. We show that apoptosis, but not differentiation, is p53 dependent, demonstrating that these are distinct outcomes of mitotic delay. Together our findings reveal that prolonged mitosis is sufficient to alter fates of radial glia progeny and define a new paradigm to understand how mitosis perturbations underlie brain size disorders such as microcephaly. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Xenopus LAP2β protein knockdown affects location of lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability.

    Science.gov (United States)

    Dubińska-Magiera, Magda; Chmielewska, Magdalena; Kozioł, Katarzyna; Machowska, Magdalena; Hutchison, Christopher J; Goldberg, Martin W; Rzepecki, Ryszard

    2016-05-01

    Xenopus LAP2β protein is the single isoform expressed in XTC cells. The protein localizes on heterochromatin clusters both at the nuclear envelope and inside a cell nucleus. The majority of XLAP2β fraction neither colocalizes with TPX2 protein during interphase nor can be immunoprecipitated with XLAP2β antibody. Knockdown of the XLAP2β protein expression in XTC cells by synthetic siRNA and plasmid encoded siRNA resulted in nuclear abnormalities including changes in shape of nuclei, abnormal chromatin structure, loss of nuclear envelope, mislocalization of integral membrane proteins of INM such as lamin B2, mislocalization of nucleoporins, and cell death. Based on timing of cell death, we suggest mechanism associated with nucleus reassembly or with entry into mitosis. This confirms that Xenopus LAP2 protein is essential for the maintenance of cell nucleus integrity and the process of its reassembly after mitosis.

  9. Actomyosin contractility rotates the cell nucleus.

    Science.gov (United States)

    Kumar, Abhishek; Maitra, Ananyo; Sumit, Madhuresh; Ramaswamy, Sriram; Shivashankar, G V

    2014-01-21

    The cell nucleus functions amidst active cytoskeletal filaments, but its response to their contractile stresses is largely unexplored. We study the dynamics of the nuclei of single fibroblasts, with cell migration suppressed by plating onto micro-fabricated patterns. We find the nucleus undergoes noisy but coherent rotational motion. We account for this observation through a hydrodynamic approach, treating the nucleus as a highly viscous inclusion residing in a less viscous fluid of orientable filaments endowed with active stresses. Lowering actin contractility selectively by introducing blebbistatin at low concentrations drastically reduced the speed and coherence of the angular motion of the nucleus. Time-lapse imaging of actin revealed a correlated hydrodynamic flow around the nucleus, with profile and magnitude consistent with the results of our theoretical approach. Coherent intracellular flows and consequent nuclear rotation thus appear to be an intrinsic property of cells.

  10. 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.

  11. 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...

  12. [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.

  13. TMBP200, a XMAP215 homologue of tobacco BY-2 cells, has an essential role in plant mitosis.

    Science.gov (United States)

    Yasuhara, Hiroki; Oe, Yuki

    2011-07-01

    TMBP200 from tobacco BY-2 cells is a member of the highly conserved family of microtubule-associated proteins that includes Xenopus XMAP215, human TOGp, and Arabidopsis MOR1/GEM1. XMAP215 homologues have an essential role in spindle assembly and function in animals and yeast, but their role in plant mitosis is not fully clarified. Here, we show by immunoblot analysis that TMBP200 levels in synchronously cultured BY-2 cells increased when the cells entered mitosis, thus indicating that TMBP200 plays an important role in mitosis in tobacco. To investigate the role of TMBP200 in mitosis, we employed inducible RNA interference to silence TMBP200 expression in BY-2 cells. The resulting depletion of TMBP200 caused severe defects in bipolar spindle formation and resulted in the appearance of multinucleated cells with variable-sized nuclei. This finding indicates that TMBP200 has an essential role in bipolar spindle formation and function.

  14. 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. © 2016. Published by The Company of Biologists Ltd.

  15. Restriction endonucleases from invasive Neisseria gonorrhoeae cause double-strand breaks and distort mitosis in epithelial cells during infection.

    Directory of Open Access Journals (Sweden)

    Linda Weyler

    Full Text Available The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

  16. Restriction endonucleases from invasive Neisseria gonorrhoeae cause double-strand breaks and distort mitosis in epithelial cells during infection.

    Science.gov (United States)

    Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

    2014-01-01

    The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

  17. 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

    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...

  18. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Effects of radiation and porphyrin on mitosis and chromosomes in human hematopoietic cell lines

    International Nuclear Information System (INIS)

    Tan, J.C.; Huang, C.C.; Fiel, R.J.

    1976-01-01

    The effect on mitosis of a human hematopoietic cell line RPMI-1788 treated with a metal chelate (Zn ++ ) of meso-tetra (p-carboxyphenyl) porphine (Zn-TCPP) alone at various concentrations or in combination with gamma-irradiation at various doses were studied. The results showed that both Zn-TCPP and radiation were effective in interfering with normal mitosis and that the effect of radiation was relatively more effective. Data also suggest interacting effects between Zn-TCPP and gamma-irradiation. At low doses of radiation, Zn-TCPP potentiated the effect of radiation. The reverse seemed to be true at a high dose of radiation. The effects of two porphyrins (Zn-TCPP and hematoporphyrin) and radiation on chromosomes were also studied. Chromosomal aberrations characteristic of radiation were observed. The porphyrins were found not to be effective chromosome-breaking agents under the experimental conditions tested

  20. O-Linked N-Acetylglucosamine Transiently Elevates in HeLa Cells during Mitosis

    Directory of Open Access Journals (Sweden)

    Viktória Fisi

    2018-05-01

    Full Text Available O-linked N-acetylglucosamine (O-GlcNAc is a dynamic post-translational modification of serine and threonine residues on nuclear and cytoplasmic proteins. O-GlcNAc modification influences many cellular mechanisms, including carbohydrate metabolism, signal transduction and protein degradation. Multiple studies also showed that cell cycle might be modulated by O-GlcNAc. Although the role of O-GlcNAc in the regulation of some cell cycle processes such as mitotic spindle organization or histone phosphorylation is well established, the general behaviour of O-GlcNAc regulation during cell cycle is still controversial. In this study, we analysed the dynamic changes of overall O-GlcNAc levels in HeLa cells using double thymidine block. O-GlcNAc levels in G1, S, G2 and M phase were measured. We observed that O-GlcNAc levels are significantly increased during mitosis in comparison to the other cell cycle phases. However, this change could only be detected when mitotic cells were enriched by harvesting round shaped cells from the G2/M fraction of the synchronized cells. Our data verify that O-GlcNAc is elevated during mitosis, but also emphasize that O-GlcNAc levels can significantly change in a short period of time. Thus, selection and collection of cells at specific cell-cycle checkpoints is a challenging, but necessary requirement for O-GlcNAc studies.

  1. Localization of spindle checkpoint proteins in cells undergoing mitosis with unreplicated genomes.

    Science.gov (United States)

    Johnson, Mary Kathrine; Cooksey, Amanda M; Wise, Dwayne A

    2008-11-01

    CHO cells can be arrested with hydoxyurea at the beginning of the DNA synthesis phase of the cell cycle. Subsequent treatment with the xanthine, caffeine, induces cells to bypass the S-phase checkpoint and enter unscheduled mitosis [Schlegel and Pardee,1986, Science 232:1264-1266]. These treated cells build a normal spindle and distribute kinetochores, unattached to chromosomes, to their daughter cells [Brinkley et al.,1988, Nature 336:251-254; Zinkowski et al.,1991, J Cell Biol 113:1091-1110; Wise and Brinkley,1997, Cell Motil Cytoskeleton 36:291-302; Balczon et al.,2003, Chromosoma 112:96-102]. To investigate how these cells distribute kinetochores to daughter cells, we analyzed the spindle checkpoint components, Mad2, CENP-E, and the 3F3 phosphoepitope, using immunofluorescence and digital microscopy. Even though the kinetochores were unpaired and DNA was fragmented, the tension, alignment, and motor components of the checkpoint were found to be present and localized as predicted in prometaphase and metaphase. This unusual mitosis proves that a cell can successfully localize checkpoint proteins and divide even when kinetochores are unpaired and fragmented. (c) 2008 Wiley-Liss, Inc.

  2. The dynamic landscape of the cell nucleus.

    Science.gov (United States)

    Austin, Christopher M; Bellini, Michel

    2010-01-01

    While the cell nucleus was described for the first time almost two centuries ago, our modern view of the nuclear architecture is primarily based on studies from the last two decades. This surprising late start coincides with the development of new, powerful strategies to probe for the spatial organization of nuclear activities in both fixed and live cells. As a result, three major principles have emerged: first, the nucleus is not just a bag filled with nucleic acids and proteins. Rather, many distinct functional domains, including the chromosomes, resides within the confines of the nuclear envelope. Second, all these nuclear domains are highly dynamic, with molecules exchanging rapidly between them and the surrounding nucleoplasm. Finally, the motion of molecules within the nucleoplasm appears to be mostly driven by random diffusion. Here, the emerging roles of several subnuclear domains are discussed in the context of the dynamic functions of the cell nucleus.

  3. Mitosis in storage cells of the eutardigrade Richtersius coronifer

    Czech Academy of Sciences Publication Activity Database

    Czerneková, Michaela; Jönsson, K. I.

    2016-01-01

    Roč. 178, č. 4 (2016), s. 888-896 ISSN 0024-4082 Institutional support: RVO:67985823 Keywords : cell division * coelomocytes * development * mitotic index * moulting * tardigrada Subject RIV: ED - Physiology Impact factor: 2.711, year: 2016

  4. 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)

  5. Structural dynamics of the cell nucleus

    Science.gov (United States)

    Wiegert, Simon; Bading, Hilmar

    2011-01-01

    Neuronal morphology plays an essential role in signal processing in the brain. Individual neurons can undergo use-dependent changes in their shape and connectivity, which affects how intracellular processes are regulated and how signals are transferred from one cell to another in a neuronal network. Calcium is one of the most important intracellular second messengers regulating cellular morphologies and functions. In neurons, intracellular calcium levels are controlled by ion channels in the plasma membrane such as NMDA receptors (NMDARs), voltage-gated calcium channels (VGCCs) and certain α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) as well as by calcium exchange pathways between the cytosol and internal calcium stores including the endoplasmic reticulum and mitochondria. Synaptic activity and the subsequent opening of ligand and/or voltage-gated calcium channels can initiate cytosolic calcium transients which propagate towards the cell soma and enter the nucleus via its nuclear pore complexes (NPCs) embedded in the nuclear envelope. We recently described the discovery that in hippocampal neurons the morphology of the nucleus affects the calcium dynamics within the nucleus. Here we propose that nuclear infoldings determine whether a nucleus functions as an integrator or detector of oscillating calcium signals. We outline possible ties between nuclear mophology and transcriptional activity and discuss the importance of extending the approach to whole cell calcium signal modeling in order to understand synapse-to-nucleus communication in healthy and dysfunctional neurons. PMID:21738832

  6. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis.

    Science.gov (United States)

    Terzaghi, L; Tessaro, I; Raucci, F; Merico, V; Mazzini, G; Garagna, S; Zuccotti, M; Franciosi, F; Lodde, V

    2016-08-02

    Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC1's localization at the mid-zone and mid-body of the mitotic and meiotic spindle.

  8. Preferential Phosphorylation on Old Histones during Early Mitosis in Human Cells*

    Science.gov (United States)

    Lin, Shu; Yuan, Zuo-Fei; Han, Yumiao; Marchione, Dylan M.; Garcia, Benjamin A.

    2016-01-01

    How histone post-translational modifications (PTMs) are inherited through the cell cycle remains poorly understood. Canonical histones are made in the S phase of the cell cycle. Combining mass spectrometry-based technologies and stable isotope labeling by amino acids in cell culture, we question the distribution of multiple histone PTMs on old versus new histones in synchronized human cells. We show that histone PTMs can be grouped into three categories according to their distributions. Most lysine mono-methylation and acetylation PTMs are either symmetrically distributed on old and new histones or are enriched on new histones. In contrast, most di- and tri-methylation PTMs are enriched on old histones, suggesting that the inheritance of different PTMs is regulated distinctly. Intriguingly, old and new histones are distinct in their phosphorylation status during early mitosis in the following three human cell types: HeLa, 293T, and human foreskin fibroblast cells. The mitotic hallmark H3S10ph is predominantly associated with old H3 at early mitosis and becomes symmetric with the progression of mitosis. This same distribution was observed with other mitotic phosphorylation marks, including H3T3/T6ph, H3.1/2S28ph, and H1.4S26ph but not S28/S31ph on the H3 variant H3.3. Although H3S10ph often associates with the neighboring Lys-9 di- or tri-methylations, they are not required for the asymmetric distribution of Ser-10 phosphorylation on the same H3 tail. Inhibition of the kinase Aurora B does not change the distribution despite significant reduction of H3S10ph levels. However, K9me2 abundance on the new H3 is significantly reduced after Aurora B inhibition, suggesting a cross-talk between H3S10ph and H3K9me2. PMID:27226594

  9. 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. ©2014 American Association for Cancer Research.

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

    International Nuclear Information System (INIS)

    Fernandez, Carlos; Lobo, Maria del Val T.; Gomez-Coronado, Diego; Lasuncion, Miguel A.

    2004-01-01

    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 14α-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

  11. Arrest of irradiated G1, S, or G2 cells at mitosis using nocodazole promotes repair of potentially lethal damage

    International Nuclear Information System (INIS)

    Iliakis, G.; Nuesse, M.

    1984-01-01

    The ability of synchronized Ehrlich ascites tumor cells, irradiated in G1, S, and G2 phases, to repair potentially lethal damage when arrested at mitosis by using 0.4 μg/ml nocodazole, a specific inhibitor of microtubule polymerization, has been studied. Cells irradiated in these phases were found to repair potentially lethal damage at mitosis. The extent of this repair was similar to that observed for cells irradiated at the same stages in the cell cycle but allowed to repair potentially lethal damage by incubating in balanced salt solution for 6 hr after X irradiation

  12. 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…

  13. Preferential Phosphorylation on Old Histones during Early Mitosis in Human Cells.

    Science.gov (United States)

    Lin, Shu; Yuan, Zuo-Fei; Han, Yumiao; Marchione, Dylan M; Garcia, Benjamin A

    2016-07-15

    How histone post-translational modifications (PTMs) are inherited through the cell cycle remains poorly understood. Canonical histones are made in the S phase of the cell cycle. Combining mass spectrometry-based technologies and stable isotope labeling by amino acids in cell culture, we question the distribution of multiple histone PTMs on old versus new histones in synchronized human cells. We show that histone PTMs can be grouped into three categories according to their distributions. Most lysine mono-methylation and acetylation PTMs are either symmetrically distributed on old and new histones or are enriched on new histones. In contrast, most di- and tri-methylation PTMs are enriched on old histones, suggesting that the inheritance of different PTMs is regulated distinctly. Intriguingly, old and new histones are distinct in their phosphorylation status during early mitosis in the following three human cell types: HeLa, 293T, and human foreskin fibroblast cells. The mitotic hallmark H3S10ph is predominantly associated with old H3 at early mitosis and becomes symmetric with the progression of mitosis. This same distribution was observed with other mitotic phosphorylation marks, including H3T3/T6ph, H3.1/2S28ph, and H1.4S26ph but not S28/S31ph on the H3 variant H3.3. Although H3S10ph often associates with the neighboring Lys-9 di- or tri-methylations, they are not required for the asymmetric distribution of Ser-10 phosphorylation on the same H3 tail. Inhibition of the kinase Aurora B does not change the distribution despite significant reduction of H3S10ph levels. However, K9me2 abundance on the new H3 is significantly reduced after Aurora B inhibition, suggesting a cross-talk between H3S10ph and H3K9me2. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. 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...

  15. MIIP, a cytoskeleton regulator that blocks cell migration and invasion, delays mitosis, and suppresses tumorogenesis.

    Science.gov (United States)

    Wang, Yingmei; Wen, Jing; Zhang, Wei

    2011-02-01

    The migration and invasion inhibitory protein (MIIP) was initially discovered in a yeast two-hybrid screen for proteins that interact and inhibit the migration and invasion-promoting protein insulin-like growth factor binding protein 2 (IGFBP2). Recent studies have shown that MIIP not only modulates IGFBP2 but also regulates microtubule by binding to and inhibiting HDAC6, a class 2 histone deacetylase that deacetylates α-tubulin, heat-shock protein 90 (HSP90), and cortactin. In addition, MIIP also regulates the mitosis checkpoint, another microtubule-associated process. The location of the MIIP gene in chromosomal region 1p36, a commonly deleted region in a broad spectrum of human cancers, and the observation that MIIP attenuates tumorigenesis in a mouse model suggest that it functions as a tumor-suppressor gene. This review summarizes the recent progress in characterizing this novel protein, which regulates cell migration and mitosis, two processes that rely on the highly coordinated dynamics of the microtubule and cytoskeleton systems.

  16. [The perichromatin compartment of the cell nucleus].

    Science.gov (United States)

    Bogoliubov, D S

    2014-01-01

    In this review, the data on the structure and composition of the perichromatin compartment, a special border area between the condensed chromatin and the interchromatin space of the cell nucleus, are discussed in the light of the concept of nuclear functions in complex nuclear architectonics. Morphological features, molecular composition and functions of main extrachromosomal structures of the perichromatin compartment, perichromatin fibrils (PFs) and perichromatin granules (PGs) including nuclear stress-bodies (nSBs) that are derivates of the PGs under heat shock, are presented. A special attention was paid to the features of the molecular compositions of PFs and PGs in different cell types and at different physiological conditions.

  17. Automatic Detection of Mitosis and Nuclei From Cytogenetic Images by CellProfiler Software for Mitotic Index Estimation.

    Science.gov (United States)

    González, Jorge Ernesto; Radl, Analía; Romero, Ivonne; Barquinero, Joan Francesc; García, Omar; Di Giorgio, Marina

    2016-12-01

    Mitotic Index (MI) estimation expressed as percentage of mitosis plays an important role as quality control endpoint. To this end, MI is applied to check the lot of media and reagents to be used throughout the assay and also to check cellular viability after blood sample shipping, indicating satisfactory/unsatisfactory conditions for the progression of cell culture. The objective of this paper was to apply the CellProfiler open-source software for automatic detection of mitotic and nuclei figures from digitized images of cultured human lymphocytes for MI assessment, and to compare its performance to that performed through semi-automatic and visual detection. Lymphocytes were irradiated and cultured for mitosis detection. Sets of images from cultures were analyzed visually and findings were compared with those using CellProfiler software. The CellProfiler pipeline includes the detection of nuclei and mitosis with 80% sensitivity and more than 99% specificity. We conclude that CellProfiler is a reliable tool for counting mitosis and nuclei from cytogenetic images, saves considerable time compared to manual operation and reduces the variability derived from the scoring criteria of different scorers. The CellProfiler automated pipeline achieves good agreement with visual counting workflow, i.e. it allows fully automated mitotic and nuclei scoring in cytogenetic images yielding reliable information with minimal user intervention. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Automatic Detection of Mitosis and Nuclei from Cytogenetic Images by CellProfiler Software for Mitotic Index Estimation

    International Nuclear Information System (INIS)

    Gonzalez, Jorge Ernesto; Romero, Ivonne; Garcia, Omar; Radl, Analia; Di Giorgio, Marina; Barquinero, Joan Francesc

    2016-01-01

    Mitotic Index (MI) estimation expressed as percentage of mitosis plays an important role as quality control endpoint. To this end, MI is applied to check the lot of media and reagents to be used throughout the assay and also to check cellular viability after blood sample shipping, indicating satisfactory/unsatisfactory conditions for the progression of cell culture. The objective of this paper was to apply the CellProfiler open-source software for automatic detection of mitotic and nuclei figures from digitized images of cultured human lymphocytes for MI assessment, and to compare its performance to that performed through semi-automatic and visual detection. Lymphocytes were irradiated and cultured for mitosis detection. Sets of images from cultures were analyzed visually and findings were compared with those using CellProfiler software. The CellProfiler pipeline includes the detection of nuclei and mitosis with 80% sensitivity and more than 99% specificity. We conclude that CellProfiler is a reliable tool for counting mitosis and nuclei from cytogenetic images, saves considerable time compared to manual operation and reduces the variability derived from the scoring criteria of different scorers. The CellProfiler automated pipeline achieves good agreement with visual counting workflow, i.e. it allows fully automated mitotic and nuclei scoring in cytogenetic images yielding reliable information with minimal user intervention. (authors)

  19. Histochemical applications of x-ray microanalysis: the simultaneous assessment of mitosis and cell death

    International Nuclear Information System (INIS)

    Bowen, I.D.; Lewis, G.H.

    1980-01-01

    The principles of x-ray microanalytical histochemistry are reviewed. The use of labelling and precipitation techniques are examined, and particular attention is paid to the localization of enzymatic activity. A new method is described for the simultaneous assessment of mitosis as represented by the incorporation of ( 3 H) thymidine, and cell death as represented by the localization of free acid phosphatase, in the same tissue section. The thymidine incorporation is demonstrated by the appearance of topographically and microanalytically detectable silver grains in an overlying emulsion and the cell lysis associated acid phosphatase activity is detected optically and microanalytically by means of a bromine-rich azo dye deposited as a result of coupling naphthol AS BI, enzymatically released from naphthyl AS BI phosphoric acid, with diazotized 2,5-dibromoaniline

  20. Progesterone Receptor Membrane Component 1 (PGRMC1 in cell division: its role in bovine granulosa cells mitosis

    Directory of Open Access Journals (Sweden)

    Laura Terzaghi

    2015-07-01

    Full Text Available The present studies were aimed to assess Progesterone Receptor Membrane Component-1 (PGRMC1 role in regulating bovine granulosa cells (bGC mitosis. First, we performed immunofluorescence studies on in vitro cultured bGC collected from antral follicles, which showed that PGRMC1 localizes to the spindle apparatus in mitotic cells. Then, to evaluate PGRMC1 effect on cell proliferation we silenced its expression with RNA interference technique (RNAi. Quantitative RT-PCR and immunoblotting confirmed down-regulation of PGRMC1 expression, when compared to CTRL-RNAi treated bGC (p<0.05. After 72h of culture, PGRMC1 silencing determined a lower growth rate (p<0.05 and a higher percentage of cells arrested at G2/M phase as assessed by flowcytometry (p<0.05. Accordingly, live imaging studies revealed more aberrant mitosis and a delayed M-phase in PGRMC1-RNAi treated cells compared to CTRL-RNAi group (p<0.05. These data confirmed that PGRMC1 is directly involved in bGC mitosis and ongoing preliminary studies are aimed to elucidate its putative mechanisms of action. Since PGRMC1 is a membrane protein, we hypothesize its possible involvement in vesicular trafficking and endocytosis, which is in turn an important process to assure proper cell division. To assess this hypothesis, we have preliminarily conducted immunofluorescence and in situ proximity ligation assay experiments that showed PGRMC1 co-localization and direct interaction with clathrin. This is important since clathrin is an essential protein for both endosomes formation, and cell division acting directly on the spindle apparatus. Thus our studies set the stage for analysis aimed to further characterize PGRMC1’s mechanism of action in mitotic cell.

  1. Nonanaplastic follicular cell-derived thyroid carcinoma: mitosis and necrosis in long-term follow-up.

    Science.gov (United States)

    Skansing, Daniel Bräuner; Londero, Stefano Christian; Asschenfeldt, Pia; Larsen, Stine Rosenkilde; Godballe, Christian

    2017-06-01

    Nonanaplastic follicular cell-derived thyroid carcinoma (NAFCTC) includes differentiated- (DTC) and poorly differentiated thyroid carcinoma (PDTC). DTC has an excellent prognosis, while PDTC is situated between DTC and anaplastic carcinomas. Short-term studies suggest that PDTC patients diagnosed only on tumor necrosis and/or mitosis have a prognosis similar to those diagnosed according to the TURIN proposal. The purpose of this study was to evaluate prognosis for NAFCTC based on long-term follow-up illuminating the significance of tumor necrosis and mitosis. A cohort of 225 patients with NAFCTC was followed more than 20 years. Age, sex, distant metastasis, histology, tumor size, extrathyroidal invasion, lymph node metastasis, tumor necrosis and mitosis were examined as possible prognostic factors. Median follow-up time for patients alive was 28 years (range 20-43 years). Age, distant metastasis, extrathyroidal invasion, tumor size, tumor necrosis and mitosis were independent prognostic factors in multivariate analysis for overall survival (OS). In disease specific survival (DSS) age was not significant. Using only necrosis and/or mitosis as criteria for PDTC the 5-, 10- and 20-year OS for DTC was 87, 79 and 69%, respectively. In DSS it was 95, 92 and 90%. For PDTC the 5-, 10- and 20-year OS was 57, 40 and 25%, respectively. In DSS it was 71, 55 and 48%. Tumor necrosis and mitosis are highly significant prognostic indicators in analysis of long time survival of nonanaplastic follicular cell-derived thyroid carcinoma indicating that a simplification of the actually used criteria for poorly differentiated carcinomas may be justified.

  2. Chinese hamster ovary cell mitosis and its response to ionizing radiation: A morphological analysis of the living cell

    International Nuclear Information System (INIS)

    Carlson, J.G.

    1989-01-01

    Repeated microscopic observations of exponentially growing Chinese hamster ovary cells were made and the times and mitotic stages were recorded in control and irradiated cultures at 37 degree C. As determined by autoradiography, the time from the end of S phase to early prophase (the G2 phase) was 46 min, to breakdown of the nuclear envelope was 91 min, and to restoration of the nuclear envelope was 116 min. The time spent in morphologically distinguishable phases of mitosis and the effects of 0.5, 1.0, 1.5, 2.0, and 4.0 Gy of gamma or X radiation on cells at each phase were determined. Affected cells were found to be delayed without or with reversion to an earlier mitotic stage before recovering and advancing through mitosis. Cells were timed in the five steps comprising delay with reversion: inertia, cessation I, regression, cessation II, and reprogression. No cells treated in late prophase, i.e., within 8-10 min of nuclear envelope breakdown, were delayed by the doses used; therefore the critical or transition point must be situated in middle prophase. Cells irradiated in this stage were not delayed by 0.5 or 1.0 Gy, but suffered a dose-dependent delay with or without reversion after 1.5, 2.0, and 4.0 Gy. Cells irradiated in early prophase and very late interphase responded similarly, but a greater percentage of the latter reverted

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

    Science.gov (United States)

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

    2011-01-01

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

  4. 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.

  5. 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…

  6. Plant WEE1 kinase is cell cycle regulated and removed at mitosis via the 26S proteasome machinery

    Science.gov (United States)

    Cook, Gemma S.; Grønlund, Anne Lentz; Siciliano, Ilario; Spadafora, Natasha; Amini, Maryam; Herbert, Robert J.; Bitonti, M. Beatrice; Graumann, Katja; Francis, Dennis; Rogers, Hilary J.

    2013-01-01

    In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of cyclin-dependent kinase (CDK) by WEE1 kinase, and, at mitosis, WEE1 protein is removed through the action of the 26S proteasome. Although in higher plants WEE1 function has been confirmed in the DNA replication checkpoint, Arabidopsis wee1 insertion mutants grow normally, and a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be confirmed. Here data are presented showing that the inhibitory effect of WEE1 on CDK activity in tobacco BY-2 cell cultures is cell cycle regulated independently of the DNA replication checkpoint: it is high during S-phase but drops as cells traverse G2 and enter mitosis. To investigate this mechanism further, a yeast two-hybrid screen was undertaken to identify proteins interacting with Arabidopsis WEE1. Three F-box proteins and a subunit of the proteasome complex were identified, and bimolecular fluorescence complementation confirmed an interaction between AtWEE1 and the F-box protein SKP1 INTERACTING PARTNER 1 (SKIP1). Furthermore, the AtWEE1–green fluorescent protein (GFP) signal in Arabidopsis primary roots treated with the proteasome inhibitor MG132 was significantly increased compared with mock-treated controls. Expression of AtWEE1–YFPC (C-terminal portion of yellow fluorescent protein) or AtWEE1 per se in tobacco BY-2 cells resulted in a premature increase in the mitotic index compared with controls, whereas co-expression of AtSKIP1–YFPN negated this effect. These data support a role for WEE1 in a normal plant cell cycle and its removal at mitosis via the 26S proteasome. PMID:23536609

  7. Regulation of mitosis-meiosis transition by the ubiquitin ligase β-TrCP in male germ cells.

    Science.gov (United States)

    Nakagawa, Tadashi; Zhang, Teng; Kushi, Ryo; Nakano, Seiji; Endo, Takahiro; Nakagawa, Makiko; Yanagihara, Noriko; Zarkower, David; Nakayama, Keiko

    2017-11-15

    The mitosis-meiosis transition is essential for spermatogenesis. Specific and timely downregulation of the transcription factor DMRT1, and consequent induction of Stra8 expression, is required for this process in mammals, but the molecular mechanism has remained unclear. Here, we show that β-TrCP, the substrate recognition component of an E3 ubiquitin ligase complex, targets DMRT1 for degradation and thereby controls the mitosis-meiosis transition in mouse male germ cells. Conditional inactivation of β-TrCP2 in male germ cells of β-TrCP1 knockout mice resulted in sterility due to a lack of mature sperm. The β-TrCP-deficient male germ cells did not enter meiosis, but instead underwent apoptosis. The induction of Stra8 expression was also attenuated in association with the accumulation of DMRT1 at the Stra8 promoter in β-TrCP-deficient testes. DMRT1 contains a consensus β-TrCP degron sequence that was found to bind β-TrCP. Overexpression of β-TrCP induced the ubiquitylation and degradation of DMRT1. Heterozygous deletion of Dmrt1 in β-TrCP-deficient spermatogonia increased meiotic cells with a concomitant reduction of apoptosis. Collectively, our data indicate that β-TrCP regulates the transition from mitosis to meiosis in male germ cells by targeting DMRT1 for degradation. © 2017. Published by The Company of Biologists Ltd.

  8. 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

    2015-01-01

    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. PMID:26283799

  9. Dorsal raphe nucleus projecting retinal ganglion cells: Why Y cells?

    Science.gov (United States)

    Pickard, Gary E.; So, Kwok-Fai; Pu, Mingliang

    2015-01-01

    Retinal ganglion Y (alpha) cells are found in retinas ranging from frogs to mice to primates. The highly conserved nature of the large, fast conducting retinal Y cell is a testament to its fundamental task, although precisely what this task is remained ill-defined. The recent discovery that Y-alpha retinal ganglion cells send axon collaterals to the serotonergic dorsal raphe nucleus (DRN) in addition to the lateral geniculate nucleus (LGN), medial interlaminar nucleus (MIN), pretectum and the superior colliculus (SC) has offered new insights into the important survival tasks performed by these cells with highly branched axons. We propose that in addition to its role in visual perception, the Y-alpha retinal ganglion cell provides concurrent signals via axon collaterals to the DRN, the major source of serotonergic afferents to the forebrain, to dramatically inhibit 5-HT activity during orientation or alerting/escape responses, which dis-facilitates ongoing tonic motor activity while dis-inhibiting sensory information processing throughout the visual system. The new data provide a fresh view of these evolutionarily old retinal ganglion cells. PMID:26363667

  10. Altering the Rate of Mitosis by Introducing Low-Gigahertz Radiation to Saccharomyces cerevisiae Cells

    Science.gov (United States)

    Garg, S.; Ashby, C.

    2017-12-01

    This experiment aims to assess the impact of low-frequency radiation (from common technological tools such as cell phones, scanners, and wifi) on the mitotic rates of cells. In particular, the focus of the study was on the growth and development of Saccharomyces cerevisiae cultures that were exposed to radio waves from a wifi router, which were then compared to a cohort of the same species without exposure. Though routers emit a low gigahertz frequency, they are categorized as Group 2B radiation (possibly carcinogenic) by the International Agency for Research on Cancer of the World Health Organization, signifying that constant exposure poses a potential risk to humans. Twelve agar dishes of active Saccharomyces cerevisiae solution were prepared, with six dishes acting as the control under no added radiation and six acting as the experimental group under 2.4 GHz of radiation due to their proximity to the router. Data on how many cultures proliferated in each dish was collected every three days, with the experiment running for a total of twelve days. All subjects experienced growth curves until day 9 when the experimental group's growth peaked with an average of 62 colonies/dish. Three of the six dishes in this group lost colonies in the following three days, leaving the experimental group with an average of 61 colonies/dish on day 12, while the control group was still increasing by day 12 with an average of 48 colonies/dish, with only one dish undergoing a loss of colonies. Exposing the Saccharomyces cerevisiae cells to low grade radiation resulted in accelerated mitosis, and though the experimental group faced colony death after nine days, the loss was likely due to overpopulation in the dish.

  11. 53BP1 and USP28 mediate p53-dependent cell cycle arrest in response to centrosome loss and prolonged mitosis.

    Science.gov (United States)

    Fong, Chii Shyang; Mazo, Gregory; Das, Tuhin; Goodman, Joshua; Kim, Minhee; O'Rourke, Brian P; Izquierdo, Denisse; Tsou, Meng-Fu Bryan

    2016-07-02

    Mitosis occurs efficiently, but when it is disturbed or delayed, p53-dependent cell death or senescence is often triggered after mitotic exit. To characterize this process, we conducted CRISPR-mediated loss-of-function screens using a cell-based assay in which mitosis is consistently disturbed by centrosome loss. We identified 53BP1 and USP28 as essential components acting upstream of p53, evoking p21-dependent cell cycle arrest in response not only to centrosome loss, but also to other distinct defects causing prolonged mitosis. Intriguingly, 53BP1 mediates p53 activation independently of its DNA repair activity, but requiring its interacting protein USP28 that can directly deubiquitinate p53 in vitro and ectopically stabilize p53 in vivo. Moreover, 53BP1 can transduce prolonged mitosis to cell cycle arrest independently of the spindle assembly checkpoint (SAC), suggesting that while SAC protects mitotic accuracy by slowing down mitosis, 53BP1 and USP28 function in parallel to select against disturbed or delayed mitosis, promoting mitotic efficiency.

  12. Adhesion of axolemmal fragments to Schwann cells: a signal- and target-specific process closely linked to axolemmal induction of Schwann cell mitosis

    International Nuclear Information System (INIS)

    Sobue, G.; Pleasure, D.

    1985-01-01

    Radioiodinated rat CNS axolemmal fragments adhered to cultured rat Schwann cells by a time-, temperature-, and concentration-dependent process independent of extracellular ionized calcium. Adhesion showed target and signal specificity; axolemmal fragments adhered to endoneurial or dermal fibroblasts to a much lesser extent than to Schwann cells, and plasma membrane fragments from skeletal muscle, erythrocytes, or PNS myelin adhered to Schwann cells to a lesser extent than did axolemmal fragments. Brief trypsinization removed 94 to 97% of bound radioactivity from Schwann cells previously incubated with 125 I-axolemmal fragments for up to 24 hr, indicating that adhesion was largely a surface phenomenon rather than the result of rapid internalization of axolemmal fragments by the Schwann cells. When adhesion was compared to the axolemmal mitogenic response of Schwann cells, the concentration of axolemmal fragments yielding half-maximal adhesion was the same as the concentration producing half-maximal stimulation of Schwann cell mitosis. Trypsin digestion, homogenization, or heating of axolemmal fragments before application to cultured Schwann cells diminished adhesion and axolemmal fragment-induced stimulation of Schwann cell mitosis in a parallel fashion. Whereas adhesion of axolemmal fragments to the surfaces of the cultured Schwann cells reached completion within 4 hr in this assay system, induction of Schwann cell mitosis by the fragments required contact with Schwann cells for a minimum of 6 to 8 hr and reached a maximum when the axolemmal fragments had adhered to the Schwann cells for 24 hr or more

  13. Novel functions of plant cyclin-dependent kinase inhibitors, ICK1/KRP1, can act non-cell-autonomously and inhibit entry into mitosis

    DEFF Research Database (Denmark)

    Weinl, Christina; Marquardt, Sebastian; Kuijt, Suzanne J H

    2005-01-01

    numbers of cells consistent with a function of CKIs in blocking the G1-S cell cycle transition. Here, we demonstrate that at least one inhibitor from Arabidopsis, ICK1/KRP1, can also block entry into mitosis but allows S-phase progression causing endoreplication. Our data suggest that plant CKIs act...... independently from ICK1/KRP1-induced endoreplication. Strikingly, we found that endoreplicated cells were able to reenter mitosis, emphasizing the high degree of flexibility of plant cells during development. Moreover, we show that in contrast with animal CDK inhibitors, ICK1/KRP1 can move between cells...

  14. 3D Protein Dynamics in the Cell Nucleus.

    Science.gov (United States)

    Singh, Anand P; Galland, Rémi; Finch-Edmondson, Megan L; Grenci, Gianluca; Sibarita, Jean-Baptiste; Studer, Vincent; Viasnoff, Virgile; Saunders, Timothy E

    2017-01-10

    The three-dimensional (3D) architecture of the cell nucleus plays an important role in protein dynamics and in regulating gene expression. However, protein dynamics within the 3D nucleus are poorly understood. Here, we present, to our knowledge, a novel combination of 1) single-objective based light-sheet microscopy, 2) photoconvertible proteins, and 3) fluorescence correlation microscopy, to quantitatively measure 3D protein dynamics in the nucleus. We are able to acquire >3400 autocorrelation functions at multiple spatial positions within a nucleus, without significant photobleaching, allowing us to make reliable estimates of diffusion dynamics. Using this tool, we demonstrate spatial heterogeneity in Polymerase II dynamics in live U2OS cells. Further, we provide detailed measurements of human-Yes-associated protein diffusion dynamics in a human gastric cancer epithelial cell line. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  15. JNK Controls the Onset of Mitosis in Planarian Stem Cells and Triggers Apoptotic Cell Death Required for Regeneration and Remodeling

    Science.gov (United States)

    Almuedo-Castillo, María; Crespo, Xenia; Seebeck, Florian; Bartscherer, Kerstin; Salò, Emili; Adell, Teresa

    2014-01-01

    Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique model in which regeneration and tissue renewal can be comprehensively studied in vivo. The presence of a population of adult pluripotent stem cells combined with the ability to decode signaling after wounding enable planarians to regenerate a complete, correctly proportioned animal within a few days after any kind of amputation, and to adapt their size to nutritional changes without compromising functionality. Here, we demonstrate that the stress-activated c-jun–NH2–kinase (JNK) links wound-induced apoptosis to the stem cell response during planarian regeneration. We show that JNK modulates the expression of wound-related genes, triggers apoptosis and attenuates the onset of mitosis in stem cells specifically after tissue loss. Furthermore, in pre-existing body regions, JNK activity is required to establish a positive balance between cell death and stem cell proliferation to enable tissue renewal, remodeling and the maintenance of proportionality. During homeostatic degrowth, JNK RNAi blocks apoptosis, resulting in impaired organ remodeling and rescaling. Our findings indicate that JNK-dependent apoptotic cell death is crucial to coordinate tissue renewal and remodeling required to regenerate and to maintain a correctly proportioned animal. Hence, JNK might act as a hub, translating wound signals into apoptotic cell death, controlled stem cell proliferation and differentiation, all of which are required to coordinate regeneration and tissue renewal. PMID:24922054

  16. Cell Nucleus-Targeting Zwitterionic Carbon Dots.

    Science.gov (United States)

    Jung, Yun Kyung; Shin, Eeseul; Kim, Byeong-Su

    2015-12-22

    An innovative nucleus-targeting zwitterionic carbon dot (CD) vehicle has been developed for anticancer drug delivery and optical monitoring. The zwitterionic functional groups of the CDs introduced by a simple one-step synthesis using β-alanine as a passivating and zwitterionic ligand allow cytoplasmic uptake and subsequent nuclear translocation of the CDs. Moreover, multicolor fluorescence improves the accuracy of the CDs as an optical code. The CD-based drug delivery system constructed by non-covalent grafting of doxorubicin, exhibits superior antitumor efficacy owing to enhanced nuclear delivery in vitro and tumor accumulation in vivo, resulting in highly effective tumor growth inhibition. Since the zwitterionic CDs are highly biocompatible and effectively translocated into the nucleus, it provides a compelling solution to a multifunctional nanoparticle for substantially enhanced nuclear uptake of drugs and optical monitoring of translocation.

  17. Nucleus and nucleus-cytoskeleton connections in 3D cell migration

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Lingling, E-mail: liulingling2012@163.com; Luo, Qing, E-mail: qing.luo@cqu.edu.cn; Sun, Jinghui, E-mail: sunjhemail@163.com; Song, Guanbin, E-mail: song@cqu.edu.cn

    2016-10-15

    Cell migration plays an important role in many physiological and pathological settings, ranging from embryonic development to cancer metastasis. Currently, accumulating data suggest that cells migrating in three-dimensional (3D) environments show well-defined differences compared to their well-established two-dimensional (2D) counterparts. During 3D migration, the cell body and nucleus must deform to allow cellular passage through the available spaces, and the deformability of the relatively rigid nucleus may constitute a limiting step. Here, we highlight the key evidence regarding the role of the nuclear mechanics in 3D migration, including the molecular components that govern the stiffness of the nucleus and review how the nuclear dynamics are connected to and controlled by cytoskeleton-based migration machinery. Intriguingly, nuclear movement must be coordinated with the cytoskeletal dynamics at the leading and trailing edges, which in turn impact the cytoplasmic dynamics that affect the migration efficiency. Thus, we suggest that alterations in the nuclear structure may facilitate cellular reorganizations that are necessary for efficient migration. - Graphical abstract: Schematic representations of a cell migrating on a 2D substrate and a cell migrating in a 3D extracellular matrix environment. (A) Nucleus-cytoskeleton connections are essential to 3D migration. Mechanical signals are transduced by integrins at the cell surface and channeled to cytoskeletal proteins, which generates prestress. The nucleus-cytoskeleton connections can either act as a stable skeleton to anchor the nuclei or provide active force to move the nuclei. The LINC complex is responsible for the nucleo-cytoskeletal coupling. Nesprins connect the cytoskeletal proteins to the inner nuclear membrane proteins SUN1 and SUN2. The SUN proteins connect to the lamins that form the lamina, which attaches to the chromatin. This physical connectivity transmits the mechanical signals from receptors at

  18. Nucleus and nucleus-cytoskeleton connections in 3D cell migration

    International Nuclear Information System (INIS)

    Liu, Lingling; Luo, Qing; Sun, Jinghui; Song, Guanbin

    2016-01-01

    Cell migration plays an important role in many physiological and pathological settings, ranging from embryonic development to cancer metastasis. Currently, accumulating data suggest that cells migrating in three-dimensional (3D) environments show well-defined differences compared to their well-established two-dimensional (2D) counterparts. During 3D migration, the cell body and nucleus must deform to allow cellular passage through the available spaces, and the deformability of the relatively rigid nucleus may constitute a limiting step. Here, we highlight the key evidence regarding the role of the nuclear mechanics in 3D migration, including the molecular components that govern the stiffness of the nucleus and review how the nuclear dynamics are connected to and controlled by cytoskeleton-based migration machinery. Intriguingly, nuclear movement must be coordinated with the cytoskeletal dynamics at the leading and trailing edges, which in turn impact the cytoplasmic dynamics that affect the migration efficiency. Thus, we suggest that alterations in the nuclear structure may facilitate cellular reorganizations that are necessary for efficient migration. - Graphical abstract: Schematic representations of a cell migrating on a 2D substrate and a cell migrating in a 3D extracellular matrix environment. (A) Nucleus-cytoskeleton connections are essential to 3D migration. Mechanical signals are transduced by integrins at the cell surface and channeled to cytoskeletal proteins, which generates prestress. The nucleus-cytoskeleton connections can either act as a stable skeleton to anchor the nuclei or provide active force to move the nuclei. The LINC complex is responsible for the nucleo-cytoskeletal coupling. Nesprins connect the cytoskeletal proteins to the inner nuclear membrane proteins SUN1 and SUN2. The SUN proteins connect to the lamins that form the lamina, which attaches to the chromatin. This physical connectivity transmits the mechanical signals from receptors at

  19. Mechano-adaptation of the stem cell nucleus.

    Science.gov (United States)

    Heo, Su-Jin; Cosgrove, Brian D; Dai, Eric N; Mauck, Robert L

    2018-01-01

    Exogenous mechanical forces are transmitted through the cell and to the nucleus, initiating mechanotransductive signaling cascades with profound effects on cellular function and stem cell fate. A growing body of evidence has shown that the force sensing and force-responsive elements of the nucleus adapt to these mechanotransductive events, tuning their response to future mechanical input. The mechanisms underlying this "mechano-adaptation" are only just beginning to be elucidated, and it remains poorly understood how these components act and adapt in tandem to drive stem cell differentiation. Here, we review the evidence on how the stem cell nucleus responds and adapts to physical forces, and provide a perspective on how this mechano-adaptation may function to drive and enforce stem cell differentiation.

  20. Analysis of structural and numerical chromosomal aberrations at the first and second mitosis after X irradiation of two-cell mouse embryos

    International Nuclear Information System (INIS)

    Weissenborn, U.; Streffer, C.

    1989-01-01

    Two-cell mouse embryos were X-irradiated in the late G2 phase in vivo. The first and second postradiation mitoses were analyzed for chromosomal anomalies. The majority of structural aberrations visible at the first mitosis after irradiation were chromatid breaks and chromatid gaps; only a few interchanges and dicentrics were observed. The aberration frequency resulted in a dose-effect relationship which was well described by a linear model. At the second mitosis 29% of the structural aberrations of the first mitosis were counted; the aberration quality changed only slightly. It is discussed whether these aberrations are to be considered new, derived, or unchanged transmitted aberrations. Contrary to the results obtained after irradiation of one-cell embryos, little chromosome loss was induced by radiation in two-cell embryos

  1. 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.

  2. 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.

  3. Mitosis delay in cells of the root meristem of pea seedlings in S and G2-phases when irradiated with gamma-rays

    International Nuclear Information System (INIS)

    Gudkov, I.N.; Zezina, N.V.

    1976-01-01

    Irradiation (800 rads) of pea seedlings, synchronized by a 24-hr treatment with 0.03% hydroxyurea, at the stage of G 1 →S, induced a 12-hr delay of mitosis peak; an 8-hr delay, in the early S-phase; a 4-hr delay, in the middle of S-phase; a 10-hr delay in the late S- and a 14-16-hr delay, in G 2 -phase. The number of cells having chromosome aberrations at the mitosis peak was similar in all the phases under study

  4. Relocalization of human chromatin remodeling cofactor TIP48 in mitosis

    International Nuclear Information System (INIS)

    Sigala, Barbara; Edwards, Mina; Puri, Teena; Tsaneva, Irina R.

    2005-01-01

    TIP48 is a highly conserved eukaryotic AAA + protein which is an essential cofactor for several complexes involved in chromatin acetylation and remodeling, transcriptional and developmental regulation and nucleolar organization and trafficking. We show that TIP48 abundance in HeLa cells did not change during the cell cycle, nor did its distribution in various biochemical fractions. However, we observed distinct changes in the subcellular localization of TIP48 during M phase using immunofluorescence microscopy. Our studies demonstrate that in interphase cells TIP48 was found mainly in the nucleus and exhibited a distinct localization in the nuclear periphery. As the cells entered mitosis, TIP48 was excluded from the condensing chromosomes but showed association with the mitotic apparatus. During anaphase, some TIP48 was detected in the centrosome colocalizing with tubulin but the strongest staining appeared in the mitotic equator associated with the midzone central spindle. Accumulation of TIP48 in the midzone and the midbody was observed in late telophase and cytokinesis. This redeployment of TIP48 during anaphase and cytokinesis was independent of microtubule assembly. The relocation of endogenous TIP48 to the midzone/midbody under physiological conditions suggests a novel and distinct function for TIP48 in mitosis and possible involvement in the exit of mitosis

  5. Identification of a novel centrosomal protein CrpF46 involved in cell cycle progression and mitosis

    International Nuclear Information System (INIS)

    Wei Yi; Shen Enzhi; Zhao Na; Liu Qian; Fan Jinling; Marc, Jan; Wang Yongchao; Sun Le; Liang Qianjin

    2008-01-01

    A novel centrosome-related protein Crp F46 was detected using a serum F46 from a patient suffering from progressive systemic sclerosis. We identified the protein by immunoprecipitation and Western blotting followed by tandem mass spectrometry sequencing. The protein Crp F46 has an apparent molecular mass of ∼ 60 kDa, is highly homologous to a 527 amino acid sequence of the C-terminal portion of the protein Golgin-245, and appears to be a splice variant of Golgin-245. Immunofluorescence microscopy of synchronized HeLa cells labeled with an anti-Crp F46 monoclonal antibody revealed that Crp F46 localized exclusively to the centrosome during interphase, although it dispersed throughout the cytoplasm at the onset of mitosis. Domain analysis using Crp F46 fragments in GFP-expression vectors transformed into HeLa cells revealed that centrosomal targeting is conferred by a C-terminal coiled-coil domain. Antisense Crp F46 knockdown inhibited cell growth and proliferation and the cell cycle typically stalled at S phase. The knockdown also resulted in the formation of poly-centrosomal and multinucleate cells, which finally became apoptotic. These results suggest that Crp F46 is a novel centrosome-related protein that associates with the centrosome in a cell cycle-dependent manner and is involved in the progression of the cell cycle and M phase mechanism

  6. Anther-preferential expressing gene PMR is essential for the mitosis of pollen development in rice.

    Science.gov (United States)

    Liu, Yaqin; Xu, Ya; Ling, Sheng; Liu, Shasha; Yao, Jialing

    2017-06-01

    Phenotype identification, expression examination, and function prediction declared that the anther-preferential expressing gene PMR may participate in regulation of male gametophyte development in rice. Male germline development in flowering plants produces the pair of sperm cells for double fertilization and the pollen mitosis is a key process of it. Although the structural features of male gametophyte have been defined, the molecular mechanisms regulating the mitotic cell cycle are not well elucidated in rice. Here, we reported an anther-preferential expressing gene in rice, PMR (Pollen Mitosis Relative), playing an essential role in male gametogenesis. When PMR gene was suppressed via RNAi, the mitosis of microspore was severely damaged, and the plants formed unmatured pollens containing only one or two nucleuses at the anthesis, ultimately leading to serious reduction of pollen fertility and seed-setting. The CRISPR mutants, pmr-1 and pmr-2, both showed the similar defects as the PMR-RNAi lines. Further analysis revealed that PMR together with its co-expressing genes were liable to participate in the regulation of DNA metabolism in the nucleus, and affected the activities of some enzymes related to the cell cycle. We finally discussed that unknown protein PMR contained the PHD, SWIB and Plus-3 domains and they might have coordinating functions in regulation pathway of the pollen mitosis in rice.

  7. The SUMO Pathway in Mitosis.

    Science.gov (United States)

    Mukhopadhyay, Debaditya; Dasso, Mary

    2017-01-01

    Mitosis is the stage of the cell cycle during which replicated chromosomes must be precisely divided to allow the formation of two daughter cells possessing equal genetic material. Much of the careful spatial and temporal organization of mitosis is maintained through post-translational modifications, such as phosphorylation and ubiquitination, of key cellular proteins. Here, we will review evidence that sumoylation, conjugation to the SUMO family of small ubiquitin-like modifiers, also serves essential regulatory roles during mitosis. We will discuss the basic biology of sumoylation, how the SUMO pathway has been implicated in particular mitotic functions, including chromosome condensation, centromere/kinetochore organization and cytokinesis, and what cellular proteins may be the targets underlying these phenomena.

  8. 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

  9. Control of cell nucleus shapes via micropillar patterns.

    Science.gov (United States)

    Pan, Zhen; Yan, Ce; Peng, Rong; Zhao, Yingchun; He, Yao; Ding, Jiandong

    2012-02-01

    We herein report a material technique to control the shapes of cell nuclei by the design of the microtopography of substrates to which the cells adhere. Poly(D,L-lactide-co-glycolide) (PLGA) micropillars or micropits of a series of height or depth were fabricated, and some surprising self deformation of the nuclei of bone marrow stromal cells (BMSCs) was found in the case of micropillars with a sufficient height. Despite severe nucleus deformation, BMSCs kept the ability of proliferation and differentiation. We further demonstrated that the shapes of cell nuclei could be regulated by the appropriate micropillar patterns. Besides circular and elliptoid shapes, some unusual nucleus shapes of BMSCs have been achieved, such as square, cross, dumbbell, and asymmetric sphere-protrusion. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  10. Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells.

    Science.gov (United States)

    Buschmann, H; Green, P; Sambade, A; Doonan, J H; Lloyd, C W

    2011-04-01

    Transient transformation with Agrobacterium is a widespread tool allowing rapid expression analyses in plants. However, the available methods generate expression in interphase and do not allow the routine analysis of dividing cells. Here, we present a transient transformation method (termed 'TAMBY2') to enable cell biological studies in interphase and cell division. Agrobacterium-mediated transient gene expression in tobacco BY-2 was analysed by Western blotting and quantitative fluorescence microscopy. Time-lapse microscopy of cytoskeletal markers was employed to monitor cell division. Double-labelling in interphase and mitosis enabled localization studies. We found that the transient transformation efficiency was highest when BY-2/Agrobacterium co-cultivation was performed on solid medium. Transformants produced in this way divided at high frequency. We demonstrated the utility of the method by defining the behaviour of a previously uncharacterized microtubule motor, KinG, throughout the cell cycle. Our analyses demonstrated that TAMBY2 provides a flexible tool for the transient transformation of BY-2 with Agrobacterium. Fluorescence double-labelling showed that KinG localizes to microtubules and to F-actin. In interphase, KinG accumulates on microtubule lagging ends, suggesting a minus-end-directed function in vivo. Time-lapse studies of cell division showed that GFP-KinG strongly labels preprophase band and phragmoplast, but not the metaphase spindle. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

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

    Science.gov (United States)

    Léguillier, Teddy; Vandormael-Pournin, Sandrine; Artus, Jérôme; Houlard, Martin; Picard, Christel; Bernex, Florence; Robine, Sylvie; Cohen-Tannoudji, Michel

    2012-07-15

    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.

  12. 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.

  13. Epimorphin regulates bile duct formation via effects on mitosis orientation in rat liver epithelial stem-like cells.

    Directory of Open Access Journals (Sweden)

    Junnian Zhou

    Full Text Available Understanding how hepatic precursor cells can generate differentiated bile ducts is crucial for studies on epithelial morphogenesis and for development of cell therapies for hepatobiliary diseases. Epimorphin (EPM is a key morphogen for duct morphogenesis in various epithelial organs. The role of EPM in bile duct formation (DF from hepatic precursor cells, however, is not known. To address this issue, we used WB-F344 rat epithelial stem-like cells as model for bile duct formation. A micropattern and a uniaxial static stretch device was used to investigate the effects of EPM and stress fiber bundles on the mitosis orientation (MO of WB cells. Immunohistochemistry of liver tissue sections demonstrated high EPM expression around bile ducts in vivo. In vitro, recombinant EPM selectively induced DF through upregulation of CK19 expression and suppression of HNF3alpha and HNF6, with no effects on other hepatocytic genes investigated. Our data provide evidence that EPM guides MO of WB-F344 cells via effects on stress fiber bundles and focal adhesion assembly, as supported by blockade EPM, beta1 integrin, and F-actin assembly. These blockers can also inhibit EPM-induced DF. These results demonstrate a new biophysical action of EPM in bile duct formation, during which determination of MO plays a crucial role.

  14. 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. © 2014 by the Society for the Study of Reproduction, Inc.

  15. 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.

  16. The Aurora A-HP1γ pathway regulates gene expression and mitosis in cells from the sperm lineage.

    Science.gov (United States)

    Leonard, Phoebe H; Grzenda, Adrienne; Mathison, Angela; Morbeck, Dean E; Fredrickson, Jolene R; de Assuncao, Thiago M; Christensen, Trace; Salisbury, Jeffrey; Calvo, Ezequiel; Iovanna, Juan; Coddington, Charles C; Urrutia, Raul; Lomberk, Gwen

    2015-05-29

    HP1γ, a well-known regulator of gene expression, has been recently identified to be a target of Aurora A, a mitotic kinase which is important for both gametogenesis and embryogenesis. The purpose of this study was to define whether the Aurora A-HP1γ pathway supports cell division of gametes and/or early embryos, using western blot, immunofluorescence, immunohistochemistry, electron microscopy, shRNA-based knockdown, site-directed mutagenesis, and Affymetrix-based genome-wide expression profiles. We find that the form of HP1γ phosphorylated by Aurora A, P-Ser83 HP1γ, is a passenger protein, which localizes to the spermatozoa centriole and axoneme. In addition, disruption in this pathway causes centrosomal abnormalities and aberrations in cell division. Expression profiling of male germ cell lines demonstrates that HP1γ phosphorylation is critical for the regulation of mitosis-associated gene expression networks. In female gametes, we observe that P-Ser83-HP1γ is not present in meiotic centrosomes of M2 oocytes, but after syngamy, it becomes detectable during cleavage divisions, coinciding with early embryonic genome activation. These results support the idea that phosphorylation of HP1γ by Aurora A plays a role in the regulation of gene expression and mitotic cell division in cells from the sperm lineage and in early embryos. Combined, this data is relevant to better understanding the function of HP1γ in reproductive biology.

  17. SnapShot: Phosphoregulation of Mitosis.

    Science.gov (United States)

    Burgess, Andrew; Vuong, Jenny; Rogers, Samuel; Malumbres, Marcos; O'Donoghue, Seán I

    2017-06-15

    During mitosis, a cell divides its duplicated genome into two identical daughter cells. This process must occur without errors to prevent proliferative diseases (e.g., cancer). A key mechanism controlling mitosis is the precise timing of more than 32,000 phosphorylation and dephosphorylation events by a network of kinases and counterbalancing phosphatases. The identity, magnitude, and temporal regulation of these events have emerged recently, largely from advances in mass spectrometry. Here, we show phosphoevents currently believed to be key regulators of mitosis. For an animated version of this SnapShot, please see http://www.cell.com/cell/enhanced/odonoghue2. Copyright © 2017. Published by Elsevier Inc.

  18. Direct observation of nanoparticle-cancer cell nucleus interactions.

    Science.gov (United States)

    Dam, Duncan Hieu M; Lee, Jung Heon; Sisco, Patrick N; Co, Dick T; Zhang, Ming; Wasielewski, Michael R; Odom, Teri W

    2012-04-24

    We report the direct visualization of interactions between drug-loaded nanoparticles and the cancer cell nucleus. Nanoconstructs composed of nucleolin-specific aptamers and gold nanostars were actively transported to the nucleus and induced major changes to the nuclear phenotype via nuclear envelope invaginations near the site of the construct. The number of local deformations could be increased by ultrafast, light-triggered release of the aptamers from the surface of the gold nanostars. Cancer cells with more nuclear envelope folding showed increased caspase 3 and 7 activity (apoptosis) as well as decreased cell viability. This newly revealed correlation between drug-induced changes in nuclear phenotype and increased therapeutic efficacy could provide new insight for nuclear-targeted cancer therapy.

  19. 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

  20. 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. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. Dll1 maintains quiescence of adult neural stem cells and segregates asymmetrically during mitosis

    OpenAIRE

    Kawaguchi, Daichi; Furutachi, Shohei; Kawai, Hiroki; Hozumi, Katsuto; Gotoh, Yukiko

    2013-01-01

    Stem cells often divide asymmetrically to produce one stem cell and one differentiating cell, thus maintaining the stem cell pool. Although neural stem cells (NSCs) in the adult mouse subventricular zone have been suggested to divide asymmetrically, intrinsic cell fate determinants for asymmetric NSC division are largely unknown. Stem cell niches are important for stem cell maintenance, but the niche for the maintenance of adult quiescent NSCs has remained obscure. Here we show that the Notch...

  2. Bacterial mitosis

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette

    2003-01-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system...... act together to generate the force required for plasmid movement during segregation. ParR protein binds cooperatively to the centromeric parC DNA region, thereby forming a complex that interacts with the filament-forming actin-like ParM protein in an ATP-dependent manner, suggesting that plasmid...

  3. The roles of BDNF, pCREB and Wnt3a in the latent period preceding activation of progenitor cell mitosis in the adult dentate gyrus by fluoxetine.

    Directory of Open Access Journals (Sweden)

    Scarlett B Pinnock

    2010-10-01

    Full Text Available The formation of new neurons continues into adult life in the dentate gyrus of the rat hippocampus, as in many other species. Neurogenesis itself turns out to be highly labile, and is regulated by a number of factors. One of these is the serotoninergic system: treatment with drugs (such as the SSRI fluoxetine markedly stimulates mitosis in the progenitor cells of the dentate gyrus. But this process has one remarkable feature: it takes at least 14 days of continuous treatment to be effective. This is despite the fact that the pharmacological action of fluoxetine occurs within an hour or so of first administration. This paper explores the role of BDNF in this process, using the effect of a Trk antagonist (K252a on the labelling of progenitor cells with the mitosis marker Ki67 and the associated expression of pCREB and Wnt3a. These experiments show that (i Fluoxetine increased Ki67 counts, as well as pCREB and Wnt3a expression in the dentate gyrus. The action of fluoxetine on the progenitor cells and on pCREB (but not Wnt3a depends upon Trk receptor activation, since it was prevented by icv infusion of K252a. (ii These receptors are required for both the first 7 days of fluoxetine action, during which no apparent change in progenitor mitosis occurs, as well as the second 7 days. Increased pCREB was always associated with progenitor cell mitosis, but Wnt3a expression may be necessary but not sufficient for increased progenitor cell proliferation. These results shed new light on the action of fluoxetine on neurogenesis in the adult dentate gyrus, and have both clinical and experimental interest.

  4. 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.

  5. DeepMitosis: Mitosis detection via deep detection, verification and segmentation networks.

    Science.gov (United States)

    Li, Chao; Wang, Xinggang; Liu, Wenyu; Latecki, Longin Jan

    2018-04-01

    Mitotic count is a critical predictor of tumor aggressiveness in the breast cancer diagnosis. Nowadays mitosis counting is mainly performed by pathologists manually, which is extremely arduous and time-consuming. In this paper, we propose an accurate method for detecting the mitotic cells from histopathological slides using a novel multi-stage deep learning framework. Our method consists of a deep segmentation network for generating mitosis region when only a weak label is given (i.e., only the centroid pixel of mitosis is annotated), an elaborately designed deep detection network for localizing mitosis by using contextual region information, and a deep verification network for improving detection accuracy by removing false positives. We validate the proposed deep learning method on two widely used Mitosis Detection in Breast Cancer Histological Images (MITOSIS) datasets. Experimental results show that we can achieve the highest F-score on the MITOSIS dataset from ICPR 2012 grand challenge merely using the deep detection network. For the ICPR 2014 MITOSIS dataset that only provides the centroid location of mitosis, we employ the segmentation model to estimate the bounding box annotation for training the deep detection network. We also apply the verification model to eliminate some false positives produced from the detection model. By fusing scores of the detection and verification models, we achieve the state-of-the-art results. Moreover, our method is very fast with GPU computing, which makes it feasible for clinical practice. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Induction of Chromosomal Aberrations at Fluences of Less Than One HZE Particle per Cell Nucleus

    Science.gov (United States)

    Hada, Megumi; Chappell, Lori J.; Wang, Minli; George, Kerry A.; Cucinotta, Francis A.

    2014-01-01

    The assumption of a linear dose response used to describe the biological effects of high LET radiation is fundamental in radiation protection methodologies. We investigated the dose response for chromosomal aberrations for exposures corresponding to less than one particle traversal per cell nucleus by high energy and charge (HZE) nuclei. Human fibroblast and lymphocyte cells where irradiated with several low doses of <0.1 Gy, and several higher doses of up to 1 Gy with O (77 keV/ (long-s)m), Si (99 keV/ (long-s)m), Fe (175 keV/ (long-s)m), Fe (195 keV/ (long-s)m) or Fe (240 keV/ (long-s)m) particles. Chromosomal aberrations at first mitosis were scored using fluorescence in situ hybridization (FISH) with chromosome specific paints for chromosomes 1, 2 and 4 and DAPI staining of background chromosomes. Non-linear regression models were used to evaluate possible linear and non-linear dose response models based on these data. Dose responses for simple exchanges for human fibroblast irradiated under confluent culture conditions were best fit by non-linear models motivated by a non-targeted effect (NTE). Best fits for the dose response data for human lymphocytes irradiated in blood tubes were a NTE model for O and a linear response model fit best for Si and Fe particles. Additional evidence for NTE were found in low dose experiments measuring gamma-H2AX foci, a marker of double strand breaks (DSB), and split-dose experiments with human fibroblasts. Our results suggest that simple exchanges in normal human fibroblasts have an important NTE contribution at low particle fluence. The current and prior experimental studies provide important evidence against the linear dose response assumption used in radiation protection for HZE particles and other high LET radiation at the relevant range of low doses.

  7. Volume regulation and shape bifurcation in the cell nucleus.

    Science.gov (United States)

    Kim, Dong-Hwee; Li, Bo; Si, Fangwei; Phillip, Jude M; Wirtz, Denis; Sun, Sean X

    2015-09-15

    Alterations in nuclear morphology are closely associated with essential cell functions, such as cell motility and polarization, and correlate with a wide range of human diseases, including cancer, muscular dystrophy, dilated cardiomyopathy and progeria. However, the mechanics and forces that shape the nucleus are not well understood. Here, we demonstrate that when an adherent cell is detached from its substratum, the nucleus undergoes a large volumetric reduction accompanied by a morphological transition from an almost smooth to a heavily folded surface. We develop a mathematical model that systematically analyzes the evolution of nuclear shape and volume. The analysis suggests that the pressure difference across the nuclear envelope, which is influenced by changes in cell volume and regulated by microtubules and actin filaments, is a major factor determining nuclear morphology. Our results show that physical and chemical properties of the extracellular microenvironment directly influence nuclear morphology and suggest that there is a direct link between the environment and gene regulation. © 2015. Published by The Company of Biologists Ltd.

  8. Imaging Mitosis in the Moss Physcomitrella patens.

    Science.gov (United States)

    Yamada, Moé; Miki, Tomohiro; Goshima, Gohta

    2016-01-01

    At first glance, mitosis in plants looks quite different from that in animals. In fact, terrestrial plants have lost the centrosome during evolution, and the mitotic spindle is assembled independently of a strong microtubule organizing center. The phragmoplast is a plant-specific mitotic apparatus formed after anaphase, which expands centrifugally towards the cell cortex. However, the extent to which plant mitosis differs from that of animals at the level of the protein repertoire is uncertain, largely because of the difficulty in the identification and in vivo characterization of mitotic genes of plants. Here, we discuss protocols for mitosis imaging that can be combined with endogenous green fluorescent protein (GFP) tagging or conditional RNA interference (RNAi) in the moss Physcomitrella patens, which is an emergent model plant for cell and developmental biology. This system has potential for use in the high-throughput study of mitosis and other intracellular processes, as is being done with various animal cell lines.

  9. Tools for visualization of phosphoinositides in the cell nucleus.

    Science.gov (United States)

    Kalasova, Ilona; Fáberová, Veronika; Kalendová, Alžběta; Yildirim, Sukriye; Uličná, Lívia; Venit, Tomáš; Hozák, Pavel

    2016-04-01

    Phosphoinositides (PIs) are glycerol-based phospholipids containing hydrophilic inositol ring. The inositol ring is mono-, bis-, or tris-phosphorylated yielding seven PIs members. Ample evidence shows that PIs localize both to the cytoplasm and to the nucleus. However, tools for direct visualization of nuclear PIs are limited and many studies thus employ indirect approaches, such as staining of their metabolic enzymes. Since localization and mobility of PIs differ from their metabolic enzymes, these approaches may result in incomplete data. In this paper, we tested commercially available PIs antibodies by light microscopy on fixed cells, tested their specificity using protein-lipid overlay assay and blocking assay, and compared their staining patterns. Additionally, we prepared recombinant PIs-binding domains and tested them on both fixed and live cells by light microscopy. The results provide a useful overview of usability of the tools tested and stress that the selection of adequate tools is critical. Knowing the localization of individual PIs in various functional compartments should enable us to better understand the roles of PIs in the cell nucleus.

  10. 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.

  11. 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.

  12. The DNA damage response during mitosis

    International Nuclear Information System (INIS)

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

    2013-01-01

    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

  13. 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.

  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. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. 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

  16. [Role of helminth antigens in the abnormal mitosis of bone marrow cells in laboratory animals].

    Science.gov (United States)

    Sivkova, T N; Tatarnikova, N A; Berezhko, V K; Benediktov, I I

    2013-01-01

    The intraabdominal administration of somatic extracts of the cestodes Hydatigera taeniaformis Batsch 1786, Lamarck, 1816 and Diphyllobothrium latum Linnaeus, 1758 and the nematodes Anisakis simplex larva Rudolphi 1809, Toxocara canis Railliet et Henry, 1912 in albino mice proved that these helminths had a karyopathic effect on the bone marrow cells of the animals. The antigenic composition of these extracts was investigated using the agar gel immunodiffusion test. The antigenic composition of the parasites was ascertained to affect their karyopathic properties. The amount of antigens and their foreignness caused a marked karyopathic effect on the bone marrow cells of laboratory animals during intraabdominal administration.

  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

    International Nuclear Information System (INIS)

    Liu, Tongzheng; Schneider, Ryan A.; Hoyt, Dale G.

    2011-01-01

    Highlights: ► Depletion of PIN1 increases inhibitory effect of calpastatin against calpain in endothelial cells. ► PIN1 associates with calpastatin. ► PIN1, but not mutants, reduces the inhibitory activity of calpastatin in vitro. ► 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 μ- 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 μ- 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 calpastatin is required for PIN1 depletion to lower calpain activity. Thus, PIN1 apparently restrains

  19. 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

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

    NARCIS (Netherlands)

    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

    2012-01-01

    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

  1. Abnormal mitosis in root meristem cells of Allium cepa L. induced by ...

    African Journals Online (AJOL)

    This investigation was aimed to find mitotic abnormalities as cytological evidence induced by the dye in root tip cells of onion (Allium cepa L.) grown in different concentrations: 0.01, 0.05, 0.1, 0.5 and 1.0% (weight per volume) prepared in distilled water in separate treatment schedules for 24 and 48 h. Mitotic aberrations ...

  2. Mitosis and cell death in the optic lobes of workers, queens and ...

    Indian Academy of Sciences (India)

    Prakash

    of white, pink, brown and black eyes, and the beginning of tegument ... Our purpose was to investigate cell division and death in the optic lobes (OL) of workers, queens and males during ... new ones in each caste and sex. Nevertheless, most ...

  3. Au nanoinjectors for electrotriggered gene delivery into the cell nucleus.

    Science.gov (United States)

    Kang, Mijeong; Kim, Bongsoo

    2015-01-01

    Intracellular delivery of exogenous materials is an essential technique required for many fundamental biological researches and medical treatments. As our understanding of cell structure and function has been improved and diverse therapeutic agents with a subcellular site of action have been continuously developed, there is a demand to enhance the performance of delivering devices. Ideal intracellular delivery devices should convey various kinds of exogenous materials without deteriorating cell viability regardless of cell type and, furthermore, precisely control the location and the timing of delivery as well as the amount of delivered materials for advanced researches.In this chapter the development of a new intracellular delivery device, a nanoinjector made of a Au (gold) nanowire (a Au nanoinjector) is described in which delivery is triggered by external application of an electric pulse. As a model study, a gene was delivered directly into the nucleus of a neuroblastoma cell, and successful delivery without cell damage was confirmed by the expression of the delivered gene. The insertion of a Au nanoinjector directly into a cell can be generally applied to any kind of cell, and a high degree of surface modification of Au allows attachment of diverse materials such as proteins, small molecules, or nanoparticles as well as genes on Au nanoinjectors. This expands their applicability, and it is expected that they will provide important information on the effects of delivered exogenous materials and consequently contribute to the development of related therapeutic or clinical technologies.

  4. 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

  5. On the origin of shape fluctuations of the cell nucleus.

    Science.gov (United States)

    Chu, Fang-Yi; Haley, Shannon C; Zidovska, Alexandra

    2017-09-26

    The nuclear envelope (NE) presents a physical boundary between the cytoplasm and the nucleoplasm, sandwiched in between two highly active systems inside the cell: cytoskeleton and chromatin. NE defines the shape and size of the cell nucleus, which increases during the cell cycle, accommodating for chromosome decondensation followed by genome duplication. In this work, we study nuclear shape fluctuations at short time scales of seconds in human cells. Using spinning disk confocal microscopy, we observe fast fluctuations of the NE, visualized by fluorescently labeled lamin A, and of the chromatin globule surface (CGS) underneath the NE, visualized by fluorescently labeled histone H2B. Our findings reveal that fluctuation amplitudes of both CGS and NE monotonously decrease during the cell cycle, serving as a reliable cell cycle stage indicator. Remarkably, we find that, while CGS and NE typically fluctuate in phase, they do exhibit localized regions of out-of-phase motion, which lead to separation of NE and CGS. To explore the mechanism behind these shape fluctuations, we use biochemical perturbations. We find the shape fluctuations of CGS and NE to be both thermally and actively driven, the latter caused by forces from chromatin and cytoskeleton. Such undulations might affect gene regulation as well as contribute to the anomalously high rates of nuclear transport by, e.g., stirring of molecules next to NE, or increasing flux of molecules through the nuclear pores.

  6. Positioning of the NOR-bearing chromosomes in relation to nucleoli in daughter cells after mitosis

    Czech Academy of Sciences Publication Activity Database

    Kalmárová, Markéta; Smirnov, Evgeny; Kováčik, Lubomír; Popov, Alexey; Raška, Ivan

    2008-01-01

    Roč. 57, č. 3 (2008), s. 421-425 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GA304/06/1691 Grant - others:GA ČR(CZ) GA304/06/1662; GA MŠk(CZ) LC535; Wellcome Trust(XE) 075834/04/Z Program:LC Institutional research plan: CEZ:AV0Z50110509 Keywords : chromosome positioning * NORs * daughter cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.653, year: 2008

  7. Microtubules move the nucleus to quiescence.

    Science.gov (United States)

    Laporte, Damien; Sagot, Isabelle

    2014-01-01

    The nucleus is a cellular compartment that hosts several macro-molecular machines displaying a highly complex spatial organization. This tight architectural orchestration determines not only DNA replication and repair but also regulates gene expression. In budding yeast microtubules play a key role in structuring the nucleus since they condition the Rabl arrangement in G1 and chromosome partitioning during mitosis through their attachment to centromeres via the kinetochore proteins. Recently, we have shown that upon quiescence entry, intranuclear microtubules emanating from the spindle pole body elongate to form a highly stable bundle that spans the entire nucleus. Here, we examine some molecular mechanisms that may underlie the formation of this structure. As the intranuclear microtubule bundle causes a profound re-organization of the yeast nucleus and is required for cell survival during quiescence, we discuss the possibility that the assembly of such a structure participates in quiescence establishment.

  8. 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)

  9. 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)

  10. Models of chromatin spatial organisation in the cell nucleus

    Science.gov (United States)

    Nicodemi, Mario

    2014-03-01

    In the cell nucleus chromosomes have a complex architecture serving vital functional purposes. Recent experiments have started unveiling the interaction map of DNA sites genome-wide, revealing different levels of organisation at different scales. The principles, though, which orchestrate such a complex 3D structure remain still mysterious. I will overview the scenario emerging from some classical polymer physics models of the general aspect of chromatin spatial organisation. The available experimental data, which can be rationalised in a single framework, support a picture where chromatin is a complex mixture of differently folded regions, self-organised across spatial scales according to basic physical mechanisms. I will also discuss applications to specific DNA loci, e.g. the HoxB locus, where models informed with biological details, and tested against targeted experiments, can help identifying the determinants of folding.

  11. APC senses cell-cell contacts and moves to the nucleus upon their disruption.

    Science.gov (United States)

    Brocardo, M G; Bianchini, M; Radrizzani, M; Reyes, G B; Dugour, A V; Taminelli, G L; Gonzalez Solveyra, C; Santa-Coloma, T A

    2001-06-22

    The adenomatous polyposis coli (APC) tumor suppressor protein is involved in the Wnt/wingless pathway, modulating beta-catenin activity. We report the development of a highly specific, chemically synthesized oligobody (oligonucleotide-based synthetic antibody), directed against the N-terminal region of APC. Using this reagent, we found that within 16 h of disrupting HT-29 cell-cell contacts by harvesting cells with trypsin/EDTA treatment and replating, APC was translocated from the cytoplasm to the nucleus. Five days after plating the cells, when the cells had returned to their normal confluent phenotype and cell-cell contacts were reestablished, APC returned to the cytoplasm. These results suggest that APC functions as part of a "sensor" system, and responds to the loss of cell-cell contacts by moving to the nucleus, and returning to the cytoplasm when the contacts are fully restored. Copyright 2001 Academic Press.

  12. Meiosis: An Overview of Key Differences from Mitosis

    Science.gov (United States)

    Ohkura, Hiroyuki

    2015-01-01

    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. PMID:25605710

  13. Kinases Involved in Both Autophagy and Mitosis.

    Science.gov (United States)

    Li, Zhiyuan; Zhang, Xin

    2017-08-31

    Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases), Aurora kinases, PLK-1 (polo-like kinase 1), BUB1 (budding uninhibited by benzimidazoles 1), MAPKs (mitogen-activated protein kinases), mTORC1 (mechanistic target of rapamycin complex 1), AMPK (AMP-activated protein kinase), PI3K (phosphoinositide-3 kinase) and protein kinase B (AKT). By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  14. Kinases Involved in Both Autophagy and Mitosis

    Directory of Open Access Journals (Sweden)

    Zhiyuan Li

    2017-08-01

    Full Text Available Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases, Aurora kinases, PLK-1 (polo-like kinase 1, BUB1 (budding uninhibited by benzimidazoles 1, MAPKs (mitogen-activated protein kinases, mTORC1 (mechanistic target of rapamycin complex 1, AMPK (AMP-activated protein kinase, PI3K (phosphoinositide-3 kinase and protein kinase B (AKT. By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  15. 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

  16. 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

  17. 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.

  18. A 2D/3D image analysis system to track fluorescently labeled structures in rod-shaped cells: application to measure spindle pole asymmetry during mitosis.

    Science.gov (United States)

    Schmitter, Daniel; Wachowicz, Paulina; Sage, Daniel; Chasapi, Anastasia; Xenarios, Ioannis; Simanis; Unser, Michael

    2013-01-01

    The yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined. We present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and

  19. 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)

    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. PMID:23300382

  20. 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.

    2012-01-01

    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. PMID:22941662

  1. [Analysis of the Effect of Non-phacoemulsification Cataract Operation on Corneal Endothelial Cell Nucleus Division].

    Science.gov (United States)

    Huang, Zufeng; Miao, Xiaoqing

    2015-09-01

    To investigate the effect of non-phacoemulsification cataract operation in two different patterns of nucleus delivery on the quantity and morphology of corneal endothelial cells and postoperative visual acuity. Forty patients diagnosed with cataract underwent cataract surgery and were assigned into the direct nuclear delivery and semi-nuclear delivery groups. Lens density was measured and divided into the hard and soft lenses according to Emery-little lens nucleus grading system. Non-phacoemulsification cataract operation was performed. At 3 d after surgery, the quantity and morphology of corneal endothelium were counted and observed under corneal endothelial microscope. During 3-month postoperative follow-up, the endothelial cell loss rate, morphological changes and visual acuity were compared among four groups. Corneal endothelial cell loss rate in the direct delivery of hard nucleus group significantly differed from those in the other three groups before and 3 months after operation (P nucleus, semi-delivery of hard nucleus and semi-delivery soft nucleus groups (all P > 0.05). Preoperative and postoperative 2-d visual acuity did not differ between the semi-delivery of hard nucleus and direct delivery of soft nucleus groups (P = 0.49), significantly differed from those in the semi-delivery of soft nucleus (P = 0.03) and direct delivery of hard nucleus groups (P = 0.14). Visual acuity at postoperative four months did not differ among four groups (P = 0.067). During non-phacoemulsification cataract surgery, direct delivery of hard nucleus caused severe injury to corneal endothelium and semi-delivery of soft nucleus yielded mild corneal endothelial injury. Slight corneal endothelial injury exerted no apparent effect upon visual acuity and corneal endothelial morphology at three months after surgery.

  2. 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.)

  3. Identification of different subsets of lung cells using Raman microspectroscopy and whole cell nucleus isolation.

    Science.gov (United States)

    Pijanka, Jacek K; Stone, Nicholas; Rutter, Abigail V; Forsyth, Nicholas; Sockalingum, Ganesh D; Yang, Ying; Sulé-Suso, Josep

    2013-09-07

    Raman spectroscopy has been widely used to study its possible clinical application in cancer diagnosis. However, in order to make it into clinical practice, it is important that this technique is able not only to identify cancer cells from their normal counterparts, but also from the array of cells present in human tissues. To this purpose, we used Raman spectroscopy to assess whether this technique was able to differentiate not only between lung cancer cells and lung epithelial cells but also from lung fibroblasts. Furthermore, we studied whether the differences were due to cell lineage (epithelial versus fibroblast) or to different proliferative characteristics of cells, and where in the cell compartment these differences might reside. To answer these questions we studied cell cytoplasm, cell nucleus and isolated whole cell nuclei. Our data suggests that Raman spectroscopy can differentiate between lung cancer, lung epithelial cells and lung fibroblasts. More important, it can also differentiate between 2 cells from the same lineage (fibroblast) but with one of them rendered immortal and with an increased proliferative activity. Finally, it seems that the main spectral differences reside in the cell nucleus and that the study of isolated nuclei strengthens the differences between cells.

  4. Hoechst tagging: a modular strategy to design synthetic fluorescent probes for live-cell nucleus imaging.

    Science.gov (United States)

    Nakamura, Akinobu; Takigawa, Kazumasa; Kurishita, Yasutaka; Kuwata, Keiko; Ishida, Manabu; Shimoda, Yasushi; Hamachi, Itaru; Tsukiji, Shinya

    2014-06-11

    We report a general strategy to create small-molecule fluorescent probes for the nucleus in living cells. Our strategy is based on the attachment of the DNA-binding Hoechst compound to a fluorophore of interest. Using this approach, simple fluorescein, BODIPY, and rhodamine dyes were readily converted to novel turn-on fluorescent nucleus-imaging probes.

  5. 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.

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

    Science.gov (United States)

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

    1999-08-01

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

  7. The reorientation of cell nucleus promotes the establishment of front-rear polarity in migrating fibroblasts.

    Science.gov (United States)

    Maninová, Miloslava; Klímová, Zuzana; Parsons, J Thomas; Weber, Michael J; Iwanicki, Marcin P; Vomastek, Tomáš

    2013-06-12

    The establishment of cell polarity is an essential step in the process of cell migration. This process requires precise spatiotemporal coordination of signaling pathways that in most cells create the typical asymmetrical profile of a polarized cell with nucleus located at the cell rear and the microtubule organizing center (MTOC) positioned between the nucleus and the leading edge. During cell polarization, nucleus rearward positioning promotes correct microtubule organizing center localization and thus the establishment of front-rear polarity and directional migration. We found that cell polarization and directional migration require also the reorientation of the nucleus. Nuclear reorientation is manifested as temporally restricted nuclear rotation that aligns the nuclear axis with the axis of cell migration. We also found that nuclear reorientation requires physical connection between the nucleus and cytoskeleton mediated by the LINC (linker of nucleoskeleton and cytoskeleton) complex. Nuclear reorientation is controlled by coordinated activity of lysophosphatidic acid (LPA)-mediated activation of GTPase Rho and the activation of integrin, FAK (focal adhesion kinase), Src, and p190RhoGAP signaling pathway. Integrin signaling is spatially induced at the leading edge as FAK and p190RhoGAP are predominantly activated or localized at this location. We suggest that integrin activation within lamellipodia defines cell front, and subsequent FAK, Src, and p190RhoGAP signaling represents the polarity signal that induces reorientation of the nucleus and thus promotes the establishment of front-rear polarity. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Attributed relational graphs for cell nucleus segmentation in fluorescence microscopy images.

    Science.gov (United States)

    Arslan, Salim; Ersahin, Tulin; Cetin-Atalay, Rengul; Gunduz-Demir, Cigdem

    2013-06-01

    More rapid and accurate high-throughput screening in molecular cellular biology research has become possible with the development of automated microscopy imaging, for which cell nucleus segmentation commonly constitutes the core step. Although several promising methods exist for segmenting the nuclei of monolayer isolated and less-confluent cells, it still remains an open problem to segment the nuclei of more-confluent cells, which tend to grow in overlayers. To address this problem, we propose a new model-based nucleus segmentation algorithm. This algorithm models how a human locates a nucleus by identifying the nucleus boundaries and piecing them together. In this algorithm, we define four types of primitives to represent nucleus boundaries at different orientations and construct an attributed relational graph on the primitives to represent their spatial relations. Then, we reduce the nucleus identification problem to finding predefined structural patterns in the constructed graph and also use the primitives in region growing to delineate the nucleus borders. Working with fluorescence microscopy images, our experiments demonstrate that the proposed algorithm identifies nuclei better than previous nucleus segmentation algorithms.

  9. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Robust Nucleus/Cell Detection and Segmentation in Digital Pathology and Microscopy Images: A Comprehensive Review.

    Science.gov (United States)

    Xing, Fuyong; Yang, Lin

    2016-01-01

    Digital pathology and microscopy image analysis is widely used for comprehensive studies of cell morphology or tissue structure. Manual assessment is labor intensive and prone to interobserver variations. Computer-aided methods, which can significantly improve the objectivity and reproducibility, have attracted a great deal of interest in recent literature. Among the pipeline of building a computer-aided diagnosis system, nucleus or cell detection and segmentation play a very important role to describe the molecular morphological information. In the past few decades, many efforts have been devoted to automated nucleus/cell detection and segmentation. In this review, we provide a comprehensive summary of the recent state-of-the-art nucleus/cell segmentation approaches on different types of microscopy images including bright-field, phase-contrast, differential interference contrast, fluorescence, and electron microscopies. In addition, we discuss the challenges for the current methods and the potential future work of nucleus/cell detection and segmentation.

  11. β-catenin at the centrosome: discrete pools of β-catenin communicate during mitosis and may co-ordinate centrosome functions and cell cycle progression.

    Science.gov (United States)

    Mbom, Bertrade C; Nelson, W James; Barth, Angela

    2013-09-01

    Beta-catenin is a multifunctional protein with critical roles in cell-cell adhesion, Wnt-signaling and the centrosome cycle. Whereas the roles of β-catenin in cell-cell adhesion and Wnt-signaling have been studied extensively, the mechanism(s) involving β-catenin in centrosome functions are poorly understood. β-Catenin localizes to centrosomes and promotes mitotic progression. NIMA-related protein kinase 2 (Nek2), which stimulates centrosome separation, binds to and phosphorylates β-catenin. β-Catenin interacting proteins involved in Wnt signaling such as adenomatous polyposis coli, Axin, and GSK3β, are also localized at centrosomes and play roles in promoting mitotic progression. Additionally, proteins associated with cell-cell adhesion sites, such as dynein, regulate mitotic spindle positioning. These roles of proteins at the cell cortex and Wnt signaling that involve β-catenin indicate a cross-talk between different sub-cellular sites in the cell at mitosis, and that different pools of β-catenin may co-ordinate centrosome functions and cell cycle progression. © 2013 WILEY Periodicals, Inc.

  12. Spatial signals link exit from mitosis to spindle position.

    Science.gov (United States)

    Falk, Jill Elaine; Tsuchiya, Dai; Verdaasdonk, Jolien; Lacefield, Soni; Bloom, Kerry; Amon, Angelika

    2016-05-11

    In budding yeast, if the spindle becomes mispositioned, cells prevent exit from mitosis by inhibiting the mitotic exit network (MEN). The MEN is a signaling cascade that localizes to spindle pole bodies (SPBs) and activates the phosphatase Cdc14. There are two competing models that explain MEN regulation by spindle position. In the 'zone model', exit from mitosis occurs when a MEN-bearing SPB enters the bud. The 'cMT-bud neck model' posits that cytoplasmic microtubule (cMT)-bud neck interactions prevent MEN activity. Here we find that 1) eliminating cMT- bud neck interactions does not trigger exit from mitosis and 2) loss of these interactions does not precede Cdc14 activation. Furthermore, using binucleate cells, we show that exit from mitosis occurs when one SPB enters the bud despite the presence of a mispositioned spindle. We conclude that exit from mitosis is triggered by a correctly positioned spindle rather than inhibited by improper spindle position.

  13. Rescue from replication stress during mitosis.

    Science.gov (United States)

    Fragkos, Michalis; Naim, Valeria

    2017-04-03

    Genomic instability is a hallmark of cancer and a common feature of human disorders, characterized by growth defects, neurodegeneration, cancer predisposition, and aging. Recent evidence has shown that DNA replication stress is a major driver of genomic instability and tumorigenesis. Cells can undergo mitosis with under-replicated DNA or unresolved DNA structures, and specific pathways are dedicated to resolving these structures during mitosis, suggesting that mitotic rescue from replication stress (MRRS) is a key process influencing genome stability and cellular homeostasis. Deregulation of MRRS following oncogene activation or loss-of-function of caretaker genes may be the cause of chromosomal aberrations that promote cancer initiation and progression. In this review, we discuss the causes and consequences of replication stress, focusing on its persistence in mitosis as well as the mechanisms and factors involved in its resolution, and the potential impact of incomplete replication or aberrant MRRS on tumorigenesis, aging and disease.

  14. 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.

  15. Arabidopsis ASYMMETRIC LEAVES2 protein required for leaf morphogenesis consistently forms speckles during mitosis of tobacco BY-2 cells via signals in its specific sequence.

    Science.gov (United States)

    Luo, Lilan; Ando, Sayuri; Sasabe, Michiko; Machida, Chiyoko; Kurihara, Daisuke; Higashiyama, Tetsuya; Machida, Yasunori

    2012-09-01

    Leaf primordia with high division and developmental competencies are generated around the periphery of stem cells at the shoot apex. Arabidopsis ASYMMETRIC-LEAVES2 (AS2) protein plays a key role in the regulation of many genes responsible for flat symmetric leaf formation. The AS2 gene, expressed in leaf primordia, encodes a plant-specific nuclear protein containing an AS2/LOB domain with cysteine repeats (C-motif). AS2 proteins are present in speckles in and around the nucleoli, and in the nucleoplasm of some leaf epidermal cells. We used the tobacco cultured cell line BY-2 expressing the AS2-fused yellow fluorescent protein to examine subnuclear localization of AS2 in dividing cells. AS2 mainly localized to speckles (designated AS2 bodies) in cells undergoing mitosis and distributed in a pairwise manner during the separation of sets of daughter chromosomes. Few interphase cells contained AS2 bodies. Deletion analyses showed that a short stretch of the AS2 amino-terminal sequence and the C-motif play negative and positive roles, respectively, in localizing AS2 to the bodies. These results suggest that AS2 bodies function to properly distribute AS2 to daughter cells during cell division in leaf primordia; and this process is controlled at least partially by signals encoded by the AS2 sequence itself.

  16. Circadian pacemaking in cells and circuits of the suprachiasmatic nucleus.

    Science.gov (United States)

    Hastings, M H; Brancaccio, M; Maywood, E S

    2014-01-01

    The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal circadian pacemaker of the brain. It co-ordinates the daily rhythms of sleep and wakefulness, as well as physiology and behaviour, that set the tempo to our lives. Disturbance of this daily pattern, most acutely with jet-lag but more insidiously with rotational shift-work, can have severely deleterious effects for mental function and long-term health. The present review considers recent developments in our understanding of the properties of the SCN that make it a robust circadian time-keeper. It first focuses on the intracellular transcriptional/ translational feedback loops (TTFL) that constitute the cellular clockwork of the SCN neurone. Daily timing by these loops pivots around the negative regulation of the Period (Per) and Cryptochrome (Cry) genes by their protein products. The period of the circadian cycle is set by the relative stability of Per and Cry proteins, and this can be controlled by both genetic and pharmacological interventions. It then considers the function of these feedback loops in the context of cytosolic signalling by cAMP and intracellular calcium ([Ca(2+) ]i ), which are both outputs from, and inputs to, the TTFL, as well as the critical role of vasoactive intestinal peptide (VIP) signalling in synchronising cellular clocks across the SCN. Synchronisation by VIP in the SCN is paracrine, operating over an unconventionally long time frame (i.e. 24 h) and wide spatial domain, mediated via the cytosolic pathways upstream of the TTFL. Finally, we show how intersectional pharmacogenetics can be used to control G-protein-coupled signalling in individual SCN neurones, and how manipulation of Gq/[Ca(2+) ]i -signalling in VIP neurones can re-programme the circuit-level encoding of circadian time. Circadian pacemaking in the SCN therefore provides an unrivalled context in which to understand how a complex, adaptive behaviour can be organised by the dynamic activity of a relatively

  17. 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

    Nucleus transfer for the production of multiple embryos derived from a donor embryo relies upon the reprogramming of the donor nucleus so that it behaves similar to a zygotic nucleus. One indication of nucleus reprogramming is the RNA synthetic activity. In normal bovine embryogenesis, the embryo....... NTE were produced using either a MII phase (nonactivated) cytoplasts at 32 hr of maturation or S-phase (activated) cytoplasts activated with calcium ionophore A23187 and cycloheximide treatment approximately 8 hr prior to fusion with a blastomere from an in-vitro-produced morula stage embryo at 32 hr...... 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...

  18. ''Protective'' effect of cells gamma-irradiation at the metaphase of mitosis after UV-irradiation at the S-period

    Energy Technology Data Exchange (ETDEWEB)

    Lebedeva, L I; Chubykin, V L [AN SSSR, Novosibirsk. Inst. Tsitologii i Genetiki

    1975-10-01

    As a result of the ultraviolet irradiation in vitro of the embryo fibroblasts of BALB mice in the S-stage with an incident dose of 40 erg/mm/sup 2/, 20.1% cells showed chromosome aberrations. Additional gamma irradiation of cells in the metaphase of the first mitosis with a dose of 5 krad leads with a high degree of certainty to a decrease to 11.7% in the frequency of aberrant cells observed in the same mitotic stage. The frequency of spontaneous aberrations does not change during the first few minutes after the gamma irradiation of intact cells. The ''protective'' effect of gamma rays cannot be attributed to non-uniform changes in the duration of the mitotic stages for aberrant and normal cells, to the adhesion of chromosome fragments or to the breaking of bridges in the anaphase. The destruction of cells during irradiation is also an unlikely explanation of the observed effect. It is assumed that the decrease in the frequency of aberrations is a result of the previously predicted modification of the processes involved, when potential chromosome damage becomes visible abberations during metaphase.

  19. MiR-509-3-5p causes aberrant mitosis and anti-proliferative effect by suppression of PLK1 in human lung cancer A549 cells

    International Nuclear Information System (INIS)

    Wang, Xian-Hui; Lu, Yao; Liang, Jing-Jing; Cao, Ji-Xiang; Jin, Ya-Qiong; An, Guo-Shun; Ni, Ju-Hua; Jia, Hong-Ti; Li, Shu-Yan

    2016-01-01

    MicroRNAs (miRNAs) are potent post-transcriptional regulators of gene expression and play roles in DNA damage response (DDR). PLK1 is identified as a modulator of DNA damage checkpoint. Although down-regulation of PLK1 by certain microRNAs has been reported, little is known about the interplay between PLK1 and miR-509-3-5p in DDR. Here we have demonstrated that miR-509-3-5p repressed PLK1 expression by targeting PLK1 3′-UTR, thereby causing mitotic aberration and growth arrest of human lung cancer A549 cells. Repression of PLK1 by miR-509-3-5p was further evidenced by over-expression of miR-509-3-5p in A549, HepG2 and HCT116p53 −/− cancer cells, in which PLK1 protein was suppressed. Consistently, miR-509-3-5p was stimulated, while PLK1 protein was down-regulated in A549 cells exposed to CIS and ADR, suggesting that suppression of PLK1 by miR-509-3-5p is a component of CIS/ADR-induced DDR pathway. Flow cytometry and immunofluorescence labeling showed that over-expression of miR-509-3-5p in A549 induced G2/M arrest and aberrant mitosis characterized by abnormal bipolar mitotic spindles, condensed chromosomes, lagging DNA and chromosome bridges. In addition, over-expression of miR-509-3-5p markedly blocked A549 cell proliferation and sensitized the cells to CIS and ADR treatment. Taken together, miR-509-3-5p is a feasible suppressor for cancer by targeting PLK1. Our data may provide aid in potential design of combined chemotherapy and in our better understanding of the roles of microRNAs in response to DNA damage. - Highlights: • MiR-509-3-5p represses PLK1 expression by targeting PLK1 3ГЉВ№-UTR. • Expression of miR-509-3-5p is induced and PLK1 repressed upon DNA damage. • Overexpression of miR-509-3-5p induces G2/M arrest and aberrant mitosis. • MiR-509-3-5p inhibits cell proliferation and sensitizes cells to DNA damage agents.

  20. Requirements of cyclin a for mitosis are independent of its subcellular localization.

    Science.gov (United States)

    Dienemann, Axel; Sprenger, Frank

    2004-06-22

    Cyclin A (CycA), the only essential mitotic cyclin in Drosophila, is cytoplasmic during interphase and accumulates in the nucleus during prophase. We show that interphase localization is mediated by Leptomycin B (LMB)-sensitive nuclear export. This is a feature shared with human CyclinB1, and it is assumed that nuclear accumulation is necessary for mitotic entry. Here, we tested if the unique mitotic function of CycA requires nuclear accumulation. We fused subcellular localization signals to CycA and tested their mitotic capability. Surprisingly, nuclear accumulation was not required, and even a membrane-tethered form of CycA was able to induce mitosis. We noted that Cyclin B (CycB) protein disappears prematurely in CycA mutants, reminiscent of rca1 mutants. Rca1 is an inhibitor of Fizzy-related-APC/C activity, and in rca1 mutants, mitotic cyclins are degraded in G2 of the 16(th) embryonic cell cycle. Overexpression of Rca1 can restore mitosis in CycA mutants, indicating that the mitotic failure of CycA mutants is caused by premature activation of the APC/C. The essential mitotic function of CycA is therefore not the activation of numerous mitotic substrates by Cdk1-dependent phosphorylation. Rather, CycA-dependent kinase activity is required to inhibit one inhibitor of mitosis, the Fzr protein.

  1. Continuous nucleus extraction by optically-induced cell lysis on a batch-type microfluidic platform.

    Science.gov (United States)

    Huang, Shih-Hsuan; Hung, Lien-Yu; Lee, Gwo-Bin

    2016-04-21

    The extraction of a cell's nucleus is an essential technique required for a number of procedures, such as disease diagnosis, genetic replication, and animal cloning. However, existing nucleus extraction techniques are relatively inefficient and labor-intensive. Therefore, this study presents an innovative, microfluidics-based approach featuring optically-induced cell lysis (OICL) for nucleus extraction and collection in an automatic format. In comparison to previous micro-devices designed for nucleus extraction, the new OICL device designed herein is superior in terms of flexibility, selectivity, and efficiency. To facilitate this OICL module for continuous nucleus extraction, we further integrated an optically-induced dielectrophoresis (ODEP) module with the OICL device within the microfluidic chip. This on-chip integration circumvents the need for highly trained personnel and expensive, cumbersome equipment. Specifically, this microfluidic system automates four steps by 1) automatically focusing and transporting cells, 2) releasing the nuclei on the OICL module, 3) isolating the nuclei on the ODEP module, and 4) collecting the nuclei in the outlet chamber. The efficiency of cell membrane lysis and the ODEP nucleus separation was measured to be 78.04 ± 5.70% and 80.90 ± 5.98%, respectively, leading to an overall nucleus extraction efficiency of 58.21 ± 2.21%. These results demonstrate that this microfluidics-based system can successfully perform nucleus extraction, and the integrated platform is therefore promising in cell fusion technology with the goal of achieving genetic replication, or even animal cloning, in the near future.

  2. Linking abnormal mitosis to the acquisition of DNA damage

    Science.gov (United States)

    Pellman, David

    2012-01-01

    Cellular defects that impair the fidelity of mitosis promote chromosome missegregation and aneuploidy. Increasing evidence reveals that errors in mitosis can also promote the direct and indirect acquisition of DNA damage and chromosome breaks. Consequently, deregulated cell division can devastate the integrity of the normal genome and unleash a variety of oncogenic stimuli that may promote transformation. Recent work has shed light on the mechanisms that link abnormal mitosis with the development of DNA damage, how cells respond to such affronts, and the potential impact on tumorigenesis. PMID:23229895

  3. The nucleus is irreversibly shaped by motion of cell boundaries in cancer and non-cancer cells.

    Science.gov (United States)

    Tocco, Vincent J; Li, Yuan; Christopher, Keith G; Matthews, James H; Aggarwal, Varun; Paschall, Lauren; Luesch, Hendrik; Licht, Jonathan D; Dickinson, Richard B; Lele, Tanmay P

    2018-02-01

    Actomyosin stress fibers impinge on the nucleus and can exert compressive forces on it. These compressive forces have been proposed to elongate nuclei in fibroblasts, and lead to abnormally shaped nuclei in cancer cells. In these models, the elongated or flattened nuclear shape is proposed to store elastic energy. However, we found that deformed shapes of nuclei are unchanged even after removal of the cell with micro-dissection, both for smooth, elongated nuclei in fibroblasts and abnormally shaped nuclei in breast cancer cells. The lack of shape relaxation implies that the nuclear shape in spread cells does not store any elastic energy, and the cellular stresses that deform the nucleus are dissipative, not static. During cell spreading, the deviation of the nucleus from a convex shape increased in MDA-MB-231 cancer cells, but decreased in MCF-10A cells. Tracking changes of nuclear and cellular shape on micropatterned substrata revealed that fibroblast nuclei deform only during deformations in cell shape and only in the direction of nearby moving cell boundaries. We propose that motion of cell boundaries exert a stress on the nucleus, which allows the nucleus to mimic cell shape. The lack of elastic energy in the nuclear shape suggests that nuclear shape changes in cells occur at constant surface area and volume. © 2017 Wiley Periodicals, Inc.

  4. Foci of cyclin A2 interact with actin and RhoA in mitosis.

    Science.gov (United States)

    Loukil, Abdelhalim; Izard, Fanny; Georgieva, Mariya; Mashayekhan, Shaereh; Blanchard, Jean-Marie; Parmeggiani, Andrea; Peter, Marion

    2016-06-09

    Cyclin A2 is a key player in the regulation of the cell cycle. Its degradation in mid-mitosis depends primarily on the ubiquitin-proteasome system (UPS), while autophagy also contributes. However, a fraction of cyclin A2 persists beyond metaphase. In this work, we focus on cyclin A2-rich foci detected in mitosis by high resolution imaging and analyse their movements. We demonstrate that cyclin A2 interacts with actin and RhoA during mitosis, and that cyclin A2 depletion induces a dramatic decrease in active RhoA in mitosis. Our data suggest cyclin A2 participation in RhoA activation in late mitosis.

  5. Tools for visualization of phosphoinositides in the cell nucleus

    Czech Academy of Sciences Publication Activity Database

    Kalasová, Ilona; Fáberová, Veronika; Kalendová, Alžběta; Uličná, Lívia; Yildirim, Sukriye; Venit, Tomáš; Hozák, Pavel

    2016-01-01

    Roč. 145, č. 4 (2016), s. 485-496 ISSN 0948-6143 R&D Projects: GA ČR GA16-03403S; GA ČR GAP305/11/2232; GA MŠk(CZ) ED1.1.00/02.0109; GA CR GA16-03403S Grant - others: Human Frontier Science Program(FR) RGP0017/2013 Institutional support: RVO:68378050 Keywords : Nucleus * Phosphoinositides * PI(4,5)P2 * PI(4)P Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.553, year: 2016

  6. The self-orientation of mammalian cells in optical tweezers—the importance of the nucleus

    International Nuclear Information System (INIS)

    Perney, Nicolas M B; Horak, Peter; Melvin, Tracy; Hanley, Neil A

    2012-01-01

    Here we present the first evidence showing that eukaryotic cells can be stably trapped in a single focused Gaussian beam with an orientation that is defined by the nucleus. A mammalian eukaryotic cell (in suspension) is trapped and is re-oriented in the focus of a linearly polarized Gaussian beam with a waist of dimension smaller than the radius of the nucleus. The cell reaches a position relative to the focus that is dictated by the nucleus and nuclear components. Our studies illustrate that the force exerted by the optical tweezers at locations within the cell can be predicted theoretically; the data obtained in this way is consistent with the experimental observations. (communication)

  7. Formation of newly synthesized adeno-associated virus capsids in the cell nucleus.

    Science.gov (United States)

    Bell, Peter; Vandenberghe, Luk H; Wilson, James M

    2014-06-01

    Adeno-associated virus (AAV) particles inside the nucleus of a HEK 293 cell are shown by electron microscopy. Cells have been triple-transfected for vector production and were analyzed for capsid formation three days later. Newly assembled particle are visible as seemingly unstructured conglomerates or crystal-like arrays.

  8. Effects of copper on mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Kostal, L

    1971-01-01

    The author deals with the effects of copper on mitosis. He found that a Cu concentration of 1 mg per liter is very toxic and strongly inhibits the course of mitosis in Vicia fabia. The effects of 0.5 mg and 0.25 mg Cu concentrations per liter were similar but a much weaker character.

  9. Role of the Nucleus as a Sensor of Cell Environment Topography.

    Science.gov (United States)

    Anselme, Karine; Wakhloo, Nayana Tusamda; Rougerie, Pablo; Pieuchot, Laurent

    2018-04-01

    The proper integration of biophysical cues from the cell vicinity is crucial for cells to maintain homeostasis, cooperate with other cells within the tissues, and properly fulfill their biological function. It is therefore crucial to fully understand how cells integrate these extracellular signals for tissue engineering and regenerative medicine. Topography has emerged as a prominent component of the cellular microenvironment that has pleiotropic effects on cell behavior. This progress report focuses on the recent advances in the understanding of the topography sensing mechanism with a special emphasis on the role of the nucleus. Here, recent techniques developed for monitoring the nuclear mechanics are reviewed and the impact of various topographies and their consequences on nuclear organization, gene regulation, and stem cell fate is summarized. The role of the cell nucleus as a sensor of cell-scale topography is further discussed. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. 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. © 2015 Hsiung et al.; Published by

  11. 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.

  12. Nuclear matrix and structural and functional compartmentalization of the eucaryotic cell nucleus.

    Science.gov (United States)

    Razin, S V; Borunova, V V; Iarovaia, O V; Vassetzky, Y S

    2014-07-01

    Becoming popular at the end of the 20th century, the concept of the nuclear matrix implies the existence of a nuclear skeleton that organizes functional elements in the cell nucleus. This review presents a critical analysis of the results obtained in the study of nuclear matrix in the light of current views on the organization of the cell nucleus. Numerous studies of nuclear matrix have failed to provide evidence of the existence of such a structure. Moreover, the existence of a filamentous structure that supports the nuclear compartmentalization appears to be unnecessary, since this function is performed by the folded genome itself.

  13. Microdosimetric study for nanosecond pulsed electric fields on a cell circuit model with nucleus.

    Science.gov (United States)

    Denzi, Agnese; Merla, Caterina; Camilleri, Paola; Paffi, Alessandra; d'Inzeo, Guglielmo; Apollonio, Francesca; Liberti, Micaela

    2013-10-01

    Recently, scientific interest in electric pulses, always more intense and shorter and able to induce biological effects on both plasma and nuclear membranes, has greatly increased. Hence, microdosimetric models that include internal organelles like the nucleus have assumed increasing importance. In this work, a circuit model of the cell including the nucleus is proposed, which accounts for the dielectric dispersion of all cell compartments. The setup of the dielectric model of the nucleus is of fundamental importance in determining the transmembrane potential (TMP) induced on the nuclear membrane; here, this is demonstrated by comparing results for three different sets of nuclear dielectric properties present in the literature. The results have been compared, even including or disregarding the dielectric dispersion of the nucleus. The main differences have been found when using pulses shorter than 10 ns. This is due to the fact that the high spectral components of the shortest pulses are differently taken into account by the nuclear membrane transfer functions computed with and without nuclear dielectric dispersion. The shortest pulses are also the most effective in porating the intracellular structures, as confirmed by the time courses of the TMP calculated across the plasma and nuclear membranes. We show how dispersive nucleus models are unavoidable when dealing with pulses shorter than 10 ns because of the large spectral contents arriving above 100 MHz, i.e., over the typical relaxation frequencies of the dipolar mechanism of the molecules constituting the nuclear membrane and the subcellular cell compartments.

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

    International Nuclear Information System (INIS)

    Miyagawa, Ryu; Mizuno, Rie; Watanabe, Kazunori; Ijiri, Kenichi

    2012-01-01

    Highlights: ► tRNAs are tranlocated into the nucleus in heat-induced HeLa cells. ► tRNAs form the unique granules in the nucleus. ► 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 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 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.

  15. In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes.

    Science.gov (United States)

    Ding, Pan; Wang, Houyu; Song, Bin; Ji, Xiaoyuan; Su, Yuanyuan; He, Yao

    2017-08-01

    Fluorescent imaging techniques for visualization of nuclear structure and function in live cells are fundamentally important for exploring major cellular events. The ideal cellular labeling method is capable of realizing label-free, in situ, real-time, and long-term nucleus labeling in live cells, which can fully obtain the nucleus-relative information and effectively alleviate negative effects of alien probes on cellular metabolism. However, current established fluorescent probes-based strategies (e.g., fluorescent proteins-, organic dyes-, fluorescent organic/inorganic nanoparticles-based imaging techniques) are unable to simultaneously realize label-free, in situ, long-term, and real-time nucleus labeling, resulting in inevitable difficulties in fully visualizing nuclear structure and function in live cells. To this end, we present a type of bioinspired fluorescent probes, which are highly efficacious for in situ and label-free tracking of nucleus in long-term and real-time manners. Typically, the bioinspired polydopamine (PDA) nanoparticles, served as fluorescent probes, can be readily synthesized in situ within live cell nucleus without any further modifications under physiological conditions (37 °C, pH ∼7.4). Compared with other conventional nuclear dyes (e.g., propidium iodide (PI), Hoechst), superior spectroscopic properties (e.g., quantum yield of ∼35.8% and high photostability) and low cytotoxicity of PDA-based probes enable long-term (e.g., 3 h) fluorescence tracking of nucleus. We also demonstrate the generality of this type of bioinspired fluorescent probes in different cell lines and complex biological samples.

  16. Heat-induced alterations in the cell nucleus

    International Nuclear Information System (INIS)

    Kampinga, H.H.

    1989-01-01

    Hyperthermia may kill eukaryotic cells and may also enhance the radiosensitivity of those cells that survive the heat treatment. Clinically, the possible use of hyperthermia as an adjuvant in the radiotherapeutic treatment of cancer needs the understanding of mechanisms that underlay heat-induced cell death and radiosensitization. By in vitro heating of established human (HeLaS3) and rodent (Ehrlich Ascites Tumor and LM fibroblast) cell lines, both killing and radiosensitization were investigated. (author). 1067 refs.; 76 figs.; 19 tabs

  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. Influence of collagen type II and nucleus pulposus cells on aggregation and differentiation of adipose tissue-derived stem cells

    NARCIS (Netherlands)

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

    2008-01-01

    Tissue microenvironment plays a critical role in guiding local stem cell differentiation. Within the intervertebral disc, collagen type II and nucleus pulposus (NP) cells are two major components. This study aimed to investigate how collagen type II and NP cells affect adipose tissue-derived stem

  19. Testing of mitosis and meiosis in female and male gametes

    Directory of Open Access Journals (Sweden)

    L. F. Kurilo

    2016-01-01

    Full Text Available Method of quantitative evaluation of the immature germ cells, their pathology in mitosis and meiosis (in semen, embryo and fetal ovaries, of gonad biopsies or fragments of sectioned material is informative method and should be introduced into the clinical practice in andrology and gynecology and fundamental research. Quantitative analysis of mitosis and female meiosis development was initiated on experimental animals and fetal gonads from spontaneous or therapeutic abortions.

  20. The small molecule inhibitor YK-4-279 disrupts mitotic progression of neuroblastoma cells, overcomes drug resistance and synergizes with inhibitors of mitosis.

    Science.gov (United States)

    Kollareddy, Madhu; Sherrard, Alice; Park, Ji Hyun; Szemes, Marianna; Gallacher, Kelli; Melegh, Zsombor; Oltean, Sebastian; Michaelis, Martin; Cinatl, Jindrich; Kaidi, Abderrahmane; Malik, Karim

    2017-09-10

    Neuroblastoma is a biologically and clinically heterogeneous pediatric malignancy that includes a high-risk subset for which new therapeutic agents are urgently required. As well as MYCN amplification, activating point mutations of ALK and NRAS are associated with high-risk and relapsing neuroblastoma. As both ALK and RAS signal through the MEK/ERK pathway, we sought to evaluate two previously reported inhibitors of ETS-related transcription factors, which are transcriptional mediators of the Ras-MEK/ERK pathway in other cancers. Here we show that YK-4-279 suppressed growth and triggered apoptosis in nine neuroblastoma cell lines, while BRD32048, another ETV1 inhibitor, was ineffective. These results suggest that YK-4-279 acts independently of ETS-related transcription factors. Further analysis reveals that YK-4-279 induces mitotic arrest in prometaphase, resulting in subsequent cell death. Mechanistically, we show that YK-4-279 inhibits the formation of kinetochore microtubules, with treated cells showing a broad range of abnormalities including multipolar, fragmented and unseparated spindles, together leading to disrupted progression through mitosis. Notably, YK-4-279 does not affect microtubule acetylation, unlike the conventional mitotic poisons paclitaxel and vincristine. Consistent with this, we demonstrate that YK-4-279 overcomes vincristine-induced resistance in two neuroblastoma cell-line models. Furthermore, combinations of YK-4-279 with vincristine, paclitaxel or the Aurora kinase A inhibitor MLN8237/Alisertib show strong synergy, particularly at low doses. Thus, YK-4-279 could potentially be used as a single-agent or in combination therapies for the treatment of high-risk and relapsing neuroblastoma, as well as other cancers. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  1. Robust Nucleus/Cell Detection and Segmentation in Digital Pathology and Microscopy Images: A Comprehensive Review

    Science.gov (United States)

    Xing, Fuyong; Yang, Lin

    2016-01-01

    Digital pathology and microscopy image analysis is widely used for comprehensive studies of cell morphology or tissue structure. Manual assessment is labor intensive and prone to inter-observer variations. Computer-aided methods, which can significantly improve the objectivity and reproducibility, have attracted a great deal of interest in recent literatures. Among the pipeline of building a computer-aided diagnosis system, nucleus or cell detection and segmentation play a very important role to describe the molecular morphological information. In the past few decades, many efforts have been devoted to automated nucleus/cell detection and segmentation. In this review, we provide a comprehensive summary of the recent state-of-the-art nucleus/cell segmentation approaches on different types of microscopy images including bright-field, phase-contrast, differential interference contrast (DIC), fluorescence, and electron microscopies. In addition, we discuss the challenges for the current methods and the potential future work of nucleus/cell detection and segmentation. PMID:26742143

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. Different surface sensing of the cell body and nucleus in healthy primary cells and in a cancerous cell line on nanogrooves.

    Science.gov (United States)

    Davidson, Patricia M; Bigerelle, Maxence; Reiter, Günter; Anselme, Karine

    2015-10-01

    Cancer cells are known to have alterations compared to healthy cells, but can these differences extend to the way cells interact with their environment? Here, the authors focused on the alignment on an array of grooves of nanometer depth using two cell types: healthy osteoprogenitor primary cells (HOP) and a cancerous osteosarcoma (SaOs-2) cell line. Another concern was how this alignment affects the cell's interior, namely, the nucleus. Based on the results, it is proposed that these two cell types respond to different size regimes: SaOs-2 cells are more sensitive to shallow grooves while HOP cells are strongly aligned with deep grooves. As a measure of the impact of cell alignment on the nucleus the orientation and elongation of the nucleus were determined. Compared to HOP cells, the cell nucleus of SaOs-2 cells is more aligned and elongated in response to grooves, suggesting a softer nucleus and/or increased force transmission. These results support the hypothesis that cancer cells have reduced nucleus rigidity compared to healthy ones and further indicate differences in sensing, which may be important during metastasis.

  9. Low dose combinations of 2-methoxyestradiol and docetaxel block prostate cancer cells in mitosis and increase apoptosis.

    Science.gov (United States)

    Reiner, Teresita; de las Pozas, Alicia; Gomez, Lourdes A; Perez-Stable, Carlos

    2009-04-08

    Clinical trials have shown that chemotherapy with docetaxel (Doc) combined with prednisone can improve survival of patients with androgen-independent prostate cancer (AI-PC). It is likely that the combination of Doc with other novel agents would also improve the survival of AI-PC patients. We investigated whether the combination of Doc and 2-methoxyestradiol (2ME2), an endogenous metabolite of estradiol promising for cancer therapy, can increase apoptotic cell death in prostate cancer cells. Low concentration 2ME2 (0.5-1 microM)+Doc (0.05-0.1 nM) combinations inhibit cell growth, increase G2/M cell cycle arrest, and increase apoptosis more effectively than the single concentrations in a variety of human AI-PC cells. Effects on apoptosis were associated with an increase in p53 protein and a decrease in cyclin A-dependent kinase activity. We then investigated whether the combination of 2ME2+Doc can increase apoptotic cell death and inhibit the growth of prostate tumors in the FG/Tag transgenic mouse model of AI-PC. Doses of 2ME2 and Doc that increase mitotic cell cycle arrest result in an increase in apoptosis and lower primary prostate tumor weights in FG/Tag mice. High dose 2ME2+Doc combinations did not increase G2/M cell cycle arrest or apoptosis in AI-PC cell lines and in the FG/Tag mice more than the single drugs. Overall, our data indicate that low dose 2ME2+Doc combinations may provide a treatment strategy that can improve therapeutic efficacy against AI-PC while reducing toxicity often seen in patients treated with Doc.

  10. Structural-Functional Organization of the Eukaryotic Cell Nucleus and Transcription Regulation: Introduction to This Special Issue of Biochemistry (Moscow).

    Science.gov (United States)

    Razin, S V

    2018-04-01

    This issue of Biochemistry (Moscow) is devoted to the cell nucleus and mechanisms of transcription regulation. Over the years, biochemical processes in the cell nucleus have been studied in isolation, outside the context of their spatial organization. Now it is clear that segregation of functional processes within a compartmentalized cell nucleus is very important for the implementation of basic genetic processes. The functional compartmentalization of the cell nucleus is closely related to the spatial organization of the genome, which in turn plays a key role in the operation of epigenetic mechanisms. In this issue of Biochemistry (Moscow), we present a selection of review articles covering the functional architecture of the eukaryotic cell nucleus, the mechanisms of genome folding, the role of stochastic processes in establishing 3D architecture of the genome, and the impact of genome spatial organization on transcription regulation.

  11. Crowding, Entropic Forces, and Confinement: Crucial Factors for Structures and Functions in the Cell Nucleus.

    Science.gov (United States)

    Hancock, R

    2018-04-01

    The view of the cell nucleus as a crowded system of colloid particles and that chromosomes are giant self-avoiding polymers is stimulating rapid advances in our understanding of its structure and activities, thanks to concepts and experimental methods from colloid, polymer, soft matter, and nano sciences and to increased computational power for simulating macromolecules and polymers. This review summarizes current understanding of some characteristics of the molecular environment in the nucleus, of how intranuclear compartments are formed, and of how the genome is highly but precisely compacted, and underlines the crucial, subtle, and sometimes unintuitive effects on structures and reactions of entropic forces caused by the high concentration of macromolecules in the nucleus.

  12. 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.

  13. 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. © 2016 Hsiung et al.; Published by Cold Spring Harbor Laboratory Press.

  14. Raman spectroscopy for DNA quantification in cell nucleus.

    Science.gov (United States)

    Okotrub, K A; Surovtsev, N V; Semeshin, V F; Omelyanchuk, L V

    2015-01-01

    Here we demonstrate the feasibility of a novel approach to quantify DNA in cell nuclei. This approach is based on spectroscopy analysis of Raman light scattering, and avoids the problem of nonstoichiometric binding of dyes to DNA, as it directly measures the signal from DNA. Quantitative analysis of nuclear DNA contribution to Raman spectrum could be reliably performed using intensity of a phosphate mode at 1096 cm(-1) . When compared to the known DNA standards from cells of different animals, our results matched those values at error of 10%. We therefore suggest that this approach will be useful to expand the list of DNA standards, to properly adjust the duration of hydrolysis in Feulgen staining, to assay the applicability of fuchsines for DNA quantification, as well as to measure DNA content in cells with complex hydrolysis patterns, when Feulgen densitometry is inappropriate. © 2014 International Society for Advancement of Cytometry.

  15. Tracking the Oxygen Status in the Cell Nucleus with a Hoechst-Tagged Phosphorescent Ruthenium Complex.

    Science.gov (United States)

    Hara, Daiki; Umehara, Yui; Son, Aoi; Asahi, Wataru; Misu, Sotaro; Kurihara, Ryohsuke; Kondo, Teruyuki; Tanabe, Kazuhito

    2018-05-04

    Molecular oxygen in living cells is distributed and consumed inhomogeneously, depending on the activity of each organelle. Therefore, tractable methods that can be used to monitor the oxygen status in each organelle are needed to understand cellular function. Here we report the design of a new oxygen-sensing probe for use in the cell nucleus. We prepared "Ru-Hoechsts", each consisting of a phosphorescent ruthenium complex linked to a Hoechst 33258 moiety, and characterized their properties as oxygen sensors. The Hoechst unit shows strong DNA-binding properties in the nucleus, and the ruthenium complex shows oxygen-dependent phosphorescence. Thus, Ru-Hoechsts accumulated in the cell nucleus and showed oxygen-dependent signals that could be monitored. Of the Ru-Hoechsts prepared in this study, Ru-Hoechst b, in which the ruthenium complex and the Hoechst unit were linked through a hexyl chain, showed the most suitable properties for monitoring the oxygen status. Ru-Hoechsts are probes with high potential for visualizing oxygen fluctuations in the nucleus. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. A CO-FISH assay to assess sister chromatid segregation patterns in mitosis of mouse embryonic stem cells.

    Science.gov (United States)

    Sauer, Stephan; Burkett, Sandra S; Lewandoski, Mark; Klar, Amar J S

    2013-05-01

    Sister chromatids contain identical DNA sequence but are chiral with respect to both their helical handedness and their replication history. Emerging evidence from various model organisms suggests that certain stem cells segregate sister chromatids nonrandomly to either maintain genome integrity or to bias cellular differentiation in asymmetric cell divisions. Conventional methods for tracing of old vs. newly synthesized DNA strands generally lack resolution for individual chromosomes and employ halogenated thymidine analogs with profound cytotoxic effects on rapidly dividing cells. Here, we present a modified chromosome orientation fluorescence in situ hybridization (CO-FISH) assay, where identification of individual chromosomes and their replication history is achieved in subsequent hybridization steps with chromosome-specific DNA probes and PNA telomere probes. Importantly, we tackle the issue of BrdU cytotoxicity and show that our method is compatible with normal mouse ES cell biology, unlike a recently published related protocol. Results from our CO-FISH assay show that mitotic segregation of mouse chromosome 7 is random in ES cells, which contrasts previously published results from our laboratory and settles a controversy. Our straightforward protocol represents a useful resource for future studies on chromatid segregation patterns of in vitro-cultured cells from distinct model organisms.

  17. Mechanical stability of the cell nucleus: roles played by the cytoskeleton in nuclear deformation and strain recovery.

    Science.gov (United States)

    Wang, Xian; Liu, Haijiao; Zhu, Min; Cao, Changhong; Xu, Zhensong; Tsatskis, Yonit; Lau, Kimberly; Kuok, Chikin; Filleter, Tobin; McNeill, Helen; Simmons, Craig A; Hopyan, Sevan; Sun, Yu

    2018-05-18

    Extracellular forces transmitted through the cytoskeleton can deform the cell nucleus. Large nuclear deformation increases the risk of disrupting the nuclear envelope's integrity and causing DNA damage. Mechanical stability of the nucleus defines its capability of maintaining nuclear shape by minimizing nuclear deformation and recovering strain when deformed. Understanding the deformation and recovery behavior of the nucleus requires characterization of nuclear viscoelastic properties. Here, we quantified the decoupled viscoelastic parameters of the cell membrane, cytoskeleton, and the nucleus. The results indicate that the cytoskeleton enhances nuclear mechanical stability by lowering the effective deformability of the nucleus while maintaining nuclear sensitivity to mechanical stimuli. Additionally, the cytoskeleton decreases the strain energy release rate of the nucleus and might thus prevent shape change-induced structural damage to chromatin. © 2018. Published by The Company of Biologists Ltd.

  18. 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

  19. 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

    25 dual-specificity phosphatase family members. In somatic cells, Wee1 is downregulated by phosphorylation and ubiquitin-mediated degradation to ensure rapid activation of CDK1 at the beginning of M phase. Here, we show that downregulation of the regulatory beta-subunit of protein kinase CK2 by RNA...

  20. Heavy metals in the cell nucleus - role in pathogenesis.

    Science.gov (United States)

    Sas-Nowosielska, Hanna; Pawlas, Natalia

    2015-01-01

    People are exposed to heavy metals both in an occupational and natural environment. The most pronounced effects of heavy metals result from their interaction with cellular genetic material packed in form of chromatin. Heavy metals influence chromatin, mimicking and substituting natural microelements in various processes taking place in the cell, or interacting chemically with nuclear components: nucleic acids, proteins and lipids. This paper is a review of current knowledge on the effects of heavy metals on chromatin, exerted at the level of various nuclear components.

  1. 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. PMID:25689737

  2. A Nuclear Attack on Traumatic Brain Injury: Sequestration of Cell Death in the Nucleus.

    Science.gov (United States)

    Tajiri, Naoki; De La Peña, Ike; Acosta, Sandra A; Kaneko, Yuji; Tamir, Sharon; Landesman, Yosef; Carlson, Robert; Shacham, Sharon; Borlongan, Cesar V

    2016-04-01

    Exportin 1 (XPO1/CRM1) plays prominent roles in the regulation of nuclear protein export. Selective inhibitors of nuclear export (SINE) are small orally bioavailable molecules that serve as drug-like inhibitors of XPO1, with potent anti-cancer properties. Traumatic brain injury (TBI) presents with a secondary cell death characterized by neuroinflammation that is putatively regulated by nuclear receptors. Here, we report that the SINE compounds (KPT-350 or KPT-335) sequestered TBI-induced neuroinflammation-related proteins (NF-(k)B, AKT, FOXP1) within the nucleus of cultured primary rat cortical neurons, which coincided with protection against TNF-α (20 ng/mL)-induced neurotoxicity as shown by at least 50% and 100% increments in preservation of cell viability and cellular enzymatic activity, respectively, compared to non-treated neuronal cells (P's nucleus as an efficacious treatment for TBI. © 2016 John Wiley & Sons Ltd.

  3. 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.

  4. The effects of osmotic stress on the structure and function of the cell nucleus.

    Science.gov (United States)

    Finan, John D; Guilak, Farshid

    2010-02-15

    Osmotic stress is a potent regulator of the normal function of cells that are exposed to osmotically active environments under physiologic or pathologic conditions. The ability of cells to alter gene expression and metabolic activity in response to changes in the osmotic environment provides an additional regulatory mechanism for a diverse array of tissues and organs in the human body. In addition to the activation of various osmotically- or volume-activated ion channels, osmotic stress may also act on the genome via a direct biophysical pathway. Changes in extracellular osmolality alter cell volume, and therefore, the concentration of intracellular macromolecules. In turn, intracellular macromolecule concentration is a key physical parameter affecting the spatial organization and pressurization of the nucleus. Hyper-osmotic stress shrinks the nucleus and causes it to assume a convoluted shape, whereas hypo-osmotic stress swells the nucleus to a size that is limited by stretch of the nuclear lamina and induces a smooth, round shape of the nucleus. These behaviors are consistent with a model of the nucleus as a charged core/shell structure pressurized by uneven partition of macromolecules between the nucleoplasm and the cytoplasm. These osmotically-induced alterations in the internal structure and arrangement of chromatin, as well as potential changes in the nuclear membrane and pores are hypothesized to influence gene transcription and/or nucleocytoplasmic transport. A further understanding of the biophysical and biochemical mechanisms involved in these processes would have important ramifications for a range of fields including differentiation, migration, mechanotransduction, DNA repair, and tumorigenesis. (c) 2009 Wiley-Liss, Inc.

  5. Unsuccessful mitosis in multicellular tumour spheroids.

    Science.gov (United States)

    Molla, Annie; Couvet, Morgane; Coll, Jean-Luc

    2017-04-25

    Multicellular spheroids are very attractive models in oncology because they mimic the 3D organization of the tumour cells with their microenvironment. We show here using 3 different cell types (mammary TSA/pc, embryonic kidney Hek293 and cervical cancer HeLa), that when the cells are growing as spheroids the frequency of binucleated cells is augmented as occurs in some human tumours.We therefore describe mitosis in multicellular spheroids by following mitotic markers and by time-lapse experiments. Chromosomes alignment appears to be correct on the metaphasic plate and the passenger complex is well localized on centromere. Moreover aurora kinases are fully active and histone H3 is phosphorylated on Ser 10. Consequently, the mitotic spindle checkpoint is satisfied and, anaphase proceeds as illustrated by the transfer of survivin on the spindle and by the segregation of the two lots of chromosomes. However, the segregation plane is not well defined and oscillations of the dividing cells are observed. Finally, cytokinesis fails and the absence of separation of the two daughter cells gives rise to binucleated cells.Division orientation is specified during interphase and persists throughout mitosis. Our data indicate that the cancer cells, in multicellular spheroids, lose their ability to regulate their orientation, a feature commonly encountered in tumours.Moreover, multicellular spheroid expansion is still sensitive to mitotic drugs as pactlitaxel and aurora kinase inhibitors. The spheroids thus represent a highly relevant model for studying drug efficiency in tumours.

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

    DEFF Research Database (Denmark)

    Ebersbach, G.; Gerdes, Kenn

    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......A-GFP oscillated in spiral-shaped structures. Amino acid substitutions in ParA simultaneously abolished ParA spiral formation, oscillation and either plasmid localization or plasmid separation at mid-cell. Therefore, our results suggest that ParA spirals position plasmids at the middle of the bacterial nucleoid...

  7. Serial interactome capture of the human cell nucleus.

    Science.gov (United States)

    Conrad, Thomas; Albrecht, Anne-Susann; de Melo Costa, Veronica Rodrigues; Sauer, Sascha; Meierhofer, David; Ørom, Ulf Andersson

    2016-04-04

    Novel RNA-guided cellular functions are paralleled by an increasing number of RNA-binding proteins (RBPs). Here we present 'serial RNA interactome capture' (serIC), a multiple purification procedure of ultraviolet-crosslinked poly(A)-RNA-protein complexes that enables global RBP detection with high specificity. We apply serIC to the nuclei of proliferating K562 cells to obtain the first human nuclear RNA interactome. The domain composition of the 382 identified nuclear RBPs markedly differs from previous IC experiments, including few factors without known RNA-binding domains that are in good agreement with computationally predicted RNA binding. serIC extends the number of DNA-RNA-binding proteins (DRBPs), and reveals a network of RBPs involved in p53 signalling and double-strand break repair. serIC is an effective tool to couple global RBP capture with additional selection or labelling steps for specific detection of highly purified RBPs.

  8. 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. © 2014 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.

  9. Direct projection from the suprachiasmatic nucleus to hypophysiotrophic corticotropin-releasing factor immunoreactive cells in the paraventricular nucleus of the hypothalamus demonstrated...

    DEFF Research Database (Denmark)

    Vrang, N.; Larsen, P.J.; Mikkelsen, J.D.

    1995-01-01

    Suprachiasmatic nucleus, paraventricular nucleus, circadian rhythms, phaseolus vulgaris-leucoagglutinin, corticotropin-releasing factor, dual immunocytochemistry......Suprachiasmatic nucleus, paraventricular nucleus, circadian rhythms, phaseolus vulgaris-leucoagglutinin, corticotropin-releasing factor, dual immunocytochemistry...

  10. Imaging and quantification of amyloid fibrillation in the cell nucleus.

    Science.gov (United States)

    Arnhold, Florian; Scharf, Andrea; von Mikecz, Anna

    2015-01-01

    Xenobiotics, as well as intrinsic processes such as cellular aging, contribute to an environment that constantly challenges nuclear organization and function. While it becomes increasingly clear that proteasome-dependent proteolysis is a major player, the topology and molecular mechanisms of nuclear protein homeostasis remain largely unknown. We have shown previously that (1) proteasome-dependent protein degradation is organized in focal microenvironments throughout the nucleoplasm and (2) heavy metals as well as nanoparticles induce nuclear protein fibrillation with amyloid characteristics. Here, we describe methods to characterize the landscape of intranuclear amyloid on the global and local level in different systems such as cultures of mammalian cells and the soil nematode Caenorhabditis elegans. Application of discrete mathematics to imaging data is introduced as a tool to develop pattern recognition of intracellular protein fibrillation. Since stepwise fibrillation of otherwise soluble proteins to insoluble amyloid-like protein aggregates is a hallmark of neurodegenerative protein-misfolding disorders including Alzheimer's disease, CAG repeat diseases, and the prion encephalopathies, investigation of intracellular amyloid may likewise aid to a better understanding of the pathomechanisms involved. We consider aggregate profiling as an important experimental approach to determine if nuclear amyloid has toxic or protective roles in various disease processes.

  11. Nanoneedle insertion into the cell nucleus does not induce double-strand breaks in chromosomal DNA.

    Science.gov (United States)

    Ryu, Seunghwan; Kawamura, Ryuzo; Naka, Ryohei; Silberberg, Yaron R; Nakamura, Noriyuki; Nakamura, Chikashi

    2013-09-01

    An atomic force microscope probe can be formed into an ultra-sharp cylindrical shape (a nanoneedle) using micro-fabrication techniques such as focused ion beam etching. This nanoneedle can be effectively inserted through the plasma membrane of a living cell to not only access the cytosol, but also to penetrate through the nuclear membrane. This technique shows great potential as a tool for performing intranuclear measurements and manipulations. Repeated insertions of a nanoneedle into a live cell were previously shown not to affect cell viability. However, the effect of nanoneedle insertion on the nucleus and nuclear components is still unknown. DNA is the most crucial component of the nucleus for proper cell function and may be physically damaged by a nanoneedle. To investigate the integrity of DNA following nanoneedle insertion, the occurrence of DNA double-strand breaks (DSBs) was assessed. The results showed that there was no chromosomal DNA damage due to nanoneedle insertion into the nucleus, as indicated by the expression level of γ-H2AX, a molecular marker of DSBs. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. 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...

  13. 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. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  14. Bubbling cell death: A hot air balloon released from the nucleus in the cold.

    Science.gov (United States)

    Chang, Nan-Shan

    2016-06-01

    Cell death emanating from the nucleus is largely unknown. In our recent study, we determined that when temperature is lowered in the surrounding environment, apoptosis stops and bubbling cell death (BCD) occurs. The study concerns the severity of frostbite. When exposed to severe cold and strong ultraviolet (UV) irradiation, people may suffer serious damages to the skin and internal organs. This ultimately leads to limb amputations, organ failure, and death. BCD is defined as "formation of a single bubble from the nucleus per cell and release of this swelling bubble from the cell surface to extracellular space that causes cell death." When cells are subjected to UV irradiation and/or brief cold shock (4℃ for 5 min) and then incubated at room temperature or 4℃ for time-lapse microscopy, each cell releases an enlarging nuclear gas bubble containing nitric oxide. Certain cells may simultaneously eject hundreds or thousands of exosome-like particles. Unlike apoptosis, no phosphatidylserine flip-over, mitochondrial apoptosis, damage to Golgi complex, and chromosomal DNA fragmentation are shown in BCD. When the temperature is increased back at 37℃, bubble formation stops and apoptosis restarts. Mechanistically, proapoptotic WW domain-containing oxidoreductase and p53 block the protective TNF receptor adaptor factor 2 that allows nitric oxide synthase 2 to synthesize nitric oxide and bubble formation. In this mini-review, updated knowledge in cell death and the proposed molecular mechanism for BCD are provided. © 2016 by the Society for Experimental Biology and Medicine.

  15. Translocation of the papillomavirus L2/vDNA complex across the limiting membrane requires the onset of mitosis.

    Science.gov (United States)

    Calton, Christine M; Bronnimann, Matthew P; Manson, Ariana R; Li, Shuaizhi; Chapman, Janice A; Suarez-Berumen, Marcela; Williamson, Tatum R; Molugu, Sudheer K; Bernal, Ricardo A; Campos, Samuel K

    2017-05-01

    The human papillomavirus type 16 (HPV16) L2 protein acts as a chaperone to ensure that the viral genome (vDNA) traffics from endosomes to the trans-Golgi network (TGN) and eventually the nucleus, where HPV replication occurs. En route to the nucleus, the L2/vDNA complex must translocate across limiting intracellular membranes. The details of this critical process remain poorly characterized. We have developed a system based on subcellular compartmentalization of the enzyme BirA and its cognate substrate to detect membrane translocation of L2-BirA from incoming virions. We find that L2 translocation requires transport to the TGN and is strictly dependent on entry into mitosis, coinciding with mitotic entry in synchronized cells. Cell cycle arrest causes retention of L2/vDNA at the TGN; only release and progression past G2/M enables translocation across the limiting membrane and subsequent infection. Microscopy of EdU-labeled vDNA reveals a rapid and dramatic shift in vDNA localization during early mitosis. At late G2/early prophase vDNA egresses from the TGN to a pericentriolar location, accumulating there through prometaphase where it begins to associate with condensed chromosomes. By metaphase and throughout anaphase the vDNA is seen bound to the mitotic chromosomes, ensuring distribution into both daughter nuclei. Mutations in a newly defined chromatin binding region of L2 potently blocked translocation, suggesting that translocation is dependent on chromatin binding during prometaphase. This represents the first time a virus has been shown to functionally couple the penetration of limiting membranes to cellular mitosis, explaining in part the tropism of HPV for mitotic basal keratinocytes.

  16. Protective effect of cannabidiol on hydrogen peroxide‑induced apoptosis, inflammation and oxidative stress in nucleus pulposus cells.

    Science.gov (United States)

    Chen, Jie; Hou, Chen; Chen, Xin; Wang, Dong; Yang, Pinglin; He, Xijing; Zhou, Jinsong; Li, Haopeng

    2016-09-01

    Cannabidiol, a major component of marijuana, protects nerves, and exerts antispasmodic, anti-inflammatory and anti‑anxiety effects. In the current study, the protective effect of cannabidiol was observed to prevent hydrogen peroxide (H2O2)‑induced apoptosis, inflammation and oxidative stress in nucleus pulposus cells. Nucleus pulposus cells were isolated from rats and cultured in vitro, and H2O2 was used to construct the nucleus pulposus cell model. Cell viability of the nucleus pulposus cells was assessed using a 3‑(4,5-dimethylthiazol-2-yl)-2,5‑diphenyltetrazolium bromide assay. The ratio of apoptotic cells, and caspase‑3 or cyclooxygenase‑2 (COX‑2) mRNA expression was analyzed by annexin V‑fluorescein isothiocyanate/propidium‑iodide staining and reverse transcription‑quantitative polymerase chain reaction, respectively. The quantities of interleukin (IL)‑1β and interleukin‑6 were measured using a series of assay kits. B-cell lymphoma 2 (Bcl‑2) and inducible nitric oxide synthase (iNOS) protein expression levels were analyzed using western blotting. The present study identified that cannabidiol enhanced cell viability and reduced apoptosis in H2O2‑treated nucleus pulposus cells in vitro using a lumbar disc herniation (LDH) model. In addition, cannabidiol reduced caspase‑3 gene expression and augmented the Bcl‑2 protein expression levels in the nucleus pulposus cells following H2O2 exposure. Pre‑treatment with cannabidiol suppressed the promotion of COX‑2, iNOS, IL‑1β and IL‑6 expression in the nucleus pulposus cells following H2O2 exposure. Taken together, these results suggest that cannabidiol potentially exerts its protective effect on LDH via the suppression of anti‑apoptosis, anti‑inflammation and anti‑oxidative activities in nucleus pulposus cells.

  17. 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.

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

    International Nuclear Information System (INIS)

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei

    2014-01-01

    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.

  19. Edaravone ameliorates compression-induced damage in rat nucleus pulposus cells.

    Science.gov (United States)

    Lin, Hui; Ma, Xuan; Wang, Bai-Chuan; Zhao, Lei; Liu, Jian-Xiang; Pu, Fei-Fei; Hu, Yi-Qiang; Hu, Hong-Zhi; Shao, Zeng-Wu

    2017-11-15

    Edaravone is a strong free radical scavenger most used for treating acute ischemic stroke. In this study we investigated the protective effects and underlying mechanisms of edaravone on compression-induced damage in rat nucleus pulposus (NP) cells. Cell viability was determined using MTT assay methods. NP cell apoptosis was measured by Hoechst 33,258 staining and Annexin V/PI double staining. Intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and intracellular calcium ([Ca 2+ ] i ) were determined by fluorescent probes DCFH-DA, JC-1 and Fluo-3/AM, respectively. Apoptosis-related proteins (cleaved caspase-3, cytosolic cytochrome c, Bax and Bcl-2) and extracellular matrix proteins (aggrecan and collagen II) were analyzed by western blot. Edaravone attenuated the compression-induced decrease in viability of NP cells in a dose-dependent manner. 33,258 and Annexin V/PI double staining showed that edaravone protected NP cells from compression-induced apoptosis. Further studies confirmed that edaravone protected NP cells against compression-induced mitochondrial pathway of apoptosis by inhibiting overproduction of ROS, collapse of MMP and overload of [Ca 2+ ] i . In addition, edaravone promoted the expression of aggrecan and collagen II in compression-treated NP cells. These results strongly indicate that edaravone ameliorates compression-induced damage in rat nucleus pulposus cells. Edaravone could be a potential new drug for treatment of IDD. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Barley disease susceptibility factor RACB acts in epidermal cell polarity and positioning of the nucleus.

    Science.gov (United States)

    Scheler, Björn; Schnepf, Vera; Galgenmüller, Carolina; Ranf, Stefanie; Hückelhoven, Ralph

    2016-05-01

    RHO GTPases are regulators of cell polarity and immunity in eukaryotes. In plants, RHO-like RAC/ROP GTPases are regulators of cell shaping, hormone responses, and responses to microbial pathogens. The barley (Hordeum vulgare L.) RAC/ROP protein RACB is required for full susceptibility to penetration by Blumeria graminis f.sp. hordei (Bgh), the barley powdery mildew fungus. Disease susceptibility factors often control host immune responses. Here we show that RACB does not interfere with early microbe-associated molecular pattern-triggered immune responses such as the oxidative burst or activation of mitogen-activated protein kinases. RACB also supports rather than restricts expression of defence-related genes in barley. Instead, silencing of RACB expression by RNAi leads to defects in cell polarity. In particular, initiation and maintenance of root hair growth and development of stomatal subsidiary cells by asymmetric cell division is affected by silencing expression of RACB. Nucleus migration is a common factor of developmental cell polarity and cell-autonomous interaction with Bgh RACB is required for positioning of the nucleus near the site of attack from Bgh We therefore suggest that Bgh profits from RACB's function in cell polarity rather than from immunity-regulating functions of RACB. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  1. Atypical growth, abonormal mitosis and polyploidy induced by ethyl-mercury-chloride

    Energy Technology Data Exchange (ETDEWEB)

    Kostoff, D

    1940-01-01

    Experiments were performed to study the effect of ethyl mercury chloride upon the atypical growths of plants. Seeds of peas, flax, rye, and wheat were treated with 2% ethyl mercury chloride. The fungicide suppressed the development of the seedlings. Cytological studies revealed that the fungicide had also significantly affected the procedure of mitosis in most of the treated seedlings. The chromosomes did not become arranged into a proper equatorial plate when the nucleus membrane and the nucleolei disappeared, but occupied a position similar to that which they had during the prophase. The chromosomes split, then the centromeres divided so that from each one chromosome, two chromosomes originated, situated side by side without polar separation, chiefly due to the absence of normal achromatic figures. Thus, chromosome division without cell division takes place.

  2. Thrombopoietin-induced Polyploidization of Bone Marrow Megakaryocytes Is Due to a Unique Regulatory Mechanism in Late Mitosis

    Science.gov (United States)

    Nagata, Yuka; Muro, Yoshinao; Todokoro, Kazuo

    1997-01-01

    Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. However, the mechanism underlying this polyploidization remains totally unknown. It has been postulated that polyploidization is due to a skipping of mitosis after each round of DNA replication. We carried out immunohistochemical studies on mouse bone marrow megakaryocytes during thrombopoietin- induced polyploidization and found that during this process megakaryocytes indeed enter mitosis and progress through normal prophase, prometaphase, metaphase, and up to anaphase A, but not to anaphase B, telophase, or cytokinesis. It was clearly observed that multiple spindle poles were formed as the polyploid megakaryocytes entered mitosis; the nuclear membrane broke down during prophase; the sister chromatids were aligned on a multifaced plate, and the centrosomes were symmetrically located on either side of each face of the plate at metaphase; and a set of sister chromatids moved into the multiple centrosomes during anaphase A. We further noted that the pair of spindle poles in anaphase were located in close proximity to each other, probably because of the lack of outward movement of spindle poles during anaphase B. Thus, the reassembling nuclear envelope may enclose all the sister chromatids in a single nucleus at anaphase and then skip telophase and cytokinesis. These observations clearly indicate that polyploidization of megakaryocytes is not simply due to a skipping of mitosis, and that the megakaryocytes must have a unique regulatory mechanism in anaphase, e.g., factors regulating anaphase such as microtubule motor proteins might be involved in this polyploidization process. PMID:9334347

  3. Tight coupling between nucleus and cell migration through the perinuclear actin cap

    Science.gov (United States)

    Kim, Dong-Hwee; Cho, Sangkyun; Wirtz, Denis

    2014-01-01

    ABSTRACT Although eukaryotic cells are known to alternate between ‘advancing’ episodes of fast and persistent movement and ‘hesitation’ episodes of low speed and low persistence, the molecular mechanism that controls the dynamic changes in morphology, speed and persistence of eukaryotic migratory cells remains unclear. Here, we show that the movement of the interphase nucleus during random cell migration switches intermittently between two distinct modes – rotation and translocation – that follow with high fidelity the sequential rounded and elongated morphologies of the nucleus and cell body, respectively. Nuclear rotation and translocation mediate the stop-and-go motion of the cell through the dynamic formation and dissolution, respectively, of the contractile perinuclear actin cap, which is dynamically coupled to the nuclear lamina and the nuclear envelope through LINC complexes. A persistent cell movement and nuclear translocation driven by the actin cap are halted following the disruption of the actin cap, which in turn allows the cell to repolarize for its next persistent move owing to nuclear rotation mediated by cytoplasmic dynein light intermediate chain 2. PMID:24639463

  4. Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy.

    Science.gov (United States)

    Stortz, Martin; Angiolini, Juan; Mocskos, Esteban; Wolosiuk, Alejandro; Pecci, Adali; Levi, Valeria

    2018-05-01

    The hierarchical organization of the cell nucleus into specialized open reservoirs and the nucleoplasm overcrowding impose restrictions to the mobility of biomolecules and their interactions with nuclear targets. These properties determine that many nuclear functions such as transcription, replication, splicing or DNA repair are regulated by complex, dynamical processes that do not follow simple rules. Advanced fluorescence microscopy tools and, in particular, fluorescence correlation spectroscopy (FCS) provide complementary and exquisite information on the dynamics of fluorescent labeled molecules moving through the nuclear space and are helping us to comprehend the complexity of the nuclear structure. Here, we describe how FCS methods can be applied to reveal the dynamical organization of the nucleus in live cells. Specifically, we provide instructions for the preparation of cellular samples with fluorescent tagged proteins and detail how FCS can be easily instrumented in commercial confocal microscopes. In addition, we describe general rules to set the parameters for one and two-color experiments and the required controls for these experiments. Finally, we review the statistical analysis of the FCS data and summarize the use of numerical simulations as a complementary approach that helps us to understand the complex matrix of molecular interactions network within the nucleus. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. A new F-actin structure in fungi: actin ring formation around the cell nucleus of Cryptococcus neoformans.

    Science.gov (United States)

    Kopecká, Marie; Kawamoto, Susumu; Yamaguchi, Masashi

    2013-04-01

    The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as 'funicular cabin' for the cell nucleus, and actin cables as intracellular 'funicular' suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.

  6. Empirical Derivation of Correction Factors for Human Spiral Ganglion Cell Nucleus and Nucleolus Count Units.

    Science.gov (United States)

    Robert, Mark E; Linthicum, Fred H

    2016-01-01

    Profile count method for estimating cell number in sectioned tissue applies a correction factor for double count (resulting from transection during sectioning) of count units selected to represent the cell. For human spiral ganglion cell counts, we attempted to address apparent confusion between published correction factors for nucleus and nucleolus count units that are identical despite the role of count unit diameter in a commonly used correction factor formula. We examined a portion of human cochlea to empirically derive correction factors for the 2 count units, using 3-dimensional reconstruction software to identify double counts. The Neurotology and House Histological Temporal Bone Laboratory at University of California at Los Angeles. Using a fully sectioned and stained human temporal bone, we identified and generated digital images of sections of the modiolar region of the lower first turn of cochlea, identified count units with a light microscope, labeled them on corresponding digital sections, and used 3-dimensional reconstruction software to identify double-counted count units. For 25 consecutive sections, we determined that double-count correction factors for nucleus count unit (0.91) and nucleolus count unit (0.92) matched the published factors. We discovered that nuclei and, therefore, spiral ganglion cells were undercounted by 6.3% when using nucleolus count units. We determined that correction factors for count units must include an element for undercounting spiral ganglion cells as well as the double-count element. We recommend a correction factor of 0.91 for the nucleus count unit and 0.98 for the nucleolus count unit when using 20-µm sections. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2015.

  7. 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. © 2016 Federation of European Biochemical Societies.

  8. Survival of alpha particle irradiated cells as a function of the shape and size of the sensitive volume (nucleus)

    International Nuclear Information System (INIS)

    Stinchcomb, T.G.; Roeske, J.C.

    1995-01-01

    Microdosimetry is the study of the stochastic variation of energy deposited within sub-cellular targets. As such, the size and shape of the critical target (i.e. cell nucleus) are essential when considering microdosimetric quantities. In this work, a microdosimetric analysis examines the expected cell survival as a function of the size and shape of the cell nucleus under conditions of irradiation emitting alpha particles. The results indicate that, in general, cell survival is relatively insensitive to changes in the shape of the cell nucleus when the volume is held constant. However, cell survival is a strong function of the variation in the size of the target. These results are useful when analysing the results of cell survival experiments for alpha particle emitters. (Author)

  9. Differential response of nucleus pulposus intervertebral disc cells to high salt, sorbitol, and urea.

    Science.gov (United States)

    Mavrogonatou, Eleni; Kletsas, Dimitris

    2012-03-01

    Nucleus pulposus intervertebral disc cells are routinely confronted with high osmolality in their microenvironment and respond to this stress in vitro by regulating cell cycle progression and by activating a DNA repair machinery in order to counteract its genotoxic effect. In the present study, we attempted to identify the origin of this osmo-regulatory response, by using an ionic NaCl/KCl solution, the compatible osmolyte sorbitol, and the readily permeant urea. High salt and sorbitol were found to activate similar molecular pathways, including the p38 MAPK and the p53-p21(WAF1)-pRb axis, that were not stimulated by high urea. On the other hand, only high urea led to the phosphorylation of ERKs and JNKs. Furthermore, salt- and sorbitol-treated cells were able to phosphorylate histone H2A.X on Ser139, in contrast to cells exposed to urea, indicating a common mechanism for DNA repair, which was achieved by a p53-dependent activation of the G1 checkpoint by both solutes. DNA repair, as directly measured by a host cell reactivation assay, occurred under conditions of hyperosmolar salt and sorbitol, although to a lesser extent in sorbitol-treated cells than in cells exposed to high salinity. Taken as a whole, our findings suggest that the hyperosmolality-provoked DNA damage and the responses of nucleus pulposus cells induced by this genotoxic stress most probably originate from cell volume alterations mediated by hypertonicity and not from increased intracellular ionic concentration. Copyright © 2011 Wiley Periodicals, Inc.

  10. Mechanical response and buckling of a polymer simulation model of the cell nucleus

    Science.gov (United States)

    Banigan, Edward; Stephens, Andrew; Marko, John

    The cell nucleus must robustly resist extra- and intracellular forces to maintain genome architecture. Micromanipulation experiments measuring nuclear mechanical response reveal that the nucleus has two force response regimes: a linear short-extension response due to the chromatin interior and a stiffer long-extension response from lamin A, comprising the intermediate filament protein shell. To explain these results, we developed a quantitative simulation model with realistic parameters for chromatin and the lamina. Our model predicts that crosslinking between chromatin and the lamina is essential for responding to small strains and that changes to the interior topological organization can alter the mechanical response of the whole nucleus. Thus, chromatin polymer elasticity, not osmotic pressure, is the dominant regulator of this force response. Our model reveals a novel buckling transition for polymer shells: as force increases, the shell buckles transverse to the applied force. This transition, which arises from topological constrains in the lamina, can be mitigated by tuning the properties of the chromatin interior. Thus, we find that the genome is a resistive mechanical element that can be tuned by its organization and connectivity to the lamina.

  11. Is the Cell Nucleus a Necessary Component in Precise Temporal Patterning?

    Science.gov (United States)

    Albert, Jaroslav; Rooman, Marianne

    2015-01-01

    One of the functions of the cell nucleus is to help regulate gene expression by controlling molecular traffic across the nuclear envelope. Here we investigate, via stochastic simulation, what effects, if any, does segregation of a system into the nuclear and cytoplasmic compartments have on the stochastic properties of a motif with a negative feedback. One of the effects of the nuclear barrier is to delay the nuclear protein concentration, allowing it to behave in a switch-like manner. We found that this delay, defined as the time for the nuclear protein concentration to reach a certain threshold, has an extremely narrow distribution. To show this, we considered two models. In the first one, the proteins could diffuse freely from cytoplasm to nucleus (simple model); and in the second one, the proteins required assistance from a special class of proteins called importins. For each model, we generated fifty parameter sets, chosen such that the temporal profiles they effectuated were very similar, and whose average threshold time was approximately 150 minutes. The standard deviation of the threshold times computed over one hundred realizations were found to be between 1.8 and 7.16 minutes across both models. To see whether a genetic motif in a prokaryotic cell can achieve this degree of precision, we also simulated five variations on the coherent feed-forward motif (CFFM), three of which contained a negative feedback. We found that the performance of these motifs was nowhere near as impressive as the one found in the eukaryotic cell; the best standard deviation was 6.6 minutes. We argue that the significance of these results, the fact and necessity of spatio-temporal precision in the developmental stages of eukaryotes, and the absence of such a precision in prokaryotes, all suggest that the nucleus has evolved, in part, under the selective pressure to achieve highly predictable phenotypes.

  12. Is the Cell Nucleus a Necessary Component in Precise Temporal Patterning?

    Directory of Open Access Journals (Sweden)

    Jaroslav Albert

    Full Text Available One of the functions of the cell nucleus is to help regulate gene expression by controlling molecular traffic across the nuclear envelope. Here we investigate, via stochastic simulation, what effects, if any, does segregation of a system into the nuclear and cytoplasmic compartments have on the stochastic properties of a motif with a negative feedback. One of the effects of the nuclear barrier is to delay the nuclear protein concentration, allowing it to behave in a switch-like manner. We found that this delay, defined as the time for the nuclear protein concentration to reach a certain threshold, has an extremely narrow distribution. To show this, we considered two models. In the first one, the proteins could diffuse freely from cytoplasm to nucleus (simple model; and in the second one, the proteins required assistance from a special class of proteins called importins. For each model, we generated fifty parameter sets, chosen such that the temporal profiles they effectuated were very similar, and whose average threshold time was approximately 150 minutes. The standard deviation of the threshold times computed over one hundred realizations were found to be between 1.8 and 7.16 minutes across both models. To see whether a genetic motif in a prokaryotic cell can achieve this degree of precision, we also simulated five variations on the coherent feed-forward motif (CFFM, three of which contained a negative feedback. We found that the performance of these motifs was nowhere near as impressive as the one found in the eukaryotic cell; the best standard deviation was 6.6 minutes. We argue that the significance of these results, the fact and necessity of spatio-temporal precision in the developmental stages of eukaryotes, and the absence of such a precision in prokaryotes, all suggest that the nucleus has evolved, in part, under the selective pressure to achieve highly predictable phenotypes.

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

    Science.gov (United States)

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

    2016-09-02

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

  14. N-Cadherin Maintains the Healthy Biology of Nucleus Pulposus Cells under High-Magnitude Compression.

    Science.gov (United States)

    Wang, Zhenyu; Leng, Jiali; Zhao, Yuguang; Yu, Dehai; Xu, Feng; Song, Qingxu; Qu, Zhigang; Zhuang, Xinming; Liu, Yi

    2017-01-01

    Mechanical load can regulate disc nucleus pulposus (NP) biology in terms of cell viability, matrix homeostasis and cell phenotype. N-cadherin (N-CDH) is a molecular marker of NP cells. This study investigated the role of N-CDH in maintaining NP cell phenotype, NP matrix synthesis and NP cell viability under high-magnitude compression. Rat NP cells seeded on scaffolds were perfusion-cultured using a self-developed perfusion bioreactor for 5 days. NP cell biology in terms of cell apoptosis, matrix biosynthesis and cell phenotype was studied after the cells were subjected to different compressive magnitudes (low- and high-magnitudes: 2% and 20% compressive deformation, respectively). Non-loaded NP cells were used as controls. Lentivirus-mediated N-CDH overexpression was used to further investigate the role of N-CDH under high-magnitude compression. The 20% deformation compression condition significantly decreased N-CDH expression compared with the 2% deformation compression and control conditions. Meanwhile, 20% deformation compression increased the number of apoptotic NP cells, up-regulated the expression of Bax and cleaved-caspase-3 and down-regulated the expression of Bcl-2, matrix macromolecules (aggrecan and collagen II) and NP cell markers (glypican-3, CAXII and keratin-19) compared with 2% deformation compression. Additionally, N-CDH overexpression attenuated the effects of 20% deformation compression on NP cell biology in relation to the designated parameters. N-CDH helps to restore the cell viability, matrix biosynthesis and cellular phenotype of NP cells under high-magnitude compression. © 2017 The Author(s). Published by S. Karger AG, Basel.

  15. [Compartmentalization of the cell nucleus and spatial organization of the genome].

    Science.gov (United States)

    Gavrilov, A A; Razin, S V

    2015-01-01

    The eukaryotic cell nucleus is one of the most complex cell organelles. Despite the absence of membranes, the nuclear space is divided into numerous compartments where different processes in- volved in the genome activity take place. The most important nuclear compartments include nucleoli, nuclear speckles, PML bodies, Cajal bodies, histone locus bodies, Polycomb bodies, insulator bodies, transcription and replication factories. The structural basis for the nuclear compartmentalization is provided by genomic DNA that occupies most of the nuclear volume. Nuclear compartments, in turn, guide the chromosome folding by providing a platform for the spatial interaction of individual genomic loci. In this review, we discuss fundamental principles of higher order genome organization with a focus on chromosome territories and chromosome domains, as well as consider the structure and function of the key nuclear compartments. We show that the func- tional compartmentalization of the cell nucleus and genome spatial organization are tightly interconnected, and that this form of organization is highly dynamic and is based on stochastic processes.

  16. [Ultrastructural study on the nucleus pulposus of the inter-vertebral disc--the behavior of the cells in the nucleus pulposus and their autolytic changes in the monkey].

    Science.gov (United States)

    Hase, H

    1985-08-01

    Ultrastructural studies were carried out to examine the normal structure and postmortem autolytic changes of the cells and matrix of nucleus pulposus using adult monkey. Two kinds of cells were observed in the nucleus pulposus. The one was chondrocyte, which contained normal organelles and that was characterized by large halo. The halo was composed of numerous "crista like structures", that were regarded to form the matrix of nucleus pulposus. The other was notochordal cell, most of which appeared in grouping or separately, and yet had cell activity. In addition, there were the intermediate type of cells between chondrocyte and notochordal cell. The ultrastructural autolytic changes were rarely seen in the cells of 6 hours after death, but the changes in the halo and in the cytoplasm were remarkable after more than 12 hours. The autopsied nucleus pulposus for electron microscopical examination should be used within 6 hours after death in usual room temperature.

  17. Onuf's nucleus X

    DEFF Research Database (Denmark)

    Schrøder, H D

    1981-01-01

    in the length of the nucleus was observed. Based on the cytoarchitecture the nucleus could be divided in three parts, a cranial, a dorsomedial and a ventrolateral. All parts of the nucleus consisted of chromatin-rich medium-sized neurons, and apparent direct appositions between different cells bodies as well...

  18. 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.

  19. 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.

    2014-01-01

    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 use whole-cell recordings, two-photon microscopy, optogenetics and pharmacogenetics to show how repeated cocaine alters connectivity in the mouse NAc medial shell. We first determine that cocaine selectively enhances amygdala innervation of D1-MSNs relative to D2-MSNs. We then show that amygdala activity is required for cocaine-induced changes to behavior and connectivity. Finally, we establish how heightened amygdala innervation can explain the structural and functional changes induced by cocaine. Our findings reveal how exposure to drugs of abuse fundamentally reorganizes cell-type and input-specific connectivity in the NAc. PMID:25108911

  20. Raptor is phosphorylated by cdc2 during mitosis.

    Directory of Open Access Journals (Sweden)

    Dana M Gwinn

    2010-02-01

    Full Text Available The appropriate control of mitotic entry and exit is reliant on a series of interlocking signaling events that coordinately drive the biological processes required for accurate cell division. Overlaid onto these signals that promote orchestrated cell division are checkpoints that ensure appropriate mitotic spindle formation, a lack of DNA damage, kinetochore attachment, and that each daughter cell has the appropriate complement of DNA. We recently discovered that AMP-activated protein kinase (AMPK modulates the G2/M phase of cell cycle progression in part through its suppression of mammalian target of rapamycin (mTOR signaling. AMPK directly phosphorylates the critical mTOR binding partner raptor inhibiting mTORC1 (mTOR-raptor rapamycin sensitive mTOR kinase complex 1. As mTOR has been previously tied to mitotic control, we examined further how raptor may contribute to this process.We have discovered that raptor becomes highly phosphorylated in cells in mitosis. Utilizing tandem mass spectrometry, we identified a number of novel phosphorylation sites in raptor, and using phospho-specific antibodies demonstrated that raptor becomes phosphorylated on phospho-serine/threonine-proline sites in mitosis. A combination of site-directed mutagenesis in a tagged raptor cDNA and analysis with a series of new phospho-specific antibodies generated against different sites in raptor revealed that Serine 696 and Threonine 706 represent two key sites in raptor phosphorylated in mitosis. We demonstrate that the mitotic cyclin-dependent kinase cdc2/CDK1 is the kinase responsible for phosphorylating these sites, and its mitotic partner Cyclin B efficiently coimmunoprecipitates with raptor in mitotic cells.This study demonstrates that the key mTOR binding partner raptor is directly phosphorylated during mitosis by cdc2. This reinforces previous studies suggesting that mTOR activity is highly regulated and important for mitotic progression, and points to a direct

  1. The average number of alpha-particle hits to the cell nucleus required to eradicate a tumour cell population

    International Nuclear Information System (INIS)

    Roeske, John C; Stinchcomb, Thomas G

    2006-01-01

    Alpha-particle emitters are currently being considered for the treatment of micrometastatic disease. Based on in vitro studies, it has been speculated that only a few alpha-particle hits to the cell nucleus are considered lethal. However, such estimates do not consider the stochastic variations in the number of alpha-particle hits, energy deposited, or in the cell survival process itself. Using a tumour control probability (TCP) model for alpha-particle emitters, we derive an estimate of the average number of hits to the cell nucleus required to provide a high probability of eradicating a tumour cell population. In simulation studies, our results demonstrate that the average number of hits required to achieve a 90% TCP for 10 4 clonogenic cells ranges from 18 to 108. Those cells that have large cell nuclei, high radiosensitivities and alpha-particle emissions occurring primarily in the nuclei tended to require more hits. As the clinical implementation of alpha-particle emitters is considered, this type of analysis may be useful in interpreting clinical results and in designing treatment strategies to achieve a favourable therapeutic outcome. (note)

  2. 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.

  3. Comparative proteomics of mitosis and meiosis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kumar, Ravinder; Dhali, Snigdha; Srikanth, Rapole; Ghosh, Santanu Kumar; Srivastava, Sanjeeva

    2014-09-23

    Precise and timely segregation of genetic material and conservation of ploidy are the two foremost requirements for survival of a eukaryotic organism. Two highly regulated cell division processes, namely mitosis and meiosis are central to achieve this objective. The modes of chromosome segregation are distinct in these two processes that generate progeny cells of equal ploidy and half the ploidy in mitosis and meiosis, respectively. Additionally, the nutritional requirement and intracellular processing of biological cue also differ in these two processes. From this, it can be envisaged that proteome of mitotic and meiotic cells will differ significantly. Therefore, identification of proteins that differ in their level of expression between mitosis and meiosis would further reveal the mechanistic detail of these processes. In the present study, we have investigated the protein expression profile of mitosis and meiosis by comparing proteome of budding yeast cultures arrested at mitotic metaphase and metaphase-I of meiosis using proteomic approach. Approximately 1000 and 2000 protein spots were visualized on 2-DE and 2D-DIGE gels respectively, out of which 14 protein spots were significant in 2-DE and 22 in 2D-DIGE (pmitosis, an up-regulation of actin cytoskeleton and its negative regulator occurs in meiosis. Mitosis and meiosis are two different types of cell division cycles with entirely different outcomes with definite biological implication for almost all eukaryotic species. In this work, we investigated, for the first time, the differential proteomic profile of Saccharomyces cerevisiae culture arrested at mitotic metaphase (M) and metaphase-I (MI) of meiosis using 2-DE and 2D-DIGE. Our findings of up-regulation of actin and its negative regulator cofilin during meiosis suggest that the rate of actin cytoskeleton turnover is more in meiosis and actin cytoskeleton may play more crucial role during meiosis compared to mitosis. Present study also suggests that actin

  4. EWSR1 regulates mitosis by dynamically influencing microtubule acetylation.

    Science.gov (United States)

    Wang, Yi-Long; Chen, Hui; Zhan, Yi-Qun; Yin, Rong-Hua; Li, Chang-Yan; Ge, Chang-Hui; Yu, Miao; Yang, Xiao-Ming

    2016-08-17

    EWSR1, participating in transcription and splicing, has been identified as a translocation partner for various transcription factors, resulting in translocation, which in turn plays crucial roles in tumorigenesis. Recent studies have investigated the role of EWSR1 in mitosis. However, the effect of EWSR1 on mitosis is poorly understood. Here, we observed that depletion of EWSR1 resulted in cell cycle arrest in the mitotic phase, mainly due to an increase in the time from nuclear envelope breakdown to metaphase, resulting in a high percentage of unaligned chromosomes and multipolar spindles. We also demonstrated that EWSR1 is a spindle-associated protein that interacts with α-tubulin during mitosis. EWSR1 depletion increased the cold-sensitivity of spindle microtubules, and decreased the rate of spindle assembly. EWSR1 regulated the level of microtubule acetylation in the mitotic spindle; microtubule acetylation was rescued in EWSR1-depleted mitotic cells following suppression of HDAC6 activity by its specific inhibitor or siRNA treatment. In summary, these results suggest that EWSR1 regulates the acetylation of microtubules in a cell cycle-dependent manner through its dynamic location on spindle MTs, and may be a novel regulator for mitosis progress independent of its translocation.

  5. Iterative h-minima-based marker-controlled watershed for cell nucleus segmentation.

    Science.gov (United States)

    Koyuncu, Can Fahrettin; Akhan, Ece; Ersahin, Tulin; Cetin-Atalay, Rengul; Gunduz-Demir, Cigdem

    2016-04-01

    Automated microscopy imaging systems facilitate high-throughput screening in molecular cellular biology research. The first step of these systems is cell nucleus segmentation, which has a great impact on the success of the overall system. The marker-controlled watershed is a technique commonly used by the previous studies for nucleus segmentation. These studies define their markers finding regional minima on the intensity/gradient and/or distance transform maps. They typically use the h-minima transform beforehand to suppress noise on these maps. The selection of the h value is critical; unnecessarily small values do not sufficiently suppress the noise, resulting in false and oversegmented markers, and unnecessarily large ones suppress too many pixels, causing missing and undersegmented markers. Because cell nuclei show different characteristics within an image, the same h value may not work to define correct markers for all the nuclei. To address this issue, in this work, we propose a new watershed algorithm that iteratively identifies its markers, considering a set of different h values. In each iteration, the proposed algorithm defines a set of candidates using a particular h value and selects the markers from those candidates provided that they fulfill the size requirement. Working with widefield fluorescence microscopy images, our experiments reveal that the use of multiple h values in our iterative algorithm leads to better segmentation results, compared to its counterparts. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

  6. Gaussian fluctuation of the diffusion exponent of virus capsid in a living cell nucleus

    Science.gov (United States)

    Itto, Yuichi

    2018-05-01

    In their work [4], Bosse et al. experimentally showed that virus capsid exhibits not only normal diffusion but also anomalous diffusion in nucleus of a living cell. There, it was found that the distribution of fluctuations of the diffusion exponent characterizing them takes the Gaussian form, which is, quite remarkably, the same form for two different types of the virus. This suggests high robustness of such fluctuations. Here, the statistical property of local fluctuations of the diffusion exponent of the virus capsid in the nucleus is studied. A maximum-entropy-principle approach (originally proposed for a different virus in a different cell) is applied for obtaining the fluctuation distribution of the exponent. Largeness of the number of blocks identified with local areas of interchromatin corrals is also examined based on the experimental data. It is shown that the Gaussian distribution of the local fluctuations can be derived, in accordance with the above form. In addition, it is quantified how the fluctuation distribution on a long time scale is different from the Gaussian distribution.

  7. 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.

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

    International Nuclear Information System (INIS)

    Debenest, T.; Silvestre, J.; Coste, M.; Delmas, F.; Pinelli, E.

    2008-01-01

    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 -6 , 10 -6 , 10 -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 -6 M and 35.96 per mille , S.D. = 3.71 for 5 x 10 -6 M) was statistically significant (Tukey test, P -6 and 5 x 10 -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 effects of MH. The alterations observed could be related to the effects of MH on the synthesis of the proteins involved in frustule formation or in the regulation of the cytoskeleton of the diatom cells

  9. Characterisation of cell death inducing Phytophthora capsici CRN effectors suggests diverse activities in the host nucleus

    Directory of Open Access Journals (Sweden)

    Remco eStam

    2013-10-01

    Full Text Available Plant-Microbe interactions are complex associations that feature recognition of Pathogen Associated Molecular Patterns by the plant immune system and dampening of subsequent responses by pathogen encoded secreted effectors. With large effector repertoires now identified in a range of sequenced microbial genomes, much attention centres on understanding their roles in immunity or disease. These studies not only allow identification of pathogen virulence factors and strategies, they also provide an important molecular toolset suited for studying immunity in plants. The Phytophthora intracellular effector repertoire encodes a large class of proteins that translocate into host cells and exclusively target the host nucleus. Recent functional studies have implicated the CRN protein family as an important class of diverse effectors that target distinct subnuclear compartments and modify host cell signalling. Here, we characterised three necrosis inducing CRNs and show that there are differences in the levels of cell death. We show that only expression of CRN20_624 has an additive effect on PAMP induced cell death but not AVR3a induced ETI. Given their distinctive phenotypes, we assessed localisation of each CRN with a set of nuclear markers and found clear differences in CRN subnuclear distribution patterns. These assays also revealed that expression of CRN83_152 leads to a distinct change in nuclear chromatin organisation, suggesting a distinct series of events that leads to cell death upon over-expression. Taken together, our results suggest diverse functions carried by CRN C-termini, which can be exploited to identify novel processes that take place in the host nucleus and are required for immunity or susceptibility.

  10. 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.

  11. 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. © 2013 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

    Inesta-Vaquera, Francisco A.; Campbell, David G.; Arthur, J. Simon C.; Cuenda, Ana

    2010-01-01

    Research highlights: → hDlg is phosphorylated during mitosis in multiple residues. → Prospho-hDlg is excluded from the midbody during mitosis. → hDlg is not phosphorylated by p38γ or JNK1/2 during mitosis. → 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.

  13. Cell-type-specific role for nucleus accumbens neuroligin-2 in depression and stress susceptibility.

    Science.gov (United States)

    Heshmati, Mitra; Aleyasin, Hossein; Menard, Caroline; Christoffel, Daniel J; Flanigan, Meghan E; Pfau, Madeline L; Hodes, Georgia E; Lepack, Ashley E; Bicks, Lucy K; Takahashi, Aki; Chandra, Ramesh; Turecki, Gustavo; Lobo, Mary Kay; Maze, Ian; Golden, Sam A; Russo, Scott J

    2018-01-30

    Behavioral coping strategies are critical for active resilience to stress and depression; here we describe a role for neuroligin-2 (NLGN-2) in the nucleus accumbens (NAc). Neuroligins (NLGN) are a family of neuronal postsynaptic cell adhesion proteins that are constituents of the excitatory and inhibitory synapse. Importantly, NLGN-3 and NLGN-4 mutations are strongly implicated as candidates underlying the development of neuropsychiatric disorders with social disturbances such as autism, but the role of NLGN-2 in neuropsychiatric disease states is unclear. Here we show a reduction in NLGN-2 gene expression in the NAc of patients with major depressive disorder. Chronic social defeat stress in mice also decreases NLGN-2 selectively in dopamine D1-positive cells, but not dopamine D2-positive cells, within the NAc of stress-susceptible mice. Functional NLGN-2 knockdown produces bidirectional, cell-type-specific effects: knockdown in dopamine D1-positive cells promotes subordination and stress susceptibility, whereas knockdown in dopamine D2-positive cells mediates active defensive behavior. These findings establish a behavioral role for NAc NLGN-2 in stress and depression; provide a basis for targeted, cell-type specific therapy; and highlight the role of active behavioral coping mechanisms in stress susceptibility.

  14. DNA precursor compartmentation in mammalian cells: distribution and rates of equilibration between nucleus and cytoplasm

    International Nuclear Information System (INIS)

    Leeds, J.M.

    1986-01-01

    A rapid nuclear isolation technique was adapted in order to examine the question of DNA precursor compartmentation in mammalian cells. By using this method a reproducible proportion of the cellular nucleotides remained associated with the isolated nuclei. Examination, at several different cell densities, of exponentially growing HeLa cells showed that the nuclei contained a constant but distinct proportion of each dNTP. The nuclear dATP and dTTP concentrations were equal at all densities examined even though the dTTP pool was 150% of the dATP whole-cell pool. The nuclear portion of the whole-cell pools was roughly equal to the volume occupied by the nucleus. The nuclear-cytoplasmic dNTP pool distribution did not change throughout the cell cycle of synchronized Chinese hamster ovary (CHO) cells. The rates at which either radiolabeled cytidine or deoxycytidine equilibrated with the nuclear and whole-cell dCTP pools of G1 and S phase CHO cells were compared. Experiments comparing the labeling kinetics of 3 H-thymidine in G1, S phase, and exponentially growing cells revealed that the S phase dTTP pool equilibrated with exogenously added thymidine faster than the G1 phase pool. The rate of equilibration in exponentially growing cells appeared to be a combination of that seen in G1 and S phases. A linear rate of 3 H-thymidine incorporation into DNA occurred at the same rate in S phase and exponentially growing cells

  15. 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.

    2016-10-01

    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.

  16. Recruitment of glutathione into the nucleus during cell proliferation adjusts whole-cell redox homeostasis in Arabidopsis thaliana and lowers the oxidative defence shield.

    Science.gov (United States)

    Vivancos, Pedro Diaz; Dong, Yingping; Ziegler, Kerstin; Markovic, Jelena; Pallardó, Federico V; Pellny, Till K; Verrier, Paul J; Foyer, Christine H

    2010-12-01

    Cellular redox homeostasis and signalling are important in progression of the eukaryotic cell cycle. In animals, the low-molecular-weight thiol tripeptide glutathione (GSH) is recruited into the nucleus early in the cell proliferation cycle. To determine whether a similar process occurs in plants, we studied cell proliferation in Arabidopsis thaliana. We show that GSH co-localizes with nuclear DNA during the proliferation of A. thaliana cells in culture. Moreover, GSH localization in the nucleus was observed in dividing pericycle cells of the lateral root meristem. There was pronounced accumulation of GSH in the nucleus at points in the growth cycle at which a high percentage of the cells were in G(1) phase, as identified by flow cytometry and marker transcripts. Recruitment of GSH into the nucleus led to a high abundance of GSH in the nucleus (GSHn) and severe depletion of the cytoplasmic GSH pool (GSHc). Sequestration of GSH in the nucleus was accompanied by significant decreases in transcripts associated with oxidative signalling and stress tolerance, and an increase in the abundance of hydrogen peroxide, an effect that was enhanced when the dividing cells were treated with salicylic acid. Total cellular GSH and the abundance of GSH1 and GSH2 transcripts increased after the initial recruitment of GSH into the nucleus. We conclude that GSH recruitment into the nucleus during cell proliferation has a profound effect on the whole-cell redox state. High GSHn levels trigger redox adjustments in the cytoplasm, favouring decreased oxidative signalling and enhanced GSH synthesis. © 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.

  17. STAT3-Activated GM-CSFRα Translocates to the Nucleus and Protects CLL Cells from Apoptosis

    Science.gov (United States)

    Li, Ping; Harris, David; Liu, Zhiming; Rozovski, Uri; Ferrajoli, Alessandra; Wang, Yongtao; Bueso-Ramos, Carlos; Hazan-Halevy, Inbal; Grgurevic, Srdana; Wierda, William; Burger, Jan; O'Brien, Susan; Faderl, Stefan; Keating, Michael; Estrov, Zeev

    2014-01-01

    Here it was determined that Chronic Lymphocytic Leukemia (CLL) cells express the α-subunit but not the β-subunit of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR/CSF3R). GM-CSFRα was detected on the surface, in the cytosol, and the nucleus of CLL cells via confocal microscopy, cell fractionation, and GM-CSFRα antibody epitope mapping. Because STAT3 is frequently activated in CLL and the GM-CSFRα promoter harbors putative STAT3 consensus binding sites, MM1 cells were transfected with truncated forms of the GM-CSFRα promoter, then stimulated with IL-6 to activate STAT3 to identify STAT3 binding sites. Chromatin immunoprecipitation (ChIP) and an electoromobility shift assay (EMSA) confirmed STAT3 occupancy to those promoter regions in both IL-6 stimulated MM1 and CLL cells. Transfection of MM1 cells with STAT3 siRNA or CLL cells with STAT3 shRNA significantly down-regulated GM-CSFRα mRNA and protein levels. RNA transcripts, involved in regulating cell-survival pathways, and the proteins KAP1 (TRIM28) and ISG15 co-immunoprecipitated with GM-CSFRα. GM-CSFRα-bound KAP1 enhanced the transcriptional activity of STAT3, whereas ISG15 inhibited the NF-κB pathway. Nevertheless, overexpression of GM-CSFRα protected MM1 cells from dexamethasone-induced apoptosis, and GM-CSFRα knockdown induced apoptosis in CLL cells, suggesting that GM-CSFRα provides a ligand-independent survival advantage. PMID:24836891

  18. Virion assembly factories in the nucleus of polyomavirus-infected cells.

    Directory of Open Access Journals (Sweden)

    Kimberly D Erickson

    Full Text Available Most DNA viruses replicate in the cell nucleus, although the specific sites of virion assembly are as yet poorly defined. Electron microscopy on freeze-substituted, plastic-embedded sections of murine polyomavirus (PyV-infected 3T3 mouse fibroblasts or mouse embryonic fibroblasts (MEFs revealed tubular structures in the nucleus adjacent to clusters of assembled virions, with virions apparently "shed" or "budding" from their ends. Promyelocytic leukemia nuclear bodies (PML-NBs have been suggested as possible sites for viral replication of polyomaviruses (BKV and SV40, herpes simplex virus (HSV, and adenovirus (Ad. Immunohistochemistry and FISH demonstrated co-localization of the viral T-antigen (Tag, PyV DNA, and the host DNA repair protein MRE11, adjacent to the PML-NBs. In PML⁻/⁻ MEFs the co-localization of MRE11, Tag, and PyV DNA remained unchanged, suggesting that the PML protein itself was not responsible for their association. Furthermore, PyV-infected PML⁻/⁻ MEFs and PML⁻/⁻ mice replicated wild-type levels of infectious virus. Therefore, although the PML protein may identify sites of PyV replication, neither the observed "virus factories" nor virus assembly were dependent on PML. The ultrastructure of the tubes suggests a new model for the encapsidation of small DNA viruses.

  19. 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.

  20. Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

    Science.gov (United States)

    Ferrero, Macarena; Ferragud, Juan; Orlando, Leonardo; Valero, Luz; Sánchez del Pino, Manuel; Farràs, Rosa; Font de Mora, Jaime

    2011-01-01

    Background Although the AIB1 oncogene has an important role during the early phase of the cell cycle as a coactivator of E2F1, little is known about its function during mitosis. Methodology/Principal Findings Mitotic cells isolated by nocodazole treatment as well as by shake-off revealed a post-translational modification occurring in AIB1 specifically during mitosis. This modification was sensitive to the treatment with phosphatase, suggesting its modification by phosphorylation. Using specific inhibitors and in vitro kinase assays we demonstrate that AIB1 is phosphorylated on Ser728 and Ser867 by Cdk1/cyclin B at the onset of mitosis and remains phosphorylated until exit from M phase. Differences in the sensitivity to phosphatase inhibitors suggest that PP1 mediates dephosphorylation of AIB1 at the end of mitosis. The phosphorylation of AIB1 during mitosis was not associated with ubiquitylation or degradation, as confirmed by western blotting and flow cytometry analysis. In addition, luciferase reporter assays showed that this phosphorylation did not alter the transcriptional properties of AIB1. Importantly, fluorescence microscopy and sub-cellular fractionation showed that AIB1 phosphorylation correlated with the exclusion from the condensed chromatin, thus preventing access to the promoters of AIB1-dependent genes. Phospho-specific antibodies developed against Ser728 further demonstrated the presence of phosphorylated AIB1 only in mitotic cells where it was localized preferentially in the periphery of the cell. Conclusions Collectively, our results describe a new mechanism for the regulation of AIB1 during mitosis, whereby phosphorylation of AIB1 by Cdk1 correlates with the subcellular redistribution of AIB1 from a chromatin-associated state in interphase to a more peripheral localization during mitosis. At the exit of mitosis, AIB1 is dephosphorylated, presumably by PP1. This exclusion from chromatin during mitosis may represent a mechanism for governing the

  1. 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.

  2. EphB4 localises to the nucleus of prostate cancer cells

    International Nuclear Information System (INIS)

    Mertens-Walker, Inga; Lisle, Jessica E.; Nyberg, William A.; Stephens, Carson R.; Burke, Leslie; Rutkowski, Raphael; Herington, Adrian C.; Stephenson, Sally-Anne

    2015-01-01

    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

  3. Bi-directional exchange of membrane components occurs during co-culture of mesenchymal stem cells and nucleus pulposus cells.

    Science.gov (United States)

    Strassburg, Sandra; Hodson, Nigel W; Hill, Patrick I; Richardson, Stephen M; Hoyland, Judith A

    2012-01-01

    Mesenchymal stem cell (MSC)-based therapies have been proposed as novel treatments for intervertebral disc (IVD) degeneration. We have previously demonstrated that when MSCs are co-cultured with nucleus pulposus (NP) cells with direct cell-cell contact, they differentiate along the NP lineage and simultaneously stimulate the degenerate NP cell population to regain a normal (non-degenerate) phenotype, an effect which requires cell-cell communication. However, the mechanisms by which NP cells and MSCs interact in this system are currently unclear. Thus, in this study we investigated a range of potential mechanisms for exchange of cellular components or information that may direct these changes, including cell fusion, gap-junctional communication and exchange of membrane components by direct transfer or via microvesicle formation. Flow cytometry of fluorescently labeled MSCs and NP cells revealed evidence of some cell fusion and formation of gapjunctions, although at the three timepoints studied these phenomena were detectable only in a small proportion of cells. While these mechanisms may play a role in cell-cell communication, the data suggests they are not the predominant mechanism of interaction. However, flow cytometry of fluorescently dual-labeled cells showed that extensive bi-directional transfer of membrane components is operational during direct co-culture of MSCs and NP cells. Furthermore, there was also evidence for secretion and internalization of membrane-bound microvesicles by both cell types. Thus, this study highlights bi-directional intercellular transfer of membrane components as a possible mechanism of cellular communication between MSC and NP cells.

  4. Single-Cell Gene Expression Analysis of Cholinergic Neurons in the Arcuate Nucleus of the Hypothalamus.

    Directory of Open Access Journals (Sweden)

    Jae Hoon Jeong

    Full Text Available The cholinoceptive system in the hypothalamus, in particular in the arcuate nucleus (ARC, plays a role in regulating food intake. Neurons in the ARC contain multiple neuropeptides, amines, and neurotransmitters. To study molecular and neurochemical heterogeneity of ARC neurons, we combine single-cell qRT-PCR and single-cell whole transcriptome amplification methods to analyze expression patterns of our hand-picked 60 genes in individual neurons in the ARC. Immunohistochemical and single-cell qRT-PCR analyses show choline acetyltransferase (ChAT-expressing neurons in the ARC. Gene expression patterns are remarkably distinct in each individual cholinergic neuron. Two-thirds of cholinergic neurons express tyrosine hydroxylase (Th mRNA. A large subset of these Th-positive cholinergic neurons is GABAergic as they express the GABA synthesizing enzyme glutamate decarboxylase and vesicular GABA transporter transcripts. Some cholinergic neurons also express the vesicular glutamate transporter transcript gene. POMC and POMC-processing enzyme transcripts are found in a subpopulation of cholinergic neurons. Despite this heterogeneity, gene expression patterns in individual cholinergic cells appear to be highly regulated in a cell-specific manner. In fact, membrane receptor transcripts are clustered with their respective intracellular signaling and downstream targets. This novel population of cholinergic neurons may be part of the neural circuitries that detect homeostatic need for food and control the drive to eat.

  5. Proteinaceous and oligosaccharidic elicitors induce different calcium signatures in the nucleus of tobacco cells.

    Science.gov (United States)

    Lecourieux, David; Lamotte, Olivier; Bourque, Stéphane; Wendehenne, David; Mazars, Christian; Ranjeva, Raoul; Pugin, Alain

    2005-12-01

    We previously reported elevated cytosolic calcium levels in tobacco cells in response to elicitors [D. Lecourieux, C. Mazars, N. Pauly, R. Ranjeva, A. Pugin, Analysis and effects of cytosolic free calcium elevations in response to elicitors in Nicotiana plumbaginifolia cells, Plant Cell 14 (2002) 2627-2641]. These data suggested that in response to elicitors, Ca2+, as a second messenger, was involved in both systemic acquired resistance (RSA) and/or hypersensitive response (HR) depending on calcium signature. Here, we used transformed tobacco cells with apoaequorin expressed in the nucleus to monitor changes in free nuclear calcium concentrations ([Ca2+](nuc)) in response to elicitors. Two types of elicitors are compared: proteins leading to necrosis including four elicitins and harpin, and non-necrotic elicitors including flagellin (flg22) and two oligosaccharidic elicitors, namely the oligogalacturonides (OGs) and the beta-1,3-glucan laminarin. Our data indicate that the proteinaceous elicitors induced a pronounced and sustainable [Ca2+](nuc) elevation, relative to the small effects of oligosaccharidic elicitors. This [Ca2+](nuc) elevation, which seems insufficient to induce cell death, is unlikely to result directly from the diffusion of calcium from the cytosol. The [Ca2+](nuc) rise depends on free cytosolic calcium, IP3, and active oxygen species (AOS) but is independent of nitric oxide.

  6. Structural dynamics of the cell nucleus: basis for morphology modulation of nuclear calcium signaling and gene transcription.

    Science.gov (United States)

    Queisser, Gillian; Wiegert, Simon; Bading, Hilmar

    2011-01-01

    Neuronal morphology plays an essential role in signal processing in the brain. Individual neurons can undergo use-dependent changes in their shape and connectivity, which affects how intracellular processes are regulated and how signals are transferred from one cell to another in a neuronal network. Calcium is one of the most important intracellular second messengers regulating cellular morphologies and functions. In neurons, intracellular calcium levels are controlled by ion channels in the plasma membrane such as NMDA receptors (NMDARs), voltage-gated calcium channels (VGCCs) and certain α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) as well as by calcium exchange pathways between the cytosol and internal calcium stores including the endoplasmic reticulum and mitochondria. Synaptic activity and the subsequent opening of ligand and/or voltage-gated calcium channels can initiate cytosolic calcium transients which propagate towards the cell soma and enter the nucleus via its nuclear pore complexes (NPCs) embedded in the nuclear envelope. We recently described the discovery that in hippocampal neurons the morphology of the nucleus affects the calcium dynamics within the nucleus. Here we propose that nuclear infoldings determine whether a nucleus functions as an integrator or detector of oscillating calcium signals. We outline possible ties between nuclear mophology and transcriptional activity and discuss the importance of extending the approach to whole cell calcium signal modeling in order to understand synapse-to-nucleus communication in healthy and dysfunctional neurons.

  7. 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....

  8. Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus.

    Science.gov (United States)

    Desai, Tanay M; Marin, Mariana; Sood, Chetan; Shi, Jiong; Nawaz, Fatima; Aiken, Christopher; Melikyan, Gregory B

    2015-10-29

    HIV-1 Vpr is recruited into virions during assembly and appears to remain associated with the viral core after the reverse transcription and uncoating steps of entry. This feature has prompted the use of fluorescently labeled Vpr to visualize viral particles and to follow trafficking of post-fusion HIV-1 cores in the cytoplasm. Here, we tracked single pseudovirus entry and fusion and observed that fluorescently tagged Vpr gradually dissociates from post-fusion viral cores over the course of several minutes and accumulates in the nucleus. Kinetics measurements showed that fluorescent Vpr released from the cores very rapidly entered the cell nucleus. More than 10,000 Vpr molecules can be delivered into the cell nucleus within 45 min of infection by HIV-1 particles pseudotyped with the avian sarcoma and leukosis virus envelope glycoprotein. The fraction of Vpr from cell-bound viruses that accumulated in the nucleus was proportional to the extent of virus-cell fusion and was fully blocked by viral fusion inhibitors. Entry of virus-derived Vpr into the nucleus occurred independently of envelope glycoproteins or target cells. Fluorescence correlation spectroscopy revealed two forms of nuclear Vpr-monomers and very large complexes, likely involving host factors. The kinetics of viral Vpr entering the nucleus after fusion was not affected by point mutations in the capsid protein that alter the stability of the viral core. The independence of Vpr shedding of capsid stability and its relatively rapid dissociation from post-fusion cores suggest that this process may precede capsid uncoating, which appears to occur on a slower time scale. Our results thus demonstrate that a bulk of fluorescently labeled Vpr incorporated into HIV-1 particles is released shortly after fusion. Future studies will address the question whether the quick and efficient nuclear delivery of Vpr derived from incoming viruses can regulate subsequent steps of HIV-1 infection.

  9. 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

  10. Centrioles: active players or passengers during mitosis?

    Science.gov (United States)

    Debec, Alain; Sullivan, William; Bettencourt-Dias, Monica

    2010-07-01

    Centrioles are cylinders made of nine microtubule (MT) triplets present in many eukaryotes. Early studies, where centrosomes were seen at the poles of the mitotic spindle led to their coining as "the organ for cell division". However, a variety of subsequent observational and functional studies showed that centrosomes might not always be essential for mitosis. Here we review the arguments in this debate. We describe the centriole structure and its distribution in the eukaryotic tree of life and clarify its role in the organization of the centrosome and cilia, with an historical perspective. An important aspect of the debate addressed in this review is how centrioles are inherited and the role of the spindle in this process. In particular, germline inheritance of centrosomes, such as their de novo formation in parthenogenetic species, poses many interesting questions. We finish by discussing the most likely functions of centrioles and laying out new research avenues.

  11. Maximal Fluctuations of Confined Actomyosin Gels: Dynamics of the Cell Nucleus.

    Science.gov (United States)

    Rupprecht, J-F; Singh Vishen, A; Shivashankar, G V; Rao, M; Prost, J

    2018-03-02

    We investigate the effect of stress fluctuations on the stochastic dynamics of an inclusion embedded in a viscous gel. We show that, in nonequilibrium systems, stress fluctuations give rise to an effective attraction towards the boundaries of the confining domain, which is reminiscent of an active Casimir effect. We apply this generic result to the dynamics of deformations of the cell nucleus, and we demonstrate the appearance of a fluctuation maximum at a critical level of activity, in agreement with recent experiments [E. Makhija, D. S. Jokhun, and G. V. Shivashankar, Proc. Natl. Acad. Sci. U.S.A. 113, E32 (2016)PNASA60027-842410.1073/pnas.1513189113].

  12. Estimation of the radiation-induced DNA double-strand breaks number by considering cell cycle and absorbed dose per cell nucleus.

    Science.gov (United States)

    Mori, Ryosuke; Matsuya, Yusuke; Yoshii, Yuji; Date, Hiroyuki

    2018-05-01

    DNA double-strand breaks (DSBs) are thought to be the main cause of cell death after irradiation. In this study, we estimated the probability distribution of the number of DSBs per cell nucleus by considering the DNA amount in a cell nucleus (which depends on the cell cycle) and the statistical variation in the energy imparted to the cell nucleus by X-ray irradiation. The probability estimation of DSB induction was made following these procedures: (i) making use of the Chinese Hamster Ovary (CHO)-K1 cell line as the target example, the amounts of DNA per nucleus in the logarithmic and the plateau phases of the growth curve were measured by flow cytometry with propidium iodide (PI) dyeing; (ii) the probability distribution of the DSB number per cell nucleus for each phase after irradiation with 1.0 Gy of 200 kVp X-rays was measured by means of γ-H2AX immunofluorescent staining; (iii) the distribution of the cell-specific energy deposition via secondary electrons produced by the incident X-rays was calculated by WLTrack (in-house Monte Carlo code); (iv) according to a mathematical model for estimating the DSB number per nucleus, we deduced the induction probability density of DSBs based on the measured DNA amount (depending on the cell cycle) and the calculated dose per nucleus. The model exhibited DSB induction probabilities in good agreement with the experimental results for the two phases, suggesting that the DNA amount (depending on the cell cycle) and the statistical variation in the local energy deposition are essential for estimating the DSB induction probability after X-ray exposure.

  13. Nucleoporins redistribute inside the nucleus after cell cycle arrest induced by histone deacetylases inhibition.

    Science.gov (United States)

    Pérez-Garrastachu, Miguel; Arluzea, Jon; Andrade, Ricardo; Díez-Torre, Alejandro; Urtizberea, Marta; Silió, Margarita; Aréchaga, Juan

    2017-09-03

    Nucleoporins are the main components of the nuclear-pore complex (NPC) and were initially considered as mere structural elements embedded in the nuclear envelope, being responsible for nucleocytoplasmic transport. Nevertheless, several recent scientific reports have revealed that some nucleoporins participate in nuclear processes such as transcription, replication, DNA repair and chromosome segregation. Thus, the interaction of NPCs with chromatin could modulate the distribution of chromosome territories relying on the epigenetic state of DNA. In particular, the nuclear basket proteins Tpr and Nup153, and the FG-nucleoporin Nup98 seem to play key roles in all these novel functions. In this work, histone deacetylase inhibitors (HDACi) were used to induce a hyperacetylated state of chromatin and the behavior of the mentioned nucleoporins was studied. Our results show that, after HDACi treatment, Tpr, Nup153 and Nup98 are translocated from the nuclear pore toward the interior of the cell nucleus, accumulating as intranuclear nucleoporin clusters. These transitory structures are highly dynamic, and are mainly present in the population of cells arrested at the G0/G1 phase of the cell cycle. Our results indicate that the redistribution of these nucleoporins from the nuclear envelope to the nuclear interior may be implicated in the early events of cell cycle initialization, particularly during the G1 phase transition.

  14. Melatonin resists oxidative stress-induced apoptosis in nucleus pulposus cells.

    Science.gov (United States)

    He, Ruijun; Cui, Min; Lin, Hui; Zhao, Lei; Wang, Jiayu; Chen, Songfeng; Shao, Zengwu

    2018-04-15

    Intervertebral disc degeneration (IVDD) is thought to be the major cause of low back pain (LBP), which is still in lack of effective etiological treatment. Oxidative stress has been demonstrated to participate in the impairment of nucleus pulposus cells (NPCs). As the most important neuroendocrine hormone in biological clock regulation, melatonin (MLT) is also featured by good antioxidant effect. In this study, we investigated the effect and mechanisms of melatonin on oxidative stress-induced damage in rat NPCs. Cytotoxicity of H 2 O 2 and protecting effect of melatonin were analyzed with Cell Counting kit-8 (CCK-8). Cell apoptosis rate was detected by Annexin V-FITC/PI staining. DCFH-DA probe was used for the reactive oxygen species (ROS) detection. The mitochondrial membrane potential (MMP) changes were analyzed with JC-1 probe. Intracellular oxidation product and reductants were measured through enzymatic reactions. Extracellular matrix (ECM) and apoptosis associated proteins were analyzed with Western blot assays. Melatonin preserved cell viability of NPCs under oxidative stress. The apoptosis rate, ROS level and malonaldehyde (MDA) declined with melatonin. MLT/H 2 O 2 group showed higher activities of GSH and SOD. The fall of MMP receded and the expression of ECM protein increased with treatment of melatonin. The mitochondrial pathway of apoptosis was inhibited by melatonin. Melatonin alleviated the oxidative stress-induced apoptosis of NPCs. Melatonin could be a promising alternative in treatment of IVDD. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. 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.

  16. Specific nuclear localizing sequence directs two myosin isoforms to the cell nucleus in calmodulin-sensitive manner.

    Science.gov (United States)

    Dzijak, Rastislav; Yildirim, Sukriye; Kahle, Michal; Novák, Petr; Hnilicová, Jarmila; Venit, Tomáš; Hozák, Pavel

    2012-01-01

    Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the "cytoplasmic" myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. We have shown that the novel specific NLS brings to the cell nucleus not only the "nuclear" isoform of myosin I (NM1 protein) but also its "cytoplasmic" isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus.

  17. 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

  18. Cycling with BRCA2 from DNA repair to mitosis

    International Nuclear Information System (INIS)

    Lee, Hyunsook

    2014-01-01

    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

  19. 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.

  20. 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.

  1. Chromatin and lamin A determine two different mechanical response regimes of the cell nucleus.

    Science.gov (United States)

    Stephens, Andrew D; Banigan, Edward J; Adam, Stephen A; Goldman, Robert D; Marko, John F

    2017-07-07

    The cell nucleus must continually resist and respond to intercellular and intracellular mechanical forces to transduce mechanical signals and maintain proper genome organization and expression. Altered nuclear mechanics is associated with many human diseases, including heart disease, progeria, and cancer. Chromatin and nuclear envelope A-type lamin proteins are known to be key nuclear mechanical components perturbed in these diseases, but their distinct mechanical contributions are not known. Here we directly establish the separate roles of chromatin and lamin A/C and show that they determine two distinct mechanical regimes via micromanipulation of single isolated nuclei. Chromatin governs response to small extensions (<3 μm), and euchromatin/heterochromatin levels modulate the stiffness. In contrast, lamin A/C levels control nuclear strain stiffening at large extensions. These results can be understood through simulations of a polymeric shell and cross-linked polymer interior. Our results provide a framework for understanding the differential effects of chromatin and lamin A/C in cell nuclear mechanics and their alterations in disease. © 2017 Stephens et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  2. The Cell Nucleus Serves as a Mechanotransducer of Tissue Damage-Induced Inflammation.

    Science.gov (United States)

    Enyedi, Balázs; Jelcic, Mark; Niethammer, Philipp

    2016-05-19

    Tissue damage activates cytosolic phospholipase A2 (cPLA2), releasing arachidonic acid (AA), which is oxidized to proinflammatory eicosanoids by 5-lipoxygenase (5-LOX) on the nuclear envelope. How tissue damage is sensed to activate cPLA2 is unknown. We investigated this by live imaging in wounded zebrafish larvae, where damage of the fin tissue causes osmotic cell swelling at the wound margin and the generation of a chemotactic eicosanoid signal. Osmotic swelling of cells and their nuclei activates cPla2 by translocating it from the nucleoplasm to the nuclear envelope. Elevated cytosolic Ca(2+) was necessary but not sufficient for cPla2 translocation, and nuclear swelling was required in parallel. cPla2 translocation upon nuclear swelling was reconstituted in isolated nuclei and appears to be a simple physical process mediated by tension in the nuclear envelope. Our data suggest that the nucleus plays a mechanosensory role in inflammation by transducing cell swelling and lysis into proinflammatory eicosanoid signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. 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.; van den Hoff, M. J.; Dam, K.; Lamers, W. H.; Medema, R. H.; Clevers, H.

    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

  4. Raman spectroscopy of individual monocytes reveals that single-beam optical trapping of mononuclear cells occurs by their nucleus

    International Nuclear Information System (INIS)

    Fore, Samantha; Chan, James; Taylor, Douglas; Huser, Thomas

    2011-01-01

    We show that laser tweezers Raman spectroscopy of eukaryotic cells with a significantly larger diameter than the tight focus of a single-beam laser trap leads to optical trapping of the cell by its optically densest part, i.e. typically the cell's nucleus. Raman spectra of individual optically trapped monocytes are compared with location-specific Raman spectra of monocytes adhered to a substrate. When the cell's nucleus is stained with a fluorescent live cell stain, the Raman spectrum of the DNA-specific stain is observed only in the nucleus of individual monocytes. Optically trapped monocytes display the same behavior. We also show that the Raman spectra of individual monocytes exhibit the characteristic Raman signature of cells that have not yet fully differentiated and that individual primary monocytes can be distinguished from transformed monocytes based on their Raman spectra. This work provides further evidence that laser tweezers Raman spectroscopy of individual cells provides meaningful biochemical information in an entirely non-destructive fashion that permits discerning differences between cell types and cellular activity

  5. Nucleus--nucleus potential

    International Nuclear Information System (INIS)

    Jaqaman, H.R.

    1977-01-01

    The nucleus--nucleus interaction is studied within the framework of the generator coordinate method that permits an easy incorporation of the full effects of antisymmetrization. It is found that the interaction, as far as the elastic scattering problem is concerned, can be described by a simple effective potential that is equivalent to the original many-body (and hence non-local) problem. The potential is obtained by dividing the wavefunction into a long-range part and a short-range part and requiring the former to satisfy a Schroedinger equation. This enables avoiding dealing with the troublesome short-range part of the wavefunction and provides a direct link with the optical model so that the potential obtained here is equivalent to the real part of the optical potential (the imaginary part is not investigated). The effective potential is found to consist of three parts: an interaction term between the nucleons belonging to different nuclei, a kinetic energy term due to the change in the intrinsic kinetic energy of the system as a result of the antisymmetrization, and finally an l-dependent part. The kinetic energy term is found to be very repulsive and effectively gives a hard core, and is calculated for the α--α and 16 O-- 16 O cases. The full potential is calculated for the α--α case for the S, D, and G partial waves and then used to calculate the corresponding phase shifts that are then compared with experimental results and other microscopic calculations. Finally, some recent results and analyses of fusion and deep inelastic reactions are reviewed that seem to indicate the presence of a hard core in the nucleus--nucleus potential. Such a hard core is present in the potential obtained in the sudden approximation

  6. In situ fluorescence activation of DNA-silver nanoclusters as a label-free and general strategy for cell nucleus imaging.

    Science.gov (United States)

    Li, Duo; Qiao, Zhenzhen; Yu, Yanru; Tang, Jinlu; He, Xiaoxiao; Shi, Hui; Ye, Xiaosheng; Lei, Yanli; Wang, Kemin

    2018-01-25

    A facile, general and turn-on nucleus imaging strategy was first developed based on in situ fluorescence activation of C-rich dark silver nanoclusters by G-rich telomeres. After a simple incubation without washing, nanoclusters could selectively stain the nucleus with intense red luminescence, which was confirmed using fixed/living cells and several cell lines.

  7. Gold nanoparticle-aided brachytherapy with vascular dose painting: estimation of dose enhancement to the tumor endothelial cell nucleus.

    Science.gov (United States)

    Ngwa, Wilfred; Makrigiorgos, G Mike; Berbeco, Ross I

    2012-01-01

    Theoretical microdosimetry at the subcellular level is employed in this study to estimate the dose enhancement to tumor endothelial cell nuclei, caused by radiation-induced photo/Auger electrons originating from gold nanoparticles (AuNPs) targeting the tumor endothelium, during brachytherapy. A tumor vascular endothelial cell (EC) is modeled as a slab of 2 μm (thickness) × 10 μm (length) × 10 μm (width). The EC contains a nucleus of 5 μm diameter and thickness of 0.5-1 μm, corresponding to nucleus size 5%-10% of cellular volume, respectively. Analytic calculations based on the electron energy loss formula of Cole were carried out to estimate the dose enhancement to the nucleus caused by photo/Auger electrons from AuNPs attached to the exterior surface of the EC. The nucleus dose enhancement factor (nDEF), representing the ratio of the dose to the nucleus with and without the presence of gold nanoparticles was calculated for different AuNP local concentrations. The investigated concentration range considers the potential for significantly higher local concentration near the EC due to preferential accumulation of AuNP in the tumor vasculature. Four brachytherapy sources: I-125, Pd-103, Yb-169, and 50 kVp x-rays were investigated. For nucleus size of 10% of the cellular volume and AuNP concentrations ranging from 7 to 140 mg/g, brachytherapy sources Pd-103, I-125, 50 kVp, and Yb-169 yielded nDEF values of 5.6-73, 4.8-58.3, 4.7-56.6, and 3.2-25.8, respectively. Meanwhile, for nucleus size 5% of the cellular volume in the same concentration range, Pd-103, I-125, 50 kVp, and Yb-169 yielded nDEF values of 6.9-79.2, 5.1-63.2, 5.0-61.5, and 3.3-28.3, respectively. The results predict that a substantial dose boost to the nucleus of endothelial cells can be achieved by applying tumor vasculature-targeted AuNPs in combination with brachytherapy. Such vascular dose boosts could induce tumor vascular shutdown, prompting extensive tumor cell death.

  8. Gold nanoparticle-aided brachytherapy with vascular dose painting: Estimation of dose enhancement to the tumor endothelial cell nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Ngwa, Wilfred; Makrigiorgos, G. Mike; Berbeco, Ross I. [Department of Radiation Oncology, Division of Medical Physics and Biophysics, Brigham and Women' s Hospital, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115 (United States)

    2012-01-15

    Purpose: Theoretical microdosimetry at the subcellular level is employed in this study to estimate the dose enhancement to tumor endothelial cell nuclei, caused by radiation-induced photo/Auger electrons originating from gold nanoparticles (AuNPs) targeting the tumor endothelium, during brachytherapy. Methods: A tumor vascular endothelial cell (EC) is modeled as a slab of 2 {mu}m (thickness) x 10 {mu}m (length) x 10 {mu}m (width). The EC contains a nucleus of 5 {mu}m diameter and thickness of 0.5-1 {mu}m, corresponding to nucleus size 5%-10% of cellular volume, respectively. Analytic calculations based on the electron energy loss formula of Cole were carried out to estimate the dose enhancement to the nucleus caused by photo/Auger electrons from AuNPs attached to the exterior surface of the EC. The nucleus dose enhancement factor (nDEF), representing the ratio of the dose to the nucleus with and without the presence of gold nanoparticles was calculated for different AuNP local concentrations. The investigated concentration range considers the potential for significantly higher local concentration near the EC due to preferential accumulation of AuNP in the tumor vasculature. Four brachytherapy sources: I-125, Pd-103, Yb-169, and 50 kVp x-rays were investigated. Results: For nucleus size of 10% of the cellular volume and AuNP concentrations ranging from 7 to 140 mg/g, brachytherapy sources Pd-103, I-125, 50 kVp, and Yb-169 yielded nDEF values of 5.6-73, 4.8-58.3, 4.7-56.6, and 3.2-25.8, respectively. Meanwhile, for nucleus size 5% of the cellular volume in the same concentration range, Pd-103, I-125, 50 kVp, and Yb-169 yielded nDEF values of 6.9-79.2, 5.1-63.2, 5.0-61.5, and 3.3-28.3, respectively. Conclusions: The results predict that a substantial dose boost to the nucleus of endothelial cells can be achieved by applying tumor vasculature-targeted AuNPs in combination with brachytherapy. Such vascular dose boosts could induce tumor vascular shutdown, prompting

  9. DNA damage and cell cycle events implicate cerebellar dentate nucleus neurons as targets of Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Yang Yan

    2010-12-01

    Full Text Available Abstract Background Although the cerebellum is considered to be predominantly involved in fine motor control, emerging evidence documents its participation in language, impulsive behavior and higher cognitive functions. While the specific connections of the cerebellar deep nuclei (CDN that are responsible for these functions are still being worked out, their deficiency has been termed "cerebellar cognitive affective syndrome" - a syndrome that bears a striking similarity to many of the symptoms of Alzheimer's disease (AD. Using ectopic cell cycle events and DNA damage markers as indexes of cellular distress, we have explored the neuropathological involvement of the CDN in human AD. Results We examined the human cerebellar dentate nucleus in 22 AD cases and 19 controls for the presence of neuronal cell cycle events and DNA damage using immunohistochemistry and fluorescence in situ hybridization. Both techniques revealed several instances of highly significant correlations. By contrast, neither amyloid plaque nor neurofibrillary tangle pathology was detected in this region, consistent with previous reports of human cerebellar pathology. Five cases of early stage AD were examined and while cell cycle and DNA damage markers were well advanced in the hippocampus of all five, few indicators of either cell cycle events (1 case or a DNA damage response (1 case were found in CDN. This implies that CDN neurons are most likely affected later in the course of AD. Clinical-pathological correlations revealed that cases with moderate to high levels of cell cycle activity in their CDN are highly likely to show deficits in unorthodox cerebellar functions including speech, language and motor planning. Conclusion Our results reveal that the CDN neurons are under cellular stress in AD and suggest that some of the non-motor symptoms found in patients with AD may be partly cerebellar in origin.

  10. Chronic tinnitus and unipolar brush cell alterations in the cerebellum and dorsal cochlear nucleus.

    Science.gov (United States)

    Brozoski, Thomas; Brozoski, Daniel; Wisner, Kurt; Bauer, Carol

    2017-07-01

    Animal model research has shown that the central features of tinnitus, the perception of sound without an acoustic correlate, include elevated spontaneous and stimulus-driven activity, enhanced burst-mode firing, decreased variance of inter-spike intervals, and distortion of tonotopic frequency representation. Less well documented are cell-specific correlates of tinnitus. Unipolar brush cell (UBC) alterations in animals with psychophysical evidence of tinnitus has recently been reported. UBCs are glutamatergic interneurons that appear to function as local-circuit signal amplifiers. UBCs are abundant in the dorsal cochlear nucleus (DCN) and very abundant in the flocculus (FL) and paraflocculus (PFL) of the cerebellum. In the present research, two indicators of UBC structure and function were examined: Doublecortin (DCX) and epidermal growth factor receptor substrate 8 (Eps8). DCX is a protein that binds to microtubules where it can modify their assembly and growth. Eps8 is a cell-surface tyrosine kinase receptor mediating the response to epidermal growth factor; it appears to have a role in actin polymerization as well as cytoskeletal protein interactions. Both functions could contribute to synaptic remodeling. In the present research UBC Eps8 and DCX immunoreactivity (IR) were determined in 4 groups of rats distinguished by their exposure to high-level sound and psychophysical performance: Unexposed, exposed to high-level sound with behavioral evidence of tinnitus, and two exposed groups without behavioral evidence of tinnitus. Compared to unexposed controls, exposed animals with tinnitus had Eps8 IR elevated in their PFL; other structures were not affected, nor was DCX IR affected. This was interpreted as UBC upregulation in animals with tinnitus. Exposure that failed to produce tinnitus did not increase either Eps8 or DCX IR. Rather Eps8 IR was decreased in the FL and DCN of one subgroup (Least-Tinnitus), while DCX IR decreased in the FL of the other subgroup (No

  11. Ase1p Organizes Antiparallel Microtubule Arrays during Interphase and Mitosis in Fission YeastV⃞

    OpenAIRE

    Loïodice, Isabelle; Staub, Jayme; Setty, Thanuja Gangi; Nguyen, Nam-Phuong T.; Paoletti, Anne; Tran, P. T.

    2005-01-01

    Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ...

  12. Acute Endoplasmic Reticulum Stress-Independent Unconventional Splicing of XBP1 mRNA in the Nucleus of Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Yuanyuan Wang

    2015-06-01

    Full Text Available The regulation of expression of X-box-binding protein-1 (XBP1, a transcriptional factor, involves an unconventional mRNA splicing that removes the 26 nucleotides intron. In contrast to the conventional splicing that exclusively takes place in the nucleus, determining the location of unconventional splicing still remains controversial. This study was designed to examine whether the unconventional spicing of XBP1 mRNA could occur in the nucleus and its possible biological relevance. We use RT-PCR reverse transcription system and the expand high fidelity PCR system to detect spliced XBP1 mRNA, and fraction cells to determine the location of the unconventional splicing of XBP1 mRNA. We employ reporter constructs to show the presence of unconventional splicing machinery in mammal cells independently of acute endoplasmic reticulum (ER stress. Our results reveal the presence of basal unconventional splicing of XBP1 mRNA in the nucleus that also requires inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α and can occur independently of acute ER stress. Furthermore, we confirm that acute ER stress induces the splicing of XBP1 mRNA predominantly occurring in the cytoplasm, but it also promotes the splicing in the nucleus. The deletion of 5′-nucleotides in XBP1 mRNA significantly increases its basal unconventional splicing, suggesting that the secondary structure of XBP1 mRNA may determine the location of unconventional splicing. These results suggest that the unconventional splicing of XBP1 mRNA can take place in the nucleus and/or cytoplasm, which possibly depends on the elaborate regulation. The acute ER stress-independent unconventional splicing in the nucleus is most likely required for the maintaining of day-to-day folding protein homeostasis.

  13. The generation and functional characterization of induced pluripotent stem cells from human intervertebral disc nucleus pulposus cells.

    Science.gov (United States)

    Zhu, Yanxia; Liang, Yuhong; Zhu, Hongxia; Lian, Cuihong; Wang, Liang; Wang, Yiwei; Gu, Hongsheng; Zhou, Guangqian; Yu, Xiaoping

    2017-06-27

    Disc degenerative disease (DDD) is believed to originate in the nucleus pulposus (NP) region therefore, it is important to obtain a greater number of active NP cells for the study and therapy of DDD. Human induced pluripotent stem cells (iPSCs) are a powerful tool for modeling the development of DDD in humans, and have the potential to be applied in regenerative medicine. NP cells were isolated from DDD patients following our improved method, and then the primary NP cells were reprogramed into iPSCs with Sendai virus vectors encoding 4 factors. Successful reprogramming of iPSCs was verified by the expression of surface markers and presence of teratoma. Differentiation of iPSCs into NP-like cells was performed in a culture plate or in hydrogel, whereby skin fibroblast derived-iPSCs were used as a control. Results demonstrated that iPSCs derived from NP cells displayed a normal karyotype, expressed pluripotency markers, and formed teratoma in nude mice. NP induction of iPSCs resulted in the expression of NP cell specific matrix proteins and related genes. Non-induced NP derived-iPSCs also showed some NP-like phenotype. Furthermore, NP-derived iPSCs differentiate much better in hydrogel than that in a culture plate. This is a novel method for the generation of iPSCs from NP cells of DDD patients, and we have successfully differentiated these iPSCs into NP-like cells in hydrogel. This method provides a novel treatment of DDD by using patient-specific NP cells in a relatively simple and straightforward manner.

  14. Co-culture of Adult Mesenchymal Stem Cells and Nucleus Pulposus Cells in Bilaminar Pellets for Intervertebral Disc Regeneration.

    Science.gov (United States)

    Allon, Aliza A; Schneider, Richard A; Lotz, Jeffrey C

    2009-01-01

    Our goal is to optimize stem cell-based tissue engineering strategies in the context of the intervertebral disc environment. We explored the benefits of co-culturing nucleus pulposus cells (NPC) and adult mesenchymal stem cells (MSC) using a novel spherical bilaminar pellet culture system where one cell type is enclosed in a sphere of the other cell type. Our 3D system provides a structure that exploits embryonic processes such as tissue induction and condensation. We observed a unique phenomenon: the budding of co-culture pellets and the formation of satellite pellets that separate from the main pellet. MSC and NPC co-culture pellets were formed with three different structural organizations. The first had random organization. The other two had bilaminar organization with either MSC inside and NPC outside or NPC inside and MSC outside. By 14 days, all co-culture pellets exhibited budding and spontaneously generated satellite pellets. The satellite pellets were composed of both cell types and, surprisingly, all had the same bilaminar organization with MSC on the inside and NPC on the outside. This organization was independent of the structure of the main pellet that the satellites stemmed from. The main pellets generated satellite pellets that spontaneously organized into a bilaminar structure. This implies that structural organization occurs naturally in this cell culture system and may be inherently favorable for cell-based tissue engineering strategies. The occurrence of budding and the organization of satellite pellets may have important implications for the use of co-culture pellets in cell-based therapies for disc regeneration. From a therapeutic point of view, the generation of satellite pellets may be a beneficial feature that would serve to spread donor cells throughout the host matrix and restore normal matrix composition in a sustainable way, ultimately renewing tissue function.

  15. Apoptosis and mitosis in the small intestine at radiation injury

    International Nuclear Information System (INIS)

    Hashiguchi, Junichiro; Ito, Masahiro; Onizuka, Shinya; Sekine, Ichiro; Uchida, Shinji

    1990-01-01

    A single whole body irradiation was given at a dose rate of 0.298 Gy/min in 6-week-old male mice. Intestinal crypt apoptosis and mitosis cells were determined by delivering radiation doses of 0.4, 0.6, 1.0, 1.5, 2.0, 5.0, 10.0, and 20.0 Gy. The incidence of apoptosis was linearly increased in a dose-dependent manner up to 5.0 Gy, and thereafter, it was gradually decreased. There was a decreased tendency for mitosis with delivering higher radiation doses. The incidence of apoptosis rapidly increased 2 hours after irradiation with either 0.6 Gy or 2.0 Gy, and reached to the peak 4 hours later. It brought about a 18-fold and 28-fold increase for 0.6 Gy and 2.0 Gy, respectively, relative to that before irradiation. Mitosis cells decreased by half one hour after irradiation with 0.6 Gy, and then returned to the pre-irradiation value through synchronization 24 hours later. The number of cells positive to BrdU was 776 in the group of mice without irradiation and 479 in the group of mice irradiated with 2.0 Gy. (N.K.)

  16. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Mesenchymal Stem Cells Protect Nucleus Pulposus Cells from Compression-Induced Apoptosis by Inhibiting the Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    Sheng Chen

    2017-01-01

    Full Text Available Objective. Excessive apoptosis of nucleus pulposus cells (NPCs induced by various stresses, including compression, contributes to the development of intervertebral disc degeneration (IVDD. Mesenchymal stem cells (MSCs can benefit the regeneration of NPCs and delay IVDD, but the underlying molecular mechanism is poorly understood. This study aimed to evaluate the antiapoptosis effects of bone marrow-derived MSC (BMSC on rat NPCs exposed to compression and investigate whether the mitochondrial pathway was involved. Methods. BMSCs and NPCs were cocultured in the compression apparatus at 1.0 MPa for 36 h. Cell viability, apoptosis, mitochondrial function, and the expression of apoptosis-related proteins were evaluated. Results. The results showed that coculturing with BMSCs increased the cell viability and reduced apoptosis of NPCs exposed to compression. Meanwhile, BMSCs could relieve the compression-induced mitochondrial damage of NPCs by decreasing reactive oxygen species level and maintaining mitochondrial membrane potential as well as mitochondrial integrity. Furthermore, coculturing with BMSCs suppressed the activated caspase-3 and activated caspase-9, decreased the expressions of cytosolic cytochrome c and Bax, and increased the expression of Bcl-2. Conclusions. Our results suggest that BMSCs can protect against compression-induced apoptosis of NPCs by inhibiting the mitochondrial pathway and thus enhance our understanding on the MSC-based therapy for IVDD.

  18. Regulatory functional territory of PLK-1 and their substrates beyond mitosis.

    Science.gov (United States)

    Kumar, Shiv; Sharma, Garima; Chakraborty, Chiranjib; Sharma, Ashish Ranjan; Kim, Jaebong

    2017-06-06

    Polo-like kinase 1 (PLK-1) is a well-known (Ser/Thr) mitotic protein kinase and is considered as a proto-oncogene. As hyper-activation of PLK-1 is broadly associated with poor prognosis and cancer progression, it is one of the most extensively studied mitotic kinases. During mitosis, PLK-1 regulates various cell cycle events, such as spindle pole maturation, chromosome segregation and cytokinesis. However, studies have demonstrated that the role of PLK-1 is not only restricted to mitosis, but PLK-1 can also regulate other vital events beyond mitosis, including transcription, translation, ciliogenesis, checkpoint adaptation and recovery, apoptosis, chromosomes dynamics etc. Recent reviews have tried to define the regulatory role of PLK-1 during mitosis progression and tumorigenesis, but its' functional role beyond mitosis is still largely unexplored. PLK-1 can regulate the activity of many proteins that work outside of its conventional territory. The dysregulation of these proteins can cause diseases such as Alzheimer's disease, tumorigenesis etc. and may also lead to drug resistance. Thus, in this review, we discussed the versatile role of PLK-1 and tried to collect data to validate its' functional role in cell cycle regulation apart from mitosis.

  19. 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.

  20. Cell thickness of UV absorption by the cell: relation to UV action spectrum shift in mammalian cells in culture

    International Nuclear Information System (INIS)

    Sakharov, V.H.; Voronkova, L.N.; Blokhin, A.V.

    1985-01-01

    By means of reconstruction of series half - thin transverse sections the three - dimensional morphometry of SPEV cells for a series of their specific states in culture is performed: for exponential growth in a monolayer, in a merged monolayer, in the mitosis phase, for giant cells and suspension cells. In the monolayer the cell thickness in its central part depended mainly on the nucleus thickness and in average changed but slightly despite a wide range of changes in volumes of nuclei and cells and their density in culture. The cell thickness has noticeably increased in mitosis. For the above states of cells UV radiation absorption spectra are determined. It is shown that a certain shift of action spectrus of death of mammalian cells as compared with that for bacterial cell can be a seguence of selfshielding and not differences in the nature of active chromophores

  1. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Neurons of the rat suprachiasmatic nucleus show a circadian rhythm in membrane properties that is lost during prolonged whole-cell recording

    NARCIS (Netherlands)

    Schaap, J.; Bos, N. P.; de Jeu, M. T.; Geurtsen, A. M.; Meijer, J. H.; Pennartz, C. M.

    1999-01-01

    The suprachiasmatic nucleus is commonly considered to contain the main pacemaker of behavioral and hormonal circadian rhythms. Using whole-cell patch-clamp recordings, the membrane properties of suprachiasmatic nucleus neurons were investigated in order to get more insight in membrane physiological

  3. Strand-like structures and the nonstructural proteins 5, 3 and 1 are present in the nucleus of mosquito cells infected with dengue virus.

    Science.gov (United States)

    Reyes-Ruiz, José M; Osuna-Ramos, Juan F; Cervantes-Salazar, Margot; Lagunes Guillen, Anel E; Chávez-Munguía, Bibiana; Salas-Benito, Juan S; Del Ángel, Rosa M

    2018-02-01

    Dengue virus (DENV) is an arbovirus, which replicates in the endoplasmic reticulum. Although replicative cycle takes place in the cytoplasm, some viral proteins such as NS5 and C are translocated to the nucleus during infection in mosquitoes and mammalian cells. To localized viral proteins in DENV-infected C6/36 cells, an immunofluorescence (IF) and immunoelectron microscopy (IEM) analysis were performed. Our results indicated that C, NS1, NS3 and NS5 proteins were found in the nucleus of DENV-infected C6/36 cells. Additionally, complex structures named strand-like structures (Ss) were observed in the nucleus of infected cells. Interestingly, the NS5 protein was located in these structures. Ss were absent in mock-infected cells, suggesting that DENV induces their formation in the nucleus of infected mosquito cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Excess free histone H3 localizes to centrosomes for proteasome-mediated degradation during mitosis in metazoans.

    Science.gov (United States)

    Wike, Candice L; Graves, Hillary K; Wason, Arpit; Hawkins, Reva; Gopalakrishnan, Jay; Schumacher, Jill; Tyler, Jessica K

    2016-08-17

    The cell tightly controls histone protein levels in order to achieve proper packaging of the genome into chromatin, while avoiding the deleterious consequences of excess free histones. Our accompanying study has shown that a histone modification that loosens the intrinsic structure of the nucleosome, phosphorylation of histone H3 on threonine 118 (H3 T118ph), exists on centromeres and chromosome arms during mitosis. Here, we show that H3 T118ph localizes to centrosomes in humans, flies, and worms during all stages of mitosis. H3 abundance at the centrosome increased upon proteasome inhibition, suggesting that excess free histone H3 localizes to centrosomes for degradation during mitosis. In agreement, we find ubiquitinated H3 specifically during mitosis and within purified centrosomes. These results suggest that targeting of histone H3 to the centrosome for proteasome-mediated degradation is a novel pathway for controlling histone supply, specifically during mitosis.

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

    Science.gov (United States)

    Duro, Eris

    2015-01-01

    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. PMID:25593304

  6. FoxC2 Enhances BMP7-Mediated Anabolism in Nucleus Pulposus Cells of the Intervertebral Disc

    OpenAIRE

    Wang, Zheng; Fu, Changfeng; Chen, Yong; Xu, Feng; Wang, Zhenyu; Qu, Zhigang; Liu, Yi

    2016-01-01

    Bone-morphogenetic protein-7 (BMP-7) is a growth factor that plays a major role in mediating anabolism and anti-catabolism of the intervertebral disc matrix and cell homeostasis. In osteoblasts, Forkhead box protein C2 (FoxC2) is a downstream target of BMPs and promotes cell proliferation and differentiation. However, the role FoxC2 may play in degenerative human intervertebral disc tissue and the relationship between FoxC2 and BMP-7 in nucleus pulposus (NP) cells remain to be elucidated. Thi...

  7. Early intranuclear replication of African swine fever virus genome modifies the landscape of the host cell nucleus.

    Science.gov (United States)

    Simões, Margarida; Martins, Carlos; Ferreira, Fernando

    2015-12-02

    Although African swine fever virus (ASFV) replicates in viral cytoplasmic factories, the presence of viral DNA within the host cell nucleus has been previously reported to be essential for productive infection. Herein, we described, for the first time, the intranuclear distribution patterns of viral DNA replication events, preceding those that occur in the cytoplasmic compartment. Using BrdU pulse-labelling experiments, newly synthesized ASFV genomes were exclusively detected inside the host cell nucleus at the early phase of infection, both in swine monocyte-derived macrophages (MDMs) and Vero cells. From 8hpi onwards, BrdU labelling was only observed in ASFV cytoplasmic factories. Our results also show that ASFV specifically activates the Ataxia Telangiectasia Mutated Rad-3 related (ATR) pathway in ASFV-infected swine MDMs from the early phase of infection, most probably because ASFV genome is recognized as foreign DNA. Morphological changes of promyelocytic leukaemia nuclear bodies (PML-NBs), nuclear speckles and Cajal bodies were also found in ASFV-infected swine MDMs, strongly suggesting the viral modulation of cellular antiviral responses and cellular transcription, respectively. As described for other viral infections, the nuclear reorganization that takes place during ASFV infection may also provide an environment that favours its intranuclear replication events. Altogether, our results contribute for a better understanding of ASFV replication strategies, starting with an essential intranuclear DNA replication phase which induces host nucleus changes towards a successful viral infection. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Visualizing Stable Features in Live Cell Nucleus for Evaluation of the Cell Global Motion Compensation

    Czech Academy of Sciences Publication Activity Database

    Sorokin, D.V.; Suchánková, Jana; Bártová, Eva; Matula, P.

    2014-01-01

    Roč. 60, č. 1 (2014), s. 45-49 ISSN 0015-5500 R&D Projects: GA ČR GBP302/12/G157; GA MŠk(CZ) EE2.3.30.0030 Institutional support: RVO:68081707 Keywords : cell global motion compensation * UV laser bleaching * image registration Subject RIV: BO - Biophysics Impact factor: 1.000, year: 2014

  9. Polysialic acid enters the cell nucleus attached to a fragment of the neural cell adhesion molecule NCAM to regulate the circadian rhythm in mouse brain.

    Science.gov (United States)

    Westphal, Nina; Kleene, Ralf; Lutz, David; Theis, Thomas; Schachner, Melitta

    2016-07-01

    In the mammalian nervous system, the neural cell adhesion molecule NCAM is the major carrier of the glycan polymer polysialic acid (PSA) which confers important functions to NCAM's protein backbone. PSA attached to NCAM contributes not only to cell migration, neuritogenesis, synaptic plasticity, and behavior, but also to regulation of the circadian rhythm by yet unknown molecular mechanisms. Here, we show that a PSA-carrying transmembrane NCAM fragment enters the nucleus after stimulation of cultured neurons with surrogate NCAM ligands, a phenomenon that depends on the circadian rhythm. Enhanced nuclear import of the PSA-carrying NCAM fragment is associated with altered expression of clock-related genes, as shown by analysis of cultured neuronal cells deprived of PSA by specific enzymatic removal. In vivo, levels of nuclear PSA in different mouse brain regions depend on the circadian rhythm and clock-related gene expression in suprachiasmatic nucleus and cerebellum is affected by the presence of PSA-carrying NCAM in the cell nucleus. Our conceptually novel observations reveal that PSA attached to a transmembrane proteolytic NCAM fragment containing part of the extracellular domain enters the cell nucleus, where PSA-carrying NCAM contributes to the regulation of clock-related gene expression and of the circadian rhythm. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. In situ surface-enhanced Raman scattering spectroscopy exploring molecular changes of drug-treated cancer cell nucleus.

    Science.gov (United States)

    Liang, Lijia; Huang, Dianshuai; Wang, Hailong; Li, Haibo; Xu, Shuping; Chang, Yixin; Li, Hui; Yang, Ying-Wei; Liang, Chongyang; Xu, Weiqing

    2015-02-17

    Investigating the molecular changes of cancer cell nucleus with drugs treatment is crucial for the design of new anticancer drugs, the development of novel diagnostic strategies, and the advancement of cancer therapy efficiency. In order to better understand the action effects of drugs, accurate location and in situ acquisition of the molecular information of the cell nuclei are necessary. In this work, we report a microspectroscopic technique called dark-field and fluorescence coimaging assisted surface-enhanced Raman scattering (SERS) spectroscopy, combined with nuclear targeting nanoprobes, to in situ study Soma Gastric Cancer (SGC-7901) cell nuclei treated with two model drugs, e.g., DNA binder (Hoechst33342) and anticancer drug (doxorubicin, Dox) via spectral analysis at the molecular level. Nuclear targeting nanoprobes with an assembly structure of thiol-modified polyethylene glycol polymers (PEG) and nuclear localizing signal peptides (NLS) around gold nanorods (AuNRs) were prepared to achieve the amplified SERS signals of biomolecules in the cell nuclei. With the assistance of dark field/fluorescence imaging with simultaneous location, in situ SERS spectra in one cell nucleus were measured and analyzed to disclose the effects of Hoechst33342 and Dox on main biomolecules in the cell nuclei. The experimental results show that this method possesses great potential to investigate the targets of new anticancer drugs and the real-time monitoring of the dynamic changes of cells caused by exogenous molecules.

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

    OpenAIRE

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

    2016-01-01

    International audience; 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 ...

  12. Direct evidence that radiation induced micronuclei of early embryos require a mitosis for expression

    International Nuclear Information System (INIS)

    Mueller, W.U.; Schlusen, I.; Streffer, C.

    1991-01-01

    The naturally synchronous development of early mouse embryos was exploited to address the question, whether micronuclei require a mitosis for expression or whether they can be expressed in the same cell cycle, in which exposure to X-rays or caffeine took place. Experiments with 2-cell and with 4-cell embryos showed that micronulcei are expressed only if a mitosis is completed. There was no indication, even after doses up to 20 Gy, that micronuclei can be expressed before the mitosis was reached, which followed exposure. Furthermore, no nuclear fragmentation pointing to apoptosis could be detected in the cycle, in which cells were exposed. The same results were obtained when caffeine (5 mM) was used as micronucleus inducing agent. (orig.)

  13. 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...

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    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 < 0.05 for each) and was associated with increases in the phosphorylation and activation of EGFR, Rac1, and ERK1/2 (p < 0.05 for each). Pretreatment with the ERK1/2 selective inhibitor PD98059 reduced periodic mechanical stress-induced nucleus pulpous cell proliferation (p < 0.05 for each), while the activation levels of EGFR and Rac1 were not inhibited. Proliferation and phosphorylation of ERK1/2 were inhibited after pretreatment with the Rac1 inhibitor NSC23766 in nucleus pulpous cells in response to periodic mechanical stress (p < 0.05 for each), while the phosphorylation site of EGFR was not affected. Inhibition of EGFR activity with AG1478 abrogated nucleus pulpous cell proliferation (p < 0.05 for each) and attenuated Rac1 and ERK1/2 activation in nucleus pulpous cells subjected to periodic mechanical stress (p < 0.05 for each). These findings suggest that periodic 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. - Highlights: • The mechanism involved in nucleus pulpous cells to respond to mechanical stimuli. • Periodic mechanical stress can stimulate the phosphorylation of EGFR. • EGFR activates Rac1 and leads to rat nucleus pulpous cell proliferation. • EGFR and Rac1 activate ERK1/2 mitogenic signals in nucleus pulpous cells. • EGFR-Rac1-ERK1/2 is constitutes at least one critical signal transduction pathway.

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

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Gongming [Department of Orthopedics, The Affiliated Changzhou No. 2 Hospital of Nanjing Medical University, Changzhou 213003 (China); Shen, Nan [Department of Clinical Pharmacy, The Affiliated Jiangyin Hospital of Southeast University Medical School, Jiangyin 214400 (China); Jiang, Xuefeng; Sun, Huiqing [Department of Orthopedics, The Affiliated Jiangyin Hospital of Southeast University Medical School, Jiangyin 214400 (China); Xu, Nanwei; Zhou, Dong [Department of Orthopedics, The Affiliated Changzhou No. 2 Hospital of Nanjing Medical University, Changzhou 213003 (China); Nong, Luming, E-mail: lumingnong@hotmail.com [Department of Orthopedics, The Affiliated Changzhou No. 2 Hospital of Nanjing Medical University, Changzhou 213003 (China); Ren, Kewei, E-mail: keweiren@hotmail.com [Department of Orthopedics, The Affiliated Jiangyin Hospital of Southeast University Medical School, Jiangyin 214400 (China)

    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 < 0.05 for each) and was associated with increases in the phosphorylation and activation of EGFR, Rac1, and ERK1/2 (p < 0.05 for each). Pretreatment with the ERK1/2 selective inhibitor PD98059 reduced periodic mechanical stress-induced nucleus pulpous cell proliferation (p < 0.05 for each), while the activation levels of EGFR and Rac1 were not inhibited. Proliferation and phosphorylation of ERK1/2 were inhibited after pretreatment with the Rac1 inhibitor NSC23766 in nucleus pulpous cells in response to periodic mechanical stress (p < 0.05 for each), while the phosphorylation site of EGFR was not affected. Inhibition of EGFR activity with AG1478 abrogated nucleus pulpous cell proliferation (p < 0.05 for each) and attenuated Rac1 and ERK1/2 activation in nucleus pulpous cells subjected to periodic mechanical stress (p < 0.05 for each). These findings suggest that periodic 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. - Highlights: • The mechanism involved in nucleus pulpous cells to respond to mechanical stimuli. • Periodic mechanical stress can stimulate the phosphorylation of EGFR. • EGFR activates Rac1 and leads to rat nucleus pulpous cell proliferation. • EGFR and Rac1 activate ERK1/2 mitogenic signals in nucleus pulpous cells. • EGFR-Rac1-ERK1/2 is constitutes at least one critical signal transduction pathway.

  16. 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.

  17. Amyloid domains in the cell nucleus controlled by nucleoskeletal protein lamin B1 reveal a new pathway of mercury neurotoxicity

    Science.gov (United States)

    Arnhold, Florian; Gührs, Karl-Heinz

    2015-01-01

    Mercury (Hg) is a bioaccumulating trace metal that globally circulates the atmosphere and waters in its elemental, inorganic and organic chemical forms. While Hg represents a notorious neurotoxicant, the underlying cellular pathways are insufficiently understood. We identify amyloid protein aggregation in the cell nucleus as a novel pathway of Hg-bio-interactions. By mass spectrometry of purified protein aggregates, a subset of spliceosomal components and nucleoskeletal protein lamin B1 were detected as constituent parts of an Hg-induced nuclear aggregome network. The aggregome network was located by confocal imaging of amyloid-specific antibodies and dyes to amyloid cores within splicing-speckles that additionally recruit components of the ubiquitin-proteasome system. Hg significantly enhances global proteasomal activity in the nucleus, suggesting that formation of amyloid speckles plays a role in maintenance of protein homeostasis. RNAi knock down showed that lamin B1 for its part regulates amyloid speckle formation and thus likewise participates in nuclear protein homeostasis. As the Hg-induced cascade of interactions between the nucleoskeleton and protein homeostasis reduces neuronal signalling, amyloid fibrillation in the cell nucleus is introduced as a feature of Hg-neurotoxicity that opens new avenues of future research. Similar to protein aggregation events in the cytoplasm that are controlled by the cytoskeleton, amyloid fibrillation of nuclear proteins may be driven by the nucleoskeleton. PMID:25699204

  18. Expression of crystallins in cell cultures of lens epithelium following inhibition of mitosis by X-rays. Expression von Kristallinen in kultivierten durch Roentgenstrahlen mitotisch arretierten Linsenepithelzellen

    Energy Technology Data Exchange (ETDEWEB)

    Henrich, C.

    1988-01-18

    Cells from bovine lens epithelium (B26 line) were cultivated in vitro and examined to characterize their growth behaviour patterns. The question as to whether those cells would still show the ability of synthetizing crystallins was investigated on the basis of indirect immunofluorescene using both polyspecific and monospecific antisera. The findings revealed confirmed the results of former studies according to which dedifferentiation processes and marked reductions of crystallin synthesis occurred in rapidly proliferating epithelial cells cultivated in vitro. Inhibitions of proliferation were followed by a time-dependent reappearance of crystallins. The study described here was primarily focused on the arrest of cell proliferation after X-irradiation. It was found that an inhibition of cells during the G{sub o} phase may be achieved through hydroxyurea, indomethacin and X-rays. Beta crystallins can better be expressed than alpha crystallins and the expression of gamma crystallins was seen to be rather poor. (orig./MG).

  19. Comparative analysis in continuous expansion of bovine and human primary nucleus pulposus cells for tissue repair applications

    Directory of Open Access Journals (Sweden)

    DH Rosenzweig

    2017-03-01

    Full Text Available Autologous NP cell implantation is a potential therapeutic avenue for intervertebral disc (IVD degeneration. However, monolayer expansion of cells isolated from surgical samples may negatively impact matrix production by way of dedifferentiation. Previously, we have used a continuous expansion culture system to successfully preserve a chondrocyte phenotype. In this work, we hypothesised that continuous expansion culture could also preserve nucleus pulposus (NP phenotype. We confirmed that serial passaging drove NP dedifferentiation by significantly decreasing collagen type II, aggrecan and chondroadherin (CHAD gene expression, compared to freshly isolated cells. Proliferation, gene expression profile and matrix production in both culture conditions were compared using primary bovine NP cells. Both standard culture and continuous culture produced clinically relevant cell populations. However, continuous culture cells maintained significantly higher collagen type II, aggrecan and CHAD transcript expression levels. Also, continuous expansion cells generated greater amounts of proteoglycan, collagen type II and aggrecan protein deposition in pellet cultures. To our surprise, continuous expansion of human intervertebral disc cells – isolated from acute herniation tissue – produced less collagen type II, aggrecan and CHAD genes and proteins, compared to standard culture. Also, continuous culture of cells isolated from young non-degenerate tissue did not preserve gene and protein expression, compared to standard culture. These data indicated that primary bovine and human NP cells responded differently to continuous culture, where the positive effects observed for bovine cells did not translate to human cells. Therefore, caution must be exercised when choosing animal models and cell sources for pre-clinical studies.

  20. Importin 8 regulates the transport of mature microRNAs into the cell nucleus.

    Science.gov (United States)

    Wei, Yao; Li, Limin; Wang, Dong; Zhang, Chen-Yu; Zen, Ke

    2014-04-11

    Mature microRNAs (miRNAs), ∼ 22-nucleotide noncoding RNAs regulating target gene expression at the post-transcriptional level, have been recently shown to be transported into the nucleus where they modulate the biogenesis of other miRNAs or their own expression. However, the mechanism that governs the transport of mature miRNAs from cytoplasm to nucleus remains unknown. Here, we report that importin 8 (IPO8), a member of the karyopherin β (also named the protein import receptor importin β) family, plays a critical role in mediating the cytoplasm-to-nucleus transport of mature miRNAs. Specifically knocking down IPO8 but not other karyopherin β family proteins via siRNA significantly decreases the nuclear transport of various known nucleus-enriched miRNAs without affecting their total cellular levels. IPO8-mediated nuclear transport of mature miRNAs is also dependent on the association of IPO8 with the Argonaute 2 (Ago2) complex. Cross-immunoprecipitation and Western blot analysis show that IPO8 is physically associated with Ago2. Knocking down IPO8 via siRNA markedly decreases the nuclear transport of Ago2 but does not affect the total cellular Ago2 level. Furthermore, dissociating the binding of miRNAs with Ago2 by trypaflavine strongly reduces the IPO8-mediated nuclear transport of miRNAs.

  1. Ouabain affects cell migration via Na,K-ATPase-p130cas and via nucleus-centrosome association.

    Directory of Open Access Journals (Sweden)

    Young Ou

    Full Text Available Na,K-ATPase is a membrane protein that catalyzes ATP to maintain transmembrane sodium and potassium gradients. In addition, Na,K-ATPase also acts as a signal-transducing receptor for cardiotonic steroids such as ouabain and activates a number of signalling pathways. Several studies report that ouabain affects cell migration. Here we used ouabain at concentrations far below those required to block Na,K-ATPase pump activity and show that it significantly reduced RPE cell migration through two mechanisms. It causes dephosphorylation of a 130 kD protein, which we identify as p130cas. Src is involved, because Src inhibitors, but not inhibitors of other kinases tested, caused a similar reduction in p130cas phosphorylation and ouabain increased the association of Na,K-ATPase and Src. Knockdown of p130cas by siRNA reduced cell migration. Unexpectedly, ouabain induced separation of nucleus and centrosome, also leading to a block in cell migration. Inhibitor and siRNA experiments show that this effect is mediated by ERK1,2. This is the first report showing that ouabain can regulate cell migration by affecting nucleus-centrosome association.

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

    DEFF Research Database (Denmark)

    Maddika, S; Bay, GH; Kroczak, TJ

    2007-01-01

    OBJECTIVES: The phosphatidylinositol 3-kinase (PI3-K)/Akt pathway is well known for the regulation of cell survival, proliferation, and some metabolic routes. MATERIALS AND METHODS: In this study, we document a novel role for the PI3-K/Akt pathway during cell death induced by apoptin, a tumour-selective....... 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......, as it likely gains access to a new set of substrates in the nucleus. The implicated link between survival and cell death pathways during apoptosis opens new pharmacological opportunities to modulate apoptosis in cancer, for example through the manipulation of Akt's cellular localization....

  3. 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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  4. Phosphorylation of DEPDC1 at Ser110 is required to maintain centrosome organization during mitosis.

    Science.gov (United States)

    Chen, Dan; Ito, Satoko; Hyodo, Toshinori; Asano-Inami, Eri; Yuan, Hong; Senga, Takeshi

    2017-09-15

    DEPDC1 (DEP domain containing 1) is overexpressed in multiple cancers and is associated with cell cycle progression. In this report, we have investigated the expression, localization, phosphorylation and function of DEPDC1 during mitosis. DEPDC1 has two isoforms (isoform a and isoform b), and both of them are increased in mitosis and degraded once cells exit mitosis. DEPDC1a is localized to the centrosome in metaphase, whereas DEPDC1b is localized to the entire cell cortex during mitosis. DEPDC1a, but not DEPDC1b, was required for the integrity of centrosome and organization of the bipolar spindle. Mass spectrometry and biochemical analyses revealed phosphorylation of DEPDC1 at Ser110. The phosphorylation of Ser110 is essential for localization of DEPDC1a to the centrosome. Consistently, non-phosphorylation mutants of DEPDC1a did not rescue disruption of centrosome organization by depletion of endogenous DEPDC1. Our results show a novel role for DEPDC1 in maintaining centrosome integrity during mitosis for the accurate distribution of chromosomes. Copyright © 2017. Published by Elsevier Inc.

  5. INPP5E Preserves Genomic Stability through Regulation of Mitosis.

    Science.gov (United States)

    Sierra Potchanant, Elizabeth A; Cerabona, Donna; Sater, Zahi Abdul; He, Ying; Sun, Zejin; Gehlhausen, Jeff; Nalepa, Grzegorz

    2017-03-15

    The partially understood phosphoinositide signaling cascade regulates multiple aspects of cellular metabolism. Previous studies revealed that INPP5E, the inositol polyphosphate-5-phosphatase that is mutated in the developmental disorders Joubert and MORM syndromes, is essential for the function of the primary cilium and maintenance of phosphoinositide balance in nondividing cells. Here, we report that INPP5E further contributes to cellular homeostasis by regulating cell division. We found that silencing or genetic knockout of INPP5E in human and murine cells impairs the spindle assembly checkpoint, centrosome and spindle function, and maintenance of chromosomal integrity. Consistent with a cell cycle regulatory role, we found that INPP5E expression is cell cycle dependent, peaking at mitotic entry. INPP5E localizes to centrosomes, chromosomes, and kinetochores in early mitosis and shuttles to the midzone spindle at mitotic exit. Our findings identify the previously unknown, essential role of INPP5E in mitosis and prevention of aneuploidy, providing a new perspective on the function of this phosphoinositide phosphatase in health and development. Copyright © 2017 Sierra Potchanant et al.

  6. Kindlin1 regulates microtubule function to ensure normal mitosis.

    Science.gov (United States)

    Patel, Hitesh; Stavrou, Ifigeneia; Shrestha, Roshan L; Draviam, Viji; Frame, Margaret C; Brunton, Valerie G

    2016-08-01

    Loss of Kindlin 1 (Kin1) results in the skin blistering disorder Kindler Syndrome (KS), whose symptoms also include skin atrophy and reduced keratinocyte proliferation. Kin1 binds to integrins to modulate their activation and more recently it has been shown to regulate mitotic spindles and cell survival in a Plk1-dependent manner. Here we report that short-term Kin1 deletion in mouse skin results in impaired mitosis, which is associated with reduced acetylated tubulin (ac-tub) levels and cell proliferation. In cells, impaired mitosis and reduced ac-tub levels are also accompanied by reduced microtubule stability, all of which are rescued by HDAC6 inhibition. The ability of Kin1 to regulate HDAC6-dependent cellular ac-tub levels is dependent on its phosphorylation by Plk1. Taken together, these data define a novel role for Kin1 in microtubule acetylation and stability and offer a mechanistic insight into how certain KS phenotypes, such as skin atrophy and reduced cell proliferation, arise. © The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.

  7. Assessment of the Nucleus-to-Cytoplasmic Ratio in MCF-7 Cells Using Ultra-high Frequency Ultrasound and Photoacoustics

    Science.gov (United States)

    Moore, M. J.; Strohm, E. M.; Kolios, M. C.

    2016-12-01

    The nucleus-to-cytoplasmic (N:C) ratio of a cell is often used when assessing histology for the presence of malignant disease. In this proof of concept study, we present a new, non-optical method for determination of the N:C ratio using ultra-high Frequency ultrasound (US) and photoacoustics (PA). When using transducers in the 100 MHz-500 MHz range, backscattered US pulses and emitted PA waves are encoded with information pertaining to the dimension and morphology of micron-sized objects. If biological cells are interrogated, the diameter of the scattering or absorbing structure can be assessed by fitting the power spectra of the measured US or PA signals to theoretical models for US backscatter and PA emission from a fluid sphere. In this study, the cell and nucleus diameters of 9 MCF-7 breast cancer cells were determined using a new simplified model that calculates the theoretical values of the location of the power spectra minima for both US and PA signals. These diameters were then used to calculate the N:C ratio of the measured cells. The average cell diameter determined by US pulses from a transducer with a central frequency of 375 MHz was found to be 15.5 μ m± 1.8 μ m. The PA waves emitted by the cell nuclei were used to determine an average nuclear diameter of 12.0 μ m± 1.3 μ m. The N:C ratio for these cells was calculated to be 1.9± 1.0, which agrees well with previously reported N:C values for this cell type.

  8. 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...

  9. Continuation of mitosis after selective laser microbeam destruction of the centriolar region

    Energy Technology Data Exchange (ETDEWEB)

    Berns, N.W.; Richardson, S.M.

    1977-12-01

    The centriole regions of prophase PTK2 cells were irradiated with a laser microbeam. Cells continued through mitosis normally. Ultrastructural analysis revealed either an absence of centrioles or severely damaged centrioles at the irradiated poles. Microtubules appeared to focus into pericentriolar cloud material.

  10. Dysregulation of the mitosis-meiosis switch in testicular carcinoma in situ

    DEFF Research Database (Denmark)

    Jørgensen, Anne; Nielsen, John E; Almstrup, Kristian

    2013-01-01

    , except in spermatocytic seminoma (not derived from CIS). In conclusion, this study indicates that meiosis signalling is dysregulated in CIS cells and that a key regulator of the mitosis-meiosis switch, DMRT1, is expressed in 'early-stage' CIS cells but is down-regulated with further invasive...

  11. Continuation of mitosis after selective laser microbeam destruction of the centriolar region

    International Nuclear Information System (INIS)

    Berns, N.W.; Richardson, S.M.

    1977-01-01

    The centriole regions of prophase PTK2 cells were irradiated with a laser microbeam. Cells continued through mitosis normally. Ultrastructural analysis revealed either an absence of centrioles or severely damaged centrioles at the irradiated poles. Microtubules appeared to focus into pericentriolar cloud material

  12. DNA-damage response during mitosis induces whole-chromosome missegregation.

    Science.gov (United States)

    Bakhoum, Samuel F; Kabeche, Lilian; Murnane, John P; Zaki, Bassem I; Compton, Duane A

    2014-11-01

    Many cancers display both structural (s-CIN) and numerical (w-CIN) chromosomal instabilities. Defective chromosome segregation during mitosis has been shown to cause DNA damage that induces structural rearrangements of chromosomes (s-CIN). In contrast, whether DNA damage can disrupt mitotic processes to generate whole chromosomal instability (w-CIN) is unknown. Here, we show that activation of the DNA-damage response (DDR) during mitosis selectively stabilizes kinetochore-microtubule (k-MT) attachments to chromosomes through Aurora-A and PLK1 kinases, thereby increasing the frequency of lagging chromosomes during anaphase. Inhibition of DDR proteins, ATM or CHK2, abolishes the effect of DNA damage on k-MTs and chromosome segregation, whereas activation of the DDR in the absence of DNA damage is sufficient to induce chromosome segregation errors. Finally, inhibiting the DDR during mitosis in cancer cells with persistent DNA damage suppresses inherent chromosome segregation defects. Thus, the DDR during mitosis inappropriately stabilizes k-MTs, creating a link between s-CIN and w-CIN. The genome-protective role of the DDR depends on its ability to delay cell division until damaged DNA can be fully repaired. Here, we show that when DNA damage is induced during mitosis, the DDR unexpectedly induces errors in the segregation of entire chromosomes, thus linking structural and numerical chromosomal instabilities. ©2014 American Association for Cancer Research.

  13. Cell-type specific increases in female hamster nucleus accumbens spine density following female sexual experience.

    Science.gov (United States)

    Staffend, Nancy A; Hedges, Valerie L; Chemel, Benjamin R; Watts, Val J; Meisel, Robert L

    2014-11-01

    Female sexual behavior is an established model of a naturally motivated behavior which is regulated by activity within the mesolimbic dopamine system. Repeated activation of the mesolimbic circuit by female sexual behavior elevates dopamine release and produces persistent postsynaptic alterations to dopamine D1 receptor signaling within the nucleus accumbens. Here we demonstrate that sexual experience in female Syrian hamsters significantly increases spine density and alters morphology selectively in D1 receptor-expressing medium spiny neurons within the nucleus accumbens core, with no corresponding change in dopamine receptor binding or protein expression. Our findings demonstrate that previous life experience with a naturally motivated behavior has the capacity to induce persistent structural alterations to the mesolimbic circuit that can increase reproductive success and are analogous to the persistent structural changes following repeated exposure to many drugs of abuse.

  14. 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. © 2015. Published by The Company of Biologists Ltd.

  15. Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

    OpenAIRE

    Pilaz, Louis-Jan; Silver, Debra L.

    2014-01-01

    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 fixe...

  16. Low-intensity pulsed ultrasound stimulates cell proliferation, proteoglycan synthesis and expression of growth factor-related genes in human nucleus pulposus cell line

    Directory of Open Access Journals (Sweden)

    Y Kobayashi

    2009-06-01

    Full Text Available Low-intensity pulsed ultrasound (LIPUS stimulation has been shown to effect differentiation and activation of human chondrocytes. A study involving stimulation of rabbit disc cells with LIPUS revealed upregulation of cell proliferation and proteoglycan (PG synthesis. However, the effect of LIPUS on human nucleus pulposus cells has not been investigated. In the present study, therefore, we investigated whether LIPUS stimulation of a human nucleus pulposus cell line (HNPSV-1 exerted a positive effect on cellular activity. HNPSV-1 cells were encapsulated in 1.2% sodium alginate solution at 1x105 cells/ml and cultured at 10 beads/well in 6-well plates. The cells were stimulated for 20 min each day using a LIPUS generator, and the effects of LIPUS were evaluated by measuring DNA and PG synthesis. Furthermore, mRNA expression was analyzed by cDNA microarray using total RNA extracted from the cultured cells. Our study revealed no significant difference in cell proliferation between the control and the ultrasound treated groups. However, PG production was significantly upregulated in HNPSV cells stimulated at intensities of 15, 30, 60, and 120 mW/cm2 compared with the control. The results of cDNA array showed that LIPUS significantly stimulated the gene expression of growth factors and their receptors (BMP2, FGF7, TGFbetaR1 EGFRF1, VEGF. These findings suggest that LIPUS stimulation upregulates PG production in human nucleus pulposus cells by the enhancement of several matrix-related genes including growth factor-related genes. Safe and non-invasive stimulation using LIPUS may be a useful treatment for delaying the progression of disc degeneration.

  17. 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

  18. Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus

    Czech Academy of Sciences Publication Activity Database

    Venit, Tomáš; Dzijak, Rastislav; Kalendová, Alžběta; Kahle, Michal; Rohožková, Jana; Schmidt, V.; Rülicke, T.; Rathkolb, B.; Hans, W.; Bohla, A.; Eickelberg, O.; Stoeger, T.; Wolf, E.; Yildirim, A.Ö.; Gailus-Durner, V.; Fuchs, H.; de Angelis, M.H.; Hozák, Pavel

    2013-01-01

    Roč. 8, č. 4 (2013), e61406 E-ISSN 1932-6203 R&D Projects: GA ČR GAP305/11/2232; GA TA ČR TE01020022; GA MŠk LH12143; GA ČR(CZ) GD204/09/H084 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : nuclear myosin * myosin isoforms * cell nucleus Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.534, year: 2013

  19. 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.

  20. Purinergic A2b Receptor Activation by Extracellular Cues Affects Positioning of the Centrosome and Nucleus and Causes Reduced Cell Migration*

    Science.gov (United States)

    Ou, Young; Chan, Gordon; Zuo, Jeremy; Rattner, Jerome B.; van der Hoorn, Frans A.

    2016-01-01

    The tight, relative positioning of the nucleus and centrosome in mammalian cells is important for the regulation of cell migration. Under pathophysiological conditions, the purinergic A2b receptor can regulate cell motility, but the underlying mechanism remains unknown. Expression of A2b, normally low, is increased in tissues experiencing adverse physiological conditions, including hypoxia and inflammation. ATP is released from such cells. We investigated whether extracellular cues can regulate centrosome-nucleus positioning and cell migration. We discovered that hypoxia as well as extracellular ATP cause a reversible increase in the distance between the centrosome and nucleus and reduced cell motility. We uncovered the underlying pathway: both treatments act through the A2b receptor and specifically activate the Epac1/RapGef3 pathway. We show that cells lacking A2b do not respond in this manner to hypoxia or ATP but transfection of A2b restores this response, that Epac1 is critically involved, and that Rap1B is important for the relative positioning of the centrosome and nucleus. Our results represent, to our knowledge, the first report demonstrating that pathophysiological conditions can impact the distance between the centrosome and nucleus. Furthermore, we identify the A2b receptor as a central player in this process. PMID:27226580

  1. Correlative analysis on the relationship between PMI and DNA degradation of cell nucleus in human different tissues.

    Science.gov (United States)

    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. New perspective for GdNCT. Gd-DTPA reaches the nucleus of glioblastoma cells in culture and in vivo

    International Nuclear Information System (INIS)

    Stasio, G. de; Gilbert, B.; Frazer, B.H.

    2000-01-01

    We investigated the prospects of gadolinium as a neutron capture therapy agent by combining three independent techniques to study the uptake of Gd-DTPA in vitro, in cultured glioblastoma cells, and in vivo, in the glioblastoma tissue sections after injection of Gd-DTPA and tumor extraction. We show that gadolinium not only penetrates the plasma membrane of glioblastoma cells grown in culture, but we also observe a statistically significant higher concentration of Gd in the nucleus relative to the cytoplasm. For the in vivo experiments, Gd-DTPA was administered to 6 glioblastoma patients before neurosurgery. The extracted bioptic tissue was then analyzed with spectromictroscopy, showing Gd localized in the nuclei of glioblastoma cells in 5 patients out of the 6 analyzed. (author)

  3. (Allium cepa) root tip mitosis

    African Journals Online (AJOL)

    Aghomotsegin

    their chemical composition and genotoxic effects on cell reproduction. Two petrochemicals, air ... the chromosomes of the individual cells of the root tip could be a pointer to their ..... Chromosome technique: Theory and. Practice. Butterworths ...

  4. 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

  5. Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus.

    Science.gov (United States)

    Venit, Tomáš; Dzijak, Rastislav; Kalendová, Alžběta; Kahle, Michal; Rohožková, Jana; Schmidt, Volker; Rülicke, Thomas; Rathkolb, Birgit; Hans, Wolfgang; Bohla, Alexander; Eickelberg, Oliver; Stoeger, Tobias; Wolf, Eckhard; Yildirim, Ali Önder; Gailus-Durner, Valérie; Fuchs, Helmut; de Angelis, Martin Hrabě; Hozák, Pavel

    2013-01-01

    Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes.

  6. WE-H-BRA-08: A Monte Carlo Cell Nucleus Model for Assessing Cell Survival Probability Based On Particle Track Structure Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Lee, B [Northwestern Memorial Hospital, Chicago, IL (United States); Georgia Institute of Technology, Atlanta, GA (Georgia); Wang, C [Georgia Institute of Technology, Atlanta, GA (Georgia)

    2016-06-15

    Purpose: To correlate the damage produced by particles of different types and qualities to cell survival on the basis of nanodosimetric analysis and advanced DNA structures in the cell nucleus. Methods: A Monte Carlo code was developed to simulate subnuclear DNA chromatin fibers (CFs) of 30nm utilizing a mean-free-path approach common to radiation transport. The cell nucleus was modeled as a spherical region containing 6000 chromatin-dense domains (CDs) of 400nm diameter, with additional CFs modeled in a sparser interchromatin region. The Geant4-DNA code was utilized to produce a particle track database representing various particles at different energies and dose quantities. These tracks were used to stochastically position the DNA structures based on their mean free path to interaction with CFs. Excitation and ionization events intersecting CFs were analyzed using the DBSCAN clustering algorithm for assessment of the likelihood of producing DSBs. Simulated DSBs were then assessed based on their proximity to one another for a probability of inducing cell death. Results: Variations in energy deposition to chromatin fibers match expectations based on differences in particle track structure. The quality of damage to CFs based on different particle types indicate more severe damage by high-LET radiation than low-LET radiation of identical particles. In addition, the model indicates more severe damage by protons than of alpha particles of same LET, which is consistent with differences in their track structure. Cell survival curves have been produced showing the L-Q behavior of sparsely ionizing radiation. Conclusion: Initial results indicate the feasibility of producing cell survival curves based on the Monte Carlo cell nucleus method. Accurate correlation between simulated DNA damage to cell survival on the basis of nanodosimetric analysis can provide insight into the biological responses to various radiation types. Current efforts are directed at producing cell

  7. Strong, reliable and precise synaptic connections between thalamic relay cells and neurones of the nucleus reticularis in juvenile rats

    Science.gov (United States)

    Gentet, Luc J; Ulrich, Daniel

    2003-01-01

    The thalamic reticular nucleus (nRT) is composed entirely of GABAergic inhibitory neurones that receive input from pyramidal cortical neurones and excitatory relay cells of the ventrobasal complex of the thalamus (VB). It plays a major role in the synchrony of thalamic networks, yet the synaptic connections it receives from VB cells have never been fully physiologically characterised. Here, whole-cell current-clamp recordings were obtained from 22 synaptically connected VB-nRT cell pairs in slices of juvenile (P14–20) rats. At 34–36 °C, single presynaptic APs evoked unitary EPSPs in nRT cells with a peak amplitude of 7.4 ± 1.5 mV (mean ± s.e.m.) and a decay time constant of 15.1 ± 0.9 ms. Only four out of 22 pairs showed transmission failures at a mean rate of 6.8 ± 1.1 %. An NMDA receptor (NMDAR)-mediated component was significant at rest and subsequent EPSPs in a train were depressed. Only one out of 14 pairs tested was reciprocally connected; the observed IPSPs in the VB cell had a peak amplitude of 0.8 mV and were completely abolished in the presence of 10 μm bicuculline. Thus, synaptic connections from VB cells to nRT neurones are mainly ‘drivers’, while a small subset of cells form closed disynaptic loops. PMID:12563005

  8. 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

  9. 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. Copyright © 2014 the American Physiological Society.

  10. The chimeric eukaryote: origin of the nucleus from the karyomastigont in amitochondriate protists

    Science.gov (United States)

    Margulis, L.; Dolan, M. F.; Guerrero, R.

    2000-01-01

    We present a testable model for the origin of the nucleus, the membrane-bounded organelle that defines eukaryotes. A chimeric cell evolved via symbiogenesis by syntrophic merger between an archaebacterium and a eubacterium. The archaebacterium, a thermoacidophil resembling extant Thermoplasma, generated hydrogen sulfide to protect the eubacterium, a heterotrophic swimmer comparable to Spirochaeta or Hollandina that oxidized sulfide to sulfur. Selection pressure for speed swimming and oxygen avoidance led to an ancient analogue of the extant cosmopolitan bacterial consortium "Thiodendron latens." By eubacterial-archaebacterial genetic integration, the chimera, an amitochondriate heterotroph, evolved. This "earliest branching protist" that formed by permanent DNA recombination generated the nucleus as a component of the karyomastigont, an intracellular complex that assured genetic continuity of the former symbionts. The karyomastigont organellar system, common in extant amitochondriate protists as well as in presumed mitochondriate ancestors, minimally consists of a single nucleus, a single kinetosome and their protein connector. As predecessor of standard mitosis, the karyomastigont preceded free (unattached) nuclei. The nucleus evolved in karyomastigont ancestors by detachment at least five times (archamoebae, calonymphids, chlorophyte green algae, ciliates, foraminifera). This specific model of syntrophic chimeric fusion can be proved by sequence comparison of functional domains of motility proteins isolated from candidate taxa.

  11. 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. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. 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...

  13. Regularity of mitosis in different varieties of winter bread wheat under the action of herbicides

    Directory of Open Access Journals (Sweden)

    Tatyana Eugenivna KOPYTCHUK

    2012-05-01

    Full Text Available The influence of the most widespread herbicides on winter wheat in Ukraine was studied by anaphase test. Treatment with herbicides reduced the germination of the seeds and disturbed the regularity of mitosis in all varieties of wheat. The range of violations of mitosis was demonstrated by the formation of chromosomal aberrations and dysfunctions of cell cytoskeleton which occurred while processing herbicides. Varietal differences between investigated wheat by sensitivity to herbicides were discovered. The most resistant to herbicides was variety Fantasya Odesskaya, and the most sensitive – Nikoniya, while the most harmful herbicide for wheat was Napalm.

  14. Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

    Science.gov (United States)

    Macurek, Libor; Benada, Jan; Müllers, Erik; Halim, Vincentius A.; Krejčíková, Kateřina; Burdová, Kamila; Pecháčková, Sona; Hodný, Zdeněk; Lindqvist, Arne; Medema, René H.; Bartek, Jiri

    2013-01-01

    Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G1 phase to G2 and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G1 cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression. PMID:23255129

  15. Shape Transformation of the Nuclear Envelope during Closed Mitosis.

    Science.gov (United States)

    Zhu, Qian; Zheng, Fan; Liu, Allen P; Qian, Jin; Fu, Chuanhai; Lin, Yuan

    2016-11-15

    The nuclear envelope (NE) in lower eukaryotes such as Schizosaccharomyces pombe undergoes large morphology changes during closed mitosis. However, which physical parameters are important in governing the shape evolution of the NE, and how defects in the dividing chromosomes/microtubules are reflected in those parameters, are fundamental questions that remain unresolved. In this study, we show that improper separation of chromosomes in genetically deficient cells leads to membrane tethering or asymmetric division in contrast to the formation of two equal-sized daughter nuclei in wild-type cells. We hypothesize that the poleward force is transmitted to the nuclear membrane through its physical contact with the separated sister chromatids at the two spindle poles. A theoretical model is developed to predict the morphology evolution of the NE where key factors such as the work done by the poleward force and bending and surface energies stored in the membrane have been taken into account. Interestingly, the predicted phase diagram, summarizing the dependence of nuclear shape on the size of the load transmission regions, and the pole-to-pole distance versus surface area relationship all quantitatively agree well with our experimental observations, suggesting that this model captures the essential physics involved in closed mitosis. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  16. 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.

    2012-01-01

    The organization and physical properties of the lipid bilayer portion of intact cortical and nuclear fiber cell plasma membranes isolated from the eyes 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. PMID:22326289

  17. 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. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  18. Insulin-induced translocation of IR to the nucleus in insulin responsive cells requires a nuclear translocation sequence.

    Science.gov (United States)

    Kesten, Dov; Horovitz-Fried, Miriam; Brutman-Barazani, Tamar; Sampson, Sanford R

    2018-04-01

    Insulin binding to its cell surface receptor (IR) activates a cascade of events leading to its biological effects. The Insulin-IR complex is rapidly internalized and then is either recycled back to the plasma membrane or sent to lysosomes for degradation. Although most of the receptor is recycled or degraded, a small amount may escape this pathway and migrate to the nucleus of the cell where it might be important in promulgation of receptor signals. In this study we explored the mechanism by which insulin induces IR translocation to the cell nucleus. Experiments were performed cultured L6 myoblasts, AML liver cells and 3T3-L1 adipocytes. Insulin treatment induced a rapid increase in nuclear IR protein levels within 2 to 5 min. Treatment with WGA, an inhibitor of nuclear import, reduced insulin-induced increases nuclear IR protein; IR was, however, translocated to a perinuclear location. Bioinformatics tools predicted a potential nuclear localization sequence (NLS) on IR. Immunofluorescence staining showed that a point mutation on the predicted NLS blocked insulin-induced IR nuclear translocation. In addition, blockade of nuclear IR activation in isolated nuclei by an IR blocking antibody abrogated insulin-induced increases in IR tyrosine phosphorylation and nuclear PKCδ levels. Furthermore, over expression of mutated IR reduced insulin-induced glucose uptake and PKB phosphorylation. When added to isolated nuclei, insulin induced IR phosphorylation but had no effect on nuclear IR protein levels. These results raise questions regarding the possible role of nuclear IR in IR signaling and insulin resistance. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Molecular Therapy for Degenerative Disc Disease: Clues from Secretome Analysis of the Notochordal Cell-Rich Nucleus Pulposus

    Science.gov (United States)

    Matta, Ajay; Karim, M. Zia; Isenman, David E.; Erwin, W. Mark

    2017-01-01

    Degenerative disc disease (DDD) is associated with spinal pain often leading to long-term disability. However, the non-chondrodystrophic canine intervertebral disc is protected from the development of DDD, ostensibly due to its retention of notochordal cells (NC) in the nucleus pulposus (NP). In this study, we hypothesized that secretome analysis of the NC-rich NP will lead to the identification of key proteins that delay the onset of DDD. Using mass-spectrometry, we identified 303 proteins including components of TGFβ- and Wnt-signaling, anti-angiogeneic factors and proteins that inhibit axonal ingrowth in the bioactive fractions of serum free, notochordal cell derived conditioned medium (NCCM). Ingenuity Pathway Analysis revealed TGFβ1 and CTGF as major hubs in protein interaction networks. In vitro treatment with TGFβ1 and CTGF promoted the synthesis of healthy extra-cellular matrix proteins, increased cell proliferation and reduced cell death in human degenerative disc NP cells. A single intra-discal injection of recombinant TGFβ1 and CTGF proteins in a pre-clinical rat-tail disc injury model restored the NC and stem cell rich NP. In conclusion, we demonstrate the potential of TGFβ1 and CTGF to mitigate the progression of disc degeneration and the potential use of these molecules in a molecular therapy to treat the degenerative disc. PMID:28358123

  20. Hypoxic regulation of β-1,3-glucuronyltransferase 1 expression in nucleus pulposus cells of the rat intervertebral disc: role of hypoxia-inducible factor proteins.

    Science.gov (United States)

    Gogate, Shilpa S; Nasser, Rena; Shapiro, Irving M; Risbud, Makarand V

    2011-07-01

    To determine whether hypoxia and hypoxia-inducible factor (HIF) proteins regulate expression of β-1,3-glucuronyltransferase 1 (GlcAT-1), a key enzyme in glycosaminoglycan synthesis in nucleus pulposus cells. Real-time reverse transcriptase-polymerase chain reaction and Western blotting were used to measure GlcAT-1 expression. Transfections were performed to determine the effect of HIF-1α and HIF-2α on GlcAT-1 promoter activity. Under hypoxic conditions there was an increase in GlcAT-1 expression; a significant increase in promoter activity was seen both in nucleus pulposus cells and in N1511 chondrocytes. We investigated whether HIF controlled GlcAT-1 expression. Suppression of HIF-1α and HIF-2α induced GlcAT-1 promoter activity and expression only in nucleus pulposus cells. Transfection with CA-HIF-1α as well as with CA-HIF-2α suppressed GlcAT-1 promoter activity only in nucleus pulposus cells, suggesting a cell type-specific regulation. Site-directed mutagenesis and deletion constructs were used to further confirm the suppressive effect of HIFs on GlcAT-1 promoter function in nucleus pulposus cells. Although it was evident that interaction of HIF with hypoxia-responsive elements resulted in suppression of basal promoter activity, it was not necessary for transcriptional suppression. This result suggested both a direct and an indirect mode of regulation, possibly through recruitment of a HIF-dependent repressor. Finally, we showed that hypoxic expression of GlcAT-1 was also partially dependent on MAPK signaling. These studies demonstrate that hypoxia regulates GlcAT-1 expression through a signaling network comprising both activator and suppressor molecules, and that this regulation is unique to nucleus pulposus cells. Copyright © 2011 by the American College of Rheumatology.

  1. 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

    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

  2. A highly efficient method for generation of therapeutic quality human pluripotent stem cells by using naive induced pluripotent stem cells nucleus for nuclear transfer.

    Science.gov (United States)

    Sanal, Madhusudana Girija

    2014-01-01

    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 the question of

  3. 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.

  4. 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. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  5. Nuclear reprogramming of somatic nucleus hybridized with embryonic stem cells by electrofusion.

    Science.gov (United States)

    Tada, Masako; Tada, Takashi

    2006-01-01

    Cell fusion is a powerful tool for understanding the molecular mechanisms of epigenetic reprogramming. In hybrid cells of somatic cells and pluripotential stem cells, including embryonic stem (ES) and embryonic germ cells, somatic nuclei acquire pluripotential competence. ES and embryonic germ cells retain intrinsic trans activity to induce epigenetic reprogramming. For generating hybrid cells, we have used the technique of electrofusion. Electrofusion is a highly effective, reproducible, and biomedically safe in vitro system. For successful cell fusion, two sequential steps of electric pulse stimulation are required for the alignment (pearl chain formation) of two different types of cells between electrodes in response to alternating current stimulation and for the fusion of cytoplasmic membranes by direct current stimulation. Optimal conditions for electrofusion with a pulse generator are introduced for ES and somatic cell fusion. Topics in the field of stem cell research include the successful production of cloned animals via the epigenetic reprogramming of somatic cells and contribution of spontaneous cell fusion to generating intrinsic plasticity of tissue stem cells. Cell fusion technology may make important contributions to the fields of epigenetic reprogramming and regenerative medicine.

  6. Mitosis-associated repression in development.

    Science.gov (United States)

    Esposito, Emilia; Lim, Bomyi; Guessous, Ghita; Falahati, Hanieh; Levine, Michael

    2016-07-01

    Transcriptional repression is a pervasive feature of animal development. Here, we employ live-imaging methods to visualize the Snail repressor, which establishes the boundary between the presumptive mesoderm and neurogenic ectoderm of early Drosophila embryos. Snail target enhancers were attached to an MS2 reporter gene, permitting detection of nascent transcripts in living embryos. The transgenes exhibit initially broad patterns of transcription but are refined by repression in the mesoderm following mitosis. These observations reveal a correlation between mitotic silencing and Snail repression. We propose that mitosis and other inherent discontinuities in transcription boost the activities of sequence-specific repressors, such as Snail. © 2016 Esposito et al.; Published by Cold Spring Harbor Laboratory Press.

  7. Mechanical stress-induced apoptosis of nucleus pulposus cells: an in vitro and in vivo rat model.

    Science.gov (United States)

    Kuo, Yi-Jie; Wu, Lien-Chen; Sun, Jui-Sheng; Chen, Ming-Hong; Sun, Man-Ger; Tsuang, Yang-Hwei

    2014-03-01

    Un-physiological loads play an important role in the degenerative process of inter-vertebral discs (IVD). In this study, we used an in vitro and in vivo rat model to investigate the mechanism of nucleus pulposus (NP) cells apoptosis induced by mechanical stress. Static compressive load to IVDs of rat tails was used as the in vivo model. For the in vitro model, NP cells were tested under the physiological and un-physiological loading. For histological examination, apoptotic index study, and apoptotic gene expression, we also selected cytokines [bone morphogenetic protein (BMP)-2/7, insulin-like growth factor (IGF)-1, platelet-derived growth factor (PDGF)] to be analyzed. Under mechanical loading, cellular density was significantly decreased, but there was an increase of TUNEL positive cells and apoptosis index. In a dose-dependent manner; the necrosis became apparent in the un-physiologic strain. The selected cytokines (BMP-2/7, IGF-1, PDGF) can significantly reduce the percentage of apoptotic and necrotic cells. We conclude that the intrinsic (mitochondrial) apoptotic pathway plays an important role in the compressive load-induced apoptosis of NP cells. Combination therapy reducing the mechanical load and selected cytokines (BMP-2/7, IGF-1 and PDGF) may have considerable promise in the treatment of spine disc degeneration.

  8. Loss of HIF-1α in the notochord results in cell death and complete disappearance of the nucleus pulposus.

    Science.gov (United States)

    Merceron, Christophe; Mangiavini, Laura; Robling, Alexander; Wilson, Tremika LeShan; Giaccia, Amato J; Shapiro, Irving M; Schipani, Ernestina; Risbud, Makarand V

    2014-01-01

    The intervertebral disc (IVD) is one of the largest avascular organs in vertebrates. The nucleus pulposus (NP), a highly hydrated and proteoglycan-enriched tissue, forms the inner portion of the IVD. The NP is surrounded by a multi-lamellar fibrocartilaginous structure, the annulus fibrosus (AF). This structure is covered superior and inferior side by cartilaginous endplates (CEP). The NP is a unique tissue within the IVD as it results from the differentiation of notochordal cells, whereas, AF and CEP derive from the sclerotome. The hypoxia inducible factor-1α (HIF-1α) is expressed in NP cells but its function in NP development and homeostasis is largely unknown. We thus conditionally deleted HIF-1α in notochordal cells and investigated how loss of this transcription factor impacts NP formation and homeostasis at E15.5, birth, 1 and 4 months of age, respectively. Histological analysis, cell lineage studies, and TUNEL assay were performed. Morphologic changes of the mutant NP cells were identified as early as E15.5, followed, postnatally, by the progressive disappearance and replacement of the NP with a novel tissue that resembles fibrocartilage. Notably, lineage studies and TUNEL assay unequivocally proved that NP cells did not transdifferentiate into chondrocyte-like cells but they rather underwent massive cell death, and were completely replaced by a cell population belonging to a lineage distinct from the notochordal one. Finally, to evaluate the functional consequences of HIF-1α deletion in the NP, biomechanical testing of mutant IVD was performed. Loss of the NP in mutant mice significantly reduced the IVD biomechanical properties by decreasing its ability to absorb mechanical stress. These findings are similar to the changes usually observed during human IVD degeneration. Our study thus demonstrates that HIF-1α is essential for NP development and homeostasis, and it raises the intriguing possibility that this transcription factor could be involved in IVD

  9. MiR-210 disturbs mitotic progression through regulating a group of mitosis-related genes.

    Science.gov (United States)

    He, Jie; Wu, Jiangbin; Xu, Naihan; Xie, Weidong; Li, Mengnan; Li, Jianna; Jiang, Yuyang; Yang, Burton B; Zhang, Yaou

    2013-01-07

    MiR-210 is up-regulated in multiple cancer types but its function is disputable and further investigation is necessary. Using a bioinformatics approach, we identified the putative target genes of miR-210 in hypoxia-induced CNE cells from genome-wide scale. Two functional gene groups related to cell cycle and RNA processing were recognized as the major targets of miR-210. Here, we investigated the molecular mechanism and biological consequence of miR-210 in cell cycle regulation, particularly mitosis. Hypoxia-induced up-regulation of miR-210 was highly correlated with the down-regulation of a group of mitosis-related genes, including Plk1, Cdc25B, Cyclin F, Bub1B and Fam83D. MiR-210 suppressed the expression of these genes by directly targeting their 3'-UTRs. Over-expression of exogenous miR-210 disturbed mitotic progression and caused aberrant mitosis. Furthermore, miR-210 mimic with pharmacological doses reduced tumor formation in a mouse metastatic tumor model. Taken together, these results implicate that miR-210 disturbs mitosis through targeting multi-genes involved in mitotic progression, which may contribute to its inhibitory role on tumor formation.

  10. Disassembly of the lens fiber cell nucleus to create a clear lens: the p27 descent

    Science.gov (United States)

    The eye lens is unique among tissues: it is transparent, does not form tumors, and the majority of its cells degrade their organelles, including their cell nuclei. A mystery for over a century, there has been considerable recent progress in elucidating mechanisms of lens fiber cell denucleation (LFC...

  11. Selective deletion of cochlear hair cells causes rapid age-dependent changes in spiral ganglion and cochlear nucleus neurons.

    Science.gov (United States)

    Tong, Ling; Strong, Melissa K; Kaur, Tejbeer; Juiz, Jose M; Oesterle, Elizabeth C; Hume, Clifford; Warchol, Mark E; Palmiter, Richard D; Rubel, Edwin W

    2015-05-20

    During nervous system development, critical periods are usually defined as early periods during which manipulations dramatically change neuronal structure or function, whereas the same manipulations in mature animals have little or no effect on the same property. Neurons in the ventral cochlear nucleus (CN) are dependent on excitatory afferent input for survival during a critical period of development. Cochlear removal in young mammals and birds results in rapid death of target neurons in the CN. Cochlear removal in older animals results in little or no neuron death. However, the extent to which hair-cell-specific afferent activity prevents neuronal death in the neonatal brain is unknown. We further explore this phenomenon using a new mouse model that allows temporal control of cochlear hair cell deletion. Hair cells express the human diphtheria toxin (DT) receptor behind the Pou4f3 promoter. Injections of DT resulted in nearly complete loss of organ of Corti hair cells within 1 week of injection regardless of the age of injection. Injection of DT did not influence surrounding supporting cells directly in the sensory epithelium or spiral ganglion neurons (SGNs). Loss of hair cells in neonates resulted in rapid and profound neuronal loss in the ventral CN, but not when hair cells were eliminated at a more mature age. In addition, normal survival of SGNs was dependent on hair cell integrity early in development and less so in mature animals. This defines a previously undocumented critical period for SGN survival. Copyright © 2015 the authors 0270-6474/15/357878-14$15.00/0.

  12. 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.

  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. Replication stress activates DNA repair synthesis in mitosis

    DEFF Research Database (Denmark)

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

    2015-01-01

    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...... 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...

  15. Possible mechanisms of chromosomal aberrations: VII. Comparative dynamics of sister chromatid disjunction and realization of radiation-induced chromosomal aberrations during mitosis

    International Nuclear Information System (INIS)

    Lebedeva, L.I.; Akhmamet'eva, E.M.

    1994-01-01

    An increase in radiation-induced chromosomal aberrations during c-metaphase sister chromatid disjunction was demonstrated in murine bone marrow cells exposed to a total γ-irradiation at 0.5 Gy. Caffeine (Cf) treatment during mitosis partially suppressed the chromatid disjunction rate and increased the number of radiation-induced aberrations in this mitosis. Nalidixic acid (NA) treatment of c-metaphase cells completely suppressed chromatid disjunction and the realization of induced aberrations. Topoisomerase 2 was assumed to be involved during mitosis in both processes

  16. 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.

  17. Phosphorylation by CK2 regulates MUS81/EME1 in mitosis and after replication stress.

    Science.gov (United States)

    Palma, Anita; Pugliese, Giusj Monia; Murfuni, Ivana; Marabitti, Veronica; Malacaria, Eva; Rinalducci, Sara; Minoprio, Anna; Sanchez, Massimo; Mazzei, Filomena; Zolla, Lello; Franchitto, Annapaola; Pichierri, Pietro

    2018-06-01

    The MUS81 complex is crucial for preserving genome stability through the resolution of branched DNA intermediates in mitosis. However, untimely activation of the MUS81 complex in S-phase is dangerous. Little is known about the regulation of the human MUS81 complex and how deregulated activation affects chromosome integrity. Here, we show that the CK2 kinase phosphorylates MUS81 at Serine 87 in late-G2/mitosis, and upon mild replication stress. Phosphorylated MUS81 interacts with SLX4, and this association promotes the function of the MUS81 complex. In line with a role in mitosis, phosphorylation at Serine 87 is suppressed in S-phase and is mainly detected in the MUS81 molecules associated with EME1. Loss of CK2-dependent MUS81 phosphorylation contributes modestly to chromosome integrity, however, expression of the phosphomimic form induces DSBs accumulation in S-phase, because of unscheduled targeting of HJ-like DNA intermediates, and generates a wide chromosome instability phenotype. Collectively, our findings describe a novel regulatory mechanism controlling the MUS81 complex function in human cells. Furthermore, they indicate that, genome stability depends mainly on the ability of cells to counteract targeting of branched intermediates by the MUS81/EME1 complex in S-phase, rather than on a correct MUS81 function in mitosis.

  18. 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).

  19. Interspecies somatic cell nucleus transfer with porcine oocytes as recipients: A novel bioassay system for assessing the competence of canine somatic cells to develop into embryos.

    Science.gov (United States)

    Sugimura, S; Narita, K; Yamashiro, H; Sugawara, A; Shoji, T; Terashita, Y; Nishimori, K; Konno, T; Yoshida, M; Sato, E

    2009-09-01

    Interspecies somatic cell nucleus transfer (iSCNT) could be a useful bioassay system for assessing the ability of mammalian somatic cells to develop into embryos. To examine this possibility, we performed canine iSCNT using porcine oocytes, allowed to mature in vitro, as recipients. Canine fibroblasts from the tail tips and dewclaws of a female poodle (Fp) and a male poodle (Mp) were used as donors. We demonstrated that the use of porcine oocytes induced blastocyst formation in the iSCNT embryos cultured in porcine zygote medium-3. In Fp and Mp, the rate of blastocyst formation from cleaved embryos (Fp: 6.3% vs. 22.4%; and Mp: 26.1% vs. 52.4%) and the number of cells at the blastocyst stage (Fp: 30.7 vs. 60.0; and Mp: 27.2 vs. 40.1) were higher in the embryos derived from dewclaw cells than in those derived from tail-tip cells (Ptip cells of Fp. Only blastocysts derived from dewclaw cells of Mp developed outgrowths. However, outgrowth formation was retrieved in the embryos derived from dewclaw cells of Fp by aggregation at the 4-cell stage. We inferred that iSCNT performed using porcine oocytes as recipients could represent a novel bioassay system for evaluating the developmental competence of canine somatic cells.

  20. Mechanobiology of bone marrow stem cells: from myosin-II forces to compliance of matrix and nucleus in cell forms and fates.

    Science.gov (United States)

    Shin, Jae-Won; Swift, Joe; Ivanovska, Irena; Spinler, Kyle R; Buxboim, Amnon; Discher, Dennis E

    2013-10-01

    Adult stem cells and progenitors are of great interest for their clinical application as well as their potential to reveal deep sensitivities to microenvironmental factors. The bone marrow is a niche for at least two types of stem cells, and the prototype is the hematopoietic stem cell/progenitors (HSC/Ps), which have saved many thousands of patients for several decades now. In bone marrow, HSC/Ps interact functionally with marrow stromal cells that are often referred to as mesenchymal stem cells (MSCs) or derivatives thereof. Myosin and matrix elasticity greatly affect MSC function, and these mechanobiological factors are now being explored with HSC/Ps both in vitro and in vivo. Also emerging is a role for the nucleus as a mechanically sensitive organelle that is semi-permeable to transcription factors which are modified for nuclear entry by cytoplasmic mechanobiological pathways. Since therapies envisioned with induced pluripotent stem cells and embryonic stem cells generally involve in vitro commitment to an adult stem cell or progenitor, a very deep understanding of stem cell mechanobiology is essential to progress with these multi-potent cells. © 2013 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  1. Chromatic summation and receptive field properties of blue-on and blue-off cells in marmoset lateral geniculate nucleus.

    Science.gov (United States)

    Eiber, C D; Pietersen, A N J; Zeater, N; Solomon, S G; Martin, P R

    2017-11-22

    The "blue-on" and "blue-off" receptive fields in retina and dorsal lateral geniculate nucleus (LGN) of diurnal primates combine signals from short-wavelength sensitive (S) cone photoreceptors with signals from medium/long wavelength sensitive (ML) photoreceptors. Three questions about this combination remain unresolved. Firstly, is the combination of S and ML signals in these cells linear or non-linear? Secondly, how does the timing of S and ML inputs to these cells influence their responses? Thirdly, is there spatial antagonism within S and ML subunits of the receptive field of these cells? We measured contrast sensitivity and spatial frequency tuning for four types of drifting sine gratings: S cone isolating, ML cone isolating, achromatic (S + ML), and counterphase chromatic (S - ML), in extracellular recordings from LGN of marmoset monkeys. We found that responses to stimuli which modulate both S and ML cones are well predicted by a linear sum of S and ML signals, followed by a saturating contrast-response relation. Differences in sensitivity and timing (i.e. vector combination) between S and ML inputs are needed to explain the amplitude and phase of responses to achromatic (S + ML) and counterphase chromatic (S - ML) stimuli. Best-fit spatial receptive fields for S and/or ML subunits in most cells (>80%) required antagonistic surrounds, usually in the S subunit. The surrounds were however generally weak and had little influence on spatial tuning. The sensitivity and size of S and ML subunits were correlated on a cell-by-cell basis, adding to evidence that blue-on and blue-off receptive fields are specialised to signal chromatic but not spatial contrast. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. 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.

  3. Matrix metalloproteinase 3 promotes cellular anti-dengue virus response via interaction with transcription factor NFκB in cell nucleus.

    Science.gov (United States)

    Zuo, Xiangyang; Pan, Wen; Feng, Tingting; Shi, Xiaohong; Dai, Jianfeng

    2014-01-01

    Dengue virus (DENV), the causative agent of human Dengue hemorrhagic fever, is a mosquito-borne virus of immense global health importance. Characterization of cellular factors promoting or inhibiting DENV infection is important for understanding the mechanism of DENV infection. In this report, MMP3 (stromelysin-1), a secretory endopeptidase that degrades extracellular matrices, has been shown promoting cellular antiviral response against DENV infection. Quantitative RT-PCR and Western Blot showed that the expression of MMP3 was upregulated in DENV-infected RAW264.7 cells. The intracellular viral loads were significantly higher in MMP3 silenced cells compared with controls. The expression level of selective anti-viral cytokines were decreased in MMP3 siRNA treated cells, and the transcription factor activity of NFκB was significantly impaired upon MMP3 silencing during DENV infection. Further, we found that MMP3 moved to cell nucleus upon DENV infection and colocalized with NFκB P65 in nucleus. Co-immunoprecipitation analysis suggested that MMP3 directly interacted with NFκB in nucleus during DENV infection and the C-terminal hemopexin-like domain of MMP3 was required for the interaction. This study suggested a novel role of MMP3 in nucleus during viral infection and provided new evidence for MMPs in immunomodulation.

  4. Monte Carlo calculated microdosimetric spread for cell nucleus-sized targets exposed to brachytherapy 125I and 192Ir sources and 60Co cell irradiation.

    Science.gov (United States)

    Villegas, Fernanda; Tilly, Nina; Ahnesjö, Anders

    2013-09-07

    The stochastic nature of ionizing radiation interactions causes a microdosimetric spread in energy depositions for cell or cell nucleus-sized volumes. The magnitude of the spread may be a confounding factor in dose response analysis. The aim of this work is to give values for the microdosimetric spread for a range of doses imparted by (125)I and (192)Ir brachytherapy radionuclides, and for a (60)Co source. An upgraded version of the Monte Carlo code PENELOPE was used to obtain frequency distributions of specific energy for each of these radiation qualities and for four different cell nucleus-sized volumes. The results demonstrate that the magnitude of the microdosimetric spread increases when the target size decreases or when the energy of the radiation quality is reduced. Frequency distributions calculated according to the formalism of Kellerer and Chmelevsky using full convolution of the Monte Carlo calculated single track frequency distributions confirm that at doses exceeding 0.08 Gy for (125)I, 0.1 Gy for (192)Ir, and 0.2 Gy for (60)Co, the resulting distribution can be accurately approximated with a normal distribution. A parameterization of the width of the distribution as a function of dose and target volume of interest is presented as a convenient form for the use in response modelling or similar contexts.

  5. Type 1 IGF receptor translocates to the nucleus of human tumor cells

    OpenAIRE

    Aleksic, Tamara; Chitnis, Meenali M.; Perestenko, Olga V.; Gao, Shan; Thomas, Peter H.; Turner, Gareth D.; Protheroe, Andrew S.; Howarth, Mark; Macaulay, Valentine M.

    2010-01-01

    The type 1 insulin-like growth factor receptor (IGF-1R) is a transmembrane glycoprotein comprising two extracellular α subunits and two β subunits with tyrosine kinase activity. The IGF-1R is frequently upregulated in cancers, and signals from the cell surface to promote proliferation and cell survival. Recent attention has focused on the IGF-1R as a target for cancer treatment. Here we report that the nuclei of human tumor cells contain IGF-1R, detectable using multiple antibodies to α- and ...

  6. The nucleus

    International Nuclear Information System (INIS)

    Marano, S.

    1998-01-01

    In 1911 E.Rutherford discovered the nucleus. Since then the nucleus has been investigated with more and more powerful tools but it remains the main field of study of nuclear physics. As it is impossible to take into account the interaction of all the nucleons, a theory based on the hypothesis that each nucleon undergoes an average interaction force has been set up. 2 representations have emerged: the Skyrme force and the Gogny force. Both representations match experimental results but are unable to describe fission yields or the multi-fragmentation of very hot nuclei. The mean-field theory can predict the shape of the nuclei according to its energy level. An experimental program involving the Vivitron accelerator and the Euroball detector is due to begin to validate it. By bombarding targets with exotic nuclei nuclear physicists detect new structures and test their collision models. About ten years ago nuclear halos were observed with lithium 11 nuclei. In this nucleus 2 neutrons move in a space larger than the nucleus itself. This discovery has triggered the elaboration of new theories based on nuclear clusters. At very high temperatures the mean-field theory predicts that nuclear matter acts as a fluid. Following the nuclei temperature different ways of decay appear: first evaporation then multi-fragmentation and vaporization. This ultimate stage occurs around 100 milliard celsius degree temperature when the nuclei decays in a multitude of light particles. Isomeric states are studied and could be seen as a way of storing energy. In a very pedagogical way this article gives information to understand the challenges that face nuclear physics today and highlights the contributions of Cea in this field. (A.C.)

  7. 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. © 2014 The Author. Bioessays published by WILEY Periodicals, Inc.

  8. p21 is Responsible for Ionizing Radiation-induced Bypass of Mitosis.

    Science.gov (United States)

    Zhang, Xu Rui; Liu, Yong Ai; Sun, Fang; Li, He; Lei, Su Wen; Wang, Ju Fang

    2016-07-01

    To explore the role of p21 in ionizing radiation-induced changes in protein levels during the G2/M transition and long-term G2 arrest. 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. 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. 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. Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  9. SGT, a Hsp90β binding partner, is accumulated in the nucleus during cell apoptosis

    International Nuclear Information System (INIS)

    Yin Hongyan; Wang Hanzhou; Zong Hongliang; Chen Xiaoning; Wang Yanlin; Yun Xiaojing; Wu Yihong; Wang Jiadong; Gu Jianxin

    2006-01-01

    In this study, we reported that small glutamine-rich TPR-containing protein (SGT) interacted with not only Hsp90α but also Hsp90β. Confocal analysis showed that treatment of cells with Hsp90-specific inhibitor geldanamycin (GA) disrupted the interaction of SGT with Hsp90β and this contributed to the increase of nuclear localization of SGT in HeLa cells. The increased nuclear localization of SGT was further confirmed by the Western blotting in GA-treated HeLa cells and H1299 cells. In our previous study, SGT was found to be a new pro-apoptotic factor, so we wondered whether the sub-cellular localization of SGT was related with cell apoptosis. By confocal analysis we found that the nuclear import of SGT was significantly increased in STS-induced apoptotic HeLa cells, which implied that the sub-cellular localization of SGT was closely associated with Hsp90β and apoptosis

  10. 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. Copyright © 2010 Wiley-Liss, Inc.

  11. The regeneration of epidermal cells of Saintpaulia leaves as a new plant-tissue system for cellular radiation biology

    International Nuclear Information System (INIS)

    Engels, F.M.; Laan, F.M. van der; Leenhouts, H.P.; Chadwick, K.H.

    1980-01-01

    investigation of the nucleus of epidermal cells of the petioles of Saintpaulia leaves by cytofluorimetry revealed that all cells are in a non-cycling pre DNA synthesis phase. Cultivation of dissected leaves results in a synchronous regeneration process of a defined number of cells. Five days after onset of cultivation the cells reach the first mitosis. The nuclear development during the regeneration process is described. Irradiation of the leaves results in a directly visible inhibition of this regenerating capability which is used to quantify cell survival in a tissue. The data show that the radiation response has a similar shape to that of the survival of single cells in culture. This response can be observed before the first mitosis of the cells and its application as a new plant tissue system for cellular radiation research is discussed. (author)

  12. 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...

  13. Elimination of radiation-induced chromosome damages in human peripheral blood lymphocyte cultures. 2. The frequency of aberrations in the first-fifth post-irradiation mitosis

    International Nuclear Information System (INIS)

    Pyatkin, E.K.; Pokrovskaya, V.N.; Nugis, V.Yu.

    1982-01-01

    The number of chromosome aberrations in 1.-5. mitoses cultivated from lymphocyte PHA of peripheric man blood after gamma irradiation in vitro in 1e5; 3 and 6 Gy has been determined. For all the doses, as the cells passed 1. and successive postradiation divisiops, observed was the decrease in the number of aberrant metaphases and all the aberrations of the chromosomal typee at that their elimination rate increases with the dose increase. No considerable differences in the frequency of pair fragments in 1.-4. mitosis after irradiation in 1,5 Gy dose, in 1.-3. mitoses after irradiation in 3 Gy dose and in 1.-2. mitoses after irradiation in 6 Gy dose were found. In lymphocyte cultures irradiated in 3 and 6 Gy doses the number of dicentries in 2. mitosis was approximately 2 times smaller than in 1. mitosis and in 3. mitosis two times smaller than in 2. mitosis. In 1. mitosis almost all the dicentrics have accompanying pair fragments in 2. and 3. mitoses a share of the dicentrics without fragments constituted about 30-70 %, and in 4.-5. mitoses amounted to 95-100 %. The reduction of the number of irregular chromosomes in the process of cell passing of 1. and successive postradiation mitosis was noted only during lymphocyte investigation irradiated in 6 Gy. At 1,5 and 3 Gy doses these aberration frequency in 1.-5. and 1.-4. mitoses were nearly the same

  14. Isotachophoresis for fractionation and recovery of cytoplasmic RNA and nucleus from single cells.

    Science.gov (United States)

    Kuriyama, Kentaro; Shintaku, Hirofumi; Santiago, Juan G

    2015-07-01

    There is a substantial need for simultaneous analyses of RNA and DNA from individual single cells. Such analysis provides unique evidence of cell-to-cell differences and the correlation between gene expression and genomic mutation in highly heterogeneous cell populations. We present a novel microfluidic system that leverages isotachophoresis to fractionate and isolate cytoplasmic RNA and genomic DNA (gDNA) from single cells. The system uniquely enables independent, sequence-specific analyses of these critical markers. Our system uses a microfluidic chip with a simple geometry and four end-channel electrodes, and completes the entire process in RNA output reservoirs, each containing high quality and purity aliquots with no measurable cross-contamination of cytoplasmic RNA versus gDNA. We demonstrate our system with simultaneous, sequence-specific quantitation using off-chip RT-qPCR and qPCR for simultaneous cytoplasmic RNA and gDNA analyses, respectively. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Interaction of the Mouse Polyomavirus Capsid Proteins with Importins Is Required for Efficient Import of Viral DNA into the Cell Nucleus.

    Science.gov (United States)

    Soldatova, Irina; Prilepskaja, Terezie; Abrahamyan, Levon; Forstová, Jitka; Huérfano, Sandra

    2018-03-31

    The mechanism used by mouse polyomavirus (MPyV) overcomes the crowded cytosol to reach the nucleus has not been fully elucidated. Here, we investigated the involvement of importin α/β1 mediated transport in the delivery of MPyV genomes into the nucleus. Interactions of the virus with importin β1 were studied by co-immunoprecipitation and proximity ligation assay. For infectivity and nucleus delivery assays, the virus and its capsid proteins mutated in the nuclear localization signals (NLSs) were prepared and produced. We found that at early times post infection, virions bound importin β1 in a time dependent manner with a peak of interactions at 6 h post infection. Mutation analysis revealed that only when the NLSs of both VP1 and VP2/3 were disrupted, virus did not bind efficiently to importin β1 and its infectivity remarkably decreased (by 80%). Nuclear targeting of capsid proteins was improved when VP1 and VP2 were co-expressed. VP1 and VP2 were effectively delivered into the nucleus, even when one of the NLS, either VP1 or VP2, was disrupted. Altogether, our results showed that MPyV virions can use VP1 and/or VP2/VP3 NLSs in concert or individually to bind importins to deliver their genomes into the cell nucleus.

  16. The master Greatwall kinase, a critical regulator of mitosis and meiosis.

    Science.gov (United States)

    Vigneron, Suzanne; Robert, Perle; Hached, Khaled; Sundermann, Lena; Charrasse, Sophie; Labbé, Jean-Claude; Castro, Anna; Lorca, Thierry

    2016-01-01

    Entry into mitosis requires the coordinated activation of various protein kinases and phosphatases that together activate sequential signaling pathways allowing entry, progression and exit of mitosis. The limiting step is thought to be the activation of the mitotic Cdk1-cyclin B kinase. However, this model has recently evolved with new data showing that in addition to the Cdk1-cyclin B complex, Greatwall (Gwl) kinase is also required to enter into and maintain mitosis. This new concept proposes that entry into mitosis is now based on the combined activation of both kinases Cdk1-cyclin B and Gwl, the former promoting massive phosphorylation of mitotic substrates and the latter inhibiting PP2A-B55 phosphatase responsible for dephosphorylation of these substrates. Activated Gwl phosphorylates both Arpp19 and ENSA, which associate and inhibit PP2A-B55. This pathway seems relatively well conserved from yeast to humans, although some differences appear based on models or techniques used. While Gwl is activated by phosphorylation, its inactivation requires dephosphorylation of critical residues. Several phosphatases such as PP1, PP2A-B55 and FCP1 are required to control the dephosphorylation and inactivation of Gwl and a properly regulated mitotic exit. Gwl has also been reported to be involved in cancer processes and DNA damage recovery. These new findings support the idea that the Gwl-Arpp19/ENSA-PP2A-B55 pathway is essential to achieve an efficient division of cells and to maintain genomic stability.

  17. 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.

  18. Synergistic antitumor cytotoxic actions of ascorbate and menadione on human prostate (DU145) cancer cells in vitro: nucleus and other injuries preceding cell death by autoschizis.

    Science.gov (United States)

    Gilloteaux, Jacques; Jamison, James M; Neal, Deborah; Summers, Jack L

    2014-04-01

    Scanning (SEM) and transmission electron microscopy (TEM) were used to characterize the cytotoxic effects of ascorbate (VC), menadione (VK3), or a VC:VK3 combination on a human prostate carcinoma cell line (DU145) following a 1-h vitamin treatment and a subsequent 24-h incubation in culture medium. Cell alterations examined by light and electron microscopy were treatment-dependent with VC + VK3 >VK3 > VC > Sham. Oxidative stress-induced damage was found in most organelles. This report describes injuries in the tumor cell nucleus (chromatin and nucleolus), mitochondria, endomembranes, lysosomal bodies (autophagocytoses) and inclusions. Morphologic alterations suggest that cytoskeleton damage is likely responsible for the superficial cytoplasmic changes, including major changes in cell shape and size and the self-excising phenomena. Unlike apoptotic bodies, the excised pieces contain ribonucleoproteins, but not organelles. These deleterious events cause a progressive, significant reduction in the tumor cell size. During nuclear alterations, the nuclei maintain their envelope during chromatolysis and karyolysis until cell death, while nucleoli undergo a characteristic segregation of their components. In addition, changes in fat and glycogen storage are consistent the cytotoxic and metabolic alterations caused by the respective treatments. All cellular ultrastructural changes are consistent with cell death by autoschizis and not apoptosis or other kinds of cell death.

  19. Effects of age, replicative lifespan and growth rate of human nucleus pulposus cells on selecting age range for cell-based biological therapies for degenerative disc diseases.

    Science.gov (United States)

    Lee, J S; Lee, S M; Jeong, S W; Sung, Y G; Lee, J H; Kim, K W

    2016-07-01

    Autologous disc cell implantation, growth factors and gene therapy appear to be promising therapies for disc regeneration. Unfortunately, the replicative lifespan and growth kinetics of human nucleus pulposus (NP) cells related to host age are unclear. We investigated the potential relations among age, replicative lifespan and growth rate of NP cells, and determined the age range that is suitable for cell-based biological therapies for degenerative disc diseases. We used NP tissues classified by decade into five age groups: 30s, 40s, 50s, 60s and 70s. The mean cumulative population doubling level (PDL) and population doubling rate (PDR) of NP cells were assessed by decade. We also investigated correlations between cumulative PDL and age, and between PDR and age. The mean cumulative PDL and PDR decreased significantly in patients in their 60s. The mean cumulative PDL and PDR in the younger groups (30s, 40s and 50s) were significantly higher than those in the older groups (60s and 70s). There also were significant negative correlations between cumulative PDL and age, and between PDR and age. We found that the replicative lifespan and growth rate of human NP cells decreased with age. The replicative potential of NP cells decreased significantly in patients 60 years old and older. Young individuals less than 60 years old may be suitable candidates for NP cell-based biological therapies for treating degenerative disc diseases.

  20. Suppressor of cytokine signaling 1 (SOCS1) limits NFkappaB signaling by decreasing p65 stability within the cell nucleus.

    Science.gov (United States)

    Strebovsky, Julia; Walker, Patrick; Lang, Roland; Dalpke, Alexander H

    2011-03-01

    Suppressor of cytokine signaling (SOCS) proteins are inhibitors of cytoplasmic Janus kinases (Jak) and signal transducer and activator of transcription (STAT) signaling pathways. Previously the authors surprisingly observed that SOCS1 translocated into the nucleus, which was because of the presence of a nuclear localization sequence. This report now hypothesizes that SOCS1 mediates specific functions within the nuclear compartment because it is instantly transported into the nucleus, as shown by photoactivation and live cell imaging in human HEK293 cells. The NFκB component p65 is identified as an interaction partner for SOCS1 but not for other members of the SOCS family. SOCS1 bound to p65 only within the nucleus. By means of its SOCS box domain, SOCS1 operated as a ubiquitin ligase, leading to polyubiquitination and proteasomal degradation of nuclear p65. Thus, SOCS1 limited prolonged p65 signaling and terminated expression of NFκB inducible genes. Using mutants that lack either nuclear translocation or a functional SOCS box, this report identifies genes that are regulated in a manner dependent on the nuclear availability of SOCS1. Data show that beyond its receptor-proximal function in Jak/STAT signaling, SOCS1 also regulates the duration of NFκB signaling within the cell nucleus, thus exerting a heretofore unrecognized function.

  1. Evolutionary consequences of polyploidy in prokaryotes and the origin of mitosis and meiosis.

    Science.gov (United States)

    Markov, Alexander V; Kaznacheev, Ilya S

    2016-06-08

    The origin of eukaryote-specific traits such as mitosis and sexual reproduction remains disputable. There is growing evidence that both mitosis and eukaryotic sex (i.e., the alternation of syngamy and meiosis) may have already existed in the basal eukaryotes. The mating system of the halophilic archaeon Haloferax volcanii probably represents an intermediate stage between typical prokaryotic and eukaryotic sex. H. volcanii is highly polyploid, as well as many other Archaea. Here, we use computer simulation to explore genetic and evolutionary outcomes of polyploidy in amitotic prokaryotes and its possible role in the origin of mitosis, meiosis and eukaryotic sex. Modeling suggests that polyploidy can confer strong short-term evolutionary advantage to amitotic prokaryotes. However, it also promotes the accumulation of recessive deleterious mutations and the risk of extinction in the long term, especially in highly mutagenic environment. There are several possible strategies that amitotic polyploids can use in order to reduce the genetic costs of polyploidy while retaining its benefits. Interestingly, most of these strategies resemble different components or aspects of eukaryotic sex. They include asexual ploidy cycles, equalization of genome copies by gene conversion, high-frequency lateral gene transfer between relatives, chromosome exchange coupled with homologous recombination, and the evolution of more accurate chromosome distribution during cell division (mitosis). Acquisition of mitosis by an amitotic polyploid results in chromosome diversification and specialization. Ultimately, it transforms a polyploid cell into a functionally monoploid one with multiple unique, highly redundant chromosomes. Specialization of chromosomes makes the previously evolved modes of promiscuous chromosome shuffling deleterious. This can result in selective pressure to develop accurate mechanisms of homolog pairing, and, ultimately, meiosis. Emergence of mitosis and the first

  2. 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

  3. Extrahypothalamic vasopressin and oxytocin in the human brain; presence of vasopressin cells in the bed nucleus of the stria terminalis

    NARCIS (Netherlands)

    Fliers, E.; Guldenaar, S. E.; van de Wal, N.; Swaab, D. F.

    1986-01-01

    In the present study, the distribution of extrahypothalamic vasopressin (VP) and oxytocin (OXT) in the human brain was investigated by means of immunocytochemistry. In the septum verum, few VP fibers were found in the nucleus septalis lateralis and medialis (NSL and NSM), and in the bed nucleus of

  4. Estrogen Enhances Matrix Synthesis in Nucleus Pulposus Cell through the Estrogen Receptor β-p38 MAPK Pathway

    Directory of Open Access Journals (Sweden)

    Pei Li

    2016-11-01

    Full Text Available Background/Aims: Matrix homeostasis within the disc nucleus pulposus (NP tissue is important for disc function. Increasing evidence indicates that sex hormone can influence the severity of disc degeneration. This study was aimed to study the role of 17β-estradiol (E2 in NP matrix synthesis and its underlying mechanism. Methods: Rat NP cells were cultured with (10-5, 10-7 and 10-9 M or without (control E2 for48 hours. The estrogen receptor (ER-β antagonist PHTPP and ERβ agonist ERB 041 were used to investigate the role mediated by ERβ. The p38 MAPK inhibitor SB203580 was used to investigate the role of p38 MAPK signaling pathway. Gene and protein expression of SOX9, aggrecan and collagen II, glycosaminoglycan (GAG content, and immunostaining assay for aggrecan and collagen II were analyzed to evaluate matrix production in rat NP cells. Results: E2 enhanced NP matrix synthesis in a concentration-dependent manner regarding gene and proetin expression of SOX9, aggrecan and collagen II, protein deposition of aggrecan and collagen II, and GAG content. Moreover, activation of p38 MAPK signaling pathway was increased with elevating E2 concentration. Further analysis indicated that ERB 041 and PHTPP could respectively enhance and suppress effects of E2 on matrix synthesis in NP cells, as well as activation of p38 MAPK pathway. Additionally, inhibition of p38 MAPK signaling pathway significantly abolished the effects of E2 on matrix synthesis. Conclusion: E2 can enhance matrix synthesis of NP cells and the ERβ/p38 MAPK pathway is involved in this regulatory process.

  5. 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.

  6. 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 -

  7. The Kisspeptin/Neurokinin B/Dynorphin (KNDy) Cell Population of the Arcuate Nucleus: Sex Differences and Effects of Prenatal Testosterone in Sheep

    OpenAIRE

    Cheng, Guanliang; Coolen, Lique M.; Padmanabhan, Vasantha; Goodman, Robert L.; Lehman, Michael N.

    2009-01-01

    Recent work in sheep has identified a neuronal subpopulation in the arcuate nucleus that coexpresses kisspeptin, neurokinin B, and dynorphin (referred to here as KNDy cells) and that mediate the negative feedback influence of progesterone on GnRH secretion. We hypothesized that sex differences in progesterone negative feedback are due to sexual dimorphism of KNDy cells and compared neuropeptide and progesterone receptor immunoreactivity in this subpopulation between male and female sheep. In ...

  8. 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.

  9. 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. © 2014 Wiley Periodicals, Inc.

  10. Use of Limiting Dilution Method for Isolation of Nucleus Pulposus Mesenchymal Stem/Progenitor Cells and Effects of Plating Density on Biological Characteristics and Plasticity

    Directory of Open Access Journals (Sweden)

    Linghan Lin

    2017-01-01

    Full Text Available Objectives. To evaluate the effects of the limiting dilution method and plating density in rat nucleus pulposus mesenchymal stem/progenitor cells (NPMSCs. Materials and Methods. Nucleus pulposus tissues were isolated from 12-week-old male Sprague-Dawley rats and NPMSCs were isolated using limiting dilution method. Cells were then classified into 3 groups according to plating density. Cell morphologies were observed, and colony-forming units, migration abilities, proliferative capacities, cell cycle percentages, multilineage differentiation capacities, stem cell biomarker expression levels, and immunophenotyping were also examined in each group. Results. Low density group (LD had higher morphological homogeneity, stronger colony-forming ability, higher cell proliferation capacity, and enhanced cell migration ability relative to the other two groups (p<0.05. Moreover, LD had more cells entering S phase, with fewer cells arrested in G0/G1 phase (p<0.05. While all three density groups showed a multilineage differentiation potential, LD showed a higher degree of observed and semiquantified lineage specific staining (p<0.05. Furthermore, LD displayed higher expression levels of stem cell biomarkers (Nanog, Oct4, and Sox2 and showed higher percentages of CD29+, CD44+, and CD90+ cells (p<0.05 following flow cytometry analysis. Conclusions. Limiting dilution method is suggested when isolating NPMSCs as a means of improving cell activity and plasticity.

  11. The kisspeptin/neurokinin B/dynorphin (KNDy) cell population of the arcuate nucleus: sex differences and effects of prenatal testosterone in sheep.

    Science.gov (United States)

    Cheng, Guanliang; Coolen, Lique M; Padmanabhan, Vasantha; Goodman, Robert L; Lehman, Michael N

    2010-01-01

    Recent work in sheep has identified a neuronal subpopulation in the arcuate nucleus that coexpresses kisspeptin, neurokinin B, and dynorphin (referred to here as KNDy cells) and that mediate the negative feedback influence of progesterone on GnRH secretion. We hypothesized that sex differences in progesterone negative feedback are due to sexual dimorphism of KNDy cells and compared neuropeptide and progesterone receptor immunoreactivity in this subpopulation between male and female sheep. In addition, because sex differences in progesterone negative feedback and neurokinin B are due to the influence of testosterone (T) during fetal life, we determined whether prenatal T exposure would mimic sex differences in KNDy cells. Adult rams had nearly half the number of kisspeptin, neurokinin B, dynorphin, and progesterone receptor-positive cells in the arcuate nucleus as did females, but the percentage of KNDy cells colocalizing progesterone receptors remained high in both sexes. Prenatal T treatment also reduced the number of dynorphin, neurokinin B, and progesterone receptor-positive cells in the female arcuate nucleus; however, the number of kisspeptin cells remained high and at levels comparable to control females. Thus, sex differences in kisspeptin in the arcuate nucleus, unlike that of dynorphin and neurokinin B, are not due solely to exposure to prenatal T, suggesting the existence of different critical periods for multiple peptides coexpressed within the same neuron. In addition, the imbalance between inhibitory (dynorphin) and stimulatory (kisspeptin) neuropeptides in this subpopulation provides a potential explanation for the decreased ability of progesterone to inhibit GnRH neurons in prenatal T-treated ewes.

  12. Nucleus-nucleus total reaction cross sections

    International Nuclear Information System (INIS)

    DeVries, R.M.; Peng, J.C.

    1980-01-01

    We compare sigma/sub R/(E) for nucleus-nucleus systems (obtained from existing direct measurements and derived from elastic scattering data) with nucleon-nucleon and nucleon-nucleus data. The energy dependence of sigma/sub R/(E) for nucleus-nucleus systems is found to be quite rapid; there appears to be no evidence for an energy independent, geometric sigma/sub R/. Simple parameter free microscopic calculations are able to quantitatively reproduce the data and thus, emphasize the dominance of nucleon-nucleon interactions in medium energy nucleus-nucleus collisions

  13. Centromere separation and association in the nuclei of an interspecific hybrid between Torenia fournieri and T. baillonii (Scrophulariaceae) during mitosis and meiosis.

    Science.gov (United States)

    Kikuchi, Shinji; Tanaka, Hiroyuki; Wako, Toshiyuki; Tsujimoto, Hisashi

    2007-10-01

    In the nuclei of some interspecific hybrid and allopolyploid plant species, each genome occupies a separate spatial domain. To analyze this phenomenon, we studied localization of the centromeres in the nuclei of a hybrid between Torenia fournieri and T. baillonii during mitosis and meiosis using three-dimensional fluorescence in situ hybridization (3D-FISH) probed with species-specific centromere repeats. Centromeres of each genome were located separately in undifferentiated cells but not differentiated cells, suggesting that cell division might be the possible force causing centromere separation. However, no remarkable difference of dividing distance was detected between chromatids with different centromeres in anaphase and telophase, indicating that tension of the spindle fiber attached to each chromatid is not the cause of centromere separation in Torenia. In differentiated cells, centromeres in both genomes were not often observed for the expected chromosome number, indicating centromere association. In addition, association of centromeres from the same genome was observed at a higher frequency than between different genomes. This finding suggests that centromeres within one genome are spatially separated from those within the other. This close position may increase possibility of association between centromeres of the same genome. In meiotic prophase, all centromeres irrespective of the genome were associated in a certain portion of the nucleus. Since centromere association in the interspecific hybrid and amphiploid was tighter than that in the diploid parents, it is possible that this phenomenon may be involved in sorting and pairing of homologous chromosomes.

  14. Carbon and nitrogen depletion-induced nucleophagy and selective autophagic sequestration of a whole nucleus in multinucleate cells of the filamentous fungus Aspergillus oryzae.

    Science.gov (United States)

    Kikuma, Takashi; Mitani, Takahiro; Kohara, Takahiro; Maruyama, Jun-Ichi; Kitamoto, Katsuhiko

    2017-05-12

    Autophagy is a conserved cellular degradation process in eukaryotes, in which cytoplasmic components and organelles are digested in vacuoles/lysosomes. Recently, autophagic degradation of nuclear materials, termed "nucleophagy", has been reported. In the multinucleate filamentous fungus Aspergillus oryzae, a whole nucleus is degraded by nucleophagy after prolonged culture. While developing an H2B-EGFP processing assay for the evaluation of nucleophagy in A. oryzae, we found that nucleophagy is efficiently induced by carbon or nitrogen depletion. Microscopic observations in a carbon depletion condition clearly demonstrated that autophagosomes selectively sequester a particular nucleus, despite the presence of multiple nuclei in the same cell. Furthermore, AoNsp1, the A. oryzae homolog of the yeast nucleoporin Nsp1p, mainly localized at the nuclear periphery, but its localization was restricted to the opposite side of the autophagosome being formed around a nucleus. In contrast, the perinuclear ER visualized with the calnexin AoClxA was not morphologically affected by nucleophagy. The findings of nucleophagy-inducing conditions enabled us to characterize the morphological process of autophagic degradation of a whole nucleus in multinucleate cells.

  15. Dissecting Cell-Type Composition and Activity-Dependent Transcriptional State in Mammalian Brains by Massively Parallel Single-Nucleus RNA-Seq.

    Science.gov (United States)

    Hu, Peng; Fabyanic, Emily; Kwon, Deborah Y; Tang, Sheng; Zhou, Zhaolan; Wu, Hao

    2017-12-07

    Massively parallel single-cell RNA sequencing can precisely resolve cellular diversity in a high-throughput manner at low cost, but unbiased isolation of intact single cells from complex tissues such as adult mammalian brains is challenging. Here, we integrate sucrose-gradient-assisted purification of nuclei with droplet microfluidics to develop a highly scalable single-nucleus RNA-seq approach (sNucDrop-seq), which is free of enzymatic dissociation and nucleus sorting. By profiling ∼18,000 nuclei isolated from cortical tissues of adult mice, we demonstrate that sNucDrop-seq not only accurately reveals neuronal and non-neuronal subtype composition with high sensitivity but also enables in-depth analysis of transient transcriptional states driven by neuronal activity, at single-cell resolution, in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Mechanosensing of matrix by stem cells: From matrix heterogeneity, contractility, and the nucleus in pore-migration to cardiogenesis and muscle stem cells in vivo.

    Science.gov (United States)

    Smith, Lucas; Cho, Sangkyun; Discher, Dennis E

    2017-11-01

    Stem cells are particularly 'plastic' cell types that are induced by various cues to become specialized, tissue-functional lineages by switching on the expression of specific gene programs. Matrix stiffness is among the cues that multiple stem cell types can sense and respond to. This seminar-style review focuses on mechanosensing of matrix elasticity in the differentiation or early maturation of a few illustrative stem cell types, with an intended audience of biologists and physical scientists. Contractile forces applied by a cell's acto-myosin cytoskeleton are often resisted by the extracellular matrix and transduced through adhesions and the cytoskeleton ultimately into the nucleus to modulate gene expression. Complexity is added by matrix heterogeneity, and careful scrutiny of the evident stiffness heterogeneity in some model systems resolves some controversies concerning matrix mechanosensing. Importantly, local stiffness tends to dominate, and 'durotaxis' of stem cells toward stiff matrix reveals a dependence of persistent migration on myosin-II force generation and also rigid microtubules that confer directionality. Stem and progenitor cell migration in 3D can be further affected by matrix porosity as well as stiffness, with nuclear size and rigidity influencing niche retention and fate choices. Cell squeezing through rigid pores can even cause DNA damage and genomic changes that contribute to de-differentiation toward stem cell-like states. Contraction of acto-myosin is the essential function of striated muscle, which also exhibit mechanosensitive differentiation and maturation as illustrated in vivo by beating heart cells and by the regenerative mobilization of skeletal muscle stem cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Autophagy is activated in compression-induced cell degeneration and is mediated by reactive oxygen species in nucleus pulposus cells exposed to compression.

    Science.gov (United States)

    Ma, K-G; Shao, Z-W; Yang, S-H; Wang, J; Wang, B-C; Xiong, L-M; Wu, Q; Chen, S-F

    2013-12-01

    To determine whether autophagy contributes to the pathogenesis of degenerative disc disease (DDD) or retards the intervertebral disc (IVD) degeneration, and investigate the possible relationship between compression-induced autophagy and intracellular reactive oxygen species (ROS) in nucleus pulposus (NP) cells in vitro. The autophagosome and autophagy-related markers were used to explore the role of autophagy in rat NP cells under compressive stress, which were measured directly by electronic microscopy, monodansylcadaverine (MDC) staining, immunofluorescence, western blot, and indirectly by analyzing the impact of pharmacological inhibitors of autophagy such as 3-methyladenine (3-MA) and chloroquine (CQ). And the relationship between autophagy and apoptosis was investigated by Annexin-V/propidium iodide (PI)-fluorescein staining. In addition, ROS were measured to determine whether these factors are responsible for the development of compression-induced autophagy. Our results indicated that rat NP cells activated autophagy in response to the same strong apoptotic stimuli that triggered apoptosis by compression. Autophagy and apoptosis were interconnected and coordinated in rat NP cells exposed to compression stimuli. Compression-induced autophagy was closely related to intracellular ROS production. Enhanced degradation of damaged components of NP cells by autophagy may be a crucial survival response against mechanical overload, and extensive autophagy may trigger autophagic cell death. Regulating autophagy and reducing the generation of intracellular ROS may retard IVD degeneration. Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  18. 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.

  19. Effect of microRNA-21 on the proliferation of human degenerated nucleus pulposus by targeting programmed cell death 4

    Directory of Open Access Journals (Sweden)

    B. Chen

    2016-01-01

    Full Text Available This study aims to explore the effect of microRNA-21 (miR-21 on the proliferation of human degenerated nucleus pulposus (NP by targeting programmed cell death 4 (PDCD4 tumor suppressor. NP tissues were collected from 20 intervertebral disc degeneration (IDD patients, and from 5 patients with traumatic spine fracture. MiR-21 expressions were tested. NP cells from IDD patients were collected and divided into blank control group, negative control group (transfected with miR-21 negative sequences, miR-21 inhibitor group (transfected with miR-21 inhibitors, miR-21 mimics group (transfected with miR-21 mimics and PDCD4 siRNA group (transfected with PDCD4 siRNAs. Cell growth was estimated by Cell Counting Kit-8; PDCD4, MMP-2,MMP-9 mRNA expressions were evaluated by qRT-PCR; PDCD4, c-Jun and p-c-Jun expressions were tested using western blot. In IDD patients, the expressions of miR-21 and PDCD4 mRNA were respectively elevated and decreased (both P<0.05. The miR-21 expressions were positively correlated with Pfirrmann grades, but negatively correlated with PDCD4 mRNA (both P<0.001. In miR-21 inhibitor group, cell growth, MMP-2 and MMP-9 mRNA expressions, and p-c-Jun protein expressions were significantly lower, while PDCD4 mRNA and protein expressions were higher than the other groups (all P<0.05. These expressions in the PDCD4 siRNA and miR-21 mimics groups was inverted compared to that in the miR-21 inhibitor group (all P<0.05. MiR-21 could promote the proliferation of human degenerated NP cells by targeting PDCD4, increasing phosphorylation of c-Jun protein, and activating AP-1-dependent transcription of MMPs, indicating that miR-21 may be a crucial biomarker in the pathogenesis of IDD.

  20. A novel branched TAT(47-57) peptide for selective Ni(2+) introduction into the human fibrosarcoma cell nucleus.

    Science.gov (United States)

    Szyrwiel, Łukasz; Shimura, Mari; Shirataki, Junko; Matsuyama, Satoshi; Matsunaga, Akihiro; Setner, Bartosz; Szczukowski, Łukasz; Szewczuk, Zbigniew; Yamauchi, Kazuto; Malinka, Wiesław; Chavatte, Laurent; Łobinski, Ryszard

    2015-07-01

    A TAT47-57 peptide was modified on the N-terminus by elongation with a 2,3-diaminopropionic acid residue and then by coupling of two histidine residues on its N-atoms. This branched peptide could bind to Ni under physiological conditions as a 1 : 1 complex. We demonstrated that the complex was quantitatively taken up by human fibrosarcoma cells, in contrast to Ni(2+) ions. Ni localization (especially at the nuclei) was confirmed by imaging using both scanning X-ray fluorescence microscopy and Newport Green fluorescence. A competitive assay with Newport Green showed that the latter displaced the peptide ligand from the Ni-complex. Ni(2+) delivered as a complex with the designed peptide induced substantially more DNA damage than when introduced as a free ion. The availability of such a construct opens up the way to investigate the importance of the nucleus as a target for the cytotoxicity, genotoxicity or carcinogenicity of Ni(2+).

  1. The intercalatus nucleus of Staderini.

    Science.gov (United States)

    Cascella, Marco

    2016-01-01

    Rutilio Staderini was one of the leading Italian anatomists of the twentieth century, together with some scientists, such as Giulio Chiarugi, Giovanni Vitali, and others. He was also a member of a new generation of anatomists. They had continued the tradition of the most famous Italian scientists, which started from the Renaissance up until the nineteenth century. Although he carried out important studies of neuroanatomy and comparative anatomy, as well as embryology, his name is rarely remembered by most medical historians. His name is linked to the nucleus he discovered: the Staderini nucleus or intercalated nucleus, a collection of nerve cells in the medulla oblongata located lateral to the hypoglossal nucleus. This article focuses on the biography of the neuroanatomist as well as the nucleus that carries his name and his other research, especially on comparative anatomy and embryology.

  2. Microdosimetry of 125I and 3H in the cell nucleus

    International Nuclear Information System (INIS)

    Booz, J.; Fidorra, J.; Smit, T.; Charlton, D.E.

    1978-01-01

    The dosimetry and microdosimetry of iodine-125 and Tritium were analyzed. In particular, a detailed examination of the number and energies of the electrons emitted by iodine-125 was performed and the result considerably changed the currently available data. In this paper, the result of this examination will be summarized and the radiobiological implications will be discussed. A new model of the radiation mechanism of iodine-125 is proposed, which takes the high energy deposition at the molecular level and the high number of emitted Auger-electrons into consideration. On the basis of this model it is concluded that about 40 primary lesions are needed in the DNA for the cellular inactivation of T-cells. (orig./AJ) [de

  3. Centrioles are freed from cilia by severing prior to mitosis.

    Science.gov (United States)

    Parker, Jeremy D K; Hilton, Laura K; Diener, Dennis R; Rasi, M Qasim; Mahjoub, Moe R; Rosenbaum, Joel L; Quarmby, Lynne M

    2010-07-01

    Cilia are necessary for normal tissue development and homeostasis and are generally present during interphase, but not in mitosis. The precise mechanism of premitotic ciliary loss has been controversial, with data supporting either sequential disassembly through the transition zone or, alternatively, a severing event at the base of the cilia. Here we show by live cell imaging and immunofluorescence microscopy that resorbing flagella of Chlamydomonas leave remnants associated with the mother cell wall. We postulated that the remnants are the product of severing of doublet microtubules between the basal bodies and the flagellar transition zone, thereby freeing the centrioles to participate in spindle organization. We show via TEM that flagellar remnants are indeed flagellar transition zones encased in vesicles derived from the flagellar membrane. This transition zone vesicle can be lodged within the cell wall or it can be expelled into the environment. This process is observable in Chlamydomonas, first because the released flagellar remnants can remain associated with the cell by virtue of attachments to the cell wall, and second because the Chlamydomonas transition zone is particularly rich with electron-dense structure. However, release of basal bodies for spindle-associated function is likely to be conserved among the eukaryotes. 2010 Wiley-Liss, Inc.

  4. Unconventional actin conformations localize on intermediate filaments in mitosis

    International Nuclear Information System (INIS)

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan

    2011-01-01

    Research highlights: → Unconventional actin conformations colocalize with vimentin on a cage-like structure in metaphase HEK 293T cells. → These conformations are detected with the anti-actin antibodies 1C7 ('lower dimer') and 2G2 ('nuclear actin'), but not C4 (monomeric actin). → Mitotic unconventional actin cables are independent of filamentous actin or microtubules. → Unconventional actin colocalizes with vimentin on a nocodazole-induced perinuclear dense mass of cables. -- Abstract: Different structural conformations of actin have been identified in cells and shown to reside in distinct subcellular locations of cells. In this report, we describe the localization of actin on a cage-like structure in metaphase HEK 293T cells. Actin was detected with the anti-actin antibodies 1C7 and 2G2, but not with the anti-actin antibody C4. Actin contained in this structure is independent of microtubules and actin filaments, and colocalizes with vimentin. Taking advantage of intermediate filament collapse into a perinuclear dense mass of cables when microtubules are depolymerized, we were able to relocalize actin to such structures. We hypothesize that phosphorylation of intermediate filaments at mitosis entry triggers the recruitment of different actin conformations to mitotic intermediate filaments. Storage and partition of the nuclear actin and antiparallel 'lower dimer' actin conformations between daughter cells possibly contribute to gene transcription and transient actin filament dynamics at G1 entry.

  5. Effects of hydrostatic pressure on microtubule organization and nucleus changes in gynogenetically activated eggs of olive flounder (Paralichthys olivaceus).

    Science.gov (United States)

    Lin, Zhengmei; Zhu, Xiangping; Zhang, Tingrong; You, Feng; Wu, Zhihao; Cao, Yuanshui

    2016-06-01

    Fluorescent double-labeled technique was used to investigate the effects of hydrostatic pressure on microtubule organization and nucleus in gynogenetically activated eggs of olive flounder (Paralichthys olivaceus). The parameter of hydrostatic pressure treatment was 600 kg/cm(2) for 6 minutes at prometaphase of the first mitosis. The data showed that nucleus and microtubule changes of the diploid control were basically similar to those of the haploid one (5 minutes behind those of the diploid control). Nuclear diameter of the haploid embryo was significantly smaller than that of the diploid one (P pressure treatment. Centrosome renucleated microtubule, and a bipolar spindle reassembled 15 minutes after treatment, leading to occurrence of the first cleavage. During the second cell cycle, about 80% treated embryo had a single centrosome and formed a unipolar spindle in both blastomeres. After prometaphase, chromosomes spread around for about 20 minutes instead of aligning on the equatorial plane, then assembled and formed one large nucleus without anaphase separation. The second cleavage was inhibited, and the chromosome set was doubled. The data indicated that the chromosome set doubling of mitogynogenetic diploid induced by hydrostatic pressure treatment, which performed at prometaphase of the first mitosis, mainly resulted from the inhibition of the second cleavage rather than the first one. This study is the first to adapt fluorescent double-labeled technique to investigate the mechanism on chromosome set doubling of mitotic gynogenesis induction. This study will offer theoretical support for mitogynogenetic diploid induction in marine fish. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Elimination of radiation-induced chromosomal damages in numan peripheral blood lymphocyte cultures. 1. The frequency of aberrations in the first and second mitosis

    International Nuclear Information System (INIS)

    Pyatkin, E.K.; Nugis, V.Yu.

    1981-01-01

    A comparative analysis of chromosomal aberrations in the first and second mitosis of cultivated human peripheral blood lymphocytes after gamma irradiation in vitro at 1-5 Gy doses has been made. Irradiated blood lymphocytes were incubated for 58 to 66 h at 37 deg with PGA and BDU (20 μg /ml). The first, second and third postradiation mitosises were identified using the distinguishing staining of sister chromatids. The share of the cells in the first mitosis fluctuated from 32 to 77 %, in the second - from 23 to 68 %, and the third - from 0 to 9 %. At all radiation doses significant differences in the frequency of the aberration cells passing the first and second mitosises were revealed as well as in the total number of chromosomal aberrations in all the cells. The frequency of pair fragments and dicentrics chromosomes in the first mitosis was on the average 1.6 and 2 times as high as in the second one, respectively. In the first mitosis almost all dicentric chromosomes occurred with accompanying pair fragments, and in the second mitosis the share of dicentric chromosomes without accompanying fragments was 25 to 50 %. The distribution of the dicentric chromosomes in the cells in the first and second mitosis did not differ from Poison distribution for the 2 to 5 Gy dose range

  7. Cell Biological Mechanisms of Activity-Dependent Synapse to Nucleus Translocation of CRTC1 in Neurons

    Directory of Open Access Journals (Sweden)

    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.

  8. Use of unstable chromosome aberrations for biological dosimetry after the first postirradiation mitosis

    International Nuclear Information System (INIS)

    Doloy, M.T.; Malarbet, J.L.; Guedeney, G.; Bourguignon, M.; Leroy, A.; Reillaudou, M.; Masse, R.

    1991-01-01

    The loss of unstable chromosome aberrations after the first postirradiation mitosis makes their use difficult in radiation dosimetry. We describe here a method which, in a cell population observed at this stage, allows retrospective estimation of the frequencies of the unstable aberrations induced at the time of irradiation, and their use as a dosimeter. The laws controlling the behavior of unstable aberrations during mitosis were defined from a large-scale experiment on irradiated human lymphocytes. For cells undergoing the first, second, or third mitosis after irradiation, relationships were determined between the frequency, at irradiation time, of acentric fragments not arising from formation of dicentrics or rings, and the ratio of dicentrics and centric rings appearing without acentric fragments to the total number of dicentrics plus rings. On the basis of this ratio, the method described here provides an assessment of the postirradiation mitotic activity in a cell population. This assessment permitted estimation of the cell distribution and frequency of dicentrics plus centric rings, and of the frequency of acentric fragments at the time of irradiation. The use of this method for retrospective dosimetry after whole-body irradiation under various conditions of exposure is illustrated

  9. Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression.

    Science.gov (United States)

    Bougdour, Alexandre; Durandau, Eric; Brenier-Pinchart, Marie-Pierre; Ortet, Philippe; Barakat, Mohamed; Kieffer, Sylvie; Curt-Varesano, Aurélie; Curt-Bertini, Rose-Laurence; Bastien, Olivier; Coute, Yohann; Pelloux, Hervé; Hakimi, Mohamed-Ali

    2013-04-17

    After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Long-term load duration induces N-cadherin down-regulation and loss of cell phenotype of nucleus pulposus cells in a disc bioreactor culture.

    Science.gov (United States)

    Li, Pei; Zhang, Ruijie; Wang, Liyuan; Gan, Yibo; Xu, Yuan; Song, Lei; Luo, Lei; Zhao, Chen; Zhang, Chengmin; Ouyang, Bin; Tu, Bing; Zhou, Qiang

    2017-04-30

    Long-term exposure to a mechanical load causes degenerative changes in the disc nucleus pulposus (NP) tissue. A previous study demonstrated that N-cadherin (N-CDH)-mediated signalling can preserve the NP cell phenotype. However, N-CDH expression and the resulting phenotype alteration in NP cells under mechanical compression remain unclear. The present study investigated the effects of the compressive duration on N-CDH expression and on the phenotype of NP cells in an ex vivo disc organ culture. Porcine discs were organ cultured in a self-developed mechanically active bioreactor for 7 days. The discs were subjected to different dynamic compression durations (1 and 8 h at a magnitude of 0.4 MPa and frequency of 1.0 Hz) once per day. Discs that were not compressed were used as controls. The results showed that long-term compression duration (8 h) significantly down-regulated the expression of N-CDH and NP-specific molecule markers (Brachyury, Laminin, Glypican-3 and Keratin 19), attenuated Alcian Blue staining intensity, decreased glycosaminoglycan (GAG) and hydroxyproline (HYP) contents and decreased matrix macromolecule (aggrecan and collagen II) expression compared with the short-term compression duration (1 h). Taken together, these findings demonstrate that long-term load duration can induce N-CDH down-regulation, loss of normal cell phenotype and result in attenuation of NP-related matrix synthesis in NP cells. © 2017 The Author(s).

  11. Experimental study of mutagenous and mitosis modifying activity of silver nanoparticles

    Directory of Open Access Journals (Sweden)

    B. S. Kirbik

    2015-01-01

    Full Text Available Mutagenous and mitosis modifying impact of silver nanoparticles has been studied on outbred mice. Nanoparticles were of round shape with dimensions of 5-50 nm, size of generated organic shell of 2-5 nm, the quantity in 1 mcm3 makes 120-270. Metaphasic analysis of mice bone marrow cells was used as a testing technique. The frequency of chromosome aberrations and mitotic index of preparations were accounted. During single intraperitoneal administration of the agent in the dose of 250 mcg/kg the silver nanoparticles demonstrated mitosis stimulating activity. No mutagenous effect of silver nanoparticles by daily administration for 4 days of 25 mcg/kg and single administration in the dose of 250 mcg/kg has been registered, but there is statistically insignificant tendency of aberrant metaphases increase. Consequently silver nanoparticles in the investigated doses demonstrated no mutagenous activity and can be considered safe for mammalian cells.

  12. Laser microirradiation of Chinese hamster cells at wavelength 365 nm: effects of psoralen and caffeine

    International Nuclear Information System (INIS)

    Cremer, T.; Peterson, S.P.; Cremer, C.; Berns, M.W.

    1981-01-01

    Cells of a V79 subline of the Chinese hamster were microirradiated at wavelength 365 nm in the presence of the psoralen derivative, trioxsalen. Microirradiation was accomplished by a pulsed argon laser microbeam either in anaphase or in interphase 3 h after mitosis. Inhibition of clonal growth and formation of micronuclei at the first postirradiation mitosis were observed after microirradiation of anaphase chromosomes and of small parts of the interphase nucleus. Microirradiation of the cytoplasm beside the interphase nucleus or between the sets of chromosomes moving apart from each other in anaphase did not produce these effects. Anaphase experiments showed that only the daughter cell which received microirradiated chromatin exhibited an abnormal growth pattern. Most interestingly, shattering of the whole chromosome complement could be induced by microirradiation of small parts of the interphase nucleus and post-treatment with caffeine. Since microirradiation of chromatin in the absence of psoralen was not effective, we consider formation of psoralen photoadducts to nucleic acids in microirradiated chromatin to be the specific cause of the effects. We suggest that DNA photolesions in chromosome segments present in the microirradiated part of the nucleus can induce shattering of all the chromosomes in the microirradiated nucleus. Several possibilities are discussed to explain this unexpected finding

  13. Specific primary sequence requirements for Aurora B kinase-mediated phosphorylation and subcellular localization of TMAP during mitosis.

    Science.gov (United States)

    Kim, Hyun-Jun; Kwon, Hye-Rim; Bae, Chang-Dae; Park, Joobae; Hong, Kyung U

    2010-05-15

    During mitosis, regulation of protein structures and functions by phosphorylation plays critical roles in orchestrating a series of complex events essential for the cell division process. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a novel player in spindle assembly and chromosome segregation. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis. However, the mechanisms and functional importance of phosphorylation at most of the sites identified are currently unknown. Here, we report that TMAP is a novel substrate of the Aurora B kinase. Ser627 of TMAP was specifically phosphorylated by Aurora B both in vitro and in vivo. Ser627 and neighboring conserved residues were strictly required for efficient phosphorylation of TMAP by Aurora B, as even minor amino acid substitutions of the phosphorylation motif significantly diminished the efficiency of the substrate phosphorylation. Nearly all mutations at the phosphorylation motif had dramatic effects on the subcellular localization of TMAP. Instead of being localized to the chromosome region during late mitosis, the mutants remained associated with microtubules and centrosomes throughout mitosis. However, the changes in the subcellular localization of these mutants could not be completely explained by the phosphorylation status on Ser627. Our findings suggest that the motif surrounding Ser627 ((625) RRSRRL (630)) is a critical part of a functionally important sequence motif which not only governs the kinase-substrate recognition, but also regulates the subcellular localization of TMAP during mitosis.

  14. Regulatory Control of the Resolution of DNA Recombination Intermediates during Meiosis and Mitosis

    OpenAIRE

    Matos, Joao; Blanco, Miguel G.; Maslen, Sarah; Skehel, J. Mark; West, Stephen C.

    2011-01-01

    The efficient and timely resolution of DNA recombination intermediates is essential for bipolar chromosome segregation. Here, we show that the specialized chromosome segregation patterns of meiosis and mitosis, which require the coordination of recombination with cell-cycle progression, are achieved by regulating the timing of activation of two crossover-promoting endonucleases. In yeast meiosis, Mus81-Mms4 and Yen1 are controlled by phosphorylation events that lead to their sequential activa...

  15. Efficient Subcellular Targeting to the Cell Nucleus of Quantum Dots Densely Decorated with a Nuclear Localization Sequence Peptide.

    Science.gov (United States)

    Maity, Amit Ranjan; Stepensky, David

    2016-01-27

    Organelle-targeted drug delivery can enhance the efficiency of the intracellularly acting drugs and reduce their toxicity. We generated core-shell type CdSe-ZnS quantum dots (QDs) densely decorated with NLS peptidic targeting residues using a 3-stage decoration approach and investigated their endocytosis and nuclear targeting efficiencies. The diameter of the generated QDs increased following the individual decoration stages (16.3, 18.9, and 21.9 nm), the ζ-potential became less negative (-33.2, -17.5, and -11.9 mV), and characteristic changes appeared in the FTIR spectra following decoration with the linker and NLS peptides. Quantitative analysis of the last decoration stage revealed that 37.9% and 33.2% of the alkyne-modified NLS groups that were added to the reaction mix became covalently attached or adsorbed to the QDs surface, respectively. These numbers correspond to 63.6 and 55.7 peptides conjugated or adsorbed to a single QD (the surface density of 42 and 37 conjugated and adsorbed peptides per 1000 nm(2) of the QDs surface), which is higher than in the majority of previous studies that reported decoration efficiencies of formulations intended for nuclear-targeted drug delivery. QDs decorated with NLS peptides undergo more efficient endocytosis, as compared to other investigated QDs formulations, and accumulated to a higher extent in the cell nucleus or in close vicinity to it (11.9%, 14.6%, and 56.1% of the QDs endocytosed by an average cell for the QD-COOH, QD-azide, and QD-NLS formulations, respectively). We conclude that dense decoration of QDs with NLS residues increased their endocytosis and led to their nuclear targeting (preferential accumulation in the cells nuclei or in close vicinity to them). The experimental system and research tools that were used in this study allow quantitative investigation of the mechanisms that govern the QDs nuclear targeting and their dependence on the formulation properties. These findings will contribute to the

  16. Proportion of collagen type II in the extracellular matrix promotes the differentiation of human adipose-derived mesenchymal stem cells into nucleus pulposus cells.

    Science.gov (United States)

    Tao, Yiqing; Zhou, Xiaopeng; Liu, Dongyu; Li, Hao; Liang, Chengzhen; Li, Fangcai; Chen, Qixin

    2016-01-01

    During degeneration process, the catabolism of collagen type II and anabolism of collagen type I in nucleus pulposus (NP) may influence the bioactivity of transplanted cells. Human adipose-derived mesenchymal stem cells (hADMSCs) were cultured as a micromass or in a series of gradual proportion hydrogels of a mix of collagen types I and II. Cell proliferation and cytotoxicity were detected using CCK-8 and LDH assays respectively. The expression of differentiation-related genes and proteins, including SOX9, aggrecan, collagen type I, and collagen type II, was examined using RT-qPCR and Western blotting. Novel phenotypic genes were also detected by RT-qPCR and western blotting. Alcian blue and dimethylmethylene blue assays were used to investigate sulfate proteoglycan expression, and PI3K/AKT, MAPK/ERK, and Smad signaling pathways were examined by Western blotting. The results showed collagen hydrogels have good biocompatibility, and cell proliferation increased after collagen type II treatment. Expressions of SOX9, aggrecan, and collagen type II were increased in a collagen type II dependent manner. Sulfate proteoglycan synthesis increased in proportion to collagen type II concentration. Only hADMSCs highly expressed NP cell marker KRT19 in collagen type II culture. Additionally, phosphorylated Smad3, which is associated with phosphorylated ERK, was increased after collagen type II-stimulation. The concentration and type of collagen affect hADMSC differentiation into NP cells. Collagen type II significantly ameliorates hADMSC differentiation into NP cells and promotes extracellular matrix synthesis. Therefore, anabolism of collagen type I and catabolism of type II may attenuate the differentiation and biosynthesis of transplanted stem cells. © 2016 International Union of Biochemistry and Molecular Biology.

  17. Role of hypoxia and growth and differentiation factor-5 on differentiation of human mesenchymal stem cells towards intervertebral nucleus pulposus-like cells

    Directory of Open Access Journals (Sweden)

    JV Stoyanov

    2011-06-01

    Full Text Available There is evidence that mesenchymal stem cells (MSCs can differentiate towards an intervertebral disc (IVD-like phenotype. We compared the standard chondrogenic protocol using transforming growth factor beta-1 (TGFß to the effects of hypoxia, growth and differentiation factor-5 (GDF5, and coculture with bovine nucleus pulposus cells (bNPC. The efficacy of molecules recently discovered as possible nucleus pulposus (NP markers to differentiate between chondrogenic and IVD-like differentiation was evaluated. MSCs were isolated from human bone marrow and encapsulated in alginate beads. Beads were cultured in DMEM (control supplemented with TGFß or GDF5 or under indirect coculture with bNPC. All groups were incubated at low (2 % or normal (20 % oxygen tension for 28 days. Hypoxia increased aggrecan and collagen II gene expression in all groups. The hypoxic GDF5 and TGFß groups demonstrated most increased aggrecan and collagen II mRNA levels and glycosaminoglycan accumulation. Collagen I and X were most up-regulated in the TGFß groups. From the NP markers, cytokeratin-19 was expressed to highest extent in the hypoxic GDF5 groups; lowest expression was observed in the TGFß group. Levels of forkhead box F1 were down-regulated by TGFß and up-regulated by coculture with bNPC. Carbonic anhydrase 12 was also down-regulated in the TGFß group and showed highest expression in the GDF5 group cocultured with bNPC under hypoxia. Trends in gene expression regulation were confirmed on the protein level using immunohistochemistry. We conclude that hypoxia and GDF5 may be suitable for directing MSCs towards the IVD-like phenotype.

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

    Science.gov (United States)

    Govindaraghavan, Meera; Lad, Alisha A.

    2014-01-01

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

  19. Proteomic analysis of polyribosomes identifies splicing factors as potential regulators of translation during mitosis.

    Science.gov (United States)

    Aviner, Ranen; Hofmann, Sarah; Elman, Tamar; Shenoy, Anjana; Geiger, Tamar; Elkon, Ran; Ehrlich, Marcelo; Elroy-Stein, Orna

    2017-06-02

    Precise regulation of mRNA translation is critical for proper cell division, but little is known about the factors that mediate it. To identify mRNA-binding proteins that regulate translation during mitosis, we analyzed the composition of polysomes from interphase and mitotic cells using unbiased quantitative mass-spectrometry (LC-MS/MS). We found that mitotic polysomes are enriched with a subset of proteins involved in RNA processing, including alternative splicing and RNA export. To demonstrate that these may indeed be regulators of translation, we focused on heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a test case and confirmed that it is recruited to elongating ribosomes during mitosis. Then, using a combination of pulsed SILAC, metabolic labeling and ribosome profiling, we showed that knockdown of hnRNP C affects both global and transcript-specific translation rates and found that hnRNP C is specifically important for translation of mRNAs that encode ribosomal proteins and translation factors. Taken together, our results demonstrate how proteomic analysis of polysomes can provide insight into translation regulation under various cellular conditions of interest and suggest that hnRNP C facilitates production of translation machinery components during mitosis to provide daughter cells with the ability to efficiently synthesize proteins as they enter G1 phase. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Large Uptake of Titania and Iron Oxide Nanoparticles in the Nucleus of Lung Epithelial Cells as Measured by Raman Imaging and Multivariate Classification

    Science.gov (United States)

    Ahlinder, Linnea; Ekstrand-Hammarström, Barbro; Geladi, Paul; Österlund, Lars

    2013-01-01

    It is a challenging task to characterize the biodistribution of nanoparticles in cells and tissue on a subcellular level. Conventional methods to study the interaction of nanoparticles with living cells rely on labeling techniques that either selectively stain the particles or selectively tag them with tracer molecules. In this work, Raman imaging, a label-free technique that requires no extensive sample preparation, was combined with multivariate classification to quantify the spatial distribution of oxide nanoparticles inside living lung epithelial cells (A549). Cells were exposed to TiO2 (titania) and/or α-FeO(OH) (goethite) nanoparticles at various incubation times (4 or 48 h). Using multivariate classification of hyperspectral Raman data with partial least-squares discriminant analysis, we show that a surprisingly large fraction of spectra, classified as belonging to the cell nucleus, show Raman bands associated with nanoparticles. Up to 40% of spectra from the cell nucleus show Raman bands associated with nanoparticles. Complementary transmission electron microscopy data for thin cell sections qualitatively support the conclusions. PMID:23870252

  1. Human umbilical cord mesenchymal stromal cells exhibit immature nucleus pulposus cell phenotype in a laminin-rich pseudo-three-dimensional culture system.

    Science.gov (United States)

    Chon, Brian H; Lee, Esther J; Jing, Liufang; Setton, Lori A; Chen, Jun

    2013-10-02

    Cell supplementation to the herniated or degenerated intervertebral disc (IVD) is a potential strategy to promote tissue regeneration and slow disc pathology. Human umbilical cord mesenchymal stromal cells (HUCMSCs) - originating from the Wharton's jelly - remain an attractive candidate for such endeavors with their ability to differentiate into multiple lineages. Previously, mesenchymal stem cells (MSCs) have been studied as a potential source for disc tissue regeneration. However, no studies have demonstrated that MSCs can regenerate matrix with unique characteristics matching that of immature nucleus pulposus (NP) tissues of the IVD. In our prior work, immature NP cells were found to express specific laminin isoforms and laminin-binding receptors that may serve as phenotypic markers for evaluating MSC differentiation to NP-like cells. The goal of this study is to evaluate these markers and matrix synthesis for HUCMSCs cultured in a laminin-rich pseudo-three-dimensional culture system. HUCMSCs were seeded on top of Transwell inserts pre-coated with Matrigel™, which contained mainly laminin-111. Cells were cultured under hypoxia environment with three differentiation conditions: NP differentiation media (containing 2.5% Matrigel™ solution to provide for a pseudo-three-dimensional laminin culture system) with no serum, or the same media supplemented with either insulin-like growth factor-1 (IGF-1) or transforming growth factor-β1 (TGF-β1). Cell clustering behavior, matrix production and the expression of NP-specific laminin and laminin-receptors were evaluated at days 1, 7, 13 and 21 of culture. Data show that a pseudo-three-dimensional culture condition (laminin-1 rich) promoted HUCMSC differentiation under no serum conditions. Starting at day 1, HUCMSCs demonstrated a cell clustering morphology similar to that of immature NP cells in situ and that observed for primary immature NP cells within the similar laminin-rich culture system (prior study

  2. The FEAR protein Slk19 restricts Cdc14 phosphatase to the nucleus until the end of anaphase, regulating its participation in mitotic exit in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Ann Marie E Faust

    Full Text Available In Saccharomyces cerevisiae mitosis, the protein Slk19 plays an important role in the initial release of Cdc14 phosphatase from the nucleolus to the nucleus in early anaphase, an event that is critical for proper anaphase progression. A role for Slk19 in later mitotic stages of Cdc14 regulation, however, has not been demonstrated. While investigating the role of Slk19 post-translational modification on Cdc14 regulation, we found that a triple point mutant of SLK19, slk19(3R (three lysine-to-arginine mutations, strongly affects Cdc14 localization during late anaphase and mitotic exit. Using fluorescence live-cell microscopy, we found that, similar to slk19Δ cells, slk19(3R cells exhibit no defect in spindle stability and only a mild defect in spindle elongation dynamics. Unlike slk19Δcells, however, slk19(3R cells exhibit no defect in Cdc14 release from the nucleolus to the nucleus. Instead, slk19(3R cells are defective in the timing of Cdc14 movement from the nucleus to the cytoplasm at the end of anaphase. This mutant has a novel phenotype: slk19(3R causes premature Cdc14 movement to the cytoplasm prior to, rather than concomitant with, spindle disassembly. One consequence of this premature Cdc14 movement is the inappropriate activation of the mitotic exit network, made evident by the fact that slk19(3R partially rescues a mutant of the mitotic exit network kinase Cdc15. In conclusion, in addition to its role in regulating Cdc14 release from the nucleolus to the nucleus, we found that Slk19 is also important for regulating Cdc14 movement from the nucleus to the cytoplasm at the end of anaphase.

  3. Cell proliferation and radiosensitivity of cow lymphocytes in culture

    International Nuclear Information System (INIS)

    Modave, C.; Fabry, L.; Leonard, A.

    1982-01-01

    The harlequin-staining technique has been used to study, after PHA-stimulation, the cell proliferation of cow lymphocytes in culture and to assess the radiosensitivity in first mitosis cells. At the 48 h fixation time, only 34% of the cells are in first mitosis whereas 55% are already in second and 11% in third mitosis. The exposure of cow lymphocytes to 200 rad X-rays result in the production of 16% dicentric chromosomes in first mitosis cells [fr

  4. Bone Morphogenetic Protein-2, but Not Mesenchymal Stromal Cells, Exert Regenerative Effects on Canine and Human Nucleus Pulposus Cells

    NARCIS (Netherlands)

    Bach, Frances C.; Miranda-Bedate, Alberto; Van Heel, Ferdi W M; Riemers, Frank M.; Müller, Margot C M E; Creemers, Laura B.; Ito, Keita; Benz, Karin; Meij, Björn P.; Tryfonidou, Marianna A.

    2017-01-01

    Chronic back pain is related to intervertebral disc (IVD) degeneration and dogs are employed as animal models to develop growth factor- and cell-based regenerative treatments. In this respect, the differential effects of transforming growth factor beta-1 (TGF-β1) and bone morphogenetic protein-2

  5. Bone morphogenetic protein-2, but not mesenchymal stromal cells, exert regenerative effects on canine and human nucleus pulposus cells

    NARCIS (Netherlands)

    Bach, Frances; Miranda-Bedate, Alberto; van Heel, Ferdi; Riemers, Frank; Muller, Margot; Creemers, Laura; Ito, Keita; Benz, Karin; Meij, Björn; Tryfonidou, M

    2017-01-01

    Chronic back pain is related to intervertebral disc (IVD) degeneration and dogs are employed as animal models to develop growth factor- and cell-based regenerative treatments. In this respect, the differential effects of transforming growth factor beta-1 (TGF-β1) and bone morphogenetic protein-2

  6. Bone morphogenetic protein-2, but not mesenchymal stromal cells, exert regenerative effects on Canine and human nucleus pulposus cells

    NARCIS (Netherlands)

    Bach, F.C.; Miranda-Bedate, A.; Van Heel, F.W.M.; Riemers, F.M.; Müller, M.C.M.E.; Creemers, L.B.; Ito, K.; Benz, K.; Meij, B.P.; Tryfonidou, M.A.

    2017-01-01

    Chronic back pain is related to intervertebral disc (IVD) degeneration and dogs are employed as animal models to develop growth factor- and cell-based regenerative treatments. In this respect, the differential effects of transforming growth factor beta-1 (TGF-β1) and bone morphogenetic protein-2

  7. Functional expression of P2 purinoceptors in a primary neuroglial cell culture of the rat arcuate nucleus.

    Science.gov (United States)

    Pollatzek, Eric; Hitzel, Norma; Ott, Daniela; Raisl, Katrin; Reuter, Bärbel; Gerstberger, Rüdiger

    2016-07-07

    The arcuate nucleus (ARC) plays an important role in the hypothalamic control of energy homeostasis. Expression of various purinoceptor subtypes in the rat ARC and physiological studies suggest a modulatory function of P2 receptors within the neuroglial ARC circuitry. A differentiated mixed neuronal and glial microculture was therefore established from postnatal rat ARC, revealing neuronal expression of ARC-specific transmitters involved in food intake regulation (neuropeptide Y (NPY), proopiomelanocortin (POMC), tyrosine hydroxylase (TH)). Some NPYergic neurons cosynthesized TH, while POMC and TH expression proved to be mutually exclusive. Stimulation with the general purinoceptor agonists 2-methylthioadenosine-5'triphosphate (2-MeSATP) and ATP but not the P2X1/P2X3 receptor subtype agonist α,β-methyleneadenosine-5'triphosphate (α,β-meATP) induced intracellular calcium signals in ARC neurons and astrocytes. Some 5-10% each of 2-MeSATP responsive neurons expressed POMC, NYP or TH. Supporting the calcium imaging data, radioligand binding studies to hypothalamic membranes showed high affinity for 2-MeSATP, ATP but not α,β-meATP to displace [α-(35)S]deoxyadenosine-5'thiotriphosphate ([(35)S]dATPαS) from P2 receptors. Repetitive superfusion with equimolar 2-MeSATP allowed categorization of ARC cells into groups with a high or low (LDD) degree of purinoceptor desensitization, the latter allowing further receptor characterization. Calcium imaging experiments performed at 37°C vs. room temperature showed further reduction of desensitization. Agonist-mediated intracellular calcium signals were suppressed in all LDD neurons but only 25% of astrocytes in the absence of extracellular calcium, suggestive of metabotropic P2Y receptor expression in the majority of ARC astrocytes. The highly P2Y1-selective receptor agonists MRS2365 and 2-methylthioadenosine-5'diphosphate (2-MeSADP) activated 75-85% of all 2-MeSATP-responsive ARC astrocytes. Taking into consideration the

  8. Heterologous expression of mammalian Plk1 in Drosophila reveals divergence from Polo during late mitosis

    International Nuclear Information System (INIS)

    Pearson, John; Godinho, Susana A.; Tavares, Alvaro; Glover, David M.

    2006-01-01

    Drosophila Polo kinase is the founder member of a conserved kinase family required for multiple stages of mitosis. We assessed the ability of mouse Polo-like kinase 1 (Plk1) to perform the multiple mitotic functions of Polo kinase, by expressing a Plk1-GFP fusion in Drosophila. Consistent with the previously reported localization of Polo kinase, Plk1-GFP was strongly localized to centrosomes and recruited to the centromeric regions of condensing chromosomes during early mitosis. However, in contrast to a functional Polo-GFP fusion, Plk1-GFP failed to localize to the central spindle midzone in both syncytial embryo mitosis and the conventional mitoses of cellularized embryos and S2 cells. Moreover, unlike endogenous Polo kinase and Polo-GFP, Plk1-GFP failed to associate with the contractile ring. Expression of Plk1-GFP enhanced the lethality of hypomorphic polo mutants and disrupted the organization of the actinomyosin cytoskeleton in a dominant-negative manner. Taken together, our results suggest that endogenous Polo kinase has specific roles in regulating actinomyosin rearrangements during Drosophila mitoses that its mammalian counterpart, Plk1, cannot fulfill. Consistent with this hypothesis, we observed defects in the cortical recruitment of myosin and myosin regulatory light chain in Polo deficient cells

  9. PLK1 Activation in Late G2 Sets Up Commitment to Mitosis.

    Science.gov (United States)

    Gheghiani, Lilia; Loew, Damarys; Lombard, Bérangère; Mansfeld, Jörg; Gavet, Olivier

    2017-06-06

    Commitment to mitosis must be tightly coordinated with DNA replication to preserve genome integrity. While we have previously established that the timely activation of CyclinB1-Cdk1 in late G2 triggers mitotic entry, the upstream regulatory mechanisms remain unclear. Here, we report that Polo-like kinase 1 (Plk1) is required for entry into mitosis during an unperturbed cell cycle and is rapidly activated shortly before CyclinB1-Cdk1. We determine that Plk1 associates with the Cdc25C1 phosphatase and induces its phosphorylation before mitotic entry. Plk1-dependent Cdc25C1 phosphosites are sufficient to promote mitotic entry, even when Plk1 activity is inhibited. Furthermore, we find that activation of Plk1 during G2 relies on CyclinA2-Cdk activity levels. Our findings thus elucidate a critical role for Plk1 in CyclinB1-Cdk1 activation and mitotic entry and outline how CyclinA2-Cdk, an S-promoting factor, poises cells for commitment to mitosis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2009-01-01

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

  11. 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.

  12. Induction of mitosis in the cultured rabbit lens initiated by the addition of insulin to medium KEI-4

    Energy Technology Data Exchange (ETDEWEB)

    Reddan, J R; Unakar, N J; Harding, C V; Bagchi, M; Saldana, G

    1975-01-01

    The epithelium of lenses cultured in KEI-4, a completely defined medium formulated with specific reference to the biochemistry and physiology of the rabbit lens, exhibits a pattern of cell division similar to that noted for the organ in situ. Initial fluctuations in mitotic activity occurred in the area of the germinative zone during the first 24 hr of culture. Mitosis decreased at 1 hr, was extremely low at 3 hr and returned to values comparable for lens in vivo by 22 hr. The precipitous drop in mitosis noted at 3 hr is in part attributable to the isolation of the lens from adjoining tissue. The addition of insulin to KEI-4 triggers a parasynchronous burst of DNA synthesis throughout the central lens epithelium. The activation requires the intact hormone; neither proinsulin nor the A and/or B chains of insulin, nor glucagon nor zinc chloride can initiate mitosis. The gamma-globulin-rich fraction of rabbit serum can also stimulate mitosis. The addition of dibutyryl adenosine 3':5' cyclic monophosphate (DBeAMP) plus theophylline to KEI-4-insulin inhibits mitosis and prevents the cells from entering the synthetic phase of the cell cycle. Theophylline alone or DBeAMP alone brings about a 90 percent reduction in the insulin-induced mitotic responses. Lenses exposed to insulin show a marked increase in RNA synthesis and also exhibit an increased binding of tritiated actinomycin D at 1 and 3 hr of culture relative to KEI-4 controls. The hormone apparently activates the genome including those genes governing cell division. The system is amenable for long-term culture of the mammalian lens and since the constituents of the medium are known it should be possible to determine the factor(s) in the medium which, in conjunction with insulin, are needed for the induction of cell division.

  13. 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

  14. Icariin Prevents H2O2-Induced Apoptosis via the PI3K/Akt Pathway in Rat Nucleus Pulposus Intervertebral Disc Cells.

    Science.gov (United States)

    Deng, Xiangyu; Chen, Sheng; Zheng, Dong; Shao, Zengwu; Liang, Hang; Hu, Hongzhi

    2017-01-01

    Icariin is a prenylated flavonol glycoside derived from the Chinese herb Epimedium sagittatum. This study investigated the mechanism by which icariin prevents H 2 O 2 -induced apoptosis in rat nucleus pulposus (NP) cells. NP cells were isolated from the rat intervertebral disc and they were divided into five groups after 3 passages: (A) blank control; (B) 200  μ M H 2 O 2 ; (C) 200  μ M H 2 O 2 + 20  μ M icariin; (D) 20  μ M icariin + 200  μ M H 2 O 2 + 25  μ M LY294002; (E) 200  μ M H 2 O 2 + 25  μ M LY294002. LY294002 is a selective inhibitor of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. NP cell viability, apoptosis rate, intracellular reactive oxygen species levels, and the expression of AKT, p-AKT, p53, Bcl-2, Bax, caspase-3 were estimated. The results show that, compared with the control group, H 2 O 2 significantly increased NP cell apoptosis and the level of intracellular ROS. Icariin pretreatment significantly decreased H 2 O 2 -induced apoptosis and intracellular ROS and upregulated p-Akt and BCL-2 and downregulated caspase-3 and Bax. LY294002 abolished the protective effects of icariin. Our results show that icariin can attenuate H2O2-induced apoptosis in rat nucleus pulposus cells and PI3K/AKT pathway is at least partly included in this protection effect.

  15. IMPACT OF BEP OR CARBOPLATIN CHEMOTHERAPY ON TESTICULAR FUNCTION AND SPERM NUCLEUS OF SUBJECTS WITH TESTICULAR GERM CELL TUMOR

    Directory of Open Access Journals (Sweden)

    Marco eGhezzi

    2016-05-01

    Full Text Available Young males have testicular germ cells tumours (TGCT as the most common malignancy and its incidence is increasing in several countries. Besides unilateral orchiectomy (UO, the treatment of TGCT may include surveillance, radiotherapy or chemotherapy (CT, basing on tumour histology and stage of disease. It is well known that both radio and CT may have negative effects on testicular function, affecting spermatogenesis and sex hormones. Many reports investigated these aspects in patients treated with bleomycin, etoposide and cisplatin (BEP, after UO. In contrast no data are available on the side effects of carboplatin treatment in these patients. We included in this study 212 consecutive subjects who undergone to sperm banking at our Andrology and Human Reproduction Unit after UO for TGCT. Hundred subjects were further treated with one or more BEP cycles (BEP-group, 54 with carboplatin (Carb group and 58 were just surveilled (S-group. All patients were evaluated for seminal parameters, sperm aneuploidy, sperm DNA, sex hormones, volume of the residual testis at baseline (T0 and after 12 (T1 and 24 months (T2 from UO or end of CT. Seminal parameters, sperm aneuploidies, DNA status, gonadic hormones and testicular volume at baseline were not different between groups. At T1 we observed a significant reduction of sperm concentration and sperm count in the BEP group versus baseline and versus both Carb and S- group. A significant increase of sperm aneuploidies was present at T1 in the BEP group. Similarly, the same group at 1 had altered sperm DNA integrity and fragmentation compared with baseline, S group and Carb group. These alterations were persistent after two years from the end of BEP treatment. Despite a slight improvement at T2, the BEP group had still higher percentages of sperm aneuploidies than other groups. No impairment of sperm aneuploidies and DNA status were observed in the Carb group both after one and two years from the end of treatment

  16. The importance of G1/S-border and mitosis in the fixation

    International Nuclear Information System (INIS)

    Iliakis, G.; Nuesse, M.

    1983-01-01

    The ability of synchronized Ehrlich ascites tumour cells to repair PLD was measured by introducing delays in their progression through the cell cycle either in the same phase as that where the irradiation was given or in a subsequent phase. Cells were incubated for this purpose either in balanced salt solution which nonspecifically delayed progression in all cell cycle phases or with 0.5 μg/ml aphidicolin which delayed cells in S-phase. Cells which had been delayed in their progression through the cell cycle were able to repair PLD irrespective of the phase at which they were held. In cases where the delay in the progression through the cell cycle was introduced in a phase subsequent to that of the exposure to irradiation, repair of PLD was observed only if the cells had not passed the G1/S-border or mitosis. Based on these results, the importance of G1/S-border and mitosis in the fixation of PLD is suggested. (orig.)

  17. Exosomes as potential alternatives to stem cell therapy for intervertebral disc degeneration: in-vitro study on exosomes in interaction of nucleus pulposus cells and bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Lu, Kang; Li, Hai-Yin; Yang, Kuang; Wu, Jun-Long; Cai, Xiao-Wei; Zhou, Yue; Li, Chang-Qing

    2017-05-10

    The stem cell-based therapies for intervertebral disc degeneration have been widely studied. However, the mechanisms of mesenchymal stem cells interacting with intervertebral disc cells, such as nucleus pulposus cells (NPCs), remain unknown. Exosomes as a vital paracrine mechanism in cell-cell communication have been highly focused on. The purpose of this study was to detect the role of exosomes derived from bone marrow mesenchymal stem cells (BM-MSCs) and NPCs in their interaction with corresponding cells. The exosomes secreted by BM-MSCs and NPCs were purified by differential centrifugation and identified by transmission electron microscope and immunoblot analysis of exosomal marker proteins. Fluorescence confocal microscopy was used to examine the uptake of exosomes by recipient cells. The effects of NPC exosomes on the migration and differentiation of BM-MSCs were determined by transwell migration assays and quantitative RT-PCR analysis of NPC phenotypic genes. Western blot analysis was performed to examine proteins such as aggrecan, sox-9, collagen II and hif-1α in the induced BM-MSCs. Proliferation and the gene expression profile of NPCs induced by BM-MSC exosomes were measured by Cell Counting Kit-8 and qRT-PCR analysis, respectively. Both the NPCs and BM-MSCs secreted exosomes, and these exosomes underwent uptake by the corresponding cells. NPC-derived exosomes promoted BM-MSC migration and induced BM-MSC differentiation to a nucleus pulposus-like phenotype. BM-MSC-derived exosomes promoted NPC proliferation and healthier extracellular matrix production in the degenerate NPCs. Our study indicates that the exosomes act as an important vehicle in information exchange between BM-MSCs and NPCs. Given a variety of functions and multiple advantages, exosomes alone or loaded with specific genes and drugs would be an appropriate option in a cell-free therapy strategy for intervertebral disc degeneration.

  18. 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.

  19. The innate immunity adaptor SARM translocates to the nucleus to stabilize lamins and prevent DNA fragmentation in response to pro-apoptotic signaling.

    Directory of Open Access Journals (Sweden)

    Chad R Sethman

    Full Text Available Sterile alpha and armadillo-motif containing protein (SARM, a highly conserved and structurally unique member of the MyD88 family of Toll-like receptor adaptors, plays an important role in innate immunity signaling and apoptosis. Its exact mechanism of intracellular action remains unclear. Apoptosis is an ancient and ubiquitous process of programmed cell death that results in disruption of the nuclear lamina and, ultimately, dismantling of the nucleus. In addition to supporting the nuclear membrane, lamins serve important roles in chromatin organization, epigenetic regulation, transcription, nuclear transport, and mitosis. Mutations and other damage that destabilize nuclear lamins (laminopathies underlie a number of intractable human diseases. Here, we report that SARM translocates to the nucleus of human embryonic kidney cells by using its amino-terminal Armadillo repeat region. Within the nucleus, SARM forms a previously unreported lattice akin to the nuclear lamina scaffold. Moreover, we show that SARM protects lamins from apoptotic degradation and reduces internucleosomal DNA fragmentation in response to signaling induced by the proinflammatory cytokine Tumor Necrosis Factor alpha. These findings indicate an important link between the innate immunity adaptor SARM and stabilization of nuclear lamins during inflammation-driven apoptosis in human cells.

  20. 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.

  1. Metabolism of the intervertebral disc: effects of low levels of oxygen, glucose, and pH on rates of energy metabolism of bovine nucleus pulposus cells.

    Science.gov (United States)

    Bibby, Susan R S; Jones, Deborah A; Ripley, Ruth M; Urban, Jill P G

    2005-03-01

    In vitro measurements of metabolic rates of isolated bovine nucleus pulposus cells at varying levels of oxygen, glucose, and pH. To obtain quantitative information on the interactions between oxygen and glucose concentrations and pH, and the rates of oxygen and glucose consumption and lactic acid production, for disc nucleus cells. Disc cells depend on diffusion from blood vessels at the disc margins for supply of nutrients. Loss of supply is thought to lead to disc degeneration, but how loss of supply affects nutrient concentrations in the disc is not known; nutrient concentrations within discs can normally only be calculated, because concentration measurements are invasive. However, realistic predictions cannot be made until there are data from measurements of metabolic rates at conditions found in the disc in vivo, i.e., at low levels of oxygen, glucose, and pH. A metabolism chamber was designed to allow simultaneous recording of oxygen and glucose concentrations and of pH. These concentrations were measured electrochemically with custom-built glucose and oxygen sensors; lactic acid was measured biochemically. Bovine nucleus pulposus cells were isolated and inserted into the chamber, and simultaneous rates of oxygen and glucose consumption and of lactic acid production were measured over a range of glucose, oxygen, and pH levels. There were strong interactions between rates of metabolism and oxygen consumption and pH. At atmospheric oxygen levels, oxygen consumption rate at pH 6.2 was 32% of that at pH 7.4. The rate fell by 60% as oxygen concentration was decreased from 21 to 5% at pH 7.4, but only by 20% at pH 6.2. Similar interactions were seen for lactic acid production and glucose consumption rates; we found that glycolysis rates fell at low oxygen and glucose concentrations and low pH. Equations were derived that satisfactorily predict the effect of nutrient and metabolite concentrations on rates of lactic acid production rate and oxygen consumption. Disc

  2. Self-organization of intracellular gradients during mitosis

    Directory of Open Access Journals (Sweden)

    Fuller Brian G

    2010-01-01

    Full Text Available Abstract Gradients are used in a number of biological systems to transmit spatial information over a range of distances. The best studied are morphogen gradients where information is transmitted over many cell lengths. Smaller mitotic gradients reflect the need to organize several distinct events along the length of the mitotic spindle. The intracellular gradients that characterize mitosis are emerging as important regulatory paradigms. Intracellular gradients utilize intrinsic auto-regulatory feedback loops and diffusion to establish stable regions of activity within the mitotic cytosol. We review three recently described intracellular mitotic gradients. The Ran GTP gradient with its elaborate cascade of nuclear transport receptors and cargoes is the best characterized, yet the dynamics underlying the robust gradient of Ran-GTP have received little attention. Gradients of phosphorylation have been observed on Aurora B kinase substrates both before and after anaphase onset. In both instances the phosphorylation gradient appears to result from a soluble gradient of Aurora B kinase activity. Regulatory properties that support gradient formation are highlighted. Intracellular activity gradients that regulate localized mitotic events bare several hallmarks of self-organizing biologic systems that designate spatial information during pattern formation. Intracellular pattern formation represents a new paradigm in mitotic regulation.

  3. Expression of SPIG1 reveals development of a retinal ganglion cell subtype projecting to the medial terminal nucleus in the mouse.

    Directory of Open Access Journals (Sweden)

    Keisuke Yonehara

    Full Text Available Visual information is transmitted to the brain by roughly a dozen distinct types of retinal ganglion cells (RGCs defined by a characteristic morphology, physiology, and central projections. However, our understanding about how these parallel pathways develop is still in its infancy, because few molecular markers corresponding to individual RGC types are available. Previously, we reported a secretory protein, SPIG1 (clone name; D/Bsp120I #1, preferentially expressed in the dorsal region in the developing chick retina. Here, we generated knock-in mice to visualize SPIG1-expressing cells with green fluorescent protein. We found that the mouse retina is subdivided into two distinct domains for SPIG1 expression and SPIG1 effectively marks a unique subtype of the retinal ganglion cells during the neonatal period. SPIG1-positive RGCs in the dorsotemporal domain project to the dorsal lateral geniculate nucleus (dLGN, superior colliculus, and accessory optic system (AOS. In contrast, in the remaining region, here named the pan-ventronasal domain, SPIG1-positive cells form a regular mosaic and project exclusively to the medial terminal nucleus (MTN of the AOS that mediates the optokinetic nystagmus as early as P1. Their dendrites costratify with ON cholinergic amacrine strata in the inner plexiform layer as early as P3. These findings suggest that these SPIG1-positive cells are the ON direction selective ganglion cells (DSGCs. Moreover, the MTN-projecting cells in the pan-ventronasal domain are apparently composed of two distinct but interdependent regular mosaics depending on the presence or absence of SPIG1, indicating that they comprise two functionally distinct subtypes of the ON DSGCs. The formation of the regular mosaic appears to be commenced at the end of the prenatal stage and completed through the peak period of the cell death at P6. SPIG1 will thus serve as a useful molecular marker for future studies on the development and function of ON DSGCs.

  4. Static compression down-regulates N-cadherin expression and facilitates loss of cell phenotype of nucleus pulposus cells in a disc perfusion culture.

    Science.gov (United States)

    Zhou, Haibo; Shi, Jianmin; Zhang, Chao; Li, Pei

    2018-02-28

    Mechanical compression often induces degenerative changes of disc nucleus pulposus (NP) tissue. It has been indicated that N-cadherin (N-CDH)-mediated signaling helps to preserve the NP cell phenotype. However, N-CDH expression and the resulting NP-specific phenotype alteration under the static compression and dynamic compression remain unclear. To study the effects of static compression and dynamic compression on N-CDH expression and NP-specific phenotype in an in vitro disc organ culture. Porcine discs were organ cultured in a self-developed mechanically active bioreactor for 7 days and subjected to static or dynamic compression (0.4 MPa for 2 h once per day). The noncompressed discs were used as controls. Compared with the dynamic compression, static compression significantly down-regulated the expression of N-CDH and NP-specific markers (laminin, brachyury, and keratin 19); decreased the Alcian Blue staining intensity, glycosaminoglycan and hydroxyproline contents; and declined the matrix macromolecule (aggrecan and collagen II) expression. Compared with the dynamic compression, static compression causes N-CDH down-regulation, loss of NP-specific phenotype, and the resulting decrease in NP matrix synthesis. © 2018 The Author(s).

  5. Eukaryotic translation initiation factor 5A induces apoptosis in colon cancer cells and associates with the nucleus in response to tumour necrosis factor α signalling

    International Nuclear Information System (INIS)

    Taylor, Catherine A.; Sun Zhong; Cliche, Dominic O.; Ming, Hong; Eshaque, Bithi; Jin Songmu; Hopkins, Marianne T.; Thai, Boun; Thompson, John E.

    2007-01-01

    Eukaryotic translation initiation factor 5A (eIF5A) is thought to function as a nucleocytoplasmic shuttle protein. There are reports of its involvement in cell proliferation, and more recently it has also been implicated in the regulation of apoptosis. In the present study, we examined the effects of eIF5A over-expression on apoptosis and of siRNA-mediated suppression of eIF5A on expression of the tumour suppressor protein, p53. Over-expression of either eIF5A or a mutant of eIF5A incapable of being hypusinated was found to induce apoptosis in colon carcinoma cells. Our results also indicate that eIF5A is required for expression of p53 following the induction of apoptosis by treatment with Actinomycin D. Depiction of eIF5A localization by indirect immunofluorescence has indicated, for the first time, that the protein is rapidly translocated from the cytoplasm to the nucleus by death receptor activation or following treatment with Actinomycin D. These findings collectively indicate that unhypusinated eIF5A may have pro-apoptotic functions and that eIF5A is rapidly translocated to the nucleus following the induction of apoptotic cell death

  6. Poly(ADP-ribosyl)ation is recognized by ECT2 during mitosis.

    Science.gov (United States)

    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.

  7. 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.

  8. Acid-Sensing Ion Channel 1a Regulates Fate of Rat Nucleus Pulposus Cells in Acid Stimulus Through Endoplasmic Reticulum Stress

    Directory of Open Access Journals (Sweden)

    Xie Zhi-Yang

    2018-02-01

    Full Text Available Acid-sensing ion channel 1a (ASIC1a participates in human intervertebral disc degeneration (IVDD and regulates the destiny of nucleus pulposus cells (NPCs in acid stimulus. However, the mechanism of ASIC1a activation and its downstream pathway remain unclear. Endoplasmic reticulum (ER stress also participates in the acid-induced apoptosis of NPCs. The main purpose of this study was to investigate whether there is any connection between ASIC1a and ER stress in an acid-induced nucleus pulposus degeneration model. The IVDs of Sprague-Dawley rats were stained by immunohistochemical staining