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Sample records for cell nucleus mitosis

  1. Mitosis.

    Science.gov (United States)

    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…

  2. Chlorpromazine inhibits mitosis of mammalian cells.

    Science.gov (United States)

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

    1983-09-01

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

  3. Mitosis.

    Science.gov (United States)

    McIntosh, J Richard

    2016-01-01

    SUMMARYAll eukaryotic cells prepare for cell division by forming a "mitotic spindle"-a bipolar machine made from microtubules (MTs) and many associated proteins. This device organizes the already duplicated DNA so one copy of each chromosome attaches to each end of the spindle. Both formation and function of the spindle require controlled MT dynamics, as well as the actions of multiple motor enzymes. Spindle-driven motions separate the duplicated chromosomes into two distinct sets that are then moved toward opposite ends of the cell. The two cells that subsequently form by cytokinesis, therefore, contain all the genes needed to grow and divide again. PMID:27587616

  4. Role of liver functions on liver cell mitosis

    Directory of Open Access Journals (Sweden)

    Takata,Tameyuki

    1974-06-01

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

  5. Cohesin is needed for bipolar mitosis in human cells.

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    Díaz-Martínez, Laura A; Beauchene, Nicole A; Furniss, Katherine; Esponda, Pedro; Giménez-Abián, Juan F; Clarke, Duncan J

    2010-05-01

    Multi-polar mitosis is strongly linked with aggressive cancers and it is a histological diagnostic of tumor-grade. However, factors that cause chromosomes to segregate to more than two spindle poles are not well understood. Here we show that cohesins Rad21, Smc1 and Smc3 are required for bipolar mitosis in human cells. After Rad21 depletion, chromosomes align at the metaphase plate and bipolar spindles assemble in most cases, but in anaphase the separated chromatids segregate to multiple poles. Time-lapse microscopy revealed that the spindle poles often become split in Rad21-depleted metaphase cells. Interestingly, exogenous expression of non-cleavable Rad21 results in multi-polar anaphase. Since cohesins are present at the spindle poles in mitosis, these data are consistent with a non-chromosomal function of cohesin. PMID:20436271

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

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

    Science.gov (United States)

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

    2014-12-01

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

  8. Dissecting mitosis by RNAi in Drosophila tissue culture cells

    Directory of Open Access Journals (Sweden)

    Maiato Helder

    2003-01-01

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

  9. Rheology of the Active Cell Cortex in Mitosis.

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    Fischer-Friedrich, Elisabeth; Toyoda, Yusuke; Cattin, Cedric J; Müller, Daniel J; Hyman, Anthony A; Jülicher, Frank

    2016-08-01

    The cell cortex is a key structure for the regulation of cell shape and tissue organization. To reach a better understanding of the mechanics and dynamics of the cortex, we study here HeLa cells in mitosis as a simple model system. In our assay, single rounded cells are dynamically compressed between two parallel plates. Our measurements indicate that the cortical layer is the dominant mechanical element in mitosis as opposed to the cytoplasmic interior. To characterize the time-dependent rheological response, we extract a complex elastic modulus that characterizes the resistance of the cortex against area dilation. In this way, we present a rheological characterization of the cortical actomyosin network in the linear regime. Furthermore, we investigate the influence of actin cross linkers and the impact of active prestress on rheological behavior. Notably, we find that cell mechanics values in mitosis are captured by a simple rheological model characterized by a single timescale on the order of 10 s, which marks the onset of fluidity in the system. PMID:27508442

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

    Science.gov (United States)

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

    2014-12-01

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

  11. Presenting Mitosis

    Science.gov (United States)

    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…

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

    Science.gov (United States)

    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.

  13. 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

    2016-01-01

    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.

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

    Science.gov (United States)

    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.

  15. Nuclear removal during terminal lens fiber cell differentiation requires CDK1 activity: appropriating mitosis-related nuclear disassembly.

    Science.gov (United States)

    Chaffee, Blake R; Shang, Fu; Chang, Min-Lee; Clement, Tracy M; Eddy, Edward M; Wagner, Brad D; Nakahara, Masaki; Nagata, Shigekazu; Robinson, Michael L; Taylor, Allen

    2014-09-01

    Lens epithelial cells and early lens fiber cells contain the typical complement of intracellular organelles. However, as lens fiber cells mature they must destroy their organelles, including nuclei, in a process that has remained enigmatic for over a century, but which is crucial for the formation of the organelle-free zone in the center of the lens that assures clarity and function to transmit light. Nuclear degradation in lens fiber cells requires the nuclease DNase IIβ (DLAD) but the mechanism by which DLAD gains access to nuclear DNA remains unknown. In eukaryotic cells, cyclin-dependent kinase 1 (CDK1), in combination with either activator cyclins A or B, stimulates mitotic entry, in part, by phosphorylating the nuclear lamin proteins leading to the disassembly of the nuclear lamina and subsequent nuclear envelope breakdown. Although most post-mitotic cells lack CDK1 and cyclins, lens fiber cells maintain these proteins. Here, we show that loss of CDK1 from the lens inhibited the phosphorylation of nuclear lamins A and C, prevented the entry of DLAD into the nucleus, and resulted in abnormal retention of nuclei. In the presence of CDK1, a single focus of the phosphonuclear mitotic apparatus is observed, but it is not focused in CDK1-deficient lenses. CDK1 deficiency inhibited mitosis, but did not prevent DNA replication, resulting in an overall reduction of lens epithelial cells, with the remaining cells possessing an abnormally large nucleus. These observations suggest that CDK1-dependent phosphorylations required for the initiation of nuclear membrane disassembly during mitosis are adapted for removal of nuclei during fiber cell differentiation.

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

    Science.gov (United States)

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

    2015-01-01

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

  17. Chfr acts with the p38 stress kinases to block entry to mitosis in mammalian cells

    OpenAIRE

    Matsusaka, Takahiro; Pines, Jonathon

    2004-01-01

    Entry into mitosis in vertebrate cells is guarded by a checkpoint that can be activated by a variety of insults, including chromosomal damage and disrupting microtubules (Rieder, C.L., and R.W. Cole. 1998. J. Cell Biol. 142:1013–1022; Rieder, C.L., and R.W. Cole. 2000. Curr. Biol. 10:1067–1070). This checkpoint acts at the end of interphase to delay cells from entering mitosis, causing cells in prophase to decondense their chromosomes and return to G2 phase. Here, we show that in response to ...

  18. 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

    2015-05-01

    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.

  19. The Ability to Survive Mitosis in the Presence of Microtubule Poisons Differs Significantly Between Human Nontransformed (RPE-1) and Cancer (U2OS, HeLa) Cells

    OpenAIRE

    Brito, Daniela A; Rieder, Conly L.

    2009-01-01

    We used live cell imaging to compare the fate of human nontransformed (RPE-1) and cancer (HeLa, U2OS) cells as they entered mitosis in nocodazole or taxol. In the same field, and in either drug, a cell in all lines could die in mitosis, exit mitosis and die within 10 h, or exit mitosis and survive ≥10 h. Relative to RPE-1 cells, significantly fewer HeLa or U2OS cells survived mitosis or remained viable after mitosis: in nocodazole concentrations that inhibit spindle microtubule assembly, or i...

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

    Science.gov (United States)

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

    2015-11-23

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

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

    OpenAIRE

    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 heterochromatin pattern is transmitted after mitosis. We additionally tested the similarity of chromatin arrangement in sister interphase nuclei. We noticed a very active cell rotation during inte...

  2. 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...

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  5. 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...

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

    Science.gov (United States)

    Sazer, Shelley; Lynch, Michael; Needleman, Daniel

    2014-11-17

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

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

    Science.gov (United States)

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

    2016-02-25

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

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

    Science.gov (United States)

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

    2016-02-25

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

  9. 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)

  10. Mitosis in diatoms: rediscovering an old model for cell division.

    Science.gov (United States)

    De Martino, Alessandra; Amato, Alberto; Bowler, Chris

    2009-08-01

    Diatoms are important protists that generate one fifth of the oxygen produced annually on earth. These aquatic organisms likely derived from a secondary endosymbiosis event, and they display peculiar genomic and structural features that reflect their chimeric origin. Diatoms were one of the first models of cell division and these early studies revealed a range of interesting features including a unique acentriolar microtubule-organising centre. Unfortunately, almost nothing is known at the molecular level, in contrast to the advances in other experimental organisms. Recently the full genome sequences of two diatoms have been annotated and molecular tools have been developed. These resources offer new possibilities to re-investigate the mechanisms of cell division in diatoms by recruiting information from more intensively studied organisms. A renaissance of the topic is further justified by the current interest in diatoms as a source of biofuels and for understanding massive diatom proliferation events in response to environmental stimuli. PMID:19572334

  11. 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

    2016-08-01

    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. PMID:27260975

  12. 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.

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

    Science.gov (United States)

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

    2015-11-23

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

  14. The fate of chrysotile-induced multipolar mitosis and aneuploid population in cultured lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Beatriz de Araujo Cortez

    Full Text Available Chrysotile is one of the six types of asbestos, and it is the only one that can still be commercialized in many countries. Exposure to other types of asbestos has been associated with serious diseases, such as lung carcinomas and pleural mesotheliomas. The association of chrysotile exposure with disease is controversial. However, in vitro studies show the mutagenic potential of chrysotile, which can induce DNA and cell damage. The present work aimed to analyze alterations in lung small cell carcinoma cultures after 48 h of chrysotile exposure, followed by 2, 4 and 8 days of recovery in fiber-free culture medium. Some alterations, such as aneuploid cell formation, increased number of cells in G2/M phase and cells in multipolar mitosis were observed even after 8 days of recovery. The presence of chrysotile fibers in the cell cultures was detected and cell morphology was observed by laser scanning confocal microscopy. After 4 and 8 days of recovery, only a few chrysotile fragments were present in some cells, and the cellular morphology was similar to that of control cells. Cells transfected with the GFP-tagged α-tubulin plasmid were treated with chrysotile for 24 or 48 h and cells in multipolar mitosis were observed by time-lapse microscopy. Fates of these cells were established: retention in metaphase, cell death, progression through M phase generating more than two daughter cells or cell fusion during telophase or cytokinesis. Some of them were related to the formation of aneuploid cells and cells with abnormal number of centrosomes.

  15. Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis.

    Science.gov (United States)

    Al Nakouzi, Nader; Wang, Chris Kedong; Beraldi, Eliana; Jager, Wolfgang; Ettinger, Susan; Fazli, Ladan; Nappi, Lucia; Bishop, Jennifer; Zhang, Fan; Chauchereau, Anne; Loriot, Yohann; Gleave, Martin

    2016-01-01

    Clusterin (CLU) is a stress-activated molecular chaperone that confers treatment resistance to taxanes when highly expressed. While CLU inhibition potentiates activity of taxanes and other anti-cancer therapies in preclinical models, progression to treatment-resistant disease still occurs implicating additional compensatory survival mechanisms. Taxanes are believed to selectively target cells in mitosis, a complex mechanism controlled in part by balancing antagonistic roles of Cdc25C and Wee1 in mitosis progression. Our data indicate that CLU silencing induces a constitutive activation of Cdc25C, which delays mitotic exit and hence sensitizes cancer cells to mitotic-targeting agents such as taxanes. Unchecked Cdc25C activation leads to mitotic catastrophe and cell death unless cells up-regulate protective mechanisms mediated through the cell cycle regulators Wee1 and Cdk1. In this study, we show that CLU silencing induces a constitutive activation of Cdc25C via the phosphatase PP2A leading to relief of negative feedback inhibition and activation of Wee1-Cdk1 to promote survival and limit therapeutic efficacy. Simultaneous inhibition of CLU-regulated cell cycle effector Wee1 may improve synergistic responses of biologically rational combinatorial regimens using taxanes and CLU inhibitors. PMID:27198502

  16. Chronic morphine induces premature mitosis of proliferating cells in the adult mouse subgranular zone.

    Science.gov (United States)

    Mandyam, Chitra D; Norris, Rebekah D; Eisch, Amelia J

    2004-06-15

    The birth of cells with neurogenic potential in the adult brain is assessed commonly by detection of exogenous S phase markers, such as bromodeoxyuridine (BrdU). Analysis of other phases of the cell cycle, however, can provide insight into how external factors, such as opiates, influence the cycling of newly born cells. To this end, we examined the expression of two endogenous cell cycle markers in relation to BrdU: proliferating cell nuclear antigen (PCNA) and phosphorylated histone H3 (pHisH3). Two hours after one intraperitoneal BrdU injection, BrdU-, PCNA-, and pHisH3-immunoreactive (IR) cells exhibited similar distribution in the adult mouse subgranular zone (SGZ). Quantitative analysis within the SGZ revealed a relative abundance of cells labeled for PCNA > BrdU > pHisH3. Similar to our reports in rat SGZ, chronic morphine treatment decreased BrdU- and PCNA-IR cells in mouse SGZ by 28 and 38%, respectively. We also show that pHisH3-IR cells are influenced by chronic morphine to a greater extent (58% decrease) than are BrdU- or PCNA-IR cells. Cell cycle phase analysis of SGZ BrdU-IR cells using triple labeling for BrdU, PCNA, and pHisH3 revealed premature mitosis in chronic morphine-treated mice. These results suggest that morphine-treated mice have a shorter Gap2/mitosis (G(2)/M) phase when compared to sham-treated mice. These findings demonstrate the power of using a combination of exogenous and endogenous cell cycle markers and nuclear morphology to track proliferating cells through different phases of the cell cycle and to reveal the regulation of cell cycle phase by chronic morphine. PMID:15160390

  17. 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…

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

    Institute of Scientific and Technical Information of China (English)

    LI XiaoFang; WARD Tarsha; YAO XueBiao; WU JiaRui

    2009-01-01

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

  19. Dual roles of Notch in regulation of apically restricted mitosis and apicobasal polarity of neuroepithelial cells.

    Science.gov (United States)

    Ohata, Shinya; Aoki, Ryo; Kinoshita, Shigeharu; Yamaguchi, Masahiro; Tsuruoka-Kinoshita, Sachiko; Tanaka, Hideomi; Wada, Hironori; Watabe, Shugo; Tsuboi, Takashi; Masai, Ichiro; Okamoto, Hitoshi

    2011-01-27

    How the mitosis of neuroepithelial stem cells is restricted to the apical ventricular area remains unclear. In zebrafish, the mosaic eyes(rw306) (moe/epb41l5(rw306)) mutation disrupts the interaction between the putative adaptor protein Moe and the apicobasal polarity regulator Crumbs (Crb), and impairs the maintenance of neuroepithelial apicobasal polarity. While Crb interacts directly with Notch and inhibits its activity, Moe reverses this inhibition. In the moe(rw306) hindbrain, Notch activity is significantly reduced, and the number of cells that proliferate basally away from the apical area is increased. Surprisingly, activation of Notch in the moe(rw306) mutant rescues not only the basally localized proliferation but also the aberrant neuroepithelial apicobasal polarity. We present evidence that the Crb⋅Moe complex and Notch play key roles in a positive feedback loop to maintain the apicobasal polarity and the apical-high basal-low gradient of Notch activity in neuroepithelial cells, both of which are essential for their apically restricted mitosis. PMID:21262462

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

    Science.gov (United States)

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

    2015-02-19

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

  1. Actomyosin contractility rotates the cell nucleus

    CERN Document Server

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

    2013-01-01

    The nucleus of the eukaryotic cell functions amidst active cytoskeletal filaments, but its response to the stresses carried by these filaments is largely unexplored. We report here the results of studies of the translational and rotational dynamics of the nuclei of single fibroblast cells, with the effects of cell migration suppressed by plating onto fibronectin-coated micro-fabricated patterns. Patterns of the same area but different shapes and/or aspect ratio were used to study the effect of cell geometry on the dynamics. On circles, squares and equilateral triangles, the nucleus undergoes persistent rotational motion, while on high-aspect-ratio rectangles of the same area it moves only back and forth. The circle and the triangle showed respectively the largest and the smallest angular speed. We show that our observations can be understood through a hydrodynamic approach in which the nucleus is treated as a highly viscous inclusion residing in a less viscous fluid of orientable filaments endowed with active...

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

    Science.gov (United States)

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

    2014-11-12

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

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

    Directory of Open Access Journals (Sweden)

    Bridget E.L. Ostrem

    2014-08-01

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

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

    Science.gov (United States)

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

    2015-08-01

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

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

    Institute of Scientific and Technical Information of China (English)

    XUSHIXIONG(SYZEE); CHUNGUILI; CHENGZHU

    1993-01-01

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

  6. HDAC3 controls gap 2/mitosis progression in adult neural stem/progenitor cells by regulating CDK1 levels

    OpenAIRE

    Jiang, Yindi; Hsieh, Jenny

    2014-01-01

    Cell cycle regulation is one of the most fundamental mechanisms to control various biological processes, including the proliferation of neural stem/progenitor cells (NSPCs) in adult mouse brain. This study shows that histone deacetylase 3 (HDAC3), a well-studied epigenetic factor, is required for the proliferation of neural stem cells. We also demonstrate that HDAC3 controls gap 2 and mitosis phase of cell cycle through stabilization of cell cycle protein cyclin-dependent kinase 1. These find...

  7. Getting In and Out of Mitosis*

    OpenAIRE

    Tim Hunt

    2011-01-01

    (Excerpt) Mitosis is the process of one cell dividing into two replicate cells. This process is a universal one, occurring throughout the phyla. Mitosis is a fundamental process. Without it, life ceases to exist.

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

    Science.gov (United States)

    Ogura, Yosuke; Sasakura, Yasunori

    2016-04-18

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

  9. Physical role for the nucleus in cell migration.

    Science.gov (United States)

    Fruleux, Antoine; Hawkins, Rhoda J

    2016-09-14

    Cell migration is important for the function of many eukaryotic cells. Recently the nucleus has been shown to play an important role in cell motility. After giving an overview of cell motility mechanisms we review what is currently known about the mechanical properties of the nucleus and the connections between it and the cytoskeleton. We also discuss connections to the extracellular matrix and mechanotransduction. We identify key physical roles of the nucleus in cell migration. PMID:27406341

  10. Physical role for the nucleus in cell migration

    Science.gov (United States)

    Fruleux, Antoine; Hawkins, Rhoda J.

    2016-09-01

    Cell migration is important for the function of many eukaryotic cells. Recently the nucleus has been shown to play an important role in cell motility. After giving an overview of cell motility mechanisms we review what is currently known about the mechanical properties of the nucleus and the connections between it and the cytoskeleton. We also discuss connections to the extracellular matrix and mechanotransduction. We identify key physical roles of the nucleus in cell migration.

  11. Effects on cell growth processes (mitosis, synthesis of nucleic acids and of proteins). Chapter 7

    International Nuclear Information System (INIS)

    A review is presented of reports of the interference of -SH radioprotective agents with cell division and with the processes of nucleic acid and protein synthesis which are a prerequisite for mitosis. Mitotic activity is inhibited to the same extent in mammalian tissues as in cultures of animal and plant cells and bacteria. With cultured cells, the toxicity and the antimitotic activity have been found to be at their highest level for intermediate concentrations of the compound and to decrease for higher and lower concentrations. Inhibition of the synthesis of nucleic acids by -SH radioprotective substances has been observed with cultures of cells and bacteria and in mammalian tissues. In vitro interactions with the structures of free DNA and nucleoprotein have also been studied. The extent to which such complexes between the protective agent and DNA or nucleoprotein occur in vivo is not known. A depression of protein synthesis has been observed, and participates in the more general inhibition of growth processes. Possible mechanisms of these effects are discussed. (U.K.)

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  13. 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…

  14. Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells.

    Directory of Open Access Journals (Sweden)

    Barry E McGuinness

    2005-03-01

    Full Text Available Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's alpha kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its alpha kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila MEI-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its alpha kleisin subunit. Centromeric

  15. 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.

  16. Suprachiasmatic Nucleus: Cell Autonomy and Network Properties

    Science.gov (United States)

    Welsh, David K.; Takahashi, Joseph S.; Kay, Steve A.

    2013-01-01

    The suprachiasmatic nucleus (SCN) is the primary circadian pacemaker in mammals. Individual SCN neurons in dispersed culture can generate independent circadian oscillations of clock gene expression and neuronal firing. However, SCN rhythmicity depends on sufficient membrane depolarization and levels of intracellular calcium and cAMP. In the intact SCN, cellular oscillations are synchronized and reinforced by rhythmic synaptic input from other cells, resulting in a reproducible topographic pattern of distinct phases and amplitudes specified by SCN circuit organization. The SCN network synchronizes its component cellular oscillators, reinforces their oscillations, responds to light input by altering their phase distribution, increases their robustness to genetic perturbations, and enhances their precision. Thus, even though individual SCN neurons can be cell-autonomous circadian oscillators, neuronal network properties are integral to normal function of the SCN. PMID:20148688

  17. Stressing Mitosis to Death

    Directory of Open Access Journals (Sweden)

    Andrew eBurgess

    2014-06-01

    Full Text Available The final stage of cell division (mitosis, involves the compaction of the duplicated genome into chromatid pairs. Each pair is captured by microtubules emanating from opposite spindle poles, aligned at the metaphase plate, and then faithfully segregated to form two identical daughter cells. Chromatids that are not correctly attached to the spindle are detected by the constitutively active spindle assembly checkpoint (SAC. Any stress that prevents correct bipolar spindle attachment, blocks the satisfaction of the SAC, and induces a prolonged mitotic arrest, providing the cell time to obtain attachment and complete segregation correctly. Unfortunately, during mitosis repairing damage is not generally possible due to the compaction of DNA into chromosomes, and subsequent suppression of gene transcription and translation. Therefore, in the presence of significant damage cell death is instigated to ensure that genomic stability is maintained. While most stresses lead to an arrest in mitosis, some promote premature mitotic exit, allowing cells to by-pass mitotic cell death. This mini-review will focus on the effects and outcomes that common stresses have on mitosis, and how this impacts on the efficacy of mitotic chemotherapies.

  18. Stressing mitosis to death.

    Science.gov (United States)

    Burgess, Andrew; Rasouli, Mina; Rogers, Samuel

    2014-01-01

    The final stage of cell division (mitosis), involves the compaction of the duplicated genome into chromatid pairs. Each pair is captured by microtubules emanating from opposite spindle poles, aligned at the metaphase plate, and then faithfully segregated to form two identical daughter cells. Chromatids that are not correctly attached to the spindle are detected by the constitutively active spindle assembly checkpoint (SAC). Any stress that prevents correct bipolar spindle attachment, blocks the satisfaction of the SAC, and induces a prolonged mitotic arrest, providing the cell time to obtain attachment and complete segregation correctly. Unfortunately, during mitosis repairing damage is not generally possible due to the compaction of DNA into chromosomes, and subsequent suppression of gene transcription and translation. Therefore, in the presence of significant damage cell death is instigated to ensure that genomic stability is maintained. While most stresses lead to an arrest in mitosis, some promote premature mitotic exit, allowing cells to bypass mitotic cell death. This mini-review will focus on the effects and outcomes that common stresses have on mitosis, and how this impacts on the efficacy of mitotic chemotherapies. PMID:24926440

  19. Mitosis is swell.

    Science.gov (United States)

    Zatulovskiy, Evgeny; Skotheim, Jan M

    2015-11-23

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

  20. Mitosis is swell

    OpenAIRE

    Zatulovskiy, Evgeny; Skotheim, Jan M.

    2015-01-01

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

  1. The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase.

    OpenAIRE

    Brandeis, M.; Hunt, T

    1996-01-01

    We have studied how the cell cycle-specific oscillations of mitotic B-type cyclins are generated in mouse fibroblasts. A reporter enzyme comprising the N-terminus of a B-type cyclin fused to bacterial chloramphenicol acetyl transferase (CAT) was degraded at the end of mitosis like endogenous cyclins. Point mutations in the destruction box of this construct completely abolished its mitotic instability. When the destructible reporter was driven by the cyclin B2 promoter, CAT activity mimicked t...

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

    Science.gov (United States)

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

    2015-08-17

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

  3. 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

    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...

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

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

    Science.gov (United States)

    S'iakste, N I

    1992-01-01

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

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    Cell-cycle transition from the G(2) phase into mitosis is regulated by the cyclin-dependent protein kinase 1 (CDK1) in complex with cyclin B. CDK1 activity is controlled by both inhibitory phosphorylation, catalysed by the Myt1 and Wee1 kinases, and activating dephosphorylation, mediated by the CDC......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...

  7. The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase.

    Science.gov (United States)

    Brandeis, M; Hunt, T

    1996-10-01

    We have studied how the cell cycle-specific oscillations of mitotic B-type cyclins are generated in mouse fibroblasts. A reporter enzyme comprising the N-terminus of a B-type cyclin fused to bacterial chloramphenicol acetyl transferase (CAT) was degraded at the end of mitosis like endogenous cyclins. Point mutations in the destruction box of this construct completely abolished its mitotic instability. When the destructible reporter was driven by the cyclin B2 promoter, CAT activity mimicked the oscillations in the level of the endogenous cyclin B2. These oscillations were largely conserved when the reporter was transcribed constitutively from the SV40 promoter. Pulse-chase experiments or addition of the proteasome inhibitors lactacystin and ALLN showed that cyclin synthesis continued after the end of mitosis. The destruction box-specific degradation of cyclins normally ceases at the onset of S phase, and is active in fibroblasts arrested in G0 and in differentiated C2 myoblasts. We were able to reproduce this proteolysis in vitro in extracts of synchronized cells. Extracts of G1 cells degraded cyclin B1 whereas p27Kip1 was stable, in contrast, cyclin B1 remained stable and p27Kip1 was degraded in extracts of S phase cells. PMID:8895573

  8. JNK controls the onset of mitosis in planarian stem cells and triggers apoptotic cell death required for regeneration and remodeling.

    Directory of Open Access Journals (Sweden)

    María Almuedo-Castillo

    2014-06-01

    Full Text Available 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.

  9. Nonthermal Fluctuations and Mechanics of the Active Cell Nucleus

    CERN Document Server

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2016-04-01

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

  11. Phosphorylated nucleolin interacts with translationally controlled tumor protein during mitosis and with Oct4 during interphase in ES cells.

    Directory of Open Access Journals (Sweden)

    Helena Johansson

    Full Text Available BACKGROUND: Reprogramming of somatic cells for derivation of either embryonic stem (ES cells, by somatic cell nuclear transfer (SCNT, or ES-like cells, by induced pluripotent stem (iPS cell procedure, provides potential routes toward non-immunogenic cell replacement therapies. Nucleolar proteins serve as markers for activation of embryonic genes, whose expression is crucial for successful reprogramming. Although Nucleolin (Ncl is one of the most abundant nucleolar proteins, its interaction partners in ES cells have remained unidentified. METHODOLOGY: Here we explored novel Ncl-interacting proteins using in situ proximity ligation assay (PLA, colocalization and immunoprecipitation (IP in ES cells. PRINCIPAL FINDINGS: We found that phosphorylated Ncl (Ncl-P interacted with translationally controlled tumor protein (Tpt1 in murine ES cells. The Ncl-P/Tpt1 complex peaked during mitosis and was reduced upon retinoic acid induced differentiation, signifying a role in cell proliferation. In addition, we showed that Ncl-P interacted with the transcription factor Oct4 during interphase in human as well as murine ES cells, indicating of a role in transcription. The Ncl-P/Oct4 complex peaked during early stages of spontaneous human ES cell differentiation and may thus be involved in the initial differentiation event(s of mammalian development. CONCLUSIONS: Here we described two novel protein-protein interactions in ES cells, which give us further insight into the complex network of interacting proteins in pluripotent cells.

  12. 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.

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

    Science.gov (United States)

    Onorati, Marco; Li, Zhen; Liu, Fuchen; Sousa, André M M; Nakagawa, Naoki; Li, Mingfeng; Dell'Anno, Maria Teresa; Gulden, Forrest O; Pochareddy, Sirisha; Tebbenkamp, Andrew T N; Han, Wenqi; Pletikos, Mihovil; Gao, Tianliuyun; Zhu, Ying; Bichsel, Candace; Varela, Luis; Szigeti-Buck, Klara; Lisgo, Steven; Zhang, Yalan; Testen, Anze; Gao, Xiao-Bing; Mlakar, Jernej; Popovic, Mara; Flamand, Marie; Strittmatter, Stephen M; Kaczmarek, Leonard K; Anton, E S; Horvath, Tamas L; Lindenbach, Brett D; Sestan, Nenad

    2016-09-01

    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. PMID:27568284

  14. Relocalization of human chromatin remodeling cofactor TIP48 in mitosis

    International Nuclear Information System (INIS)

    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

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

    Science.gov (United States)

    Lu, Meng; Boschetti, Chiara; Tunnacliffe, Alan

    2015-11-13

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

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

    Science.gov (United States)

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

    2015-11-10

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

  17. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  19. 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.

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

    Science.gov (United States)

    Jiráková, Klára; Kulda, Jaroslav; Nohýnková, Eva

    2012-05-01

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

  1. Nucleus-associated microtubules help determine the division plane of plant epidermal cells: avoidance of four-way junctions and the role of cell geometry.

    Science.gov (United States)

    Flanders, D J; Rawlins, D J; Shaw, P J; Lloyd, C W

    1990-04-01

    To investigate the spatial relationship between the nucleus and the cortical division site, epidermal cells were selected in which the separation between these two areas is large. Avoiding enzyme treatment and air drying, Datura stramonium cells were labeled with antitubulin antibodies and the three-dimensional aspect of the cytoskeletons was reconstructed using computer-aided optical sectioning. In vacuolated cells preparing for division, the nucleus migrates into the center of the cell, suspended by transvacuolar strands. These strands are now shown to contain continuous bundles of microtubules which bridge the nucleus to the cortex. These nucleus-radiating microtubules adopt different configurations in cells of different shape. In elongated cells with more or less parallel side walls, oblique strands radiating from the nucleus to the long side walls are presumably unstable, for they are progressively realigned into a transverse disc (the phragmosome) as broad, cortical, preprophase bands (PPBs) become tighter. The phragmosome and the PPB are both known predictors of the division plane and our observations indicate that they align simultaneously in elongated epidermal cells. These observations suggest another hypothesis: that the PPB may contain microtubules polymerized from the nuclear surface. In elongated cells, the majority of the radiating microtubules, therefore, come to anchor the nucleus in the transverse plane, consistent with the observed tendency of such cells to divide perpendicular to the long axis. In nonrectangular isodiametric epidermal cells, which approximate regular hexagons in section, the radial microtubular strands emanating from the nucleus tend to remain associated with the middle of each subtending cell wall. The strands are not reorganized into a single dominant transverse bar, but remain as a starlike array until mitosis. PPBs in these cells are not as tight; they may only be a sparse accumulation of microtubules, even forming along non

  2. 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

  3. 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.

  4. Influence of low-dose gamma-ray irradiation on mitosis and adaptive response of meristematic cells of pea rootlets

    International Nuclear Information System (INIS)

    Pea seeds (Pisum sativa) were exposed to gamma irradiation (60Co, dose 7 Gy) at different dose rates (from 0.3 cGy/h to 19.1 cGy/h). The beginning of irradiation coincided with the beginning of soaking. After the irradiation a fraction of the seeds was irradiated with 50 Gy (137Cs, dose rate 25 Gy/min) to examine the adaptive response. After 24 hours of soaking all the seeds were germinated at 25 deg C and rootlets were fixed. Mitotic index and the number of cells with chromosomal aberrations (CA) in first mitosis anaphases of the meristematic cells of rootlets were determined. After 7 cGy irradiation, judging by the above criteria, the radiation hormesis was identified at the dose rate 1.2 cGy/h. For 19.1 cGy/h damaging effect and decrease in the mitotic activity were observed. As the dose rate grows, the adaptive response is better expressed (by the analysis of CA). When the seeds are conserved at 13-14 % humidity, the CA level is higher and the adaptive response is better expressed compared to conserving at 10 % humidity. (author)

  5. 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.

  6. Romancing mitosis and the mitotic apparatus.

    Science.gov (United States)

    Brinkley, William B R

    2014-11-01

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

  7. Understanding mitosis in secondary school in Slovenia

    OpenAIRE

    Frank, Aleksandra

    2012-01-01

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

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

    Science.gov (United States)

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

    2016-05-27

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

  9. Human Cyclin a Is Required for Mitosis until Mid Prophase

    OpenAIRE

    Furuno, Nobuaki; den Elzen, Nicole; Pines, Jonathon

    1999-01-01

    We have used microinjection and time-lapse video microscopy to study the role of cyclin A in mitosis. We have injected purified, active cyclin A/cyclin-dependent kinase 2 (CDK2) into synchronized cells at specific points in the cell cycle and assayed its effect on cell division. We find that cyclin A/CDK2 will drive G2 phase cells into mitosis within 30 min of microinjection, up to 4 h before control cells enter mitosis. Often this premature mitosis is abnormal; the chromosomes do not complet...

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    C. Risso-Pascotto

    2005-11-01

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

  16. Myb-binding protein 1A (MYBBP1A is essential for early embryonic development, controls cell cycle and mitosis, and acts as a tumor suppressor.

    Directory of Open Access Journals (Sweden)

    Silvia Mori

    Full Text Available MYBBP1A is a predominantly nucleolar transcriptional regulator involved in rDNA synthesis and p53 activation via acetylation. However little further information is available as to its function. Here we report that MYBBP1A is developmentally essential in the mouse prior to blastocyst formation. In cell culture, down-regulation of MYBBP1A decreases the growth rate of wild type mouse embryonic stem cells, mouse embryo fibroblasts (MEFs and of human HeLa cells, where it also promotes apoptosis. HeLa cells either arrest at G2/M or undergo delayed and anomalous mitosis. At mitosis, MYBBP1A is localized to a parachromosomal region and gene-expression profiling shows that its down-regulation affects genes controlling chromosomal segregation and cell cycle. However, MYBBP1A down-regulation increases the growth rate of the immortalized NIH3T3 cells. Such Mybbp1a down-regulated NIH3T3 cells are more susceptible to Ras-induced transformation and cause more potent Ras-driven tumors. We conclude that MYBBP1A is an essential gene with novel roles at the pre-mitotic level and potential tumor suppressor activity.

  17. Unattached kinetochores rather than intrakinetochore tension arrest mitosis in taxol-treated cells.

    Science.gov (United States)

    Magidson, Valentin; He, Jie; Ault, Jeffrey G; O'Connell, Christopher B; Yang, Nachen; Tikhonenko, Irina; McEwen, Bruce F; Sui, Haixin; Khodjakov, Alexey

    2016-02-01

    Kinetochores attach chromosomes to the spindle microtubules and signal the spindle assembly checkpoint to delay mitotic exit until all chromosomes are attached. Light microscopy approaches aimed to indirectly determine distances between various proteins within the kinetochore (termed Delta) suggest that kinetochores become stretched by spindle forces and compact elastically when the force is suppressed. Low Delta is believed to arrest mitotic progression in taxol-treated cells. However, the structural basis of Delta remains unknown. By integrating same-kinetochore light microscopy and electron microscopy, we demonstrate that the value of Delta is affected by the variability in the shape and size of outer kinetochore domains. The outer kinetochore compacts when spindle forces are maximal during metaphase. When the forces are weakened by taxol treatment, the outer kinetochore expands radially and some kinetochores completely lose microtubule attachment, a condition known to arrest mitotic progression. These observations offer an alternative interpretation of intrakinetochore tension and question whether Delta plays a direct role in the control of mitotic progression. PMID:26833787

  18. Unattached kinetochores rather than intrakinetochore tension arrest mitosis in taxol-treated cells

    Science.gov (United States)

    Magidson, Valentin; He, Jie; Ault, Jeffrey G.; O’Connell, Christopher B.; Yang, Nachen; Tikhonenko, Irina; McEwen, Bruce F.

    2016-01-01

    Kinetochores attach chromosomes to the spindle microtubules and signal the spindle assembly checkpoint to delay mitotic exit until all chromosomes are attached. Light microscopy approaches aimed to indirectly determine distances between various proteins within the kinetochore (termed Delta) suggest that kinetochores become stretched by spindle forces and compact elastically when the force is suppressed. Low Delta is believed to arrest mitotic progression in taxol-treated cells. However, the structural basis of Delta remains unknown. By integrating same-kinetochore light microscopy and electron microscopy, we demonstrate that the value of Delta is affected by the variability in the shape and size of outer kinetochore domains. The outer kinetochore compacts when spindle forces are maximal during metaphase. When the forces are weakened by taxol treatment, the outer kinetochore expands radially and some kinetochores completely lose microtubule attachment, a condition known to arrest mitotic progression. These observations offer an alternative interpretation of intrakinetochore tension and question whether Delta plays a direct role in the control of mitotic progression. PMID:26833787

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

    Science.gov (United States)

    Ohkura, Hiroyuki

    2015-05-01

    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.

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

    NARCIS (Netherlands)

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

    2015-01-01

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

  1. Zero Crossing Edge Detection and Contour Tracing for Segmentation of Cervical Cell Nucleus .

    Directory of Open Access Journals (Sweden)

    B.V. Ramesh

    1993-07-01

    Full Text Available To automate the process of screening of normal and abnormal cervical cells, there is a need for automatic segmentation of the nucleus of these cells. This paper presents an algorithm using the Laplacian of Gaussian operator and contour tracer to segment the nucleus from the background. The algorithm has been tested on different kinds of images of cervical cells.

  2. Linking abnormal mitosis to the acquisition of DNA damage

    OpenAIRE

    Ganem, Neil J.; 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 develop...

  3. 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

  4. 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.

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

    NARCIS (Netherlands)

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

    2007-01-01

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

  6. EGFR/MEK/ERK/CDK5-dependent integrin-independent FAK phosphorylated on serine 732 contributes to microtubule depolymerization and mitosis in tumor cells.

    Science.gov (United States)

    Rea, K; Sensi, M; Anichini, A; Canevari, S; Tomassetti, A

    2013-01-01

    FAK is a non-receptor tyrosine kinase contributing to migration and proliferation downstream of integrin and/or growth factor receptor signaling of normal and malignant cells. In addition to well-characterized tyrosine phosphorylations, FAK is phosphorylated on several serines, whose role is not yet clarified. We observed that phosphorylated FAK on serine 732 (P-FAKSer732) is present at variable levels in vitro, in several melanoma, ovarian and thyroid tumor cell lines and in vivo, in tumor cells present in fresh ovarian cancer ascites. In vitro P-FAKSer732 was barely detectable during interphase while its levels strongly increased in mitotic cells upon activation of the EGFR/MEK/ERK axis in an integrin-independent manner. P-FAKSer732 presence was crucial for the maintenance of the proliferation rate and its levels were inversely related to the levels of acetylated α-tubulin. P-FAKSer732 localized at the microtubules (MTs) of the spindle, biochemically associated with MTs and contributed to MT depolymerization. The lack of the phosphorylation on Ser732 as well as the inhibition of CDK5 activity by roscovitine impaired mitotic spindle assembly and correct chromosome alignment during mitosis. We also identified, for the first time, that the EGF-dependent EGFR activation led to increased P-FAKSer732 and polymerized MTs. Our data shed light on the multifunctional roles of FAK in neoplastic cells, being involved not only in integrin-dependent migratory signaling but also in integrin-independent MT dynamics and mitosis control. These findings provide a new potential target for inhibiting the growth of tumor cells in which the EGFR/MEK/ERK/CDK5 pathway is active. PMID:24091658

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

    Science.gov (United States)

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

    2014-06-15

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

  8. 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.)

  9. The Cell Nucleus in Physiological and Experimentally Induced Hypometabolism

    Science.gov (United States)

    Malatesta, M.

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

  10. Thermodynamic pathways to genome spatial organization in the cell nucleus.

    Science.gov (United States)

    Nicodemi, Mario; Prisco, Antonella

    2009-03-18

    The architecture of the eukaryotic genome is characterized by a high degree of spatial organization. Chromosomes occupy preferred territories correlated to their state of activity and, yet, displace their genes to interact with remote sites in complex patterns requiring the orchestration of a huge number of DNA loci and molecular regulators. Far from random, this organization serves crucial functional purposes, but its governing principles remain elusive. By computer simulations of a statistical mechanics model, we show how architectural patterns spontaneously arise from the physical interaction between soluble binding molecules and chromosomes via collective thermodynamics mechanisms. Chromosomes colocalize, loops and territories form, and find their relative positions as stable thermodynamic states. These are selected by thermodynamic switches, which are regulated by concentrations/affinity of soluble mediators and by number/location of their attachment sites along chromosomes. Our thermodynamic switch model of nuclear architecture, thus, explains on quantitative grounds how well-known cell strategies of upregulation of DNA binding proteins or modification of chromatin structure can dynamically shape the organization of the nucleus. PMID:19289043

  11. Integration of murine leukemia virus DNA depends on mitosis.

    OpenAIRE

    Roe, T.; Reynolds, T. C.; Yu, G.; Brown, P O

    1993-01-01

    In synchronized rat or mouse cells infected with Moloney murine leukemia virus (MLV), integration of viral DNA and production of viral proteins occur only after the cells traverse mitosis. Integration is blocked when cells are prevented from progressing through mitosis. Viral nucleoprotein complexes isolated from arrested cells contain full-length viral DNA and can integrate this viral DNA in vitro, showing that the block to integration in arrested cells is not due to a lack of mature integra...

  12. 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.

  13. 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.

  14. Mitosis Methods & Protocols

    Directory of Open Access Journals (Sweden)

    CarloAlberto Redi

    2010-06-01

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

  15. Coupling mitosis to S-phase

    NARCIS (Netherlands)

    Clijsters, L.

    2014-01-01

    In this thesis, we studied the structural and biochemical findings described in the introduction, from a more cell biological perspective. We investigated how replication licensing and mitosis might be linked. We also asked, how the spindle checkpoint and the APC/C can coordinate mitotic exit and DN

  16. Estimated Radiation on Mars, Hits per Cell Nucleus

    Science.gov (United States)

    2002-01-01

    This global map of Mars shows estimates for amounts of high-energy-particle cosmic radiation reaching the surface, a serious health concern for any future human exploration of the planet.The estimates are based on cosmic-radiation measurements made on the way to Mars by the Mars radiation environment experiment, an instrument on NASA's 2001 Mars Odyssey spacecraft, plus information about Mars' surface elevations from the laser altimeter instrument on NASA's Mars Global Surveyor. The areas of Mars expected to have least radiation are where elevation is lowest, because those areas have more atmosphere above them to block out some of the radiation. Earth's thick atmosphere shields us from most cosmic radiation, but Mars has a much thinner atmosphere than Earth does.Colors in the map refer to the estimated average number of times per year each cell nucleus in a human there would be hit by a high-energy cosmic ray particle. The range is generally from two hits (color-coded green), a moderate risk level, to eight hits (coded red), a high risk level.NASA's Jet Propulsion Laboratory, Pasadena, Calif. manages the 2001 Mars Odyssey and Mars Global Surveyor missions for NASA's Office of Space Science, Washington D.C. The Mars radiation environment experiment was developed by NASA's Johnson Space Center. Lockheed Martin Astronautics, Denver, is the prime contractor for Odyssey, and developed and built the orbiter. Mission operations are conducted jointly from Lockheed Martin and from JPL, a division of the California Institute of Technology in Pasadena.

  17. Internal dynamics of a living cell nucleus investigated by dynamic light scattering

    Science.gov (United States)

    Suissa, M.; Place, C.; Goillot, E.; Freyssingeas, E.

    2008-08-01

    Recent progresses in cellular biology have shown that the nucleus of a living cell is a structured integration of many functional domains with a complex spatial organization. This organization, as well as molecular and biochemical processes, is time regulated. In the past years many investigations have been performed using fluorescent microscopy techniques to study the internal dynamics of the nucleus of a living cell. These investigations, however, have never focussed on the global internal dynamics of the nucleus, which is still unknown. In this article we present an original light scattering experimental device that we built to investigate this dynamics during biological processes. By means of this experimental set-up, we investigated the global dynamics of the nucleus of a living cell treated with a DNA replication inhibitor. This dynamics presents different and independent kinds of relaxation well separated in time that vary as a function of the cell cycle phases.

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

    Directory of Open Access Journals (Sweden)

    Paula Irles

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

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

    Institute of Scientific and Technical Information of China (English)

    李志刚; 赵洪波; 李素丽; 杨丽涛; 李杨瑞

    2008-01-01

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

  20. On the robustness of SAC silencing in closed mitosis

    Science.gov (United States)

    Ruth, Donovan; Liu, Jian

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

  1. Nanomolar pyrophosphate detection and nucleus staining in living cells with simple terpyridine–Zn(II) complexes

    OpenAIRE

    Duobin Chao; Shitan Ni

    2016-01-01

    Great efforts have been made to develop fluorescent probes for pyrophosphate (PPi) detection. Nucleus staining with fluorescence microscopy has been also widely investigated. But fluorescent probes for PPi detection with high sensitivity in water medium and nucleus staining with low–cost non–precious metal complexes in living cells are still challenging. Herein, we report simple terpyridine–Zn(II) complexes for selective nanomolar PPi detection over ATP and ADP in water based on aggregation i...

  2. 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.

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

    Science.gov (United States)

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

    2016-01-01

    Here, we describe how the rapid reversibility of the nda3-KM311 cold-sensitive β-tubulin mutation was optimized by Mitsuhiro Yanagida's laboratory to synchronize mitotic progression in an entire cell population. The inability to form microtubules following the loss of β-tubulin function at 20°C triggers the spindle assembly checkpoint, which arrests mitotic progression. Restoration of β-tubulin function by rewarming to 30°C (or higher) releases the arrest, generating a highly synchronous progression through mitosis. The viability of nda3-KM311 strains at 30°C makes it feasible to generate double mutants between nda3-KM311 and any temperature-sensitive mutant that can also grow at 30°C. These double mutants can be used in reciprocal shift analyses, in which cold-induced early mitotic arrest is relieved by a shift to 36°C, which then inactivates the product of the second mutant gene. The addition of microtubule depolymerizing drugs before the return to 36°C will maintain checkpoint signaling at 36°C transiently, permitting analysis of the impact of temperature-sensitive mutations on checkpoint function. Silencing the checkpoint of nda3-KM311-arrested cells at 20°C through chemical inhibition of aurora kinase is a powerful way to study checkpoint recovery pathways and mitotic exit without anaphase. PMID:27480719

  4. Turning meiosis into mitosis.

    Directory of Open Access Journals (Sweden)

    Isabelle d'Erfurth

    2009-06-01

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

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

    Science.gov (United States)

    Halley-Stott, Richard P

    2015-01-01

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

  6. Dissociation of FAK/p130CAS/c-Src Complex during Mitosis: Role of Mitosis-specific Serine Phosphorylation of FAK

    OpenAIRE

    Yamakita, Yoshihiko; Totsukawa, Go; Yamashiro, Shigeko; Fry, David; Zhang, Xiaoe; Hanks, Steven K.; Matsumura, Fumio

    1999-01-01

    At mitosis, focal adhesions disassemble and the signal transduction from focal adhesions is inactivated. We have found that components of focal adhesions including focal adhesion kinase (FAK), paxillin, and p130CAS (CAS) are serine/threonine phosphorylated during mitosis when all three proteins are tyrosine dephosphorylated. Mitosis-specific phosphorylation continues past cytokinesis and is reversed during post-mitotic cell spreading. We have found two significant alterations in FAK-mediated ...

  7. Mitosis. Microtubule detyrosination guides chromosomes during mitosis.

    Science.gov (United States)

    Barisic, Marin; Silva e Sousa, Ricardo; Tripathy, Suvranta K; Magiera, Maria M; Zaytsev, Anatoly V; Pereira, Ana L; Janke, Carsten; Grishchuk, Ekaterina L; Maiato, Helder

    2015-05-15

    Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination. PMID:25908662

  8. Heat-induced alterations in the cell nucleus

    International Nuclear Information System (INIS)

    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

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

    Directory of Open Access Journals (Sweden)

    Perrin Mathilde

    2004-06-01

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

  10. Effect of vertebroplasty filler materials on viability and gene expression of human nucleus pulposus cells.

    Science.gov (United States)

    Lazáry, Aron; Speer, Gábor; Varga, Péter Pál; Balla, Bernadett; Bácsi, Krisztián; Kósa, János P; Nagy, Zsolt; Takács, István; Lakatos, Péter

    2008-05-01

    Consequences of intradiscal cement leakage--often occurring after vertebral cement augmentation for the treatment of vertebral compression fractures--are still unknown. In this study, we have investigated the influences of vertebroplasty filler materials (polymethylmethacrylate-, calcium phosphate- and calcium sulfate-based bone cement) on isolated nucleus pulposus cells. Cell viability of cultured human nucleus pulposus cells were measured after treatment with vertebroplasty filler materials. Gene expression profile of selected genes was determined with quantitative real-time PCR. The widely used polymethylmethacrylate and calcium phosphate cement significantly decreased cell number in a dose- and time-dependent manner while calcium sulfate cement affected cell viability less. Expression of genes involved in matrix metabolism of nucleus pulposus--aggrecan, collagens, small proteoglycans--as well as important transcription factors have also significantly changed due to treatment (e.g., 2.5-fold decrease in aggrecan expression was determined in cultures due to polymethylmethacrylate treatment). Our results suggest that vertebroplasty filler materials--depending on the type of applied material--can accelerate the degeneration of nucleus pulposus cells resulting in a less flexible disc in case of intradiscal cement leakage. This process may increase the risk of a subsequent new vertebral fracture, the main complication of vertebral augmentation. PMID:18176942

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

    Institute of Scientific and Technical Information of China (English)

    邓骛远; 罗通; 郑田喜

    2011-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Fenghua TAO; Feng LI; Guanghui LI; Feng PAN

    2008-01-01

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

  13. Nanomolar pyrophosphate detection and nucleus staining in living cells with simple terpyridine–Zn(II) complexes

    Science.gov (United States)

    Chao, Duobin; Ni, Shitan

    2016-05-01

    Great efforts have been made to develop fluorescent probes for pyrophosphate (PPi) detection. Nucleus staining with fluorescence microscopy has been also widely investigated. But fluorescent probes for PPi detection with high sensitivity in water medium and nucleus staining with low–cost non–precious metal complexes in living cells are still challenging. Herein, we report simple terpyridine–Zn(II) complexes for selective nanomolar PPi detection over ATP and ADP in water based on aggregation induced emission (AIE) and intramolecular charge transfer (ICT). In addition, these terpyridine–Zn(II) complexes were successfully employed for nucleus staining in living cells. These results demonstrated simply obtained terpyridine–Zn(II) complexes are powerful tool for PPi detection and the development of PPi–related studies.

  14. Nanomolar pyrophosphate detection and nucleus staining in living cells with simple terpyridine-Zn(II) complexes.

    Science.gov (United States)

    Chao, Duobin; Ni, Shitan

    2016-01-01

    Great efforts have been made to develop fluorescent probes for pyrophosphate (PPi) detection. Nucleus staining with fluorescence microscopy has been also widely investigated. But fluorescent probes for PPi detection with high sensitivity in water medium and nucleus staining with low-cost non-precious metal complexes in living cells are still challenging. Herein, we report simple terpyridine-Zn(II) complexes for selective nanomolar PPi detection over ATP and ADP in water based on aggregation induced emission (AIE) and intramolecular charge transfer (ICT). In addition, these terpyridine-Zn(II) complexes were successfully employed for nucleus staining in living cells. These results demonstrated simply obtained terpyridine-Zn(II) complexes are powerful tool for PPi detection and the development of PPi-related studies. PMID:27198968

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Christopher L Gilchrist

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

  17. Computational prediction of strain-dependent diffusion of transcription factors through the cell nucleus.

    Science.gov (United States)

    Nava, Michele M; Fedele, Roberto; Raimondi, Manuela T

    2016-08-01

    Nuclear spreading plays a crucial role in stem cell fate determination. In previous works, we reported evidence of multipotency maintenance for mesenchymal stromal cells cultured on three-dimensional engineered niche substrates, fabricated via two-photon laser polymerization. We correlated maintenance of multipotency to a more roundish morphology of these cells with respect to those cultured on conventional flat substrates. To interpret these findings, here we present a multiphysics model coupling nuclear strains induced by cell adhesion to passive diffusion across the cell nucleus. Fully three-dimensional reconstructions of cultured cells were developed on the basis of confocal images: in particular, the level of nuclear spreading resulted significantly dependent on the cell localization within the niche architecture. We assumed that the cell diffusivity varies as a function of the local volumetric strain. The model predictions indicate that the higher the level of spreading of the cell, the higher the flux across the nucleus of small solutes such as transcription factors. Our results point toward nuclear spreading as a primary mechanism by which the stem cell translates its shape into a fate decision, i.e., by amplifying the diffusive flow of transcriptional activators into the nucleus. PMID:26476736

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    Cristian Campeanu

    2012-10-01

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

  20. 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

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

    DEFF Research Database (Denmark)

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

    2004-01-01

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

  2. 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.

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

    Science.gov (United States)

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

    2010-11-17

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

  4. 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.

  5. 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.

  6. 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.

  7. Monodisperse Magnetite Nanoparticles Coupled with Nuclear Localization Signal Peptide for Cell-Nucleus Targeting

    OpenAIRE

    Xu, Chenjie; Xie, Jin; Kohler, Nathan; Walsh, Edward G; Chin, Y. Eugene; Sun, Shouheng

    2008-01-01

    Functionalization of monodisperse superparamagnetic magnetite (Fe3O4) nanoparticles for cell specific targeting is crucial for cancer diagnostics and therapeutics. Targeted magnetic nanoparticles can be used to enhance the tissue contrast in magnetic resonance imaging (MRI), to improve the efficiency in anticancer drug delivery, and to eliminate tumor cells by magnetic fluid hyperthermia. Herein we report the nucleus-targeting Fe3O4 nanoparticles functionalized with protein and nuclear locali...

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

    Institute of Scientific and Technical Information of China (English)

    Hong Lin LIU; Kentaro T.HARA; Fugaku AOKI

    2005-01-01

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

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

    Science.gov (United States)

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

    2016-09-01

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

  10. The bacterium endosymbiont of Crithidia deanei undergoes coordinated division with the host cell nucleus.

    Directory of Open Access Journals (Sweden)

    Maria Cristina Machado Motta

    Full Text Available In trypanosomatids, cell division involves morphological changes and requires coordinated replication and segregation of the nucleus, kinetoplast and flagellum. In endosymbiont-containing trypanosomatids, like Crithidia deanei, this process is more complex, as each daughter cell contains only a single symbiotic bacterium, indicating that the prokaryote must replicate synchronically with the host protozoan. In this study, we used light and electron microscopy combined with three-dimensional reconstruction approaches to observe the endosymbiont shape and division during C. deanei cell cycle. We found that the bacterium replicates before the basal body and kinetoplast segregations and that the nucleus is the last organelle to divide, before cytokinesis. In addition, the endosymbiont is usually found close to the host cell nucleus, presenting different shapes during the protozoan cell cycle. Considering that the endosymbiosis in trypanosomatids is a mutualistic relationship, which resembles organelle acquisition during evolution, these findings establish an excellent model for the understanding of mechanisms related with the establishment of organelles in eukaryotic cells.

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

    Directory of Open Access Journals (Sweden)

    Ulbricht Tobias

    2010-12-01

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

  12. Monodisperse magnetite nanoparticles coupled with nuclear localization signal peptide for cell-nucleus targeting.

    Science.gov (United States)

    Xu, Chenjie; Xie, Jin; Kohler, Nathan; Walsh, Edward G; Chin, Y Eugene; Sun, Shouheng

    2008-03-01

    Functionalization of monodisperse superparamagnetic magnetite (Fe(3)O(4)) nanoparticles for cell specific targeting is crucial for cancer diagnostics and therapeutics. Targeted magnetic nanoparticles can be used to enhance the tissue contrast in magnetic resonance imaging (MRI), to improve the efficiency in anticancer drug delivery, and to eliminate tumor cells by magnetic fluid hyperthermia. Herein we report the nucleus-targeting Fe(3)O(4) nanoparticles functionalized with protein and nuclear localization signal (NLS) peptide. These NLS-coated nanoparticles were introduced into the HeLa cell cytoplasm and nucleus, where the particles were monodispersed and non-aggregated. The success of labeling was examined and identified by fluorescence microscopy and MRI. The work demonstrates that monodisperse magnetic nanoparticles can be readily functionalized and stabilized for potential diagnostic and therapeutic applications. PMID:18080259

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

    Science.gov (United States)

    Yanagida, Mitsuhiro

    2014-09-01

    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.

  14. 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.

  15. 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...

  16. On the move: organelle dynamics during mitosis.

    Science.gov (United States)

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

    2015-03-01

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

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

    Science.gov (United States)

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

    2012-03-01

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

  18. Replication stress activates DNA repair synthesis in mitosis

    DEFF Research Database (Denmark)

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

    2015-01-01

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

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

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

    International Nuclear Information System (INIS)

    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.

  1. 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.

  2. 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. PMID:27430346

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

    Institute of Scientific and Technical Information of China (English)

    李伟

    2014-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  5. Novel functions of endocytic player clathrin in mitosis

    Institute of Scientific and Technical Information of China (English)

    Wenxiang Fu; Qing Jiang; Chuanmao Zhang

    2011-01-01

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

  6. Cdc18 transcription and proteolysis couple S phase to passage through mitosis.

    OpenAIRE

    Baum, B.; Nishitani, H; Yanow, S; Nurse, P

    1998-01-01

    In fission yeast, cdc18p plays a critical role in bringing about the onset of S phase. We show that cdc18p expression is subject to a complex sequence of cell cycle controls which ensure that cdc18p levels rise dramatically as cells exit mitosis, before the appearance of CDK activity in G1. We find that transcription of cdc18, together with the transcription of other cdc10p/res1p targets, is first initiated as cells enter mitosis and continues even in cells arrested in mitosis with highly con...

  7. miR-155 Inhibits Nucleus Pulposus Cells' Degeneration through Targeting ERK 1/2

    Science.gov (United States)

    Dai, Libing; Yao, Yicun; Qin, Shengnan; Xie, Han; Wang, Wen

    2016-01-01

    We first investigated the difference in microRNA expression between normal NP cells and degenerative NP cells using gene chip. We have found that the expression of ERK1/2 was decreased with overexpression of miR-155 in normal nucleus pulposus cell. Expression of ERK1/2 was increased with inhibition of miR-155. Overexpression or inhibition of miR-155 had no effects on the expression level of mRNA ERK1/2 in nucleus pulposus cell, which showed that miR-155 affected the expression of pERK1/2 after transcription of ERK1/2 mRNA indicating that ERK1/2 was a new target protein regulated by miR-155. In the degeneration of intervertebral disc, inhibited miR-155 decreased the expressions of extracellular main matrix collagen II and glycosaminoglycan and increased expression of ERK1/2. Taken together, our data suggested that miR-155 was the identified miRNA which regulated NP cells degenerated through directly targeting ERK1/2. PMID:27635110

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

    Science.gov (United States)

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

    2016-09-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2015-01-01

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

  10. 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.

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

    Directory of Open Access Journals (Sweden)

    Ioudinkova E. S.

    2014-03-01

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

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

    Science.gov (United States)

    Jin, J; Liu, J

    2016-01-01

    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. PMID:27609478

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

    Science.gov (United States)

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

    2012-08-14

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

  14. The formation of argpyrimidine, a methylglyoxal-arginine adduct, in the nucleus of neural cells

    International Nuclear Information System (INIS)

    Methylglyoxal (MG) is an endogenous metabolite in glycolysis and forms stable adducts primarily with arginine residues of intracellular proteins. The biological role of this modification in cell function is not known. In the present study, we found that a MG-detoxification enzyme glyoxalase I (GLO1) is mainly expressed in the ventricular zone (VZ) at embryonic day 16 which neural stem and progenitor cells localize. Moreover, immunohistochemical analysis revealed that argpyrimidine, a major MG-arginine adduct, is predominantly produced in cortical plate neurons not VZ during cerebral cortex development and is exclusively located in the nucleus. Immunoblotting experiment showed that the formation of argpyrimidine occurs on some nuclear proteins of cortical neurons. To our knowledge, this is first report of the argpyrimidine formation in the nucleus of neuron. These findings suggest that GLO1, which is dominantly expressed in the embryonic VZ, reduces the intracellular level of MG and suppresses the formation of argpyrimidine in neural stem and progenitor cells. Argpyrimidine may contribute to the neural differentiation and/or the maintenance of the differentiated state via the modification of nuclear proteins.

  15. Mitosis: spindle evolution and the matrix model.

    Science.gov (United States)

    Pickett-Heaps, Jeremy; Forer, Art

    2009-03-01

    Current spindle models explain "anaphase A" (movement of chromosomes to the poles) in terms of a motility system based solely on microtubules (MTs) and that functions in a manner unique to mitosis. We find both these propositions unlikely. An evolutionary perspective suggests that when the spindle evolved, it should have come to share not only components (e.g., microtubules) of the interphase cell but also the primitive motility systems available, including those using actin and myosin. Other systems also came to be involved in the additional types of motility that now accompany mitosis in extant spindles. The resultant functional redundancy built reliability into this critical and complex process. Such multiple mechanisms are also confusing to those who seek to understand how chromosomes move. Narrowing this commentary down to just anaphase A, we argue that the spindle matrix participates with MTs in anaphase A and that this matrix may contain actin and myosin. The diatom spindle illustrates how such a system could function. This matrix may be motile and work in association with the MT cytoskeleton, as it does with the actin cytoskeleton during cell ruffling and amoeboid movement. Instead of pulling the chromosome polewards, the kinetochore fibre's role might be to slow polewards movement to allow correct chromosome attachment to the spindle. Perhaps the earliest eukaryotic cell was a cytoplast organised around a radial MT cytoskeleton. For cell division, it separated into two cytoplasts via a spindle of overlapping MTs. Cytokinesis was actin-based cleavage. As chromosomes evolved into individual entities, their interaction with the dividing cytoplast developed into attachment of the kinetochore to radial (cytoplast) MTs. We believe it most likely that cytoplasmic motility systems participated in these events. PMID:19255823

  16. Mitotic stopwatch for the blast fungus Magnaporthe oryzae during invasion of rice cells.

    Science.gov (United States)

    Jones, Kiersun; Jenkinson, Cory B; Borges Araújo, Maíra; Zhu, Jie; Kim, Rebecca Y; Kim, Dong Won; Khang, Chang Hyun

    2016-08-01

    To study nuclear dynamics of Magnaporthe oryzae, we developed a novel mitotic reporter strain with GFP-NLS (localized in nuclei during interphase but in the cytoplasm during mitosis) and H1-tdTomato (localized in nuclei throughout the cell cycle). Time-lapse confocal microscopy of the reporter strain during host cell invasion provided several new insights into nuclear division and migration in M. oryzae: (i) mitosis lasts about 5min; (ii) mitosis is semi-closed; (iii) septal pores are closed during mitosis; and (iv) a nucleus exhibits extreme constriction (approximately from 2μm to 0.5μm), elongation (over 5μm), and long migration (over 16μm). Our observations raise new questions about mechanisms controlling the mitotic dynamics, and the answers to these questions may result in new means to prevent fungal proliferation without negatively affecting the host cell cycle. PMID:27321562

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

    Science.gov (United States)

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

    2014-09-01

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

  18. 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.

  19. ABCG2 Localizes to the Nucleus and Modulates CDH1 Expression in Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Shu-Ching Liang

    2015-03-01

    Full Text Available Breast cancer resistance protein [BCRP/ATP-binding cassette subfamily G member 2 (ABCG2] is a member of the ATP-binding cassette transporter family. The presence of ABCG2 on the plasma membrane in many kinds of human cancer cells contributes to multidrug resistance during chemotherapy, and it has been used as the side population marker for identifying cancer stem cells in lung cancers. We report here that, in addition to the membranous form, ABCG2 proteins are also found inside the nucleus, where they bind to the E-box of CDH1 (E-cadherin promoter and regulate transcription of this gene. Increased expression of ABCG2 causes an increase of E-cadherin and attenuates cell migration, whereas knockdown of ABCG2 downregulates E-cadherin and enhances cell motility. In mice, xenografted A549 cells that have less ABCG2 are more likely to metastasize from the subcutaneous inoculation site to the internal organs. However, for the cancer cells that have already entered the blood circulation, an increased level of ABCG2, and correspondingly increased E-cadherin, may facilitate circulating cancer cells to colonize at a distant site and form a metastatic tumor. We propose a novel role for nuclear ABCG2 that functions as a transcription regulator and participates in modulation of cancer metastasis.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    归改霞

    2015-01-01

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

  2. Neurons and Glial Cells Are Added to the Female Rat Anteroventral Periventricular Nucleus During Puberty.

    Science.gov (United States)

    Mohr, Margaret A; Garcia, Francisca L; DonCarlos, Lydia L; Sisk, Cheryl L

    2016-06-01

    The anteroventral periventricular nucleus (AVPV) orchestrates the neuroendocrine-positive feedback response that triggers ovulation in female rodents. The AVPV is larger and more cell-dense in females than in males, and during puberty, only females develop the capacity to show a positive feedback response. We previously reported a potential new mechanism to explain this female-specific gain of function during puberty, namely a female-biased sex difference in the pubertal addition of new cells to the rat AVPV. Here we first asked whether this sex difference is due to greater cell proliferation and/or survival in females. Female and male rats received the cell birthdate marker 5-bromo-2'-deoxyuridine (BrdU; 200 mg/kg, ip) on postnatal day (P) 30; brains were collected at short and long intervals after BrdU administration to assess cell proliferation and survival, respectively. Overall, females had more BrdU-immunoreactive cells in the AVPV than did males, with no sex differences in the rate of cell attrition over time. Thus, the sex difference in pubertal addition of AVPV cells appears to be due to greater cell proliferation in females. Next, to determine the phenotype of pubertally born AVPV cells, daily BrdU injections were given to female rats on P28-56, and tissue was collected on P77 to assess colocalization of BrdU and markers for mature neurons or glia. Of the pubertally born AVPV cells, approximately 15% differentiated into neurons, approximately 19% into astrocytes, and approximately 23% into microglia. Thus, both neuro- and gliogenesis occur in the pubertal female rat AVPV and potentially contribute to maturation of female reproductive function. PMID:27145006

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

    Science.gov (United States)

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

    2015-05-01

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

  4. 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....

  5. Gastrointestinal stromal tumor and mitosis, pay attention

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2003-01-01

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

  7. Mapping and morphometric analysis of synapses and spines on fusiform cells in the dorsal cochlear nucleus.

    Science.gov (United States)

    Salloum, Rony H; Chen, Guoyou; Velet, Liliya; Manzoor, Nauman F; Elkin, Rachel; Kidd, Grahame J; Coughlin, John; Yurosko, Christopher; Bou-Anak, Stephanie; Azadi, Shirin; Gohlsch, Stephanie; Schneider, Harold; Kaltenbach, James A

    2014-01-01

    Fusiform cells are the main integrative units of the mammalian dorsal cochlear nucleus (DCN), collecting and processing inputs from auditory and other sources before transmitting information to higher levels of the auditory system. Despite much previous work describing these cells and the sources and pharmacological identity of their synaptic inputs, information on the three-dimensional organization and utltrastructure of synapses on these cells is currently very limited. This information is essential since an understanding of synaptic plasticity and remodeling and pathologies underlying disease states and hearing disorders must begin with knowledge of the normal characteristics of synapses on these cells, particularly those features that determine the strength of their influence on the various compartments of the cell. Here, we employed serial block face scanning electron microscopy (SBFSEM) followed by 3D reconstructions to map and quantitatively characterize synaptic features on DCN fusiform cells. Our results reveal a relative sparseness of synapses on the somata of fusiform cells but a dense distribution of synapses on apical and basal dendrites. Synapses on apical dendrites were smaller and more numerous than on basal dendrites. The vast majority of axosomatic terminals were found to be linked to other terminals connected by the same axon or different branches of the same axon, suggesting a high degree of divergent input to fusiform cells. The size of terminals was correlated with the number of mitochondria and with the number of active zones, which was highly correlated with the number of postsynaptic densities, suggesting that larger terminals exert more powerful influence on the cell than smaller terminals. These size differences suggest that the input to basal dendrites, most likely those from the auditory nerve, provide the most powerful sources of input to fusiform cells, while those to apical dendrites (e.g., parallel fiber) are weaker but more

  8. Mapping and Morphometric Analysis of Synapses and Spines on Fusiform Cells in the Dorsal Cochlear Nucleus.

    Directory of Open Access Journals (Sweden)

    Rony H. Salloum

    2014-09-01

    Full Text Available Fusiform cells are the main integrative units of the mammalian dorsal cochlear nucleus (DCN, collecting and processing inputs from auditory and other sources before transmitting information to higher levels of the auditory system. Despite much previous work describing these cells and the sources and pharmacological identity of their synaptic inputs, information on the 3- dimensional organization and utltrastructure of synapses on these cells is currently very limited. This information is essential since an understanding of synaptic plasticity and remodeling and pathologies underlying disease states and hearing disorders must begin with knowledge of the normal characteristics of synapses on these cells, particularly those features that determine the strength of their influence on the various compartments of the cell. Here, we employed serial block face scanning electron microscopy (SBFSEM followed by 3D reconstructions to map and quantitatively characterize synaptic features on DCN fusiform cells. Our results reveal a relative sparseness of synapses on the somata of fusiform cells but a dense distribution of synapses on apical and basal dendrites. Synapses on apical dendrites were smaller and more numerous than on basal dendrites. The vast majority of axosomatic terminals were found to be linked to other terminals connected by the same axon or different branches of the same axon, suggesting a high degree of divergent input to fusiform cells. The size of terminals was correlated with the number of mitochondria and with the number of active zones, which was highly correlated with the number of postsynaptic densities, suggesting that larger terminals exert more powerful influence on the cell than smaller terminals. These size differences suggest that the input to basal dendrites, most likely those from the auditory nerve, provide the most powerful sources of input to fusiform cells, while those to apical dendrites (e.g., parallel fiber are weaker

  9. 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.

  10. EphB4 localises to the nucleus of prostate cancer cells

    International Nuclear Information System (INIS)

    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

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

    Directory of Open Access Journals (Sweden)

    Tao eSun

    2012-09-01

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

  12. 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.

  13. Cycling with BRCA2 from DNA repair to mitosis

    International Nuclear Information System (INIS)

    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

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

    OpenAIRE

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

    2016-01-01

    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 dec...

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

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Daliri

    2012-02-01

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

  16. 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

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

    Science.gov (United States)

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

    2012-08-15

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

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

    Science.gov (United States)

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

    2015-01-01

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

  19. Agrobacterium tumefaciens and A. rhizogenes use different proteins to transport bacterial DNA into the plant cell nucleus.

    Science.gov (United States)

    Ream, Walt

    2009-07-01

    Agrobacterium tumefaciens and A. rhizogenes transport single-stranded DNA (ssDNA; T-strands) and virulence proteins into plant cells through a type IV secretion system. DNA transfer initiates when VirD2 nicks border sequences in the tumour-inducing plasmid, attaches to the 5' end, and pilots T-strands into plant cells. Agrobacterium tumefaciens translocates ssDNA-binding protein VirE2 into plant cells where it targets T-strands into the nucleus. Some A. rhizogenes strains lack VirE2 but transfer T-strands efficiently due to the GALLS gene, which complements an A. tumefaciens virE2 mutant. VirE2 and full-length GALLS (GALLS-FL) contain nuclear localization sequences that target these proteins to the plant cell nucleus. VirE2 binds cooperatively to T-strands allowing it to move ssDNA without ATP hydrolysis. Unlike VirE2, GALLS-FL contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. VirE2 may accumulate in the nucleus and pull T-strands into the nucleus using the force generated by cooperative DNA binding. GALLS-FL accumulates inside the nucleus where its predicted ATP-dependent strand transferase may pull T-strands into the nucleus. These different mechanisms for nuclear import of T-strands may affect the efficiency and quality of transgenic events in plant biotechnology applications. PMID:21255274

  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. 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.

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

    Directory of Open Access Journals (Sweden)

    Klein George

    2007-04-01

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

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

    OpenAIRE

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

    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 m...

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

    Science.gov (United States)

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

    2016-01-15

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

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

    Science.gov (United States)

    Khan, Shahanavaj; Zakariah, Mohammed; Palaniappan, Sellappan

    2016-08-01

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

  6. Hibernation as a Far-Reaching Programme for Cell Nucleus Activity Modulation

    Science.gov (United States)

    Malatesta, M.; Biggiogera, M.; Zancanaro, C.

    Maintaining part of the crew under hypometabolic conditions could help with the problems associated with long-term space missions. In the natural world, hibernators represent the most suitable model for a hypometabolic state. These animals are, in fact, able to drastically reduce all metabolic and physiological activities under adverse environmental conditions, but they can rapidly leave the depressed metabolic state as soon as the environment becomes favourable. Hibernators' cellular machinery must therefore undergo adaptive morpho-functional modifications to allow survival. Our studies on tissues of hibernating dormice revealed that the cell nucleus undergoes an important structural reorganisation during the hypometabolic period. Interestingly, despite the drastic reduction in pre-mRNA transcriptional and processing rate, cell nuclei of hibernating dormice never showed features typical of quiescence. Recent analyses revealed that pre-mRNA processing factors undergo an intranuclear redistribution which varies in different tissues. This suggests a programmed intranuclear reorganization of such molecules aimed to an efficient and rapid restoration of pre-mRNA processing upon arousal. Natural hibernation therefore appears as a highly programmed hypometabolic state rather than a simple fall of metabolic and physiological functions.

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

    Science.gov (United States)

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

    1968-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Akane Nakano

    2013-01-01

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

  9. 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

    2015-11-01

    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.

  10. 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.

  11. 孔雀石绿对蚕豆根尖细胞有丝分裂的影响%Effect of Malachite Green on Mitosis of Vicia faba Root Tip Cell

    Institute of Scientific and Technical Information of China (English)

    于淑池; 沈燕霞; 池玥兰; 吴霞

    2011-01-01

    [Objective] The research aimed to study the effect of malachite green (MG) on mitosis of Vicia faba root tip cell. [Method] Dif ferent concentrations of malachite green were used to treat Viciafaba root lip cell for 12.24,36,48 and 72 h.The mitosis of Vieia fa a root tip ceil was observed by conventional chromosome compression technology ,and the mitotic index ( MI) of each treatment group was counted. [ Re sult] As the treated time prolonged,MI presented the M-shaped variation trend,which illustrated that Vicia faba root tip had a adaptive process to MC. As the concentration of MC increased,Ml gradually rote. When the concentration of MG was during 0.50 - 1. 00 mg/L, Ml was the maximum. When the concentration of MG further increased,MI decreased. It showed that MG was favorable for the plant growth in the certain concentration range. [Conclusion] The research could provide the basis for the reasonable utilization of MG,and the reference for evaluating the environmental harm of MG.%[目的]研究孔雀石绿对蚕豆根尖细胞有丝分裂的影响.[方法]采用不同浓度的孔雀石绿溶液处理蚕豆根尖细胞12、24、36、48和72 h,通过常规染色体压片技术,观察蚕豆根尖细胞有丝分裂,并计算各处理组的有丝分裂指数.[结果]随着处理时间的延长,有丝分裂指数呈M型变化趋势,说明蚕豆根尖对孔雀石绿有一个适应过程.随着孔雀石绿浓度的升高,有丝分裂指数逐渐升高,在浓度为0.50~1.00 mg/L时,有丝分裂指数整体达到顶点;随着孔雀石绿浓度的进一步升高,有丝分裂指数有所下降,说明孔雀石绿在一定浓度范围内对植物生长有利.[结论]该研究可以为孔雀石绿的合理利用提供依据,并为评价孔雀石绿对环境的危害提供参考.

  12. 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.

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

    Science.gov (United States)

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

    2016-08-01

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

  14. Descending projections from auditory cortex to excitatory and inhibitory cells in the nucleus of the brachium of the inferior colliculus

    Directory of Open Access Journals (Sweden)

    Jeffrey Garrett Mellott

    2014-10-01

    Full Text Available Descending projections from the auditory cortex (AC terminate in subcortical auditory centers from the medial geniculate nucleus (MG to the cochlear nucleus, allowing the AC to modulate the processing of acoustic information at many levels of the auditory system. The nucleus of the brachium of the inferior colliculus (NBIC is a large midbrain auditory nucleus that is a target of these descending cortical projections. The NBIC is a source of several auditory projections, including an ascending projection to the MG. This ascending projection appears to originate from both excitatory and inhibitory NBIC cells, but whether the cortical projections contact either of these cell groups is unknown. In this study, we first combined retrograde tracing and immunochemistry for glutamic acid decarboxylase (GAD, a marker of GABAergic cells to identify GABAergic and non-GABAergic NBIC projections to the MG. Our first result is that GAD-immunopositive cells constitute ~17% of the NBIC to MG projection. We then used anterograde labeling and electron microscopy to examine the AC projection to the NBIC. Our second result is that cortical boutons in the NBIC form synapses with round vesicles and asymmetric synapses, consistent with excitatory effects. Finally, we combined fluorescent anterograde labeling of corticofugal axons with immunochemistry and retrograde labeling of NBIC cells that project to the MG. These final results suggest first that AC axons contact both GAD-negative and GAD-positive NBIC cells and, second, that some of cortically-contacted cells project to the MG. Overall, the results imply that corticofugal projections can modulate both excitatory and inhibitory ascending projections from the NBIC to the auditory thalamus.

  15. 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.

  16. Evaluation of CD24 as a marker to rapidly define the mesenchymal stem cell phenotype and its differentiation in human nucleus pulposus

    Institute of Scientific and Technical Information of China (English)

    Guan Xiaoming; Ma Xun; Zhang Li; Feng Haoyu; Ma Zhuo

    2014-01-01

    Background Recent studies have indicated that human nucleus pulposus contain mesenchymal stem cells (NP-MSCs).However,the immunophenotypic variation of NP-MSCs in vitro was unclear.The present study was conducted to address the immunophenotypic variation of mesenchymal stem cells in nucleus pulposus under continuous proliferation in vitro and show the difference between mesenchymal stem cells and nucleus pulposus cell.Methods Tissue samples were obtained from thoracolumbar burst fracture patients and degenerative disc disease patients who underwent discectomy and fusion procedures.Flow cytometric and laser scanning confocal microscope (LSCM) were used to detect the variation of mesenchymal stem cells in nucleus pulposus which were expressing CD105 and CD24 in condition with or without transforming growth factor β1 (TGF-β1).Results More than 90% of the analyzed primary cells of mesenchymal stem cells in nucleus pulposus fulfilled the general immunophenotyping criteria for MSCs,such as CD44,CD105 and CD29,but the marker of mature NP cells characterized as CD24 was negative.In continuous cultures,the proportion of mesenchymal stem cells which were expressing CD44,CD105 and CD29 in nucleus pulposus gradually decreased.The mesenchymal stem cells in nucleus pulposus cells were positive for CD105 and CD29,with slight positivity for CD44.The CD24 expression gradually increased in proliferation.Biparametric flow cytometry and laser scanning confocal microscopy confirmed the presence of cells which were expressing CD105 and CD24 independently,and only a small part of cells expressed both CD105 and CD24 simultaneously.TGF-β1 could stimulate mesenchymal stem cells in nucleus pulposus to express CD24.Conclusions Non-degenerative and degenerative NP contains mesechymal stem cells.The variation of CD24 can be used as a marker to identify the NP-MSCs differentiation into NP-like cells.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    Science.gov (United States)

    Erwin, C; Brown, D T

    1983-02-01

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

  20. Emi1 is needed to couple DNA replication with mitosis but does not regulate activation of the mitotic APC/C

    OpenAIRE

    Di Fiore, Barbara; Pines, Jonathon

    2007-01-01

    Ubiquitin-mediated proteolysis is critical for the alternation between DNA replication and mitosis and for the key regulatory events in mitosis. The anaphase-promoting complex/cyclosome (APC/C) is a conserved ubiquitin ligase that has a fundamental role in regulating mitosis and the cell cycle in all eukaryotes. In vertebrate cells, early mitotic inhibitor 1 (Emi1) has been proposed as an important APC/C inhibitor whose destruction may trigger activation of the APC/C at mitosis. However, in t...

  1. Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G₁ phase in CHO cells.

    Science.gov (United States)

    van Opstal, Angélique; Bijvelt, José; van Donselaar, Elly; Humbel, Bruno M; Boonstra, Johannes

    2012-04-01

    Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase. PMID:22251027

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

    Science.gov (United States)

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

    2016-08-01

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

  3. 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...

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

    Science.gov (United States)

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

    2013-09-01

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

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

    Science.gov (United States)

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

    2013-10-01

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

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

    Science.gov (United States)

    Gheghiani, Lilia; Gavet, Olivier

    2014-02-01

    Mitosis has been studied since the early 1880s as a key event of the cell division cycle where remarkable changes in cellular architecture take place and ultimately lead to an equal segregation of duplicated chromosomes into two daughter cells. A detailed description of the complex and highly ordered cellular events taking place is now available. Many regulators involved in key steps including entry into mitosis, nuclear envelope breakdown, microtubule (MT) spindle formation, and chromosome attachment, as well as mitotic exit and cytokinesis, have also been identified. However, understanding the precise spatio-temporal contribution of each regulator in the cell reorganization process has been technically challenging. This review will focus on a number of recent advances in our understanding of the spatial distribution of protein activities and the temporal regulation of their activation and inactivation during entry and progression through mitosis by the use of intramolecular Förster resonance energy transfer (FRET)-based biosensors. PMID:24421267

  7. Two S-phase checkpoint systems, one involving the function of both BIME and Tyr15 phosphorylation of p34cdc2, inhibit NIMA and prevent premature mitosis.

    OpenAIRE

    Ye, X S; Fincher, R R; Tang, A.; O'Donnell, K; Osmani, S A

    1996-01-01

    We demonstrate that there are at least two S-phase checkpoint mechanisms controlling mitosis in Aspergillus. The first responds to the rate of DNA replication and inhibits mitosis via tyrosine phosphorylation of p34cdc2. Cells unable to tyrosine phosphorylate p34cdc2 are therefore viable but are unable to tolerate low levels of hydroxyurea and prematurely enter lethal mitosis when S-phase is slowed. However, if the NIMA mitosis-promoting kinase is inactivated then non-tyrosine-phosphorylated ...

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    Florian Arnhold

    2015-02-01

    Full Text Available 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.

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

    Science.gov (United States)

    Paul, Angshuman; Mukherjee, Dipti Prasad

    2015-11-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

  13. A novel function of Rab5 in mitosis

    OpenAIRE

    Lanzetti, Letizia

    2012-01-01

    Several membrane trafficking proteins have been shown to participate in spindle assembly and stability during mitosis. Despite the fact that the role of some of them has been clarified, the requirement for these molecules in mitosis is still poorly understood.

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

    Science.gov (United States)

    Woo, H H; Hawes, M C

    1997-12-01

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

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

    Science.gov (United States)

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

    2016-01-01

    Transient inactivation of the cdc25(+) gene product by manipulation of the culture temperature for cdc25-22 cells is the most commonly exploited approach to mitotic synchronization in fission yeast. Because Cdc25 removes the inhibitory phosphate placed on Cdk1 by Wee1, inactivation of Cdc25 arrests cells at the G2/M boundary. Incubation at the restrictive temperature of 36°C for just over one generation time forces all cells in the culture to accumulate at the G2/M boundary. Restoration of Cdc25 function via a return to the permissive temperature or chemical inhibition of Wee1 activity at 36°C can then promote a highly synchronous wave of cell division throughout the culture. These approaches can be performed on any scale and thus support simultaneous assessment of numerous events within a single culture. After describing this simple and widely applicable procedure, we discuss frequently overlooked issues that can have a considerable impact on the interpretation of data from cdc25-22 induction-synchronized cultures. PMID:27480720

  16. 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...

  17. Nonchromosomal cytogenetic analysis of mammal somatic cells

    Directory of Open Access Journals (Sweden)

    Kovalova O. А.

    2013-01-01

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

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

    Science.gov (United States)

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

    1995-01-01

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

  19. Antioxidants in aqueous extract of Myristicafragrans (Houtt.) suppress mitosis and cyclophosphamide-induced chromosomal aberrations in Allium cepa L.cells

    Institute of Scientific and Technical Information of China (English)

    Akeem AKINBORO; Kamaruzaman Bin MOHAMED; Mohd Zaini ASMAWI; Shaida Fariza SULAIMAN; Othman Ahmad SOFIMAN

    2011-01-01

    In this study,freeze-dried water extract from the leaves of Myristica fragrans (Houtt.) was tested for mutagenic and antimutagenic potentials using the Allium cepa assay.Freeze-dried water extract alone and its combination with cyclophosphamide (CP) (50 mg/kg) were separately dissolved in tap water at 500,1000,2000,and 4000 mg/kg.Onions (A.cepa) were suspended in the solutions and controls for 48 h in the dark.Root tips were prepared for microscopic evaluation.2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radicals' scavenging power of the extract was tested using butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) as standards.Water extract of Myristica fragrans scavenged free radicals better than BHA,but worse than BHT.The extract alone,as well as in combination with CP suppressed cell division,and induced chromosomal aberrations that were insignificantly different from the negative control (P≤0.05).However,cytotoxic and mutagenic actions of CP were considerably suppressed.The observed effects on cell division and chromosomes of A.cepa may be principally connected to the antioxidant properties of the extract.The obtained results suggest mitodepressive and antimutagenic potentials of water extract of the leaves of M.fragrans as desirable properties of a promising anticancer agent.

  20. Mitosis and cell death in the optic lobes of workers, queens and drones of the honey bee (Apis mellifera) during metamorphosis

    Indian Academy of Sciences (India)

    Thaisa Cristina Roat; Carminda Da Cruz Landim

    2010-09-01

    Colonies of the honey bee, Apis mellifera, consist of males and two female castes: workers and queens. The castes and males from A. mellifera have a distinct morphology, physiology and behaviour that correlate with their roles in the society and are characterized by some brain polymorphisms. Compound eyes are one of the characteristics that differ among the castes and sexes. A. mellifera is a holometabolous insect; therefore, the development of adult organs during metamorphosis, which will produce these differences, requires the precise coordination of three main programmed cellular processes: proliferation, differentiation and death. These processes take place simultaneously during pupation. Our purpose was to investigate cell division and death in the optic lobes (OL) of workers, queens and males during pupation to identify how the differences in the compound eyes in adults of these classes are achieved. The results showed that OL differentiation follows a similar pattern in the three classes of individuals studied, without structural differences in their development. The main non-structural differences involve cell division, mortality rates and timing. The results suggest a modelling of the brain during differentiation, which contributes to the specific functions of each individual class.

  1. Mitosis and cell death in the optic lobes of workers, queens and drones of the honey bee (Apis mellifera) during metamorphosis.

    Science.gov (United States)

    Roat, Thaisa Cristina; Landim, Carminda da Cruz

    2010-09-01

    Colonies of the honey bee, Apis mellifera, consist of males and two female castes: workers and queens. The castes and males from A. mellifera have a distinct morphology, physiology and behaviour that correlate with their roles in the society and are characterized by some brain polymorphisms. Compound eyes are one of the characteristics that differ among the castes and sexes. A. mellifera is a holometabolous insect; therefore, the development of adult organs during metamorphosis, which will produce these differences, requires the precise coordination of three main programmed cellular processes: proliferation, differentiation and death. These processes take place simultaneously during pupation. Our purpose was to investigate cell division and death in the optic lobes (OL) of workers, queens and males during pupation to identify how the differences in the compound eyes in adults of these classes are achieved. The results showed that OL differentiation follows a similar pattern in the three classes of individuals studied, without structural differences in their development. The main non-structural differences involve cell division, mortality rates and timing. The results suggest a modelling of the brain during differentiation, which contributes to the specific functions of each individual class.

  2. 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

  3. PKCα translocation from mitochondria to nucleus is closely related to induction of apoptosis in gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    WU; Qiao(吴乔); LIU; Su(刘苏); DING; Liang(丁亮); YE; Xiaofeng(叶晓峰); SU; Wenjin(苏文金)

    2002-01-01

    PKCs have been implicated in the regulation of cellular differentiation, proliferation, apoptosis and signal transduction. It was demonstrated in this study that PKC? was located both at mitochondria and in cytosol in gastric cancer cell line BGC-823. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the translocation of PKCα from both mitochondria and cytosol to nucleus as clearly shown by laser-scanning-confocal microscopy, while the protein level of PKCα was not changed by TPA treatment as detected by Western blot. The results also revealed that TPA-induced translocation of PKCα was in close association with apoptosis induction, and such association was further affirmed by other experiments where various apoptotic stimuli and specific inhibitors of PKC were used. Taken together, these findings indicate that translocation of PKCα from both mitochondria and cytosol to nucleus in gastric cancer cell is accompanied by induction of apoptosis, and may imply a new mechanism of the potential linking between cell apoptosis and PKCα translocation.

  4. Differentiation of rat adipose tissue-derived mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro

    Institute of Scientific and Technical Information of China (English)

    XIE Li-wei; FANG Huang; CHEN An-min; LI Feng

    2009-01-01

    Objective: To differentiate rat adipose tissue-derived mesenchymal stem cells (ADSCs) into cells with a nucleus pulposus-like phenotype in vitro,so as to lay a foundation for the cell-based transplantation therapy of degenerated intervertebral discs.Methods: Rat ADSCs were isolated only from the subcutaneous inguinal region and purified by limited dilution.ADSCs of the third passages were analyzed by fluorescence activated cell sorter (FACS) to detect the cell surface markers (Sca-1,CD44,CD45,CD11b).To induce ADSCs towards a nucleus pulposus-like phenotype,ADSCs were immobilized in 3-dimensional alginate hydrogels and cultured in an inducing medium containing transforming growth factor-betal (TGF-β1) under hypoxia (2% O2),while control groups under normoxia (21% O2) in alginate beads in medium with or without the presence of TGF-β1.Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was carded out to evaluate phenotypic and biosynthetic activities in the process of differentiation.Meanwhile,Alcian blue staining were used to detect the formation of sulfated glycosaminoglycans (GAGs) in the differentiated cells.Results: The purified ADSCs were fibroblast-like and proliferated rapidly in vitro.The flow cytometry showed that ADSCs were positive for Sea-1 and CD44,negative for CD45 and CD11b.The results of RT-PCR manifested that the gene expressions of Sox-9,aggrecan and collagen Ⅱ,which were chondrocyte specific,were upregulated in medium containing TGF-β1 under hypoxia (2% O2).Likewise,gene expression of HIF-la,which was characteristics of intervertebral discs,was also upregulated.Simultaneously,Alcian blue staining exhibited the formation of many GAGs.Conclusions: The approach in our experiment is a simple and effective way to acquire a large quantity of homogenous ADSCs.Rat ADSCs can be differentiated into nucleus pulposus-like cells.ADSCs may replace bone marrow mesenchymal stem cells as a new kind of seed cells in regeneration of

  5. 3D/4D architecture of chromosomal break point regions in the cell nucleus following irradiation of normal cells and tumor cells

    International Nuclear Information System (INIS)

    The development of an effective analytical methodology for a correct description of oncogenic chromosomal aberrations is the challenge of medical radiobiology with respect to preventive therapeutic methods. Scope of the project was a better understanding of the behavior of break point regions dependent on the genome loci, the chromatin folding, the involved repair proteins and the beam quality with respect to an improvement and an efficient prognosis of the health consequences following radiation exposure. New microscopic insights in the normal cell nucleus are supposed to allow a better understanding of the spatial interactions on a molecular scale.

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

    DEFF Research Database (Denmark)

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

    2004-01-01

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

  7. Y-like retinal ganglion cells innervate the dorsal raphe nucleus in the Mongolian gerbil (Meriones unguiculatus.

    Directory of Open Access Journals (Sweden)

    Liju Luan

    Full Text Available BACKGROUND: The dorsal raphe nucleus (DRN of the mesencephalon is a complex multi-functional and multi-transmitter nucleus involved in a wide range of behavioral and physiological processes. The DRN receives a direct input from the retina. However little is known regarding the type of retinal ganglion cell (RGC that innervates the DRN. We examined morphological characteristics and physiological properties of these DRN projecting ganglion cells. METHODOLOGY/PRINCIPAL FINDINGS: The Mongolian gerbils are highly visual rodents with a diurnal/crepuscular activity rhythm. It has been widely used as experimental animals of various studies including seasonal affective disorders and depression. Young adult gerbils were used in the present study. DRN-projecting RGCs were identified following retrograde tracer injection into the DRN, characterized physiologically by extracellular recording and morphologically after intracellular filling. The result shows that DRN-projecting RGCs exhibit morphological characteristics typical of alpha RGCs and physiological response properties of Y-cells. Melanopsin was not detected in these RGCs and they show no evidence of intrinsic photosensitivity. CONCLUSIONS/SIGNIFICANCE: These findings suggest that RGCs with alpha-like morphology and Y-like physiology appear to perform a non-imaging forming function and thus may participate in the modulation of DRN activity which includes regulation of sleep and mood.

  8. Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis.

    Science.gov (United States)

    Pillai, Smitha; Nguyen, Jonathan; Johnson, Joseph; Haura, Eric; Coppola, Domenico; Chellappan, Srikumar

    2015-01-01

    TANK Binding Kinase 1 (TBK1) is a non-canonical IκB kinase that contributes to KRAS-driven lung cancer. Here we report that TBK1 plays essential roles in mammalian cell division. Specifically, levels of active phospho-TBK1 increase during mitosis and localize to centrosomes, mitotic spindles and midbody, and selective inhibition or silencing of TBK1 triggers defects in spindle assembly and prevents mitotic progression. TBK1 binds to the centrosomal protein CEP170 and to the mitotic apparatus protein NuMA, and both CEP170 and NuMA are TBK1 substrates. Further, TBK1 is necessary for CEP170 centrosomal localization and binding to the microtubule depolymerase Kif2b, and for NuMA binding to dynein. Finally, selective disruption of the TBK1-CEP170 complex augments microtubule stability and triggers defects in mitosis, suggesting that TBK1 functions as a mitotic kinase necessary for microtubule dynamics and mitosis.

  9. PTEN regulates EG5 to control spindle architecture and chromosome congression during mitosis.

    Science.gov (United States)

    He, Jinxue; Zhang, Zhong; Ouyang, Meng; Yang, Fan; Hao, Hongbo; Lamb, Kristy L; Yang, Jingyi; Yin, Yuxin; Shen, Wen H

    2016-01-01

    Architectural integrity of the mitotic spindle is required for efficient chromosome congression and accurate chromosome segregation to ensure mitotic fidelity. Tumour suppressor PTEN has multiple functions in maintaining genome stability. Here we report an essential role of PTEN in mitosis through regulation of the mitotic kinesin motor EG5 for proper spindle architecture and chromosome congression. PTEN depletion results in chromosome misalignment in metaphase, often leading to catastrophic mitotic failure. In addition, metaphase cells lacking PTEN exhibit defects of spindle geometry, manifested prominently by shorter spindles. PTEN is associated and co-localized with EG5 during mitosis. PTEN deficiency induces aberrant EG5 phosphorylation and abrogates EG5 recruitment to the mitotic spindle apparatus, leading to spindle disorganization. These data demonstrate the functional interplay between PTEN and EG5 in controlling mitotic spindle structure and chromosome behaviour during mitosis. We propose that PTEN functions to equilibrate mitotic phosphorylation for proper spindle formation and faithful genomic transmission. PMID:27492783

  10. 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.

  11. 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...

  12. 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.

  13. Genetic cell targeting uncovers specific neuronal types and distinct subregions in the bed nucleus of the stria terminalis.

    Science.gov (United States)

    Nguyen, Amanda Q; Dela Cruz, Julie A D; Sun, Yanjun; Holmes, Todd C; Xu, Xiangmin

    2016-08-15

    The bed nucleus of the stria terminalis (BNST) plays an important role in fear, stress, and anxiety. It contains a collection of subnuclei delineated by gross cytoarchitecture features; however, there has yet to be a systematic examination of specific BNST neuronal types and their associated neurochemical makeup. The present study focuses on improved characterization of the anterior BNST based on differing molecular and chemical expression aided by mouse genetics. Specific Cre driver lines crossed with a fluorescent reporter line were used for genetic cell targeting and immunochemical staining. Using this new approach, we were able to robustly identify specific excitatory and inhibitory cell types in the BNST. The presence and distribution of excitatory neurons were firmly established; glutamatergic neurons in the anterior BNST accounted for about 14% and 31% of dorsal and ventral BNST cells, respectively. GABAergic neurons expressing different isoforms of glutamic acid decarboxylase were found to have differential subregional distributions. Almost no parvalbumin-expressing cells were found in the BNST, while somatostatin-expressing cells and calretinin-expressing cells account for modest proportions of BNST cells. In addition, vasoactive intestinal peptide-expressing axonal plexuses were prominent in the oval and juxtacapsular subregions. In addition, we discovered that corticotropin-releasing hormone-expressing cells contain GABAergic and glutamatergic subpopulations. Together, this study reveals new information on excitatory and inhibitory neurons in the BNST, which will facilitate genetic dissection and functional studies of BNST subregions. J. Comp. Neurol. 524:2379-2399, 2016. © 2016 Wiley Periodicals, Inc. PMID:26718312

  14. Inhibitory action of oestrogen on calcium-induced mitosis in rat bone marrow and thymus.

    Science.gov (United States)

    Smith, G R; Gurson, M L; Riddell, A J; Perris, A D

    1975-04-01

    In the male rat injections of CaCl-2 and MgCl-2 stimulated mitosis in bone marrow and thymus tissue. The magnesium salt was also mitogenic in the normal female, but calcium only exerted its mitogenic effect after ovariectomy. Oestradiol, but not progesterone replacement therpy abolished calcium-induced mitosis in the ovariectomized rat. The inability of calcium to stimulate cell division was also apparent in the thyroparathyroidectomized female rat, suggesting the oestradiol blockage did not operate via some indirect action on the calcium homeostatic hormones calcitonin or parathyroid hormone. When thymic lymphocytes derived from male or female rats were isolated and maintained in suspension, increased calcium or magnesium concentrations in the culture medium stimulated the entry of cells into mitosis. Addition of oestradiol to the culture medium abolished the mitogenic effect of increased calcium levels, but had no effect on magnesium-induced proliferation. These experiments suggested that oestradiol might act at the cell surface to prevent the influx of calcium but not magnesium ions into the interior of the cell and thus to block the sequence of biochemical events which led to the initiation of DNA synthesis and culminate in mitosis.

  15. Centrosomes split in the presence of impaired DNA integrity during mitosis

    NARCIS (Netherlands)

    Hut, HMJ; Lemstra, W; Blaauw, EH; van Cappellen, GWA; Kampinga, HH; Sibon, OCM

    2003-01-01

    A well-established function of centrosomes is their role in accomplishing a successful mitosis that gives rise to a pair of identical daughter cells. We recently showed that DNA replication defects and DNA damage in Drosophila embryos trigger centrosomal changes, but it remained unclear whether comp

  16. 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

  17. 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.

  18. Observation of Mitosis and Meiosis in Rice Cells by Simple Squash Method%简易压片法观察水稻有丝分裂和减数分裂行为

    Institute of Scientific and Technical Information of China (English)

    杨旭; 代西梅

    2009-01-01

    色体复制,减数分裂II 过程中仅发生了细胞分裂.[结论]改进后的水稻染色体压片技术能够获得准确的染色体资料,可为育种和遗传操作提供依据.%[Objective] The aim of this study was to establish a feasible squashing technique for chromosome and obtain data of rice chromosome. [Method] With the materials of rice root tips and anther, the specimen was prepared by the modified squash method, and microscopic observation of mitosis and meiosis in rice cells was also carried out. [Result] Mitosis in rice cells included interphase, prophase, metaphase, anaphase and telophase. Chromosome in metaphase shortened to the minmum, which was a good time for observing and investigating chromosome. However, meiosis in rice cells included meiosis I and meiosis II. Chromosome replication appeared in meiosis I, while cell division only appeared in meiosis II. [Conclusion] The modified squashing technique for rice chromosome can obtain accurate data of rice chromosome, which provides evidence for genetic breeding.

  19. Characterization of the tight junction protein ZO-2 localized at the nucleus of epithelial cells.

    Science.gov (United States)

    Jaramillo, Blanca Estela; Ponce, Arturo; Moreno, Jacqueline; Betanzos, Abigail; Huerta, Miriam; Lopez-Bayghen, Esther; Gonzalez-Mariscal, Lorenza

    2004-07-01

    ZO-2 is a MAGUK protein that in confluent epithelial sheets localizes at tight junctions (TJ) whereas in sparse cultures accumulates in clusters at the nucleus. Here, we have characterized several nuclear properties of ZO-2. We observe that ZO-2 is present in the nuclear matrix and co-immunoprecipitates with lamin B(1) and actin from the nuclei of sparse cultures. We show that ZO-2 presents several NLS at its amino region, that when deleted, diminish the nuclear import of the ZO-2 amino segment and impair the ability of the region to regulate the transcriptional activity of promoters controlled by AP-1. Several RS repeats are detected in the ZO-2 amino segment, however, their deletion does not preclude the display of a speckled nuclear pattern. ZO-2 displays two putative NES. However, only the second one appears to be functional, as when conjugated to ovalbumin (OV), it is able to translocate this protein from the nucleus to the cytoplasm in a leptomycin B-sensitive way.

  20. Culture of nucleus pulposus cells from intervertebral disc on self-assembling KLD-12 peptide hydrogel scaffold

    International Nuclear Information System (INIS)

    KLD-12 peptide is a new self-assembling biomaterial and it has been used as cell scaffold for cartilage repair. In this study, self-assembled KLD-12 peptide nanofiber was fabricated and the biocompatibility of this scaffold for nucleus pulposus (NP) cells was evaluated. The structure of this scaffold was characterized by atomic force microscopy (AFM). This hydrogel was structurally integral and homogeneous. KLD-12 peptide was able to self-assemble into nanofibers with a diameter of 10-30 nm (mean: 13.7 ± 4.7 nm) and a length of hundreds of nanometers. Two-week culture of rabbits NP cells in this scaffold showed that the self-assembled hydrogel maintained the live cell number by 93% and the cell viability increased gradually with the culture time. The expression of type II collagen mRNA was further confirmed by reverse transcription polymerase chain reaction (RT-PCR). The expression of type II collagen was high in the hydrogel, however, type I collagen expression was observed in few cells. Furthermore, GAG content increased gradually accompanied with the extension of culture time. In conclusion, this self-assembled nanofiber scaffold provided a conducive microenvironment for NP cell to survive and proliferate in vitro.

  1. Block copolymer micelles target Auger electron radiotherapy to the nucleus of HER2-positive breast cancer cells.

    Science.gov (United States)

    Hoang, Bryan; Reilly, Raymond M; Allen, Christine

    2012-02-13

    Intracellular trafficking of Auger electron emitting radionuclides to perinuclear and nuclear regions of cells is critical to realizing their full therapeutic potential. In the present study, block copolymer micelles (BCMs) were labeled with the Auger electron emitter indium-111 ((111)In) and loaded with the radiosensitizer methotrexate. HER2 specific antibodies (trastuzumab fab) and nuclear localization signal (NLS; CGYGPKKKRKVGG) peptides were conjugated to the surface of the BCMs to direct uptake in HER2 expressing cells and subsequent localization in the cell nucleus. Cell uptake and intracellular distribution of the multifunctional BCMs were evaluated in a panel of breast cancer cell lines with different levels of HER2 expression. Indeed cell uptake was found to be HER2 density dependent, confirming receptor-mediated internalization of the BCMs. Importantly, conjugation of NLS peptides to the surface of BCMs was found to result in a significant increase in nuclear uptake of the radionuclide (111)In. Successful nuclear targeting was shown to improve the antipoliferative effect of the Auger electrons as measured by clonogenic assays. In addition, a significant radiation enhancement effect was observed by concurrent delivery of low-dose MTX and (111)In in all breast cancer cell lines evaluated.

  2. 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.

  3. The nucleoporin Mlp2 is involved in chromosomal distribution during mitosis in trypanosomatids.

    Science.gov (United States)

    Morelle, Christelle; Sterkers, Yvon; Crobu, Lucien; MBang-Benet, Diane-Ethna; Kuk, Nada; Portalès, Pierre; Bastien, Patrick; Pagès, Michel; Lachaud, Laurence

    2015-04-30

    Nucleoporins are evolutionary conserved proteins mainly involved in the constitution of the nuclear pores and trafficking between the nucleus and cytoplasm, but are also increasingly viewed as main actors in chromatin dynamics and intra-nuclear mitotic events. Here, we determined the cellular localization of the nucleoporin Mlp2 in the 'divergent' eukaryotes Leishmania major and Trypanosoma brucei. In both protozoa, Mlp2 displayed an atypical localization for a nucleoporin, essentially intranuclear, and preferentially in the periphery of the nucleolus during interphase; moreover, it relocated at the mitotic spindle poles during mitosis. In T. brucei, where most centromeres have been identified, TbMlp2 was found adjacent to the centromeric sequences, as well as to a recently described unconventional kinetochore protein, in the periphery of the nucleolus, during interphase and from the end of anaphase onwards. TbMlp2 and the centromeres/kinetochores exhibited a differential migration towards the poles during mitosis. RNAi knockdown of TbMlp2 disrupted the mitotic distribution of chromosomes, leading to a surprisingly well-tolerated aneuploidy. In addition, diploidy was restored in a complementation assay where LmMlp2, the orthologue of TbMlp2 in Leishmania, was expressed in TbMlp2-RNAi-knockdown parasites. Taken together, our results demonstrate that Mlp2 is involved in the distribution of chromosomes during mitosis in trypanosomatids.

  4. A polycomb group protein is retained at specific sites on chromatin in mitosis.

    Directory of Open Access Journals (Sweden)

    Nicole E Follmer

    Full Text Available Epigenetic regulation of gene expression, including by Polycomb Group (PcG proteins, may depend on heritable chromatin states, but how these states can be propagated through mitosis is unclear. Using immunofluorescence and biochemical fractionation, we find PcG proteins associated with mitotic chromosomes in Drosophila S2 cells. Genome-wide sequencing of chromatin immunoprecipitations (ChIP-SEQ from mitotic cells indicates that Posterior Sex Combs (PSC is not present at well-characterized PcG targets including Hox genes in mitosis, but does remain at a subset of interphase sites. Many of these persistent sites overlap with chromatin domain borders described by Sexton et al. (2012, which are genomic regions characterized by low levels of long range contacts. Persistent PSC binding sites flank both Hox gene clusters. We hypothesize that disruption of long-range chromatin contacts in mitosis contributes to PcG protein release from most sites, while persistent binding at sites with minimal long-range contacts may nucleate re-establishment of PcG binding and chromosome organization after mitosis.

  5. Live imaging of mitosis in the developing mouse embryonic cortex.

    Science.gov (United States)

    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 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.

  6. 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.

  7. On the properties and origin of the GABAB inhibitory postsynaptic potential recorded in morphologically identified projection cells of the cat dorsal lateral geniculate nucleus.

    Science.gov (United States)

    Soltesz, I; Lightowler, S; Leresche, N; Crunelli, V

    1989-01-01

    Intracellular recordings were performed from projection cells of the cat dorsal lateral geniculate nucleus in vitro to investigate the properties and origin of optic tract evoked inhibitory postsynaptic potentials mediated by GABAB receptors and their relationship to the physiologically different cell classes present in this nucleus. In all three main laminae of the dorsal lateral geniculate nucleus, stimulation of the optic tract evoked an excitatory postsynaptic potential followed by two inhibitory postsynaptic potentials. The first is a GABAA receptor mediated inhibitory postsynaptic potential since it was blocked by bicuculline, reversed in polarity following intracellular Cl- injection and had a reversal potential similar to the bicuculline sensitive hyperpolarizing effect of GABA. The second is a GABAB receptor mediated inhibitory postsynaptic potential. Its amplitude was not linearly related to membrane potential (maximal amplitude at -60 mV), it decreased when using frequencies of stimulation higher than 0.05 Hz and it was reversibly increased by addition of bicuculline to the perfusion medium. The reversal potential of GABAB inhibitory postsynaptic potentials was dependent on the extracellular K+ concentration but did not change in the presence of bicuculline or when recording with Cl- filled microelectrodes. While GABAA inhibitory postsynaptic potentials always abolished repetitive firing of projection cells, GABAB inhibitory postsynaptic potentials were able to block weak firing but unable to decrease strong activation of projection cells evoked by direct current injection. Optic tract evoked GABAB (as well as GABAA) inhibitory postsynaptic potentials could be recorded in slices which did not include the perigeniculate nucleus, thus indicating that they are generated by the interneurons of the dorsal lateral geniculate nucleus. Using intracellular injection of horseradish peroxidase, we have found that the GABAB inhibitory postsynaptic potentials are

  8. 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.

  9. Protein Phosphatases Involved in Regulating Mitosis: Facts and Hypotheses.

    Science.gov (United States)

    Kim, Hyun-Soo; Fernandes, Gary; Lee, Chang-Woo

    2016-09-01

    Almost all eukaryotic proteins are subject to post-translational modifications during mitosis and cell cycle, and in particular, reversible phosphorylation being a key event. The recent use of high-throughput experimental analyses has revealed that more than 70% of all eukaryotic proteins are regulated by phosphorylation; however, the mechanism of dephosphorylation, counteracting phosphorylation, is relatively unknown. Recent discoveries have shown that many of the protein phosphatases are involved in the temporal and spatial control of mitotic events, such as mitotic entry, mitotic spindle assembly, chromosome architecture changes and cohesion, and mitotic exit. This implies that certain phosphatases are tightly regulated for timely dephosphorylation of key mitotic phosphoproteins and are essential for control of various mitotic processes. This review describes the physiological and pathological roles of mitotic phosphatases, as well as the versatile role of various protein phosphatases in several mitotic events.

  10. Protein Phosphatases Involved in Regulating Mitosis: Facts and Hypotheses.

    Science.gov (United States)

    Kim, Hyun-Soo; Fernandes, Gary; Lee, Chang-Woo

    2016-09-01

    Almost all eukaryotic proteins are subject to post-translational modifications during mitosis and cell cycle, and in particular, reversible phosphorylation being a key event. The recent use of high-throughput experimental analyses has revealed that more than 70% of all eukaryotic proteins are regulated by phosphorylation; however, the mechanism of dephosphorylation, counteracting phosphorylation, is relatively unknown. Recent discoveries have shown that many of the protein phosphatases are involved in the temporal and spatial control of mitotic events, such as mitotic entry, mitotic spindle assembly, chromosome architecture changes and cohesion, and mitotic exit. This implies that certain phosphatases are tightly regulated for timely dephosphorylation of key mitotic phosphoproteins and are essential for control of various mitotic processes. This review describes the physiological and pathological roles of mitotic phosphatases, as well as the versatile role of various protein phosphatases in several mitotic events. PMID:27669825

  11. Transcriptional Timers Regulating Mitosis in Early Drosophila Embryos.

    Science.gov (United States)

    Momen-Roknabadi, Amir; Di Talia, Stefano; Wieschaus, Eric

    2016-09-13

    The development of an embryo requires precise spatiotemporal regulation of cellular processes. During Drosophila gastrulation, a precise temporal pattern of cell division is encoded through transcriptional regulation of cdc25(string) in 25 distinct mitotic domains. Using a genetic screen, we demonstrate that the same transcription factors that regulate the spatial pattern of cdc25(string) transcription encode its temporal activation. We identify buttonhead and empty spiracles as the major activators of cdc25(string) expression in mitotic domain 2. The effect of these activators is balanced through repression by hairy, sloppy paired 1, and huckebein. Within the mitotic domain, temporal precision of mitosis is robust and unaffected by changing dosage of rate-limiting transcriptional factors. However, precision can be disrupted by altering the levels of the two activators or two repressors. We propose that the additive and balanced action of activators and repressors is a general strategy for precise temporal regulation of cellular transitions during development. PMID:27626650

  12. The ups and downs of somatic cell nucleus transfer (SCNT) in humans

    OpenAIRE

    Fulka, Josef; Langerova, Alena; Loi, Pasqualino; Ptak, Grazyna; Albertini, David; Fulka, Helena

    2013-01-01

    Achieving successful somatic cell nuclear transfer (SCNT) in the human and subhuman primate relative to other mammals has been questioned for a variety of technical and logistical issues. Here we summarize the gradual evolution of SCNT technology from the perspective of oocyte quality and cell cycle status that has recently led to the demonstration of feasibility in the human for deriving chromosomally normal stem cells lines. With these advances in hand, prospects for therapeutic cloning mus...

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

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xu Rui; LIU Yong Ai; SUN Fang; LI He; LEI Su Wen; WANG Ju Fang

    2016-01-01

    Objective To explore the role of p21 in ionizing radiation-induced changes in protein levels during the G2/M transition and long-term G2 arrest. Methods Protein expression levels were assessed by western blot in the human uveal melanoma 92-1 cells after treatment with ionizing radiation. Depletion of p21 was carried out by employing the siRNA technique. Cell cycle distribution was determined by flow cytometry combined with histone H3 phosphorylation at Ser28, an M-phase marker. Senescence was assessed by senescence-associated-β-galactosidase (SA-β-gal) staining combined with Ki67 staining, a cell proliferation marker. Results Accompanying increased p21, the protein levels of G2/M transition genes declined significantly in 92-1 cells irradiated with 5 Gy of X-rays. Furthermore, these irradiated cells were blocked at the G2 phase followed by cellular senescence. Depletion of p21 rescued radiation-induced G2 arrest as demonstrated by the upregulation of G2/M transition kinases, as well as the high expression of histone H3 phosphorylated at Ser28. Knockdown of p21 resulted in entry into mitosis of irradiated 92-1 cells. However, cells with serious DNA damage failed to undergo cytokinesis, leading to the accumulation of multinucleated cells. Conclusion Our results indicated that p21 was responsible for the downregulation of G2/M transition regulatory proteins and the bypass of mitosis induced by irradiation. Downregulation of p21 by siRNA resulted in G2-arrested cells entering into mitosis with serious DNA damage. This is the first report on elucidating the role of p21 in the bypass of mitosis.

  14. Transfer of a eubacteria-type cell division site-determining factor CrMinD gene to the nucleus from the chloroplast genome in Chlamydomonas reinhardtii

    Institute of Scientific and Technical Information of China (English)

    LIU WeiZhong; HU Yong; ZHANG RunJie; ZHOU WeiWei; ZHU JiaYing; LIU XiangLin; HE YiKun

    2007-01-01

    MinD is a ubiquitous ATPase that plays a crucial role in selection of the division site in eubacteria, chloroplasts, and probably Archaea. In four green algae, Mesostigma viride, Nephroselmis olivacea, Chlorella vulgaris and Prototheca wickerhamii, MinD homologues are encoded in the plastid genome. However, in Arabidopsis, MinD is a nucleus-encoded, chloroplast-targeted protein involved in chloroplast division, which suggests that MinD has been transferred to the nucleus in higher land plants. Yet the lateral gene transfer (LGT) of MinD from plastid to nucleus during plastid evolution remains poorly understood. Here, we identified a nucleus-encoded MinD homologue from unicellular green alga Chlamydomonas reinhardtii, a basal species in the green plant lineage. Overexpression of CrMinD in wild type E. coli inhibited cell division and resulted in the filamentous cell formation, clearly demonstrated the conservation of the MinD protein during the evolution of photosynthetic eukaryotes. The transient expression of CrMinD-egfp confirmed the role of CrMinD protein in the regulation of plastid division. Searching all the published plastid genomic sequences of land plants, no MinD homologues were found, which suggests that the transfer of MinD from plastid to nucleus might have occurred before the evolution of land plants.

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

    Science.gov (United States)

    Nielsen, Christian F; Huttner, Diana; Bizard, Anna H; Hirano, Seiki; Li, Tian-Neng; Palmai-Pallag, Timea; Bjerregaard, Victoria A; Liu, Ying; Nigg, Erich A; Wang, Lily Hui-Ching; Hickson, Ian D

    2015-01-01

    PICH is a SNF2 family DNA translocase that binds to ultra-fine DNA bridges (UFBs) in mitosis. Numerous roles for PICH have been proposed from protein depletion experiments, but a consensus has failed to emerge. Here, we report that deletion of PICH in avian cells causes chromosome structural abnormalities, and hypersensitivity to an inhibitor of Topoisomerase II (Topo II), ICRF-193. ICRF-193-treated PICH(-/-) cells undergo sister chromatid non-disjunction in anaphase, and frequently abort cytokinesis. PICH co-localizes with Topo IIα on UFBs and at the ribosomal DNA locus, and the timely resolution of both structures depends on the ATPase activity of PICH. Purified PICH protein strongly stimulates the catalytic activity of Topo II in vitro. Consistent with this, a human PICH(-/-) cell line exhibits chromosome instability and chromosome condensation and decatenation defects similar to those of ICRF-193-treated cells. We propose that PICH and Topo II cooperate to prevent chromosome missegregation events in mitosis.

  16. Mechanical trapping of the nucleus on micropillared surfaces inhibits the proliferation of vascular smooth muscle cells but not cervical cancer HeLa cells.

    Science.gov (United States)

    Nagayama, Kazuaki; Hamaji, Yumi; Sato, Yuji; Matsumoto, Takeo

    2015-07-16

    The interaction between cells and the extracellular matrix on a topographically patterned surface can result in changes in cell shape and many cellular functions. In the present study, we demonstrated the mechanical deformation and trapping of the intracellular nucleus using polydimethylsiloxane (PDMS)-based microfabricated substrates with an array of micropillars. We investigated the differential effects of nuclear deformation on the proliferation of healthy vascular smooth muscle cells (SMCs) and cervical cancer HeLa cells. Both types of cell spread normally in the space between micropillars and completely invaded the extracellular microstructures, including parts of their cytoplasm and their nuclei. We found that the proliferation of SMCs but not HeLa cells was dramatically inhibited by cultivation on the micropillar substrates, even though remarkable deformation of nuclei was observed in both types of cells. Mechanical testing with an atomic force microscope and a detailed image analysis with confocal microscopy revealed that SMC nuclei had a thicker nuclear lamina and greater expression of lamin A/C than those of HeLa cells, which consequently increased the elastic modulus of the SMC nuclei and their nuclear mechanical resistance against extracellular microstructures. These results indicate that the inhibition of cell proliferation resulted from deformation of the mature lamin structures, which might be exposed to higher internal stress during nuclear deformation. This nuclear stress-induced inhibition of cell proliferation occurred rarely in cancer cells with deformable nuclei. PMID:26054426

  17. DNA damage response during mitosis induces whole chromosome mis-segregation

    Science.gov (United States)

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

    2014-01-01

    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, DDR during mitosis inappropriately stabilizes k-MTs creating a link between s-CIN and w-CIN. PMID:25107667

  18. Activation of the c-fos gene in prodynorphin- and proenkephalin-expressing cells of nucleus tractus solitarius after seizures.

    Science.gov (United States)

    Kanter, R K; Erickson, J T; Millhorn, D E

    1994-10-01

    We performed studies to determine the anatomical regions and chemical phenotypes of neurons within the rat medulla oblongata activated by pentylenetetrazole-induced seizures. Activated cells were identified by their expression of the c-fos gene, detected by in situ hybridization for c-fos mRNA and immunocytochemistry for Fos protein. Activated cells were located predominantly in nucleus tractus solitarius (NTS), with c-fos mRNA appearing within 20 min after seizures (peak at 1-2 h), followed by Fos immunoreactivity visible at 1 h (peak at 2-4 h). Neither nonspecific noxious stimulation by intraperitoneal injection of saline nor brief exposure to hypoxic or hypercapnic gas mixtures to stimulate chemoreceptors reproduced this pattern of labeling. Prodynorphin or proenkephalin mRNA, detected by in situ hybridization, was colocalized with Fos immunoreactivity in many NTS cells. Thus, seizures activate neuronal pathways in the medulla oblongata which express genes for endogenous opioids. Potential long-term effects of seizures are suggested by the in situ hybridization finding that NTS prodynorphin mRNA increased 24 h after seizures compared to control levels. PMID:7957742

  19. Selective hair cell ablation and noise exposure lead to different patterns of changes in the cochlea and the cochlear nucleus.

    Science.gov (United States)

    Kurioka, Takaomi; Lee, Min Young; Heeringa, Amarins N; Beyer, Lisa A; Swiderski, Donald L; Kanicki, Ariane C; Kabara, Lisa L; Dolan, David F; Shore, Susan E; Raphael, Yehoash

    2016-09-22

    In experimental animal models of auditory hair cell (HC) loss, insults such as noise or ototoxic drugs often lead to secondary changes or degeneration in non-sensory cells and neural components, including reduced density of spiral ganglion neurons, demyelination of auditory nerve fibers and altered cell numbers and innervation patterns in the cochlear nucleus (CN). However, it is not clear whether loss of HCs alone leads to secondary degeneration in these neural components of the auditory pathway. To elucidate this issue, we investigated changes of central components after cochlear insults specific to HCs using diphtheria toxin receptor (DTR) mice expressing DTR only in HCs and exhibiting complete HC loss when injected with diphtheria toxin (DT). We showed that DT-induced HC ablation has no significant impacts on the survival of auditory neurons, central synaptic terminals, and myelin, despite complete HC loss and profound deafness. In contrast, noise exposure induced significant changes in synapses, myelin and CN organization even without loss of inner HCs. We observed a decrease of neuronal size in the auditory pathway, including peripheral axons, spiral ganglion neurons, and CN neurons, likely due to loss of input from the cochlea. Taken together, selective HC ablation and noise exposure showed different patterns of pathology in the auditory pathway and the presence of HCs is not essential for the maintenance of central synaptic connectivity and myelination. PMID:27403879

  20. 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...

  1. Electric fields generated by synchronized oscillations of microtubules, centrosomes and chromosomes regulate the dynamics of mitosis and meiosis

    Directory of Open Access Journals (Sweden)

    Zhao Yue

    2012-07-01

    Full Text Available Abstract Super-macromolecular complexes play many important roles in eukaryotic cells. Classical structural biological studies focus on their complicated molecular structures, physical interactions and biochemical modifications. Recent advances concerning intracellular electric fields generated by cell organelles and super-macromolecular complexes shed new light on the mechanisms that govern the dynamics of mitosis and meiosis. In this review we synthesize this knowledge to provide an integrated theoretical model of these cellular events. We suggest that the electric fields generated by synchronized oscillation of microtubules, centrosomes, and chromatin fibers facilitate several events during mitosis and meiosis, including centrosome trafficking, chromosome congression in mitosis and synapsis between homologous chromosomes in meiosis. These intracellular electric fields are generated under energy excitation through the synchronized electric oscillations of the dipolar structures of microtubules, centrosomes and chromosomes, three of the super-macromolecular complexes within an animal cell.

  2. Influence of cell geometry on division-plane positioning.

    Science.gov (United States)

    Minc, Nicolas; Burgess, David; Chang, Fred

    2011-02-01

    The spatial organization of cells depends on their ability to sense their own shape and size. Here, we investigate how cell shape affects the positioning of the nucleus, spindle and subsequent cell division plane. To manipulate geometrical parameters in a systematic manner, we place individual sea urchin eggs into microfabricated chambers of defined geometry (e.g., triangles, rectangles, and ellipses). In each shape, the nucleus is positioned at the center of mass and is stretched by microtubules along an axis maintained through mitosis and predictive of the future division plane. We develop a simple computational model that posits that microtubules sense cell geometry by probing cellular space and orient the nucleus by exerting pulling forces that scale to microtubule length. This model quantitatively predicts division-axis orientation probability for a wide variety of cell shapes, even in multicellular contexts, and estimates scaling exponents for length-dependent microtubule forces. PMID:21295701

  3. Differential expression of extracellular-signal-regulated kinase 5 (ERK5) in normal and degenerated human nucleus pulposus tissues and cells

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Weiguo, E-mail: liangweiguo@tom.com [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); Fang, Dejian [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); Ye, Dongping [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); School of Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia 6009 (Australia); Zou, Longqiang; Shen, Yan; Dai, Libing [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); Xu, Jiake, E-mail: jiake.xu@uwa.edu.au [Guangzhou Institute of Traumatic Surgery, The Fourth Affiliated Hospital of Medical College, Jinan University, Guangzhou 510220 (China); School of Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia 6009 (Australia)

    2014-07-11

    Highlights: • ERK5 involved in NP cells. • ERK5 involved in NP tissue. • It was important modulator. - Abstract: Extracellular-signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and regulates a wide variety of cellular processes such as proliferation, differentiation, necrosis, apoptosis and degeneration. However, the expression of ERK5 and its role in degenerated human nucleus pulposus (NP) is hitherto unknown. In this study, we observed the differential expression of ERK5 in normal and degenerated human nucleus pulposus tissues by using immunohistochemical staining and Western blot. Treatment of NP cells with Pro-inflammatory cytokine, TNF-α decreased ERK5 gene expression as well as NP marker gene expression; including the type II collagen and aggrecan. Suppression of ERK5 gene expression in NP cells by ERK5 siRNA resulted in decreased gene expression of type II collagen and aggrecan. Furthermore, inhibition of ERK5 activation by BIX02188 (5 μM) decreased the gene expression of type II collagen and aggrecan in NP cells. Our results document the expression of ERK5 in degenerated nucleus pulposus tissues, and suggest a potential involvement of ERK5 in human degenerated nucleus pulposus.

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

    International Nuclear Information System (INIS)

    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

  5. 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.

  6. 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.

  7. A fraction of neurofibromin interacts with PML bodies in the nucleus of the CCF astrocytoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Godin, Fabienne; Villette, Sandrine; Vallee, Beatrice; Doudeau, Michel; Morisset-Lopez, Severine [Centre de Biophysique Moleculaire, Centre National de la Recherche Scientifique (CNRS), UPR 4301, Universite d' Orleans et INSERM, rue Charles Sadron, 45071 Orleans Cedex 2 (France); Ardourel, Maryvonne; Hevor, Tobias [Laboratoire de Neurobiologie, Universite d' Orleans, BP 6759, 45067 Orleans Cedex 2 (France); Pichon, Chantal [Centre de Biophysique Moleculaire, Centre National de la Recherche Scientifique (CNRS), UPR 4301, Universite d' Orleans et INSERM, rue Charles Sadron, 45071 Orleans Cedex 2 (France); Benedetti, Helene, E-mail: helene.benedetti@cnrs-orleans.fr [Centre de Biophysique Moleculaire, Centre National de la Recherche Scientifique (CNRS), UPR 4301, Universite d' Orleans et INSERM, rue Charles Sadron, 45071 Orleans Cedex 2 (France)

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer We validate the use of specific anti-Nf1 antibodies for immunofluorescence studies. Black-Right-Pointing-Pointer We detect Nf1 in the cytoplasm and nucleus of CCF cells. Black-Right-Pointing-Pointer We demonstrate that Nf1 partially colocalizes with PML nuclear bodies. Black-Right-Pointing-Pointer We demonstrate that there is a direct interaction between a fraction of Nf1 and the PML bodies. -- Abstract: Neurofibromatosis type 1 is a common genetic disease that causes nervous system tumors, and cognitive deficits. It is due to mutations within the NF1 gene, which encodes the Nf1 protein. Nf1 has been shown to be involved in the regulation of Ras, cAMP and actin cytoskeleton dynamics. In this study, using immunofluorescence experiments, we have shown a partial nuclear localization of Nf1 in the astrocytoma cell line: CCF and we have demonstrated that Nf1 partially colocalizes with PML (promyelocytic leukemia) nuclear bodies. A direct interaction between Nf1 and the multiprotein complex has further been demonstrated using 'in situ' proximity ligation assay (PLA).

  8. 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.

  9. β-Adrenergic activation enhances NMDA-induced current in pyramidal cells of the basolateral nucleus of amygdala

    Institute of Scientific and Technical Information of China (English)

    LIU Xinqiu; CAO Xiaohua; LI Bao-ming

    2005-01-01

    NMDA receptor (NMDA-R) in the amygdala complex is critical for both long-term potentiation (LTP) and formation of conditioned fear memory. It is reported that activation of β-adrenoceptors (β-AR) in the amygdala facilitates LTP and enhances memory consolidation. The present study examined the regulatory effect of β-AR activation on NMDA-R mediated current in pyramidal cells of the basolateral nucleus of amygdala (BLA), using whole-cell recording technique. Bath application of the β-AR agonist isoproterenol enhanced NMDA-induced current, and this facilitatory effect was blocked by co-administered propranolol, a β-AR antagonist. The facilitatory effect of isoproterenol on NMDA-induced current could not be induced when the protein kinase A (PKA) inhibitor Rp-cAMPs was added in electrode internal solution.The present results suggest that β-AR activation in the BLA could modulate NMDA-R activity directly and positively, probably via PKA.

  10. Unequal distribution of plastids during generative cell formation in Impatiens.

    Science.gov (United States)

    van Went, J L

    1984-07-01

    This paper describes the unequal distribution of plastids in the developing microspores of Impatiens walleriana and Impatiens glandulifera which leads to the exclusion of plastids from the generative cell. During the development from young microspore to the onset of mitosis a change in the organization of the cytoplasm and distribution of organelles is gradually established. This includes the formation of vacuoles at the poles of the elongate-shaped microspores, the movement of the nucleus to a position near the microspore wall in the central part of the cell, and the accumulation of the plastids to a position near the wall at the opposite side of the cell. In Impatiens walleriana, the accumulated plastids are separated from each other by ER cisterns, and some mitochondria are also accumulated. In both Impatiens species, the portion of the microspore in which the generative cell will be formed is completely devoid of plastids at the time mitosis starts. PMID:24257638

  11. Increased number of TH-immunoreactive cells in the ventral tegmental area after deep brain stimulation of the anterior nucleus of the thalamus.

    Science.gov (United States)

    Dela Cruz, J A D; Hescham, S; Adriaanse, B; Campos, F L; Steinbusch, H W M; Rutten, B P F; Temel, Y; Jahanshahi, A

    2015-09-01

    Dopamine (DA) has been long implicated with the processes of memory. In long-term memory, the hippocampus and ventral tegmental area (VTA) use DA to enhance long-term potentiation, while prefrontal DA D1 receptors are involved in working memory. Deep brain stimulation (DBS) of specific brain areas have been shown to affect memory impairments in animal models. Here, we tested the hypothesis that DBS could reverse memory impairments by increasing the number of dopaminergic cells in the VTA. Rats received DBS at the level of the mammillothalamic tract, the anterior nucleus of the thalamus, and entorhinal cortex before euthanasia. These regions are part of the so-called memory circuit. Brain sections were processed for c-Fos and tyrosine hydroxylase (TH) immunocytochemistry in the VTA and the substantia nigra pars compacta (SNc). c-Fos, TH and c-Fos/TH immunoreactive cells were analyzed by means of stereology and confocal microscopy. Our results showed that DBS of the anterior nucleus of the thalamus induced substantial higher numbers of TH-immunoreactive cells in the VTA, while there were no significant differences between the experimental groups in the number of TH immunoreactive cells in the SNc, c-Fos immunoreactive cells and c-Fos/TH double-labeled cells in both the SNc and VTA. Our findings suggest a phenotypic switch, or neurotransmitter respecification, of DAergic cells specifically in the VTA which may be induced by DBS in the anterior nucleus of the thalamus.

  12. Effect of corticosterone and adrenalectomy on NMDA-induced cholinergic cell death in rat magnocellular nucleus basalis

    NARCIS (Netherlands)

    Abraham, [No Value; Veenema, AH; Nyakas, C; Harkany, T; Bohus, BGJ; Luiten, PGM; Ábrahám, I.

    1997-01-01

    The present study demonstrates the effects of adrenalectomy and subcutaneously administered corticosterone on N-methyl-D-aspartate-induced neurodegeneration in the cholinergic magnocellular basal nucleus of the rat, NMDA was unilaterally injected into the nucleus basalis at different plasma corticos

  13. Zebrafish Noxa promotes mitosis in early embryonic development and regulates apoptosis in subsequent embryogenesis.

    Science.gov (United States)

    Zhong, J-X; Zhou, L; Li, Z; Wang, Y; Gui, J-F

    2014-06-01

    Noxa functions in apoptosis and immune system of vertebrates, but its activities in embryo development remain unclear. In this study, we have studied the role of zebrafish Noxa (zNoxa) by using zNoxa-specifc morpholino knockdown and overexpression approaches in developing zebrafish embryos. Expression pattern analysis indicates that zNoxa transcript is of maternal origin, which displays a uniform distribution in early embryonic development until shield stage, and the zygote zNoxa transcription is initiated from this stage and mainly localized in YSL of the embryos. The zNoxa expression alterations result in strong embryonic development defects, demonstrating that zNoxa regulates apoptosis from 75% epiboly stage of development onward, in which zNoxa firstly induces the expression of zBik, and then cooperates with zBik to regulate apoptosis. Moreover, zNoxa knockdown also causes a reduction in number of mitotic cells before 8 h.p.f., suggesting that zNoxa also promotes mitosis before 75% epiboly stage. The effect of zNoxa on mitosis is mediated by zWnt4b in early embryos, whereas zMcl1a and zMcl1b suppress the ability of zNoxa to regulate mitosis and apoptosis at different developmental stages. In addition, mammalian mouse Noxa (mNoxa) mRNA was demonstrated to rescue the arrest of mitosis when zNoxa was knocked down, suggesting that mouse and zebrafish Noxa might have similar dual functions. Therefore, the current findings indicate that Noxa is a novel regulator of early mitosis before 75% epiboly stage when it translates into a key mediator of apoptosis in subsequent embryogenesis.

  14. p53 Prevents Entry into Mitosis with Uncapped Telomeres

    Science.gov (United States)

    Thanasoula, Maria; Escandell, Jose Miguel; Martinez, Paula; Badie, Sophie; Muñoz, Purificacion; Blasco, María A.; Tarsounas, Madalena

    2016-01-01

    Summary Telomeres are protected by capping structures consisting of core protein complexes that bind with sequence specificity to telomeric DNA (reviewed in [1]). In their absence, telomeres trigger a DNA damage response, materialized in accumulation at the telomere of damage response proteins, e.g., phosphorylated histone H2AX (γH2AX), into telomere-dysfunction-induced foci [2, 3]. Telomere uncapping occurs transiently in every cell cycle in G2 [4], following DNA replication, but little is known about how protective structures are reassembled or whether this process is controlled by the cell-cycle surveillance machinery. Here, we report that telomere capping is monitored at the G2/M transition by the p53/p21 damage response pathway. Unlike their wild-type counterparts, human and mouse cells lacking p53 or p21 progress into mitosis prematurely with persisting uncapped telomeres. Furthermore, artificially uncapped telomeres delay mitotic entry in a p53- and p21-dependent manner. Uncapped telomeres that persist in mitotic p53-deficient cells are shorter than average and religate to generate end-to-end fusions. These results suggest that a p53-dependent pathway monitors telomere capping after DNA replication and delays G2/M progression in the presence of unprotected telomeres. This mechanism maintains a cell-cycle stage conducive for capping reactions and prevents progression into stages during which uncapped telomeres are prone to deleterious end fusions. PMID:20226664

  15. SDF-1/CXCR4 axis induces apoptosis of human degenerative nucleus pulposus cells via the NF-κB pathway

    Science.gov (United States)

    LIU, ZONGCHAO; MA, CHUAN; SHEN, JIELIANG; WANG, DAWU; HAO, JIE; HU, ZHENMING

    2016-01-01

    Intervertebral disc degeneration (IVDD) is a major cause of lower back pain, and increased cell apoptosis is a key characteristic of IVDD. The present study aimed to investigate the effects and mechanism of the stromal cell-derived factor-1 (SDF-1)/C-X-C motif chemokine receptor 4 (CXCR4) axis on apoptosis in human degenerative nucleus pulposus cells (NPCs). The expression levels of SDF-1 and CXCR4 in human intervertebral discs (IVD) were determined using immunohistochemistry and western blot analysis. Apoptosis of primary cultured NPCs was quantified by Annexin V/propidium iodide staining following stimulation with SDF-1 and knockdown of CXCR4 using small interfering RNA (siRNA). The association with the nuclear factor-κB (NF-κB) signaling pathway was investigated using CXCR4-siRNA and NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), treatment. The results demonstrated that SDF-1 and its receptor, CXCR4, were upregulated in degenerative IVD samples compared with normal samples. Stimulation with SDF-1 increased the level of apoptosis in cultured NPCs, and conversely, the apoptosis level was suppressed post-transfection with CXCR4 siRNA compared with SDF-1 stimulation alone. Furthermore, SDF-1 treatment increased the level of phosphorylated NF-κB subunit P65, which was downregulated following CXCR4 siRNA and PDTC treatment. In addition, CXCR4 siRNA and PDTC inhibited the nuclear translocation of P65, which was induced by SDF-1. Taken together, SDF-1-mediated apoptosis was suppressed by NF-κB inhibition using PDTC. In conclusion, the SDF-1/CXCR4 axis promoted cell apoptosis in human degenerative NPCs via the NF-κB pathway, thus suggesting that SDF-1/CXCR signaling may be a therapeutic target for the treatment of degenerative IVD diseases. PMID:27220474

  16. 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. PMID:27149303

  17. A novel human gene spindlin1,encoding a protein localized in the cell nucleus and inducing NIH3T3 cell's transformation

    Institute of Scientific and Technical Information of China (English)

    GAO Yanhong; QIN Lipeng; ZHANG Peng; CHEN Lin; YUAN Hongfeng; BAI Cixian; YAN Fang; YUE Wen; PEI Xuetao

    2004-01-01

    A novel human gene, spindlin1, recently cloned in our laboratory, is highly expressed in the tissue of ovary cancer. To study its biological function, a vector expressing green fluorescent-spindlin1 fusion protein was constructed and transfected into COS-7 and NIH3T3 cells by lipofectamine methods. The results showed that the fusion protein pEGFP-N1-spindlin1 was localized in the nucleus of COS-7 and NIH3T3 cells. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the parental NIH3T3 cells displayed a complete morphological change, improved the cell growth and increased the percentage of cells in G2/M phase (12.6% vs control cells at 3.4%). Furthermore, overexpressed spindlin1 cells formed colonies in soft agar, more motile in migration assay in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may be a prooncogene which is associated with tumorigenesis.

  18. 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

  19. An organism arises from every nucleus.

    OpenAIRE

    Nurullah Keklikoglu

    2009-01-01

    The fact that, cloning using somatic cell nuclear transfer (SCNT) method has been performed, opened new horizons for cloning, and changed the way of our understanding and approach to cell and nucleus. The progress in cloning technology, brought the anticipation of the ability to clone an organism from each somatic cell nucleus. Therefore, the 'Cell Theory' is about to take the additional statement as "An organism arises from every nucleus". The development of gene targeting procedures which c...

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

    Science.gov (United States)

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

    1980-09-01

    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. PMID:7012060

  1. Angular velocity and head direction signals recorded from the dorsal tegmental nucleus of gudden in the rat: implications for path integration in the head direction cell circuit.

    Science.gov (United States)

    Sharp, P E; Tinkelman, A; Cho, J

    2001-06-01

    When a rat navigates through space, head direction (HD) cells provide an ongoing signal of the rat's directional heading. It is thought that these cells rely, in part, on angular path integration of the rat's head movements. This integration requires that the HD cell system receive information about angular head movements and that this information be combined with the current directional signal, to generate the next "predicted" direction. Recent data suggest that the dorsal tegmental nucleus (DTN) may play a critical role in helping to generate the HD cell signal. To test this, recordings were made from cells in the DTN in freely moving rats. The following cell types were found: (a) "classic" HD cells, (b) angular velocity cells, and (c) cells that fired as a function of both head direction and angular velocity. Thus, DTN cells exhibit firing characteristics that are critical to the neural circuit hypothesized for generation of the HD cell signal. PMID:11439447

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

    Science.gov (United States)

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

    2015-04-01

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

  3. The acetyllysine reader BRD3R promotes human nuclear reprogramming and regulates mitosis.

    Science.gov (United States)

    Shao, Zhicheng; Zhang, Ruowen; Khodadadi-Jamayran, Alireza; Chen, Bo; Crowley, Michael R; Festok, Muhamad A; Crossman, David K; Townes, Tim M; Hu, Kejin

    2016-01-01

    It is well known that both recipient cells and donor nuclei demonstrate a mitotic advantage as observed in the traditional reprogramming with somatic cell nuclear transfer (SCNT). However, it is not known whether a specific mitotic factor plays a critical role in reprogramming. Here we identify an isoform of human bromodomain-containing 3 (BRD3), BRD3R (BRD3 with Reprogramming activity), as a reprogramming factor. BRD3R positively regulates mitosis during reprogramming, upregulates a large set of mitotic genes at early stages of reprogramming, and associates with mitotic chromatin. Interestingly, a set of the mitotic genes upregulated by BRD3R constitutes a pluripotent molecular signature. The two BRD3 isoforms display differential binding to acetylated histones. Our results suggest a molecular interpretation for the mitotic advantage in reprogramming and show that mitosis may be a driving force of reprogramming.

  4. Cytological evidence for assortment mitosis leading to loss of heterozygosity in rice.

    Science.gov (United States)

    Wang, Richard R-C; Li, Xiaomei; Chatterton, N Jerry

    2006-05-01

    In the root meristem cells of the rice line AMR, which causes loss of heterozygosity in its hybrids, both normal and assortment mitoses were observed. During normal mitosis, chromosomes did not form homologous pairs at metaphase; all chromosomes lined up at the equatorial plate and 2 chromatids of each chromosome disjoined at the centromere and moved toward opposite poles. During assortment mitosis, varying numbers of paired homologues were observed at mitotic metaphase. Two groups of 12 chromosomes separated and moved towards the opposite poles of daughter cells with few chromosomes having their chromatids separated at anaphase. These observations support the proposed mechanism that is responsible for early genotype fixation in rice hybrids involving AMR.

  5. SDF‑1/CXCR4 axis induces apoptosis of human degenerative nucleus pulposus cells via the NF‑κB pathway.

    Science.gov (United States)

    Liu, Zongchao; Ma, Chuan; Shen, Jieliang; Wang, Dawu; Hao, Jie; Hu, Zhenming

    2016-07-01

    Intervertebral disc degeneration (IVDD) is a major cause of lower back pain, and increased cell apoptosis is a key characteristic of IVDD. The present study aimed to investigate the effects and mechanism of the stromal cell‑derived factor‑1 (SDF‑1)/C‑X‑C motif chemokine receptor 4 (CXCR4) axis on apoptosis in human degenerative nucleus pulposus cells (NPCs). The expression levels of SDF‑1 and CXCR4 in human intervertebral discs (IVD) were determined using immunohistochemistry and western blot analysis. Apoptosis of primary cultured NPCs was quantified by Annexin V/propidium iodide staining following stimulation with SDF‑1 and knockdown of CXCR4 using small interfering RNA (siRNA). The association with the nuclear factor‑κB (NF‑κB) signaling pathway was investigated using CXCR4‑siRNA and NF‑κB inhibitor, pyrrolidine dithiocarbamate (PDTC), treatment. The results demonstrated that SDF‑1 and its receptor, CXCR4, were upregulated in degenerative IVD samples compared with normal samples. Stimulation with SDF‑1 increased the level of apoptosis in cultured NPCs, and conversely, the apoptosis level was suppressed post‑transfection with CXCR4 siRNA compared with SDF‑1 stimulation alone. Furthermore, SDF‑1 treatment increased the level of phosphorylated NF‑κB subunit P65, which was downregulated following CXCR4 siRNA and PDTC treatment. In addition, CXCR4 siRNA and PDTC inhibited the nuclear translocation of P65, which was induced by SDF‑1. Taken together, SDF‑1‑mediated apoptosis was suppressed by NF‑κB inhibition using PDTC. In conclusion, the SDF‑1/CXCR4 axis promoted cell apoptosis in human degenerative NPCs via the NF‑κB pathway, thus suggesting that SDF‑1/CXCR signaling may be a therapeutic target for the treatment of degenerative IVD diseases. PMID:27220474

  6. Replication stress activates DNA repair synthesis in mitosis.

    Science.gov (United States)

    Minocherhomji, Sheroy; Ying, Songmin; Bjerregaard, Victoria A; Bursomanno, Sara; Aleliunaite, Aiste; Wu, Wei; Mankouri, Hocine W; Shen, Huahao; Liu, Ying; Hickson, Ian D

    2015-12-10

    Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis. Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs). CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS 'expression'), particularly when cells have been exposed to replicative stress. The MUS81-EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress. Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach.

  7. [Transport of newly synthesized rRNA from the nucleus to the cytoplasm in freely suspended cells of parsley (Petroselinum sativum)].

    Science.gov (United States)

    Seitz, U; Seitz, U

    1972-06-01

    A rapidly labelled rRNA precursor can be detected in callus cells of Petroselinum sativum grown on a liquid synthetic medium. Its molecular weight has been calculated to be 2.3×10(6). This value agrees with that of the rRNA precursor from other plant material. In order to follow the synthesis and processing of rRNA in time and to correlate single steps in this process with cell organelles it was necessary to obtain pure fractions of nuclei and ribosomes. The isolation method for nuclei is given in detail. The nucleic acids are separated on polyacrylamide gels of low acrylamide concentration. Pulse-chase experiments show that the rRNA precursor is split into two fragments within the nucleus: an 18S and a 25S component. The 18S RNA leaves the nucleus rapidly. It is already found quantitatively in the ribosomal fraction after 30-60 min chase. At that time the 25S RNA is still within the nucleus; it appears much later in the ribosomes. Since the increase in ribosomal label occurs simultaneously with the decrease in nuclear label, it is concluded that there is no degradation of 18S RNA within the nucleus. Apparently there are two distinct transport mechanisms with different kinetics for the two RNA components. PMID:24477955

  8. Membrane-to-nucleus signaling links insulin-like growth factor-1- and stem cell factor-activated pathways.

    Directory of Open Access Journals (Sweden)

    Yujiro Hayashi

    Full Text Available Stem cell factor (mouse: Kitl, human: KITLG and insulin-like growth factor-1 (IGF1, acting via KIT and IGF1 receptor (IGF1R, respectively, are critical for the development and integrity of several tissues. Autocrine/paracrine KITLG-KIT and IGF1-IGF1R signaling are also activated in several cancers including gastrointestinal stromal tumors (GIST, the most common sarcoma. In murine gastric muscles, IGF1 promotes Kitl-dependent development of interstitial cells of Cajal (ICC, the non-neoplastic counterpart of GIST, suggesting cooperation between these pathways. Here, we report a novel mechanism linking IGF1-IGF1R and KITLG-KIT signaling in both normal and neoplastic cells. In murine gastric muscles, the microenvironment for ICC and GIST, human hepatic stellate cells (LX-2, a model for cancer niches, and GIST cells, IGF1 stimulated Kitl/KITLG protein and mRNA expression and promoter activity by activating several signaling pathways including AKT-mediated glycogen synthase kinase-3β inhibition (GSK3i. GSK3i alone also stimulated Kitl/KITLG expression without activating mitogenic pathways. Both IGF1 and GSK3i induced chromatin-level changes favoring transcriptional activation at the Kitl promoter including increased histone H3/H4 acetylation and H3 lysine (K 4 methylation, reduced H3K9 and H3K27 methylation and reduced occupancy by the H3K27 methyltransferase EZH2. By pharmacological or RNA interference-mediated inhibition of chromatin modifiers we demonstrated that these changes have the predicted impact on KITLG expression. KITLG knock-down and immunoneutralization inhibited the proliferation of GIST cells expressing wild-type KIT, signifying oncogenic autocrine/paracrine KITLG-KIT signaling. We conclude that membrane-to-nucleus signaling involving GSK3i establishes a previously unrecognized link between the IGF1-IGF1R and KITLG-KIT pathways, which is active in both physiologic and oncogenic contexts and can be exploited for therapeutic purposes.

  9. Membrane-to-nucleus signaling links insulin-like growth factor-1- and stem cell factor-activated pathways.

    Science.gov (United States)

    Hayashi, Yujiro; Asuzu, David T; Gibbons, Simon J; Aarsvold, Kirsten H; Bardsley, Michael R; Lomberk, Gwen A; Mathison, Angela J; Kendrick, Michael L; Shen, K Robert; Taguchi, Takahiro; Gupta, Anu; Rubin, Brian P; Fletcher, Jonathan A; Farrugia, Gianrico; Urrutia, Raul A; Ordog, Tamas

    2013-01-01

    Stem cell factor (mouse: Kitl, human: KITLG) and insulin-like growth factor-1 (IGF1), acting via KIT and IGF1 receptor (IGF1R), respectively, are critical for the development and integrity of several tissues. Autocrine/paracrine KITLG-KIT and IGF1-IGF1R signaling are also activated in several cancers including gastrointestinal stromal tumors (GIST), the most common sarcoma. In murine gastric muscles, IGF1 promotes Kitl-dependent development of interstitial cells of Cajal (ICC), the non-neoplastic counterpart of GIST, suggesting cooperation between these pathways. Here, we report a novel mechanism linking IGF1-IGF1R and KITLG-KIT signaling in both normal and neoplastic cells. In murine gastric muscles, the microenvironment for ICC and GIST, human hepatic stellate cells (LX-2), a model for cancer niches, and GIST cells, IGF1 stimulated Kitl/KITLG protein and mRNA expression and promoter activity by activating several signaling pathways including AKT-mediated glycogen synthase kinase-3β inhibition (GSK3i). GSK3i alone also stimulated Kitl/KITLG expression without activating mitogenic pathways. Both IGF1 and GSK3i induced chromatin-level changes favoring transcriptional activation at the Kitl promoter including increased histone H3/H4 acetylation and H3 lysine (K) 4 methylation, reduced H3K9 and H3K27 methylation and reduced occupancy by the H3K27 methyltransferase EZH2. By pharmacological or RNA interference-mediated inhibition of chromatin modifiers we demonstrated that these changes have the predicted impact on KITLG expression. KITLG knock-down and immunoneutralization inhibited the proliferation of GIST cells expressing wild-type KIT, signifying oncogenic autocrine/paracrine KITLG-KIT signaling. We conclude that membrane-to-nucleus signaling involving GSK3i establishes a previously unrecognized link between the IGF1-IGF1R and KITLG-KIT pathways, which is active in both physiologic and oncogenic contexts and can be exploited for therapeutic purposes. PMID:24116170

  10. 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.

  11. Reversal of morphine-induced cell-type-specific synaptic plasticity in the nucleus accumbens shell blocks reinstatement.

    Science.gov (United States)

    Hearing, Matthew C; Jedynak, Jakub; Ebner, Stephanie R; Ingebretson, Anna; Asp, Anders J; Fischer, Rachel A; Schmidt, Clare; Larson, Erin B; Thomas, Mark John

    2016-01-19

    Drug-evoked plasticity at excitatory synapses on medium spiny neurons (MSNs) of the nucleus accumbens (NAc) drives behavioral adaptations in addiction. MSNs expressing dopamine D1 (D1R-MSN) vs. D2 receptors (D2R-MSN) can exert antagonistic effects in drug-related behaviors, and display distinct alterations in glutamate signaling following repeated exposure to psychostimulants; however, little is known of cell-type-specific plasticity induced by opiates. Here, we find that repeated morphine potentiates excitatory transmission and increases GluA2-lacking AMPA receptor expression in D1R-MSNs, while reducing signaling in D2-MSNs following 10-14 d of forced abstinence. In vivo reversal of this pathophysiology with optogenetic stimulation of infralimbic cortex-accumbens shell (ILC-NAc shell) inputs or treatment with the antibiotic, ceftriaxone, blocked reinstatement of morphine-evoked conditioned place preference. These findings confirm the presence of overlapping and distinct plasticity produced by classes of abused drugs within subpopulations of MSNs that may provide targetable molecular mechanisms for future pharmacotherapies. PMID:26739562

  12. DNA replication and spindle checkpoints cooperate during S phase to delay mitosis and preserve genome integrity.

    Science.gov (United States)

    Magiera, Maria M; Gueydon, Elisabeth; Schwob, Etienne

    2014-01-20

    Deoxyribonucleic acid (DNA) replication and chromosome segregation must occur in ordered sequence to maintain genome integrity during cell proliferation. Checkpoint mechanisms delay mitosis when DNA is damaged or upon replication stress, but little is known on the coupling of S and M phases in unperturbed conditions. To address this issue, we postponed replication onset in budding yeast so that DNA synthesis is still underway when cells should enter mitosis. This delayed mitotic entry and progression by transient activation of the S phase, G2/M, and spindle assembly checkpoints. Disabling both Mec1/ATR- and Mad2-dependent controls caused lethality in cells with deferred S phase, accompanied by Rad52 foci and chromosome missegregation. Thus, in contrast to acute replication stress that triggers a sustained Mec1/ATR response, multiple pathways cooperate to restrain mitosis transiently when replication forks progress unhindered. We suggest that these surveillance mechanisms arose when both S and M phases were coincidently set into motion by a unique ancestral cyclin-Cdk1 complex.

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

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    Toh Hean eCh'ng

    2015-09-01

    Full Text Available Previous studies have revealed a critical role for CREB-regulated transcriptional coactivator (CRTC1 in regulating neuronal gene expression during learning and memory. CRTC1 localizes to synapses but undergoes activity-dependent nuclear translocation to regulate the transcription of CREB target genes. Here we investigate the long-distance retrograde transport of CRTC1 in hippocampal neurons. We show that local elevations in calcium, triggered by activation of synaptic glutamate receptors and L-type voltage-gated calcium channels, initiate active, dynein-mediated retrograde transport of CRTC1 along microtubules. We identify a nuclear localization signal within CRTC1, and characterize three conserved serine residues whose dephosphorylation is required for nuclear import. Domain analysis reveals that the amino-terminal third of CRTC1 contains all of the signals required for regulated nucleocytoplasmic trafficking. We fuse this region to Dendra2 to generate a reporter construct and perform live-cell imaging coupled with local uncaging of glutamate and photoconversion to characterize the dynamics of stimulus-induced retrograde transport and nuclear accumulation.

  14. Cell biological mechanisms of activity-dependent synapse to nucleus translocation of CRTC1 in neurons

    Science.gov (United States)

    Ch'ng, Toh Hean; DeSalvo, Martina; Lin, Peter; Vashisht, Ajay; Wohlschlegel, James A.; Martin, Kelsey C.

    2015-01-01

    Previous studies have revealed a critical role for CREB-regulated transcriptional coactivator (CRTC1) in regulating neuronal gene expression during learning and memory. CRTC1 localizes to synapses but undergoes activity-dependent nuclear translocation to regulate the transcription of CREB target genes. Here we investigate the long-distance retrograde transport of CRTC1 in hippocampal neurons. We show that local elevations in calcium, triggered by activation of glutamate receptors and L-type voltage-gated calcium channels, initiate active, dynein-mediated retrograde transport of CRTC1 along microtubules. We identify a nuclear localization signal within CRTC1, and characterize three conserved serine residues whose dephosphorylation is required for nuclear import. Domain analysis reveals that the amino-terminal third of CRTC1 contains all of the signals required for regulated nucleocytoplasmic trafficking. We fuse this region to Dendra2 to generate a reporter construct and perform live-cell imaging coupled with local uncaging of glutamate and photoconversion to characterize the dynamics of stimulus-induced retrograde transport and nuclear accumulation. PMID:26388727

  15. Temporal coding by cochlear nucleus bushy cells in DBA/2J mice with early onset hearing loss.

    Science.gov (United States)

    Wang, Yong; Manis, Paul B

    2006-12-01

    The bushy cells of the anterior ventral cochlear nucleus (AVCN) preserve or improve the temporal coding of sound information arriving from auditory nerve fibers (ANF). The critical cellular mechanisms entailed in this process include the specialized nerve terminals, the endbulbs of Held, and the membrane conductance configuration of the bushy cell. In one strain of mice (DBA/2J), an early-onset hearing loss can cause a reduction in neurotransmitter release probability, and a smaller and slower spontaneous miniature excitatory postsynaptic current (EPSC) at the endbulb synapse. In the present study, by using a brain slice preparation, we tested the hypothesis that these changes in synaptic transmission would degrade the transmission of timing information from the ANF to the AVCN bushy neuron. We show that the electrical excitability of bushy cells in hearing-impaired old DBA mice was different from that in young, normal-hearing DBA mice. We found an increase in the action potential (AP) firing threshold with current injection; a larger AP afterhyperpolarization; and an increase in the number of spikes produced by large depolarizing currents. We also tested the temporal precision of bushy cell responses to high-frequency stimulation of the ANF. The standard deviation of spikes (spike jitter) produced by ANF-evoked excitatory postsynaptic potentials (EPSPs) was largely unaffected in old DBA mice. However, spike entrainment during a 100-Hz volley of EPSPs was significantly reduced. This was not a limitation of the ability of bushy cells to fire APs at this stimulus frequency, because entrainment to trains of current pulses was unaffected. Moreover, the decrease in entrainment is not attributable to increased synaptic depression. Surprisingly, the spike latency was 0.46 ms shorter in old DBA mice, and was apparently attributable to a faster conduction velocity, since the evoked excitatory postsynaptic current (EPSC) latency was shorter in old DBA mice as well. We also

  16. Polo kinase regulates the localization and activity of the chromosomal passenger complex in meiosis and mitosis in Drosophila melanogaster.

    Science.gov (United States)

    Carmena, Mar; Lombardia, Miguel Ortiz; Ogawa, Hiromi; Earnshaw, William C

    2014-11-01

    Cell cycle progression is regulated by members of the cyclin-dependent kinase (CDK), Polo and Aurora families of protein kinases. The levels of expression and localization of the key regulatory kinases are themselves subject to very tight control. There is increasing evidence that crosstalk between the mitotic kinases provides for an additional level of regulation. We have previously shown that Aurora B activates Polo kinase at the centromere in mitosis, and that the interaction between Polo and the chromosomal passenger complex (CPC) component INCENP is essential in this activation. In this report, we show that Polo kinase is required for the correct localization and activity of the CPC in meiosis and mitosis. Study of the phenotype of different polo allele combinations compared to the effect of chemical inhibition revealed significant differences in the localization and activity of the CPC in diploid tissues. Our results shed new light on the mechanisms that control the activity of Aurora B in meiosis and mitosis.

  17. Phosphorylation of the centrosomal protein, Cep169, by Cdk1 promotes its dissociation from centrosomes in mitosis.

    Science.gov (United States)

    Mori, Yusuke; Inoue, Yoko; Taniyama, Yuki; Tanaka, Sayori; Terada, Yasuhiko

    2015-12-25

    Cep169 is a centrosomal protein conserved among vertebrates. In our previous reports, we showed that mammalian Cep169 interacts and collaborates with CDK5RAP2 to regulate microtubule (MT) dynamics and stabilization. Although Cep169 is required for MT regulation, its precise cellular function remains largely elusive. Here we show that Cep169 associates with centrosomes during interphase, but dissociates from these structures from the onset of mitosis, although CDK5RAP2 (Cep215) is continuously located at the centrosomes throughout cell cycle. Interestingly, treatment with purvalanol A, a Cdk1 inhibitor, nearly completely blocked the dissociation of Cep169 from centrosomes during mitosis. In addition, mass spectrometry analyses identified 7 phosphorylated residues of Cep169 corresponding to consensus phosphorylation sequence for Cdk1. These data suggest that the dissociation of Cep169 from centrosomes is controlled by Cdk1/Cyclin B during mitosis, and that Cep169 might regulate MT dynamics of mitotic spindle.

  18. INHIBITING MAP KINASE ACTIVITY PREVENTS CALCIUM TRANSIENTS AND MITOSIS ENTRY IN EARLY SEA URCHIN EMBRYOS

    OpenAIRE

    Philipova, Rada; Larman, Mark G.; Leckie, Calum P.; Harrison, Patrick K.; Groigno, Laurence; Whitaker, Michael

    2005-01-01

    A transient calcium increase triggers nuclear envelope breakdown (mitosis entry) in sea urchin embryos. Cdk1/cyclin B kinase activation is also known to be required for mitosis entry. More recently MAP kinase activity has also been shown to increase during mitosis. In sea urchin embryos both kinases show a similar activation profile, peaking at the time of mitosis entry.

  19. A gibberellin-induced nuclease is localized in the nucleus of wheat aleurone cells undergoing programmed cell death

    OpenAIRE

    Domínguez, Fernándo; Moreno Onorato, Francisco Javier; Cejudo Fernández, Francisco Javier

    2003-01-01

    The aleurone layer of cereal grains undergoes a gibberellin-regulated process of programmed cell death (PCD) following germination. We have applied a combination of ultrastructural and biochemical approaches to analyze aleurone PCD in intact wheat grains. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay revealed that PCD was initiated in aleurone cells proximal to the embryo and then extended to distal cells. DNA fragmentation and terminal deoxynucleotidyl trans...

  20. 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

  1. Cytotoxic Effects of (5 Medicinal Plants on Mitosis in Allium cepa Root Tips

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    I.J. Udo

    2014-03-01

    Full Text Available The study was conducted to investigate the effects that plant extracts from 5 medicinal plants may have on mitosis in Allium cepa. Root of A .cepa were immersed in alcoholic extracts at the concentrations of 0, 25, 50, 75 and 100 mg/mL, respectively for each of the following plants: Gnetum africanum Welw., Lasianther aafricana P. Beauv, Ocimum gratissimum Linn., Telfairia occidentalis Hook F. and Vernonia amygdalina Del. Leafy vegetable which are commonly used in herbal medicine. Results obtained show that the various concentrations of the extracts from test plants had toxic effects on the cells, which caused significant reduction (p<0.05 in the mitotic index when compared with the control. Other effects were prophase inhibition, the delay of mitosis and nuclear lesion. The cytotoxic effect makes a case for a precaution in the use of the leafy extracts in herbal medicine practice.

  2. Construction of a tissue engineered intervertebral disc with high biological activity using an allogeneic intervertebral disc supplemented with transfected nucleus pulposus cells expressing exogenous dopamine beta-hydroxylase.

    Science.gov (United States)

    Bai, M; Wang, Y H; Yin, H P; Li, S W

    2015-09-09

    This study addressed the in vitro construction and biological activity of tissue engineered intervertebral discs with exogenous human dopamine beta-hydroxylase (DBH) nucleus pulposus cells. pSNAV2.0-DBH expression plasmids were utilized to enhance the survival rates of intervertebral disc tissue cells. Various concentrations of transfected nucleus pulposus cells were injected into the discs, and DBH mRNA expression was determined using polymerase chain reaction amplification. Polysaccharide content and total collagen protein content in the engineered disc nucleus pulposus tissue were determined. The visible fluorescence intensities of the 1 x 10(5) and 1 x 10(6) groups vs the 1 x 10(4) group were significantly increased (P 0.05) at 7 days after injection. DBH mRNA expression could be detected in the all but the EGFP control group at 14 days culture. No significant difference was observed in the protein content between the 1 x 10(4) and the control groups at various times, while the protein content was significantly higher in the 1 x 10(5) vs the control and the 1 x 10(4) groups at 7-, 14-, and 21-day cultures. These results demonstrate that a tissue engineered intervertebral disc with high biological activity can be constructed by utilizing allogeneic intervertebral discs stored in liquid nitrogen and a 1 x 10(5) transfected nucleus pulposus cell complex with in vitro culture for 14 days. This model can be used in animal experiments to study the biological activity of the engineered discs.

  3. ANKRD53 interacts with DDA3 and regulates chromosome integrity during mitosis.

    Science.gov (United States)

    Kim, Seul; Jang, Chang-Young

    2016-02-12

    Spindle dynamics drives chromosome movement and mitotic progression during mitosis. Microtubule (MT)-associated proteins (MAPs) regulate MT stabilization/destabilization and MT polymerization/depolymerization for congression of sister chromatids at the mitotic equator and subsequent segregation toward the spindle poles. Here, we identified ANKRD53 as a novel DDA3-interacting protein through proteomic analysis. Based on expression profiles, ANKRD53 is phosphorylated by mitotic kinases during mitosis. In ANKRD53-depleted HeLa cells, the progression of mitosis was delayed and the number of unaligned chromosomes increased substantially. In addition, spindle MT polymerization decreased and the spindle assembly checkpoint (SAC) was concomitantly activated by the decreased spindle dynamics in ANKRD53-depleted cells. Although ANKRD53 is recruited to the mitotic spindle by DDA3, it counteracts the activity of DDA3 for spindle MT polymerization. Furthermore, ANKRD53 depletion increased the number of bi-nuclei and polylobed nuclei. Thus, ANKRD53 is recruited to the mitotic spindle by DDA3 and acts as a regulator of spindle dynamics and cytokinesis.

  4. Dynamic Alterations to α-Actinin Accompanying Sarcomere Disassembly and Reassembly during Cardiomyocyte Mitosis.

    Science.gov (United States)

    Fan, Xiaohu; Hughes, Bryan G; Ali, Mohammad A M; Cho, Woo Jung; Lopez, Waleska; Schulz, Richard

    2015-01-01

    Although mammals are thought to lose their capacity to regenerate heart muscle shortly after birth, embryonic and neonatal cardiomyocytes in mammals are hyperplastic. During proliferation these cells need to selectively disassemble their myofibrils for successful cytokinesis. The mechanism of sarcomere disassembly is, however, not understood. To study this, we performed a series of immunofluorescence studies of multiple sarcomeric proteins in proliferating neonatal rat ventricular myocytes and correlated these observations with biochemical changes at different cell cycle stages. During myocyte mitosis, α-actinin and titin were disassembled as early as prometaphase. α-actinin (representing the sarcomeric Z-disk) disassembly precedes that of titin (M-line), suggesting that titin disassembly occurs secondary to the collapse of the Z-disk. Sarcomere disassembly was concurrent with the dissolution of the nuclear envelope. Inhibitors of several intracellular proteases could not block the disassembly of α-actinin or titin. There was a dramatic increase in both cytosolic (soluble) and sarcomeric α-actinin during mitosis, and cytosolic α-actinin exhibited decreased phosphorylation compared to sarcomeric α-actinin. Inhibition of cyclin-dependent kinase 1 (CDK1) induced the quick reassembly of the sarcomere. Sarcomere dis- and re-assembly in cardiomyocyte mitosis is CDK1-dependent and features dynamic differential post-translational modifications of sarcomeric and cytosolic α-actinin.

  5. Dynamic Alterations to α-Actinin Accompanying Sarcomere Disassembly and Reassembly during Cardiomyocyte Mitosis.

    Directory of Open Access Journals (Sweden)

    Xiaohu Fan

    Full Text Available Although mammals are thought to lose their capacity to regenerate heart muscle shortly after birth, embryonic and neonatal cardiomyocytes in mammals are hyperplastic. During proliferation these cells need to selectively disassemble their myofibrils for successful cytokinesis. The mechanism of sarcomere disassembly is, however, not understood. To study this, we performed a series of immunofluorescence studies of multiple sarcomeric proteins in proliferating neonatal rat ventricular myocytes and correlated these observations with biochemical changes at different cell cycle stages. During myocyte mitosis, α-actinin and titin were disassembled as early as prometaphase. α-actinin (representing the sarcomeric Z-disk disassembly precedes that of titin (M-line, suggesting that titin disassembly occurs secondary to the collapse of the Z-disk. Sarcomere disassembly was concurrent with the dissolution of the nuclear envelope. Inhibitors of several intracellular proteases could not block the disassembly of α-actinin or titin. There was a dramatic increase in both cytosolic (soluble and sarcomeric α-actinin during mitosis, and cytosolic α-actinin exhibited decreased phosphorylation compared to sarcomeric α-actinin. Inhibition of cyclin-dependent kinase 1 (CDK1 induced the quick reassembly of the sarcomere. Sarcomere dis- and re-assembly in cardiomyocyte mitosis is CDK1-dependent and features dynamic differential post-translational modifications of sarcomeric and cytosolic α-actinin.

  6. Dynamic Alterations to α-Actinin Accompanying Sarcomere Disassembly and Reassembly during Cardiomyocyte Mitosis

    Science.gov (United States)

    Ali, Mohammad A. M.; Cho, Woo Jung; Lopez, Waleska; Schulz, Richard

    2015-01-01

    Although mammals are thought to lose their capacity to regenerate heart muscle shortly after birth, embryonic and neonatal cardiomyocytes in mammals are hyperplastic. During proliferation these cells need to selectively disassemble their myofibrils for successful cytokinesis. The mechanism of sarcomere disassembly is, however, not understood. To study this, we performed a series of immunofluorescence studies of multiple sarcomeric proteins in proliferating neonatal rat ventricular myocytes and correlated these observations with biochemical changes at different cell cycle stages. During myocyte mitosis, α-actinin and titin were disassembled as early as prometaphase. α-actinin (representing the sarcomeric Z-disk) disassembly precedes that of titin (M-line), suggesting that titin disassembly occurs secondary to the collapse of the Z-disk. Sarcomere disassembly was concurrent with the dissolution of the nuclear envelope. Inhibitors of several intracellular proteases could not block the disassembly of α-actinin or titin. There was a dramatic increase in both cytosolic (soluble) and sarcomeric α-actinin during mitosis, and cytosolic α-actinin exhibited decreased phosphorylation compared to sarcomeric α-actinin. Inhibition of cyclin-dependent kinase 1 (CDK1) induced the quick reassembly of the sarcomere. Sarcomere dis- and re-assembly in cardiomyocyte mitosis is CDK1-dependent and features dynamic differential post-translational modifications of sarcomeric and cytosolic α-actinin. PMID:26076379

  7. Neurons of human nucleus accumbens

    Directory of Open Access Journals (Sweden)

    Sazdanović Maja

    2011-01-01

    Full Text Available Background/Aim. Nucleus accumbens is a part of the ventral striatum also known as a drug active brain region, especially related with drug addiction. The aim of the study was to investigate the Golgi morphology of the nucleus accumbens neurons. Methods. The study was performed on the frontal and sagittal sections of 15 human brains by the Golgi Kopsch method. We classified neurons in the human nucleus accumbens according to their morphology and size into four types: type I - fusiform neurons; type II - fusiform neurons with lateral dendrite, arising from a part of the cell body; type III - pyramidal-like neuron; type IV - multipolar neuron. The medium spiny neurons, which are mostly noted regarding to the drug addictive conditions of the brain, correspond to the type IV - multipolar neurons. Results. Two regions of human nucleus accumbens could be clearly recognized on Nissl and Golgi preparations each containing different predominant neuronal types. Central part of nucleus accumbens, core region, has a low density of impregnated neurons with predominant type III, pyramidal-like neurons, with spines on secondary branches and rare type IV, multipolar neurons. Contrary to the core, peripheral region, shell of nucleus, has a high density of impregnated neurons predominantly contained of type I and type IV - multipolar neurons, which all are rich in spines on secondary and tertiary dendritic branches. Conclusion. Our results indicate great morphological variability of human nucleus accumbens neurons. This requires further investigations and clarifying clinical significance of this important brain region.

  8. Low-concentration uranium enters the HepG2 cell nucleus rapidly and induces cell stress response.

    Science.gov (United States)

    Guéguen, Yann; Suhard, David; Poisson, Clémentine; Manens, Line; Elie, Christelle; Landon, Géraldine; Bouvier-Capely, Céline; Rouas, Caroline; Benderitter, Marc; Tessier, Christine

    2015-12-25

    This study aimed to compare the cell stress effects of low and high uranium concentrations and relate them to its localization, precipitate formation, and exposure time. The time-course analysis shows that uranium appears in cell nuclei as a soluble form within 5 min of exposure, and quickly induces expression of antioxidant and DNA repair genes. On the other hand, precipitate formations began at the very beginning of exposure at the 300-μM concentration, but took longer to appear at lower concentrations. Adaptive response might occur at low concentrations but are overwhelmed at high concentrations, especially when uranium precipitates are abundant.

  9. Cytotoxic effects of cylindrospermopsin in mitotic and non-mitotic Vicia faba cells.

    Science.gov (United States)

    Garda, Tamás; Riba, Milán; Vasas, Gábor; Beyer, Dániel; M-Hamvas, Márta; Hajdu, Gréta; Tándor, Ildikó; Máthé, Csaba

    2015-02-01

    Cylindrospermopsin (CYN) is a cyanobacterial toxin known as a eukaryotic protein synthesis inhibitor. We aimed to study its effects on growth, stress responses and mitosis of a eukaryotic model, Vicia faba (broad bean). Growth responses depended on exposure time (3 or 6d), cyanotoxin concentration, culture conditions (dark or continuous light) and V. faba cultivar ("Standard" or "ARC Egypt Cross"). At 6d of exposure, CYN had a transient stimulatory effect on root system growth, roots being possibly capable of detoxification. The toxin induced nucleus fragmentation, blebbing and chromosomal breaks indicating double stranded DNA breaks and programmed cell death. Root necrotic tissue was observed at 0.1-20 μg mL(-1) CYN that probably impeded toxin uptake into vascular tissue. Growth and cell death processes observed were general stress responses. In lateral root tip meristems, lower CYN concentrations (0.01-0.1 μg mL(-1)) induced the stimulation of mitosis and distinct mitotic phases, irrespective of culture conditions or the cultivar used. Higher cyanotoxin concentrations inhibited mitosis. Short-term exposure of hydroxylurea-synchronized roots to 5 μg mL(-1) CYN induced delay of mitosis that might have been related to a delay of de novo protein synthesis. CYN induced the formation of double, split and asymmetric preprophase bands (PPBs), in parallel with the alteration of cell division planes, related to the interference of cyanotoxin with protein synthesis, thus it was a plant- and CYN specific alteration.

  10. 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.

  11. Actin microfilaments are associated with the migrating nucleus and the cell cortex in the green alga Micrasterias. Studies on living cells.

    Science.gov (United States)

    Meindl, U; Zhang, D; Hepler, P K

    1994-07-01

    Rhodamine-phalloidin or FITC-phalloidin has been injected in small amounts into living, developing cells of Micrasterias denticulata and the stained microfilaments visualized by confocal laser scanning microscopy. The results reveal that two different actin filament systems are present in a growing cell: a cortical actin network that covers the inner surface of the cell and is extended far into the tips of the lobes in both the growing and the nongrowing semicell; it is also associated with the surface of the chloroplast. The second actin system ensheathes the nucleus at the isthmus-facing side during nuclear migration. Its arrangement corresponds to that of the microtubule system that has been described in earlier electron microscopic investigations. The spatial correspondence between the distribution of actin filaments and microtubules suggests a cooperation between both cytoskeleton elements in generating the motive force for nuclear migration. The function of the cortical actin network is not yet clear. It may be involved in processes like transport and fusion of secretory vesicles and may also function in shaping and anchoring the chloroplast. PMID:7983159

  12. 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

    2016-07-01

    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

  13. Interaction with DNA and different effect on the nucleus of cancer cells for copper(II) complexes of N-benzyl di(pyridylmethyl)amine.

    Science.gov (United States)

    Chen, Qiu-Yun; Fu, Hai-Jian; Zhu, Wei-Hua; Qi, Yan; Ma, Zheng-Ping; Zhao, Kai-Di; Gao, Jing

    2011-05-01

    Three new copper(II) complexes of N-benzyl di(pyridylmethyl)amine (phdpa) were synthesized and characterized by spectroscopic methods. The interaction between CT-DNA and the complexes was studied by UV and fluorescence titration methods. It was found that the complex [(phdpa)Cu(H(2)O)Ac)](Ac), with the non-planar aromatic heterocyclic ring ligand (phdpa), showed good anticancer properties and could cause the fragmentation of the nucleus, although its interaction with CT-DNA was weaker than that of 1,10-phenanthroline (phen)-based copper(II) complexes. The anticancer activities of copper(II) complexes with phdpa and phen based ligands are correlated to their binding constants with DNA, but phen-based copper(II) complexes did not cause the nucleus fragmentation of HeLa cells. [(phdpa)Cu(H(2)O)Ac)](Ac) can noticeably decrease the oxygen content of a culture solution and of HeLa cells, which make it a new nucleus and oxygen related anticancer copper(II) complex. Information obtained here would be helpful in the design of new antitumor complexes in oxidative therapy.

  14. Using a GFP-gene fusion technique to study the cell cycle-dependent distribution of calmodulin in living cells

    Institute of Scientific and Technical Information of China (English)

    李朝军; 吕品; 张东才

    1999-01-01

    In this study, a green fluorescent protein (GFP)-calmodulin (CaM) fusion gene method was used to examine the distribution of calmodulin during various stages of cell cycle. First, it was found that the distribution of CaM in living cells changes with the cell cycle. CaM was found mainly in the cytoplasm during G1 phase. It began to move into the nucleus when the cell entered S phase. At G2 phase, CaM became more concentrated in the nucleus than in cytoplasm. Second, the accumulation of CaM in the nucleus during G2 phase appeared to be related to the onset of mitosis, since inhibiting the activation of CaM at this stage resulted in blocking the nuclear membrane breakdown and chromatin condensation. Finally, after the cell entered mitosis, a high concentration of CaM was found at the polar regions of the mitotic spindle. At this time, inhibiting the activity of CaM would cause a disruption of the spindle structure. The relationship between the stage-specific distribution of CaM and its function in regulat

  15. A SAP domain-containing protein shuttles between the nucleus and cell membranes and plays a role in adhesion and migration in D. discoideum

    Directory of Open Access Journals (Sweden)

    Jessica S. Kelsey

    2013-02-01

    The AmpA protein reduces cell adhesion, thereby influencing cell migration in Dictyostelium. To understand how ampA influences cell migration, second site suppressors of an AmpA overexpressing cell line were created by REMI mutagenesis. Mutant candidates were identified by their ability to suppress the large plaques that the AmpA overexpressing cells form on bacterial lawns as a result of their increased rate of migration. One suppressor gene, sma, encodes an uncharacterized protein, which contains a SAP DNA-binding domain and a PTEN-like domain. Using sma gene knockouts and Sma-mRFP expressing cell lines, a role for sma in influencing cell migration was uncovered. Knockouts of the sma gene in a wild-type background enhanced chemotaxis. An additional role for Sma in influencing cell–cell adhesion was also demonstrated. Sma protein transitions between cytosolic and nuclear localizations as a function of cell density. In growing cells migrating to folic acid it is localized to regions of actin polymerization and absent from the nucleus. A role for Sma in influencing ampA mRNA levels is also demonstrated. Sma additionally appears to be involved in ampA pathways regulating cell size, actin polymerization, and cell substrate adhesion. We present insights to the SAP domain-containing group of proteins in Dictyostelium and provide evidence of a role for a SAP domain-containing protein shuttling from the nucleus to sites of actin polymerization during chemotaxis to folic acid and influencing the efficiency of migration.

  16. Phosphorylation of human Sgo1 by NEK2A is essential for chromosome congression in mitosis

    Institute of Scientific and Technical Information of China (English)

    Guosheng Fu; Xia Ding; Kai Yuan; Felix Aikhionbare; Jianhui Yao; Xin Cai; Kai Jiang; Xuebiao Yao

    2007-01-01

    Chromosome segregation in mitosis is orchestrated by the interaction of the kinetochore with spindle microtubules. Our recent study shows that NEK2A interacts with MAD1 at the kinetochore and possibly functions as a novel integrator of spindle checkpoint signaling. However, it is unclear how NEK2 A regulates kinetochore-microtubule attachment in mitosis. Here we show that NEK2A phosphorylates human Sgol and such phosphorylation is essential for faithful chromosome congression in mitosis. NEK2A binds directly to HsSgol in vitro and co-distributes with HsSgol to the kinetochore of mitotic cells. Our in vitro phosphorylation experiment demonstrated that HsSgol is a substrate of NEK2A and the phosphorylation sites were mapped to Ser14 and Ser507 as judged by the incorporation of 32P. Although such phosphorylation is not required for assembly of HsSgol to the kinetochore, expression of non-phosphorylatable mutant HsSgol perturbed chromosome congression and resulted in a dramatic increase in microtubule attachment errors, including syntelic and monotelic attachments. These findings reveal a key role for the NEK2A-mediated phosphorylation of HsSgol in orchestrating dynamic kinetochore-microtubule interaction. We propose that NEK2A-mediated phosphorylation of human Sgol provides a link between centromeric cohesion and spindle microtubule attachment at the kinetochores.

  17. Chromatids segregate without centrosomes during Caenorhabditis elegans mitosis in a Ran- and CLASP-dependent manner.

    Science.gov (United States)

    Nahaboo, Wallis; Zouak, Melissa; Askjaer, Peter; Delattre, Marie

    2015-06-01

    During mitosis, chromosomes are connected to a microtubule-based spindle. Current models propose that displacement of the spindle poles and/or the activity of kinetochore microtubules generate mechanical forces that segregate sister chromatids. Using laser destruction of the centrosomes during Caenorhabditis elegans mitosis, we show that neither of these mechanisms is necessary to achieve proper chromatid segregation. Our results strongly suggest that an outward force generated by the spindle midzone, independently of centrosomes, is sufficient to segregate chromosomes in mitotic cells. Using mutant and RNAi analysis, we show that the microtubule-bundling protein SPD-1/MAP-65 and BMK-1/kinesin-5 act as a brake opposing the force generated by the spindle midzone. Conversely, we identify a novel role for two microtubule-growth and nucleation agents, Ran and CLASP, in the establishment of the centrosome-independent force during anaphase. Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is continuously required to sustain chromosome segregation during mitosis.

  18. Spatial organization of the Ran pathway by microtubules in mitosis.

    Science.gov (United States)

    Oh, Doogie; Yu, Che-Hang; Needleman, Daniel J

    2016-08-01

    Concentration gradients of soluble proteins are believed to be responsible for control of morphogenesis of subcellular systems, but the mechanisms that generate the spatial organization of these subcellular gradients remain poorly understood. Here, we use a newly developed multipoint fluorescence fluctuation spectroscopy technique to study the ras-related nuclear protein (Ran) pathway, which forms soluble gradients around chromosomes in mitosis and is thought to spatially regulate microtubule behaviors during spindle assembly. We found that the distribution of components of the Ran pathway that influence microtubule behaviors is determined by their interactions with microtubules, resulting in microtubule nucleators being localized by the microtubules whose formation they stimulate. Modeling and perturbation experiments show that this feedback makes the length of the spindle insensitive to the length scale of the Ran gradient, allows the spindle to assemble outside the peak of the Ran gradient, and explains the scaling of the spindle with cell size. Such feedback between soluble signaling pathways and the mechanics of the cytoskeleton may be a general feature of subcellular organization. PMID:27439876

  19. EB1 is required for spindle symmetry in mammalian mitosis.

    Directory of Open Access Journals (Sweden)

    Anke Brüning-Richardson

    Full Text Available Most information about the roles of the adenomatous polyposis coli protein (APC and its binding partner EB1 in mitotic cells has come from siRNA studies. These suggest functions in chromosomal segregation and spindle positioning whose loss might contribute to tumourigenesis in cancers initiated by APC mutation. However, siRNA-based approaches have drawbacks associated with the time taken to achieve significant expression knockdown and the pleiotropic effects of EB1 and APC gene knockdown. Here we describe the effects of microinjecting APC- or EB1- specific monoclonal antibodies and a dominant-negative EB1 protein fragment into mammalian mitotic cells. The phenotypes observed were consistent with the roles proposed for EB1 and APC in chromosomal segregation in previous work. However, EB1 antibody injection also revealed two novel mitotic phenotypes, anaphase-specific cortical blebbing and asymmetric spindle pole movement. The daughters of microinjected cells displayed inequalities in microtubule content, with the greatest differences seen in the products of mitoses that showed the severest asymmetry in spindle pole movement. Daughters that inherited the least mobile pole contained the fewest microtubules, consistent with a role for EB1 in processes that promote equality of astral microtubule function at both poles in a spindle. We propose that these novel phenotypes represent APC-independent roles for EB1 in spindle pole function and the regulation of cortical contractility in the later stages of mitosis. Our work confirms that EB1 and APC have important mitotic roles, the loss of which could contribute to CIN in colorectal tumour cells.

  20. Three-Dimensional Organization of Chromosome Territories and the Human Cell Nucleus: Comparison between simulated Parameters and Experiments

    NARCIS (Netherlands)

    T.A. Knoch (Tobias)

    2000-01-01

    textabstractDespite the successful linear sequencing of the human genome its three-dimensional structure is widely unknown, although it is important for gene regulation and replication. For a long time the interphase nucleus has been viewed as a 'spaghetti soup' of DNA without much internal stru

  1. A decrease in the addition of new cells in the nucleus accumbens and prefrontal cortex between puberty and adulthood in male rats.

    Science.gov (United States)

    Staffend, Nancy A; Mohr, Margaret A; DonCarlos, Lydia L; Sisk, Cheryl L

    2014-06-01

    Adolescence involves shifts in social behaviors, behavioral flexibility, and adaptive risk-taking that coincide with structural remodeling of the brain. We previously showed that new cells are added to brain regions associated with sexual behaviors, suggesting that cytogenesis may be a mechanism for acquiring adult-typical behaviors during adolescence. Whether pubertal cell addition occurs in brain regions associated with behavioral flexibility or motivation and whether these patterns differ between pubertal and adult animals had not been determined. Therefore, we assessed patterns of cell proliferation or survival in the prefrontal cortex and nucleus accumbens. Pubertal and adult male rats were given injections of bromo-deoxyuridine (BrdU). To assess cell proliferation, half of the animals from each group were sacrificed 24 h following the last injection. The remaining animals were sacrificed at Day 30 following the last injection to evaluate cell survival. Adult animals had significantly lower densities of BrdU-immunoreactive (ir) cells in the prefrontal cortex, irrespective of post-BrdU survival time, whereas in the nucleus accumbens, adult animals had a lower density of BrdU-ir cells at the short survival time; however, the density of BrdU-ir cells was equivalent in pubertal and adult animals at the longer survival time. These data provide evidence that cell addition during puberty may contribute to the remodeling of brain regions associated with behavioral flexibility and motivation, and this cell addition continues into adulthood, albeit at lower levels. Higher levels of cell proliferation or survival in younger animals may reflect a higher level of plasticity, possibly contributing to the dynamic remodeling of the pubertal brain. PMID:24339170

  2. Nuclear localization of P-glycoprotein is responsible for protection of the nucleus from doxorubicin in the resistant LoVo cell line.

    Science.gov (United States)

    Szaflarski, Witold; Sujka-Kordowska, Patrycja; Januchowski, Radosław; Wojtowicz, Karolina; Andrzejewska, Małgorzata; Nowicki, Michał; Zabel, Maciej

    2013-07-01

    The high expression of P-glycoprotein (P-gp) belongs to one of the most important factors causing multidrug-resistant (MDR) of cancer cells. P-gp is primarily associated with plasma membrane; however, small fraction of that protein is present in the nuclear envelope. Such phenomenon is observed in cancer cells and may result in the selection of MDR cells as the secondary tumor and/or resistant metastasis that significantly shorten patient survival rate. Here, we confirmed nuclear localization of P-gp in resistant LoVo cells and demonstrated its impact on doxorubicin efflux from the nucleus to cytoplasm. Furthermore, we showed that P-gp located at the nuclear envelope might have a different glycoside chain when compared to the form located in the cytoplasm. It suggests that the glycoside chain plays a role in the intracellular trafficking of P-gp and may decide about the destination place in the cell. PMID:23602050

  3. Nuclear localization of P-glycoprotein is responsible for protection of the nucleus from doxorubicin in the resistant LoVo cell line.

    Science.gov (United States)

    Szaflarski, Witold; Sujka-Kordowska, Patrycja; Januchowski, Radosław; Wojtowicz, Karolina; Andrzejewska, Małgorzata; Nowicki, Michał; Zabel, Maciej

    2013-07-01

    The high expression of P-glycoprotein (P-gp) belongs to one of the most important factors causing multidrug-resistant (MDR) of cancer cells. P-gp is primarily associated with plasma membrane; however, small fraction of that protein is present in the nuclear envelope. Such phenomenon is observed in cancer cells and may result in the selection of MDR cells as the secondary tumor and/or resistant metastasis that significantly shorten patient survival rate. Here, we confirmed nuclear localization of P-gp in resistant LoVo cells and demonstrated its impact on doxorubicin efflux from the nucleus to cytoplasm. Furthermore, we showed that P-gp located at the nuclear envelope might have a different glycoside chain when compared to the form located in the cytoplasm. It suggests that the glycoside chain plays a role in the intracellular trafficking of P-gp and may decide about the destination place in the cell.

  4. Impact of Bep or Carboplatin Chemotherapy on Testicular Function and Sperm Nucleus of Subjects with Testicular Germ Cell Tumor.

    Science.gov (United States)

    Ghezzi, Marco; Berretta, Massimiliano; Bottacin, Alberto; Palego, Pierfrancesco; Sartini, Barbara; Cosci, Ilaria; Finos, Livio; Selice, Riccardo; Foresta, Carlo; Garolla, Andrea

    2016-01-01

    Young males have testicular germ cells tumors (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 tumor 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 2 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 1 and 2 years from the end of treatment. Despite

  5. 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

  6. Amaranth lunasin-like peptide internalizes into the cell nucleus and inhibits chemical carcinogen-induced transformation of NIH-3T3 cells.

    Science.gov (United States)

    Maldonado-Cervantes, Enrique; Jeong, Hyung Jin; León-Galván, Fabiola; Barrera-Pacheco, Alberto; De León-Rodríguez, Antonio; González de Mejia, Elvira; de Lumen, Ben O; Barba de la Rosa, Ana P

    2010-09-01

    Because an unbalanced diet is an important risk factor for several illnesses, interest has increased in finding novel health-promoting foods. Amaranth produces seeds that not only have substantial nutritional properties but that also contain phytochemical compounds as rutin and nicotiflorin and peptides with antihypertensive and anticarcinogenic activities. We report that a cancer-preventive peptide in amaranth has activities similar to those of soybean lunasin. The amaranth lunasin-like peptide, however, requires less time than the soybean lunasin to internalize into the nucleus of NIH-3T3 cells, and inhibits histone acetylation (H(3) and H(4) in a 70 and 77%, respectively). The amaranth lunasin-like peptide inhibited the transformation of NIH-3T3 cells to cancerous foci. The open reading frame (ORF) of amaranth lunasin corresponds to a bifunctional inhibitor/lipid-transfer protein (LTP). LTPs are a family of proteins that in plants are implicated in different functions, albeit all linked to developmental processes and biotic and abiotic stress resistance. Our results open new intriguing questions about the function of lunasin in plants and support that amaranth is a food alternative containing natural peptides with health-promoting benefits.

  7. TFIIIB is phosphorylated, disrupted and selectively released from tRNA promoters during mitosis in vivo

    OpenAIRE

    Fairley, Jennifer A.; Scott, Pamela H.; White, Robert J.

    2003-01-01

    Mitosis involves a generalized repression of gene expression. In the case of RNA polymerase III transcription, this is due to phosphorylation-mediated inactivation of TFIIIB, an essential complex comprising the TATA-binding protein TBP and the TAF subunits Brf1 and Bdp1. In HeLa cells, this repression is mediated by a mitotic kinase other than cdc2–cyclin B and is antagonized by protein phosphatase 2A. Brf1 is hyperphosphorylated in metaphase-arrested cells, but remains associated with promot...

  8. Regulation of mRNA translation during mitosis.

    Science.gov (United States)

    Tanenbaum, Marvin E; Stern-Ginossar, Noam; Weissman, Jonathan S; Vale, Ronald D

    2015-01-01

    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.

  9. Matefin/SUN-1 Phosphorylation on Serine 43 Is Mediated by CDK-1 and Required for Its Localization to Centrosomes and Normal Mitosis in C. elegans Embryos

    Directory of Open Access Journals (Sweden)

    Noam Zuela

    2016-02-01

    Full Text Available Matefin/SUN-1 is an evolutionary conserved C. elegans inner nuclear membrane SUN-domain protein. By creating a bridge with the KASH-domain protein ZYG-12, it connects the nucleus to cytoplasmic filaments and organelles. Matefin/SUN-1 is expressed in the germline where it undergoes specific phosphorylation at its N-terminal domain, which is required for germline development and homologous chromosome pairing. The maternally deposited matefin/SUN-1 is then essential for embryonic development. Here, we show that in embryos, serine 43 of matefin/SUN-1 (S43 is phosphorylated in a CDK-1 dependent manner and is localized throughout the cell cycle mostly to centrosomes. By generating animals expressing phosphodead S43A and phosphomimetic S43E mutations, we show that phosphorylation of S43 is required to maintain centrosome integrity and function, as well as for the localization of ZYG-12 and lamin. Expression of S43E in early embryos also leads to an increase in chromatin structural changes, decreased progeny and to almost complete embryonic lethality. Down regulation of emerin further increases the occurrence of chromatin organization abnormalities, indicating possible collaborative roles for these proteins that is regulated by S43 phosphorylation. Taken together, these results support a role for phosphorylation of serine 43 in matefin/SUN-1 in mitosis.

  10. In silico prediction of escherichia coli proteins targeting the host cell nucleus, with special reference to their role in colon cancer etiology.

    Science.gov (United States)

    Khan, Abdul Arif

    2014-06-01

    The potential role of Escherichia coli in the development of colorectal carcinoma (CRC) has been investigated in many studies. Although the exact mechanism is not clear, chronic inflammation caused by E. coli and other related events are suggested as possible causes behind E. coli-induced colon cancer. It has been found that CRC cells, but not normal cells, are colonized by an intracellular form of E. coli. We predicted nuclear targeting of bacterial proteins in the host cell through computational tools nuclear localization signal (NLS) mapper and balanced subcellular localization predictor (BaCeILo). During intracellular E. coli residence, such targeting is highly likely and may have a possible role in colon cancer etiology. We observed that several gene expression-associated proteins of E. coli can migrate to the host nucleus during intracellular infections. This situation provides an opportunity for competitive interaction of host and pathogen proteins with similar cellular substrates, thereby increasing the chances of development of colon cancer. Moreover, the results indicated that proteins localized in the membrane of E. coli mostly act as secretary proteins in host cells. No exact correlation was observed between NLS prediction and nuclear localization prediction by BaCeILo. This is partly because of a number of reasons, including that only 30% of nuclear proteins carry NLS and that proteins <40 kDa molecular weight can passively target the host nucleus. This study concludes that detection of gene expression-specific E. coli proteins and their targeting of the nucleus may have a profound impact on CRC etiology. PMID:24611522

  11. 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.

  12. 微柱对细胞核形态影响的研究%Studys on Effects of Micropillars on Cell Nucleus Shapes

    Institute of Scientific and Technical Information of China (English)

    安燕飞; 王进义

    2015-01-01

    目的::探讨微柱对3种细胞细胞核形态的影响。方法:在高度10μm,直径18μm,间距15μm 的 PDMS 微柱上分别培养3种细胞(表皮细胞、内皮细胞和成纤维细胞),24 h 后对其细胞核形态参数包括平均面积、周长、纵横比以及圆度值进行测量,并与其在平面上的相应值进行比较分析。结果:微柱上表皮细胞和内皮细胞的细胞核变形明显,核平均面积较平面上小很多,出现了弯月形和哑铃形;而成纤维细胞核则变形较小,仍为椭圆形,只是纵横比增大。结论:本实验采用的微柱尺度会使得3种细胞有不同程度的细胞核变形。%Objective:Effects of micropillar on cell nucleus shapes of three different kinds of cells was discussed.Methods:First, these three different kinds of cells(epithelial cell,fibroblast and endothelial cell)were cultured on an array of PDMS micropillars which were 10 μm in height,18 μm in diameter and 15 μm in edge to edge spacing.Then,a quantitative and comparative analysis of the cell nucleus shapes was given by measuring the average area,aspect ratio and their circularity of the cells both on the micropillars and on the flat substrate.Results:Distinct nuclear deformation such as crescent,and dumbbell was observed of epithelial cells and endothelial cells on the micropillar substrate and their nuclear average areas were much smaller than that of the the cells on the flat substrate,when nucleus shape of fibroblasts was still in oval but just with a larger aspect ratio.Conclusion:Different degree of nuclear deformation would be induced by the micropillars that we used in this paper.

  13. Activation of the nimA protein kinase plays a unique role during mitosis that cannot be bypassed by absence of the bimE checkpoint.

    OpenAIRE

    Osmani, A H; O'Donnell, K; Pu, R T; Osmani, S A

    1991-01-01

    Mutation of nimA reversibly arrests cells in late G2 and nimA overexpression promotes premature mitosis. Here we demonstrate that the product of nimA (designated NIMA) has protein kinase activity that can phosphorylate beta-casein but not histone proteins. NIMA kinase activity is cell cycle regulated being 20-fold higher at mitosis when compared to S-phase arrested cells. NIMA activation is normally required in G2 to initiate chromosome condensation, to nucleate spindle pole body microtubules...

  14. 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.

  15. Insulin Phosphorylates Tyrosine Residue 464 of Tub and Translocates Tubby into the Nucleus in HIRcB Cells

    OpenAIRE

    Kim, Jin Wook; Kim, Hyeon Soo; Kim, Sang Dae; Park, Jung Yul

    2014-01-01

    Background The tubby protein has a motif that might be relevant for its action in the insulin signaling pathway. Previous studies have indicated that tubby undergoes phosphorylation on tyrosine residues in response to several stimuli and is known to localize in the nucleus as well as in the plasma membrane. However, the relationship between phosphorylation and nuclear translocation is not well understood. Here, we report that insulin directly phosphorylates tubby, which translocates into the ...

  16. Nucleoporin MOS7/Nup88 is required for mitosis in gametogenesis and seed development in Arabidopsis.

    Science.gov (United States)

    Park, Guen Tae; Frost, Jennifer M; Park, Jin-Sup; Kim, Tae Ho; Lee, Jong Seob; Oh, Sung Aeong; Twell, David; Brooks, Janie Sue; Fischer, Robert L; Choi, Yeonhee

    2014-12-23

    Angiosperm reproduction is characterized by alternate diploid sporophytic and haploid gametophytic generations. Gametogenesis shares similarities with that of animals except for the formation of the gametophyte, whereby haploid cells undergo several rounds of postmeiotic mitosis to form gametes and the accessory cells required for successful reproduction. The mechanisms regulating gametophyte development in angiosperms are incompletely understood. Here, we show that the nucleoporin Nup88-homolog MOS7 (Modifier of Snc1,7) plays a crucial role in mitosis during both male and female gametophyte formation in Arabidopsis thaliana. Using a mutagenesis screen, we identify the mos7-5 mutant allele, which causes ovule and pollen abortion in MOS7/mos7-5 heterozygous plants, and preglobular stage embryonic lethality in homozygous mos7-5 seeds. During interphase, we show that MOS7 is localized to the nuclear membrane but, like many nucleoporins, is associated with the spindle apparatus during mitosis. We detect interactions between MOS7 and several nucleoporins known to control spindle dynamics, and find that in pollen from MOS7/mos7-5 heterozygotes, abortion is accompanied by a failure of spindle formation, cell fate specification, and phragmoplast activity. Most intriguingly, we show that following gamete formation by MOS7/mos7-5 heterozygous spores, inheritance of either the MOS7 or the mos7-5 allele by a given gamete does not correlate with its respective survival or abortion. Instead, we suggest a model whereby MOS7, which is highly expressed in the Pollen- and Megaspore Mother Cells, enacts a dosage-limiting effect on the gametes to enable their progression through subsequent mitoses.

  17. Identification of Candidate Cyclin-dependent kinase 1 (Cdk1) Substrates in Mitosis by Quantitative Phosphoproteomics.

    Science.gov (United States)

    Petrone, Adam; Adamo, Mark E; Cheng, Chao; Kettenbach, Arminja N

    2016-07-01

    Cyclin-dependent kinase 1 (Cdk1) is an essential regulator of many mitotic processes including the reorganization of the cytoskeleton, chromosome segregation, and formation and separation of daughter cells. Deregulation of Cdk1 activity results in severe defects in these processes. Although the role of Cdk1 in mitosis is well established, only a limited number of Cdk1 substrates have been identified in mammalian cells. To increase our understanding of Cdk1-dependent phosphorylation pathways in mitosis, we conducted a quantitative phosphoproteomics analysis in mitotic HeLa cells using two small molecule inhibitors of Cdk1, Flavopiridol and RO-3306. In these analyses, we identified a total of 24,840 phosphopeptides on 4,273 proteins, of which 1,215 phosphopeptides on 551 proteins were significantly reduced by 2.5-fold or more upon Cdk1 inhibitor addition. Comparison of phosphopeptide quantification upon either inhibitor treatment revealed a high degree of correlation (R(2) value of 0.87) between the different datasets. Motif enrichment analysis of significantly regulated phosphopeptides revealed enrichment of canonical Cdk1 kinase motifs. Interestingly, the majority of proteins identified in this analysis contained two or more Cdk1 inhibitor-sensitive phosphorylation sites, were highly connected with other candidate Cdk1 substrates, were enriched at specific subcellular structures, or were part of protein complexes as identified by the CORUM database. Furthermore, candidate Cdk1 substrates were enriched in G2 and M phase-specific genes. Finally, we validated a subset of candidate Cdk1 substrates by in vitro kinase assays. Our findings provide a valuable resource for the cell signaling and mitosis research communities and greatly increase our knowledge of Cdk1 substrates and Cdk1-dependent signaling pathways. PMID:27134283

  18. Penetration of short fluorescence-labeled peptides into the nucleus in HeLa cells and in vitro specific interaction of the peptides with deoxyribooligonucleotides and DNA.

    Science.gov (United States)

    Fedoreyeva, L I; Kireev, I I; Khavinson, V Kh; Vanyushin, B F

    2011-11-01

    Marked fluorescence in cytoplasm, nucleus, and nucleolus was observed in HeLa cells after incubation with each of several fluorescein isothiocyanate-labeled peptides (epithalon, Ala-Glu-Asp-Gly; pinealon, Glu-Asp-Arg; testagen, Lys-Glu-Asp-Gly). This means that short biologically active peptides are able to penetrate into an animal cell and its nucleus and, in principle they may interact with various components of cytoplasm and nucleus including DNA and RNA. It was established that various initial (intact) peptides differently affect the fluorescence of the 5,6-carboxyfluorescein-labeled deoxyribooligonucleotides and DNA-ethidium bromide complexes. The Stern-Volmer constants characterizing the degree of fluorescence quenching of various single- and double-stranded fluorescence-labeled deoxyribooligonucleotides with short peptides used were different depending on the peptide primary structures. This indicates the specific interaction between short biologically active peptides and nucleic acid structures. On binding to them, the peptides discriminate between different nucleotide sequences and recognize even their cytosine methylation status. Judging from corresponding constants of the fluorescence quenching, the epithalon, pinealon, and bronchogen (Ala-Glu-Asp-Leu) bind preferentially with deoxyribooligonucleotides containing CNG sequence (CNG sites are targets for cytosine DNA methylation in eukaryotes). Epithalon, testagen, and pinealon seem to preferentially bind with CAG- but bronchogen with CTG-containing sequences. The site-specific interactions of peptides with DNA can control epigenetically the cell genetic functions, and they seem to play an important role in regulation of gene activity even at the earliest stages of life origin and in evolution.

  19. GSK3β-Dzip1-Rab8 cascade regulates ciliogenesis after mitosis.

    Science.gov (United States)

    Zhang, Boyan; Zhang, Tingting; Wang, Guopeng; Wang, Gang; Chi, Wangfei; Jiang, Qing; Zhang, Chuanmao

    2015-04-01

    The primary cilium, which disassembles before mitotic entry and reassembles after mitosis, organizes many signal transduction pathways that are crucial for cell life and individual development. However, how ciliogenesis is regulated during the cell cycle remains largely unknown. Here we show that GSK3β, Dzip1, and Rab8 co-regulate ciliogenesis by promoting the assembly of the ciliary membrane after mitosis. Immunofluorescence and super-resolution microscopy showed that Dzip1 was localized to the periciliary diffusion barrier and enriched at the mother centriole. Knockdown of Dzip1 by short hairpin RNAs led to failed ciliary localization of Rab8, and Rab8 accumulation at the basal body. Dzip1 preferentially bound to Rab8GDP and promoted its dissociation from its inhibitor GDI2 at the pericentriolar region, as demonstrated by sucrose gradient centrifugation of purified basal bodies, immunoprecipitation, and acceptor-bleaching fluorescence resonance energy transfer assays. By means of in vitro phosphorylation, in vivo gel shift, phospho-peptide identification by mass spectrometry, and GST pulldown assays, we demonstrated that Dzip1 was phosphorylated by GSK3β at S520 in G0 phase, which increased its binding to GDI2 to promote the release of Rab8GDP at the cilium base. Moreover, ciliogenesis was inhibited by overexpression of the GSK3β-nonphosphorylatable Dzip1 mutant or by disabling of GSK3β by specific inhibitors or knockout of GSK3β in cells. Collectively, our data reveal a unique cascade consisting of GSK3β, Dzip1, and Rab8 that regulates ciliogenesis after mitosis.

  20. Fanconi anaemia proteins are associated with sister chromatid bridging in mitosis

    DEFF Research Database (Denmark)

    Ying, Songmin; Hickson, Ian D

    2011-01-01

    The maintenance of genome stability is critical for the suppression of cancer and premature ageing. The maintenance of the human genome requires hundreds of proteins involved in DNA repair, DNA replication, chromosome segregation and cell cycle checkpoint responses. A number of genetic disorders...... that specifically occur during chromosome segregation in mitosis. The BS protein, BLM, was shown recently to define a novel class of anaphase DNA bridge structures that, in some cases, also contain FA proteins. We will discuss the possible source of these bridges and the role that FA proteins and BLM might play...

  1. Specialization of B-Type Cyclins for Mitosis or Meiosis in S. Cerevisiae

    OpenAIRE

    Dahmann, C.; Futcher, B.

    1995-01-01

    The CLB1, CLB2, and CLB3 genes encode B-type cyclins important for mitosis in Saccharomyces cerevisiae, while a fourth B-type cyclin gene, CLB4, has no clear role. The effects of homozygous clb mutations on meiosis were examined. Mutants homozygous for clb1 clb3, or for clb1 clb4, gave high levels of sporulation, but produced mainly two-spored asci instead of four-spored asci. The cells had completed meiosis I but not meiosis II, producing viable diploid ascospores. CLB1 and CLB4 seem to be m...

  2. LOX is a novel mitotic spindle-associated protein essential for mitosis

    Science.gov (United States)

    Boufraqech, Myriem; Wei, Darmood; Weyemi, Urbain; Zhang, Lisa; Quezado, Martha; Kalab, Petr; Kebebew, Electron

    2016-01-01

    LOX regulates cancer progression in a variety of human malignancies. It is overexpressed in aggressive cancers and higher expression of LOX is associated with higher cancer mortality. Here, we report a new function of LOX in mitosis. We show that LOX co-localizes to mitotic spindles from metaphase to telophase, and p-H3(Ser10)-positive cells harbor strong LOX staining. Further, purification of mitotic spindles from synchronized cells show that LOX fails to bind to microtubules in the presence of nocodazole, whereas paclitaxel treated samples showed enrichment in LOX expression, suggesting that LOX binds to stabilized microtubules. LOX knockdown leads to G2/M phase arrest; reduced p-H3(Ser10), cyclin B1, CDK1, and Aurora B. Moreover, LOX knockdown significantly increased sensitivity of cancer cells to chemotherapeutic agents that target microtubules. Our findings suggest that LOX has a role in cancer cell mitosis and may be targeted to enhance the activity of microtubule inhibitors for cancer therapy. PMID:27296552

  3. Breast cancer mitosis detection in histopathological images with spatial feature extraction

    Science.gov (United States)

    Albayrak, Abdülkadir; Bilgin, Gökhan

    2013-12-01

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

  4. 50 ways to build a spindle: the complexity of microtubule generation during mitosis.

    Science.gov (United States)

    Duncan, Tommy; Wakefield, James G

    2011-04-01

    The accurate segregation of duplicated chromosomes, essential for the development and viability of a eukaryotic organism, requires the formation of a robust microtubule (MT)-based spindle apparatus. Entry into mitosis or meiosis precipitates a cascade of signalling events which result in the activation of pathways responsible for a dramatic reorganisation of the MT cytoskeleton: through changes in the properties of MT-associated proteins, local concentrations of free tubulin dimer and through enhanced MT nucleation. The latter is generally thought to be driven by localisation and activation of γ-tubulin-containing complexes (γ-TuSC and γ-TuRC) at specific subcellular locations. For example, upon entering mitosis, animal cells concentrate γ-tubulin at centrosomes to tenfold the normal level during interphase, resulting in an aster-driven search and capture of chromosomes and bipolar mitotic spindle formation. Thus, in these cells, centrosomes have traditionally been perceived as the primary microtubule organising centre during spindle formation. However, studies in meiotic cells, plants and cell-free extracts have revealed the existence of complementary mechanisms of spindle formation, mitotic chromatin, kinetochores and nucleation from existing MTs or the cytoplasm can all contribute to a bipolar spindle apparatus. Here, we outline the individual known mechanisms responsible for spindle formation and formulate ideas regarding the relationship between them in assembling a functional spindle apparatus. PMID:21484448

  5. The DNA damage response during mitosis

    NARCIS (Netherlands)

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

    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

  6. Treatment of peritoneal carcinomatosis by targeted delivery of the radio-labeled tumor homing peptide bi-DTPA-[F3]2 into the nucleus of tumor cells.

    Directory of Open Access Journals (Sweden)

    Enken Drecoll

    Full Text Available BACKGROUND: Alpha-particle emitting isotopes are effective novel tools in cancer therapy, but targeted delivery into tumors is a prerequisite of their application to avoid toxic side effects. Peritoneal carcinomatosis is a widespread dissemination of tumors throughout the peritoneal cavity. As peritoneal carcinomatosis is fatal in most cases, novel therapies are needed. F3 is a tumor homing peptide which is internalized into the nucleus of tumor cells upon binding to nucleolin on the cell surface. Therefore, F3 may be an appropriate carrier for alpha-particle emitting isotopes facilitating selective tumor therapies. PRINCIPAL FINDINGS: A dimer of the vascular tumor homing peptide F3 was chemically coupled to the alpha-emitter (213Bi ((213Bi-DTPA-[F3](2. We found (213Bi-DTPA-[F3](2 to accumulate in the nucleus of tumor cells in vitro and in intraperitoneally growing tumors in vivo. To study the anti-tumor activity of (213Bi-DTPA-[F3](2 we treated mice bearing intraperitoneally growing xenograft tumors with (213Bi-DTPA-[F3](2. In a tumor prevention study between the days 4-14 after inoculation of tumor cells 6x1.85 MBq (50 microCi of (213Bi-DTPA-[F3](2 were injected. In a tumor reduction study between the days 16-26 after inoculation of tumor cells 6x1.85 MBq of (213Bi-DTPA-[F3](2 were injected. The survival time of the animals was increased from 51 to 93.5 days in the prevention study and from 57 days to 78 days in the tumor reduction study. No toxicity of the treatment was observed. In bio-distribution studies we found (213Bi-DTPA-[F3](2 to accumulate in tumors but only low activities were found in control organs except for the kidneys, where (213Bi-DTPA-[F3](2 is found due to renal excretion. CONCLUSIONS/SIGNIFICANCE: In conclusion we report that (213Bi-DTPA-[F3](2 is a novel tool for the targeted delivery of alpha-emitters into the nucleus of tumor cells that effectively controls peritoneal carcinomatosis in preclinical models and may also be

  7. Students as "Humans Chromosomes" in Role-Playing Mitosis and Meiosis

    Science.gov (United States)

    Chinnici, Joseph P.; Yue, Joyce W.; Torres, Kieron M.

    2004-01-01

    Students often find it challenging to understand mitosis and meiosis and determine their processes. To develop an easier way to understand these terms, students are asked to role-play mitosis and meiosis and students themselves act as human chromosomes, which help students to learn differences between mitosis and meiosis.

  8. Using pool noodles to teach mitosis and meiosis.

    Science.gov (United States)

    Locke, John; McDermid, Heather E

    2005-05-01

    Although mitosis and meiosis are fundamental to understanding genetics, students often find them difficult to learn. We suggest using common "pool noodles" as teaching aids to represent chromatids in classroom demonstrations. Students use these noodles to demonstrate the processes of synapsis, segregation, and recombination. Student feedback has been overwhelmingly positive.

  9. Mitosis: Too Much of a Good Thing (Can Be Bad)

    OpenAIRE

    Khodjakov, Alexey; Rieder, Conly L.

    2009-01-01

    Recent studies reveal that the precise regulation of microtubule dynamics is essential for an error-free mitosis. Kinetochore microtubule attachments that are too stable increase the rate of chromosome mis-segregation, a leading cause of chromosomal instability in tumors.

  10. Using Pool Noodles to Teach Mitosis and Meiosis

    OpenAIRE

    Locke, John; McDermid, Heather E.

    2005-01-01

    Although mitosis and meiosis are fundamental to understanding genetics, students often find them difficult to learn. We suggest using common “pool noodles” as teaching aids to represent chromatids in classroom demonstrations. Students use these noodles to demonstrate the processes of synapsis, segregation, and recombination. Student feedback has been overwhelmingly positive.

  11. The Yb body, a major site for Piwi-associated RNA biogenesis and a gateway for Piwi expression and transport to the nucleus in somatic cells.

    Science.gov (United States)

    Qi, Hongying; Watanabe, Toshiaki; Ku, Hsueh-Yen; Liu, Na; Zhong, Mei; Lin, Haifan

    2011-02-01

    Despite exciting progress in understanding the Piwi-interacting RNA (piRNA) pathway in the germ line, less is known about this pathway in somatic cells. We showed previously that Piwi, a key component of the piRNA pathway in Drosophila, is regulated in somatic cells by Yb, a novel protein containing an RNA helicase-like motif and a Tudor-like domain. Yb is specifically expressed in gonadal somatic cells and regulates piwi in somatic niche cells to control germ line and somatic stem cell self-renewal. However, the molecular basis of the regulation remains elusive. Here, we report that Yb recruits Armitage (Armi), a putative RNA helicase involved in the piRNA pathway, to the Yb body, a cytoplasmic sphere to which Yb is exclusively localized. Moreover, co-immunoprecipitation experiments show that Yb forms a complex with Armi. In Yb mutants, Armi is dispersed throughout the cytoplasm, and Piwi fails to enter the nucleus and is rarely detectable in the cytoplasm. Furthermore, somatic piRNAs are drastically diminished, and soma-expressing transposons are desilenced. These observations indicate a crucial role of Yb and the Yb body in piRNA biogenesis, possibly by regulating the activity of Armi that controls the entry of Piwi into the nucleus for its function. Finally, we discovered putative endo-siRNAs in the flamenco locus and the Yb dependence of their expression. These observations further implicate a role for Yb in transposon silencing via both the piRNA and endo-siRNA pathways. PMID:21106531

  12. Differentiation of adipose-derived stem cells toward nucleus pulposuslike cells induced by hypoxia and a three-dimensional chitosan-alginate gel scaffold in vitro

    Institute of Scientific and Technical Information of China (English)

    Zhang Zhicheng; Li Fang; Tian Haiquan; Guan Kai; Zhao Guangmin; Shan Jianlin; Ren Dajiang

    2014-01-01

    Background Injectable three-dimensional (3D) scaffolds have the advantages of fluidity and moldability to fill irregularshaped defects,simple incorporation of bioactive factors,and limited surgical invasiveness.Adipose-derived stem cells (ADSCs) are multipotent and can be differentiated toward nucleus pulposus (NP)-Iike cells.A hypoxic environment may be important for differentiation to NP-like cells because the intervertebral disc is an avascular tissue.Hence,we investigated the induction effects of hypoxia and an injectable 3D chitosan-alginate (C/A) gel scaffold on ADSCs.Methods The C/A gel scaffold consisted of medical-grade chitosan and alginate.Gel porosity was calculated by liquid displacement method.Pore microstructure was analyzed by light and scanning electron microscopy.ADSCs were isolated and cultured by conventional methods.Passage 2 BrdU-labeled ADSCs were co-cultured with the C/A gel.ADSCs were divided into three groups (control,normoxia-induced,and hypoxia-induced groups).In the control group,cells were cultured in 10% FBS/DMEM.Hypoxia-induced and normoxia-induced groups were induced by adding transforming growth factor-β1,dexamethasone,vitamin C,sodium pyruvate,proline,bone morphogenetic protein-7,and 1% ITS-plus to the culture medium and maintaining in 2% and 20% O2,respectively.Histological and morphological changes were observed by light and electron microscopy.ADSCs were characterized by flow cytometry.Cell viability was investigated by BrdU incorporation.Proteoglycan and type Ⅱ collagen were measured by safranin O staining and the Sicool method,respectively.mRNA expression of hypoxia-inducing factor-1α (HIF-1α),aggrecan,and Type Ⅱ collagen was determined by reverse transcription-polymerase chain reaction.Results C/A gels had porous exterior surfaces with 80.57% porosity and 50-200 μm pore size.Flow cytometric analysis of passage 2 rabbit ADSCs showed high CD90 expression,while CD45 expression was very low.The morphology of

  13. 3D/4D architecture of chromosomal break point regions in the cell nucleus following irradiation of normal cells and tumor cells; 3D/4D Architektur von chromosomalen Bruchpunktregionen im Zellkern nach Bestrahlung von Normalzellen und Tumorzellen

    Energy Technology Data Exchange (ETDEWEB)

    Hausmann, M.; Cremer, C.; Friedl, A.; Dollinger, G.; Loebrich, M.; Friedland, W.

    2015-01-15

    The development of an effective analytical methodology for a correct description of oncogenic chromosomal aberrations is the challenge of medical radiobiology with respect to preventive therapeutic methods. Scope of the project was a better understanding of the behavior of break point regions dependent on the genome loci, the chromatin folding, the involved repair proteins and the beam quality with respect to an improvement and an efficient prognosis of the health consequences following radiation exposure. New microscopic insights in the normal cell nucleus are supposed to allow a better understanding of the spatial interactions on a molecular scale.

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

    Science.gov (United States)

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

    2015-01-01

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

  15. An organelle-exclusion envelope assists mitosis and underlies distinct molecular crowding in the spindle region.

    Science.gov (United States)

    Schweizer, Nina; Pawar, Nisha; Weiss, Matthias; Maiato, Helder

    2015-08-31

    The mitotic spindle is a microtubular assembly required for chromosome segregation during mitosis. Additionally, a spindle matrix has long been proposed to assist this process, but its nature has remained elusive. By combining live-cell imaging with laser microsurgery, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy in Drosophila melanogaster S2 cells, we uncovered a microtubule-independent mechanism that underlies the accumulation of molecules in the spindle region. This mechanism relies on a membranous system surrounding the mitotic spindle that defines an organelle-exclusion zone that is conserved in human cells. Supported by mathematical modeling, we demonstrate that organelle exclusion by a membrane system causes spatio-temporal differences in molecular crowding states that are sufficient to drive accumulation of mitotic regulators, such as Mad2 and Megator/Tpr, as well as soluble tubulin, in the spindle region. This membranous "spindle envelope" confined spindle assembly, and its mechanical disruption compromised faithful chromosome segregation. Thus, cytoplasmic compartmentalization persists during early mitosis to promote spindle assembly and function.

  16. Nucleoporin translocated promoter region (Tpr) associates with dynein complex, preventing chromosome lagging formation during mitosis.

    Science.gov (United States)

    Nakano, Hiroshi; Funasaka, Tatsuyoshi; Hashizume, Chieko; Wong, Richard W

    2010-04-01

    Gain or loss of whole chromosomes is often observed in cancer cells and is thought to be due to aberrant chromosome segregation during mitosis. Proper chromosome segregation depends on a faithful interaction between spindle microtubules and kinetochores. Several components of the nuclear pore complex/nucleoporins play critical roles in orchestrating the rapid remodeling events that occur during mitosis. Our recent studies revealed that the nucleoporin, Rae1, plays critical roles in maintaining spindle bipolarity. Here, we show association of another nucleoporin, termed Tpr (translocated promoter region), with the molecular motors dynein and dynactin, which both orchestrate with the spindle checkpoints Mad1 and Mad2 during cell division. Overexpression of Tpr enhanced multinucleated cell formation. RNA interference-mediated knockdown of Tpr caused a severe lagging chromosome phenotype and disrupted spindle checkpoint proteins expression and localization. Next, we performed a series of rescue and dominant negative experiments to confirm that Tpr orchestrates proper chromosome segregation through interaction with dynein light chain. Our data indicate that Tpr functions as a spatial and temporal regulator of spindle checkpoints, ensuring the efficient recruitment of checkpoint proteins to the molecular motor dynein to promote proper anaphase formation.

  17. Simple Laboratory methods to measure cell proliferation using DNA synthesis property

    Directory of Open Access Journals (Sweden)

    Madhavan H N

    2007-01-01

    Full Text Available This is a mini-review on the techniques to measure proliferation of cells by estimation of DNA synthesis. This is not an exhaustive review of literature, but a bird’s eye view of a few selected articles which may provide the technical details to the readers.The nucleus of a cell occupies about 10-30% of the cells space, depends on the type of genetic material (DNA -DeoxyriboNucleic Acid. DNA is a long, double-stranded, helical molecule which carries the genetic information. Duplication of the DNA takes place by the phenomena of replication. One copy of double-stranded DNA molecule forms two double-stranded DNA molecules. DNA replication is the fundamental process used in all living organisms as it is the basis for biological inheritance. This process is known also as Mitosis in somatic cells. In Mitosis, the duplication process results in two genetically identical "daughter" cells from a single "parent" cell. The resulting double-stranded DNA molecules are identical; proof reading and error-checking mechanisms exist to ensure near perfect pair. Mitosis is divided into six phases: prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis.

  18. Mitosis detection in breast cancer histological images An ICPR 2012 contest

    Directory of Open Access Journals (Sweden)

    Ludovic Roux

    2013-01-01

    Full Text Available Introduction: In the framework of the Cognitive Microscope (MICO project, we have set up a contest about mitosis detection in images of H and E stained slides of breast cancer for the conference ICPR 2012. Mitotic count is an important parameter for the prognosis of breast cancer. However, mitosis detection in digital histopathology is a challenging problem that needs a deeper study. Indeed, mitosis detection is difficult because mitosis are small objects with a large variety of shapes, and they can thus be easily confused with some other objects or artefacts present in the image. We added a further dimension to the contest by using two different slide scanners having different resolutions and producing red-green-blue (RGB images, and a multi-spectral microscope producing images in 10 different spectral bands and 17 layers Z-stack. 17 teams participated in the study and the best team achieved a recall rate of 0.7 and precision of 0.89. Context: Several studies on automatic tools to process digitized slides have been reported focusing mainly on nuclei or tubule detection. Mitosis detection is a challenging problem that has not yet been addressed well in the literature. Aims: Mitotic count is an important parameter in breast cancer grading as it gives an evaluation of the aggressiveness of the tumor. However, consistency, reproducibility and agreement on mitotic count for the same slide can vary largely among pathologists. An automatic tool for this task may help for reaching a better consistency, and at the same time reducing the burden of this demanding task for the pathologists. Subjects and Methods: Professor Frιdιrique Capron team of the pathology department at Pitiι-Salpκtriθre Hospital in Paris, France, has selected a set of five slides of breast cancer. The slides are stained with H and E. They have been scanned by three different equipments: Aperio ScanScope XT slide scanner, Hamamatsu NanoZoomer 2.0-HT slide scanner and 10 bands

  19. ChAcNLS, a Novel Modification to Antibody-Conjugates Permitting Target Cell-Specific Endosomal Escape, Localization to the Nucleus, and Enhanced Total Intracellular Accumulation.

    Science.gov (United States)

    Beaudoin, Simon; Rondeau, Andreanne; Martel, Olivier; Bonin, Marc-Andre; van Lier, Johan E; Leyton, Jeffrey V

    2016-06-01

    The design of antibody-conjugates (ACs) for delivering molecules for targeted applications in humans has sufficiently progressed to demonstrate clinical efficacy in certain malignancies and reduced systemic toxicity that occurs with standard nontargeted therapies. One area that can advance clinical success for ACs will be to increase their intracellular accumulation. However, entrapment and degradation in the endosomal-lysosomal pathway, on which ACs are reliant for the depositing of their molecular payload inside target cells, leads to reduced intracellular accumulation. Innovative approaches that can manipulate this pathway may provide a strategy for increasing accumulation. We hypothesized that escape from entrapment inside the endosomal-lysosomal pathway and redirected trafficking to the nucleus could be an effective approach to increase intracellular AC accumulation in target cells. Cholic acid (ChAc) was coupled to the peptide CGYGPKKKRKVGG containing the nuclear localization sequence (NLS) from SV-40 large T-antigen, which is termed ChAcNLS. ChAcNLS was conjugated to the mAb 7G3 (7G3-ChAcNLS), which has nanomolar affinity for the cell-surface leukemic antigen interleukin-3 receptor-α (IL-3Rα). Our aim was to determine whether 7G3-ChAcNLS increased intracellular accumulation while retaining nanomolar affinity and IL-3Rα-positive cell selectivity. Competition ELISA and cell treatment assays were performed. Cell fractionation, confocal microscopy, flow cytometry, and Western blot techniques were used to determine the level of antibody accumulation inside cells and in corresponding nuclei. In addition, the radioisotope copper-64 ((64)Cu) was also utilized as a surrogate molecular cargo to evaluate nuclear and intracellular accumulation by radioactivity counting. 7G3-ChAcNLS effectively escaped endosome entrapment and degradation resulting in a unique intracellular distribution pattern. mAb modification with ChAcNLS maintained 7G3 nM affinity and produced high

  20. RPL41, a Small Ribosomal Peptide Deregulated in Tumors, Is Essential for Mitosis and Centrosome Integrity

    Directory of Open Access Journals (Sweden)

    Shan Wang

    2010-03-01

    Full Text Available Ribosomal large subunit protein RPL41 is a basic (positively charged peptide consisting of only 25 amino acids. An antisense-based functional screening revealed that the down-regulation of RPL41 led to an anchorage-independent growth of NIH3T3 cells in soft agar plates. RPL41 depletion with gene-specific small interfering RNA also resulted in malignant transformation of NIH3T3 cells including increased tumor growth in mice. RPL41 deletion was detected in 59% of tumor cell lines by fluorescence in situ hybridization analyses and RPL41 down-regulation in 75% of primary breast cancers by real-time quantitative reverse transcription-polymerase chain reaction. These studies suggest a tumor suppression role for RPL41. By mass spectrometry, RPL41 was associated with several cytoskeleton components including tubulin β, γ, and myosin IIA, which was confirmed by Western blot analysis on both cellular lysis and individually in vitro-expressed proteins. RPL41 also bound directly to polymerized tubulins. Cells overexpressing a GFP-RPL41 were resistant to nocodazole-induced microtubule depolymerization. A synthetic RPL41 induced cellular α-tubulin acetylation and G2/M cell cycle arrest. These results indicate a stabilizing role of RPL41 on microtubule. Microtubule spindles are essential for chromosome segregation during mitosis. Cells with RPL41 knock-down showed abnormal spindles, frequent failure of cytokinesis, and formation of polynuclear cells. In interphase cells, RPL41-depleted cells had premature splitting of centrosome. Our results provide evidence that RPL41 is a microtubule-associated protein essential for functional spindles and for the integrity of centrosome and that the abnormal mitosis and disrupted centrosome associated with the RPL41 down-regulation may be related to malignant transformation.

  1. THE MORPHOLOGICAL CHANGES IN MUSCLE SPINDLES AND ALTERATIONS IN CELL ACTIVITY OF THE RATS' RED NUCLEUS AFTER 2 WEEKS' SIMULATED WEIGHTLESSNESS

    Institute of Scientific and Technical Information of China (English)

    Zhu Yongjin; Fan Xiaoli; Wu Sudi; Li Qiang

    2006-01-01

    Objective To study the morphological changes of soleus muscle spindle and electrical activity of neurons in Red Nucleus(RN) of the rat after 2 weeks' simulated weightlessness, and to reveal the interaction between proprioceptive inputs of muscle spindles and reciprocal alterations in RN under simulated weightlessness. Methods Twenty female rats were exposed to weightlessness simulated by tail-suspension for 14 days (SW-14d). Body weight(200-220g) matched female rats were control group(Con). The morphological changes in isolated muscle spindle of soleus muscle, the discharges of red nucleus neurons were observed after 14d tail-suspensions by silver staining and extracellular recording respectively. Results Compared with control group ,the nerve ending of muscle spindle in SW-14d was distorted, degenerated and dissolved; the diameters of intrafusal fibers and capsule in equatorial region of soleus muscle spindles were diminished(P<0.05). The spontaneous cell activity and discharge of RN neurons (spikes/s) induced by afferent firing from muscle spindles after injection of succinylcholine were reduced after 2 weeks' simulated weightlessness respectively (18.44±5.96 vs. 10.19±6.88, 32.50±8.08 vs. 16.86±5.97, P<0.01). Conclusion The degeneration of muscle spindle induced by simulated weightlessness may be one of the causes that led to alterations in discharges of RN.

  2. CS/PAA@TPGS/PLGA nanoparticles with intracellular pH-sensitive sequential release for delivering drug to the nucleus of MDR cells.

    Science.gov (United States)

    Wang, Ying-Ying; Zhang, Dan-Dan; Kong, Yan-Yan; Shao, Luan-Luan; Zhang, Fen-Yi; Gao, Yu; Mu, Xu; Wang, Jie; Li, Hao-Fan; Yu, Shu-Qin; Xu, Qian

    2016-09-01

    Development of novel nano-drug delivery systems (NDDS) that can transport anticancer drugs into cell nuclei is still a highly desirable strategy for reversing multi-drug resistance (MDR) in cancer therapy. Herein, we designed and prepared a novel NDDS, designated S@L NPs, in which several smaller nanoparticles are contained within a larger nanoparticle. Our S@L NPs (CS/PAA/VP-16@TPGS/PLGA NPs) possess a structure in which smaller nanoparticles (Chitosan-Poly(acrylic acid) nanoparticles, CS/PAA NPs) containing the drug etoposide (VP-16) are loaded within a larger nanoparticle (Vitamin E d-a-tocopheryl polyethylene glycol 1000 succinate-modified poly(lactic-co-glycolic acid) nanoparticles, TPGS/PLGA NPs). The system utilizes intracellular pH gradients to achieve pH-sensitive sequential release within different intracellular domains of MDR cells. S@L NPs could be triggered to degrade and release CS/PAA/VP-16 NPs in the acid environment of the cytosol, endosomes or lysosomes, and CS/PAA/VP-16 NPs were capable of entering the nucleus through nucleopores. It is significant that CS/PAA/VP-16 NPs exhibit disaggregation in the alkaline environment of the nucleus and thereby release the contained anticancer drug. Further mechanistic studies showed that CS/PAA/VP-16 NPs escaped retention and degradation within lysosomes and protected the drug from P-glycoprotein-induced efflux. Simultaneously, S@L NPs enhanced the anticancer effect of the loaded drug by inducing autophagy and apoptosis of MDR cells. This novel NDDS may provide a promising platform for nuclear drug delivery for reversing MDR. PMID:27289313

  3. Label-free mitosis detection in tumor spheroids using tissue dynamics imaging

    Science.gov (United States)

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

    2012-03-01

    The detection of cellular mitosis inside three-dimensional living tissue at depths up to 1 mm has been beyond the detection limits of conventional microscopies. In this paper, we demonstrate the use of motility contrast imaging and fluctuation spectroscopy to detect motional signatures that we attribute to mitotic events within groups of 100 cells in multicellular tumor spheroids. Motility contrast imaging is a coherence-domain speckle-imaging technique that uses low-coherence off-axis holography as a coherence gate to localize dynamic light scattering from selected depths inside tissue. Fluctuation spectroscopy is performed on a pervoxel basis to generate micro-spectrograms that display frequency content vs. time. Mitosis, especially in Telophase and Cytokinesis, is a relatively fast and high-amplitude phenomenon that should display energetic features within the micro-spectrograms. By choosing an appropriate frequency range and threshold, we detect energetic events with a density and rate that are comparable to the expected mitotic fraction in the UMR cell line. By studying these mitotic events in tumors of two different sizes, we show that micro-spectrograms contain characteristically different information content than macro-spectrograms (averaged over many voxels) in which the mitotic signatures (which are overall a low-probability event) are averaged out. The detection of mitotic fraction in thick living tissue has important consequences for the use of tissue-based assays for drug discovery.

  4. Cell cycle-dependent alteration in NAC1 nuclear body dynamics and morphology

    Science.gov (United States)

    Wu, Pei-Hsun; Hung, Shen-Hsiu; Ren, Tina; Shih, Ie-Ming; Tseng, Yiider

    2011-02-01

    NAC1, a BTB/POZ family member, has been suggested to participate in maintaining the stemness of embryonic stem cells and has been implicated in the pathogenesis of human cancer. In ovarian cancer, NAC1 upregulation is associated with disease aggressiveness and with the development of chemoresistance. Like other BTB/POZ proteins, NAC1 forms discrete nuclear bodies in non-dividing cells. To investigate the biological role of NAC1 nuclear bodies, we characterized the expression dynamics of NAC1 nuclear bodies during different phases of the cell cycle. Fluorescence recovery after photobleaching assays revealed that NAC1 was rapidly exchanged between the nucleoplasm and NAC1 nuclear bodies in interphase cells. The number of NAC1 bodies significantly increased and their size decreased in the S phase as compared to the G0/G1 and G2 phases. NAC1 nuclear bodies disappeared and NAC1 became diffuse during mitosis. NAC1 nuclear bodies reappeared immediately after completion of mitosis. These results indicate that a cell cycle-dependent regulatory mechanism controls NAC1 body formation in the nucleus and suggest that NAC1 body dynamics are associated with mitosis or cytokinesis.

  5. A cell cycle role for the epigenetic factor CTCF-L/BORIS.

    Directory of Open Access Journals (Sweden)

    Manuel Rosa-Garrido

    Full Text Available CTCF is a ubiquitous epigenetic regulator that has been proposed as a master keeper of chromatin organisation. CTCF-like, or BORIS, is thought to antagonise CTCF and has been found in normal testis, ovary and a large variety of tumour cells. The cellular function of BORIS remains intriguing although it might be involved in developmental reprogramming of gene expression patterns. We here unravel the expression of CTCF and BORIS proteins throughout human epidermis. While CTCF is widely distributed within the nucleus, BORIS is confined to the nucleolus and other euchromatin domains. Nascent RNA experiments in primary keratinocytes revealed that endogenous BORIS is present in active transcription sites. Interestingly, BORIS also localises to interphase centrosomes suggesting a role in the cell cycle. Blocking the cell cycle at S phase or mitosis, or causing DNA damage, produced a striking accumulation of BORIS. Consistently, ectopic expression of wild type or GFP- BORIS provoked a higher rate of S phase cells as well as genomic instability by mitosis failure. Furthermore, down-regulation of endogenous BORIS by specific shRNAs inhibited both RNA transcription and cell cycle progression. The results altogether suggest a role for BORIS in coordinating S phase events with mitosis.

  6. Microtubule detyrosination guides chromosomes during mitosis

    OpenAIRE

    Barisic, Marin; Silva e Sousa, Ricardo; Tripathy, Suvranta K.; Magiera, Maria M.; Zaytsev, Anatoly V.; Pereira, Ana L.; Janke, Carsten; Grishchuk, Ekaterina L.; Maiato, Helder

    2015-01-01

    Before chromosomes segregate into daughter cells they align at the mitotic spindle equator, a process known as chromosome congression. CENP-E/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically towards the equator. Here we found that congression of pole-proximal c...

  7. Mitosis detection in breast cancer pathology images by combining handcrafted and convolutional neural network features.

    Science.gov (United States)

    Wang, Haibo; Cruz-Roa, Angel; Basavanhally, Ajay; Gilmore, Hannah; Shih, Natalie; Feldman, Mike; Tomaszewski, John; Gonzalez, Fabio; Madabhushi, Anant

    2014-10-01

    Breast cancer (BCa) grading plays an important role in predicting disease aggressiveness and patient outcome. A key component of BCa grade is the mitotic count, which involves quantifying the number of cells in the process of dividing (i.e., undergoing mitosis) at a specific point in time. Currently, mitosis counting is done manually by a pathologist looking at multiple high power fields (HPFs) on a glass slide under a microscope, an extremely laborious and time consuming process. The development of computerized systems for automated detection of mitotic nuclei, while highly desirable, is confounded by the highly variable shape and appearance of mitoses. Existing methods use either handcrafted features that capture certain morphological, statistical, or textural attributes of mitoses or features learned with convolutional neural networks (CNN). Although handcrafted features are inspired by the domain and the particular application, the data-driven CNN models tend to be domain agnostic and attempt to learn additional feature bases that cannot be represented through any of the handcrafted features. On the other hand, CNN is computationally more complex and needs a large number of labeled training instances. Since handcrafted features attempt to model domain pertinent attributes and CNN approaches are largely supervised feature generation methods, there is an appeal in attempting to combine these two distinct classes of feature generation strategies to create an integrated set of attributes that can potentially outperform either class of feature extraction strategies individually. We present a cascaded approach for mitosis detection that intelligently combines a CNN model and handcrafted features (morphology, color, and texture features). By employing a light CNN model, the proposed approach is far less demanding computationally, and the cascaded strategy of combining handcrafted features and CNN-derived features enables the possibility of maximizing the performance

  8. Cascaded ensemble of convolutional neural networks and handcrafted features for mitosis detection

    Science.gov (United States)

    Wang, Haibo; Cruz-Roa, Angel; Basavanhally, Ajay; Gilmore, Hannah; Shih, Natalie; Feldman, Mike; Tomaszewski, John; Gonzalez, Fabio; Madabhushi, Anant

    2014-03-01

    Breast cancer (BCa) grading plays an important role in predicting disease aggressiveness and patient outcome. A key component of BCa grade is mitotic count, which involves quantifying the number of cells in the process of dividing (i.e. undergoing mitosis) at a specific point in time. Currently mitosis counting is done manually by a pathologist looking at multiple high power fields on a glass slide under a microscope, an extremely laborious and time consuming process. The development of computerized systems for automated detection of mitotic nuclei, while highly desirable, is confounded by the highly variable shape and appearance of mitoses. Existing methods use either handcrafted features that capture certain morphological, statistical or textural attributes of mitoses or features learned with convolutional neural networks (CNN). While handcrafted features are inspired by the domain and the particular application, the data-driven CNN models tend to be domain agnostic and attempt to learn additional feature bases that cannot be represented through any of the handcrafted features. On the other hand, CNN is computationally more complex and needs a large number of labeled training instances. Since handcrafted features attempt to model domain pertinent attributes and CNN approaches are largely unsupervised feature generation methods, there is an appeal to attempting to combine these two distinct classes of feature generation strategies to create an integrated set of attributes that can potentially outperform either class of feature extraction strategies individually. In this paper, we present a cascaded approach for mitosis detection that intelligently combines a CNN model and handcrafted features (morphology, color and texture features). By employing a light CNN model, the proposed approach is far less demanding computationally, and the cascaded strategy of combining handcrafted features and CNN-derived features enables the possibility of maximizing performance by

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

    Science.gov (United States)

    Griffin, Brian; Kilfoil, Maria L.

    2013-03-01

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

  10. The Significance of Studying the Origin of the Cell Nucleus and the Way for Studying%细胞核起源研究的意义和研究途径的探讨

    Institute of Scientific and Technical Information of China (English)

    李靖炎

    2001-01-01

    The cell nucleus as the controller of all genetic and physiological activities within the cell,is the most prominent marker of eukaryotic cells.The formation of the cell nucleus is the key event during the origin of eukaryotic cells.The first appearance of the primitive cell nucleus indicates the emergence of the first eukaryotic cell.The research on the origin of the cell nucleus not only enriches the modern cell biology and evolutionary biology,but might also even influence the further development of the molecular cell biology by stimulating cell biologists to consider the significance of the viewpoint of evolution to cell researches.   However,for a long time,on the origin of the cell nucleus there were only several reckless and assumptions and a few earnest efforts which were unsuccessful or only indirectly related to the formation of the nucleus.One of the main reasons for this situation seems to be the lack of a practical way for the study.   Through searching for a long time we found a way.The essential point of the way is to consider the comprehension of the primitive nucleus as the key link for understanding the whole process of and early evolution of the cell nucleus.We have already known that the prokaryotic ancestor of eukaryotes must be one kind of ancient archaea (Li,1999).The primitive cell nucleus should be an intermediate link between the archaeal nucleoid and the typical cell nucleus.In order to obtain some features of the original nucleus,We' d better study the most primitive protists we can find today and investigate all aspects of their cell nucleus thoroughly.Then,combined with the present knowledge on archaea we would be able to propose a hypothetical model for the primitive cell nucleus,and arrange various possible experiments to examine it from various aspects in order to test,to modify,to improve it,or replace it with a new one.Along this way,we would finally obtain a convincing model of the primitive cell

  11. [CHROMATIN ORGANIZATION IN CELL CYCLE OF AMOEBA PROTEUS ACCORDING TO OPTICAL TOMOGRAPHY DATA].

    Science.gov (United States)

    Demin, S Yu; Berdieva, M A; Podlipaeva, Yu I; Yudin, A L; Goodkov, A V

    2015-01-01

    For the first time the nuclear cycle of large freshwater amoeba Amoeba proteus was studied by the method of optical tomography. The nuclei were fixed in situ in the cells of synchronized culture, stained by DAPI and examined by confocal laser scanning microscope. 3D-images of intranuclear chromatin were studied in details at different stages of nuclear cycle. The obtained data, together with literary ones allow represent the dynamics of structural organization of the nucleus in Amoeba proteus cell cycle in a new fashion. It was concluded that in this species the two-stage interphase takes place, as well as mitosis of peculiar type which does not correspond to any known type of mitosis according to classification existing now. It is presumed that in the course of nuclear cycle the chromosomes and/or their fragments are amplified, this presumption being in a good correspondence with the data about nuclear DNA hyperreplication in the cell cycle of A. proteus. As a result of chromosomes amplification their number may vary at different stages of cell cycle, and it allows to explain the contradictory data concerning the exact number of chromosomes in this species. The elimination of extra-DNA occurs mainly at the stage between prophase and prometaphase. We presume the majority of chromosomes, or may be even all of them to be referred to cholocentric type according to their behaviour during the mitosis.

  12. Lamin A reassembly at the end of mitosis is regulated by its SUMO-interacting motif.

    Science.gov (United States)

    Moriuchi, Takanobu; Kuroda, Masaki; Kusumoto, Fumiya; Osumi, Takashi; Hirose, Fumiko

    2016-03-01

    Modification of proteins with small ubiquitin-related modifier (SUMO; SUMOylation) is involved in the regulation of various biological processes. Recent studies have demonstrated that noncovalent associations between SUMOylated proteins and co-operative proteins containing SUMO-interacting motifs (SIMs) are important for the spatiotemporal organization of many protein complexes. In this study, we demonstrate that interactions between lamin A, a major component of the nuclear lamina, and SUMO isoforms are dependent on one of the four SIMs (SIM3) resided in lamin A polypeptide in vitro. Live cell imaging and immunofluorescence staining showed that SIM3 is required for accumulation of lamin A on the chromosomes during telophase, and subsequent evaluation of a panel of deletion mutants determined that a 156-amino acid region spanning the carboxyl-terminal Ig-fold domain of lamin A is sufficient for this accumulation. Notably, mutation of SIM3 abrogated the dephosphorylation of mitosis-specific phosphorylation at Ser-22 of lamin A, which normally occurs during telophase, and the subsequent nuclear lamina reorganization. Furthermore, expression of a conjugation-defective SUMO2 mutant, which was previously shown to inhibit endogenous SUMOylation in a dominant-negative manner, also impaired the accumulation of wild type lamin A on telophase chromosomes. These findings suggest that interactions between SIM3 of lamin A and a putative SUMO2-modified protein plays an important role in the reorganization of the nuclear lamina at the end of mitosis.

  13. Matrix elasticity, cytoskeletal forces and physics of the nucleus: how deeply do cells ‘feel’ outside and in?

    OpenAIRE

    Buxboim, Amnon; Ivanovska, Irena L.; Discher, Dennis E.

    2010-01-01

    Cellular organization within a multicellular organism requires that a cell assess its relative location, taking in multiple cues from its microenvironment. Given that the extracellular matrix (ECM) consists of the most abundant proteins in animals and contributes both structure and elasticity to tissues, ECM probably provides key physical cues to cells. In vivo, in the vicinity of many tissue cell types, fibrous characteristics of the ECM are less discernible than the measurably distinct elas...

  14. Human Tissue Factor Pathway Inhibitor-2 is Internalized by Cells and Translocated to the Nucleus by the Importin System

    OpenAIRE

    Kempaiah, Prakasha; Chand, Hitendra S.; Kisiel, Walter

    2008-01-01

    Tissue factor pathway inhibitor-2 (TFPI-2) is a serine proteinase inhibitor that induces caspase-mediated apoptosis when offered to a variety of tumor cells. In order to investigate the mechanism of TFPI-2-induced apoptosis, we initially studied the uptake and trafficking of TFPI-2 by HT-1080 cells. Exogenously offered TFPI-2 was rapidly internalized and distributed in both the cytosolic and nuclear fractions. Nuclear localization of TFPI-2 was also detected in a variety of endothelial cells ...

  15. Three-dimensional scaffold of type II collagen promote the differentiation of adipose-derived stem cells into a nucleus pulposus-like phenotype.

    Science.gov (United States)

    Zhou, Xiaopeng; Tao, Yiqing; Wang, Jingkai; Liu, Dongyu; Liang, Chengzhen; Li, Hao; Chen, Qixin

    2016-07-01

    Type II collagen is reported to have the capability of guiding adipose-derived stem cells (ADSCs) to differentiate towards a nucleus pulposus (NP)-like phenotype. So this study aimed to establish a three-dimensional (3D) collagen scaffold using N,N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide and N-hydroxysuccinimide (EDAC/NHS) to increase the efficiency of ADSC differentiation into NP-like cells. Physical properties, such as porosity, biodegradation, and microstructure, and biological characteristics such as cytotoxicity, cell proliferation, and expression of relevant genes and proteins were measured to evaluate the efficacy of different scaffolds. Collagen scaffolds cross-linked with EDAC/NHS exhibited higher biological stability, better spatial structure, and higher gene and protein expression of functional markers such as aggrecan, SOX9 and COL2 than those of other groups. Based on the results, freeze-dried type II collagen cross-linked with EDAC/NHS formed the best 3D scaffold, for inducing ADSC proliferation and differentiation toward a NP-like phenotype. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1687-1693, 2016. PMID:26940048

  16. Levofloxacin increases the effect of serum deprivation on anoikis of rat nucleus pulposus cells via Bax/Bcl-2/caspase-3 pathway.

    Science.gov (United States)

    Yang, Si-Dong; Bai, Zhi-Long; Zhang, Feng; Ma, Lei; Yang, Da-Long; Ding, Wen-Yuan

    2014-12-01

    Levofloxacin, a fluoroquinolone, is a widely-used and effective antibiotic. However, various adverse side effects are associated with levofloxacin. The purpose of this study was to further explore the effects of levofloxacin on rat nucleus pulposus cells (NPCs). Inverted phase-contrast microscopy, flow cytometry and caspase-3 activity assays were used and revealed that serum deprivation induced apoptosis, which was markedly increased by levofloxacin in a dose-dependent manner. Simultaneously, levofloxacin decreased cell binding to type II collagen (COL2). Thus, levofloxacin-induced apoptosis exhibits characteristics of anoikis, the process by which cell death is triggered by separation from the extracellular matrix, which contains COL2. Furthermore, real-time quantitative RT-PCR was used to further confirm that levofloxacin downregulates COL2 expression in a dose-dependent manner. At last, western blot was used to find that levofloxacin increased the ratio of Bax/Bcl-2 and active caspase-3 in a dose-dependent manner. Levofloxacin therefore increases the effects of serum deprivation on anoikis by downregulating COL2 in rat NPCs in vitro via Bax/Bcl-2/caspase-3 pathway. This research provides a novel insight into the mechanisms of levofloxacin-induced toxicity and may potentially lead to a better understanding of the clinical effects of levofloxacin, especially in terms of intervertebral disc degeneration. PMID:25224805

  17. High Glucose-Induced Oxidative Stress Mediates Apoptosis and Extracellular Matrix Metabolic Imbalances Possibly via p38 MAPK Activation in Rat Nucleus Pulposus Cells

    Directory of Open Access Journals (Sweden)

    Xiaofei Cheng

    2016-01-01

    Full Text Available Objectives. To investigate whether high glucose-induced oxidative stress is implicated in apoptosis of rat nucleus pulposus cells (NPCs and abnormal expression of critical genes involved in the metabolic balance of extracellular matrix (ECM. Methods. NPCs were cultured with various concentrations of glucose to detect cell viability and apoptosis. Cells cultured with high glucose (25 mM were untreated or pretreated with N-acetylcysteine or a p38 MAPK inhibitor SB 202190. Reactive oxygen species (ROS production was evaluated. Activation of p38 MAPK was measured by Western blot. The expression of ECM metabolism-related genes, including type II collagen, aggrecan, SRY-related high-mobility-group box 9 (Sox-9, matrix metalloproteinase 3 (MMP-3, and tissue inhibitor of metalloproteinase 1 (TIMP-1, was analyzed by semiquantitative RT-PCR. Results. High glucose reduced viability of NPCs and induced apoptosis. High glucose resulted in increased ROS generation and p38 MAPK activation. In addition, it negatively regulated the expression of type II collagen, aggrecan, Sox-9, and TIMP-1 and positively regulated MMP-3 expression. These results were changed by pretreatment with N-acetylcysteine or SB 202190. Conclusions. High glucose might promote apoptosis of NPCs, trigger ECM catabolic pathways, and inhibit its anabolic activities, possibly through a p38 MAPK-dependent oxidative stress mechanism.

  18. Cell bodies of the trigeminal proprioceptive neurons that transmit reflex contraction of the levator muscle are located in the mesencephalic trigeminal nucleus in rats.

    Science.gov (United States)

    Fujita, Kenya; Matsuo, Kiyoshi; Yuzuriha, Shunsuke; Kawagishi, Kyutaro; Moriizumi, Tetsuji

    2012-12-01

    Since the levator and frontalis muscles lack interior muscle spindles despite being antigravity mixed muscles to involuntarily sustain eyelid opening and eyebrow lifting, this study has proposed a hypothetical mechanism to compensate for this anatomical defect. The voluntary contraction of fast-twitch fibres of the levator muscle stretches the mechanoreceptors in Müller's muscle to evoke proprioception, which continuously induces reflex contraction of slow-twitch fibres of the levator and frontalis muscles. This study confirmed the presence of cell bodies of the trigeminal proprioceptive neurons that transmit reflex contraction of the levator and frontalis muscles. After confirming that severing the trigeminal proprioceptive fibres that innervate the mechanoreceptors in Müller's muscle induced ipsilateral eyelid ptosis, Fluorogold was applied as a tracer to the proximal stump of the trigeminal proprioceptive nerve in rats. Fluorogold labelled the cell bodies of the trigeminal proprioceptive neurons, not in any regions of the rat brain including the trigeminal ganglion, but in the ipsilateral mesencephalic trigeminal nucleus neighbouring the locus ceruleus. Some Fluorogold particles accumulated in the area of the locus ceruleus. The trigeminal proprioceptive neurons could be considered centrally displaced ganglion cells to transmit afferent signal from the mechanoreceptors in Müller's muscle to the mesencephalon, where they may be able to make excitatory synaptic connections with both the oculomotor neurons and the frontalis muscle motoneurons for the involuntary coordination of the eyelid and eyebrow activities, and potentially to the locus ceruleus.

  19. High Glucose-Induced Oxidative Stress Mediates Apoptosis and Extracellular Matrix Metabolic Imbalances Possibly via p38 MAPK Activation in Rat Nucleus Pulposus Cells.

    Science.gov (United States)

    Cheng, Xiaofei; Ni, Bin; Zhang, Feng; Hu, Ying; Zhao, Jie

    2016-01-01

    Objectives. To investigate whether high glucose-induced oxidative stress is implicated in apoptosis of rat nucleus pulposus cells (NPCs) and abnormal expression of critical genes involved in the metabolic balance of extracellular matrix (ECM). Methods. NPCs were cultured with various concentrations of glucose to detect cell viability and apoptosis. Cells cultured with high glucose (25 mM) were untreated or pretreated with N-acetylcysteine or a p38 MAPK inhibitor SB 202190. Reactive oxygen species (ROS) production was evaluated. Activation of p38 MAPK was measured by Western blot. The expression of ECM metabolism-related genes, including type II collagen, aggrecan, SRY-related high-mobility-group box 9 (Sox-9), matrix metalloproteinase 3 (MMP-3), and tissue inhibitor of metalloproteinase 1 (TIMP-1), was analyzed by semiquantitative RT-PCR. Results. High glucose reduced viability of NPCs and induced apoptosis. High glucose resulted in increased ROS generation and p38 MAPK activation. In addition, it negatively regulated the expression of type II collagen, aggrecan, Sox-9, and TIMP-1 and positively regulated MMP-3 expression. These results were changed by pretreatment with N-acetylcysteine or SB 202190. Conclusions. High glucose might promote apoptosis of NPCs, trigger ECM catabolic pathways, and inhibit its anabolic activities, possibly through a p38 MAPK-dependent oxidative stress mechanism. PMID:27635402

  20. High Glucose-Induced Oxidative Stress Mediates Apoptosis and Extracellular Matrix Metabolic Imbalances Possibly via p38 MAPK Activation in Rat Nucleus Pulposus Cells

    Science.gov (United States)

    Cheng, Xiaofei; Ni, Bin; Zhang, Feng; Hu, Ying

    2016-01-01

    Objectives. To investigate whether high glucose-induced oxidative stress is implicated in apoptosis of rat nucleus pulposus cells (NPCs) and abnormal expression of critical genes involved in the metabolic balance of extracellular matrix (ECM). Methods. NPCs were cultured with various concentrations of glucose to detect cell viability and apoptosis. Cells cultured with high glucose (25 mM) were untreated or pretreated with N-acetylcysteine or a p38 MAPK inhibitor SB 202190. Reactive oxygen species (ROS) production was evaluated. Activation of p38 MAPK was measured by Western blot. The expression of ECM metabolism-related genes, including type II collagen, aggrecan, SRY-related high-mobility-group box 9 (Sox-9), matrix metalloproteinase 3 (MMP-3), and tissue inhibitor of metalloproteinase 1 (TIMP-1), was analyzed by semiquantitative RT-PCR. Results. High glucose reduced viability of NPCs and induced apoptosis. High glucose resulted in increased ROS generation and p38 MAPK activation. In addition, it negatively regulated the expression of type II collagen, aggrecan, Sox-9, and TIMP-1 and positively regulated MMP-3 expression. These results were changed by pretreatment with N-acetylcysteine or SB 202190. Conclusions. High glucose might promote apoptosis of NPCs, trigger ECM catabolic pathways, and inhibit its anabolic activities, possibly through a p38 MAPK-dependent oxidative stress mechanism.

  1. Autophagy attenuates the catabolic effect during inflammatory conditions in nucleus pulposus cells, as sustained by NF-κB and JNK inhibition.

    Science.gov (United States)

    Xu, Kang; Chen, Weijian; Wang, Xiaofei; Peng, Yan; Liang, Anjing; Huang, Dongsheng; Li, Chunhai; Ye, Wei

    2015-09-01

    Proteoglycan degradation contributing to the pathogenesis of intervertebral disc (IVD) degeneration is induced by inflammatory cytokines, such as tumor necrosis factor‑α (TNF‑α) and interleukin‑1β (IL‑1β). Cell autophagy exists in degenerative diseases, including osteoarthritis and intervertebral disc degeneration. However, the autophagy induced by TNF‑α and IL‑1β and the corresponding molecular mechanism appear to be cell‑type dependent. The effect and mechanism of autophagy regulated by TNF‑α and IL‑1β in IVDs remains unclear. Additionally, the impact of autophagy on the catabolic effect in inflammatory conditions also remains elusive. In the present study, autophagy activator and inhibitor were used to demonstrate the impact of autophagy on the catabolic effect induced by TNF‑α. A critical role of autophagy was identified in rat nucleus pulposus (NP) cells: Inhibition of autophagy suppresses, while activation of autophagy enhances, the catabolic effect of cytokines. Subsequently, the autophagy‑related gene expression in rat NP cells following TNF‑α and IL‑1β treatment was observed using immunofluorescence, quantitative polymerase chain reaction and western blot analysis; however, no association was present. In addition, nuclear factor κB (NF‑κB), c‑Jun N‑terminal kinase (JNK), extracellular signal‑regulated kinases and p38 mitogen‑activated protein kinase inhibitors and TNF‑α were used to determine the molecular mechanism of autophagy during the inflammatory conditions, and only the NF‑κB and JNK inhibitor were found to enhance the autophagy of rat NP cells. Finally, IKKβ knockdown was used to further confirm the effect of the NF‑κB signal on human NP cells autophagy, and the data showed that IKKβ knockdown upregulated the autophagy of NP cells during inflammatory conditions.

  2. Zona occludens-2 inhibits cyclin D1 expression and cell proliferation and exhibits changes in localization along the cell cycle.

    Science.gov (United States)

    Tapia, Rocio; Huerta, Miriam; Islas, Socorro; Avila-Flores, Antonia; Lopez-Bayghen, Esther; Weiske, Jörg; Huber, Otmar; González-Mariscal, Lorenza

    2009-02-01

    Here, we have studied the effect of the tight junction protein zona occludens (ZO)-2 on cyclin D1 (CD1) protein expression. CD1 is essential for cell progression through the G1 phase of the cell cycle. We have found that in cultures of synchronized Madin-Darby canine kidney cells, ZO-2 inhibits cell proliferation at G0/G1 and decreases CD1 protein level. These effects occur in response to a diminished CD1 translation and an augmented CD1 degradation at the proteosome triggered by ZO-2. ZO-2 overexpression decreases the amount of Glycogen synthase kinase-3beta phosphorylated at Ser9 and represses beta-catenin target gene expression. We have also explored the expression of ZO-2 through the cell cycle and demonstrate that ZO-2 enters the nucleus at the late G1 phase and leaves the nucleus when the cell is in mitosis. These results thus explain why in confluent quiescent epithelia ZO-2 is absent from the nucleus and localizes at the cellular borders, whereas in sparse proliferating cultures ZO-2 is conspicuously present at the nucleus.

  3. 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...... neoplasms arise frequently in undervirilized and dysgenetic gonads and the striking physiological difference between meiotic entry in ovaries (fetal life) versus testes (at puberty), this study aimed to investigate whether errors in regulation of meiosis may be implicated in the pathogenesis of CIS or its...... invasive progression to TGCT. The main focus was on a key sex differentiation and meiosis regulator, DMRT1, which has also been linked to TGCT risk in recent genetic association studies. Expression patterns of DMRT1 and other meiosis regulators (SCP3, DMC1, STRA8, CYP26B1, NANOS2, NANOS3) were investigated...

  4. Distribution pattern of histone H3 phosphorylation at serine 10 during mitosis and meiosis in Brachiaria species

    Indian Academy of Sciences (India)

    C. M. P. Paula; V. H. Techio; F. Souza Sobrinho; A. S. Freitas

    2013-08-01

    Histones are the major eukaryotic DNA-binding proteins. Posttranslational modifications on N-terminal tails of histones that form nucleosomes are often associated with distinct biological functions. Some theories suggest that one of these modifications, the phosphorylation of histone H3 at serine 10 (H3S10ph) plays a role in both chromosome condensation and sister chromatid cohesion. Although histones and some of their modifications are highly conserved, studies have shown that role and distribution of H3S10ph may differ between species. We evaluated the pattern of H3 phosphorylation using immunodetection during mitosis and meiosis in both diploid and tetraploid genotypes of Brachiaria species. Results revealed differences in chromosome distribution of H3S10ph when mitosis and meiosis were compared. Whole chromosomes were phosphorylated during meiosis I, whereas phosphorylation was restricted to the pericentromeric region in both meiosis II and mitosis. There was no variation in phosphorylation patterns between Brachiaria species and diploid and tetraploid genotypes. Regarding spatiotemporal coordination in the Brachiaria species evaluated, H3S10ph is related to maintenance of sister chromatid cohesion during cell divisions.

  5. Comparative analysis and molecular characterization of a gene BANF1 encoded a DNA-binding protein during mitosis from the Giant Panda and Black Bear.

    Science.gov (United States)

    Zeng, Yichun; Hou, Yi-Ling; Ding, Xiang; Hou, Wan-Ru; Li, Jian

    2014-01-01

    Barrier to autointegration factor 1 (BANF1) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mitosis. The cDNA and the genomic sequence of BANF1 were cloned from the Giant Panda (Ailuropoda melanoleuca) and Black Bear (Ursus thibetanus mupinensis) using RT-PCR technology and Touchdown-PCR, respectively. The cDNA of the BANF1 cloned from Giant Panda and Black Bear is 297 bp in size, containing an open reading frame of 270 bp encoding 89 amino acids. The length of the genomic sequence from Giant Panda is 521 bp, from Black Bear is 536 bp, which were found both to possess 2 exons. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to some mammalian species studied. Topology prediction showed there is one Protein kinase C phosphorylation site, one Casein kinase II phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Giant Panda, and there is one Protein kinase C phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Black Bear. The BANF1 gene can be readily expressed in E. coli. Results showed that the protein BANF1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 14 kD polypeptide that formed inclusion bodies. The expression products obtained could be used to purify the proteins and study their function further. PMID:25009988

  6. Temporomandibular joint inflammation activates glial and immune cells in both the trigeminal ganglia and in the spinal trigeminal nucleus

    Directory of Open Access Journals (Sweden)

    Jasmin Luc

    2010-12-01

    Full Text Available Abstract Background Glial cells have been shown to directly participate to the genesis and maintenance of chronic pain in both the sensory ganglia and the central nervous system (CNS. Indeed, glial cell activation has been reported in both the dorsal root ganglia and the spinal cord following injury or inflammation of the sciatic nerve, but no data are currently available in animal models of trigeminal sensitization. Therefore, in the present study, we evaluated glial cell activation in the trigeminal-spinal system following injection of the Complete Freund's Adjuvant (CFA into the temporomandibular joint, which generates inflammatory pain and trigeminal hypersensitivity. Results CFA-injected animals showed ipsilateral mechanical allodynia and temporomandibular joint edema, accompanied in the trigeminal ganglion by a strong increase in the number of GFAP-positive satellite glial cells encircling neurons and by the activation of resident macrophages. Seventy-two hours after CFA injection, activated microglial cells were observed in the ipsilateral trigeminal subnucleus caudalis and in the cervical dorsal horn, with a significant up-regulation of Iba1 immunoreactivity, but no signs of reactive astrogliosis were detected in the same areas. Since the purinergic system has been implicated in the activation of microglial cells during neuropathic pain, we have also evaluated the expression of the microglial-specific P2Y12 receptor subtype. No upregulation of this receptor was detected following induction of TMJ inflammation, suggesting that any possible role of P2Y12 in this paradigm of inflammatory pain does not involve changes in receptor expression. Conclusions Our data indicate that specific glial cell populations become activated in both the trigeminal ganglia and the CNS following induction of temporomandibular joint inflammation, and suggest that they might represent innovative targets for controlling pain during trigeminal nerve sensitization.

  7. Automated mitosis detection using texture, SIFT features and HMAX biologically inspired approach

    OpenAIRE

    Humayun Irshad; Sepehr Jalali; Ludovic Roux; Daniel Racoceanu; Lim Joo Hwee; Gilles Le Naour; Frédérique Capron

    2013-01-01

    Context: According to Nottingham grading system, mitosis count in breast cancer histopathology is one of three components required for cancer grading and prognosis. Manual counting of mitosis is tedious and subject to considerable inter- and intra-reader variations. Aims: The aim is to investigate the various texture features and Hierarchical Model and X (HMAX) biologically inspired approach for mitosis detection using machine-learning techniques. Materials and Methods: We propose an approach...

  8. Checkpoint Defects Leading to Premature Mitosis Also Cause Endoreplication of DNA in Aspergillus nidulans

    OpenAIRE

    De Souza, Colin P. C.; Ye, Xiang S.; Osmani, Stephen A.

    1999-01-01

    The G2 DNA damage and slowing of S-phase checkpoints over mitosis function through tyrosine phosphorylation of NIMXcdc2 in Aspergillus nidulans. We demonstrate that breaking these checkpoints leads to a defective premature mitosis followed by dramatic rereplication of genomic DNA. Two additional checkpoint functions, uvsB and uvsD, also cause the rereplication phenotype after their mutation allows premature mitosis in the presence of low concentrations of hydroxyurea. uvsB is shown to encode ...

  9. In vitro culture of mouse nucleus pulposus cells of intervertebral disc by multiple enzymatic digestions%多次胶原酶消化法培养小鼠椎间盘髓核细胞

    Institute of Scientific and Technical Information of China (English)

    张兴凯; 曹鹏; 王君; 梁裕; 吴文坚

    2011-01-01

    BACKGROUND: In vitro culture of nucleus pulposus cells of intervertebral disc is an important method to study degeneration of intervertebral disc. However, it is difficult to culture nucleus pulposus cells in vitro because the intervertebral disc is an avascular organ and nucleus pulposus cells poorly differentiate and proliferate.OBJECTIVE: To establish the method for in vitro culture of nucleus pulposus cells of intervertebral disc, and provide a reliable tool for research of phenotype changes of nucleus pulposus cells in degenerative disc and disc cell transplantation for treatment of degenerative disc diseases.METHODS: Mouse nucleus pulposus tissue was collected and repeatedly digested using collagenase. Cells were cultured and subcultured. The secretion of collagen Ⅱ and aggrecan of passage 2 cells were detected by immunohistochemistry and RT -PCR,The results were compared with other types of cells.RESULTS AND CONCLUSION: Nucleus pulposus cells of intervertebral disc exhibit a chondrocyte-like shape after adhesion.Immunohistochemistry study showed that the cells were positive for collagen Ⅱ and aggrecan staining. RT -PCR study showed that secretions of collagen Ⅱ and aggrecan in cultured cells were equal to those of chondrocyes and had significant difference from those of osteoblasts and fibroblasts. Multiple enzymatic digestions of nucleus pulposus can release a large amount of pure nucleus pulposus cells which had stable phenotype and can settle basis for research and treatment of intervertebal disc diseases.%背景:椎间盘为无血运组织,椎间盘髓核细胞为分化终末细胞,细胞增殖能力较差,体外培养难度较大.目的:探索小鼠椎间盘髓核细胞体外分离培养的方法.方法:取小鼠椎间盘髓核组织,使用多次胶原酶消化的方法,分离培养髓核组织细胞,接种,传代,取第2代细胞,分别采用免疫细胞化学和RT-PCR方法检测椎间盘髓核细胞特征性分泌物Ⅱ型胶原和聚合蛋白

  10. A novel big protein TPRBK possessing 25 units of TPR motif is essential for the progress of mitosis and cytokinesis.

    Science.gov (United States)

    Izumiyama, Tomohiro; Minoshima, Shinsei; Yoshida, Tetsuhiko; Shimizu, Nobuyoshi

    2012-12-15

    Through the comprehensive analysis of the genomic DNA sequence of human chromosome 22, we identified a novel gene of 702 kb encoding a big protein of 2481 amino acid residues, and named it as TPRBK (TPR containing big gene cloned at Keio). A novel protein TPRBK possesses 25 units of the TPR motif, which has been known to associate with a diverse range of biological functions. Orthologous genes of human TPRBK were found widely in animal species, from insecta to mammal, but not found in plants, fungi and nematoda. Northern blotting and RT-PCR analyses revealed that TPRBK gene is expressed ubiquitously in the human and mouse fetal tissues and various cell lines of human, monkey and mouse. Immunofluorescent staining of the synchronized monkey COS-7 cells with several relevant antibodies indicated that TPRBK changes its subcellular localization during the cell cycle: at interphase TPRBK locates on the centrosomes, during mitosis it translocates from spindle poles to mitotic spindles then to spindle midzone, and through a period of cytokinesis it stays on the midbody. Co-immunoprecipitation assay and immunofluorescent staining with adequate antibodies revealed that TPRBK binds to Aurora B, and those proteins together translocate throughout mitosis and cytokinesis. Treatments of cells with two drugs (Blebbistatin and Y-27632), that are known to inhibit the contractility of actin-myosin, disturbed the proper intracellular localization of TPRBK. Moreover, the knockdown of TPRBK expression by small interfering RNA (siRNA) suppressed the bundling of spindle midzone microtubules and disrupted the midbody formation, arresting the cells at G(2)+M phase. These observations indicated that a novel big protein TPRBK is essential for the formation and integrity of the midbody, hence we postulated that TPRBK plays a critical role in the progress of mitosis and cytokinesis during mammalian cell cycle.

  11. A pericentrin-related protein homolog in Aspergillus nidulans plays important roles in nucleus positioning and cell polarity by affecting microtubule organization.

    Science.gov (United States)

    Chen, Peiying; Gao, Rongsui; Chen, Shaochun; Pu, Li; Li, Pin; Huang, Ying; Lu, Ling

    2012-12-01

    Pericentrin is a large coiled-coil protein in mammalian centrosomes that serves as a multifunctional scaffold for anchoring numerous proteins. Recent studies have linked numerous human disorders with mutated or elevated levels of pericentrin, suggesting unrecognized contributions of pericentrin-related proteins to the development of these disorders. In this study, we characterized AnPcpA, a putative homolog of pericentrin-related protein in the model filamentous fungus Aspergillus nidulans, and found that it is essential for conidial germination and hyphal development. Compared to the hyphal apex localization pattern of calmodulin (CaM), which has been identified as an interactive partner of the pericentrin homolog, GFP-AnPcpA fluorescence dots are associated mainly with nuclei, while the accumulation of CaM at the hyphal apex depends on the function of AnPcpA. In addition, the depletion of AnPcpA by an inducible alcA promoter repression results in severe growth defects and abnormal nuclear segregation. Most interestingly, in mature hyphal cells, knockdown of pericentrin was able to significantly induce changes in cell shape and cytoskeletal remodeling; it resulted in some enlarged compartments with condensed nuclei and anucleate small compartments as well. Moreover, defects in AnPcpA significantly disrupted the microtubule organization and nucleation, suggesting that AnPcpA may affect nucleus positioning by influencing microtubule organization.

  12. Modulation of synaptic potentials and cell excitability by dendritic KIR and KAS channels in nucleus accumbens medium spiny neurons: A computational study

    Indian Academy of Sciences (India)

    Jessy John; Rohit Manchanda

    2011-06-01

    The nucleus accumbens (NAc), a critical structure of the brain reward circuit, is implicated in normal goal-directed behaviour and learning as well as pathological conditions like schizophrenia and addiction. Its major cellular substrates, the medium spiny (MS) neurons, possess a wide variety of dendritic active conductances that may modulate the excitatory post synaptic potentials (EPSPs) and cell excitability. We examine this issue using a biophysically detailed 189-compartment stylized model of the NAc MS neuron, incorporating all the known active conductances. We find that, of all the active channels, inward rectifying K+ (KIR) channels play the primary role in modulating the resting membrane potential (RMP) and EPSPs in the down-state of the neuron. Reduction in the conductance of KIR channels evokes facilitatory effects on EPSPs accompanied by rises in local input resistance and membrane time constant. At depolarized membrane potentials closer to up-state levels, the slowly inactivating A-type potassium channel (KAs) conductance also plays a strong role in determining synaptic potential parameters and cell excitability. We discuss the implications of our results for the regulation of accumbal MS neuron biophysics and synaptic integration by intrinsic factors and extrinsic agents such as dopamine.

  13. Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

    OpenAIRE

    Yujiro Hayashi; Asuzu, David T.; Gibbons, Simon J.; Aarsvold, Kirsten H.; Bardsley, Michael R.; Lomberk, Gwen A.; Angela J Mathison; Michael L Kendrick; K Robert Shen; Takahiro Taguchi; Anu Gupta; Rubin, Brian P.; Fletcher, Jonathan A.; Gianrico Farrugia; Urrutia, Raul A.

    2013-01-01

    Stem cell factor (mouse: Kitl, human: KITLG) and insulin-like growth factor-1 (IGF1), acting via KIT and IGF1 receptor (IGF1R), respectively, are critical for the development and integrity of several tissues. Autocrine/paracrine KITLG-KIT and IGF1-IGF1R signaling are also activated in several cancers including gastrointestinal stromal tumors (GIST), the most common sarcoma. In murine gastric muscles, IGF1 promotes Kitl-dependent development of interstitial cells of Cajal (ICC), the non-neopla...

  14. Summary: The nucleus--a close-knit community of dynamic structures.

    Science.gov (United States)

    Henikoff, S

    2010-01-01

    The 75th Cold Spring Harbor Symposium on Nuclear Organization and Function explored topics ranging from nucleosomes to nuclear pores. Exciting new genomic and imaging technologies have been used to characterize the nuclear interior, which consists of stable chromatin territories, dynamic domains, and self-organizing nuclear bodies. Histone variants and chaperones, posttranslational modifications, and ATP-dependent remodelers mediate nucleosome dynamics, regulated by Polycomb and other chromatin-associated proteins. Epigenetic memory is an emergent property of chromatin dynamics that is key to understanding how differentiated cells can become reprogrammed. Nuclear body composition and structure are becoming increasingly well understood, although their functions, if any, remain speculative. The nuclear envelope is strengthened by a fibrous lamin network that anchors chromosomes and represses gene expression, and disruption can lead to disease. Nuclear pores regulate the flow of substrates and products, using unstructured polypeptides to filter small molecules and flexible walls that allow large macromolecular assemblages to pass through. At mitosis, nucleosomes collapse into tightly packed nonfibrous cylinders that are then pulled to opposite poles at their kinetochores, where novel centromeric nucleosomes, mitotic motors, and spindle microtubules come together. By considering these complex processes in the context of the nucleus, the Symposium provided a coherent view of the genome in its native habitat.

  15. Poly(ADP-ribosyl)ation is recognized by ECT2 during mitosis

    OpenAIRE

    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 recogni...

  16. Focal adhesions control cleavage furrow shape and spindle tilt during mitosis

    Science.gov (United States)

    Taneja, Nilay; Fenix, Aidan M.; Rathbun, Lindsay; Millis, Bryan A.; Tyska, Matthew J.; Hehnly, Heidi; Burnette, Dylan T.

    2016-01-01

    The geometry of the cleavage furrow during mitosis is often asymmetric in vivo and plays a critical role in stem cell differentiation and the relative positioning of daughter cells during development. Early observations of adhesive cell lines revealed asymmetry in the shape of the cleavage furrow, where the bottom (i.e., substrate attached side) of the cleavage furrow ingressed less than the top (i.e., unattached side). This data suggested substrate attachment could be regulating furrow ingression. Here we report a population of mitotic focal adhesions (FAs) controls the symmetry of the cleavage furrow. In single HeLa cells, stronger adhesion to the substrate directed less ingression from the bottom of the cell through a pathway including paxillin, focal adhesion kinase (FAK) and vinculin. Cell-cell contacts also direct ingression of the cleavage furrow in coordination with FAs in epithelial cells—MDCK—within monolayers and polarized cysts. In addition, mitotic FAs established 3D orientation of the mitotic spindle and the relative positioning of mother and daughter centrosomes. Therefore, our data reveals mitotic FAs as a key link between mitotic cell shape and spindle orientation, and may have important implications in our understanding stem cell homeostasis and tumorigenesis. PMID:27432211

  17. The multiple roles of Bub1 in chromosome segregation during mitosis and meiosis

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Venkatachalam, Sundaresan

    2009-06-19

    Aneuploidy, any deviation from an exact multiple of the haploid number of chromosomes, is a common occurrence in cancer and represents the most frequent chromosomal disorder in newborns. Eukaryotes have evolved mechanisms to assure the fidelity of chromosome segregation during cell division that include a multiplicity of checks and controls. One of the main cell division control mechanisms is the spindle assembly checkpoint (SAC) that monitors the proper attachment of chromosomes to spindle fibers and prevents anaphase until all kinetochores are properly attached. The mammalian SAC is composed by at least 14 evolutionary-conserved proteins that work in a coordinated fashion to monitor the establishment of amphitelic attachment of all chromosomes before allowing cell division to occur. Among the SAC proteins, the budding uninhibited by benzimidazole protein 1 (Bub1), is a highly conserved protein of prominent importance for the proper functioning of the SAC. Studies have revealed many roles for Bub1 in both mitosis and meiosis, including the localization of other SAC proteins to the kinetochore, SAC signaling, metaphase congression and the protection of the sister chromatid cohesion. Recent data show striking sex specific differences in the response to alterations in Bub1 activity. Proper Bub1 functioning is particularly important during oogenesis in preventing the generation of aneuploid gametes that can have detrimental effects on the health status of the fetus and the newborn. These data suggest that Bub1 is a master regulator of SAC and chromosomal segregation in both mitosis and meiosis. Elucidating its many essential functions in regulating proper chromosome segregation can have important consequences for preventing tumorigenesis and developmental abnormalities.

  18. Photon Exchange in Nucleus-Nucleus Collisions

    OpenAIRE

    Bertulani, Carlos A.

    2002-01-01

    The strong electromagnetic fields in peripheral heavy ion collisions give rise to photon-photon and photon-nucleus interactions. I present a general survey of the photon-photon and photon-hadron physics accessible in these collisions. Among these processes I discuss the nuclear fragmentation through the excitation of giant resonances, the Coulomb dissociation method for application in nuclear astrophysics, and the production of particles.

  19. Periodic Solutions of a Model of Mitosis in Frog Eggs

    Institute of Scientific and Technical Information of China (English)

    Bei-ye Feng; Zuo-huan Zheng

    2002-01-01

    In this paper,we discuss a simplified model of mitosis in frog eggs proposed by M.T. Borisuk and J.J.Tyson in [1]. By using rigorous qualitative analysis, we prove the existence of the periodic solutions on a large scale and present the space region of the periodic solutions and the parameter region coresponding to the periodic solution. We also present the space region and the parameter region where there are no periodic solutions. The results are in accordance with the numerical results in [1] up to the qualitative property.

  20. PGC-1 family coactivators and cell fate: roles in cancer, neurodegeneration, cardiovascular disease and retrograde mitochondria-nucleus signalling.

    OpenAIRE

    Jones, A W; Z. Yao; Vicencio, J. M.; Karkucinska-Wieckowska, A.; Szabadkai, G.

    2012-01-01

    Over the past two decades, a complex nuclear transcriptional machinery controlling mitochondrial biogenesis and function has been described. Central to this network are the PGC-1 family coactivators, characterised as master regulators of mitochondrial biogenesis. Recent literature has identified a broader role for PGC-1 coactivators in both cell death and cellular adaptation under conditions of stress, here reviewed in the context of the pathology associated with cancer, neurodegeneration and...

  1. Different generations of rabbit nucleus pulposus cells:Morphological and biological properties%不同代次兔髓核细胞的形态学特征和生物学性状*★

    Institute of Scientific and Technical Information of China (English)

    武海军; 银和平; 李树文; 白明; 杜志才; 曹振华

    2013-01-01

    properties of nucleus pulposus cells can provide theoretical basis for studying the mechanism underlying intervertebral disc degeneration, construction of the intervertebral disc by tissue engineering, and gene therapy. OBJECTIVE: To investigate the characteristics of different generations of rabbit nucleus pulposus cells, searching for the best suitable seed cells to treat degenerative disc diseases. METHODS: Nucleus pulposus cells from New Zealand white rabbits were separated, cultured and then passaged. The morphological changes of primary, passages 3 and 4 nucleus pulposus cells were observed by hematoxylin-eosin staining under an inverted microscope. The biological properties of rabbit nucleus pulposus cells were observed. Aggrecan and type Ⅱ colagen expressions w ere detected by toluidine blue and immunocytochemistry staining, respectively. Type nucleus pulposus cells were detected by reverse transcription-PCR. RESULTS AND CONCLUSION: Rabbit nucleus pulposus cells were successful y cultured and passaged in vitro. Primary nucleus pulposus cells were round or polygonal, and the average adherence time was 7 days. The first and third generations of nucleus pulposus cells were round or polygonal, and have strong vitality. Hematoxylin-eosin staining showed that nuclei were in a uniform blue-black, and cytoplasm showed light pink. The cytoplasm of nucleus pulposus cells was sky blue stained for toluidine blue staining, and type II col agen immunohistochemical staining showed the cytoplasm of nucleus pulposus cells displayed yel owish-brown. Passage 4 nucleus pulposus cells appeared with degeneration, and type mRNA expression was significantly decreased compared with previous generations. The first three generations of nucleus pulposus cells were exuberant in metabolism and showed consistent phenotypes and normal expression of aggrecan and type Ⅱ col agen. Passage 4 nucleus pulposus cells began to age and degenerate.

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    Microglial population expansion occurs in response to neural damage via processes that involve mitosis and immigration of bone marrow-derived cells. However, little is known of the mechanisms that regulate clearance of reactive microglia, when microgliosis diminishes days to weeks later. We have...... microglia often occurred in clusters, some having recently incorporated bromodeoxyuridine, showing that proliferation had occurred. Annexin V labeling and staining for activated caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling showed that apoptotic mechanisms participate...... in dissolution of the microglial response. Using bone marrow chimeric mice, we found that the lesion-induced proliferative capacity of resident microglia superseded that of immigrant microglia, whereas lesion-induced kinetics of apoptosis were comparable. Microglial numbers and responses were severely reduced...

  3. Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development.

    Science.gov (United States)

    Loponte, Sara; Segré, Chiara V; Senese, Silvia; Miccolo, Claudia; Santaguida, Stefano; Deflorian, Gianluca; Citro, Simona; Mattoscio, Domenico; Pisati, Federica; Moser, Mirjam A; Visintin, Rosella; Seiser, Christian; Chiocca, Susanna

    2016-01-01

    Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology. PMID:27458029

  4. An integrated overview of spatiotemporal organization and regulation in mitosis in terms of the proteins in the functional supercomplexes

    Directory of Open Access Journals (Sweden)

    Yueyuan eZheng

    2014-10-01

    Full Text Available Eukaryotic cells may divide via the critical cellular process of cell division/mitosis, resulting in two daughter cells with the same genetic information. A large number of dedicated proteins are involved in this process and spatiotemporally assembled into three distinct super-complex structures/organelles, including the centrosome/spindle pole body, kinetochore/centromere and cleavage furrow/midbody/bud neck, so as to precisely modulate the cell division/mitosis events of chromosome alignment, chromosome segregation and cytokinesis in an orderly fashion. In recent years, many efforts have been made to identify the protein components and architecture of these subcellular organelles, aiming to uncover the organelle assembly pathways, determine the molecular mechanisms underlying the organelle functions, and thereby provide new therapeutic strategies for a variety of diseases. However, the organelles are highly dynamic structures, making it difficult to identify the entire components. Here, we review the current knowledge of the identified protein components governing the organization and functioning of organelles, especially in human and yeast cells, and discuss the multi-localized protein components mediating the communication between organelles during cell division.

  5. Thymosin Beta 4 May Translocate from the Cytoplasm in to the Nucleus in HepG2 Cells following Serum Starvation. An Ultrastructural Study

    Science.gov (United States)

    Piludu, Marco; Piras, Monica; Pichiri, Giuseppina; Coni, Pierpaolo; Orrù, Germano; Cabras, Tiziana; Messana, Irene; Faa, Gavino; Castagnola, Massimo

    2015-01-01

    Due to its actin-sequestering properties, thymosin beta-4 (Tβ4) is considered to play a significant role in the cellular metabolism. Several physiological properties of Tβ4 have been reported;, however, many questions concerning its cellular function remain to be ascertained. To better understand the role of this small peptide we have analyzed by means of transmission immunoelectron microscopy techniques the ultrastructural localization of Tβ4 in HepG2 cells. Samples of HepG2 cells were fixed in a mixture of 3% formaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer and processed for standard electron microscopic techniques. The samples were dehydrated in a cold graded methanol series and embedded in LR gold resin. Ultrathin sections were labeled with rabbit antibodies to Tβ4, followed by gold-labeled goat anti-rabbit, stained with uranyl acetate and bismuth subnitrate, observed and photographed in a JEOL 100S transmission electron microscope. High-resolution electron microscopy showed that Tβ4 was mainly restricted to the cytoplasm of HepG2 growing in complete medium. A strong Tβ4 reactivity was detected in the perinuclear region of the cytoplasmic compartment where gold particles appeared strictly associated to the nuclear membrane. In the nucleus specific Tβ4 labeling was observed in the nucleolus. The above electron microscopic results confirm and extend previous observations at light microscopic level, highlighting the subcellular distribution of Tβ4 in both cytoplasmic and nuclear compartments of HepG2 cells. The meaning of Tβ4 presence in the nucleolus is not on the best of our knowledge clarified yet. It could account for the interaction of Tβ4 with nucleolar actin and according with this hypothesis, Tβ4 could contribute together with the other nucleolar acting binding proteins to modulate the transcription activity of the RNA polymerases. PMID:25835495

  6. Mitosis Phase Enrichment with Identification of Mitotic Centromere-Associated Kinesin As a Therapeutic Target in Castration-Resistant Prostate Cancer

    Science.gov (United States)

    Sircar, Kanishka; Huang, Heng; Hu, Limei; Liu, Yuexin; Dhillon, Jasreman; Cogdell, David; Aprikian, Armen; Efstathiou, Eleni; Navone, Nora; Troncoso, Patricia; Zhang, Wei

    2012-01-01

    The recently described transcriptomic switch to a mitosis program in castration-resistant prostate cancer (CRPC) suggests that mitotic proteins may be rationally targeted at this lethal stage of the disease. In this study, we showed upregulation of the mitosis-phase at the protein level in our cohort of 51 clinical CRPC cases and found centrosomal aberrations to also occur preferentially in CRPC compared with untreated, high Gleason–grade hormone-sensitive prostate cancer (P<0.0001). Expression profiling of chemotherapy-resistant CRPC samples (n = 25) was performed, and the results were compared with data from primary chemotherapy-naïve CRPC (n = 10) and hormone-sensitive prostate cancer cases (n = 108). Our results showed enrichment of mitosis-phase genes and pathways, with progression to both castration-resistant and chemotherapy-resistant disease. The mitotic centromere-associated kinesin (MCAK) was identified as a novel mitosis-phase target in prostate cancer that was overexpressed in multiple CRPC gene-expression datasets. We found concordant gene expression of MCAK between our parent and murine CRPC xenograft pairs and increased MCAK protein expression with clinical progression of prostate cancer to a castration-resistant disease stage. Knockdown of MCAK arrested the growth of prostate cancer cells suggesting its utility as a potential therapeutic target. PMID:22363599

  7. Mitosis phase enrichment with identification of mitotic centromere-associated kinesin as a therapeutic target in castration-resistant prostate cancer.

    Directory of Open Access Journals (Sweden)

    Kanishka Sircar

    Full Text Available The recently described transcriptomic switch to a mitosis program in castration-resistant prostate cancer (CRPC suggests that mitotic proteins may be rationally targeted at this lethal stage of the disease. In this study, we showed upregulation of the mitosis-phase at the protein level in our cohort of 51 clinical CRPC cases and found centrosomal aberrations to also occur preferentially in CRPC compared with untreated, high Gleason-grade hormone-sensitive prostate cancer (P<0.0001. Expression profiling of chemotherapy-resistant CRPC samples (n = 25 was performed, and the results were compared with data from primary chemotherapy-naïve CRPC (n = 10 and hormone-sensitive prostate cancer cases (n = 108. Our results showed enrichment of mitosis-phase genes and pathways, with progression to both castration-resistant and chemotherapy-resistant disease. The mitotic centromere-associated kinesin (MCAK was identified as a novel mitosis-phase target in prostate cancer that was overexpressed in multiple CRPC gene-expression datasets. We found concordant gene expression of MCAK between our parent and murine CRPC xenograft pairs and increased MCAK protein expression with clinical progression of prostate cancer to a castration-resistant disease stage. Knockdown of MCAK arrested the growth of prostate cancer cells suggesting its utility as a potential therapeutic target.

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

    OpenAIRE

    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 e...

  9. Mitosis detection in breast cancer histology images with deep neural networks.

    Science.gov (United States)

    Cireşan, Dan C; Giusti, Alessandro; Gambardella, Luca M; Schmidhuber, Jürgen

    2013-01-01

    We use deep max-pooling convolutional neural networks to detect mitosis in breast histology images. The networks are trained to classify each pixel in the images, using as context a patch centered on the pixel. Simple postprocessing is then applied to the network output. Our approach won the ICPR 2012 mitosis detection competition, outperforming other contestants by a significant margin.

  10. Regulated pH-Responsive Polymeric Micelles for Doxorubicin Delivery to the Nucleus of Liver Cancer Cells.

    Science.gov (United States)

    Li, Hao; Li, Xian; Zhang, Chao; Sun, Qiquan; Yi, Wei; Wang, Xuan; Cheng, Du; Chen, Shupeng; Liang, Biling; Shuai, Xintao

    2016-06-01

    A diblock copolymer of poly(ethylene glycol) (PEG) and poly(γ-benzyl L-glutamate) (PBLG), PEG-PBLG, was synthesized via the ring-opening polymerization of γ-benzyl L-glutamate N-carboxyanhydride (BLG-NCA) using allyl-PEG-NH2 as a macroinitiator. After deprotection of the benzyl groups, N,N-diisopropyl ethylenediamine (DIP) was conjugated to poly(L-glutamic acid) (PGA) blocks as side groups. The pendant DIP groups on the PGA blocks greatly enhance the pH-sensitivity of poly(ethylene glycol)-block-poly[N-(N',N'-diisopropylaminoethyl) glutamide] [PEG-PGA(DIP)] micelles, and a higher grafting percentage of DIP favors a faster acid-response. In neutral aqueous solution, the PEG-PGA(DIP) can self-assemble into stable micelles featuring an acid-responsive PGA(DIP) core with the encapsulated anticancer drug doxorubicin (DOX). In an acidic environment, the hydrophobic-hydrophilic transition of the PGA block leads to the gradual expansion and disassembly of these micelles and, consequently, an accelerated release of DOX. Thus, DOX transported by PEG-PGA(DIP) micelles can be entrapped more efficiently into the nuclei of hepatoma Bel 7402 cells.

  11. Nucleus Accumbens-Associated Protein 1 Expression Has Potential as a Marker for Distinguishing Oral Epithelial Dysplasia and Squamous Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    Joji Sekine

    Full Text Available Oral epithelial dysplasia (OED and carcinoma in situ (CIS are defined by dysplastic cells in the epithelium. Over a third of oral squamous cell carcinoma (OSCC patients present with associated OED. However, accurate histopathological diagnosis of such lesions is difficult. Nucleus accumbens-associated protein 1 (NAC1 is a member of the Pox virus and Zinc finger/Bric-a-brac Tramtrack Broad complex family of proteins, and is overexpressed in OSCC. This study aimed to determine whether NAC1 has the potential to be used as a marker to distinguish OED and OSCC.The study included 114 patients (64 men, 50 women. There were 67, 10, and 37 patients with OED, CIS, and OSCC, respectively. NAC1 labeling indices (LIs and immunoreactivity intensities (IRI were evaluated. The patients' pathological classification was significantly associated with age, sex, NAC1 LIs, and NAC1 IRI (p = 0.025, p = 0.022, p 50% positivity the sensitivity, specificity, positive predictive value (PPV, and negative predictive value (NPV were 0.766, 0.910, 0.857, and 0.847, respectively. For NAC1 IRI with ≤ 124 positive pixels, the sensitivity, specificity, PPV, and NPV were 0.787, 0.866, 0.804, and 0.853, respectively. Though there are several potential limitations to this study and the results were obtained from a retrospective analysis of a single site cohort, the data suggest that the NAC1 LIs/IRI is a strong predictor of CIS/OSCC.NAC1 has potential as a marker for distinguishing OED from CIS/OSCC.

  12. Viscoelastic properties of human normal nucleus pulposus cells%正常髓核细胞的黏弹性研究

    Institute of Scientific and Technical Information of China (English)

    任龙韬; 卫陈刚; 牛建鹏; 郭志坚; 郝海虎

    2011-01-01

    Objective To study the viscoelastic properties of nucleus pulposus (NP) cells from human in vitro. Methods NP was obtained from discarded NP tissue of 3 scoliosis patients aged from 13 to 16 years. Pancreatin and collagenase type Ⅱ were used to digest NP and cells were isolated from NP. Type Ⅱ collagen immunofluorescence and Fan seaing were used to identify NP cells. The micropipette aspiration test was used in combination with a three-parameter viscoelastic solid model to measure the mechanical properties of NP cells. Results The mean diameter of the digested NP cells was ( 15.40 ± 1.83) μm. In response to a prescribed pressure, the NP cells exhibited viscolastic solid creep behavior, which was characterized initially by a jump in displacement followed by a monotony decreasing rate of deformation that generally reached an equilibrium. NP cells were deformed to a length as much as 2 times the radius of the micropipette without completely entering the micropipette. The viscolastic parameters were k1 (0. 101 ±0. 052) kPa, k2 (0. 353 ± 0. 199) kPa, and μ ( 3. 034 ± 1. 843 ) kPa· s, respectively. Only the k1 was positively correlated to the cell diameter (r =-0. 389, P < 0. 05 ). Conclusion Human normal NP cells behave as a typical viscolastic solid creep. Micropipette aspiration technique is a valid method for the study on biomechanics of NP cells.%目的 观察正常髓核细胞的黏弹性。方法 髓核组织取材于3例脊柱侧凸矫形手术者术中取出废弃的髓核组织,用胰蛋白酶和Ⅱ型胶原酶消化分离细胞,Ⅱ型胶原免疫荧光组化和蕃红染色进行细胞鉴定,测量细胞直径,采用微管吸吮技术分析髓核细胞的黏弹性特性。结果 髓核细胞直径为(15.40±1.83)μm,正常髓核细胞的黏弹性参数k1(0.101 ±0.052) kPn、k2(0.353±0.199) kPa和μ(3.034±1.843) kPa·s。直线相关性分析表明,仅k1与髓核细胞直径明显相关(r=-0.389,P<0.05)。结论 正常髓核细胞表现为典

  13. 外侧膝状体细胞对边缘的响应模型%Response of Lateral Geniculate Nucleus Cells to Edges

    Institute of Scientific and Technical Information of China (English)

    任远

    2014-01-01

    Edges composing of stimuli of different intensities are common in both natural scenes and digital images.Edge detection is a basic step for machine visual systems,particularly machine biological visual systems.This paper establishes a model of lateral geniculate nucleus (LGN)cells in a primary visual pathway,describes the LGN cell response to edges,and provides feature representations for designing image processing approaches based on neural mechanism. According to physiological characteristics of retinal ganglion cells,the paper uses the classical model of difference of Gaussians to describe the LGN cell response to stimuli,and obtains a re-sponse function via reasonable simplifications.Through simple analyses,several mathematical properties of the response function are obtained,which agree with the physiological characteristics of neurons.By further simplifying the contrast of a stimulus,a normalized response function is obtained.Numeric experiments show that similarities exist between the function’s response curve and the physiological curve discovered in a previous neural science research,showing validity of the described model.%边缘检测是机器视觉系统与生物视觉系统处理视觉信息的基础阶段。为初级视觉通路中的外侧膝状体(LGN)细胞建立一个模型,描述其对边缘的响应,为构建基于神经机制的图像处理方法提供特征表征。根据神经节细胞感受野的生理特性,用经典的高斯差模型描述 LGN 细胞对刺激的响应,通过合理地简化得到相对简单的响应函数。通过简单数学分析,能够得到函数的几点数学性质,且这些性质都与神经元的生理特性相符。进一步简化刺激的对比度,得到归一化的响应函数。数值实验发现,函数的响应曲线和神经科学研究得到的生理曲线具有相似性,说明该数学模型的合理性。

  14. Oncogenic KRAS triggers MAPK-dependent errors in mitosis and MYC-dependent sensitivity to anti-mitotic agents.

    Science.gov (United States)

    Perera, David; Venkitaraman, Ashok R

    2016-07-14

    Oncogenic KRAS induces cell proliferation and transformation, but little is known about its effects on cell division. Functional genetic screens have recently revealed that cancer cell lines expressing oncogenic KRAS are sensitive to interference with mitosis, but neither the mechanism nor the uniformity of anti-mitotic drug sensitivity connected with mutant KRAS expression are yet clear. Here, we report that acute expression of oncogenic KRAS in HeLa cells induces mitotic delay and defects in chromosome segregation through mitogen-activated protein kinase (MAPK) pathway activation and de-regulated expression of several mitosis-related genes. These anomalies are accompanied by increased sensitivity to anti-mitotic agents, a phenotype dependent on the transcription factor MYC and its downstream target anti-apoptotic protein BCL-XL. Unexpectedly, we find no correlation between KRAS mutational status or MYC expression levels and anti-mitotic drug sensitivity when surveying a large database of anti-cancer drug responses. However, we report that the co-existence of KRAS mutations and high MYC expression predicts anti-mitotic drug sensitivity. Our findings reveal a novel function of oncogenic KRAS in regulating accurate mitotic progression and suggest new avenues to therapeutically target KRAS-mutant tumours and stratify patients in ongoing clinical trials of anti-mitotic drugs.

  15. 兔髓核与纤维环细胞生物学特性差异的研究%Different biological characteristics between nucleus pulposus and annulus fibrosus cells in rabbits

    Institute of Scientific and Technical Information of China (English)

    谢健; 童培建; 肖鲁伟; 金红婷; 吴承亮; 单乐天; 毛强; 潘佳菲

    2013-01-01

    Objective:To compare biological characteristics between nucleus pulposus and annulus fibrosus cells in vitro model.Methods:Five New Zealand white rabbits (2 to 3 kg,either gender) were isolated nucleus pulposus and annulus fibrosus under sterilized condition,then cultured in nutrient solution with 15% FBS and DMEM/F12 (1∶1) by enzyme digestion combined with tissue block method.When 90% cells fused,subcultring were performed.Cell morphology were observed by inverted phase contrast microscope,cell viability were detected by trypan blue staining,histological were observed by a toluidine blue and HE staining,cell proliferation were tested by MTT method,then the cell morphology,viability,proliferation between nucleus pulposus and annulus fibrosus were compared.Results:There were no obvioualy differences between nucleus pulposus and annulus fibrosus in original and the first strain.Physalides were appeared in annulus fibrosus on the second generation.The strapping time was later,and activity was lower in nucleus pulposus than annulus fibrosus.The growth of cell proliferation in nucleus pulposus was lower than annulus fibrosus from the ninth day.Conclusion:The cell activity in annulus fibrosus is higher than nucleus pulposus.Digenerative disc disease may caused by recession of nucleus pulposus,local biomechnical changes,furether caused structure change and function loss of annulus fibrosus.%目的:同时建立兔髓核细胞与纤维环细胞体外培养模型,比较两者生物学特性差异.方法:新西兰大白兔5只(2~3 kg,雌雄不限),无菌条件下分离髓核及纤维环,酶消化法联合组织块法含15%FBS的DMEM/F12(1∶1)培养液培养,当细胞90%融合后进行传代培养.通过倒置相差显微镜观测细胞形态,台盼蓝染色测定细胞活力,甲苯胺蓝和HE染色进行组织学观察,MTT法测定细胞增殖,分析比较髓核细胞与纤维环细胞形态、活力、增殖的差异.结果:原代及第1代髓核细胞和纤维

  16. Rho proteins − the key regulators of cytoskeleton in the progression of mitosis and cytokinesis

    Directory of Open Access Journals (Sweden)

    Anna Klimaszewska

    2011-11-01

    Full Text Available The Rho proteins are members of the Ras superfamily of small GTPases. They are thought to be crucial regulators of multiple signal transduction pathways that influence a wide range of cellular functions, including migration, membrane trafficking, adhesion, polarity and cell shape changes. Thanks to their ability to control the assembly and organization of the actin and microtubule cytoskeletons, Rho GTPases are known to regulate mitosis and cytokinesis progression. These proteins are required for formation and rigidity of the cortex during mitotic cell rounding, mitotic spindle formation and attachment of the spindle microtubules to the kinetochore. In addition, during cytokinesis, they are involved in promoting division plane determination, contractile ring and cleavage furrow formation and abscission. They are also known as regulators of cell cycle progression at the G1/S and G2/M transition. Thus, the signal transduction pathways in which Rho proteins participate, appear to connect dynamics of actin and microtubule cytoskeletons to cell cycle progression. We review the current state of knowledge concerning the molecular mechanisms by which Rho GTPase signaling regulates remodeling of actin and microtubule cytoskeletons in order to control cell division progression.

  17. An organism arises from every nucleus.

    Directory of Open Access Journals (Sweden)

    Nurullah Keklikoglu

    2009-12-01

    Full Text Available The fact that, cloning using somatic cell nuclear transfer (SCNT method has been performed, opened new horizons for cloning, and changed the way of our understanding and approach to cell and nucleus. The progress in cloning technology, brought the anticipation of the ability to clone an organism from each somatic cell nucleus. Therefore, the 'Cell Theory' is about to take the additional statement as "An organism arises from every nucleus". The development of gene targeting procedures which can be applied with SCNT, showed us that it may be possible to obtain different versions of the original genetic constitution of a cell. Because of this opportunity which is provided by SCNT, in reproductive cloning, it would be possible to clone enhanced organisms which can adapt to different environmental conditions and survive. Furthermore, regaining the genetic characteristics of ancestors or reverse herediter variations would be possible. On the other hand, in therapeutic cloning, more precise and easily obtainable alternatives for cell replacement therapy could be presented. However, while producing healthier or different organisms from a nucleus, it is hard to foresee the side effects influencing natural processes in long term is rather difficult.

  18. Human umbilical cord mesenchymal stromal cells exhibit immature nucleus pulposus cell phenotype in a laminin-rich pseudo-three-dimensional culture system

    OpenAIRE

    Chon, Brian H; Lee, Esther J.; Jing, Liufang; Lori A Setton; Chen, Jun

    2013-01-01

    Introduction 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. Howev...

  19. Fibrillarin redistributes to the spindle poles and partially colocalizes with NuMA during mitosis

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Fibrillarin, a major protein in the nucleolus, is known to redistribute during mitosis from the nucleolus to the cytosol, and is related to the dynamics of post-mitotic reassembly of the nucleolus. To better understand the dynamic behavior and the relationship with other cytoplasmic structures, we have now expressed fibrillarin-pDsRed1 fusion protein in HeLa cells. The results showed that a part of fibrillarin was associated with mitotic spindle poles in the mitotic cells. Nocodazole-induced microtubule depolymerization resulted in fibrillarin redistribution throughout the cytoplasm, and removal of nocodazole resulted in relocalization of fibrillarin at the polar region during the mitotic spindles reassembly. In a mitotic cell free system, fibrillarin was found in the center of taxol-induced microtubule asters. Moreover, fibrillarin was found to colocalize with the nuclear mitotic apparatus protein (NuMA) at the poles of mitotic cells. Therefore, it is postulated that the polar redistribution of fibrillarin is mediated by microtubules.

  20. An Arabidopsis tissue-specific RNAi method for studying genes essential to mitosis.

    Directory of Open Access Journals (Sweden)

    Brunilís Burgos-Rivera

    Full Text Available A large fraction of the genes in plants can be considered essential in the sense that when absent the plant fails to develop past the first few cell divisions. The fact that angiosperms pass through a haploid gametophyte stage can make it challenging to propagate such mutants even in the heterozygous condition. Here we describe a tissue-specific RNAi method that allows us to visualize cell division phenotypes in petals, which are large dispensable organs. Portions of the APETALA (AP3 and PISTILLATA (PI promoters confer early petal-specific expression. We show that when either promoter is used to drive the expression of a beta-glucuronidase (GUS RNAi transgene in plants uniformly expressing GUS, GUS expression is knocked down specifically in petals. We further tested the system by targeting the essential kinetochore protein CENPC and two different components of the Spindle Assembly Checkpoint (MAD2 and BUBR1. Plant lines expressing petal-specific RNAi hairpins targeting these genes exhibited an array of petal phenotypes. Cytological analyses of the affected flower buds confirmed that CENPC knockdown causes cell cycle arrest but provided no evidence that either MAD2 or BUBR1 are required for mitosis (although both genes are required for petal growth by this assay. A key benefit of the petal-specific RNAi method is that the phenotypes are not expressed in the lineages leading to germ cells, and the phenotypes are faithfully transmitted for at least four generations despite their pronounced effects on growth.

  1. Enhancement of φ Mesons in Relativistic Nucleus-Nucleus Collisions

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The фmeson production in relativistic nucleus-nucleus collisions is investigated systematically usinga hadron-string cascade model LUCIAE. Within the framework of the model and relying on the collective

  2. Trichostatin A and 5-azacytidine both cause an increase in global histone H4 acetylation and a decrease in global DNA and H3K9 methylation during mitosis in maize

    Directory of Open Access Journals (Sweden)

    Yang Fei

    2010-08-01

    Full Text Available Abstract Background Modifications of DNA and histones in various combinations are correlated with many cellular processes. In this study, we investigated the possible relationship between histone H4 tetraacetylation, DNA methylation and histone H3 dimethylation at lysine 9 during mitosis in maize root meristems. Results Treatment with trichostatin A, which inhibits histone deacetylases, resulted in increased histone H4 acetylation accompanied by the decondensation of interphase chromatin and a decrease in both global H3K9 dimethylation and DNA methylation during mitosis in maize root tip cells. These observations suggest that histone acetylation may affect DNA and histone methylation during mitosis. Treatment with 5-azacytidine, a cytosine analog that reduces DNA methylation, caused chromatin decondensation and mediated an increase in H4 acetylation, in addition to reduced DNA methylation and H3K9 dimethylation during interphase and mitosis. These results suggest that decreased DNA methylation causes a reduction in H3K9 dimethylation and an increase in H4 acetylation. Conclusions The interchangeable effects of 5-azacytidine and trichostatin A on H4 acetylation, DNA methylation and H3K9 dimethylation indicate a mutually reinforcing action between histone acetylation, DNA methylation and histone methylation with respect to chromatin modification. Treatment with trichostatin A and 5-azacytidine treatment caused a decrease in the mitotic index, suggesting that H4 deacetylation and DNA and H3K9 methylation may contain the necessary information for triggering mitosis in maize root tips.

  3. Trichostatin A and 5-azacytidine both cause an increase in global histone H4 acetylation and a decrease in global DNA and H3K9 methylation during mitosis in maize

    Science.gov (United States)

    2010-01-01

    Background Modifications of DNA and histones in various combinations are correlated with many cellular processes. In this study, we investigated the possible relationship between histone H4 tetraacetylation, DNA methylation and histone H3 dimethylation at lysine 9 during mitosis in maize root meristems. Results Treatment with trichostatin A, which inhibits histone deacetylases, resulted in increased histone H4 acetylation accompanied by the decondensation of interphase chromatin and a decrease in both global H3K9 dimethylation and DNA methylation during mitosis in maize root tip cells. These observations suggest that histone acetylation may affect DNA and histone methylation during mitosis. Treatment with 5-azacytidine, a cytosine analog that reduces DNA methylation, caused chromatin decondensation and mediated an increase in H4 acetylation, in addition to reduced DNA methylation and H3K9 dimethylation during interphase and mitosis. These results suggest that decreased DNA methylation causes a reduction in H3K9 dimethylation and an increase in H4 acetylation. Conclusions The interchangeable effects of 5-azacytidine and trichostatin A on H4 acetylation, DNA methylation and H3K9 dimethylation indicate a mutually reinforcing action between histone acetylation, DNA methylation and histone methylation with respect to chromatin modification. Treatment with trichostatin A and 5-azacytidine treatment caused a decrease in the mitotic index, suggesting that H4 deacetylation and DNA and H3K9 methylation may contain the necessary information for triggering mitosis in maize root tips. PMID:20718950

  4. A thalamic input to the nucleus accumbens mediates opiate dependence.

    Science.gov (United States)

    Zhu, Yingjie; Wienecke, Carl F R; Nachtrab, Gregory; Chen, Xiaoke

    2016-02-11

    Chronic opiate use induces opiate dependence, which is characterized by extremely unpleasant physical and emotional feelings after drug use is terminated. Both the rewarding effects of a drug and the desire to avoid withdrawal symptoms motivate continued drug use, and the nucleus accumbens is important for orchestrating both processes. While multiple inputs to the nucleus accumbens regulate reward, little is known about the nucleus accumbens circuitry underlying withdrawal. Here we identify the paraventricular nucleus of the thalamus as a prominent input to the nucleus accumbens mediating the expression of opiate-withdrawal-induced physical signs and aversive memory. Activity in the paraventricular nucleus of the thalamus to nucleus accumbens pathway is necessary and sufficient to mediate behavioural aversion. Selectively silencing this pathway abolishes aversive symptoms in two different mouse models of opiate withdrawal. Chronic morphine exposure selectively potentiates excitatory transmission between the paraventricular nucleus of the thalamus and D2-receptor-expressing medium spiny neurons via synaptic insertion of GluA2-lacking AMPA receptors. Notably, in vivo optogenetic depotentiation restores normal transmission at these synapses and robustly suppresses morphine withdrawal symptoms. This links morphine-evoked pathway- and cell-type-specific plasticity in the paraventricular nucleus of the thalamus to nucleus accumbens circuit to opiate dependence, and suggests that reprogramming this circuit holds promise for treating opiate addiction.

  5. The Slx4-Dpb11 scaffold complex: coordinating the response to replication fork stalling in S-phase and the subsequent mitosis.

    Science.gov (United States)

    Princz, Lissa N; Gritenaite, Dalia; Pfander, Boris

    2015-01-01

    Replication fork stalling at DNA lesions is a common problem during the process of DNA replication. One way to allow the bypass of these lesions is via specific recombination-based mechanisms that involve switching of the replication template to the sister chromatid. Inherent to these mechanisms is the formation of DNA joint molecules (JMs) between sister chromatids. Such JMs need to be disentangled before chromatid separation in mitosis and the activity of JM resolution enzymes, which is under stringent cell cycle control, is therefore up-regulated in mitosis. An additional layer of control is facilitated by scaffold proteins. In budding yeast, specifically during mitosis, Slx4 and Dpb11 form a cell cycle kinase-dependent complex with the Mus81-Mms4 structure-selective endonuclease, which allows efficient JM resolution by Mus81. Furthermore, Slx4 and Dpb11 interact even prior to joining Mus81 and respond to replication fork stalling in S-phase. This S-phase complex is involved in the regulation of the DNA damage checkpoint as well as in early steps of template switch recombination. Similar interactions and regulatory principles are found in human cells suggesting that Slx4 and Dpb11 may have an evolutionary conserved role organizing the cellular response to replication fork stalling.

  6. Human chromokinesins promote chromosome congression and spindle microtubule dynamics during mitosis.

    Science.gov (United States)

    Wandke, Cornelia; Barisic, Marin; Sigl, Reinhard; Rauch, Veronika; Wolf, Frank; Amaro, Ana C; Tan, Chia H; Pereira, Antonio J; Kutay, Ulrike; Maiato, Helder; Meraldi, Patrick; Geley, Stephan

    2012-09-01

    Chromokinesins are microtubule plus end-directed motor proteins that bind to chromosome arms. In Xenopus egg cell-free extracts, Xkid and Xklp1 are essential for bipolar spindle formation but the functions of the human homologues, hKID (KIF22) and KIF4A, are poorly understood. By using RNAi-mediated protein knockdown in human cells, we find that only co-depletion delayed progression through mitosis in a Mad2-dependent manner. Depletion of hKID caused abnormal chromosome arm orientation, delayed chromosome congression, and sensitized cells to nocodazole. Knockdown of KIF4A increased the number and length of microtubules, altered kinetochore oscillations, and decreased kinetochore microtubule flux. These changes were associated with failures in establishing a tight metaphase plate and an increase in anaphase lagging chromosomes. Co-depletion of both chromokinesins aggravated chromosome attachment failures, which led to mitotic arrest. Thus, hKID and KIF4A contribute independently to the rapid and correct attachment of chromosomes by controlling the positioning of chromosome arms and the dynamics of microtubules, respectively. PMID:22945934

  7. ULTRASTRUCTURE OF THE RAT MESENCEPHALIC TRIGEMINAL NUCLEUS

    NARCIS (Netherlands)

    LIEM, RSB; COPRAY, JCVM; VANWILLIGEN, JD

    1991-01-01

    The subcellular morphology of the mesencephalic trigeminal (Me5) nucleus in the rat was studied by transmission electron microscopy. Most neurons in the thin rostral as well as in the major caudal part of Me5 appeared as large (40-50-mu-m), round-to ovoid-shaped unipolar cells. A few neurons (estima

  8. Nuclear transport factor directs localization of protein synthesis during mitosis

    NARCIS (Netherlands)

    Bogaart, Geert van den; Meinema, Anne C.; Krasnikov, Viktor; Veenhoff, Liesbeth M.; Poolman, Bert

    2009-01-01

    Export of messenger RNA from the transcription site in the nucleus and mRNA targeting to the translation site in the cytoplasm are key regulatory processes in protein synthesis. In yeast, the mRNA-binding proteins Nab2p and Nab4p/Hrp1p accompany transcripts to their translation site, where the karyo

  9. Multiplication of human NHIK 3025 cells exposed to porphyrins in combination with light.

    OpenAIRE

    Christensen, T.

    1981-01-01

    Cells from the established line NHIK 3025 were exposed to haematoporphyrin derivative and light. After this photodynamic treatment the first interphase of surviving cells was prolonged. Furthermore, a pronounced effect on the progression through the first mitosis was observed. Mainly the duration of metaphase was increased. Some of the cells were irreversibly arrested in mitosis and the cells that were able to complete mitosis after treatment multiplied in the subsequent generations at the sa...

  10. Neuroprotection Against NMDA Induced Cell Death in Rat Nucleus Basalis by Ca2+ Antagonist Nimodipine, Influence of Aging and Developmental Drug Treatment

    NARCIS (Netherlands)

    Luiten, P.G.M.; Douma, B.R.K.; Zee, E.A. van der; Nyakas, C.

    1995-01-01

    In the current study the neuroprotective effect of the L-type calcium channel antagonist nimodipine in rat brain was investigated in N-methyl-D-aspartate-induced neuronal degeneration in vivo. In the present model NMDA was unilaterally injected in the magnocellular nucleus basalis and the neurotoxic

  11. Neutrino nucleus cross sections

    CERN Document Server

    Athar, M Sajjad; Singh, S K; Vacas, M J Vicente

    2008-01-01

    We present the results of our calculation which has been performed to study the nuclear effects in the quasielastic, inelastic and deep inelastic scattering of neutrinos(antineutrinos) from nuclear targets. These calculations are done in the local density approximation. We take into account the effect of Pauli blocking, Fermi motion, Coulomb effect, renormalization of weak transition strengths in the nuclear medium in the case of the quasielastic reaction. The inelastic reaction leading to production of pions is calculated in a $\\Delta $- dominance model taking into account the renormalization of $\\Delta$ properties in the nuclear medium and the final state interaction effects of the outgoing pions with the residual nucleus. We discuss the nuclear effects in the $F_{3}^{A}(x)$ structure function in the deep inelastic neutrino(antineutrino) reaction using a relativistic framework to describe the nucleon spectral function in the nucleus.

  12. Polarized Proton Nucleus Scattering

    OpenAIRE

    Kopeliovich, B. Z.; Trueman, T. L.

    2000-01-01

    We show that, to a very good approximation, the ratio of the spin-flip to the non-flip parts of the elastic proton-nucleus amplitude is the same as for proton-nucleon scattering at very high energy. The result is used to do a realistic calculation of the analyzing power A_N for pC scattering in the Coulomb-nuclear interference (CNI) region of momentum transfer.

  13. Hadron nucleus interactions

    International Nuclear Information System (INIS)

    The elastic and inelastic scattering of intermediate energy (less than or equal to 1 GeV) protons by nuclei is considered first. The discussion focuses on the determination of the proton-nucleus optical potential in terms of the elementary nucleon-nucleon scattering amplitudes and the properties of the target and residual nucleus. The result is a series of terms for the optical potential. Then the interaction of pions with nuclei for energies in the neighborhood of the Δ-resonance is discussed. In this energy domain an incident pion will with high probability be absorbed by a nucleon to produce the Δ-resonance and thus form a Δ-particle hole state in the nucleus. Next, the subject of hypernuclei is taken up. The Λ hypernuclei and a recently observed Σ hypernuclei comprise situations in which the core nucleus can be probed by a baryon of roughly the same mass as a nucleon, with similar albeit not identical interactions with nucleons. But the Λ (or Σ) does not need to satisfy the Pauli exclusion principle with respect to the nucleons, and therefore can be in orbits forbidden to it if it were a nucleon. As the energy of the projectile increases, it becomes correspondingly more important to take relativistic effects into account. The importance of these effects is strikingly revealed by experiments involving the collision of ultrarelativistic hadrons, protons, pions, kaons (up to Fermilab energies) with nuclei. This phenomenon forms part of the final topic, which includes as well as the collision of relativistic heavy ion projectiles with nuclei. A nuclear Weiszaecker-Williams method developed for dealing with peripheral collisions is described. 32 figures, 10 tables

  14. Antinucleon-nucleus interactions

    Energy Technology Data Exchange (ETDEWEB)

    Dover, C.B.

    1987-01-01

    Recent experimental and theoretical results on anti p-nucleus interactions are reviewed. We focus on determinations of the anti p optical potential from elastic scattering, the use of (anti p, anti p') inelastic scattering to reveal aspects of the spin-isospin dependence of N anti N amplitudes, and some puzzling features of (anti p, anti n) charge exchange reactions on nuclei. 47 refs., 7 figs.

  15. Parkin Regulates Mitosis and Genomic Stability through Cdc20/Cdh1.

    Science.gov (United States)

    Lee, Seung Baek; Kim, Jung Jin; Nam, Hyun-Ja; Gao, Bowen; Yin, Ping; Qin, Bo; Yi, Sang-Yeop; Ham, Hyoungjun; Evans, Debra; Kim, Sun-Hyun; Zhang, Jun; Deng, Min; Liu, Tongzheng; Zhang, Haoxing; Billadeau, Daniel D; Wang, Liewei; Giaime, Emilie; Shen, Jie; Pang, Yuan-Ping; Jen, Jin; van Deursen, Jan M; Lou, Zhenkun

    2015-10-01

    Mutations in the E3 ubiquitin ligase Parkin have been linked to familial Parkinson's disease. Parkin has also been implicated in mitosis through mechanisms that are unclear. Here we show that Parkin interacts with anaphase promoting complex/cyclosome (APC/C) coactivators Cdc20 and Cdh1 to mediate the degradation of several key mitotic regulators independent of APC/C. We demonstrate that ordered progression through mitosis is orchestrated by two distinct E3 ligases through the shared use of Cdc20 and Cdh1. Furthermore, Parkin is phosphorylated and activated by polo-like kinase 1 (Plk1) during mitosis. Parkin deficiency results in overexpression of its substrates, mitotic defects, genomic instability, and tumorigenesis. These results suggest that the Parkin-Cdc20/Cdh1 complex is an important regulator of mitosis.

  16. Protein quality control in the nucleus.

    Science.gov (United States)

    Jones, Ramon D; Gardner, Richard G

    2016-06-01

    The nucleus is the repository for the eukaryotic cell's genetic blueprint, which must be protected from harm to ensure survival. Multiple quality control (QC) pathways operate in the nucleus to maintain the integrity of the DNA, the fidelity of the DNA code during replication, its transcription into mRNA, and the functional structure of the proteins that are required for DNA maintenance, mRNA transcription, and other important nuclear processes. Although we understand a great deal about DNA and RNA QC mechanisms, we know far less about nuclear protein quality control (PQC) mechanisms despite that fact that many human diseases are causally linked to protein misfolding in the nucleus. In this review, we discuss what is known about nuclear PQC and we highlight new questions that have emerged from recent developments in nuclear PQC studies. PMID:27015023

  17. The nucleus: a black box being opened.

    Science.gov (United States)

    van Driel, R; Humbel, B; de Jong, L

    1991-12-01

    Until recently our knowledge about the structural and functional organization of the cell nucleus was very limited. Recent technical developments in the field of ultrastructural analysis, combined with ongoing research on the properties of the nuclear matrix, give new insight into how the nucleus is structured. Two types of observations shape our ideas about nuclear organization. First, most nuclear functions (replication, transcription, RNA processing, and RNA transport) are highly localized within the nucleus, rather than diffusely distributed. Moreover, they are associated with the nuclear matrix. Second, chromatin is organized in discrete loops, bordered by nuclear matrix attachment sequences (MARs). Each loop may contain one or several genes. The arrangement of chromatin in loops has profound consequences for the regulation of gene expression.

  18. Importance of the CEP215-Pericentrin Interaction for Centrosome Maturation during Mitosis

    OpenAIRE

    Seongjae Kim; Kunsoo Rhee

    2014-01-01

    At the onset of mitosis, the centrosome undergoes maturation, which is characterized by a drastic expansion of the pericentriolar material (PCM) and a robust increase in microtubule-organizing activity. CEP215 is one of the major PCM components which accumulates at the centrosome during mitosis. The depletion phenotypes indicate that CEP215 is essential for centrosome maturation and bipolar spindle formation. Here, we performed a series of knockdown-rescue experiments to link the protein-prot...

  19. Wound-induced endogenous jasmonates stunt plant growth by inhibiting mitosis.

    Directory of Open Access Journals (Sweden)

    Yi Zhang

    Full Text Available When plants are repeatedly injured their growth is stunted and the size of organs such as leaves is greatly reduced. The basis of this effect is not well-understood however, even though it reduces yield of crops injured by herbivory, and produces dramatic effects exemplified in ornamental bonsai plants. We have investigated the genetic and physiological basis of this "bonsai effect" by repeatedly wounding leaves of the model plant Arabidopsis. This treatment stunted growth by 50% and increased the endogenous content of jasmonate (JA, a growth inhibitor, by seven-fold. Significantly, repeated wounding did not stunt the growth of the leaves of mutants unable to synthesise JA, or unable to respond to JA including coi1, jai3, myc2, but not jar1. The stunted growth did not result from reduced cell size, but resulted instead from reduced cell number, and was associated with reduced expression of CycB1;2. Wounding caused systemic disappearance of constitutively expressed JAZ1::GUS. Wounding also activates plant immunity. We show that a gene, 12-oxo-phytodienoate reductase, which catalyses a step in JA biosynthesis, and which we confirm is not required for defence, is however required for wound-induced stunting. Our data suggest that intermediates in the JA biosynthetic pathway activate defence, but a primary function of wound-induced JA is to stunt growth through the suppression of mitosis.

  20. Pin1-mediated Sp1 phosphorylation by CDK1 increases Sp1 stability and decreases its DNA-binding activity during mitosis.

    Science.gov (United States)

    Yang, Hang-Che; Chuang, Jian-Ying; Jeng, Wen-Yih; Liu, Chia-I; Wang, Andrew H-J; Lu, Pei-Jung; Chang, Wen-Chang; Hung, Jan-Jong

    2014-12-16

    We have shown that Sp1 phosphorylation at Thr739 decreases its DNA-binding activity. In this study, we found that phosphorylation of Sp1 at Thr739 alone is necessary, but not sufficient for the inhibition of its DNA-binding activity during mitosis. We demonstrated that Pin1 could be recruited to the Thr739(p)-Pro motif of Sp1 to modulate the interaction between phospho-Sp1 and CDK1, thereby facilitating CDK1-mediated phosphorylation of Sp1 at Ser720, Thr723 and Thr737 during mitosis. Loss of the C-terminal end of Sp1 (amino acids 741-785) significantly increased Sp1 phosphorylation, implying that the C-terminus inhibits CDK1-mediated Sp1 phosphorylation. Binding analysis of Sp1 peptides to Pin1 by isothermal titration calorimetry indicated that Pin1 interacts with Thr739(p)-Sp1 peptide but not with Thr739-Sp1 peptide. X-ray crystallography data showed that the Thr739(p)-Sp1 peptide occupies the active site of Pin1. Increased Sp1 phosphorylation by CDK1 during mitosis not only stabilized Sp1 levels by decreasing interaction with ubiquitin E3-ligase RNF4 but also caused Sp1 to move out of the chromosomes completely by decreasing its DNA-binding activity, thereby facilitating cell cycle progression. Thus, Pin1-mediated conformational changes in the C-terminal region of Sp1 are critical for increased CDK1-mediated Sp1 phosphorylation to facilitate cell cycle progression during mitosis.

  1. Multi-channels statistical and morphological features based mitosis detection in breast cancer histopathology.

    Science.gov (United States)

    Irshad, Humayun; Roux, Ludovic; Racoceanu, Daniel

    2013-01-01

    Accurate counting of mitosis in breast cancer histopathology plays a critical role in the grading process. Manual counting of mitosis is tedious and subject to considerable inter- and intra-reader variations. This work aims at improving the accuracy of mitosis detection by selecting the color channels that better capture the statistical and morphological features having mitosis discrimination from other objects. The proposed framework includes comprehensive analysis of first and second order statistical features together with morphological features in selected color channels and a study on balancing the skewed dataset using SMOTE method for increasing the predictive accuracy of mitosis classification. The proposed framework has been evaluated on MITOS data set during an ICPR 2012 contest and ranked second from 17 finalists. The proposed framework achieved 74% detection rate, 70% precision and 72% F-Measure. In future work, we plan to apply our mitosis detection tool to images produced by different types of slide scanners, including multi-spectral and multi-focal microscopy.

  2. TATA-binding protein-related factor 2 is localized in the cytoplasm of mammalian cells and much of it migrates to the nucleus in response to genotoxic agents.

    Science.gov (United States)

    Park, Kyoung-ae; Tanaka, Yuji; Suenaga, Yusuke; Tamura, Taka-aki

    2006-10-31

    TBP (TATA-binding protein)-related factor 2 (TRF2) regulates transcription during a nuber of cellular processes. We previously demonstrated that it is localized in the cytoplasm and is translocated to the nucleus by DNA-damaging agents. However, the cytoplasmic localization of TRF2 is controversial. In this study, we reconfirmed its cytoplasmic localization in various ways and examined its nuclear migration. Stresses such as heat shock, redox agents, heavy metals, and osmotic shock did not affect localization whereas genotoxins such as methyl methanesulfonate (MMS), cisplatin, etoposide, and hydroxyurea caused it to migrate to the nucleus. Adriamycin, mitomycin C and gamma-rays had no obvious effect. We determined optimal conditions for the nuclear migration. The proportions of cells with nuclei enriched for TRF2 were 25-60% and 5-10% for stressed cells and control cells, respectively. Nuclear translocation was observed after 1 h, 4 h and 12 h for cisplatin, etoposide and MMS and hydroxyurea, respectively. The association of TRF2 with the chromatin and promoter region of the proliferating cell nuclear antigen (PCNA) gene, a putative target of TRF2, was increased by MMS treatment. Thus TRF2 may be involved in genotoxin-induced transcriptional regulation. PMID:17085973

  3. Do nuclear envelope and intranuclear proteins reorganize during mitosis to form an elastic, hydrogel-like spindle matrix?

    Science.gov (United States)

    Johansen, Kristen M; Forer, Arthur; Yao, Changfu; Girton, Jack; Johansen, Jørgen

    2011-04-01

    The idea of a spindle matrix has long been proposed in order to account for poorly understood features of mitosis. However, its molecular nature and structural composition have remained elusive. Here, we propose that the spindle matrix may be constituted by mainly nuclear-derived proteins that reorganize during the cell cycle to form an elastic gel-like matrix. We discuss this hypothesis in the context of recent observations from phylogenetically diverse organisms that nuclear envelope and intranuclear proteins form a highly dynamic and malleable structure that contributes to mitotic spindle function. We suggest that the viscoelastic properties of such a matrix may constrain spindle length while at the same time facilitating microtubule growth and dynamics as well as chromosome movement. A corollary to this hypothesis is that a key determinant of spindle size may be the amount of nuclear proteins available to form the spindle matrix. Such a matrix could also serve as a spatial regulator of spindle assembly checkpoint proteins during open and semi-open mitosis. PMID:21274615

  4. ATF7 is stabilized during mitosis in a CDK1-dependent manner and contributes to cyclin D1 expression.

    Science.gov (United States)

    Schaeffer, Etienne; Vigneron, Marc; Sibler, Annie-Paule; Oulad-Abdelghani, Mustapha; Chatton, Bruno; Donzeau, Mariel

    2015-01-01

    The transcription factor ATF7 undergoes multiple post-translational modifications, each of which has distinct effects upon ATF7 function. Here, we show that ATF7 phosphorylation on residue Thr112 exclusively occurs during mitosis, and that ATF7 is excluded from the condensed chromatin. Both processes are CDK1/cyclin B dependent. Using a transduced neutralizing monoclonal antibody directed against the Thr112 epitope in living cells, we could demonstrate that Thr112 phosphorylation protects endogenous ATF7 protein from degradation, while it has no effect on the displacement of ATF7 from the condensed chromatin. The crucial role of Thr112 phosphorylation in stabilizing ATF7 protein during mitosis was confirmed using phospho-mimetic and phospho-deficient mutants. Finally, silencing ATF7 by CRISPR/Cas9 technology leads to a decrease of cyclin D1 protein expression levels. We propose that mitotic stabilized ATF7 protein re-localizes onto chromatin at the end of telophase and contributes to induce the cyclin D1 gene expression.

  5. Characterization of ring-like F-actin structure as a mechanical partner for spindle positioning in mitosis.

    Directory of Open Access Journals (Sweden)

    Huan Lu

    Full Text Available Proper spindle positioning and orientation are essential for accurate mitosis which requires dynamic interactions between microtubule and actin filament (F-actin. Although mounting evidence demonstrates the role of F-actin in cortical cytoskeleton dynamics, it remains elusive as to the structure and function of F-actin-based networks in spindle geometry. Here we showed a ring-like F-actin structure surrounding the mitotic spindle which forms since metaphase and maintains in MG132-arrested metaphase HeLa cells. This cytoplasmic F-actin structure is relatively isotropic and less dynamic. Our computational modeling of spindle position process suggests a possible mechanism by which the ring-like F-actin structure can regulate astral microtubule dynamics and thus mitotic spindle orientation. We further demonstrated that inhibiting Plk1, Mps1 or Myosin, and disruption of microtubules or F-actin polymerization perturbs the formation of the ring-like F-actin structure and alters spindle position and symmetric division. These findings reveal a previously unrecognized but important link between mitotic spindle and ring-like F-actin network in accurate mitosis and enables the development of a method to theoretically illustrate the relationship between mitotic spindle and cytoplasmic F-actin.

  6. Neutrino-nucleus interactions

    Energy Technology Data Exchange (ETDEWEB)

    Gallagher, H.; /Tufts U.; Garvey, G.; /Los Alamos; Zeller, G.P.; /Fermilab

    2011-01-01

    The study of neutrino oscillations has necessitated a new generation of neutrino experiments that are exploring neutrino-nuclear scattering processes. We focus in particular on charged-current quasi-elastic scattering, a particularly important channel that has been extensively investigated both in the bubble-chamber era and by current experiments. Recent results have led to theoretical reexamination of this process. We review the standard picture of quasi-elastic scattering as developed in electron scattering, review and discuss experimental results, and discuss additional nuclear effects such as exchange currents and short-range correlations that may play a significant role in neutrino-nucleus scattering.

  7. Antineutron-nucleus annihilation

    CERN Document Server

    Botta, E

    2001-01-01

    The n-nucleus annihilation process has been studied by the OBELIX experiment at the CERN Low Energy Antiproton Ring (LEAR) in the (50-400) MeV/c projectile momentum range on C, Al, Cu, Ag, Sn, and Pb nuclear targets. A systematic survey of the annihilation cross- section, sigma /sub alpha /(A, p/sub n/), has been performed, obtaining information on its dependence on the target mass number and on the incoming n momentum. For the first time the mass number dependence of the (inclusive) final state composition of the process has been analyzed. Production of the rho vector meson has also been examined. (13 refs).

  8. Nucleus-nucleus potential with shell-correction contribution

    CERN Document Server

    Denisov, V Yu

    2015-01-01

    The full relaxed-density potential between spherical nuclei is considered as a sum of the macroscopic and shell-correction contributions. The macroscopic part of the potential is related to a nucleus-nucleus potential obtained in the framework of the extended Thomas-Fermi approach with the Skyrme and Coulomb forces and the relaxed-density ansatz for evaluation of proton and neutron densities of interacting nuclei. A simple prescription for the shell-correction part of the total potential is discussed. The parameters of the shell-correction and macroscopic parts of the relaxed-density potential are found by fitting the empirical barrier heights of the 89 nucleus-nucleus systems as well as macroscopic potentials evaluated for 1485 nucleus-nucleus systems at 12 distances around touching points.

  9. Higgs-Boson Production in Nucleus-Nucleus Collisions

    Science.gov (United States)

    Norbury, John W.

    1992-01-01

    Cross section calculations are presented for the production of intermediate-mass Higgs bosons produced in ultrarelativistic nucleus-nucleus collisions via two photon fusion. The calculations are performed in position space using Baur's method for folding together the Weizsacker-Williams virtual-photon spectra of the two colliding nuclei. It is found that two photon fusion in nucleus-nucleus collisions is a plausible way of finding intermediate-mass Higgs bosons at the Superconducting Super Collider or the CERN Large Hadron Collider.

  10. Noninvasive three-dimensional live imaging methodology for the spindles at meiosis and mitosis

    Science.gov (United States)

    Zheng, Jing-gao; Huo, Tiancheng; Tian, Ning; Chen, Tianyuan; Wang, Chengming; Zhang, Ning; Zhao, Fengying; Lu, Danyu; Chen, Dieyan; Ma, Wanyun; Sun, Jia-lin; Xue, Ping

    2013-05-01

    The spindle plays a crucial role in normal chromosome alignment and segregation during meiosis and mitosis. Studying spindles in living cells noninvasively is of great value in assisted reproduction technology (ART). Here, we present a novel spindle imaging methodology, full-field optical coherence tomography (FF-OCT). Without any dye labeling and fixation, we demonstrate the first successful application of FF-OCT to noninvasive three-dimensional (3-D) live imaging of the meiotic spindles within the mouse living oocytes at metaphase II as well as the mitotic spindles in the living zygotes at metaphase and telophase. By post-processing of the 3-D dataset obtained with FF-OCT, the important morphological and spatial parameters of the spindles, such as short and long axes, spatial localization, and the angle of meiotic spindle deviation from the first polar body in the oocyte were precisely measured with the spatial resolution of 0.7 μm. Our results reveal the potential of FF-OCT as an imaging tool capable of noninvasive 3-D live morphological analysis for spindles, which might be useful to ART related procedures and many other spindle related studies.

  11. A second tubulin binding site on the kinesin-13 motor head domain is important during mitosis.

    Directory of Open Access Journals (Sweden)

    Dong Zhang

    Full Text Available Kinesin-13s are microtubule (MT depolymerases different from most other kinesins that move along MTs. Like other kinesins, they have a motor or head domain (HD containing a tubulin and an ATP binding site. Interestingly, kinesin-13s have an additional binding site (Kin-Tub-2 on the opposite side of the HD that contains several family conserved positively charged residues. The role of this site in kinesin-13 function is not clear. To address this issue, we investigated the in-vitro and in-vivo effects of mutating Kin-Tub-2 family conserved residues on the Drosophila melanogaster kinesin-13, KLP10A. We show that the Kin-Tub-2 site enhances tubulin cross-linking and MT bundling properties of KLP10A in-vitro. Disruption of the Kin-Tub-2 site, despite not having a deleterious effect on MT depolymerization, results in abnormal mitotic spindles and lagging chromosomes during mitosis in Drosophila S2 cells. The results suggest that the additional Kin-Tub-2 tubulin biding site plays a direct MT attachment role in-vivo.

  12. Cyclin CYB-3 controls both S-phase and mitosis and is asymmetrically distributed in the early C. elegans embryo.

    Science.gov (United States)

    Michael, W Matthew

    2016-09-01

    In early C. elegans embryos the timing of cell division is both invariant and developmentally regulated, yet how the cell cycle is controlled in the embryo and how cell cycle timing impacts early development remain important, unanswered questions. Here, I focus on the cyclin B3 ortholog CYB-3, and show that this cyclin has the unusual property of controlling both the timely progression through S-phase and mitotic entry, suggesting that CYB-3 is both an S-phase-promoting and mitosis-promoting factor. Furthermore, I find that CYB-3 is asymmetrically distributed in the two-cell embryo, such that the somatic precursor AB cell contains ∼2.5-fold more CYB-3 than its sister cell, the germline progenitor P1 CYB-3 is not only physically limited in P1 but also functionally limited, and this asymmetry is controlled by the par polarity network. These findings highlight the importance of the CYB-3 B3-type cyclin in cell cycle regulation in the early embryo and suggest that CYB-3 asymmetry helps establish the well-documented cell cycle asynchrony that occurs during cell division within the P-lineage.

  13. Cyclin CYB-3 controls both S-phase and mitosis and is asymmetrically distributed in the early C. elegans embryo.

    Science.gov (United States)

    Michael, W Matthew

    2016-09-01

    In early C. elegans embryos the timing of cell division is both invariant and developmentally regulated, yet how the cell cycle is controlled in the embryo and how cell cycle timing impacts early development remain important, unanswered questions. Here, I focus on the cyclin B3 ortholog CYB-3, and show that this cyclin has the unusual property of controlling both the timely progression through S-phase and mitotic entry, suggesting that CYB-3 is both an S-phase-promoting and mitosis-promoting factor. Furthermore, I find that CYB-3 is asymmetrically distributed in the two-cell embryo, such that the somatic precursor AB cell contains ∼2.5-fold more CYB-3 than its sister cell, the germline progenitor P1 CYB-3 is not only physically limited in P1 but also functionally limited, and this asymmetry is controlled by the par polarity network. These findings highlight the importance of the CYB-3 B3-type cyclin in cell cycle regulation in the early embryo and suggest that CYB-3 asymmetry helps establish the well-documented cell cycle asynchrony that occurs during cell division within the P-lineage. PMID:27578178

  14. Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins.

    Science.gov (United States)

    Colin, Didier J; Hain, Karolina O; Allan, Lindsey A; Clarke, Paul R

    2015-03-01

    Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.

  15. Inhibition of Wnt signaling by cucurbitacin B in breast cancer cells: reduction of Wnt-associated proteins and reduced translocation of galectin-3-mediated β-catenin to the nucleus.

    Science.gov (United States)

    Dakeng, Sumana; Duangmano, Suwit; Jiratchariyakul, Weena; U-Pratya, Yaowalak; Bögler, Oliver; Patmasiriwat, Pimpicha

    2012-01-01

    The cucurbitacins are tetracyclic triterpenes found in plants of the family Cucurbitaceae. Cucurbitacins have been shown to have anti-cancer and anti-inflamatory activities. We investigated the anti-cancer activity of cucurbitacin B extracted from Thai medicinal plant Trichosanthes cucumerina Linn. Cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Results indicated that cucurbitacin B from T. cucumerina Linn. has a cytotoxic effect on breast cancer cell lines SKBR-3 and MCF-7 with an IC50 of 4.60 and 88.75 µg/ml, respectively. Growth inhibition was attributed to G2/M phase arrest and apoptosis. Cyclin D1, c-Myc, and β-catenin expression levels were reduced. Western blot analysis showed increased PARP cleavage and decreased Wnt-associated signaling molecules β-catenin, galectin-3, cyclin D1 and c-Myc, and corresponding changes in phosphorylated GSK-3β levels. Cucurbitacin B treatment inhibited translocation to the nucleus of β-catenin and galectin-3. The depletion of β-catenin and galectin-3 in the nucleus was confirmed by cellular protein fractionation. T-cell factor (TCF)/lymphoid enhancer factor (LEF)-dependent transcriptional activity was disrupted in cucurbitacin B treated cells as tested by a TCF reporter assay. The relative luciferase activity was reduced when we treated cells with cucurbitacin B compound for 24 h. Our data suggest that cucurbitacin B may in part induce apoptosis and exert growth inhibitory effect via interruption the Wnt signaling.

  16. Tumor necrosis factor alpha promotes the proliferation of human nucleus pulposus cells via nuclear factor-κB, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase.

    Science.gov (United States)

    Wang, Xiao-Hu; Hong, Xin; Zhu, Lei; Wang, Yun-Tao; Bao, Jun-Ping; Liu, Lei; Wang, Feng; Wu, Xiao-Tao

    2015-04-01

    Although tumor necrosis factor alpha (TNF-α) is known to play a critical role in intervertebral disc (IVD) degeneration, the effect of TNF-α on nucleus pulposus (NP) cells has not yet been elucidated. The aim of this study was to explore the effect of TNF-α on proliferation of human NP cells. NP cells were treated with different concentrations of TNF-α. Cell proliferation was determined by cell counting kit-8 (CCK-8) analysis and Ki67 immunofluorescence staining, and expression of cyclin B1 was studied by quantitative real-time RT-PCR. Cell cycle was measured by flow cytometry and cell apoptosis was analyzed using an Annexin V-fluorescein isothiocyanate (FITC) & propidium iodide (PI) apoptosis detection kit. To identify the mechanism by which TNF-α induced proliferation of NP cells, selective inhibitors of major signaling pathways were used and Western blotting was carried out. Treatment with TNF-α increased cell viability (as determined by CCK-8 analysis) and expression of cyclin B1 and the number of Ki67-positive and S-phase NP cells, indicating enhancement of proliferation. Consistent with this, NP cell apoptosis was suppressed by TNF-α treatment. Moreover, inhibition of NF-κB, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) blocked TNF-α-stimulated proliferation of NP cells. In conclusion, the current findings suggest that the effect of TNF-α on IVD degeneration involves promotion of the proliferation of human NP cells via the NF-κB, JNK, and p38 MAPK pathways.

  17. Protein quality control in the nucleus

    DEFF Research Database (Denmark)

    Nielsen, Sofie V.; Poulsen, Esben Guldahl; Rebula, Caio A.;

    2014-01-01

    to aggregate, cells have evolved several elaborate quality control systems to deal with these potentially toxic proteins. First, various molecular chaperones will seize the misfolded protein and either attempt to refold the protein or target it for degradation via the ubiquitin-proteasome system...... to be particularly active in protein quality control. Thus, specific ubiquitin-protein ligases located in the nucleus, target not only misfolded nuclear proteins, but also various misfolded cytosolic proteins which are transported to the nucleus prior to their degradation. In comparison, much less is known about...... these mechanisms in mammalian cells. Here we highlight recent advances in our understanding of nuclear protein quality control, in particular regarding substrate recognition and proteasomal degradation....

  18. The retrotrapezoid nucleus and breathing.

    Science.gov (United States)

    Guyenet, Patrice G; Stornetta, Ruth L; Abbott, Stephen B G; Depuy, Seth D; Kanbar, Roy

    2012-01-01

    The retrotrapezoid nucleus (RTN) is located in the rostral medulla oblongata close to the ventral surface and consists of a bilateral cluster of glutamatergic neurons that are non-aminergic and express homeodomain transcription factor Phox2b throughout life. These neurons respond vigorously to increases in local pCO(2) via cell-autonomous and paracrine (glial) mechanisms and receive additional chemosensory information from the carotid bodies. RTN neurons exclusively innervate the regions of the brainstem that contain the respiratory pattern generator (RPG). Lesion or inhibition of RTN neurons largely attenuates the respiratory chemoreflex of adult rats whereas their activation increases respiratory rate, inspiratory amplitude and active expiration. Phox2b mutations that cause congenital central hypoventilation syndrome in humans prevent the development of RTN neurons in mice. Selective deletion of the RTN Phox2b-VGLUT2 neurons by genetic means in mice eliminates the respiratory chemoreflex in neonates.In short, RTN Phox2b-VGLUT2 neurons are a major nodal point of the CNS network that regulates pCO(2) via breathing and these cells are probable central chemoreceptors. PMID:23080151

  19. Resumption of mitosis in frozen-thawed embryos is not related to the chromosomal constitution

    DEFF Research Database (Denmark)

    Agerholm, Inge E; Kølvrå, Steen; Crüger, Dorthe G;

    2007-01-01

    OBJECTIVE: To study the relation between the resumption of mitosis after thaw and chromosomal constitution in frozen-thawed embryos. In addition, to evaluate the correlation among the three parameters of resumption of mitosis after thaw, postthaw blastomere loss, and multinucleation. DESIGN: Frozen......-thawed embryos were morphologically evaluated at thaw and after 24 hours of culture. Then, fluorescence in situ hybridization (FISH) analysis, including enumeration of 13 chromosomes, was performed by using a combination of peptide nucleic acid and DNA probes. SETTING: In vitro fertilization laboratory. PATIENT......(S): Forty IVF and/or intracytoplasmic sperm injection patients. INTERVENTION(S): Embryo thawing, morphological evaluation, and fluorescence in situ hybridization analysis for aneuploidy screening. MAIN OUTCOME MEASURE(S): Resumption of mitosis, blastomere loss, multinucleation, and chromosome enumeration...

  20. 1,2:3,4-Diepoxybutane Induces Multipolar Mitosis in Cultured Human Lymphocytes.

    Science.gov (United States)

    Barajas Torres, Reyna Lucía; Domínguez Cruz, Martín Daniel; Borjas Gutiérrez, César; Ramírez Dueñas, María de Lourdes; Magaña Torres, María Teresa; González García, Juan Ramón

    2016-01-01

    1,3-Butadiene, a colorless gas regularly used in the production of plastics, thermoplastic resins, and styrene-butadiene rubber, poses an increased leukemia mortality risk to workers in this field. 1,3-Butadiene is also produced by incomplete combustion of motor fuels or by tobacco smoking. It is absorbed principally through the respiratory system and metabolized by several enzymes rendering 1,2:3,4-diepoxybutane (DEB), which has the highest genotoxic potency of all metabolites of 1,3-butadiene. DEB is considered a carcinogen mainly due to its high potential as clastogen, which induces structural chromosome aberrations such as sister chromatid exchanges, chromosomal breaks, and micronuclei. Due to its clastogenic effect, DEB is one of the most used agents for diagnostic studies of Fanconi anemia, a recessively inherited disease related to mutations affecting several genes involved in a common DNA repair pathway. When performing Fanconi anemia diagnostic tests in our laboratory, we have observed occasional multipolar mitosis (MM) in lymphocyte cultures exposed to 0.1 μg/ml of DEB and harvested in the absence of any mitotic spindle inhibitor. Although previous studies reported an aneugenic effect (i.e. it induces aneuploidy) of DEB, no mechanism was suggested to explain such observations. Therefore, the aim of this study was to investigate whether exposure to 0.1 μg/ml of DEB is significantly associated with the occurrence of MM. We blindly assessed the frequency of MM in lymphocyte cultures from 10 nonsmoking healthy individuals. Two series of 3 cultures were performed from each sample under different conditions: A, without DEB; B, with 0.1 μg/ml of DEB, and C, with 25 μM of mitomycin C as positive control. Cultures exposed to DEB showed higher frequencies of MM (23 of 2,000 cells) than did the unexposed ones (3 of 2,000 cells). PMID:27193269

  1. Overexpression of the human MNB/DYRK1A gene induces formation of multinucleate cells through overduplication of the centrosome

    Directory of Open Access Journals (Sweden)

    Hiraoka Yasushi

    2003-09-01

    Full Text Available Abstract Background Previously we cloned the human MNB/DYRK1A gene from the "Down syndrome critical region" on chromosome 21. This gene encodes a dual specificity protein kinase that catalyzes its autophosphorylation on serine/threonine and tyrosine residues. But, the functions of the MNB/DYRK1A gene in cellular processes are unknown. Results In this study, we examined HeLa cells transfected with cDNA encoding a green fluorescent protein (GFP-MNB/DYRK1A fusion protein and found 2 patterns of expression: In one group of transfected cells, GFP-MNB/DYRK1A was localized as dots within the nucleus; and in the other group, it was overexpressed and had accumulated all over the nucleus. In the cells overexpressing GFP-MNB/DYRK1A, multinucleation was clearly observed; whereas in those with the nuclear dots, such aberrant nuclei were not found. Furthermore, in the latter cells, essential processes such as mitosis and cytokinesis occurred normally. Multinucleation was dependent on the kinase activity of MNB/DYRK1A, because it was not observed in cells overexpressing kinase activity-negative mutants, GFP-MNB/DYRK1A (K179R and GFP-MNB/DYRK1A (Y310F/Y312F. Immunostaining of GFP-MNB/DYRK1A-overexpressing cells with specific antibodies against α- and γ-tubulin revealed that multiple copies of centrosomes and aberrant multipolar spindles were generated in these cells. Conclusions These results indicate that overexpression of MNB/DYRK1A induces multinucleation in HeLa cells through overduplication of the centrosome during interphase and production of aberrant spindles and missegregation of chromosomes during mitosis.

  2. M-31 mutant (virA::Tn5) of Agrobacterium tumefaciens is capable of transferring its T-DNA into the nucleus of host cell, but incapable of integrating it into the chromosome.

    Science.gov (United States)

    Majumder, P; Yoshida, H; Shioiri, H; Nozue, M; Kojima, M

    2000-01-01

    An avirulent mutant (M-31 strain) was produced by the transposon (Tn5) mutagenesis of Agrobacterium tumefaciens (A-208 strain). A binary vector, pIG121-Hm, containing a kanamycin resistance gene (nptII) and beta-glucuronidase (GUS) gene with an intron, was introduced into M-31 and A-208 strains. The resultant Agrobacteria were inoculated onto leaves of Kalanchoe daigremontiana and to tobacco BY-2 cells to assay GUS activity to monitor the T-DNA transfer into the nuclei of host cells. The results indicated that T-DNA was transferred into the nuclei of cells of both host plants inoculated with the M-31 mutant. The M-31 mutant strain had an insertion of Tn5 in the virA gene on its Ti plasmid. The introduction of the virA gene in the M-31 mutant complemented its avirulent phenotype. No kanamycin-resistant cells were observed when the M-31 mutant harboring the pIG121-Hm was inoculated to tobacco BY-2 cells. The M-31 mutant (virA::Tn5) seems to transfer T-DNA into the nucleus of the host cell, but is unable to integrate it to the chromosome. PMID:16232864

  3. Role of substrate concentration in mitosis and hyphal extension of Aspergillus

    DEFF Research Database (Denmark)

    Müller, Christian; Spohr, Anders Bendsen; Nielsen, Jens

    2000-01-01

    The filamentous fungi Aspergillus oryzae and A. niger grow by apical extension of multinucleate hyphae that are subdivided into compartments by cross-walls called septa. Submerged cultivation, image analysis, and fluorescence microscopy were used to study the role of the carbon source on mitosis...... of a high glucose concentration, whereas a short apical compartment with few nuclei was the result of a low glucose concentration. This is the first study of the influence of glucose concentration on nuclear mitosis and septation in filamentous fungi grown submerged. In addition, this is the first time...

  4. Caudal topographic nucleus isthmi and the rostral nontopographic nucleus isthmi in the turtle, Pseudemys scripta.

    Science.gov (United States)

    Sereno, M I; Ulinski, P S

    1987-07-15

    Isthmotectal projections in turtles were examined by making serial section reconstructions of axonal and dendritic arborizations that were anterogradely or retrogradely filled with HRP. Two prominent tectal-recipient isthmic nuclei--the caudal magnocellular nucleus isthmi (Imc) and the rostral magnocellular nucleus isthmi (Imr)--exhibited strikingly different patterns of organization. Imc cells have flattened, bipolar dendritic fields that cover a few percent of the area of the cell plate constituting the nucleus and they project topographically to the ipsilateral tectum without local axon branches. The topography was examined explicitly at the single-cell level by using cases with two injections at widely separated tectal loci. Each Imc axon terminates as a compact swarm of several thousand boutons placed mainly in the upper central gray and superficial gray layers. One Imc terminal spans less that 1% of the tectal surface. Imr cells, by contrast, have large, sparsely branched dendritic fields overlapped by local axon collaterals while distally, their axons nontopographically innervate not only the deeper layers of the ipsilateral tectum but also ipsilateral Imc. Imr receives a nontopographic tectal input that contrasts with the topographic tectal input to Imc. Previous work on nucleus isthmi emphasized the role of the contralateral isthmotectal projection (which originates from a third isthmic nucleus in turtles) in mediating binocular interactions in the tectum. The present results on the two different but overlapping ipsilateral tecto-isthmo-tectal circuits set up by Imc and Imr are discussed in the light of physiological evidence for selective attention effects and local-global interactions in the tectum.

  5. Formation of light particles in nucleus-nucleus collisions

    International Nuclear Information System (INIS)

    The principal experimental results on the yield of the light charged particles in nucleus-nucleus collisions at the low and intermediate energies are reviewed. Inclusive spectra of light particles and their coincidences with the characteristic KX-rays, γ-rays, neutrons, projectile-like fragments, other light particles, fission fragments, and evaporation residues are analyzed. The main theoretical models used for the description of the light particle formation are briefly outlined together with their merits and shortcomings. The unsolved problems of fast light particle formation, in particular, and of nucleus-nucleus interaction dynamics, on the whole, are discussed with the outlooks of new experiments able to clear up some of these problems. (author) 144 refs., 40 figs., 2 tabs

  6. Co-expression of mitosis-regulating genes contributes to malignant progression and prognosis in oligodendrogliomas.

    Science.gov (United States)

    Liu, Yanwei; Hu, Huimin; Zhang, Chuanbao; Wang, Haoyuan; Zhang, Wenlong; Wang, Zheng; Li, Mingyang; Zhang, Wei; Zhou, Dabiao; Jiang, Tao

    2015-11-10

    The clinical prognosis of patients with glioma is determined by tumor grades, but tumors of different subtypes with equal malignancy grade usually have different prognosis that is largely determined by genetic abnormalities. Oligodendrogliomas (ODs) are the second most common type of gliomas. In this study, integrative analyses found that distribution of TCGA transcriptomic subtypes was associated with grade progression in ODs. To identify critical gene(s) associated with tumor grades and TCGA subtypes, we analyzed 34 normal brain tissue (NBT), 146 WHO grade II and 130 grade III ODs by microarray and RNA sequencing, and identified a co-expression network of six genes (AURKA, NDC80, CENPK, KIAA0101, TIMELESS and MELK) that was associated with tumor grades and TCGA subtypes as well as Ki-67 expression. Validation of the six genes was performed by qPCR in additional 28 ODs. Importantly, these genes also were validated in four high-grade recurrent gliomas and the initial lower-grade gliomas resected from the same patients. Finally, the RNA data on two genes with the highest discrimination potential (AURKA and NDC80) and Ki-67 were validated on an independent cohort (5 NBTs and 86 ODs) by immunohistochemistry. Knockdown of AURKA and NDC80 by siRNAs suppressed Ki-67 expression and proliferation of gliomas cells. Survival analysis showed that high expression of the six genes corporately indicated a poor survival outcome. Correlation and protein interaction analysis provided further evidence for this co-expression network. These data suggest that the co-expression of the six mitosis-regulating genes was associated with malignant progression and prognosis in ODs.

  7. Move, Stop, Learn: Illustrating Mitosis through Stop-Motion Animation

    Science.gov (United States)

    Kamp, Brandi L.; Deaton, Cynthia C. M.

    2013-01-01

    Learning about microscopic things, such as cells, can often be mundane to students because they are not able to see or manipulate what they are learning about. Students often recall learning about cell division through memorization--thus they find it tedious and dull. Few opportunities exist that allow students to explore and manipulate cells or…

  8. Using "Chromosomal Socks" to Demonstrate Ploidy in Mitosis and Meiosis

    Science.gov (United States)

    Chinnici, Joseph P.; Neth, Somalin Zaroh; Sherman, Leah R.

    2006-01-01

    Today, many biology instructors use visual models to help students understand abstract concepts like cell division. For all biology instructors, dealing with student misconceptions of cell division may seem hopeless at times--even after using visual models. Although student errors in cell division are built around the three key events of cell…

  9. Dynamic Bcl-xL (S49) and (S62) Phosphorylation/Dephosphorylation during Mitosis Prevents Chromosome Instability and Aneuploidy in Normal Human Diploid Fibroblasts

    Science.gov (United States)

    Baruah, Prasamit Saurav; Beauchemin, Myriam; Hébert, Josée; Bertrand, Richard

    2016-01-01

    Bcl-xL proteins undergo dynamic phosphorylation/dephosphorylation on Ser49 and Ser62 residues during mitosis. The expression of Bcl-xL(S49A), (S62A) and dual (S49/62A) phosphorylation mutants in tumor cells lead to severe mitotic defects associated with multipolar spindle, chromosome lagging and bridging, and micro-, bi- and multi-nucleated cells. Because the above observations were made in tumor cells which already display genomic instability, we now address the question: will similar effects occur in normal human diploid cells? We studied normal human diploid BJ foreskin fibroblast cells expressing Bcl-xL (wild type), (S49A), (S49D), (S62A), (S62D) and the dual-site (S49/62A) and (S49/62D) mutants. Cells expressing S49 and/or S62 phosphorylation mutants showed reduced kinetics of cell population doubling. These effects on cell population doubling kinetics correlated with early outbreak of senescence with no impact on the cell death rate. Senescent cells displayed typical senescence-associated phenotypes including high-level of senescence-associated β-galactosidase activity, interleukin-6 (IL-6) secretion, tumor suppressor p53 and cyclin-dependent kinase inhibitor p21Waf1/Cip1 activation as well as γH2A.X-associated nuclear chromatin foci. Fluorescence in situ hybridization analysis and Giemsa-banded karyotypes revealed that the expression of Bcl-xL phosphorylation mutants in normal diploid BJ cells provoked chromosome instability and aneuploidy. These findings suggest that dynamic Bcl-xL(S49) and (S62) phosphorylation/dephosphorylation cycles are important in the maintenance of chromosome integrity during mitosis in normal cells. They could impact future strategies aiming to develop and identify compounds that could target not only the anti-apoptotic domain of Bcl-xL protein, but also its mitotic domain for cancer therapy. PMID:27398719

  10. M-CSF TARGETING INTO LCL NUCLEUS BEHAVES AS A MALIGNANCY PROMOTOR

    Institute of Scientific and Technical Information of China (English)

    曹震宇; 吴克复; 宋玉华; 李戈; 林永敏; 饶青; 马小彤

    2003-01-01

    Objective: To investigate the functions of nM-CSF in malignant cells. Methods: recombinant M-CSF was targeted into cell nucleus by employing a eukaryotic expression plasmid vector pCMV/myc/nuc. The constructed plasmid was transfected into cells of EBV transformed lymphoblastoid cell line (LCL). RT-PCR, Western blot and immunofluorescent staining showed that recombinant M-CSF was localized into LCL cell nucleus. The transgenic cells showed elevated proliferation potential, enhanced resistance to apoptosis and increased ability of in vitro migration. Conclusion: Nucleus presenting M-CSF might act as a promoting factor in the processes of cell malignancy.

  11. 肝细胞生长因子对体外培养的人退变髓核细胞生物学活性影响%Effect of hepatocyte growth factor on the biological activity of the human degeneration nucleus pulposus cells cultured in vitro

    Institute of Scientific and Technical Information of China (English)

    满孝旭; 马迅; 关晓明; 张丽

    2011-01-01

    Objective To explore the effect of hepatocyte growth factor (HGF) on the biological activity of the human degeneration nucleus pulposus cells cultured in vitro. Methods The human nucleus pulposus were collected ,and the nucleus pulposus cells were isolated and cultured . To observe HGF receptor expression in nucleus pulposus cells by immunohistochemical staining . The experimental groups were cultured with 0,5,50,500 ng/ml HGF,the optimal concentration was selected by MTT. The 3rd generation of nucleus pulposus cells were selected aid divided into HGF ,HGF + IG,IGF and control group, reverse transcription-polymerase chain reaction ( RT-PCR) was used to determine levels of type II collagen, proteoglycan and SOX9; flow cytometer were used to detect cell cycle. Results Nucleus pulposus cells expressed HGF receptor. HGF could significantly promote the proliferation of the human degeneration nucleus pulposus cells , which had a significant different compared with the control group . Various concentrations of HGF were effective to promote the nucleus pulposus cells proliferation and the optimal HGF concentration was 50 ng/ ml. The human degeneration nucleus pulposus cells under HGF ,IGF and HGF + IGF had shown to keep type II collagen, proteoglycan and SOX9 expression positive and increasing , and the proportion of cells into the proliferative phase increased compared with control group , which HGF + IGF was more effective. Conclusions Nucleus pulposus cells expressed HGF receptor. HGF can promote the proliferation of the human degeneration nucleus pulposus cells cultured in vitro, enhance the expression of extracellular matrix .The combined effects of HGF and IGF are better than alone.%目的 探讨肝细胞生长因子(HGF)对体外培养的人退变髓核细胞的生物学作用.方法 收集人退变髓核组织标本,分离培养髓核细胞;免疫组化染色观察HGF受体在髓核细胞中表达;设立不同浓度HGF组(0 ng/ml,5 ng/ml,50 ng/ml,500 ng/ml),采

  12. Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis

    NARCIS (Netherlands)

    Yamaguchi, Tomoya; Goto, Hidemasa; Yokoyama, Tomoya; Silljé, Herman; Hanisch, Anja; Uldschmid, Andreas; Takai, Yasushi; Oguri, Takashi; Nigg, Erich A; Inagaki, Masaki

    2005-01-01

    Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its si

  13. Dance of the Chromosomes: A Kinetic Learning Approach to Mitosis and Meiosis

    Science.gov (United States)

    Kreiser, Brian; Hairston, Rosalina

    2007-01-01

    Understanding mitosis and meiosis is fundamental to understanding the basics of Mendelian inheritance, yet many students find these concepts challenging or confusing. Here we present a visually and physically stimulating activity using minimal supplies to supplement traditional instruction in order to engage the students and facilitate…

  14. Parkin Regulates Mitosis and Genomic Stability through Cdc20/Cdh1

    NARCIS (Netherlands)

    Lee, S.B.; Kim, J.J.; Nam, H.J.; Gao, B.; Yin, P.; Qin, B.; Yi, S.Y.; Ham, H.; Evans, D.; Kim, S.H.; Zhang, Jun; Deng, M.; Liu, T.; Zhang, H.; Billadeau, D.D.; Wang, L.; Giaime, E.; Shen, J.; Pang, Y.P.; Jen, J.; Deursen, J.M.A. van; Lou, Z.

    2015-01-01

    Mutations in the E3 ubiquitin ligase Parkin have been linked to familial Parkinson's disease. Parkin has also been implicated in mitosis through mechanisms that are unclear. Here we show that Parkin interacts with anaphase promoting complex/cyclosome (APC/C) coactivators Cdc20 and Cdh1 to mediate th

  15. Creating a Double-Spring Model to Teach Chromosome Movement during Mitosis & Meiosis

    Science.gov (United States)

    Luo, Peigao

    2012-01-01

    The comprehension of chromosome movement during mitosis and meiosis is essential for understanding genetic transmission, but students often find this process difficult to grasp in a classroom setting. I propose a "double-spring model" that incorporates a physical demonstration and can be used as a teaching tool to help students understand this…

  16. Mitosis Counting in Breast Cancer : Object-Level Interobserver Agreement and Comparison to an Automatic Method

    NARCIS (Netherlands)

    Veta, Mitko; van Diest, Paul J; Jiwa, Mehdi; Al-Janabi, Shaimaa; Pluim, JPW

    2016-01-01

    BACKGROUND: Tumor proliferation speed, most commonly assessed by counting of mitotic figures in histological slide preparations, is an important biomarker for breast cancer. Although mitosis counting is routinely performed by pathologists, it is a tedious and subjective task with poor reproducibilit

  17. Assessment of algorithms for mitosis detection in breast cancer histopathology images

    DEFF Research Database (Denmark)

    Veta, Mitko; van Diest, Paul J.; Willems, Stefan M.;

    2014-01-01

    The proliferative activity of breast tumors, which is routinely estimated by counting of mitotic figures in hematoxylin and eosin stained histology sections, is considered to be one of the most important prognostic markers. However, mitosis counting is laborious, subjective and may suffer from lo...

  18. Phosphorylation of CPAP by Aurora-A Maintains Spindle Pole Integrity during Mitosis

    Directory of Open Access Journals (Sweden)

    En-Ju Chou

    2016-03-01

    Full Text Available CPAP is required for centriole elongation during S/G2 phase, but the role of CPAP in mitosis is incompletely understood. Here, we show that CPAP maintains spindle pole integrity through its phosphorylation by Aurora-A during mitosis. Depletion of CPAP induced a prolonged delay in mitosis, pericentriolar material (PCM dispersion, and multiple mitotic abnormalities. Further studies demonstrated that CPAP directly interacts with and is phosphorylated by Aurora-A at serine 467 during mitosis. Interestingly, the dispersal of the PCM was effectively rescued by ectopic expression of wild-type CPAP or a phospho-mimic CPAP-S467D mutant, but not a non-phosphorylated CPAP-S467A mutant. Finally, we found that CPAP-S467D has a low affinity for microtubule binding but a high affinity for PCM proteins. Together, our results support a model wherein CPAP is required for proper mitotic progression, and phosphorylation of CPAP by Aurora-A is essential for maintaining spindle pole integrity.

  19. Phosphorylation of CPAP by Aurora-A Maintains Spindle Pole Integrity during Mitosis.

    Science.gov (United States)

    Chou, En-Ju; Hung, Liang-Yi; Tang, Chieh-Ju C; Hsu, Wen-Bin; Wu, Hsin-Yi; Liao, Pao-Chi; Tang, Tang K

    2016-03-29

    CPAP is required for centriole elongation during S/G2 phase, but the role of CPAP in mitosis is incompletely understood. Here, we show that CPAP maintains spindle pole integrity through its phosphorylation by Aurora-A during mitosis. Depletion of CPAP induced a prolonged delay in mitosis, pericentriolar material (PCM) dispersion, and multiple mitotic abnormalities. Further studies demonstrated that CPAP directly interacts with and is phosphorylated by Aurora-A at serine 467 during mitosis. Interestingly, the dispersal of the PCM was effectively rescued by ectopic expression of wild-type CPAP or a phospho-mimic CPAP-S467D mutant, but not a non-phosphorylated CPAP-S467A mutant. Finally, we found that CPAP-S467D has a low affinity for microtubule binding but a high affinity for PCM proteins. Together, our results support a model wherein CPAP is required for proper mitotic progression, and phosphorylation of CPAP by Aurora-A is essential for maintaining spindle pole integrity.

  20. Transcripts of the MHM region on the chicken Z chromosome accumulate as non-coding RNA in the nucleus of female cells adjacent to the DMRT1 locus.

    Science.gov (United States)

    Teranishi, M; Shimada, Y; Hori, T; Nakabayashi, O; Kikuchi, T; Macleod, T; Pym, R; Sheldon, B; Solovei, I; Macgregor, H; Mizuno, S

    2001-01-01

    The male hypermethylated (MHM) region, located near the middle of the short arm of the Z chromosome of chickens, consists of approximately 210 tandem repeats of a BamHI 2.2-kb sequence unit. Cytosines of the CpG dinucleotides of this region are extensively methylated on the two Z chromosomes in the male but much less methylated on the single Z chromosome in the female. The state of methylation of the MHM region is established after fertilization by about the 1-day embryonic stage. The MHM region is transcribed only in the female from the particular strand into heterogeneous, high molecular-mass, non-coding RNA, which is accumulated at the site of transcription, adjacent to the DMRT1 locus, in the nucleus. The transcriptional silence of the MHM region in the male is most likely caused by the CpG methylation, since treatment of the male embryonic fibroblasts with 5-azacytidine results in hypo-methylation and active transcription of this region. In ZZW triploid chickens, MHM regions are hypomethylated and transcribed on the two Z chromosomes, whereas MHM regions are hypermethylated and transcriptionally inactive on the three Z chromosomes in ZZZ triploid chickens, suggesting a possible role of the W chromosome on the state of the MHM region. PMID:11321370