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Sample records for drosophila embryo mitosis

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

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    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.

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

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

    2015-01-01

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

  3. The Ran pathway in Drosophila melanogaster mitosis

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    James G Wakefield

    2015-11-01

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

  4. Spindle mechanics and dynamics during mitosis in Drosophila.

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    Kwon, Mijung; Scholey, Jonathan M

    2004-04-01

    Drosophila melanogaster is an excellent model for studying mitosis. Syncytial embryos are amenable to time-lapse imaging of hundreds of synchronously dividing spindles, allowing the quantitation of spindle and chromosome dynamics with unprecedented fidelity. Other Drosophila cell types, including neuroblasts, cultured cells, spermatocytes and oocytes, contain spindles that differ in their design, providing cells amenable to different types of experiments and allowing identification of common core mechanisms. The function of mitotic proteins can be studied using mutants, inhibitor microinjection and RNA interference (RNAi) to identify the full inventory of mitotic proteins encoded by the genome. Here, we review recent advances in understanding how ensembles of mitotic proteins coordinate spindle assembly and chromosome motion in this system.

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

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    Powers, James A

    2010-06-01

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

  6. The syncytial Drosophila embryo as a mechanically excitable medium

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    Idema, Timon; Manning, M Lisa; Nelson, Philip C; Liu, Andrea J

    2013-01-01

    Mitosis in the early syncytial Drosophila embryo is highly correlated in space and time, as manifested in mitotic wavefronts that propagate across the embryo. In this paper we investigate the idea that the embryo can be considered a mechanically-excitable medium, and that mitotic wavefronts can be understood as nonlinear wavefronts that propagate through this medium. We study the wavefronts via both image analysis of confocal microscopy videos and theoretical models. We find that the mitotic wavefront can be resolved into two distinct wavefronts in each cycle, corresponding to metaphase and anaphase, respectively. The two wavefronts have the same speed and are separated by a time interval that is independent of cycle, supporting the idea that they are two different markers for the same process. To understand the wavefronts theoretically we analyze wavefront propagation in excitable media. We study two classes of models, one with biochemical signaling and one with mechanical signaling. We find that the depende...

  7. Dissecting mitosis by RNAi in Drosophila tissue culture cells

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    Maiato Helder

    2003-01-01

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

  8. 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....... RESULT(S): No difference was observed in the chromosomal constitution of embryos with and without resumption of mitosis. Neither was the postthaw blastomere loss connected to the chromosomal constitution. The resumption of mitosis was not associated with postthaw loss of blastomeres...

  9. In-vivo Centrifugation of Drosophila Embryos

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    Tran, Susan L.; Welte, Michael A.

    2010-01-01

    A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, an...

  10. Centrosome splitting during nuclear elongation in the Drosophila embryo.

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    Callaini, G; Anselmi, F

    1988-10-01

    In the early Drosophila embryo, nuclear elongation occurs during cellularization of the syncytial blastoderm. This process is closely related to the presence of microtubular bundles forming a basket-like structure surrounding the nuclei. In immunofluorescence observations with antibodies against alpha-tubulin, the microtubules appear to radiate from two bright foci widely separated from each other. We used electron microscopy to show that these foci are true centrosomes constituted by daughter and parent centrioles orthogonally disposed and surrounded by pericentriolar electrondense material. The centrosomes may be observed in the apical region of the blastoderm cells from the beginning of cellularization until the reestablishment of the first postblastodermic mitosis, when they organize the spindle poles. Until this time the dimensions of the procentrioles remain unchanged. The significance of these results is discussed in relation to the known behavior of centrioles in the cell cycle.

  11. Maternal effect mutations of the sponge locus affect actin cytoskeletal rearrangements in Drosophila melanogaster embryos

    OpenAIRE

    1992-01-01

    In the syncytial blastoderm stage of Drosophila embryogenesis, dome- shaped actin "caps" are observed above the interphase nuclei. During mitosis, this actin rearranges to participate in the formation of pseudocleavage furrows, transient membranous invaginations between dividing nuclei. Embryos laid by homozygous sponge mothers lack these characteristic actin structures, but retain other actin associated structures and processes. Our results indicate that the sponge product is specifically re...

  12. DAPI Staining of Drosophila Embryos.

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    Rothwell, Wendy F; Sullivan, William

    2007-10-01

    INTRODUCTIONDrosophila embryos can be stained with specific fluorescent probes or antibodies through either direct or indirect immunofluorescence. In particular, several effective probes exist for visualizing DNA. 4',6-diamidino-2-phenylindole (DAPI) is a commonly used DNA-binding dye. Because it is specific for double-stranded DNA, no prior RNase treatment is required. While the embryo staining method described here uses DAPI, other fluorescent DNA probes can be processed similarly.

  13. In-vivo centrifugation of Drosophila embryos.

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    Tran, Susan L; Welte, Michael A

    2010-06-23

    A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000 g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

  14. Microfluidic system with integrated microinjector for automated Drosophila embryo injection.

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    Delubac, Daniel; Highley, Christopher B; Witzberger-Krajcovic, Melissa; Ayoob, Joseph C; Furbee, Emily C; Minden, Jonathan S; Zappe, Stefan

    2012-11-21

    Drosophila is one of the most important model organisms in biology. Knowledge derived from the recently sequenced 12 genomes of various Drosophila species can today be combined with the results of more than 100 years of research to systematically investigate Drosophila biology at the molecular level. In order to enable automated, high-throughput manipulation of Drosophila embryos, we have developed a microfluidic system based on a Pyrex-silicon-Pyrex sandwich structure with integrated, surface-micromachined silicon nitride injector for automated injection of reagents. Our system automatically retrieves embryos from an external reservoir, separates potentially clustered embryos through a sheath flow mechanisms, passively aligns an embryo with the integrated injector through geometric constraints, and pushes the embryo onto the injector through flow drag forces. Automated detection of an embryo at injection position through an external camera triggers injection of reagents and subsequent ejection of the embryo to an external reservoir. Our technology can support automated screens based on Drosophila embryos as well as creation of transgenic Drosophila lines. Apart from Drosophila embryos, the layout of our system can be easily modified to accommodate injection of oocytes, embryos, larvae, or adults of other species and fills an important technological gap with regard to automated manipulation of multicellular organisms.

  15. Gradual meiosis-to-mitosis transition in the early mouse embryo.

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    Courtois, Aurélien; Hiiragi, Takashi

    2012-01-01

    The transition from meiosis to mitosis is a fundamental process to guarantee the successful development of the embryo. In the mouse, the transition includes extensive reorganisation of the division machinery, centrosome establishment and changes in spindle proprieties and characteristic. Recent findings indicate that this transition is gradual and lasts until the late blastocyst stage. In-depth knowledge of the mechanisms underlying the transition would provide new insight into de novo centrosome formation and regulation of spindle size and proprieties. Here, we review recent advances in the understanding of acentrosomal spindle formation, centriole establishment and the meiosis-to-mitosis transition in the mouse pre-implantation embryo.

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

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    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.

  17. Mutations in Drosophila Greatwall/Scant reveal its roles in mitosis and meiosis and interdependence with Polo kinase.

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    Vincent Archambault

    2007-11-01

    Full Text Available Polo is a conserved kinase that coordinates many events of mitosis and meiosis, but how it is regulated remains unclear. Drosophila females having only one wild-type allele of the polo kinase gene and the dominant Scant mutation produce embryos in which one of the centrosomes detaches from the nuclear envelope in late prophase. We show that Scant creates a hyperactive form of Greatwall (Gwl with altered specificity in vitro, another protein kinase recently implicated in mitotic entry in Drosophila and Xenopus. Excess Gwl activity in embryos causes developmental failure that can be rescued by increasing maternal Polo dosage, indicating that coordination between the two mitotic kinases is crucial for mitotic progression. Revertant alleles of Scant that restore fertility to polo-Scant heterozygous females are recessive alleles or deficiencies of gwl; they show chromatin condensation defects and anaphase bridges in larval neuroblasts. One recessive mutant allele specifically disrupts a Gwl isoform strongly expressed during vitellogenesis. Females hemizygous for this allele are sterile, and their oocytes fail to arrest in metaphase I of meiosis; both homologues and sister chromatids separate on elongated meiotic spindles with little or no segregation. This allelic series of gwl mutants highlights the multiple roles of Gwl in both mitotic and meiotic progression. Our results indicate that Gwl activity antagonizes Polo and thus identify an important regulatory interaction of the cell cycle.

  18. Mitosis.

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    Henderson, Paula

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

  19. Drosophila chem mutations disrupt epithelial polarity in Drosophila embryos

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    José M. Zamudio-Arroyo

    2016-12-01

    Full Text Available Drosophila embryogenesis has proven to be an extremely powerful system for developmental gene discovery and characterization. We isolated five new EMS-induced alleles that do not complement the l(3R5G83 lethal line isolated in the Nüsslein-Volhard and Wieschaus screens. We have named this locus chem. Lethality of the new alleles as homozygous zygotic mutants is not completely penetrant, and they have an extended phenocritical period. Like the original allele, a fraction of mutant embryos die with cuticular defects, notably head involution and dorsal closure defects. Embryonic defects are much more extreme in germline clones, where the majority of mutant embryos die during embryogenesis and do not form cuticle, implying a strong chem maternal contribution. chem mutations genetically interact with mutations in cytoskeletal genes (arm and with mutations in the epithelial polarity genes coracle, crumbs, and yurt. chem mutants dorsal open defects are similar to those present in yurt mutants, and, likewise, they have epithelial polarity defects. chem1 and chem3 mutations suppress yurt3, and chem3 mutants suppress crumbs1 mutations. In contrast, chem1 and coracle2 mutations enhance each other. Compared to controls, in chem mutants in embryonic lateral epithelia Crumbs expression is mislocalized and reduced, Coracle is increased and mislocalized basally at embryonic stages 13–14, then reduced at stage 16. Arm expression has a similar pattern but levels are reduced.

  20. Tracking individual nanodiamonds in Drosophila melanogaster embryos

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    Simpson, David A; Kowarsky, Mark; Zeeshan, Nida F; Barson, Michael S J; Hall, Liam; Yan, Yan; Kaufmann, Stefan; Johnson, Brett C; Ohshima, Takeshi; Caruso, Frank; Scholten, Robert; Saint, Robert B; Murray, Michael J; Hollenberg, Lloyd C L

    2013-01-01

    Tracking the dynamics of fluorescent nanoparticles during embryonic development allows insights into the physical state of the embryo and, potentially, molecular processes governing developmental mechanisms. In this work, we investigate the motion of individual fluorescent nanodiamonds micro-injected into Drosophila melanogaster embryos prior to cellularisation. Fluorescence correlation spectroscopy and wide-field imaging techniques are applied to individual fluorescent nanodiamonds in blastoderm cells during stage 5 of development to a depth of ~40 \\mu m. The majority of nanodiamonds in the blastoderm cells during cellularisation exhibit free diffusion with an average diffusion coefficient of (6 $\\pm$ 3) x 10$^{-3}$ \\mu m$^2$/s, (mean $\\pm$ SD). Driven motion in the blastoderm cells was also observed with an average velocity of 0.13 $\\pm$ 0.10 \\mu m/s (mean $\\pm$ SD) \\mu m/s and an average applied force of 0.07 $\\pm$ 0.05 pN (mean $\\pm$ SD). Nanodiamonds in the periplasm between the nuclei and yolk were also...

  1. A spindle checkpoint functions during mitosis in the early Caenorhabditis elegans embryo.

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    Encalada, Sandra E; Willis, John; Lyczak, Rebecca; Bowerman, Bruce

    2005-03-01

    During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis.

  2. Waves of Cdk1 Activity in S Phase Synchronize the Cell Cycle in Drosophila Embryos.

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    Deneke, Victoria E; Melbinger, Anna; Vergassola, Massimo; Di Talia, Stefano

    2016-08-22

    Embryos of most metazoans undergo rapid and synchronous cell cycles following fertilization. While diffusion is too slow for synchronization of mitosis across large spatial scales, waves of Cdk1 activity represent a possible process of synchronization. However, the mechanisms regulating Cdk1 waves during embryonic development remain poorly understood. Using biosensors of Cdk1 and Chk1 activities, we dissect the regulation of Cdk1 waves in the Drosophila syncytial blastoderm. We show that Cdk1 waves are not controlled by the mitotic switch but by a double-negative feedback between Cdk1 and Chk1. Using mathematical modeling and surgical ligations, we demonstrate a fundamental distinction between S phase Cdk1 waves, which propagate as active trigger waves in an excitable medium, and mitotic Cdk1 waves, which propagate as passive phase waves. Our findings show that in Drosophila embryos, Cdk1 positive feedback serves primarily to ensure the rapid onset of mitosis, while wave propagation is regulated by S phase events. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Methylmercury as a mitosis disturbing agent. [Allium cepa; Drosophila melanogaster

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    Ramel, C.

    1969-01-01

    Experiments were performed to investigate the genetic effects of mercurials. These investigations included both cytological and genetical analyses. One of the main purposes of the investigations was to establish the lowest dose of the mercurials, which was genetically active. For the cytological work root tips cells of Allium cepa were used, while the genetical analyses were preformed on Drosophila melanogaster. The cytological tests on Allium included methyl mercury hydroxide, methyl mercury dicyandiamide, phenyl mercury hydroxide, and methoxyethylmercury chloride. The pesticide Panogen was also tested. The results from these studies are summarized.

  4. The embryo as a laboratory: quantifying transcription in Drosophila

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    Gregor, Thomas; Garcia, Hernan G.; Little, Shawn C.

    2014-01-01

    Transcriptional regulation of gene expression is fundamental to most cellular processes, including determination of cellular fates. Quantitative studies of transcription in cultured cells have led to significant advances in identifying mechanisms underlying transcriptional control. Recent progress allowed implementation of these same quantitative methods in multicellular organisms to ask how transcriptional regulation unfolds both in vivo and at the single molecule level in the context of embryonic development. Here we review some of these advances in early Drosophila development, which bring the embryo on par with its single-celled counterparts. In particular, we discuss progress in methods to measure mRNA and protein distributions in fixed and living embryos, and we highlight some initial applications that lead to fundamental new insights about molecular transcription processes. We end with an outlook on how to further exploit the unique advantages that come with investigating transcriptional control in the developmental context of the embryo. PMID:25005921

  5. Embryo-scale tissue mechanics during Drosophila gastrulation movements

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    Rauzi, Matteo; Krzic, Uros; Saunders, Timothy E.; Krajnc, Matej; Ziherl, Primož; Hufnagel, Lars; Leptin, Maria

    2015-01-01

    Morphogenesis of an organism requires the development of its parts to be coordinated in time and space. While past studies concentrated on defined cell populations, a synthetic view of the coordination of these events in a whole organism is needed for a full understanding. Drosophila gastrulation begins with the embryo forming a ventral furrow, which is eventually internalized. It is not understood how the rest of the embryo participates in this process. Here we use multiview selective plane illumination microscopy coupled with infrared laser manipulation and mutant analysis to dissect embryo-scale cell interactions during early gastrulation. Lateral cells have a denser medial–apical actomyosin network and shift ventrally as a compact cohort, whereas dorsal cells become stretched. We show that the behaviour of these cells affects furrow internalization. A computational model predicts different mechanical properties associated with tissue behaviour: lateral cells are stiff, whereas dorsal cells are soft. Experimental analysis confirms these properties in vivo. PMID:26497898

  6. Chromatin proteins and RNA are associated with DNA during all phases of mitosis

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    L Black, Kathryn; Petruk, Svetlana; Fenstermaker, Tyler K.; Hodgson, Jacob W.; Caplan, Jeffrey L.; Brock, Hugh W; Mazo, Alexander

    2016-01-01

    Mitosis brings about major changes to chromosome and nuclear structure. We used recently developed proximity ligation assay-based techniques to investigate the association with DNA of chromatin-associated proteins and RNAs in Drosophila embryos during mitosis. All groups of tested proteins, histone-modifying and chromatin-remodeling proteins and methylated histones remained in close proximity to DNA during all phases of mitosis. We also found that RNA transcripts are associated with DNA durin...

  7. Multiplex detection of RNA expression in Drosophila embryos.

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    Kosman, Dave; Mizutani, Claudia M; Lemons, Derek; Cox, W Gregory; McGinnis, William; Bier, Ethan

    2004-08-06

    We present a fluorescence-based, multiplex in situ hybridization method that permits the simultaneous detection of five differently labeled antisense RNA probes and up to seven differ-ent transcripts in a single Drosophila embryo. We also show that it should be possible to increase the number of detected transcripts substantially with nascent transcript multiplex fluorescent in situ hybridization. These multiplex methods fill a current technological gap between high-resolution in situ hybridization with one or two fluorescently labeled probes and low-resolution but genome-wide microarray RNA profiling and should be of great utility in establishing gene networks.

  8. Tracing myoblast fusion in Drosophila embryos by fluorescent actin probes.

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    Haralalka, Shruti; Abmayr, Susan M

    2015-01-01

    Myoblast fusion in the Drosophila embryo is a highly elaborate process that is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs). It occurs through an asymmetric event in which actin foci assemble in the FCMs at points of cell-cell contact and direct the formation of membrane protrusions that drive fusion. Herein, we describe the approach that we have used to image in living embryos the highly dynamic actin foci and actin-rich projections that precede myoblast fusion. We discuss resources currently available for imaging actin and myogenesis, and our experience with these resources if available. This technical report is not intended to be comprehensive on providing instruction on standard microscopy practices or software utilization. However, we discuss microscope parameters that we have used in data collection, and our experience with image processing tools in data analysis.

  9. Optical Tweezing Nuclei in the Cellular Blastoderm of Drosophila Embryos

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    Schoetz, Eva-Maria; Chaikin, Paul M.; Wieschaus, Eric F.

    2004-03-01

    Optical tweezers are used to manipulate nuclei in the syncytial blastoderm of Drosophila embryos. Our aim is to move a nucleus in a living embryo and study the reactions of its nearest neighbors to this displacement. Effects on the surrounding nuclei may allow us to test models in which actin-microtubule networks connect individual nuclei and keep them in place. In our experiments we use video analysis to follow individual nuclei using GFP-labeled histone protein. In a first approach, we were able to move nuclei in embryonic homogenates suspended in oil. Although the squashing destroys the cell, mitotic nuclear divisions continue, implying that the cytoskeleton, which connects the nuclei to the cortex, is still functioning. We will present studies of nuclear interactions in these squashes and in intact syncytial blastoderms.

  10. Kebab: kinetochore and EB1 associated basic protein that dynamically changes its localisation during Drosophila mitosis.

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    Meireles, Ana M; Dzhindzhev, Nikola S; Ohkura, Hiroyuki

    2011-01-01

    Microtubule plus ends are dynamic ends that interact with other cellular structures. Microtubule plus end tracking proteins are considered to play important roles in the regulation of microtubule plus ends. Recent studies revealed that EB1 is the central regulator for microtubule plus end tracking proteins by recruiting them to microtubule plus ends through direct interaction. Here we report the identification of a novel Drosophila protein, which we call Kebab (kinetochore and EB1 associated basic protein), through in vitro expression screening for EB1-interacting proteins. Kebab fused to GFP shows a novel pattern of dynamic localisation in mitosis. It localises to kinetochores weakly in metaphase and accumulates progressively during anaphase. In telophase, it associates with microtubules in central-spindle and centrosomal regions. The localisation to kinetochores depends on microtubules. The protein has a domain most similar to the atypical CH domain of Ndc80, and a coiled-coil domain. The interaction with EB1 is mediated by two SxIP motifs but is not required for the localisation. Depletion of Kebab in cultured cells by RNA interference did not show obvious defects in mitotic progression or microtubule organisation. Generation of mutants lacking the kebab gene indicated that Kebab is dispensable for viability and fertility.

  11. Kebab: kinetochore and EB1 associated basic protein that dynamically changes its localisation during Drosophila mitosis.

    Directory of Open Access Journals (Sweden)

    Ana M Meireles

    Full Text Available Microtubule plus ends are dynamic ends that interact with other cellular structures. Microtubule plus end tracking proteins are considered to play important roles in the regulation of microtubule plus ends. Recent studies revealed that EB1 is the central regulator for microtubule plus end tracking proteins by recruiting them to microtubule plus ends through direct interaction. Here we report the identification of a novel Drosophila protein, which we call Kebab (kinetochore and EB1 associated basic protein, through in vitro expression screening for EB1-interacting proteins. Kebab fused to GFP shows a novel pattern of dynamic localisation in mitosis. It localises to kinetochores weakly in metaphase and accumulates progressively during anaphase. In telophase, it associates with microtubules in central-spindle and centrosomal regions. The localisation to kinetochores depends on microtubules. The protein has a domain most similar to the atypical CH domain of Ndc80, and a coiled-coil domain. The interaction with EB1 is mediated by two SxIP motifs but is not required for the localisation. Depletion of Kebab in cultured cells by RNA interference did not show obvious defects in mitotic progression or microtubule organisation. Generation of mutants lacking the kebab gene indicated that Kebab is dispensable for viability and fertility.

  12. Function and dynamics of slam in furrow formation in early Drosophila embryo.

    Science.gov (United States)

    Acharya, Sreemukta; Laupsien, Philip; Wenzl, Christian; Yan, Shuling; Großhans, Jörg

    2014-02-15

    The Drosophila embryo undergoes a developmental transition in the blastoderm stage switching from syncytial to cellular development. The cleavage furrow, which encloses nuclei into cells, is a prominent morphological feature of this transition. It is not clear how the pattern of the furrow array is defined and how zygotic genes trigger the formation and invagination of interphase furrows. A key to these questions is provided by the gene slam, which has been previously implicated in controlling furrow invagination. Here we investigate the null phenotype of slam, the dynamics of Slam protein, and its control by the recycling endosome. We find that slam is essential for furrow invagination during cellularisation and together with nullo, for specification of the furrow. During cellularisation, Slam marks first the furrow, which is derived from the metaphase furrow of the previous mitosis. Slightly later, Slam accumulates at new furrows between daughter cells early in interphase. Slam is stably associated with the furrow canal except for the onset of cellularisation as revealed by FRAP experiments. Restriction of Slam to the furrow canal and Slam mobility during cellularisation is controlled by the recycling endosome and centrosomes. We propose a three step model. The retracting metaphase furrow leaves an initial mark. This mark and the border between corresponding daughter nuclei are refined by vesicular transport away from pericentrosomal recycling endosome towards the margins of the somatic buds. Following the onset of zygotic gene expression, Slam and Nullo together stabilise this mark and Slam triggers invagination of the cleavage furrow.

  13. Procedure for the permeabilization and cryobiological preservation of Drosophila embryos

    Energy Technology Data Exchange (ETDEWEB)

    Cole, K.W. [Oak Ridge National Lab., TN (United States). Biology Div.; Schreuders, P.D. [Univ. of Tennessee-Oak Ridge Graduate School of Biomedical Science, TN (United States); Mahowald, A.P. [Univ. of Chicago, IL (United States). Dept. of Molecular Genetics and Cell Biology; Mazur, P. [Oak Ridge National Lab., TN (United States). Biology Div.]|[Univ. of Tennessee-Oak Ridge Graduate School of Biomedical Science, TN (United States)

    1993-05-06

    The authors describe the detailed protocol developed in their laboratory at Oak Ridge for the permeabilization and cryobiological preservation of embryos of Drosophila melanogaster, Oregon R strain. The protocol is supplemented by notes containing two sorts of information. One category includes references to the appropriate portions of their published papers giving the scientific rationale and experimental basis for important steps. The other category is concerned with the criticality of certain steps and the precision with which they need to be performed. As an aid to investigators, the authors list even ordinary pieces of equipment. Brand names and model numbers are given where it is either important or convenient for readers to know precisely what is used.

  14. Shadow enhancers enable Hunchback bifunctionality in the Drosophila embryo.

    Science.gov (United States)

    Staller, Max V; Vincent, Ben J; Bragdon, Meghan D J; Lydiard-Martin, Tara; Wunderlich, Zeba; Estrada, Javier; DePace, Angela H

    2015-01-20

    Hunchback (Hb) is a bifunctional transcription factor that activates and represses distinct enhancers. Here, we investigate the hypothesis that Hb can activate and repress the same enhancer. Computational models predicted that Hb bifunctionally regulates the even-skipped (eve) stripe 3+7 enhancer (eve3+7) in Drosophila blastoderm embryos. We measured and modeled eve expression at cellular resolution under multiple genetic perturbations and found that the eve3+7 enhancer could not explain endogenous eve stripe 7 behavior. Instead, we found that eve stripe 7 is controlled by two enhancers: the canonical eve3+7 and a sequence encompassing the minimal eve stripe 2 enhancer (eve2+7). Hb bifunctionally regulates eve stripe 7, but it executes these two activities on different pieces of regulatory DNA--it activates the eve2+7 enhancer and represses the eve3+7 enhancer. These two "shadow enhancers" use different regulatory logic to create the same pattern.

  15. PS2 integrin requirements in Drosophila embryo and wing morphogenesis.

    Science.gov (United States)

    Brabant, M C; Brower, D L

    1993-05-01

    The Drosophila inflated (if) gene encodes the alpha PS2 subunit of the PS integrins. We describe the generation of new if mutations, their lethal embryonic phenotype, and experiments that examine the spatial and temporal requirements for integrins in adult wing morphogenesis. Embryos hemizygous for either new allele, ifA7 or ifB2, make reduced amounts of alpha PS2. In a variety of genetic tests, these alleles behave similarly to ifk27e, which makes no detectable alpha PS2, and all three alleles display the same embryonic phenotype. We therefore conclude that all of the lethal alleles retain little or no wild-type alpha PS2 function. As seen for strong mutations at the myospheroid (mys) locus, which encodes the beta PS integrin subunit, if mutants show extreme defects in somatic muscle attachments and in midgut morphogenesis. Unlike mys, however, there is no dorsal herniation of the if mutant embryos. With respect to wing morphogenesis, clonal analysis experiments demonstrate that if+ function is required only in cells of the ventral wing surface. We have rescued the wing blister phenotype of double mutants for the hypomorphic mysnj42 and if3 alleles using a heat shock-inducible mys+ transgene. By varying times of transgene induction, we find that integrin function is required from very early in metamorphosis until at least the last 24-48 hr of wing development.

  16. 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.

  17. Ndae1 expression and regulation in Drosophila embryos.

    Directory of Open Access Journals (Sweden)

    Maria Florencia Tevy

    Full Text Available The construction and prediction of cell fate maps at the whole embryo level require the establishment of an accurate atlas of gene expression patterns throughout development and the identification of the corresponding cis-regulatory sequences. However, while the expression and regulation of genes encoding upstream developmental regulators such as transcription factors or signaling pathway components have been analyzed in detail, up to date the number of cis-regulatory sequences identified for downstream effector genes, like ion channels, pumps and exchangers, is very low. The control and regulation of ion homeostasis in each cell, including at blastoderm stages, are essential for normal embryonic development. In this study, we analyzed in detail the embryonic expression pattern and cis-regulatory modules of the Drosophila Na+-driven anion exchanger 1 (Ndae1 gene, involved in the regulation of pH homeostasis. We show that Ndae1 is expressed in a tight and complex spatial-temporal pattern. In particular, we report that this downstream effector gene is under the control of the canonical dorsal-ventral patterning cascade through dorsal, Toll, twist and snail at early embryogenesis. Moreover, we identify several cis-regulatory modules, some of which control discrete and non-overlapping aspects of endogenous gene expression throughout development.

  18. Ena drives invasive macrophage migration in Drosophila embryos.

    Science.gov (United States)

    Tucker, Philippa K; Evans, Iwan R; Wood, Will

    2011-01-01

    It is seldom the primary tumour that proves fatal in cancer, with metastasis the fundamental pathological process for disease progression. Upregulation of Mena, a member of the evolutionarily conserved Ena/VASP family of actin cytoskeletal regulators, promotes metastasis and invasive motility of breast cancer cells in vivo. To complement in vitro studies of Ena/VASP function in fibroblasts, we manipulated levels of Ena, the Drosophila homologue of Mena, in migrating embryonic macrophages (haemocytes). Consistent with data from fibroblasts in vitro, Ena localises to regions of actin dynamics within migrating haemocytes, stimulates lamellipodial dynamics and positively regulates the number and length of filopodia. However, whereas Ena overexpression in fibroblasts reduces migration speeds, overexpressing Ena in haemocytes leads to a dramatic increase in migration speeds, more closely resembling the increased motility of breast cancer cells that overexpress Mena. We provide evidence that this key difference is due to spatial constraints imposed on cells within the three-dimensional environment of the embryo; this might explain how Mena can be used to promote aggressive migratory behaviour during cancer progression.

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

    Science.gov (United States)

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

    1985-01-01

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

  20. Mitosis-associated repression in development.

    Science.gov (United States)

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

    2016-07-01

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

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

    NARCIS (Netherlands)

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

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

  2. 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

  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. The Biochemistry of Mitosis

    Science.gov (United States)

    Wieser, Samuel; Pines, Jonathon

    2015-01-01

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

  5. Stable, precise, and reproducible patterning of bicoid and hunchback molecules in the early Drosophila embryo.

    Directory of Open Access Journals (Sweden)

    Yurie Okabe-Oho

    2009-08-01

    Full Text Available Precise patterning of morphogen molecules and their accurate reading out are of key importance in embryonic development. Recent experiments have visualized distributions of proteins in developing embryos and shown that the gradient of concentration of Bicoid morphogen in Drosophila embryos is established rapidly after fertilization and remains stable through syncytial mitoses. This stable Bicoid gradient is read out in a precise way to distribute Hunchback with small fluctuations in each embryo and in a reproducible way, with small embryo-to-embryo fluctuation. The mechanisms of such stable, precise, and reproducible patterning through noisy cellular processes, however, still remain mysterious. To address these issues, here we develop the one- and three-dimensional stochastic models of the early Drosophila embryo. The simulated results show that the fluctuation in expression of the hunchback gene is dominated by the random arrival of Bicoid at the hunchback enhancer. Slow diffusion of Hunchback protein, however, averages out this intense fluctuation, leading to the precise patterning of distribution of Hunchback without loss of sharpness of the boundary of its distribution. The coordinated rates of diffusion and transport of input Bicoid and output Hunchback play decisive roles in suppressing fluctuations arising from the dynamical structure change in embryos and those arising from the random diffusion of molecules, and give rise to the stable, precise, and reproducible patterning of Bicoid and Hunchback distributions.

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

    Science.gov (United States)

    Mills, C L; Bellairs, R

    1989-01-01

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

  7. Dynamin regulates metaphase furrow formation and plasma membrane compartmentalization in the syncytial Drosophila embryo

    Directory of Open Access Journals (Sweden)

    Richa Rikhy

    2015-02-01

    Full Text Available The successive nuclear division cycles in the syncytial Drosophila embryo are accompanied by ingression and regression of plasma membrane furrows, which surround individual nuclei at the embryo periphery, playing a central role in embryo compartmentalization prior to cellularization. Here, we demonstrate that cell cycle changes in dynamin localization and activity at the plasma membrane (PM regulate metaphase furrow formation and PM organization in the syncytial embryo. Dynamin was localized on short PM furrows during interphase, mediating endocytosis of PM components. Dynamin redistributed off ingressed PM furrows in metaphase, correlating with stabilized PM components and the associated actin regulatory machinery on long furrows. Acute inhibition of dynamin in the temperature sensitive shibire mutant embryo resulted in morphogenetic consequences in the syncytial division cycle. These included inhibition of metaphase furrow ingression, randomization of proteins normally polarized to intercap PM and disruption of the diffusion barrier separating PM domains above nuclei. Based on these findings, we propose that cell cycle changes in dynamin orchestrate recruitment of actin regulatory machinery for PM furrow dynamics during the early mitotic cycles in the Drosophila embryo.

  8. Dynamin regulates metaphase furrow formation and plasma membrane compartmentalization in the syncytial Drosophila embryo

    Science.gov (United States)

    Rikhy, Richa; Mavrakis, Manos; Lippincott-Schwartz, Jennifer

    2015-01-01

    ABSTRACT The successive nuclear division cycles in the syncytial Drosophila embryo are accompanied by ingression and regression of plasma membrane furrows, which surround individual nuclei at the embryo periphery, playing a central role in embryo compartmentalization prior to cellularization. Here, we demonstrate that cell cycle changes in dynamin localization and activity at the plasma membrane (PM) regulate metaphase furrow formation and PM organization in the syncytial embryo. Dynamin was localized on short PM furrows during interphase, mediating endocytosis of PM components. Dynamin redistributed off ingressed PM furrows in metaphase, correlating with stabilized PM components and the associated actin regulatory machinery on long furrows. Acute inhibition of dynamin in the temperature sensitive shibire mutant embryo resulted in morphogenetic consequences in the syncytial division cycle. These included inhibition of metaphase furrow ingression, randomization of proteins normally polarized to intercap PM and disruption of the diffusion barrier separating PM domains above nuclei. Based on these findings, we propose that cell cycle changes in dynamin orchestrate recruitment of actin regulatory machinery for PM furrow dynamics during the early mitotic cycles in the Drosophila embryo. PMID:25661871

  9. Chromator is required for proper microtubule spindle formation and mitosis in Drosophila.

    Science.gov (United States)

    Ding, Yun; Yao, Changfu; Lince-Faria, Mariana; Rath, Uttama; Cai, Weili; Maiato, Helder; Girton, Jack; Johansen, Kristen M; Johansen, Jørgen

    2009-10-01

    The chromodomain protein, Chromator, has been shown to have multiple functions that include regulation of chromatin structure as well as coordination of muscle remodeling during metamorphosis depending on the developmental context. In this study we show that mitotic neuroblasts from brain squash preparations from larvae heteroallelic for the two Chromator loss-of-function alleles Chro(71) and Chro(612) have severe microtubule spindle and chromosome segregation defects that were associated with a reduction in brain size. The microtubule spindles formed were incomplete, unfocused, and/or without clear spindle poles and at anaphase chromosomes were lagging and scattered. Time-lapse analysis of mitosis in S2 cells depleted of Chromator by RNAi treatment suggested that the lagging and scattered chromosome phenotypes were caused by incomplete alignment of chromosomes at the metaphase plate, possibly due to a defective spindle-assembly checkpoint, as well as of frayed and unstable microtubule spindles during anaphase. Expression of full-length Chromator transgenes under endogenous promoter control restored both microtubule spindle morphology as well as brain size strongly indicating that the observed mutant defects were directly attributable to lack of Chromator function.

  10. The methyltransferase Setdb1 is essential for meiosis and mitosis in mouse oocytes and early embryos.

    Science.gov (United States)

    Eymery, Angeline; Liu, Zichuan; Ozonov, Evgeniy A; Stadler, Michael B; Peters, Antoine H F M

    2016-08-01

    Oocytes develop the competence for meiosis and early embryogenesis during their growth. Setdb1 is a histone H3 lysine 9 (H3K9) methyltransferase required for post-implantation development and has been implicated in the transcriptional silencing of genes and endogenous retroviral elements (ERVs). To address its role in oogenesis and pre-implantation development, we conditionally deleted Setdb1 in growing oocytes. Loss of Setdb1 expression greatly impaired meiosis. It delayed meiotic resumption, altered the dynamics of chromatin condensation, and impaired kinetochore-spindle interactions, bipolar spindle organization and chromosome segregation in more mature oocytes. The observed phenotypes related to changes in abundance of specific transcripts in mutant oocytes. Setdb1 maternally deficient embryos arrested during pre-implantation development and showed comparable defects during cell cycle progression and in chromosome segregation. Finally, transcriptional profiling data indicate that Setdb1 downregulates rather than silences expression of ERVK and ERVL-MaLR retrotransposons and associated chimearic transcripts during oogenesis. Our results identify Setdb1 as a newly discovered meiotic and embryonic competence factor safeguarding genome integrity at the onset of life. © 2016. Published by The Company of Biologists Ltd.

  11. Roles for two partially redundant alpha-tubulins during mitosis in early Caenorhabditis elegans embryos.

    Science.gov (United States)

    Phillips, Jennifer B; Lyczak, Rebecca; Ellis, Gregory C; Bowerman, Bruce

    2004-06-01

    The Caenorhabditis elegans genome encodes multiple isotypes of alpha-tubulin and beta-tubulin. Roles for a number of these tubulins in neuronal development have been described, but less is known about the isoforms that function during early embryonic development. Microtubules are required for multiple events after fertilization produces a one-cell zygote in C. elegans, including pronuclear migration, mitotic spindle assembly and function, and proper spindle positioning. Here we describe a conditional and dominant mis-sense mutation in the C. elegans alpha-tubulin gene tba-1 that disrupts pronuclear migration and positioning of the first mitotic spindle, and results in a highly penetrant embryonic lethality, at the restrictive temperature of 26 degrees C. Our analysis of the dominant tba-1 (or346ts) allele suggests that TBA-1 assembles into microtubules in early embryonic cells. However, we also show that reduction of tba-1 function using RNA interference results in defects much less severe than those caused by the dominant or346ts mutation, due to partial redundancy of TBA-1 and another alpha-tubulin called TBA-2. Reducing the function of both TBA-1 and TBA-2 results in severe defects in microtubule-dependent processes. We conclude that microtubules in the early C. elegans embryo are composed of both TBA-1 and TBA-2, and that the dominant tba-1(or346ts) mutation disrupts MT assembly or stability. Cell Motil.

  12. Fluorescently labeled inhibitors detect localized serine protease activities in Drosophila melanogaster pole cells, embryos, and ovarian egg chambers

    DEFF Research Database (Denmark)

    Jakobsen, Rasmus Kragh; Ono, S.; Powers, J. C.

    2005-01-01

    processes that they mediate. Until only recently, the tools to conveniently address the question of where and when serine proteases are active within complex tissues have been lacking. In order to detect spatially restricted serine protease activities in Drosophila embryos and ovaries we introduce...... activity localized to the oocyte-somatic follicle cell interface of the developing egg chamber. Our results suggest that this technique holds promise to identify new spatially restricted activities in adult Drosophila tissues and developing embryos....

  13. The endo-siRNA pathway is essential for robust development of the Drosophila embryo.

    Directory of Open Access Journals (Sweden)

    Elena M Lucchetta

    Full Text Available BACKGROUND: Robustness to natural temperature fluctuations is critical to proper development in embryos and to cellular functions in adult organisms. However, mechanisms and pathways which govern temperature compensation remain largely unknown beyond circadian rhythms. Pathways which ensure robustness against temperature fluctuations may appear to be nonessential under favorable, uniform environmental conditions used in conventional laboratory experiments where there is little variation for which to compensate. The endo-siRNA pathway, which produces small double-stranded RNAs in Drosophila, appears to be nonessential for robust development of the embryo under ambient uniform temperature and to be necessary only for viral defense. Embryos lacking a functional endo-siRNA pathway develop into phenotypically normal adults. However, we hypothesized that small RNAs may regulate the embryo's response to temperature, as a ribonucleoprotein complex has been previously shown to mediate mammalian cell response to heat shock. PRINCIPAL FINDINGS: Here, we show that the genes DICER-2 and ARGONAUTE2, which code for integral protein components of the endo-siRNA pathway, are essential for robust development and temperature compensation in the Drosophila embryo when exposed to temperature perturbations. The regulatory functions of DICER-2 and ARGONAUTE2 were uncovered by using microfluidics to expose developing Drosophila embryos to a temperature step, in which each half of the embryo develops at a different temperature through developmental cycle 14. Under this temperature perturbation, dicer-2 or argonaute2 embryos displayed abnormal segmentation. The abnormalities in segmentation are presumably due to the inability of the embryo to compensate for temperature-induced differences in rate of development and to coordinate developmental timing in the anterior and posterior halves. A deregulation of the length of nuclear division cycles 10-14 is also observed in

  14. On the mechanics of cardiac function of Drosophila embryo.

    Science.gov (United States)

    Wu, Mingming; Sato, Thomas N

    2008-01-01

    The heart is a vital organ that provides essential circulation throughout the body. Malfunction of cardiac pumping, thus, leads to serious and most of the times, to fatal diseases. Mechanics of cardiac pumping is a complex process, and many experimental and theoretical approaches have been undertaken to understand this process. We have taken advantage of the simplicity of the embryonic heart of an invertebrate, Drosophila melanogaster, to understand the fundamental mechanics of the beating heart. We applied a live imaging technique to the beating embryonic heart combined with analytical imaging tools to study the dynamic mechanics of the pumping. Furthermore, we have identified one mutant line that exhibits aberrant pumping mechanics. The Drosophila embryonic heart consists of only 104 cardiac cells forming a simple straight tube that can be easily accessed for real-time imaging. Therefore, combined with the wealth of available genetic tools, the embryonic Drosophila heart may serve as a powerful model system for studies of human heart diseases, such as arrhythmia and congenital heart diseases. We, furthermore, believe our mechanistic data provides important information that is useful for our further understanding of the design of biological structure and function and for engineering the pumps for medical uses.

  15. High resolution mapping of Twist to DNA in Drosophila embryos: Efficient functional analysis and evolutionary conservation

    OpenAIRE

    Ozdemir, Anil; Fisher-Aylor, Katherine I.; Pepke, Shirley; Samanta, Manoj; Dunipace, Leslie; McCue, Kenneth; Zeng, Lucy; Ogawa, Nobuo; Wold, Barbara J; Stathopoulos, Angelike

    2011-01-01

    Cis-regulatory modules (CRMs) function by binding sequence specific transcription factors, but the relationship between in vivo physical binding and the regulatory capacity of factor-bound DNA elements remains uncertain. We investigate this relationship for the well-studied Twist factor in Drosophila melanogaster embryos by analyzing genome-wide factor occupancy and testing the functional significance of Twist occupied regions and motifs within regions. Twist ChIP-seq data efficiently identif...

  16. Determination of gene expression patterns using high-throughput RNA in situ hybridizaion to whole-mount Drosophila embryos

    Energy Technology Data Exchange (ETDEWEB)

    Weiszmann, R.; Hammonds, A.S.; Celniker, S.E.

    2009-04-09

    We describe a high-throughput protocol for RNA in situ hybridization (ISH) to Drosophila embryos in a 96-well format. cDNA or genomic DNA templates are amplified by PCR and then digoxigenin-labeled ribonucleotides are incorporated into antisense RNA probes by in vitro transcription. The quality of each probe is evaluated before ISH using a RNA probe quantification (dot blot) assay. RNA probes are hybridized to fixed, mixed-staged Drosophila embryos in 96-well plates. The resulting stained embryos can be examined and photographed immediately or stored at 4oC for later analysis. Starting with fixed, staged embryos, the protocol takes 6 d from probe template production through hybridization. Preparation of fixed embryos requires a minimum of 2 weeks to collect embryos representing all stages. The method has been used to determine the expression patterns of over 6,000 genes throughout embryogenesis.

  17. Stochastic model for gene transcription on Drosophila melanogaster embryos

    Science.gov (United States)

    Prata, Guilherme N.; Hornos, José Eduardo M.; Ramos, Alexandre F.

    2016-02-01

    We examine immunostaining experimental data for the formation of stripe 2 of even-skipped (eve) transcripts on D. melanogaster embryos. An estimate of the factor converting immunofluorescence intensity units into molecular numbers is given. The analysis of the eve dynamics at the region of stripe 2 suggests that the promoter site of the gene has two distinct regimes: an earlier phase when it is predominantly activated until a critical time when it becomes mainly repressed. That suggests proposing a stochastic binary model for gene transcription on D. melanogaster embryos. Our model has two random variables: the transcripts number and the state of the source of mRNAs given as active or repressed. We are able to reproduce available experimental data for the average number of transcripts. An analysis of the random fluctuations on the number of eves and their consequences on the spatial precision of stripe 2 is presented. We show that the position of the anterior or posterior borders fluctuate around their average position by ˜1 % of the embryo length, which is similar to what is found experimentally. The fitting of data by such a simple model suggests that it can be useful to understand the functions of randomness during developmental processes.

  18. Parallel imaging of Drosophila embryos for quantitative analysis of genetic perturbations of the Ras pathway

    Directory of Open Access Journals (Sweden)

    Yogesh Goyal

    2017-07-01

    Full Text Available The Ras pathway patterns the poles of the Drosophila embryo by downregulating the levels and activity of a DNA-binding transcriptional repressor Capicua (Cic. We demonstrate that the spatiotemporal pattern of Cic during this signaling event can be harnessed for functional studies of mutations in the Ras pathway in human diseases. Our approach relies on a new microfluidic device that enables parallel imaging of Cic dynamics in dozens of live embryos. We found that although the pattern of Cic in early embryos is complex, it can be accurately approximated by a product of one spatial profile and one time-dependent amplitude. Analysis of these functions of space and time alone reveals the differential effects of mutations within the Ras pathway. Given the highly conserved nature of Ras-dependent control of Cic, our approach provides new opportunities for functional analysis of multiple sequence variants from developmental abnormalities and cancers.

  19. An integrated platform for large-scale data collection and precise perturbation of live Drosophila embryos

    Science.gov (United States)

    Levario, Thomas J.; Zhao, Charles; Rouse, Tel; Shvartsman, Stanislav Y.; Lu, Hang

    2016-02-01

    Understanding the fundamental principles governing embryogenesis is a key goal of developmental biology. Direct observation of embryogenesis via in vivo live imaging is vital to understanding embryogenesis; yet, tedious sample preparation makes it difficult to acquire large-scale imaging data that is often required to overcome experimental and biological noises for quantitative studies. Furthermore, it is often difficult, and sometimes impossible, to incorporate environmental perturbation for understanding developmental responses to external stimuli. To address this issue, we have developed a method for high-throughput imaging of live embryos, delivering precise environmental perturbations, and unbiased data extraction. This platform includes an optimized microfluidic device specifically for live embryos and also for precise perturbations in the microenvironment of the developing embryos. In addition, we developed software for simple, yet accurate, automated segmentation of fluorescent images, and automated data extraction. Using a quantitative assessment we find that embryos develop normally within the microfluidic device. Finally, we show an application of the high-throughput assay for monitoring developmental responses to external stimuli: anoxia-induced developmental arrest in Drosophila embryos. With slight modifications, the method developed in this work can be applied to many other models of development and other stimulus-response behaviors during development.

  20. Drosophila embryos as model systems for monitoring bacterial infection in real time.

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    Isabella Vlisidou

    2009-07-01

    Full Text Available Drosophila embryos are well studied developmental microcosms that have been used extensively as models for early development and more recently wound repair. Here we extend this work by looking at embryos as model systems for following bacterial infection in real time. We examine the behaviour of injected pathogenic (Photorhabdus asymbiotica and non-pathogenic (Escherichia coli bacteria and their interaction with embryonic hemocytes using time-lapse confocal microscopy. We find that embryonic hemocytes both recognise and phagocytose injected wild type, non-pathogenic E. coli in a Dscam independent manner, proving that embryonic hemocytes are phagocytically competent. In contrast, injection of bacterial cells of the insect pathogen Photorhabdus leads to a rapid 'freezing' phenotype of the hemocytes associated with significant rearrangement of the actin cytoskeleton. This freezing phenotype can be phenocopied by either injection of the purified insecticidal toxin Makes Caterpillars Floppy 1 (Mcf1 or by recombinant E. coli expressing the mcf1 gene. Mcf1 mediated hemocyte freezing is shibire dependent, suggesting that endocytosis is required for Mcf1 toxicity and can be modulated by dominant negative or constitutively active Rac expression, suggesting early and unexpected effects of Mcf1 on the actin cytoskeleton. Together these data show how Drosophila embryos can be used to track bacterial infection in real time and how mutant analysis can be used to genetically dissect the effects of specific bacterial virulence factors.

  1. Mid-embryo patterning and precision in Drosophila segmentation: Kruppel dual regulation of hunchback.

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    David M Holloway

    Full Text Available In early development, genes are expressed in spatial patterns which later define cellular identities and tissue locations. The mechanisms of such pattern formation have been studied extensively in early Drosophila (fruit fly embryos. The gap gene hunchback (hb is one of the earliest genes to be expressed in anterior-posterior (AP body segmentation. As a transcriptional regulator for a number of downstream genes, the spatial precision of hb expression can have significant effects in the development of the body plan. To investigate the factors contributing to hb precision, we used fine spatial and temporal resolution data to develop a quantitative model for the regulation of hb expression in the mid-embryo. In particular, modelling hb pattern refinement in mid nuclear cleavage cycle 14 (NC14 reveals some of the regulatory contributions of simultaneously-expressed gap genes. Matching the model to recent data from wild-type (WT embryos and mutants of the gap gene Krüppel (Kr indicates that a mid-embryo Hb concentration peak important in thoracic development (at parasegment 4, PS4 is regulated in a dual manner by Kr, with low Kr concentration activating hb and high Kr concentration repressing hb. The processes of gene expression (transcription, translation, transport are intrinsically random. We used stochastic simulations to characterize the noise generated in hb expression. We find that Kr regulation can limit the positional variability of the Hb mid-embryo border. This has been recently corroborated in experimental comparisons of WT and Kr- mutant embryos. Further, Kr regulation can decrease uncertainty in mid-embryo hb expression (i.e. contribute to a smooth Hb boundary and decrease between-copy transcriptional variability within nuclei. Since many tissue boundaries are first established by interactions between neighbouring gene expression domains, these properties of Hb-Kr dynamics to diminish the effects of intrinsic expression noise may

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

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    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.

  3. Wnt, Hedgehog and junctional Armadillo/beta-catenin establish planar polarity in the Drosophila embryo.

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    Pamela F Colosimo

    Full Text Available To generate specialized structures, cells must obtain positional and directional information. In multi-cellular organisms, cells use the non-canonical Wnt or planar cell polarity (PCP signaling pathway to establish directionality within a cell. In vertebrates, several Wnt molecules have been proposed as permissible polarity signals, but none has been shown to provide a directional cue. While PCP signaling components are conserved from human to fly, no PCP ligands have been reported in Drosophila. Here we report that in the epidermis of the Drosophila embryo two signaling molecules, Hedgehog (Hh and Wingless (Wg or Wnt1, provide directional cues that induce the proper orientation of Actin-rich structures in the larval cuticle. We further find that proper polarity in the late embryo also involves the asymmetric distribution and phosphorylation of Armadillo (Arm or beta-catenin at the membrane and that interference with this Arm phosphorylation leads to polarity defects. Our results suggest new roles for Hh and Wg as instructive polarizing cues that help establish directionality within a cell sheet, and a new polarity-signaling role for the membrane fraction of the oncoprotein Arm.

  4. Regulation of the BMP Signaling-Responsive Transcriptional Network in the Drosophila Embryo.

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    Deignan, Lisa; Pinheiro, Marco T; Sutcliffe, Catherine; Saunders, Abbie; Wilcockson, Scott G; Zeef, Leo A H; Donaldson, Ian J; Ashe, Hilary L

    2016-07-01

    The BMP signaling pathway has a conserved role in dorsal-ventral axis patterning during embryonic development. In Drosophila, graded BMP signaling is transduced by the Mad transcription factor and opposed by the Brinker repressor. In this study, using the Drosophila embryo as a model, we combine RNA-seq with Mad and Brinker ChIP-seq to decipher the BMP-responsive transcriptional network underpinning differentiation of the dorsal ectoderm during dorsal-ventral axis patterning. We identify multiple new BMP target genes, including positive and negative regulators of EGF signaling. Manipulation of EGF signaling levels by loss- and gain-of-function studies reveals that EGF signaling negatively regulates embryonic BMP-responsive transcription. Therefore, the BMP gene network has a self-regulating property in that it establishes a balance between its activity and that of the antagonistic EGF signaling pathway to facilitate correct patterning. In terms of BMP-dependent transcription, we identify key roles for the Zelda and Zerknüllt transcription factors in establishing the resulting expression domain, and find widespread binding of insulator proteins to the Mad and Brinker-bound genomic regions. Analysis of embryos lacking the BEAF-32 insulator protein shows reduced transcription of a peak BMP target gene and a reduction in the number of amnioserosa cells, the fate specified by peak BMP signaling. We incorporate our findings into a model for Mad-dependent activation, and discuss its relevance to BMP signal interpretation in vertebrates.

  5. Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay

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    Merabet Samir

    2011-01-01

    Full Text Available Abstract Background Protein interactions control the regulatory networks underlying developmental processes. The understanding of developmental complexity will, therefore, require the characterization of protein interactions within their proper environment. The bimolecular fluorescence complementation (BiFC technology offers this possibility as it enables the direct visualization of protein interactions in living cells. However, its potential has rarely been applied in embryos of animal model organisms and was only performed under transient protein expression levels. Results Using a Hox protein partnership as a test case, we investigated the suitability of BiFC for the study of protein interactions in the living Drosophila embryo. Importantly, all BiFC parameters were established with constructs that were stably expressed under the control of endogenous promoters. Under these physiological conditions, we showed that BiFC is specific and sensitive enough to analyse dynamic protein interactions. We next used BiFC in a candidate interaction screen, which led to the identification of several Hox protein partners. Conclusion Our results establish the general suitability of BiFC for revealing and studying protein interactions in their physiological context during the rapid course of Drosophila embryonic development.

  6. Drosophila embryos as model to assess cellular and developmental toxicity of multi-walled carbon nanotubes (MWCNT in living organisms.

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    Boyin Liu

    Full Text Available Different toxicity tests for carbon nanotubes (CNT have been developed to assess their impact on human health and on aquatic and terrestrial animal and plant life. We present a new model, the fruit fly Drosophila embryo offering the opportunity for rapid, inexpensive and detailed analysis of CNTs toxicity during embryonic development. We show that injected DiI labelled multi-walled carbon nanotubes (MWCNTs become incorporated into cells in early Drosophila embryos, allowing the study of the consequences of cellular uptake of CNTs on cell communication, tissue and organ formation in living embryos. Fluorescently labelled subcellular structures showed that MWCNTs remained cytoplasmic and were excluded from the nucleus. Analysis of developing ectodermal and neural stem cells in MWCNTs injected embryos revealed normal division patterns and differentiation capacity. However, an increase in cell death of ectodermal but not of neural stem cells was observed, indicating stem cell-specific vulnerability to MWCNT exposure. The ease of CNT embryo injections, the possibility of detailed morphological and genomic analysis and the low costs make Drosophila embryos a system of choice to assess potential developmental and cellular effects of CNTs and test their use in future CNT based new therapies including drug delivery.

  7. Human BMP sequences can confer normal dorsal-ventral patterning in the Drosophila embryo.

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    Padgett, R W; Wozney, J M; Gelbart, W M

    1993-04-01

    The type beta transforming growth factor family is composed of a series of processed, secreted growth factors, several of which have been implicated in important regulatory roles in cell determination, inductive interactions, and tissue differentiation. Among these factors, the sequence of the DPP protein from Drosophila is most similar to two of the vertebrate bone morphogenetic proteins, BMP2 and BMP4. Here we report that the human BMP4 ligand sequences can function in lieu of DPP in Drosophila embryos. We introduced the ligand region from human BMP4 into a genomic fragment of the dpp gene in place of the Drosophila ligand sequences and recovered transgenic flies by P-element transformation. We find that this chimeric dpp-BMP4 transgene can completely rescue the embryonic dorsal-ventral patterning defect of null dpp mutant genotypes. We infer that the chimeric DPP-BMP4 protein can be processed properly and, by analogy with the action of other family members, can activate the endogenous DPP receptor to carry out the events necessary for dorsal-ventral patterning. Our evidence suggests that the DPP-BMP4 signal transduction pathway has been functionally conserved for at least 600 million years.

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

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    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.

  9. Medea SUMOylation restricts the signaling range of the Dpp morphogen in the Drosophila embryo.

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    Miles, Wayne O; Jaffray, Ellis; Campbell, Susan G; Takeda, Shugaku; Bayston, Laura J; Basu, Sanjay P; Li, Mingfa; Raftery, Laurel A; Ashe, Mark P; Hay, Ronald T; Ashe, Hilary L

    2008-09-15

    Morphogens are secreted signaling molecules that form concentration gradients and control cell fate in developing tissues. During development, it is essential that morphogen range is strictly regulated in order for correct cell type specification to occur. One of the best characterized morphogens is Drosophila Decapentaplegic (Dpp), a BMP signaling molecule that patterns the dorsal ectoderm of the embryo by activating the Mad and Medea (Med) transcription factors. We demonstrate that there is a spatial and temporal expansion of the expression patterns of Dpp target genes in SUMO pathway mutant embryos. We identify Med as the primary SUMOylation target in the Dpp pathway, and show that failure to SUMOylate Med leads to the increased Dpp signaling range observed in the SUMO pathway mutant embryos. Med is SUMO modified in the nucleus, and we provide evidence that SUMOylation triggers Med nuclear export. Hence, Med SUMOylation provides a mechanism by which nuclei can continue to monitor the presence of extracellular Dpp signal to activate target gene expression for an appropriate duration. Overall, our results identify an unusual strategy for regulating morphogen range that, rather than impacting on the morphogen itself, targets an intracellular transducer.

  10. 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

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    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.

  11. The Atg1-Tor pathway regulates yolk catabolism in Drosophila embryos.

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    Kuhn, Hallie; Sopko, Richelle; Coughlin, Margaret; Perrimon, Norbert; Mitchison, Tim

    2015-11-15

    Yolk provides an important source of nutrients during the early development of oviparous organisms. It is composed mainly of vitellogenin proteins packed into membrane-bound compartments called yolk platelets. Catabolism of yolk is initiated by acidification of the yolk platelet, leading to the activation of Cathepsin-like proteinases, but it is unknown how this process is triggered. Yolk catabolism initiates at cellularization in Drosophila melanogaster embryos. Using maternal shRNA technology we found that yolk catabolism depends on the Tor pathway and on the autophagy-initiating kinase Atg1. Whereas Atg1 was required for a burst of spatially regulated autophagy during late cellularization, autophagy was not required for initiating yolk catabolism. We propose that the conserved Tor metabolic sensing pathway regulates yolk catabolism, similar to Tor-dependent metabolic regulation on the lysosome.

  12. SWATH-MS dataset of heat-shock treated Drosophila melanogaster embryos.

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    Fabre, Bertrand; Korona, Dagmara; Nightingale, Daniel J H; Russell, Steven; Lilley, Kathryn S

    2016-12-01

    Data independent acquisition (DIA) has emerged as a promising mass spectrometry based approach, combining the advantages of shotgun and targeted proteomics. Here we applied a DIA approach (termed SWATH) to monitor the dynamics of the Drosophila melanogaster embryonic proteome upon heat-shock treatment. Embryos were incubated for 0.5, 1 or 3 h at 37 °C to induce heat-shock or maintained at 25 °C. The present dataset contains SWATH files acquired on a Sciex Triple-TOF 6600. A spectral library built in-house was used to analyse these data and led to the quantification of more than 2500 proteins at every timepoint. The files presented here are permanent digital maps and can be reanalysed to search for new questions. The data have been deposited with the ProteomeXchange Consortium with the dataset identifier PRIDE: PXD004753.

  13. SWATH-MS dataset of heat-shock treated Drosophila melanogaster embryos

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    Bertrand Fabre

    2016-12-01

    Full Text Available Data independent acquisition (DIA has emerged as a promising mass spectrometry based approach, combining the advantages of shotgun and targeted proteomics. Here we applied a DIA approach (termed SWATH to monitor the dynamics of the Drosophila melanogaster embryonic proteome upon heat-shock treatment. Embryos were incubated for 0.5, 1 or 3 h at 37 °C to induce heat-shock or maintained at 25 °C. The present dataset contains SWATH files acquired on a Sciex Triple-TOF 6600. A spectral library built in-house was used to analyse these data and led to the quantification of more than 2500 proteins at every timepoint. The files presented here are permanent digital maps and can be reanalysed to search for new questions. The data have been deposited with the ProteomeXchange Consortium with the dataset identifier PRIDE: PXD004753.

  14. Syndapin promotes pseudocleavage furrow formation by actin organization in the syncytial Drosophila embryo.

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    Sherlekar, Aparna; Rikhy, Richa

    2016-07-01

    Coordinated membrane and cytoskeletal remodeling activities are required for membrane extension in processes such as cytokinesis and syncytial nuclear division cycles in Drosophila Pseudocleavage furrow membranes in the syncytial Drosophila blastoderm embryo show rapid extension and retraction regulated by actin-remodeling proteins. The F-BAR domain protein Syndapin (Synd) is involved in membrane tubulation, endocytosis, and, uniquely, in F-actin stability. Here we report a role for Synd in actin-regulated pseudocleavage furrow formation. Synd localized to these furrows, and its loss resulted in short, disorganized furrows. Synd presence was important for the recruitment of the septin Peanut and distribution of Diaphanous and F-actin at furrows. Synd and Peanut were both absent in furrow-initiation mutants of RhoGEF2 and Diaphanous and in furrow-progression mutants of Anillin. Synd overexpression in rhogef2 mutants reversed its furrow-extension phenotypes, Peanut and Diaphanous recruitment, and F-actin organization. We conclude that Synd plays an important role in pseudocleavage furrow extension, and this role is also likely to be crucial in cleavage furrow formation during cell division.

  15. Overlapping functions of argonaute proteins in patterning and morphogenesis of Drosophila embryos.

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    Wibke J Meyer

    2006-08-01

    Full Text Available Argonaute proteins are essential components of the molecular machinery that drives RNA silencing. In Drosophila, different members of the Argonaute family of proteins have been assigned to distinct RNA silencing pathways. While Ago1 is required for microRNA function, Ago2 is a crucial component of the RNA-induced silencing complex in siRNA-triggered RNA interference. Drosophila Ago2 contains an unusual amino-terminus with two types of imperfect glutamine-rich repeats (GRRs of unknown function. Here we show that the GRRs of Ago2 are essential for the normal function of the protein. Alleles with reduced numbers of GRRs cause specific disruptions in two morphogenetic processes associated with the midblastula transition: membrane growth and microtubule-based organelle transport. These defects do not appear to result from disruption of siRNA-dependent processes but rather suggest an interference of the mutant Ago2 proteins in an Ago1-dependent pathway. Using loss-of-function alleles, we further demonstrate that Ago1 and Ago2 act in a partially redundant manner to control the expression of the segment-polarity gene wingless in the early embryo. Our findings argue against a strict separation of Ago1 and Ago2 functions and suggest that these proteins act in concert to control key steps of the midblastula transition and of segmental patterning.

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

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

    2012-01-01

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

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

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    Elaine M Dunleavy

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

  18. The Cell Cycle Timing of Centromeric Chromatin Assembly in Drosophila Meiosis Is Distinct from Mitosis Yet Requires CAL1 and CENP-C

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    Gorgescu, Walter; Tang, Jonathan; Costes, Sylvain V.; Karpen, Gary H.

    2012-01-01

    CENP-A (CID in flies) is the histone H3 variant essential for centromere specification, kinetochore formation, and chromosome segregation during cell division. Recent studies have elucidated major cell cycle mechanisms and factors critical for CENP-A incorporation in mitosis, predominantly in cultured cells. However, we do not understand the roles, regulation, and cell cycle timing of CENP-A assembly in somatic tissues in multicellular organisms and in meiosis, the specialized cell division cycle that gives rise to haploid gametes. Here we investigate the timing and requirements for CID assembly in mitotic tissues and male and female meiosis in Drosophila melanogaster, using fixed and live imaging combined with genetic approaches. We find that CID assembly initiates at late telophase and continues during G1 phase in somatic tissues in the organism, later than the metaphase assembly observed in cultured cells. Furthermore, CID assembly occurs at two distinct cell cycle phases during male meiosis: prophase of meiosis I and after exit from meiosis II, in spermatids. CID assembly in prophase I is also conserved in female meiosis. Interestingly, we observe a novel decrease in CID levels after the end of meiosis I and before meiosis II, which correlates temporally with changes in kinetochore organization and orientation. We also demonstrate that CID is retained on mature sperm despite the gross chromatin remodeling that occurs during protamine exchange. Finally, we show that the centromere proteins CAL1 and CENP-C are both required for CID assembly in meiosis and normal progression through spermatogenesis. We conclude that the cell cycle timing of CID assembly in meiosis is different from mitosis and that the efficient propagation of CID through meiotic divisions and on sperm is likely to be important for centromere specification in the developing zygote. PMID:23300382

  19. Early development of Drosophila embryos requires Smc5/6 function during oogenesis.

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    Tran, Martin; Tsarouhas, Vasilios; Kegel, Andreas

    2016-07-15

    Mutations in structural maintenance of chromosomes (Smc) proteins are frequently associated with chromosomal abnormalities commonly observed in developmental disorders. However, the role of Smc proteins in development still remains elusive. To investigate Smc5/6 function during early embryogenesis we examined smc5 and smc6 mutants of the fruit fly Drosophila melanogaster using a combination of reverse genetics and microscopy approaches. Smc5/6 exhibited a maternally contributed function in maintaining chromosome stability during early embryo development, which manifested as female subfertility in its absence. Loss of Smc5/6 caused an arrest and a considerable delay in embryo development accompanied by fragmented nuclei and increased anaphase-bridge formation, respectively. Surprisingly, early embryonic arrest was attributable to the absence of Smc5/6 during oogenesis, which resulted in insufficient repair of pre-meiotic and meiotic DNA double-strand breaks. Thus, our findings contribute to the understanding of Smc proteins in higher eukaryotic development by highlighting a maternal function in chromosome maintenance and a link between oogenesis and early embryogenesis.

  20. Early development of Drosophila embryos requires Smc5/6 function during oogenesis

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    Martin Tran

    2016-07-01

    Full Text Available Mutations in structural maintenance of chromosomes (Smc proteins are frequently associated with chromosomal abnormalities commonly observed in developmental disorders. However, the role of Smc proteins in development still remains elusive. To investigate Smc5/6 function during early embryogenesis we examined smc5 and smc6 mutants of the fruit fly Drosophila melanogaster using a combination of reverse genetics and microscopy approaches. Smc5/6 exhibited a maternally contributed function in maintaining chromosome stability during early embryo development, which manifested as female subfertility in its absence. Loss of Smc5/6 caused an arrest and a considerable delay in embryo development accompanied by fragmented nuclei and increased anaphase-bridge formation, respectively. Surprisingly, early embryonic arrest was attributable to the absence of Smc5/6 during oogenesis, which resulted in insufficient repair of pre-meiotic and meiotic DNA double-strand breaks. Thus, our findings contribute to the understanding of Smc proteins in higher eukaryotic development by highlighting a maternal function in chromosome maintenance and a link between oogenesis and early embryogenesis.

  1. Mitigating phototoxicity during multiphoton microscopy of live Drosophila embryos in the 1.0-1.2 µm wavelength range.

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    Delphine Débarre

    Full Text Available Light-induced toxicity is a fundamental bottleneck in microscopic imaging of live embryos. In this article, after a review of photodamage mechanisms in cells and tissues, we assess photo-perturbation under illumination conditions relevant for point-scanning multiphoton imaging of live Drosophila embryos. We use third-harmonic generation (THG imaging of developmental processes in embryos excited by pulsed near-infrared light in the 1.0-1.2 µm range. We study the influence of imaging rate, wavelength, and pulse duration on the short-term and long-term perturbation of development and define criteria for safe imaging. We show that under illumination conditions typical for multiphoton imaging, photodamage in this system arises through 2- and/or 3-photon absorption processes and in a cumulative manner. Based on this analysis, we derive general guidelines for improving the signal-to-damage ratio in two-photon (2PEF/SHG or THG imaging by adjusting the pulse duration and/or the imaging rate. Finally, we report label-free time-lapse 3D THG imaging of gastrulating Drosophila embryos with sampling appropriate for the visualisation of morphogenetic movements in wild-type and mutant embryos, and long-term multiharmonic (THG-SHG imaging of development until hatching.

  2. Preferential genome targeting of the CBP co-activator by Rel and Smad proteins in early Drosophila melanogaster embryos.

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    Per-Henrik Holmqvist

    Full Text Available CBP and the related p300 protein are widely used transcriptional co-activators in metazoans that interact with multiple transcription factors. Whether CBP/p300 occupies the genome equally with all factors or preferentially binds together with some factors is not known. We therefore compared Drosophila melanogaster CBP (nejire ChIP-seq peaks with regions bound by 40 different transcription factors in early embryos, and we found high co-occupancy with the Rel-family protein Dorsal. Dorsal is required for CBP occupancy in the embryo, but only at regions where few other factors are present. CBP peaks in mutant embryos lacking nuclear Dorsal are best correlated with TGF-ß/Dpp-signaling and Smad-protein binding. Differences in CBP occupancy in mutant embryos reflect gene expression changes genome-wide, but CBP also occupies some non-expressed genes. The presence of CBP at silent genes does not result in histone acetylation. We find that Polycomb-repressed H3K27me3 chromatin does not preclude CBP binding, but restricts histone acetylation at CBP-bound genomic sites. We conclude that CBP occupancy in Drosophila embryos preferentially overlaps factors controlling dorso-ventral patterning and that CBP binds silent genes without causing histone hyperacetylation.

  3. Preferential Genome Targeting of the CBP Co-Activator by Rel and Smad Proteins in Early Drosophila melanogaster Embryos

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    Holmqvist, Per-Henrik; Boija, Ann; Philip, Philge; Crona, Filip; Stenberg, Per; Mannervik, Mattias

    2012-01-01

    CBP and the related p300 protein are widely used transcriptional co-activators in metazoans that interact with multiple transcription factors. Whether CBP/p300 occupies the genome equally with all factors or preferentially binds together with some factors is not known. We therefore compared Drosophila melanogaster CBP (nejire) ChIP–seq peaks with regions bound by 40 different transcription factors in early embryos, and we found high co-occupancy with the Rel-family protein Dorsal. Dorsal is required for CBP occupancy in the embryo, but only at regions where few other factors are present. CBP peaks in mutant embryos lacking nuclear Dorsal are best correlated with TGF-ß/Dpp-signaling and Smad-protein binding. Differences in CBP occupancy in mutant embryos reflect gene expression changes genome-wide, but CBP also occupies some non-expressed genes. The presence of CBP at silent genes does not result in histone acetylation. We find that Polycomb-repressed H3K27me3 chromatin does not preclude CBP binding, but restricts histone acetylation at CBP-bound genomic sites. We conclude that CBP occupancy in Drosophila embryos preferentially overlaps factors controlling dorso-ventral patterning and that CBP binds silent genes without causing histone hyperacetylation. PMID:22737084

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

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    Robert Ruggiero

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

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

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    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.

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

    Science.gov (United States)

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

    2012-01-01

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

  7. CLOCK expression identifies developing circadian oscillator neurons in the brains of Drosophila embryos

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    Ng Fanny

    2008-12-01

    Full Text Available Abstract Background The Drosophila circadian oscillator is composed of transcriptional feedback loops in which CLOCK-CYCLE (CLK-CYC heterodimers activate their feedback regulators period (per and timeless (tim via E-box mediated transcription. These feedback loop oscillators are present in distinct clusters of dorsal and lateral neurons in the adult brain, but how this pattern of expression is established during development is not known. Since CLK is required to initiate feedback loop function, defining the pattern of CLK expression in embryos and larvae will shed light on oscillator neuron development. Results A novel CLK antiserum is used to show that CLK expression in the larval CNS and adult brain is limited to circadian oscillator cells. CLK is initially expressed in presumptive small ventral lateral neurons (s-LNvs, dorsal neurons 2 s (DN2s, and dorsal neuron 1 s (DN1s at embryonic stage (ES 16, and this CLK expression pattern persists through larval development. PER then accumulates in all CLK-expressing cells except presumptive DN2s during late ES 16 and ES 17, consistent with the delayed accumulation of PER in adult oscillator neurons and antiphase cycling of PER in larval DN2s. PER is also expressed in non-CLK-expressing cells in the embryonic CNS starting at ES 12. Although PER expression in CLK-negative cells continues in ClkJrk embryos, PER expression in cells that co-express PER and CLK is eliminated. Conclusion These data demonstrate that brain oscillator neurons begin development during embryogenesis, that PER expression in non-oscillator cells is CLK-independent, and that oscillator phase is an intrinsic characteristic of brain oscillator neurons. These results define the temporal and spatial coordinates of factors that initiate Clk expression, imply that circadian photoreceptors are not activated until the end of embryogenesis, and suggest that PER functions in a different capacity before oscillator cell development is

  8. 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…

  9. 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…

  10. 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…

  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. A gene expression atlas of a bicoid-depleted Drosophila embryo reveals early canalization of cell fate.

    Science.gov (United States)

    Staller, Max V; Fowlkes, Charless C; Bragdon, Meghan D J; Wunderlich, Zeba; Estrada, Javier; DePace, Angela H

    2015-02-01

    In developing embryos, gene regulatory networks drive cells towards discrete terminal fates, a process called canalization. We studied the behavior of the anterior-posterior segmentation network in Drosophila melanogaster embryos by depleting a key maternal input, bicoid (bcd), and measuring gene expression patterns of the network at cellular resolution. This method results in a gene expression atlas containing the levels of mRNA or protein expression of 13 core patterning genes over six time points for every cell of the blastoderm embryo. This is the first cellular resolution dataset of a genetically perturbed Drosophila embryo that captures all cells in 3D. We describe the technical developments required to build this atlas and how the method can be employed and extended by others. We also analyze this novel dataset to characterize the degree and timing of cell fate canalization in the segmentation network. We find that in two layers of this gene regulatory network, following depletion of bcd, individual cells rapidly canalize towards normal cell fates. This result supports the hypothesis that the segmentation network directly canalizes cell fate, rather than an alternative hypothesis whereby cells are initially mis-specified and later eliminated by apoptosis. Our gene expression atlas provides a high resolution picture of a classic perturbation and will enable further computational modeling of canalization and gene regulation in this transcriptional network. © 2015. Published by The Company of Biologists Ltd.

  13. Mitosis Methods & Protocols

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    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..... 

  14. A rapid, membrane-dependent pathway directs furrow formation through RalA in the early Drosophila embryo.

    Science.gov (United States)

    Holly, Ryan M; Mavor, Lauren M; Zuo, Zhongyuan; Blankenship, J Todd

    2015-07-01

    Plasma membrane furrow formation is crucial in cell division and cytokinesis. Furrow formation in early syncytial Drosophila embryos is exceptionally rapid, with furrows forming in as little as 3.75 min. Here, we use 4D imaging to identify furrow formation, stabilization, and regression periods, and identify a rapid, membrane-dependent pathway that is essential for plasma membrane furrow formation in vivo. Myosin II function is thought to provide the ingression force for cytokinetic furrows, but the role of membrane trafficking pathways in guiding furrow formation is less clear. We demonstrate that a membrane trafficking pathway centered on Ras-like protein A (RalA) is required for fast furrow ingression in the early fly embryo. RalA function is absolutely required for furrow formation and initiation. In the absence of RalA and furrow function, chromosomal segregation is aberrant and polyploid nuclei are observed. RalA localizes to syncytial furrows, and mediates the movement of exocytic vesicles to the plasma membrane. Sec5, which is an exocyst complex subunit and localizes to ingressing furrows in wild-type embryos, becomes punctate and loses its cortical association in the absence of RalA function. Rab8 also fails to traffic to the plasma membrane and accumulates aberrantly in the cytoplasm in RalA disrupted embryos. RalA localization precedes F-actin recruitment to the furrow tip, suggesting that membrane trafficking might function upstream of cytoskeletal remodeling. These studies identify a pathway, which stretches from Rab8 to RalA and the exocyst complex, that mediates rapid furrow formation in early Drosophila embryos.

  15. Signalling crosstalk at the leading edge controls tissue closure dynamics in the Drosophila embryo

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    Carballès, Fabrice; Parassol, Nadège; Schaub, Sébastien; Cérézo, Delphine; Noselli, Stéphane

    2017-01-01

    Tissue morphogenesis relies on proper differentiation of morphogenetic domains, adopting specific cell behaviours. Yet, how signalling pathways interact to determine and coordinate these domains remains poorly understood. Dorsal closure (DC) of the Drosophila embryo represents a powerful model to study epithelial cell sheet sealing. In this process, JNK (JUN N-terminal Kinase) signalling controls leading edge (LE) differentiation generating local forces and cell shape changes essential for DC. The LE represents a key morphogenetic domain in which, in addition to JNK, a number of signalling pathways converges and interacts (anterior/posterior -AP- determination; segmentation genes, such as Wnt/Wingless; TGFβ/Decapentaplegic). To better characterize properties of the LE morphogenetic domain, we sought out new JNK target genes through a genomic approach: 25 were identified of which 8 are specifically expressed in the LE, similarly to decapentaplegic or puckered. Quantitative in situ gene profiling of this new set of LE genes reveals complex patterning of the LE along the AP axis, involving a three-way interplay between the JNK pathway, segmentation and HOX genes. Patterning of the LE into discrete domains appears essential for coordination of tissue sealing dynamics. Loss of anterior or posterior HOX gene function leads to strongly delayed and asymmetric DC, due to incorrect zipping in their respective functional domain. Therefore, in addition to significantly increasing the number of JNK target genes identified so far, our results reveal that the LE is a highly heterogeneous morphogenetic organizer, sculpted through crosstalk between JNK, segmental and AP signalling. This fine-tuning regulatory mechanism is essential to coordinate morphogenesis and dynamics of tissue sealing. PMID:28231245

  16. Comparative gene expression analysis of Dtg, a novel target gene of Dpp signaling pathway in the early Drosophila melanogaster embryo.

    Science.gov (United States)

    Hodar, Christian; Zuñiga, Alejandro; Pulgar, Rodrigo; Travisany, Dante; Chacon, Carlos; Pino, Michael; Maass, Alejandro; Cambiazo, Verónica

    2014-02-10

    In the early Drosophila melanogaster embryo, Dpp, a secreted molecule that belongs to the TGF-β superfamily of growth factors, activates a set of downstream genes to subdivide the dorsal region into amnioserosa and dorsal epidermis. Here, we examined the expression pattern and transcriptional regulation of Dtg, a new target gene of Dpp signaling pathway that is required for proper amnioserosa differentiation. We showed that the expression of Dtg was controlled by Dpp and characterized a 524-bp enhancer that mediated expression in the dorsal midline, as well as, in the differentiated amnioserosa in transgenic reporter embryos. This enhancer contained a highly conserved region of 48-bp in which bioinformatic predictions and in vitro assays identified three Mad binding motifs. Mutational analysis revealed that these three motifs were necessary for proper expression of a reporter gene in transgenic embryos, suggesting that short and highly conserved genomic sequences may be indicative of functional regulatory regions in D. melanogaster genes. Dtg orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa, nevertheless Dtg orthologs were identified in the transcriptome of Musca domestica, in which dorsal ectoderm patterning leads to the formation of a single extra-embryonic membrane. These results suggest that Dtg was recruited as a new component of the network that controls dorsal ectoderm patterning in the lineage leading to higher Cyclorrhaphan flies, such as D. melanogaster and M. domestica. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. The presence of nuclear cactus in the early Drosophila embryo may extend the dynamic range of the dorsal gradient.

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    Michael D O'Connell

    2015-04-01

    Full Text Available In a developing embryo, the spatial distribution of a signaling molecule, or a morphogen gradient, has been hypothesized to carry positional information to pattern tissues. Recent measurements of morphogen distribution have allowed us to subject this hypothesis to rigorous physical testing. In the early Drosophila embryo, measurements of the morphogen Dorsal, which is a transcription factor responsible for initiating the earliest zygotic patterns along the dorsal-ventral axis, have revealed a gradient that is too narrow to pattern the entire axis. In this study, we use a mathematical model of Dorsal dynamics, fit to experimental data, to determine the ability of the Dorsal gradient to regulate gene expression across the entire dorsal-ventral axis. We found that two assumptions are required for the model to match experimental data in both Dorsal distribution and gene expression patterns. First, we assume that Cactus, an inhibitor that binds to Dorsal and prevents it from entering the nuclei, must itself be present in the nuclei. And second, we assume that fluorescence measurements of Dorsal reflect both free Dorsal and Cactus-bound Dorsal. Our model explains the dynamic behavior of the Dorsal gradient at lateral and dorsal positions of the embryo, the ability of Dorsal to regulate gene expression across the entire dorsal-ventral axis, and the robustness of gene expression to stochastic effects. Our results have a general implication for interpreting fluorescence-based measurements of signaling molecules.

  18. The Presence of Nuclear Cactus in the Early Drosophila Embryo May Extend the Dynamic Range of the Dorsal Gradient

    Science.gov (United States)

    O’Connell, Michael D.; Reeves, Gregory T.

    2015-01-01

    In a developing embryo, the spatial distribution of a signaling molecule, or a morphogen gradient, has been hypothesized to carry positional information to pattern tissues. Recent measurements of morphogen distribution have allowed us to subject this hypothesis to rigorous physical testing. In the early Drosophila embryo, measurements of the morphogen Dorsal, which is a transcription factor responsible for initiating the earliest zygotic patterns along the dorsal-ventral axis, have revealed a gradient that is too narrow to pattern the entire axis. In this study, we use a mathematical model of Dorsal dynamics, fit to experimental data, to determine the ability of the Dorsal gradient to regulate gene expression across the entire dorsal-ventral axis. We found that two assumptions are required for the model to match experimental data in both Dorsal distribution and gene expression patterns. First, we assume that Cactus, an inhibitor that binds to Dorsal and prevents it from entering the nuclei, must itself be present in the nuclei. And second, we assume that fluorescence measurements of Dorsal reflect both free Dorsal and Cactus-bound Dorsal. Our model explains the dynamic behavior of the Dorsal gradient at lateral and dorsal positions of the embryo, the ability of Dorsal to regulate gene expression across the entire dorsal-ventral axis, and the robustness of gene expression to stochastic effects. Our results have a general implication for interpreting fluorescence-based measurements of signaling molecules. PMID:25879657

  19. DNA damage leads to a Cyclin A-dependent delay in metaphase-anaphase transition in the Drosophila gastrula.

    Science.gov (United States)

    Su, T T; Jaklevic, B

    2001-01-09

    In response to DNA damage, fission yeast, mammalian cells, and cells of the Drosophila gastrula inhibit Cdk1 to delay the entry into mitosis. In contrast, budding yeast delays metaphase-anaphase transition by stabilization of an anaphase inhibitor, Pds1p. A variation of the second response is seen in Drosophila cleavage embryos; when nuclei enter mitosis with damaged DNA, centrosomes lose gamma-tubulin, spindles lose astral microtubules, chromosomes fail to reach a metaphase configuration, and interphase resumes without an intervening anaphase. The resulting polyploid nuclei are eliminated. The cells of the Drosophila gastrula can also delay metaphase-anaphase transition in response to DNA damage. This delay accompanies the stabilization of Cyclin A, a known inhibitor of sister chromosome separation in Drosophila. Unlike in cleavage embryos, gamma-tubulin remains at the spindle poles, and anaphase always occurs after the delay. Cyclin A mutants fail to delay metaphase-anaphase transition after irradiation and show an increased frequency of chromosome breakage in the subsequent anaphase. DNA damage delays metaphase-anaphase transition in Drosophila by stabilizing Cyclin A. This delay may normally serve to preserve chromosomal integrity during segregation. To our knowledge this is the first report of a metazoan metaphase-anaphase transition being delayed in response to DNA damage. Though mitotic progression is modulated in response to DNA damage in both cleaving and gastruating embryos of Drosophila, different mechanisms operate. These differences are discussed in the context of differential cell cycle regulation in cleavage and gastrula stages.

  20. Noncanonical compensation of zygotic X transcription in early Drosophila melanogaster development revealed through single-embryo RNA-seq.

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    Susan E Lott

    Full Text Available When Drosophila melanogaster embryos initiate zygotic transcription around mitotic cycle 10, the dose-sensitive expression of specialized genes on the X chromosome triggers a sex-determination cascade that, among other things, compensates for differences in sex chromosome dose by hypertranscribing the single X chromosome in males. However, there is an approximately 1 hour delay between the onset of zygotic transcription and the establishment of canonical dosage compensation near the end of mitotic cycle 14. During this time, zygotic transcription drives segmentation, cellularization, and other important developmental events. Since many of the genes involved in these processes are on the X chromosome, we wondered whether they are transcribed at higher levels in females and whether this might lead to sex-specific early embryonic patterning. To investigate this possibility, we developed methods to precisely stage, sex, and characterize the transcriptomes of individual embryos. We measured genome-wide mRNA abundance in male and female embryos at eight timepoints, spanning mitotic cycle 10 through late cycle 14, using polymorphisms between parental lines to distinguish maternal and zygotic transcription. We found limited sex-specific zygotic transcription, with a weak tendency for genes on the X to be expressed at higher levels in females. However, transcripts derived from the single X chromosome in males were more abundant that those derived from either X chromosome in females, demonstrating that there is widespread dosage compensation prior to the activation of the canonical MSL-mediated dosage compensation system. Crucially, this new system of early zygotic dosage compensation results in nearly identical transcript levels for key X-linked developmental regulators, including giant (gt, brinker (brk, buttonhead (btd, and short gastrulation (sog, in male and female embryos.

  1. In vivo imaging and tracking of individual nanodiamonds in drosophila melanogaster embryos

    OpenAIRE

    Simpson, David A.; Thompson, Amelia J.; Kowarsky, Mark; Zeeshan, Nida F.; Barson, Michael S. J.; Hall, Liam T.; Yan, Yan; Kaufmann, Stefan; Johnson, Brett C.; Ohshima, Takeshi; Caruso, Frank; Scholten, Robert E.; Robert B Saint; Murray, Michael J.; Hollenberg, Lloyd C. L.

    2014-01-01

    In this work, we incorporate and image individual fluorescent nanodiamonds in the powerful genetic model system Drosophila melanogaster. Fluorescence correlation spectroscopy and wide-field imaging techniques are applied to individual fluorescent nanodiamonds in blastoderm cells during stage 5 of development, up to a depth of 40 µm. The majority of nanodiamonds in the blastoderm cells during cellularization exhibit free diffusion with an average diffusion coefficient of (6 ± 3) × 10−3 µm2/s, ...

  2. Loss of PTB or negative regulation of Notch mRNA reveals distinct zones of Notch and actin protein accumulation in Drosophila embryo.

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    Cedric S Wesley

    Full Text Available Polypyrimidine Tract Binding (PTB protein is a regulator of mRNA processing and translation. Genetic screens and studies of wing and bristle development during the post-embryonic stages of Drosophila suggest that it is a negative regulator of the Notch pathway. How PTB regulates the Notch pathway is unknown. Our studies of Drosophila embryogenesis indicate that (1 the Notch mRNA is a potential target of PTB, (2 PTB and Notch functions in the dorso-lateral regions of the Drosophila embryo are linked to actin regulation but not their functions in the ventral region, and (3 the actin-related Notch activity in the dorso-lateral regions might require a Notch activity at or near the cell surface that is different from the nuclear Notch activity involved in cell fate specification in the ventral region. These data raise the possibility that the Drosophila embryo is divided into zones of different PTB and Notch activities based on whether or not they are linked to actin regulation. They also provide clues to the almost forgotten role of Notch in cell adhesion and reveal a role for the Notch pathway in cell fusions.

  3. The pathology of embryo death caused by the male-killing Spiroplasma bacterium in Drosophila nebulosa

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    Heraty Joseph

    2007-03-01

    Full Text Available Abstract Background Inherited bacteria that kill male offspring, male-killers, are known to be common in insects, but little is understood about the mechanisms used by male-killing bacteria to kill males. In this paper we describe the tempo and changes that occur during male-killing by Spiroplasma bacteria in the host Drosophila nebulosa. Results Spiroplasma infected D. nebulosa males were developmentally retarded from 6–8 h into embryonic development at 25°C, and arrested at between stages 12 and 13 of embryogenesis (10–12 h. Dying males were characterized by a failure to form segments, and ultimately disintegration of the normal oval embryonic shape. Prior to death, dying males exhibited widespread apoptosis, as testified by TUNEL staining. Conclusion The Spiroplasma kills male Drosophila in a narrow developmental period, shortly after the formation of the host dosage compensation complex that is required for male-killing. Male death is preceded by widespread apoptosis, but it is uncertain if this is primary or secondary apoptosis.

  4. P-TEFb, the super elongation complex and mediator regulate a subset of non-paused genes during early Drosophila embryo development.

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    Olle Dahlberg

    2015-02-01

    Full Text Available Positive Transcription Elongation Factor b (P-TEFb is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II into active elongation. It can assemble into a larger Super Elongation Complex (SEC consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-α. We also demonstrate that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3' end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos.

  5. The L1-type cell adhesion molecule Neuroglian is necessary for maintenance of sensory axon advance in the Drosophila embryo

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    Martin Veronica

    2008-04-01

    Full Text Available Abstract Background Cell adhesion molecules have long been implicated in the regulation of axon growth, but the precise cellular roles played by individual cell adhesion molecules and the molecular basis for their action are still not well understood. We have used the sensory system of the Drosophila embryo to shed light on the mechanism by which the L1-type cell adhesion molecule Neuroglian regulates axon growth. Results We have found a highly penetrant sensory axon stalling phenotype in neuroglian mutant embryos. Axons stalled at a variety of positions along their normal trajectory, but most commonly in the periphery some distance along the peripheral nerve. All lateral and dorsal cluster sensory neurons examined, except for the dorsal cluster neuron dbd, showed stalling. Sensory axons were never seen to project along inappropriate pathways in neuroglian mutants and stalled axons showed normal patterns of fasciculation within nerves. The growth cones of stalled axons possessed a simple morphology, similar to their appearance in wild-type embryos when advancing along nerves. Driving expression of the wild-type form of Neuroglian in sensory neurons alone rescued the neuroglian mutant phenotype of both pioneering and follower neurons. A partial rescue was achieved by expressing the Neuroglian extracellular domain. Over/mis-expression of Neuroglian in all neurons, oenocytes or trachea had no apparent effect on sensory axon growth. Conclusion We conclude that Neuroglian is necessary to maintain axon advance along axonal substrates, but is not required for initiation of axon outgrowth, axon fasciculation or recognition of correct growth substrates. Expression of Neuroglian in sensory neurons alone is sufficient to promote axon advance and the intracellular region of the molecule is largely dispensable for this function. It is unlikely, therefore, that Nrg acts as a molecular 'clutch' to couple adhesion of F-actin within the growth cone to the

  6. Depleting Gene Activities in Early Drosophila Embryos with the “Maternal-Gal4–shRNA” System

    Science.gov (United States)

    Staller, Max V.; Yan, Dong; Randklev, Sakara; Bragdon, Meghan D.; Wunderlich, Zeba B.; Tao, Rong; Perkins, Lizabeth A.; DePace, Angela H.; Perrimon, Norbert

    2013-01-01

    In a developing Drosophila melanogaster embryo, mRNAs have a maternal origin, a zygotic origin, or both. During the maternal–zygotic transition, maternal products are degraded and gene expression comes under the control of the zygotic genome. To interrogate the function of mRNAs that are both maternally and zygotically expressed, it is common to examine the embryonic phenotypes derived from female germline mosaics. Recently, the development of RNAi vectors based on short hairpin RNAs (shRNAs) effective during oogenesis has provided an alternative to producing germline clones. Here, we evaluate the efficacies of: (1) maternally loaded shRNAs to knockdown zygotic transcripts and (2) maternally loaded Gal4 protein to drive zygotic shRNA expression. We show that, while Gal4-driven shRNAs in the female germline very effectively generate phenotypes for genes expressed maternally, maternally loaded shRNAs are not very effective at generating phenotypes for early zygotic genes. However, maternally loaded Gal4 protein is very efficient at generating phenotypes for zygotic genes expressed during mid-embryogenesis. We apply this powerful and simple method to unravel the embryonic functions of a number of pleiotropic genes. PMID:23105012

  7. Depleting gene activities in early Drosophila embryos with the "maternal-Gal4-shRNA" system.

    Science.gov (United States)

    Staller, Max V; Yan, Dong; Randklev, Sakara; Bragdon, Meghan D; Wunderlich, Zeba B; Tao, Rong; Perkins, Lizabeth A; Depace, Angela H; Perrimon, Norbert

    2013-01-01

    In a developing Drosophila melanogaster embryo, mRNAs have a maternal origin, a zygotic origin, or both. During the maternal-zygotic transition, maternal products are degraded and gene expression comes under the control of the zygotic genome. To interrogate the function of mRNAs that are both maternally and zygotically expressed, it is common to examine the embryonic phenotypes derived from female germline mosaics. Recently, the development of RNAi vectors based on short hairpin RNAs (shRNAs) effective during oogenesis has provided an alternative to producing germline clones. Here, we evaluate the efficacies of: (1) maternally loaded shRNAs to knockdown zygotic transcripts and (2) maternally loaded Gal4 protein to drive zygotic shRNA expression. We show that, while Gal4-driven shRNAs in the female germline very effectively generate phenotypes for genes expressed maternally, maternally loaded shRNAs are not very effective at generating phenotypes for early zygotic genes. However, maternally loaded Gal4 protein is very efficient at generating phenotypes for zygotic genes expressed during mid-embryogenesis. We apply this powerful and simple method to unravel the embryonic functions of a number of pleiotropic genes.

  8. From Embryo to Adult: piRNA-Mediated Silencing throughout Germline Development in Drosophila

    Science.gov (United States)

    Marie, Pauline P.; Ronsseray, Stéphane; Boivin, Antoine

    2016-01-01

    In metazoan germ cells, transposable element activity is repressed by small noncoding PIWI-associated RNAs (piRNAs). Numerous studies in Drosophila have elucidated the mechanism of this repression in the adult germline. However, when and how transposable element repression is established during germline development has not been addressed. Here, we show that homology-dependent trans silencing is active in female primordial germ cells from late embryogenesis through pupal stages, and that genes related to the adult piRNA pathway are required for silencing during development. In larval gonads, we detect rhino-dependent piRNAs indicating de novo biogenesis of functional piRNAs during development. Those piRNAs exhibit the molecular signature of the “ping-pong” amplification step. Moreover, we show that Heterochromatin Protein 1a is required for the production of piRNAs coming from telomeric transposable elements. Furthermore, as in adult ovaries, incomplete, bimodal, and stochastic repression resembling variegation can occur at all developmental stages. Clonal analysis indicates that the repression status established in embryonic germ cells is maintained until the adult stage, suggesting the implication of a cellular memory mechanism. Taken together, data presented here show that piRNAs and their associated proteins are epigenetic components of a continuous repression system throughout germ cell development. PMID:27932388

  9. From Embryo to Adult: piRNA-Mediated Silencing throughout Germline Development in Drosophila

    Directory of Open Access Journals (Sweden)

    Pauline P. Marie

    2017-02-01

    Full Text Available In metazoan germ cells, transposable element activity is repressed by small noncoding PIWI-associated RNAs (piRNAs. Numerous studies in Drosophila have elucidated the mechanism of this repression in the adult germline. However, when and how transposable element repression is established during germline development has not been addressed. Here, we show that homology-dependent trans silencing is active in female primordial germ cells from late embryogenesis through pupal stages, and that genes related to the adult piRNA pathway are required for silencing during development. In larval gonads, we detect rhino-dependent piRNAs indicating de novo biogenesis of functional piRNAs during development. Those piRNAs exhibit the molecular signature of the “ping-pong” amplification step. Moreover, we show that Heterochromatin Protein 1a is required for the production of piRNAs coming from telomeric transposable elements. Furthermore, as in adult ovaries, incomplete, bimodal, and stochastic repression resembling variegation can occur at all developmental stages. Clonal analysis indicates that the repression status established in embryonic germ cells is maintained until the adult stage, suggesting the implication of a cellular memory mechanism. Taken together, data presented here show that piRNAs and their associated proteins are epigenetic components of a continuous repression system throughout germ cell development.

  10. The Toll pathway is required in the epidermis for muscle development in the Drosophila embryo

    Science.gov (United States)

    Halfon, M. S.; Keshishian, H.

    1998-01-01

    The Toll signaling pathway functions in several Drosophila processes, including dorsal-ventral pattern formation and the immune response. Here, we demonstrate that this pathway is required in the epidermis for proper muscle development. Previously, we showed that the zygotic Toll protein is necessary for normal muscle development; in the absence of zygotic Toll, close to 50% of hemisegments have muscle patterning defects consisting of missing, duplicated and misinserted muscle fibers (Halfon, M.S., Hashimoto, C., and Keshishian, H., Dev. Biol. 169, 151-167, 1995). We have now also analyzed the requirements for easter, spatzle, tube, and pelle, all of which function in the Toll-mediated dorsal-ventral patterning pathway. We find that spatzle, tube, and pelle, but not easter, are necessary for muscle development. Mutations in these genes give a phenotype identical to that seen in Toll mutants, suggesting that elements of the same pathway used for Toll signaling in dorsal-ventral development are used during muscle development. By expressing the Toll cDNA under the control of distinct Toll enhancer elements in Toll mutant flies, we have examined the spatial requirements for Toll expression during muscle development. Expression of Toll in a subset of epidermal cells that includes the epidermal muscle attachment cells, but not Toll expression in the musculature, is necessary for proper muscle development. Our results suggest that signals received by the epidermis early during muscle development are an important part of the muscle patterning process.

  11. Cellular resolution models for even skipped regulation in the entire Drosophila embryo

    Science.gov (United States)

    Ilsley, Garth R; Fisher, Jasmin; Apweiler, Rolf; DePace, Angela H; Luscombe, Nicholas M

    2013-01-01

    Transcriptional control ensures genes are expressed in the right amounts at the correct times and locations. Understanding quantitatively how regulatory systems convert input signals to appropriate outputs remains a challenge. For the first time, we successfully model even skipped (eve) stripes 2 and 3+7 across the entire fly embryo at cellular resolution. A straightforward statistical relationship explains how transcription factor (TF) concentrations define eve’s complex spatial expression, without the need for pairwise interactions or cross-regulatory dynamics. Simulating thousands of TF combinations, we recover known regulators and suggest new candidates. Finally, we accurately predict the intricate effects of perturbations including TF mutations and misexpression. Our approach imposes minimal assumptions about regulatory function; instead we infer underlying mechanisms from models that best fit the data, like the lack of TF-specific thresholds and the positional value of homotypic interactions. Our study provides a general and quantitative method for elucidating the regulation of diverse biological systems. DOI: http://dx.doi.org/10.7554/eLife.00522.001 PMID:23930223

  12. Embryo-larval exposure to atrazine reduces viability and alters oxidative stress parameters in Drosophila melanogaster.

    Science.gov (United States)

    Figueira, Fernanda Hernandes; Aguiar, Lais Mattos de; Rosa, Carlos Eduardo da

    2017-01-01

    The herbicide atrazine has been used worldwide with subsequent residual contamination of water and food, which may cause adverse effects on non-target organisms. Animal exposure to this herbicide may affect development, reproduction and energy metabolism. Here, the effects of atrazine regarding survival and redox metabolism were assessed in the fruit fly D. melanogaster exposed during embryonic and larval development. The embryos (newly fertilized eggs) were exposed to different atrazine concentrations (10μM and 100μM) in the diet until the adult fly emerged. Pupation and emergence rates, developmental time and sex ratio were determined as well as oxidative stress parameters and gene expression of the antioxidant defence system were evaluated in newly emerged male and female flies. Atrazine exposure reduced pupation and emergence rates in fruit flies without alterations to developmental time and sex ratio. Different redox imbalance patterns were observed between males and females exposed to atrazine. Atrazine caused an increase in oxidative damage, reactive oxygen species generation and antioxidant capacity and decreased thiol-containing molecules. Further, atrazine exposure altered the mRNA expression of antioxidant genes (keap1, sod, sod2, cat, irc, gss, gclm, gclc, trxt, trxr-1 and trxr-2). Reductions in fruit fly larval and pupal viability observed here are likely consequences of the oxidative stress induced by atrazine exposure. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Calcium and Mitosis

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    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.

  14. 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.

  15. Collective synchronization of divisions in Drosophila development

    Science.gov (United States)

    Vergassola, Massimo

    Mitoses in the early development of most metazoans are rapid and synchronized across the entire embryo. While diffusion is too slow, in vitro experiments have shown that waves of the cell-cycle regulator Cdk1 can transfer information rapidly across hundreds of microns. However, the signaling dynamics and the physical properties of chemical waves during embryonic development remain unclear. We develop FRET biosensors for the activity of Cdk1 and the checkpoint kinase Chk1 in Drosophila embryos and exploit them to measure waves in vivo. We demonstrate that Cdk1 chemical waves control mitotic waves and that their speed is regulated by the activity of Cdk1 during the S-phase (and not mitosis). We quantify the progressive slowdown of the waves with developmental cycles and identify its underlying control mechanism by the DNA replication checkpoint through the Chk1/Wee1 pathway. The global dynamics of the mitotic signaling network illustrates a novel control principle: the S-phase activity of Cdk1 regulates the speed of the mitotic wave, while the Cdk1 positive feedback ensures an invariantly rapid onset of mitosis. Mathematical modeling captures the speed of the waves and predicts a fundamental distinction between the S-phase Cdk1 trigger waves and the mitotic phase waves, which is illustrated by embryonic ablation experiments. In collaboration with Victoria Deneke1, Anna Melbinger2, and Stefano Di Talia1 1 Department of Cell Biology, Duke University Medical Center 2 Department of Physics, University of California San Diego.

  16. Lateral gene expression in Drosophila early embryos is supported by Grainyhead-mediated activation and tiers of dorsally-localized repression.

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    Mayra Garcia

    Full Text Available The general consensus in the field is that limiting amounts of the transcription factor Dorsal establish dorsal boundaries of genes expressed along the dorsal-ventral (DV axis of early Drosophila embryos, while repressors establish ventral boundaries. Yet recent studies have provided evidence that repressors act to specify the dorsal boundary of intermediate neuroblasts defective (ind, a gene expressed in a stripe along the DV axis in lateral regions of the embryo. Here we show that a short 12 base pair sequence ("the A-box" present twice within the ind CRM is both necessary and sufficient to support transcriptional repression in dorsal regions of embryos. To identify binding factors, we conducted affinity chromatography using the A-box element and found a number of DNA-binding proteins and chromatin-associated factors using mass spectroscopy. Only Grainyhead (Grh, a CP2 transcription factor with a unique DNA-binding domain, was found to bind the A-box sequence. Our results suggest that Grh acts as an activator to support expression of ind, which was surprising as we identified this factor using an element that mediates dorsally-localized repression. Grh and Dorsal both contribute to ind transcriptional activation. However, another recent study found that the repressor Capicua (Cic also binds to the A-box sequence. While Cic was not identified through our A-box affinity chromatography, utilization of the same site, the A-box, by both factors Grh (activator and Cic (repressor may also support a "switch-like" response that helps to sharpen the ind dorsal boundary. Furthermore, our results also demonstrate that TGF-β signaling acts to refine ind CRM expression in an A-box independent manner in dorsal-most regions, suggesting that tiers of repression act in dorsal regions of the embryo.

  17. Lateral gene expression in Drosophila early embryos is supported by Grainyhead-mediated activation and tiers of dorsally-localized repression.

    Science.gov (United States)

    Garcia, Mayra; Stathopoulos, Angelike

    2011-01-01

    The general consensus in the field is that limiting amounts of the transcription factor Dorsal establish dorsal boundaries of genes expressed along the dorsal-ventral (DV) axis of early Drosophila embryos, while repressors establish ventral boundaries. Yet recent studies have provided evidence that repressors act to specify the dorsal boundary of intermediate neuroblasts defective (ind), a gene expressed in a stripe along the DV axis in lateral regions of the embryo. Here we show that a short 12 base pair sequence ("the A-box") present twice within the ind CRM is both necessary and sufficient to support transcriptional repression in dorsal regions of embryos. To identify binding factors, we conducted affinity chromatography using the A-box element and found a number of DNA-binding proteins and chromatin-associated factors using mass spectroscopy. Only Grainyhead (Grh), a CP2 transcription factor with a unique DNA-binding domain, was found to bind the A-box sequence. Our results suggest that Grh acts as an activator to support expression of ind, which was surprising as we identified this factor using an element that mediates dorsally-localized repression. Grh and Dorsal both contribute to ind transcriptional activation. However, another recent study found that the repressor Capicua (Cic) also binds to the A-box sequence. While Cic was not identified through our A-box affinity chromatography, utilization of the same site, the A-box, by both factors Grh (activator) and Cic (repressor) may also support a "switch-like" response that helps to sharpen the ind dorsal boundary. Furthermore, our results also demonstrate that TGF-β signaling acts to refine ind CRM expression in an A-box independent manner in dorsal-most regions, suggesting that tiers of repression act in dorsal regions of the embryo.

  18. A microtubule interactome: complexes with roles in cell cycle and mitosis.

    Directory of Open Access Journals (Sweden)

    Julian R Hughes

    2008-04-01

    Full Text Available The microtubule (MT cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, the maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs, which work in concert with each other, binding MTs and altering their properties. We have used a MT cosedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to identify over 250 MAPs from early Drosophila embryos. We have taken two complementary approaches to analyse the cellular function of novel MAPs isolated using this approach. First, we have carried out an RNA interference (RNAi screen, identifying 21 previously uncharacterised genes involved in MT organisation. Second, we have undertaken a bioinformatics analysis based on binary protein interaction data to produce putative interaction networks of MAPs. By combining both approaches, we have identified and validated MAP complexes with potentially important roles in cell cycle regulation and mitosis. This study therefore demonstrates that biologically relevant data can be harvested using such a multidisciplinary approach, and identifies new MAPs, many of which appear to be important in cell division.

  19. Mitosis and its regulation

    Directory of Open Access Journals (Sweden)

    Frías Vázquez Sara

    2014-07-01

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

  20. 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)

  1. 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)

  2. A functional genomic screen combined with time-lapse microscopy uncovers a novel set of genes involved in dorsal closure of Drosophila embryos.

    Directory of Open Access Journals (Sweden)

    Ferenc Jankovics

    Full Text Available Morphogenesis, the establishment of the animal body, requires the coordinated rearrangement of cells and tissues regulated by a very strictly-determined genetic program. Dorsal closure of the epithelium in the Drosophila melanogaster embryo is one of the best models for such a complex morphogenetic event. To explore the genetic regulation of dorsal closure, we carried out a large-scale RNA interference-based screen in combination with in vivo time-lapse microscopy and identified several genes essential for the closure or affecting its dynamics. One of the novel dorsal closure genes, the small GTPase activator pebble (pbl, was selected for detailed analysis. We show that pbl regulates actin accumulation and protrusion dynamics in the leading edge of the migrating epithelial cells. In addition, pbl affects dorsal closure dynamics by regulating head involution, a morphogenetic process mechanically coupled with dorsal closure. Finally, we provide evidence that pbl is involved in closure of the adult thorax, suggesting its general requirement in epithelial closure processes.

  3. 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…

  4. 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…

  5. The core planar cell polarity gene prickle interacts with flamingo to promote sensory axon advance in the Drosophila embryo.

    Science.gov (United States)

    Mrkusich, Eli M; Flanagan, Dustin J; Whitington, Paul M

    2011-10-01

    The atypical cadherin Drosophila protein Flamingo and its vertebrate homologues play widespread roles in the regulation of both dendrite and axon growth. However, little is understood about the molecular mechanisms that underpin these functions. Whereas flamingo interacts with a well-defined group of genes in regulating planar cell polarity, previous studies have uncovered little evidence that the other core planar cell polarity genes are involved in regulation of neurite growth. We present data in this study showing that the planar cell polarity gene prickle interacts with flamingo in regulating sensory axon advance at a key choice point - the transition between the peripheral nervous system and the central nervous system. The cytoplasmic tail of the Flamingo protein is not required for this interaction. Overexpression of another core planar cell polarity gene dishevelled produces a similar phenotype to prickle mutants, suggesting that this gene may also play a role in regulation of sensory axon advance.

  6. 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.

  7. 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.

  8. 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.

  9. The SUMO Pathway in Mitosis.

    Science.gov (United States)

    Mukhopadhyay, Debaditya; Dasso, Mary

    2017-01-01

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

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

    Science.gov (United States)

    Clift, Dean; Schuh, Melina

    2013-09-01

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

  11. Hypoxia transiently sequesters mps1 and polo to collagenase-sensitive filaments in Drosophila prometaphase oocytes.

    Directory of Open Access Journals (Sweden)

    William D Gilliland

    Full Text Available BACKGROUND: The protein kinases Mps1 and Polo, which are required for proper cell cycle regulation in meiosis and mitosis, localize to numerous ooplasmic filaments during prometaphase in Drosophila oocytes. These filaments first appear throughout the oocyte at the end of prophase and are disassembled after egg activation. METHODOLOGY/PRINCIPAL FINDINGS: We showed here that Mps1 and Polo proteins undergo dynamic and reversible localization to static ooplasmic filaments as part of an oocyte-specific response to hypoxia. The observation that Mps1- and Polo-associated filaments reappear in the same locations through multiple cycles of oxygen deprivation demonstrates that underlying structural components of the filaments must still be present during normoxic conditions. Using immuno-electron microscopy, we observed triple-helical binding of Mps1 to numerous electron-dense filaments, with the gold label wrapped around the outside of the filaments like a garland. In addition, we showed that in live oocytes the relocalization of Mps1 and Polo to filaments is sensitive to injection of collagenase, suggesting that the structural components of the filaments are composed of collagen-like fibrils. However, the collagen-like genes we have been able to test so far (vkg and CG42453 did not appear to be associated with the filaments, demonstrating that the collagenase-sensitive component of the filaments is one of a number of other Drosophila proteins bearing a collagenase cleavage site. Finally, as hypoxia is known to cause Mps1 protein to accumulate at kinetochores in syncytial embryos, we also show that GFP-Polo accumulates at both kinetochores and centrosomes in hypoxic syncytial embryos. CONCLUSIONS/SIGNIFICANCE: These findings identify both a novel cellular structure (the ooplasmic filaments as well as a new localization pattern for Mps1 and Polo and demonstrate that hypoxia affects Polo localization in Drosophila.

  12. Tissue-specific direct microtransfer of nanomaterials into Drosophila embryos as a versatile in vivo test bed for nanomaterial toxicity assessment

    Directory of Open Access Journals (Sweden)

    Vega-Alvarez S

    2014-04-01

    Full Text Available Sasha Vega-Alvarez,1 Adriana Herrera,2 Carlos Rinaldi,2–4 Franklin A Carrero-Martínez1,5 1Department of Biology, 2Department of Chemical Engineering, University of Puerto Rico-Mayagüez, Mayagüez, Puerto Rico; 3J Crayton Pruitt Family Department of Biomedical Engineering, 4Department of Chemical Engineering, University of Florida, Gainesville, FL, USA; 5Department of Anatomy and Neuroscience, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico Abstract: Nanomaterials are the subject of intense research, focused on their synthesis, modification, and biomedical applications. Increased nanomaterial production and their wide range of applications imply a higher risk of human and environmental exposure. Unfortunately, neither environmental effects nor toxicity of nanomaterials to organisms are fully understood. Cost-effective, rapid toxicity assays requiring minimal amounts of materials are needed to establish both their biomedical potential and environmental safety standards. Drosophila exemplifies an efficient and cost-effective model organism with a vast repertoire of in vivo tools and techniques, all with high-throughput scalability and screening feasibility throughout its life cycle. Here we report tissue specific nanomaterial assessment through direct microtransfer into target tissues. We tested several nanomaterials with potential biomedical applications such as single-wall carbon nanotubes, multiwall carbon nanotubes, silver, gold, titanium dioxide, and iron oxide nanoparticles. Assessment of nanomaterial toxicity was conducted by evaluating progression through developmental morphological milestones in Drosophila. This cost-effective assessment method is amenable to high-throughput screening. Keywords: nanotoxicity, Drosophila, microtransfer, nanoparticle, iron oxide, silver, gold, titanium dioxide, carbon nanotube

  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. Tetraploid induction by inhibiting mitosis I in scallop Chlamys farreri

    Science.gov (United States)

    Yang, Hui-Ping; Guo, Xi-Ming; Chen, Zai-Zhong; Wang, Yong-Ping

    1999-12-01

    Tetraploid induction was carried out by inhibiting mitosis I in fertilized eggs of Chlamys farreri. Mitosis I was blocked with cold shock (5 7°C), Cytochalasin B (0.75 mg/L) and 6-dimethylaminopurine (6-DMAP) (60 75 mg/L) when 60% fertilized eggs released polar body II at 20°C. At 4-cells embryo stage, the ploidy was determined by counting chromosome number. In control groups, most embryos were diploids (72.22%) and aneuploids (24.78%). In Cytochalasin B, cold shock and 6-DMAP treated groups, tetraploids were respectively 10.51%, 4.08%, and 13.34%; aneuploids were 43.10%, 35.93% and 29.16%, and triploids were 7.84%, 8.52% and 18.33%. At D-larva stage, ploidy was determined by flow cytometry (FCM). The ploidy analysis of day 2 larvae showed diploids in control group and also in three treated groups. Juvenile scallops (0.2 0.3cm) which were harvested in two control groups and two CB treated groups were all diploids through checking ploidy individually by FCM.

  15. Embryo splitting

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    Karl Illmensee

    2010-04-01

    Full Text Available Mammalian embryo splitting has successfully been established in farm animals. Embryo splitting is safely and efficiently used for assisted reproduction in several livestock species. In the mouse, efficient embryo splitting as well as single blastomere cloning have been developed in this animal system. In nonhuman primates embryo splitting has resulted in several pregnancies. Human embryo splitting has been reported recently. Microsurgical embryo splitting under Institutional Review Board approval has been carried out to determine its efficiency for blastocyst development. Embryo splitting at the 6–8 cell stage provided a much higher developmental efficiency compared to splitting at the 2–5 cell stage. Embryo splitting may be advantageous for providing additional embryos to be cryopreserved and for patients with low response to hormonal stimulation in assisted reproduction programs. Social and ethical issues concerning embryo splitting are included regarding ethics committee guidelines. Prognostic perspectives are presented for human embryo splitting in reproductive medicine.

  16. myFX: a turn-key software for laboratory desktops to analyze spatial patterns of gene expression in Drosophila embryos.

    Science.gov (United States)

    Montiel, Ivan; Konikoff, Charlotte; Braun, Bremen; Packard, Mary; Gramates, Sian L; Sun, Qian; Ye, Jieping; Kumar, Sudhir

    2014-05-01

    Spatial patterns of gene expression are of key importance in understanding developmental networks. Using in situ hybridization, many laboratories are generating images to describe these spatial patterns and to test biological hypotheses. To facilitate such analyses, we have developed biologist-centric software (myFX) that contains computational methods to automatically process and analyze images depicting embryonic gene expression in the fruit fly Drosophila melanogaster. It facilitates creating digital descriptions of spatial patterns in images and enables measurements of pattern similarity and visualization of expression across genes and developmental stages. myFX interacts directly with the online FlyExpress database, which allows users to search thousands of existing patterns to find co-expressed genes by image comparison.

  17. Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells.

    Science.gov (United States)

    Gallaud, Emmanuel; Caous, Renaud; Pascal, Aude; Bazile, Franck; Gagné, Jean-Philippe; Huet, Sébastien; Poirier, Guy G; Chrétien, Denis; Richard-Parpaillon, Laurent; Giet, Régis

    2014-03-31

    The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.

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

    Directory of Open Access Journals (Sweden)

    Anne-Laure Bougé

    2016-03-01

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

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

    Science.gov (United States)

    Bougé, Anne-Laure; Parmentier, Marie-Laure

    2016-03-01

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

  20. Cappuccino, a Drosophila maternal effect gene required for polarity of the egg and embryo, is related to the vertebrate limb deformity locus.

    Science.gov (United States)

    Emmons, S; Phan, H; Calley, J; Chen, W; James, B; Manseau, L

    1995-10-15

    We report the molecular isolation of cappuccino (capu), a gene required for localization of molecular determinants within the developing Drosophila oocyte. The carboxy-terminal half of the capu protein is closely related to that of the vertebrate limb deformity locus, which is known to function in polarity determination in the developing vertebrate limb. In addition, capu shares both a proline-rich region and a 70-amino-acid domain with a number of other genes, two of which also function in pattern formation, the Saccharomyes cerevisiae BNI1 gene and the Aspergillus FigA gene. We also show that capu mutant oocytes have abnormal microtubule distributions and premature microtubule-based cytoplasmic streaming within the oocyte, but that neither the speed nor the timing of the cytoplasmic streaming correlates with the strength of the mutant allele. This suggests that the premature cytoplasmic streaming in capu mutant oocytes does not suffice to explain the patterning defects. By inducing cytoplasmic streaming in wild-type oocytes during mid-oogenesis, we show that premature cytoplasmic streaming can displace staufen protein from the posterior pole, but not gurken mRNA from around the oocyte nucleus.

  1. 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.

  2. Killing cells by targeting mitosis.

    Science.gov (United States)

    Manchado, E; Guillamot, M; Malumbres, M

    2012-03-01

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

  3. Insights into centromeric transcription in mitosis.

    Science.gov (United States)

    Liu, Hong

    2016-01-01

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

  4. 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.

  5. Homolog pairing and segregation in Drosophila meiosis.

    Science.gov (United States)

    McKee, B D

    2009-01-01

    Pairing of homologous chromosomes is fundamental to their reliable segregation during meiosis I and thus underlies sexual reproduction. In most eukaryotes homolog pairing is confined to prophase of meiosis I and is accompanied by frequent exchanges, known as crossovers, between homologous chromatids. Crossovers give rise to chiasmata, stable interhomolog connectors that are required for bipolar orientation (orientation to opposite poles) of homologs during meiosis I. Drosophila is unique among model eukaryotes in exhibiting regular homolog pairing in mitotic as well as meiotic cells. I review the results of recent molecular studies of pairing in both mitosis and meiosis in Drosophila. These studies show that homolog pairing is continuous between pre-meiotic mitosis and meiosis but that pairing frequencies and patterns are altered during the mitotic-meiotic transition. They also show that, with the exception of X-Y pairing in male meiosis, which is mediated specifically by the 240-bp rDNA spacer repeats, chromosome pairing is not restricted to specific sites in either mitosis or meiosis. Instead, virtually all chromosome regions, both heterochromatic and euchromatic, exhibit autonomous pairing capacity. Mutations that reduce the frequencies of both mitotic and meiotic pairing have been recently described, but no mutations that abolish pairing completely have been discovered, and the genetic control of pairing in Drosophila remains to be elucidated.

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

    Science.gov (United States)

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

    2016-11-01

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

  7. Myoblast fusion in Drosophila

    Energy Technology Data Exchange (ETDEWEB)

    Haralalka, Shruti [Stowers Institute for Medical Research, Kansas City, MO 64110 (United States); Abmayr, Susan M., E-mail: sma@stowers.org [Stowers Institute for Medical Research, Kansas City, MO 64110 (United States); Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, MO 66160 (United States)

    2010-11-01

    The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process . With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.

  8. 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.

  9. A Polycomb Group Protein Is Retained at Specific Sites on Chromatin in Mitosis

    Science.gov (United States)

    Follmer, Nicole E.; Wani, Ajazul H.; Francis, Nicole J.

    2012-01-01

    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. PMID:23284300

  10. SnapShot: Phosphoregulation of Mitosis.

    Science.gov (United States)

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

    2017-06-15

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

  11. 自然流产胚胎组织中人类纺锤体有丝分裂俘获缺陷基因的表达及意义%Expression of human spindle mitosis arrest deficiency gene in spontaneous abortion embryo tissues

    Institute of Scientific and Technical Information of China (English)

    蔡燕; 王箭; 袁泰先; 施琼; 翁亚光; 王应雄; 蒋洪彦; 刘子杰

    2008-01-01

    Objective To investigate the expression of human spindle mitosis arrest deficiency gene (hsMAD2)in spontaneous abortion embryos and the relationship between low expression of hsMAD2 and numerical chromosomal aberration. Methods Spontaneous abortion embryo tissues were collected,including 23 cases of once spontaneous abortion tissue and 10 cases of twice or more spontaneous abortion tissue and induced abortion embryos(35 cases)from the Department of Gynaecology and Obstetrics of the Affilisted Hospitals of Chongqing University of Medical Science during the period of March 2006 to March 2007.FQ-PCR and western blot were used to evaluate the endogenous expression level of hsMAD2 mRNA and hsMAD2 protein;primary culturing of cells from the induced abortion embryos was conducted and 5 embryonic cells were selected by chromosomes karyotype analysis.Recombinant shRNA plasmids targeting hsMAD2 gene were constructed to inhibit the expression of endogenous hsMAIY2 genes in embryonic cells which have normal karyotypes;the groups were defined as the first experimental group(transfeeted with pshRNA-hsMAD2-1),the second experimental group(transfected with pshRNA-hsMAD2-2),the third experimental group(transfected with pshRNA-hsMAD2-3),the first control group(transfected with nothing),the second control group(transfected with pTZU6+1)and the independent group(transfected with pshRNA-N1).Interference efficiency was demonstrated by FQ-PCR and western blot:cell prolireration was meagured by methyl thiazolyl tetrazolium(MTT)assay;cell-cycle was assessed by flow cytometry (FCM):the chromosome numbers were calculated to analyze the variation of chromosomes.Results(1) The mRNA levels of hsMAD2 in the once spontaneous abortion tissue,twice or more spontaneous abortion tissue and indueed abortion tissue were 0.00879±0.00035.0.00901±0.00033 and 0.00941±0.00026 respectively,and there Wag no significant ditierence(P>0.05)compared with each other;however,the protein levels of hsMAD2 in

  12. 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.

  13. Molecular cloning, functional expression, and gene silencing of two Drosophila receptors for the Drosophila neuropeptide pyrokinin-2

    DEFF Research Database (Denmark)

    Rosenkilde, Carina; Cazzamali, Giuseppe; Williamson, Michael

    2003-01-01

    embryos and first instar larvae. In addition to the two Drosophila receptors, we also identified two probable pyrokinin receptors in the genomic database from the malaria mosquito Anopheles gambiae. The two Drosophila pyrokinin receptors are, to our knowledge, the first invertebrate pyrokinin receptors...

  14. 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...

  15. 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.

  16. A role for Caenorhabditis elegans importin IMA-2 in germ line and embryonic mitosis.

    Science.gov (United States)

    Geles, Kenneth G; Johnson, Jeffrey J; Jong, Sena; Adam, Stephen A

    2002-09-01

    The importin alpha family of nuclear-cytoplasmic transport factors mediates the nuclear localization of proteins containing classical nuclear localization signals. Metazoan animals express multiple importin alpha proteins, suggesting their possible roles in cell differentiation and development. Adult Caenorhabditis elegans hermaphrodites express three importin alpha proteins, IMA-1, IMA-2, and IMA-3, each with a distinct expression and localization pattern. IMA-2 was expressed exclusively in germ line cells from the early embryonic through adult stages. The protein has a dynamic pattern of localization dependent on the stage of the cell cycle. In interphase germ cells and embryonic cells, IMA-2 is cytoplasmic and nuclear envelope associated, whereas in developing oocytes, the protein is cytoplasmic and intranuclear. During mitosis in germ line cells and embryos, IMA-2 surrounded the condensed chromosomes but was not directly associated with the mitotic spindle. The timing of IMA-2 nuclear localization suggested that the protein surrounded the chromosomes after fenestration of the nuclear envelope in prometaphase. Depletion of IMA-2 by RNA-mediated gene interference (RNAi) resulted in embryonic lethality and a terminal aneuploid phenotype. ima-2(RNAi) embryos have severe defects in nuclear envelope formation, accumulating nucleoporins and lamin in the cytoplasm. We conclude that IMA-2 is required for proper chromosome dynamics in germ line and early embryonic mitosis and is involved in nuclear envelope assembly at the conclusion of mitosis.

  17. mars and tousled-like kinase act in parallel to ensure chromosome fidelity in Drosophila

    Directory of Open Access Journals (Sweden)

    Huang Hsiao-Yu

    2009-06-01

    Full Text Available Abstract Background High levels of Hepatoma Up-Regulated Protein (HURP and Tousled-Like Kinase (TLK transcripts are found in hepatocellular carcinoma. HURP overexpression induces anchorage-independent growth of 293-T cells and enhances a rough-eye phenotype resulting from tlk overexpression in Drosophila. In addition, both HURP and Mars, a Drosophila HURP sequence homologue, promote polymerization of mitotic spindles. Thus, the genetic interaction of mars with tlk might be required for accurate chromosome segregation. Methods To reveal whether chromosome fidelity was decreased, the frequency of gynandromorphy, an individual with both male and female characteristics, and of non-disjunction were measured in the progeny from parents with reduced mars and/or tlk activities and analyzed by Student's t-test. To show that the genetic interaction between mars and tlk is epistatic or parallel, a cytological analysis of embryos with either reduced or increased activities of mars and/or tlk was used to reveal defects in mitotic-spindle morphology and chromosome segregation. Results A significant but small fraction of the progeny from parents with reduced mars activity showed gynandromorphy and non-disjunction. Results of cytological analysis revealed that the decrease in chromosome fidelity was a result of delayed polymerization of the mitotic spindle, which led to asynchronous chromosome segregation in embryos that had reduced mars activity. By removing one copy of tousled-like kinase (tlk from flies with reduced mars activity, chromosome fidelity was further reduced. This was indicated by an increased in the non-disjunction rate and more severe asynchrony. However, the morphology of the mitotic spindles in the embryos at metaphase where both gene activities were reduced was similar to that in mars embryos. Furthermore, tlk overexpression did not affect the morphology of the mitotic spindles and the cellular localization of Mars protein. Conclusion

  18. The DNA damage response during mitosis.

    Science.gov (United States)

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

    2013-10-01

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

  19. 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.

  20. The tumor suppressor CDKN3 controls mitosis.

    Science.gov (United States)

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

    2013-06-24

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

  1. Rescue from replication stress during mitosis.

    Science.gov (United States)

    Fragkos, Michalis; Naim, Valeria

    2017-04-03

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

  2. Dynamics of the mitochondrial network during mitosis.

    Science.gov (United States)

    Kanfer, Gil; Kornmann, Benoît

    2016-04-15

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

  3. Action of mercury in plant mitosis II

    Energy Technology Data Exchange (ETDEWEB)

    Lorente, R.

    1972-01-01

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

  4. 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.

  5. The FHA domain determines Drosophila Chk2/Mnk localization to key mitotic structures and is essential for early embryonic DNA damage responses.

    Science.gov (United States)

    Takada, Saeko; Collins, Eric R; Kurahashi, Kayo

    2015-05-15

    DNA damage responses, including mitotic centrosome inactivation, cell-cycle delay in mitosis, and nuclear dropping from embryo cortex, maintain genome integrity in syncytial Drosophila embryos. A conserved signaling kinase, Chk2, known as Mnk/Loki, is essential for the responses. Here we demonstrate that functional EGFP-Mnk expressed from a transgene localizes to the nucleus, centrosomes, interkinetochore/centromere region, midbody, and pseudocleavage furrows without DNA damage and in addition forms numerous foci/aggregates on mitotic chromosomes upon DNA damage. We expressed EGFP-tagged Mnk deletion or point mutation variants and investigated domain functions of Mnk in vivo. A triple mutation in the phosphopeptide-binding site of the forkhead-associated (FHA) domain disrupted normal Mnk localization except to the nucleus. The mutation also disrupted Mnk foci formation on chromosomes upon DNA damage. FHA mutations and deletion of the SQ/TQ-cluster domain (SCD) abolished Mnk transphosphorylations and autophosphorylations, indicative of kinase activation after DNA damage. A potent NLS was found at the C-terminus, which is required for normal Mnk function. We propose that the FHA domain in Mnk plays essential dual functions in mediating embryonic DNA damage responses by means of its phosphopeptide-binding ability: activating Mnk in the nucleus upon DNA damage and recruiting Mnk to multiple subcellular structures independently of DNA damage.

  6. Action of mercury in plant mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Lorente, R.

    1969-01-01

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

  7. Mitosis and growth in biological tissues

    OpenAIRE

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

    1993-01-01

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

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

    Science.gov (United States)

    Ohkura, Hiroyuki

    2015-01-20

    Meiosis is the specialized cell division that generates gametes. In contrast to mitosis, molecular mechanisms and regulation of meiosis are much less understood. Meiosis shares mechanisms and regulation with mitosis in many aspects, but also has critical differences from mitosis. This review highlights these differences between meiosis and mitosis. Recent studies using various model systems revealed differences in a surprisingly wide range of aspects, including cell-cycle regulation, recombination, postrecombination events, spindle assembly, chromosome-spindle interaction, and chromosome segregation. Although a great degree of diversity can be found among organisms, meiosis-specific processes, and regulation are generally conserved. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.

  9. 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.

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

    Science.gov (United States)

    Pilaz, Louis-Jan; Silver, Debra L

    2014-06-04

    Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-α-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

  11. Transgenerational propagation and quantitative maintenance of paternal centromeres depends on Cid/Cenp-A presence in Drosophila sperm.

    Directory of Open Access Journals (Sweden)

    Nitika Raychaudhuri

    Full Text Available In Drosophila melanogaster, as in many animal and plant species, centromere identity is specified epigenetically. In proliferating cells, a centromere-specific histone H3 variant (CenH3, named Cid in Drosophila and Cenp-A in humans, is a crucial component of the epigenetic centromere mark. Hence, maintenance of the amount and chromosomal location of CenH3 during mitotic proliferation is important. Interestingly, CenH3 may have different roles during meiosis and the onset of embryogenesis. In gametes of Caenorhabditis elegans, and possibly in plants, centromere marking is independent of CenH3. Moreover, male gamete differentiation in animals often includes global nucleosome for protamine exchange that potentially could remove CenH3 nucleosomes. Here we demonstrate that the control of Cid loading during male meiosis is distinct from the regulation observed during the mitotic cycles of early embryogenesis. But Cid is present in mature sperm. After strong Cid depletion in sperm, paternal centromeres fail to integrate into the gonomeric spindle of the first mitosis, resulting in gynogenetic haploid embryos. Furthermore, after moderate depletion, paternal centromeres are unable to re-acquire normal Cid levels in the next generation. We conclude that Cid in sperm is an essential component of the epigenetic centromere mark on paternal chromosomes and it exerts quantitative control over centromeric Cid levels throughout development. Hence, the amount of Cid that is loaded during each cell cycle appears to be determined primarily by the preexisting centromeric Cid, with little flexibility for compensation of accidental losses.

  12. 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.

  13. Mitosis and growth in biological tissues

    Science.gov (United States)

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

    1993-07-01

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

  14. 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.

  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. Consistency of polyamine profiles and expression of arginine decarboxylase in mitosis during zygotic embryogenesis of Scots pine.

    Science.gov (United States)

    Vuosku, Jaana; Jokela, Anne; Läärä, Esa; Sääskilahti, Mira; Muilu, Riina; Sutela, Suvi; Altabella, Teresa; Sarjala, Tytti; Häggman, Hely

    2006-11-01

    In this study, we show that both arginine decarboxylase (ADC) protein and mRNA transcript are present at different phases of mitosis in Scots pine (Pinus sylvestris) zygotic embryogenesis. We also examined the consistency of polyamine (PA) profiles with the effective temperature sum, the latter indicating the developmental stage of the embryos. PA metabolism was analyzed by fitting statistical regression models to the data of free and soluble conjugated PAs, to the enzyme activities of ADC and ornithine decarboxylase (ODC), as well as to the gene expression of ADC. According to the fitted models, PAs typically had the tendency to increase at the early stages but decrease at the late stages of embryogenesis. Only the free putrescine fraction remained stable during embryo development. The PA biosynthesis strongly preferred the ADC pathway. Both ADC gene expression and ADC enzyme activity were substantially higher than putative ODC gene expression or ODC enzyme activity, respectively. ADC gene expression and enzyme activity increased during embryogenesis, which suggests the involvement of transcriptional regulation in the expression of ADC. Both ADC mRNA and ADC protein localized in dividing cells of embryo meristems and more specifically within the mitotic spindle apparatus and close to the chromosomes, respectively. The results suggest the essential role of ADC in the mitosis of plant cells.

  17. 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.)

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

    Science.gov (United States)

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

    2012-11-01

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

  19. 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.)

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

    Science.gov (United States)

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

    2016-05-11

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

  1. P element excision in drosophila melanogaster and related drosophilids

    Science.gov (United States)

    The frequency of P element excision and the structure of the resulting excision products were determined in three drosophilid species, Drosophila melanogaster, D. virilis, and Chymomyza procnemis. A transient P element mobility assay was conducted in the cells of developing insect embryos, but unlik...

  2. The fate of frozen human embryos when transferred either on the day of thawing or after overnight culture

    Institute of Scientific and Technical Information of China (English)

    Yanhe Liu; Kelli Peirce; Kailin Yap; Kate McKenzie; Jay Natalwala; Vince Chapple; Margo Norman; Phillip Matson

    2012-01-01

    Objective:To study the performance of thawed zygotes and cleavage stage embryos transferred either on the day of thaw or after overnight culture.Methods:A retrospective study of864 frozen embryo transfer cycles.Cryosurvival rates per thawed embryo and implantation rates were analysed for embryos frozen onDay1,Day2 orDay3 relative to oocyte collection(Day0) and transferred on the day of thaw or after overnight culture, together with clinical pregnancy rates and prevalence of multiple gestations.Results:Survival ofDay3 embryos was significantly lower than those frozen onDay1(P=0.017) orDay2(P=0.015).Following overnight culture, resumption of mitosis of zygotes was more frequent thanDay2(P=0.000) which are in turn higher thanDay3(P=0.000) embryos.The implantation rate forDay2 embryos dividing overnight was significantly higher than those that did not divide for women <35 yrs(P=0.001) but not those women≥35 yrs(P=0.055).There were no differences in the implantation rates for those dividing or not after culture, for embryos frozen onDay3 for women <35 yrs(P=0.254) or≥35 yrs(P=0.403). Conclusions:Later cleavage stage post-thaw embryos survive and resume mitosis less frequently compared to earlier stages.Embryos not resuming mitosis after culture overnight can implant, particularlyDay3 embryos, suggesting that they can further increase the cumulative pregnancy rate per oocyte collection and that discarding them is wasteful.Overnight culture is best used for logistical reasons rather than a strategy to improve pregnancy rates.

  3. 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.

  4. 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.

  5. Mitosis can drive cell cannibalism through entosis.

    Science.gov (United States)

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

    2017-07-11

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

  6. Requirement for Pak3 in Rac1-induced organization of actin and myosin during Drosophila larval wound healing

    National Research Council Canada - National Science Library

    Baek, Seung Hee; Cho, Hae Weon; Kwon, Young-Chang; Lee, Hyangkyu; Lee, Ji Hyun; Kim, Moon Jong; Choe, Kwang-Min

    2012-01-01

    .... In Drosophila embryos, Cdc42 mediates filopodial projection into the wound leading edge (LE) and is important for suturing of the wound hole, while Rho1 mediates actin cable formation, which is critical during the initial stage of wound contraction [10] . In Drosophila larvae, Rac1, Cdc42, and Rho1 are essential for wound closure and organi...

  7. 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.

  8. Clathrin-mediated endocytosis is inhibited during mitosis.

    Science.gov (United States)

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

    2012-04-24

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

  9. Clathrin-mediated endocytosis is inhibited during mitosis

    Science.gov (United States)

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

    2012-01-01

    A long-standing paradigm in cell biology is the shutdown of endocytosis during mitosis. There is consensus that transferrin uptake is inhibited after entry into prophase and that it resumes in telophase. A recent study proposed that endocytosis is continuous throughout the cell cycle and that the observed inhibition of transferrin uptake is due to a decrease in available transferrin receptor at the cell surface, and not to a shutdown of endocytosis. This challenge to the established view is gradually becoming accepted. Because of this controversy, we revisited the question of endocytic activity during mitosis. Using an antibody uptake assay and controlling for potential changes in surface receptor density, we demonstrate the strong inhibition of endocytosis in mitosis of CD8 chimeras containing any of the three major internalization motifs for clathrin-mediated endocytosis (YXXΦ, [DE]XXXL[LI], or FXNPXY) or a CD8 protein with the cytoplasmic tail of the cation-independent mannose 6-phosphate receptor. The shutdown is not gradual: We describe a binary switch from endocytosis being “on” in interphase to “off” in mitosis as cells traverse the G2/M checkpoint. In addition, we show that the inhibition of transferrin uptake in mitosis occurs despite abundant transferrin receptor at the surface of HeLa cells. Our study finds no support for the recent idea that endocytosis continues during mitosis, and we conclude that endocytosis is temporarily shutdown during early mitosis. PMID:22493256

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

    Science.gov (United States)

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

    2015-02-01

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

  11. Drosophila CENP-A mutations cause a BubR1-dependent early mitotic delay without normal localization of kinetochore components.

    Directory of Open Access Journals (Sweden)

    Michael D Blower

    2006-07-01

    Full Text Available The centromere/kinetochore complex plays an essential role in cell and organismal viability by ensuring chromosome movements during mitosis and meiosis. The kinetochore also mediates the spindle attachment checkpoint (SAC, which delays anaphase initiation until all chromosomes have achieved bipolar attachment of kinetochores to the mitotic spindle. CENP-A proteins are centromere-specific chromatin components that provide both a structural and a functional foundation for kinetochore formation. Here we show that cells in Drosophila embryos homozygous for null mutations in CENP-A (CID display an early mitotic delay. This mitotic delay is not suppressed by inactivation of the DNA damage checkpoint and is unlikely to be the result of DNA damage. Surprisingly, mutation of the SAC component BUBR1 partially suppresses this mitotic delay. Furthermore, cid mutants retain an intact SAC response to spindle disruption despite the inability of many kinetochore proteins, including SAC components, to target to kinetochores. We propose that SAC components are able to monitor spindle assembly and inhibit cell cycle progression in the absence of sustained kinetochore localization.

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

    OpenAIRE

    Nurse, Paul; Thuriaux, Pierre

    1980-01-01

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

  13. Linking abnormal mitosis to the acquisition of DNA damage

    Science.gov (United States)

    Pellman, David

    2012-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  15. How frog embryos replicate their DNA reliably

    Science.gov (United States)

    Bechhoefer, John; Marshall, Brandon

    2007-03-01

    Frog embryos contain three billion base pairs of DNA. In early embryos (cycles 2-12), DNA replication is extremely rapid, about 20 min., and the entire cell cycle lasts only 25 min., meaning that mitosis (cell division) takes place in about 5 min. In this stripped-down cell cycle, there are no efficient checkpoints to prevent the cell from dividing before its DNA has finished replication - a disastrous scenario. Even worse, the many origins of replication are laid down stochastically and are also initiated stochastically throughout the replication process. Despite the very tight time constraints and despite the randomness introduced by origin stochasticity, replication is extremely reliable, with cell division failing no more than once in 10,000 tries. We discuss a recent model of DNA replication that is drawn from condensed-matter theories of 1d nucleation and growth. Using our model, we discuss different strategies of replication: should one initiate all origins as early as possible, or is it better to hold back and initiate some later on? Using concepts from extreme-value statistics, we derive the distribution of replication times given a particular scenario for the initiation of origins. We show that the experimentally observed initiation strategy for frog embryos meets the reliability constraint and is close to the one that requires the fewest resources of a cell.

  16. Pathologic mitoses and pathology of mitosis in tumorigenesis

    OpenAIRE

    RG Steinbeck

    2009-01-01

    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 a...

  17. Cell cycling and patterned cell proliferation in the wing primordium of Drosophila.

    OpenAIRE

    1996-01-01

    The pattern of cell proliferation in the Drosophila imaginal wing primordium is spatially and temporally heterogeneous. Direct visualization of cells in S, G2, and mitosis phases of the cell cycle reveals several features invariant throughout development. The fraction of cells in the disc in the different cell cycle stages is constant, the majority remaining in G1. Cells in the different phases of the cell cycle mainly appear in small synchronic clusters that are nonclonally derived but resul...

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

    Science.gov (United States)

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

    2000-11-01

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

  19. 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.

  20. Entry into mitosis without Cdc2 kinase activation.

    Science.gov (United States)

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

    1998-11-01

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

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

    Science.gov (United States)

    Nurse, P; Thuriaux, P

    1980-11-01

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

  2. Mitosis can drive cell cannibalism through entosis

    Science.gov (United States)

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

    2017-01-01

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

  3. Isolation of Drosophila egg chambers for imaging.

    Science.gov (United States)

    Parton, Richard M; Vallés, Ana Maria; Dobbie, Ian M; Davis, Ilan

    2010-04-01

    The fruit fly Drosophila melanogaster is an important model for basic research into the molecular mechanisms underlying cell function and development, as well as a major biomedical research tool. A significant advantage of Drosophila is the ability to apply live cell imaging to a variety of living tissues that can be dissected and imaged in vivo, ex vivo, or in vitro. Drosophila egg chambers, for example, have proven to be a useful model system for studying border cell migration, Golgi unit transport, the rapid movement of mRNA and protein particles, and the role of microtubules in meiosis and oocyte differentiation. A crucial first step before imaging is preparation of the experimental material to ensure physiological relevance and to achieve the best conditions for image quality. Early- to mid-stage egg chambers cannot be mounted in an aqueous-based medium, because this causes a change in microtubule organization and follicle cell morphology. Such egg chambers survive better in Halocarbon oil, which allows free diffusion of oxygen, has low viscosity, and thus prevents dehydration and hypoxia. With a refractive index similar to glycerol, Halocarbon oil also has good optical properties for imaging. It also provides a good environment for injection and is particularly useful for long-term imaging of embryos. However, unlike with aqueous solutions, changes in the medium are not possible. This protocol describes the isolation of Drosophila egg chambers.

  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. Nuclear transport factors: global regulation of mitosis.

    Science.gov (United States)

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

    2015-08-01

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

  6. Culture systems: embryo density.

    Science.gov (United States)

    Reed, Michael L

    2012-01-01

    Embryo density is defined as the embryo-to-volume ratio achieved during in vitro culture; in other words, it is the number of embryos in a defined volume of culture medium. The same density can be achieved by manipulating either the number of embryos in a given volume of medium, or manipulating the volume of the medium for a given number of embryos: for example, a microdrop with five embryos in a 50 μl volume under oil has the same embryo-to-volume ratio (1:10 μl) as a microdrop with one embryo in a 10 μl volume under oil (1:10 μl). Increased embryo density can improve mammalian embryo development in vitro; however, the mechanism(s) responsible for this effect may be different with respect to which method is used to increase embryo density.Standard, flat sterile plastic petri dishes are the most common, traditional platform for embryo culture. Microdrops under a mineral oil overlay can be prepared to control embryo density, but it is critical that dish preparation is consistent, where appropriate techniques are applied to prevent microdrop dehydration during preparation, and results of any data collection are reliable, and repeatable. There are newer dishes available from several manufacturers that are specifically designed for embryo culture; most are readily available for use with human embryos. The concept behind these newer dishes relies on fabrication of conical and smaller volume wells into the dish design, so that embryos rest at the lowest point in the wells, and where putative embryotrophic factors may concentrate.Embryo density is not usually considered by the embryologist as a technique in and of itself; rather, the decision to culture embryos in groups or individually is protocol-driven, and is based more on convenience or the need to collect data on individual embryos. Embryo density can be controlled, and as such, it can be utilized as a simple, yet effective tool to improve in vitro development of human embryos.

  7. Modeling transcriptional networks in Drosophila development at multiple scales.

    Science.gov (United States)

    Wunderlich, Zeba; DePace, Angela H

    2011-12-01

    Quantitative models of developmental processes can provide insights at multiple scales. Ultimately, models may be particularly informative for key questions about network level behavior during development such as how does the system respond to environmental perturbation, or operate reliably in different genetic backgrounds? The transcriptional networks that pattern the Drosophila embryo have been the subject of numerous quantitative experimental studies coupled to modeling frameworks in recent years. In this review, we describe three studies that consider these networks at different levels of molecular detail and therefore result in different types of insights. We also discuss other developmental transcriptional networks operating in Drosophila, with the goal of highlighting what additional insights they may provide.

  8. Drosophila homolog of the murine Int-1 protooncogene.

    OpenAIRE

    1988-01-01

    We have isolated phage clones from Drosophila melanogaster genomic and cDNA libraries containing a sequence homologous to the murine Int-1 protooncogene. The Drosophila gene is represented by a single locus at position 28A1-2 on chromosome 2. The gene is expressed as a 2.9-kilobase-long polyadenylylated mRNA in embryo, larval, and pupal stages. It is hardly detectable in adult flies. The longest open reading frame of the cDNA clone corresponds to a protein 469 amino acids long. Alignment of t...

  9. CRISPR/Cas9 mediated genome engineering in Drosophila.

    Science.gov (United States)

    Bassett, Andrew; Liu, Ji-Long

    2014-09-01

    Genome engineering has revolutionised genetic analysis in many organisms. Here we describe a simple and efficient technique to generate and detect novel mutations in desired target genes in Drosophila melanogaster. We target double strand breaks to specific sites within the genome by injecting mRNA encoding the Cas9 endonuclease and in vitro transcribed synthetic guide RNA into Drosophila embryos. The small insertion and deletion mutations that result from inefficient non-homologous end joining at this site are detected by high resolution melt analysis of whole flies and individual wings, allowing stable lines to be made within 1 month.

  10. Both Chromosome Decondensation and Condensation Are Dependent on DNA Replication in C. elegans Embryos.

    Science.gov (United States)

    Sonneville, Remi; Craig, Gillian; Labib, Karim; Gartner, Anton; Blow, J Julian

    2015-07-21

    During cell division, chromatin alternates between a condensed state to facilitate chromosome segregation and a decondensed form when DNA replicates. In most tissues, S phase and mitosis are separated by defined G1 and G2 gap phases, but early embryogenesis involves rapid oscillations between replication and mitosis. Using Caenorhabditis elegans embryos as a model system, we show that chromosome condensation and condensin II concentration on chromosomal axes require replicated DNA. In addition, we found that, during late telophase, replication initiates on condensed chromosomes and promotes the rapid decondensation of the chromatin. Upon replication initiation, the CDC-45-MCM-GINS (CMG) DNA helicase drives the release of condensin I complexes from chromatin and the activation or displacement of inactive MCM-2-7 complexes, which together with the nucleoporin MEL-28/ELYS tethers condensed chromatin to the nuclear envelope, thereby promoting chromatin decondensation. Our results show how, in an early embryo, the chromosome-condensation cycle is functionally linked with DNA replication.

  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. Regulation of mRNA translation during mitosis.

    Science.gov (United States)

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

    2015-08-25

    Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ~200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.

  13. Drosophila roadblock and Chlamydomonas Lc7

    Science.gov (United States)

    Bowman, Aaron B.; Patel-King, Ramila S.; Benashski, Sharon E.; McCaffery, J. Michael; Goldstein, Lawrence S.B.; King, Stephen M.

    1999-01-01

    Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97–amino acid polypeptide that is 57% identical (70% similar) to the 105–amino acid Chlamydomonas outer arm dynein–associated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions. PMID:10402468

  14. Mechanisms of Chromosome Congression during Mitosis

    Science.gov (United States)

    Maiato, Helder; Gomes, Ana Margarida; Sousa, Filipe; Barisic, Marin

    2017-01-01

    Chromosome congression during prometaphase culminates with the establishment of a metaphase plate, a hallmark of mitosis in metazoans. Classical views resulting from more than 100 years of research on this topic have attempted to explain chromosome congression based on the balance between opposing pulling and/or pushing forces that reach an equilibrium near the spindle equator. However, in mammalian cells, chromosome bi-orientation and force balance at kinetochores are not required for chromosome congression, whereas the mechanisms of chromosome congression are not necessarily involved in the maintenance of chromosome alignment after congression. Thus, chromosome congression and maintenance of alignment are determined by different principles. Moreover, it is now clear that not all chromosomes use the same mechanism for congressing to the spindle equator. Those chromosomes that are favorably positioned between both poles when the nuclear envelope breaks down use the so-called “direct congression” pathway in which chromosomes align after bi-orientation and the establishment of end-on kinetochore-microtubule attachments. This favors the balanced action of kinetochore pulling forces and polar ejection forces along chromosome arms that drive chromosome oscillatory movements during and after congression. The other pathway, which we call “peripheral congression”, is independent of end-on kinetochore microtubule-attachments and relies on the dominant and coordinated action of the kinetochore motors Dynein and Centromere Protein E (CENP-E) that mediate the lateral transport of peripheral chromosomes along microtubules, first towards the poles and subsequently towards the equator. How the opposite polarities of kinetochore motors are regulated in space and time to drive congression of peripheral chromosomes only now starts to be understood. This appears to be regulated by position-dependent phosphorylation of both Dynein and CENP-E and by spindle microtubule

  15. Mechanisms of Chromosome Congression during Mitosis

    Directory of Open Access Journals (Sweden)

    Helder Maiato

    2017-02-01

    Full Text Available Chromosome congression during prometaphase culminates with the establishment of a metaphase plate, a hallmark of mitosis in metazoans. Classical views resulting from more than 100 years of research on this topic have attempted to explain chromosome congression based on the balance between opposing pulling and/or pushing forces that reach an equilibrium near the spindle equator. However, in mammalian cells, chromosome bi-orientation and force balance at kinetochores are not required for chromosome congression, whereas the mechanisms of chromosome congression are not necessarily involved in the maintenance of chromosome alignment after congression. Thus, chromosome congression and maintenance of alignment are determined by different principles. Moreover, it is now clear that not all chromosomes use the same mechanism for congressing to the spindle equator. Those chromosomes that are favorably positioned between both poles when the nuclear envelope breaks down use the so-called “direct congression” pathway in which chromosomes align after bi-orientation and the establishment of end-on kinetochore-microtubule attachments. This favors the balanced action of kinetochore pulling forces and polar ejection forces along chromosome arms that drive chromosome oscillatory movements during and after congression. The other pathway, which we call “peripheral congression”, is independent of end-on kinetochore microtubule-attachments and relies on the dominant and coordinated action of the kinetochore motors Dynein and Centromere Protein E (CENP-E that mediate the lateral transport of peripheral chromosomes along microtubules, first towards the poles and subsequently towards the equator. How the opposite polarities of kinetochore motors are regulated in space and time to drive congression of peripheral chromosomes only now starts to be understood. This appears to be regulated by position-dependent phosphorylation of both Dynein and CENP-E and by spindle

  16. 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.

  17. Aurora-A regulates MCRS1 function during mitosis.

    Science.gov (United States)

    Meunier, Sylvain; Timón, Krystal; Vernos, Isabelle

    2016-07-02

    The mitotic spindle is made of microtubules (MTs) nucleated through different pathways involving the centrosomes, the chromosomes or the walls of pre-existing MTs. MCRS1 is a RanGTP target that specifically associates with the chromosome-driven MTs protecting them from MT depolymerases. MCRS1 is also needed for the control of kinetochore fiber (K-fiber) MT minus-ends dynamics in metaphase. Here, we investigated the regulation of MCRS1 activity in M-phase. We show that MCRS1 is phosphorylated by the Aurora-A kinase in mitosis on Ser35/36. Although this phosphorylation has no role on MCRS1 localization to chromosomal MTs and K-fiber minus-ends, we show that it regulates MCRS1 activity in mitosis. We conclude that Aurora-A activity is particularly important in the tuning of K-fiber minus-ends dynamics in mitosis.

  18. Mitosis in the Human Malaria Parasite Plasmodium falciparum ▿

    Science.gov (United States)

    Gerald, Noel; Mahajan, Babita; Kumar, Sanjai

    2011-01-01

    Malaria is caused by intraerythrocytic protozoan parasites belonging to Plasmodium spp. (phylum Apicomplexa) that produce significant morbidity and mortality, mostly in developing countries. Plasmodium parasites have a complex life cycle that includes multiple stages in anopheline mosquito vectors and vertebrate hosts. During the life cycle, the parasites undergo several cycles of extreme population growth within a brief span, and this is critical for their continued transmission and a contributing factor for their pathogenesis in the host. As with other eukaryotes, successful mitosis is an essential requirement for Plasmodium reproduction; however, some aspects of Plasmodium mitosis are quite distinct and not fully understood. In this review, we will discuss the current understanding of the architecture and key events of mitosis in Plasmodium falciparum and related parasites and compare them with the traditional mitotic events described for other eukaryotes. PMID:21317311

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

    Science.gov (United States)

    Meunier, Sylvain; Vernos, Isabelle

    2016-02-01

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

  20. Effectiveness of teaching mitosis through the learning cycle in secondary school

    OpenAIRE

    Perko, Irena

    2013-01-01

    We carried out research on mitosis with first year secondary school students in altogether eleven different programs. The aim of the research was to find out how much knowledge about mitosis these students already have acquired in primary school. Additionally, we wanted to test how much new information on mitosis the students acquired, after I explained with the use of computer the method of classifying images of mitosis phases according to Schields. Five weeks after this lesson, I tested the...

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

    OpenAIRE

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

    2016-01-01

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

  2. Relevance of LIF and EGF on Mouse Preimplantation Embryo Development

    Directory of Open Access Journals (Sweden)

    Iraj Amiri

    2008-01-01

    Full Text Available Objective: Recent evidence suggests that Leukemia Inhibitory Factor (LIF, a member ofinterleukin-6 family, has biological actions on preimplantation embryo development. Alsoit is established that Epidermal Growth Factor (EGF, a strong mitosis-promoting agent,improves the preimplantation embryo development by increasing the cell metabolism andproliferation. The purpose of the present study is to investigate the effects of these factors,alone and in combination together, on preimplantation and development of the embryo.Materials and Methods: Six to eight weeks old NMRI mice were super ovulated by injectionof 10IU PMSG and 10IU hCG, then the mated mice were killed 46 hours later. Theiroviducts were flushed, two-cell embryos collected and divided randomly to the four groupsas following: Control, treatment 1 (LIF, treatment 2 (EGF, treatment 3 (LIF+EGF. In eachgroup, the embryos were cultured in an incubator at 37°C with 5% CO2 and 90% humidityfor 72hrs. The state of embryo development was evaluated in 24,36,48,60 and 72hrsfollowing the embryos cultures. By the end of the cultures, cell apoptosis was studiedby the terminal deoxynucleotidyl transferas-mediated dUTP nick end-labeling (TUNELtechnique.Results: Significant difference was detected in the rate of hatching in the LIF and LIF+EGFgroups. This difference was also seen in the rate of blastocyst formation after 36hrs(p<0.05 and in the average of the total cell number (p<0.05 after 72hrs. In comparison tothe apoptotic index, there was no significant difference between the control and treatmentgroups.Conclusion: The findings in this study show a beneficial effect of LIF and EGF on theblastocyst formation, hatching and its total cell numbers in vitro.

  3. The Role of Drosophila Merlin in the Control of Mitosis Exit and Development

    Science.gov (United States)

    2008-07-01

    such a rule , and some of them crossed the vein boundary (arrows in Figure 6 point to these clones). In addition to crossing the vein, the mosaic...9A). The retina is the innermost layer of the eye and is derived embryologically from the outgrowth of the developing brain (Martinez-Morales et al

  4. The Role of Drosophila Merlin in the Control of Mitosis Exit and Development

    Science.gov (United States)

    2007-07-01

    schwannoma xenografts in severe combined immunodeficiency mice. Laryngoscope. 2006 Nov; 116(11): 2018-26. Figure Legends Figure 1: Salient features...Rhinological and Otological Society, Inc. Growth of Benign and Malignant Schwannoma Xenografts in Severe Combined Immunodeficiency Mice Long-Sheng Chang, PhD...lignant schwannoma (HMS-97) cells were implanted near the sciatic nerve in the thigh of severe combined immunodeficiency (SCID) mice. Additionally

  5. Multiscale diffusion in the mitotic Drosophila melanogaster syncytial blastoderm

    OpenAIRE

    2012-01-01

    Despite the fundamental importance of diffusion for embryonic morphogen gradient formation in the early Drosophila melanogaster embryo, there remains controversy regarding both the extent and the rate of diffusion of well-characterized morphogens. Furthermore, the recent observation of diffusional “compartmentalization” has suggested that diffusion may in fact be nonideal and mediated by an as-yet-unidentified mechanism. Here, we characterize the effects of the geometry of the early syncytial...

  6. Maternal control of the Drosophila dorsal-ventral body axis.

    Science.gov (United States)

    Stein, David S; Stevens, Leslie M

    2014-01-01

    The pathway that generates the dorsal-ventral (DV) axis of the Drosophila embryo has been the subject of intense investigation over the previous three decades. The initial asymmetric signal originates during oogenesis by the movement of the oocyte nucleus to an anterior corner of the oocyte, which establishes DV polarity within the follicle through signaling between Gurken, the Drosophila Transforming Growth Factor (TGF)-α homologue secreted from the oocyte, and the Drosophila Epidermal Growth Factor Receptor (EGFR) that is expressed by the follicular epithelium cells that envelop the oocyte. Follicle cells that are not exposed to Gurken follow a ventral fate and express Pipe, a sulfotransferase that enzymatically modifies components of the inner vitelline membrane layer of the eggshell, thereby transferring DV spatial information from the follicle to the egg. These ventrally sulfated eggshell proteins comprise a localized cue that directs the ventrally restricted formation of the active Spätzle ligand within the perivitelline space between the eggshell and the embryonic membrane. Spätzle activates Toll, a transmembrane receptor in the embryonic membrane. Transmission of the Toll signal into the embryo leads to the formation of a ventral-to-dorsal gradient of the transcription factor Dorsal within the nuclei of the syncytial blastoderm stage embryo. Dorsal controls the spatially specific expression of a large constellation of zygotic target genes, the Dorsal gene regulatory network, along the embryonic DV circumference. This article reviews classic studies and integrates them with the details of more recent work that has advanced our understanding of the complex pathway that establishes Drosophila embryo DV polarity. For further resources related to this article, please visit the WIREs website. The authors have declared no conflicts of interest for this article. © 2014 Wiley Periodicals, Inc.

  7. Maternal control of the Drosophila dorsal–ventral body axis

    Science.gov (United States)

    Stein, David S.; Stevens, Leslie M.

    2016-01-01

    The pathway that generates the dorsal–ventral (DV) axis of the Drosophila embryo has been the subject of intense investigation over the previous three decades. The initial asymmetric signal originates during oogenesis by the movement of the oocyte nucleus to an anterior corner of the oocyte, which establishes DV polarity within the follicle through signaling between Gurken, the Drosophila Transforming Growth Factor (TGF)-α homologue secreted from the oocyte, and the Drosophila Epidermal Growth Factor Receptor (EGFR) that is expressed by the follicular epithelium cells that envelop the oocyte. Follicle cells that are not exposed to Gurken follow a ventral fate and express Pipe, a sulfotransferase that enzymatically modifies components of the inner vitelline membrane layer of the eggshell, thereby transferring DV spatial information from the follicle to the egg. These ventrally sulfated eggshell proteins comprise a localized cue that directs the ventrally restricted formation of the active Spätzle ligand within the perivitelline space between the eggshell and the embryonic membrane. Spätzle activates Toll, a transmembrane receptor in the embryonic membrane. Transmission of the Toll signal into the embryo leads to the formation of a ventral-to-dorsal gradient of the transcription factor Dorsal within the nuclei of the syncytial blastoderm stage embryo. Dorsal controls the spatially specific expression of a large constellation of zygotic target genes, the Dorsal gene regulatory network, along the embryonic DV circumference. This article reviews classic studies and integrates them with the details of more recent work that has advanced our understanding of the complex pathway that establishes Drosophila embryo DV polarity. PMID:25124754

  8. 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.

  9. 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.

  10. POSH misexpression induces caspase-dependent cell death in Drosophila.

    Science.gov (United States)

    Lennox, Ashley L; Stronach, Beth

    2010-02-01

    POSH (Plenty of SH3 domains) is a scaffold for signaling proteins regulating cell survival. Specifically, POSH promotes assembly of a complex including Rac GTPase, mixed lineage kinase (MLK), MKK7, and Jun kinase (JNK). In Drosophila, genetic analysis implicated POSH in Tak1-dependent innate immune response, in part through regulation of JNK signaling. Homologs of the POSH signaling complex components, MLK and MKK7, are essential in Drosophila embryonic dorsal closure. Using a gain-of-function approach, we tested whether POSH plays a role in this process. Ectopic expression of POSH in the embryo causes dorsal closure defects due to apoptosis of the amnioserosa, but ectodermal JNK signaling is normal. Phenotypic consequences of POSH expression were found to be dependent on Drosophila Nc, the caspase-9 homolog, but only partially on Tak1 and not at all on Slpr and Hep. These results suggest that POSH may use different signaling complexes to promote cell death in distinct contexts.

  11. 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.

  12. Mitosis, diffusible crosslinkers, and the ideal gas law.

    Science.gov (United States)

    Odde, David J

    2015-03-12

    During mitosis, molecular motors hydrolyze ATP to generate sliding forces between adjacent microtubules and form the bipolar mitotic spindle. Lansky et al. now show that the diffusible microtubule crosslinker Ase1p can generate sliding forces between adjacent microtubules, and it does so without ATP hydrolysis. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. 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....

  14. 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...

  15. EWSR1 regulates mitosis by dynamically influencing microtubule acetylation.

    Science.gov (United States)

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

    2016-08-17

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

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

    Science.gov (United States)

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

    2014-09-23

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

  17. Sequential activities of Dynein, Mud and Asp in centrosome-spindle coupling maintain centrosome number upon mitosis.

    Science.gov (United States)

    Bosveld, Floris; Ainslie, Anna; Bellaïche, Yohanns

    2017-09-01

    Centrosomes nucleate microtubules and are tightly coupled to the bipolar spindle to ensure genome integrity, cell division orientation and centrosome segregation. While the mechanisms of centrosome-dependent microtubule nucleation and bipolar spindle assembly have been the focus of numerous works, less is known on the mechanisms ensuring the centrosome-spindle coupling. The conserved NuMA protein (Mud in Drosophila) is best known for its role in spindle orientation. Here we analyzed the role of Mud and two of its interactors, Asp and Dynein, in the regulation of centrosome numbers in Drosophila epithelial cells. We found that Dynein and Mud mainly initiate centrosome-spindle coupling prior to nuclear envelope breakdown (NEB) by promoting correct centrosome positioning or separation, while Asp acts largely independently of Dynein and Mud to maintain centrosome-spindle coupling. Failure in the centrosome-spindle coupling leads to mis-segregation of the two centrosomes into one daughter cell resulting in cells with supernumerary centrosomes during subsequent divisions. Together, we propose that Dynein, Mud and Asp sequentially operate during the cell cycle to ensure efficient centrosome-spindle coupling in mitosis preventing centrosome mis-segregation to maintain centrosome number. © 2017. Published by The Company of Biologists Ltd.

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

    Science.gov (United States)

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

    2016-02-02

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

  19. Specification of the somatic musculature in Drosophila.

    Science.gov (United States)

    Dobi, Krista C; Schulman, Victoria K; Baylies, Mary K

    2015-01-01

    The somatic muscle system formed during Drosophila embryogenesis is required for larvae to hatch, feed, and crawl. This system is replaced in the pupa by a new adult muscle set, responsible for activities such as feeding, walking, and flight. Both the larval and adult muscle systems are comprised of distinct muscle fibers to serve these specific motor functions. In this way, the Drosophila musculature is a valuable model for patterning within a single tissue: while all muscle cells share properties such as the contractile apparatus, properties such as size, position, and number of nuclei are unique for a particular muscle. In the embryo, diversification of muscle fibers relies first on signaling cascades that pattern the mesoderm. Subsequently, the combinatorial expression of specific transcription factors leads muscle fibers to adopt particular sizes, shapes, and orientations. Adult muscle precursors (AMPs), set aside during embryonic development, proliferate during the larval phases and seed the formation of the abdominal, leg, and flight muscles in the adult fly. Adult muscle fibers may either be formed de novo from the fusion of the AMPs, or are created by the binding of AMPs to an existing larval muscle. While less is known about adult muscle specification compared to the larva, expression of specific transcription factors is also important for its diversification. Increasingly, the mechanisms required for the diversification of fly muscle have found parallels in vertebrate systems and mark Drosophila as a robust model system to examine questions about how diverse cell types are generated within an organism.

  20. Multiscale diffusion in the mitotic Drosophila melanogaster syncytial blastoderm.

    Science.gov (United States)

    Daniels, Brian R; Rikhy, Richa; Renz, Malte; Dobrowsky, Terrence M; Lippincott-Schwartz, Jennifer

    2012-05-29

    Despite the fundamental importance of diffusion for embryonic morphogen gradient formation in the early Drosophila melanogaster embryo, there remains controversy regarding both the extent and the rate of diffusion of well-characterized morphogens. Furthermore, the recent observation of diffusional "compartmentalization" has suggested that diffusion may in fact be nonideal and mediated by an as-yet-unidentified mechanism. Here, we characterize the effects of the geometry of the early syncytial Drosophila embryo on the effective diffusivity of cytoplasmic proteins. Our results demonstrate that the presence of transient mitotic membrane furrows results in a multiscale diffusion effect that has a significant impact on effective diffusion rates across the embryo. Using a combination of live-cell experiments and computational modeling, we characterize these effects and relate effective bulk diffusion rates to instantaneous diffusion coefficients throughout the syncytial blastoderm nuclear cycle phase of the early embryo. This multiscale effect may be related to the effect of interphase nuclei on effective diffusion, and thus we propose that an as-yet-unidentified role of syncytial membrane furrows is to temporally regulate bulk embryonic diffusion rates to balance the multiscale effect of interphase nuclei, which ultimately stabilizes the shapes of various morphogen gradients.

  1. A role for CKII phosphorylation of the Cactus PEST domain in dorsoventral patterning of the Drosophila embryo

    OpenAIRE

    Liu, Zhi-Ping; Galindo, Rene L.; Wasserman, Steven A.

    1997-01-01

    Regulated proteolysis of Cactus, the cytoplasmic inhibitor of the Rel-related transcription factor Dorsal, is an essential step in patterning of the Drosophila embryo. Signal-induced Cactus degradation frees Dorsal for nuclear translocation on the ventral and lateral sides of the embryo, establishing zones of gene expression along the dorsoventral axis. Cactus stability is regulated by amino-terminal serine residues necessary for signal responsiveness, as well as by a carboxy-terminal PEST do...

  2. 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.

  3. Altered callose deposition during embryo sac formation of multi-pistil mutant (mp1) in Medicago sativa.

    Science.gov (United States)

    Zhou, H C; Jin, L; Li, J; Wang, X J

    2016-06-03

    Whether callose deposition is the cause or result of ovule sterility in Medicago sativa remains controversial, because it is unclear when and where changes in callose deposition and dissolution occur during fertile and sterile embryo sac formation. Here, alfalfa spontaneous multi-pistil mutant (mp1) and wild-type plants were used to compare the dynamics of callose deposition during embryo sac formation using microscopy. The results showed that both mutant and wild-type plants experienced megasporogenesis and megagametogenesis, and there was no significant difference during megasporogenesis. In contrast to the wild-type plants, in which the mature embryo sac was observed after three continuous cycles of mitosis, functional megaspores of mutant plants developed abnormally after the second round of mitosis, leading to degeneration of synergid, central, and antipodal cells. Callose deposition in both mutant and wild-type plants was first observed in the walls of megasporocytes, and then in the megaspore tetrad walls. After meiosis, the callose wall began to degrade as the functional megaspore underwent mitosis, and almost no callose was observed in the mature embryo sac in wild-type plants. However, callose deposition was observed in mp1 plants around the synergid, and increased with the development of the embryo sac, and was mainly deposited at the micropylar end. Our results indicate that synergid, central, and antipodal cells, which are surrounded by callose, may degrade owing to lack of nutrition. Callose accumulation around the synergid and at the micropylar end may hinder signals required for the pollen tube to enter the embryo sac, leading to abortion.

  4. Mechanical Coupling between Endoderm Invagination and Axis Extension in Drosophila.

    Directory of Open Access Journals (Sweden)

    Claire M Lye

    Full Text Available How genetic programs generate cell-intrinsic forces to shape embryos is actively studied, but less so how tissue-scale physical forces impact morphogenesis. Here we address the role of the latter during axis extension, using Drosophila germband extension (GBE as a model. We found previously that cells elongate in the anteroposterior (AP axis in the extending germband, suggesting that an extrinsic tensile force contributed to body axis extension. Here we further characterized the AP cell elongation patterns during GBE, by tracking cells and quantifying their apical cell deformation over time. AP cell elongation forms a gradient culminating at the posterior of the embryo, consistent with an AP-oriented tensile force propagating from there. To identify the morphogenetic movements that could be the source of this extrinsic force, we mapped gastrulation movements temporally using light sheet microscopy to image whole Drosophila embryos. We found that both mesoderm and endoderm invaginations are synchronous with the onset of GBE. The AP cell elongation gradient remains when mesoderm invagination is blocked but is abolished in the absence of endoderm invagination. This suggested that endoderm invagination is the source of the tensile force. We next looked for evidence of this force in a simplified system without polarized cell intercalation, in acellular embryos. Using Particle Image Velocimetry, we identify posteriorwards Myosin II flows towards the presumptive posterior endoderm, which still undergoes apical constriction in acellular embryos as in wildtype. We probed this posterior region using laser ablation and showed that tension is increased in the AP orientation, compared to dorsoventral orientation or to either orientations more anteriorly in the embryo. We propose that apical constriction leading to endoderm invagination is the source of the extrinsic force contributing to germband extension. This highlights the importance of physical

  5. 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.

  6. Circadian-independent cell mitosis in immortalized fibroblasts

    OpenAIRE

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

    2010-01-01

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

  7. Circadian-independent cell mitosis in immortalized fibroblasts.

    Science.gov (United States)

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

    2010-05-25

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

  8. Rheology of the Active Cell Cortex in Mitosis.

    Science.gov (United States)

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

    2016-08-09

    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. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  9. 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.

  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. Mitosis orientation in prostate epithelial cells changed by endocrine effect

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  12. A quantitative spatiotemporal atlas of gene expression in the Drosophila blastoderm.

    Science.gov (United States)

    Fowlkes, Charless C; Hendriks, Cris L Luengo; Keränen, Soile V E; Weber, Gunther H; Rübel, Oliver; Huang, Min-Yu; Chatoor, Sohail; DePace, Angela H; Simirenko, Lisa; Henriquez, Clara; Beaton, Amy; Weiszmann, Richard; Celniker, Susan; Hamann, Bernd; Knowles, David W; Biggin, Mark D; Eisen, Michael B; Malik, Jitendra

    2008-04-18

    To fully understand animal transcription networks, it is essential to accurately measure the spatial and temporal expression patterns of transcription factors and their targets. We describe a registration technique that takes image-based data from hundreds of Drosophila blastoderm embryos, each costained for a reference gene and one of a set of genes of interest, and builds a model VirtualEmbryo. This model captures in a common framework the average expression patterns for many genes in spite of significant variation in morphology and expression between individual embryos. We establish the method's accuracy by showing that relationships between a pair of genes' expression inferred from the model are nearly identical to those measured in embryos costained for the pair. We present a VirtualEmbryo containing data for 95 genes at six time cohorts. We show that known gene-regulatory interactions can be automatically recovered from this data set and predict hundreds of new interactions.

  13. Biolistic techniques for transfection of mosquito embryos (Anopheles gambiae).

    Science.gov (United States)

    Mialhe, E; Miller, L H

    1994-05-01

    To compensate for the extremely low rates of transformation by DNA microinjection into mosquito embryos of Anopheles gambiae, biolistic techniques were evaluated for introduction of DNA into large numbers of mosquito embryos. Biolistic experiments were first performed with a commercially available instrument intended for this purpose, according to the recommended procedure. The amount of DNA delivered was measured by the expression of luciferase under the control of the Drosophila heat shock protein (hsp) 70 promoter. Despite attempts to optimize biolistic parameters, the level of luciferase activity was low and highly variable. Two other methods of biolistic delivery of DNA-coated particles in aqueous suspension were then evaluated. One method used the gas explosion of the commercially available instrument (mentioned above) to drive an aqueous suspension of DNA-coated particles at high pressure. This method reproducibly increased the level of expression about 100-fold without greatly reducing embryo viability. Another method, which was recently described for plant transfection, uses lower pressure to deliver the aqueous suspension of DNA-coated particles. The level of expression of luciferase and the survival of embryos were equivalent to that obtained with the instrument modified for aqueous delivery of particles. Thus, both aqueous methods offer the advantages of reproducibly delivering more DNA to the embryos. Moreover, these methods could be suitable for delivering DNA mixed with proteins, such as restriction endonucleases and integrases, that may be destroyed by ethanol precipitation used in the standard PDS-1000/He method.

  14. Embryo-maternal communication

    DEFF Research Database (Denmark)

    Østrup, Esben; Hyttel, Poul; Østrup, Olga

    2011-01-01

    Communication during early pregnancy is essential for successful reproduction. In this review we address the beginning of the communication between mother and developing embryo; including morphological and transcriptional changes in the endometrium as well as epigenetic regulation mechanisms...... directing the placentation. An increasing knowledge of the embryo-maternal communication might not only help to improve the fertility of our farm animals but also our understanding of human health and reproduction....

  15. Adult Neurogenesis in Drosophila

    OpenAIRE

    Ismael Fernández-Hernández; Christa Rhiner; Eduardo Moreno

    2013-01-01

    Adult neurogenesis has been linked to several cognitive functions and neurological disorders. Description of adult neurogenesis in a model organism like Drosophila could facilitate the genetic study of normal and abnormal neurogenesis in the adult brain. So far, formation of new neurons has not been detected in adult fly brains and hence has been thought to be absent in Drosophila. Here, we used an improved lineage-labeling method to show that, surprisingly, adult neurogenesis occurs in the m...

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

    Science.gov (United States)

    2007-05-01

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

  17. 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.

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

    Science.gov (United States)

    O'Day, Danton H; Budniak, Aldona

    2015-02-01

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

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

    Science.gov (United States)

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

    2016-01-01

    Mitosis is a fast process that involves dramatic cellular remodeling and has a high energy demand. Whether autophagy is active or inactive during the early stages of mitosis in a naturally dividing cell is still debated. Here we aimed to use multiple assays to resolve this apparent discrepancy. Although the LC3 puncta number was reduced in mitosis, the four different cell lines we tested all have active autophagic flux in both interphase and mitosis. In addition, the autophagic flux was highly active in nocodazole-induced, double-thymidine synchronization released as well as naturally occurring mitosis in HeLa cells. Multiple autophagy proteins are upregulated in mitosis and the increased Beclin-1 level likely contributes to the active autophagic flux in early mitosis. It is interesting that although the autophagic flux is active throughout the cell cycle, early mitosis and S phase have relatively higher autophagic flux than G1 and late G2 phases, which might be helpful to degrade the damaged organelles and provide energy during S phase and mitosis. PMID:27213594

  20. Polarity and cell division orientation in the cleavage embryo: from worm to human

    Science.gov (United States)

    Ajduk, Anna; Zernicka-Goetz, Magdalena

    2016-01-01

    Cleavage is a period after fertilization, when a 1-cell embryo starts developing into a multicellular organism. Due to a series of mitotic divisions, the large volume of a fertilized egg is divided into numerous smaller, nucleated cells—blastomeres. Embryos of different phyla divide according to different patterns, but molecular mechanism of these early divisions remains surprisingly conserved. In the present paper, we describe how polarity cues, cytoskeleton and cell-to-cell communication interact with each other to regulate orientation of the early embryonic division planes in model animals such as Caenorhabditis elegans, Drosophila and mouse. We focus particularly on the Par pathway and the actin-driven cytoplasmic flows that accompany it. We also describe a unique interplay between Par proteins and the Hippo pathway in cleavage mammalian embryos. Moreover, we discuss the potential meaning of polarity, cytoplasmic dynamics and cell-to-cell communication as quality biomarkers of human embryos. PMID:26660321

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

    Science.gov (United States)

    Duro, Eris

    2015-01-01

    During eukaryotic cell division, chromosomes must be precisely partitioned to daughter cells. This relies on a mechanism to move chromosomes in defined directions within the parental cell. While sister chromatids are segregated from one another in mitosis and meiosis II, specific adaptations enable the segregation of homologous chromosomes during meiosis I to reduce ploidy for gamete production. Many of the factors that drive these directed chromosome movements are known, and their molecular mechanism has started to be uncovered. Here we review the mechanisms of eukaryotic chromosome segregation, with a particular emphasis on the modifications that ensure the segregation of homologous chromosomes during meiosis I. PMID:25593304

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

    Science.gov (United States)

    Duro, Eris; Marston, Adèle L

    2015-01-15

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

  3. 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.

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

    Science.gov (United States)

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

    2013-01-01

    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. 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. We propose an approach that assists pathologists in automated mitosis detection and counting. The proposed method, which is based on the most favorable texture features combination, examines the separability between different channels of color space. Blue-ratio channel provides more discriminative information for mitosis detection in histopathological images. Co-occurrence features, run-length features, and Scale-invariant feature transform (SIFT) features were extracted and used in the classification of mitosis. Finally, a classification is performed to put the candidate patch either in the mitosis class or in the non-mitosis class. Three different classifiers have been evaluated: Decision tree, linear kernel Support Vector Machine (SVM), and non-linear kernel SVM. We also evaluate the performance of the proposed framework using the modified biologically inspired model of HMAX and compare the results with other feature extraction methods such as dense SIFT. The proposed method has been tested on Mitosis detection in breast cancer histological images (MITOS) dataset provided for an International Conference on Pattern Recognition (ICPR) 2012 contest. The proposed framework achieved 76% recall, 75% precision and 76% F-measure. Different frameworks for classification have been evaluated for mitosis detection. In future work, instead of regions, we intend to compute features on the results of mitosis contour segmentation and use them to improve detection and classification rate.

  5. Cellular Mechanisms of Drosophila Heart Morphogenesis

    Directory of Open Access Journals (Sweden)

    Georg Vogler

    2015-02-01

    Full Text Available Many of the major discoveries in the fields of genetics and developmental biology have been made using the fruit fly, Drosophila melanogaster. With regard to heart development, the conserved network of core cardiac transcription factors that underlies cardiogenesis has been studied in great detail in the fly, and the importance of several signaling pathways that regulate heart morphogenesis, such as Slit/Robo, was first shown in the fly model. Recent technological advances have led to a large increase in the genomic data available from patients with congenital heart disease (CHD. This has highlighted a number of candidate genes and gene networks that are potentially involved in CHD. To validate genes and genetic interactions among candidate CHD-causing alleles and to better understand heart formation in general are major tasks. The specific limitations of the various cardiac model systems currently employed (mammalian and fish models provide a niche for the fly model, despite its evolutionary distance to vertebrates and humans. Here, we review recent advances made using the Drosophila embryo that identify factors relevant for heart formation. These underline how this model organism still is invaluable for a better understanding of CHD.

  6. Drosophila MOF controls Checkpoint protein2 and regulates genomic stability during early embryogenesis

    Directory of Open Access Journals (Sweden)

    Pushpavalli Sreerangam NCVL

    2013-01-01

    Full Text Available Abstract Background In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established. Results Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof1/+; mnkp6/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks. Conclusion mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using

  7. Drosophila MOF controls Checkpoint protein2 and regulates genomic stability during early embryogenesis.

    Science.gov (United States)

    Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Ramaiah, M Janaki; Chowdhury, Debabani Roy; Bhadra, Utpal; Pal-Bhadra, Manika

    2013-01-24

    In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established. Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof¹/+; mnkp⁶/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks. mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry out the interaction of MOF

  8. Oxygen diffusion in fish embryos

    NARCIS (Netherlands)

    Kranenbarg, S.

    2002-01-01

    All vertebrate embryos pass through a developmental period of remarkably low morphological variability. This period has been called phylotypic period. During the phylotypic period, organogenesis takes place, including blood vessel development. Before the phylotypic period, the embryos

  9. The First Human Cloned Embryo.

    Science.gov (United States)

    Cibelli, Jose B.; Lanza, Robert P.; West, Michael D.; Ezzell, Carol

    2002-01-01

    Describes a process known as parthenogenesis which produces cloned, early-stage embryos and human embryos generated only from eggs. Speculates that this technology puts therapeutic cloning within reach. (DDR)

  10. 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.

  11. Fluorescent visualization of macromolecules in Drosophila whole mounts.

    Science.gov (United States)

    Ramos, Ricardo Guelerman Pinheiro; Machado, Luciana Claudia Herculano; Moda, Livia Maria Rosatto

    2010-01-01

    The ability to determine the expression dynamics of individual genes "in situ" by visualizing the precise spatial and temporal distribution of their products in whole mounts by histochemical and immunocytochemical reactions has revolutionized our understanding of cellular processes. Drosophila developmental genetics was one of the fields that benefited most from these technologies, and a variety of fluorescent methods were specifically designed for investigating the localization of developmentally important proteins and cell markers during embryonic and post embryonic stages of this model organism. In this chapter we present detailed protocols for fluorescence immunocytochemistry of whole mount embryos, imaginal discs, pupal retinas, and salivary glands of Drosophila melanogaster, as well as methods for fluorescent visualization of specific subcellular structures in these tissues.

  12. Negative regulation of P element excision by the somatic product and terminal sequences of P in drosophila melanogaster

    Science.gov (United States)

    A transient in vivo P element excision assay was used to test the regulatory properties of putative repressor-encoding plasmids in Drosophila melanogaster embryos. The somatic expression of an unmodified transposase transcription unit under the control of a heat shock gene promoter (phsn) effectivel...

  13. Endosomal recycling controls plasma membrane area during mitosis.

    Science.gov (United States)

    Boucrot, Emmanuel; Kirchhausen, Tomas

    2007-05-08

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

  14. 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.

  15. 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.

  16. 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.

  17. Regulation of APC/C activators in mitosis and meiosis.

    Science.gov (United States)

    Pesin, Jillian A; Orr-Weaver, Terry L

    2008-01-01

    The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit E3 ubiquitin ligase that triggers the degradation of multiple substrates during mitosis. Cdc20/Fizzy and Cdh1/Fizzy-related activate the APC/C and confer substrate specificity through complex interactions with both the core APC/C and substrate proteins. The regulation of Cdc20 and Cdh1 is critical for proper APC/C activity and occurs in multiple ways: targeted protein degradation, phosphorylation, and direct binding of inhibitory proteins. During the specialized divisions of meiosis, the activity of the APC/C must be modified to achieve proper chromosome segregation. Recent studies show that one way in which APC/C activity is modified is through the use of meiosis-specific APC/C activators. Furthermore, regulation of the APC/C during meiosis is carried out by both mitotic regulators of the APC/C as well as meiosis-specific regulators. Here, we review the regulation of APC/C activators during mitosis and the role and regulation of the APC/C during female meiosis.

  18. INPP5E Preserves Genomic Stability through Regulation of Mitosis.

    Science.gov (United States)

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

    2017-03-15

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

  19. Kindlin1 regulates microtubule function to ensure normal mitosis.

    Science.gov (United States)

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

    2016-08-01

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

  20. Ovarian stimulation and embryo quality

    NARCIS (Netherlands)

    Baart, Esther; Macklon, Nick S.; Fauser, Bart J. C. M.

    2009-01-01

    To Study the effects of different ovarian stimulation approaches on oocyte and embryo quality, it is imperative to assess embryo quality with a reliable and objective method. Embryos rated as high quality by standardized morphological assessment are associated with higher implantation and pregnancy

  1. 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.

  2. 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.

  3. 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.

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

    Science.gov (United States)

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

    2016-10-01

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

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

    Science.gov (United States)

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

    2016-01-06

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

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

    Science.gov (United States)

    Jin, J; Liu, J

    2016-08-31

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

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

    DEFF Research Database (Denmark)

    Pedersen, Rune Troelsgaard

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

  8. Importance of the CEP215-pericentrin interaction for centrosome maturation during mitosis.

    Science.gov (United States)

    Kim, Seongjae; Rhee, Kunsoo

    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-protein interaction properties of CEP215 to its biological functions. The results showed that CEP215 and pericentrin, another major PCM component, is interdependent for their accumulation at the spindle poles during mitosis. As a result, The CEP215-pericentrin interaction is required for centrosome maturation and subsequent bipolar spindle formation during mitosis. On the other hand, CEP215 interaction with γ-tubulin is dispensable for centrosome maturation. Our results provide an insight how PCM components are assembled to form a spindle pole during mitosis.

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

    Science.gov (United States)

    Yanagida, Mitsuhiro

    2014-09-02

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

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

    DEFF Research Database (Denmark)

    Pedersen, Rune Troelsgaard

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

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

    Science.gov (United States)

    Ariz, Mohd; Mainpal, Rana; Subramaniam, Kuppuswamy

    2009-02-15

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

  12. The developmental transcriptome of Drosophila melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    University of Connecticut; Graveley, Brenton R.; Brooks, Angela N.; Carlson, Joseph W.; Duff, Michael O.; Landolin, Jane M.; Yang, Li; Artieri, Carlo G.; van Baren, Marijke J.; Boley, Nathan; Booth, Benjamin W.; Brown, James B.; Cherbas, Lucy; Davis, Carrie A.; Dobin, Alex; Li, Renhua; Lin, Wei; Malone, John H.; Mattiuzzo, Nicolas R.; Miller, David; Sturgill, David; Tuch, Brian B.; Zaleski, Chris; Zhang, Dayu; Blanchette, Marco; Dudoit, Sandrine; Eads, Brian; Green, Richard E.; Hammonds, Ann; Jiang, Lichun; Kapranov, Phil; Langton, Laura; Perrimon, Norbert; Sandler, Jeremy E.; Wan, Kenneth H.; Willingham, Aarron; Zhang, Yu; Zou, Yi; Andrews, Justen; Bicke, Peter J.; Brenner, Steven E.; Brent, Michael R.; Cherbas, Peter; Gingeras, Thomas R.; Hoskins, Roger A.; Kaufman, Thomas C.; Oliver, Brian; Celniker, Susan E.

    2010-12-02

    . Whereas, 20% of Drosophila genes are annotated as encoding alternatively spliced premRNAs, splice-junction microarray experiments indicate that this number is at least 40% (ref. 7). Determining the diversity of mRNAs generated by alternative promoters, alternative splicing and RNA editing will substantially increase the inferred protein repertoire. Non-coding RNA genes (ncRNAs) including short interfering RNAs (siRNAs) and microRNAS (miRNAs) (reviewed in ref. 10), and longer ncRNAs such as bxd (ref. 11) and rox (ref. 12), have important roles in gene regulation, whereas others such as small nucleolar RNAs (snoRNAs)and small nuclear RNAs (snRNAs) are important components of macromolecular machines such as the ribosome and spliceosome. The transcription and processing of these ncRNAs must also be fully documented and mapped. As part of the modENCODE project to annotate the functional elements of the D. melanogaster and Caenorhabditis elegans genomes, we used RNA-Seq and tiling microarrays to sample the Drosophila transcriptome at unprecedented depth throughout development from early embryo to ageing male and female adults. We report on a high-resolution view of the discovery, structure and dynamic expression of the D. melanogaster transcriptome.

  13. The Drosophila HOAP protein is required for telomere capping.

    Science.gov (United States)

    Cenci, Giovanni; Siriaco, Giorgia; Raffa, Grazia D; Kellum, Rebecca; Gatti, Maurizio

    2003-01-01

    HOAP (HP1/ORC-associated protein) has recently been isolated from Drosophila melanogaster embryos as part of a cytoplasmic complex that contains heterochromatin protein 1 (HP1) and the origin recognition complex subunit 2 (ORC2). Here, we show that caravaggio, a mutation in the HOAP-encoding gene, causes extensive telomere-telomere fusions in larval brain cells, indicating that HOAP is required for telomere capping. Our analyses indicate that HOAP is specifically enriched at mitotic chromosome telomeres, and strongly suggest that HP1 and HOAP form a telomere-capping complex that does not contain ORC2.

  14. The non-catalytic domains of Drosophila katanin regulate its abundance and microtubule-disassembly activity.

    Directory of Open Access Journals (Sweden)

    Kyle D Grode

    Full Text Available Microtubule severing is a biochemical reaction that generates an internal break in a microtubule and regulation of microtubule severing is critical for cellular processes such as ciliogenesis, morphogenesis, and meiosis and mitosis. Katanin is a conserved heterodimeric ATPase that severs and disassembles microtubules, but the molecular determinants for regulation of microtubule severing by katanin remain poorly defined. Here we show that the non-catalytic domains of Drosophila katanin regulate its abundance and activity in living cells. Our data indicate that the microtubule-interacting and trafficking (MIT domain and adjacent linker region of the Drosophila katanin catalytic subunit Kat60 cooperate to regulate microtubule severing in two distinct ways. First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation. The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60. Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules. On the basis of our data, we propose that the non-catalytic domains of Drosophila katanin serve as the principal sites of integration of regulatory inputs, thereby controlling its ability to sever and disassemble microtubules.

  15. Automated mitosis detection in histopathology using morphological and multi-channel statistics features.

    Science.gov (United States)

    Irshad, Humayun

    2013-01-01

    According to Nottingham grading system, mitosis count plays a critical role in cancer diagnosis and grading. Manual counting of mitosis is tedious and subject to considerable inter- and intra-reader variations. The aim is to improve the accuracy of mitosis detection by selecting the color channels that better capture the statistical and morphological features, which classify mitosis from other objects. We propose a framework that includes comprehensive analysis of statistics and morphological features in selected channels of various color spaces that assist pathologists in mitosis detection. In candidate detection phase, we perform Laplacian of Gaussian, thresholding, morphology and active contour model on blue-ratio image to detect and segment candidates. In candidate classification phase, we extract a total of 143 features including morphological, first order and second order (texture) statistics features for each candidate in selected channels and finally classify using decision tree classifier. The proposed method has been evaluated on Mitosis Detection in Breast Cancer Histological Images (MITOS) dataset provided for an International Conference on Pattern Recognition 2012 contest and achieved 74% and 71% detection rate, 70% and 56% precision and 72% and 63% F-Measure on Aperio and Hamamatsu images, respectively. The proposed multi-channel features computation scheme uses fixed image scale and extracts nuclei features in selected channels of various color spaces. This simple but robust model has proven to be highly efficient in capturing multi-channels statistical features for mitosis detection, during the MITOS international benchmark. Indeed, the mitosis detection of critical importance in cancer diagnosis is a very challenging visual task. In future work, we plan to use color deconvolution as preprocessing and Hough transform or local extrema based candidate detection in order to reduce the number of candidates in mitosis and non-mitosis classes.

  16. BMAA neurotoxicity in Drosophila.

    Science.gov (United States)

    Zhou, Xianchong; Escala, Wilfredo; Papapetropoulos, Spyridon; Bradley, Walter G; Zhai, R Grace

    2009-01-01

    We report the establishment of an in vivo model using the fruit fly Drosophila melanogaster to investigate the toxic effects of L-BMAA. We found that dietary intake of BMAA reduced the lifespan as well as the neurological functions of flies. Furthermore, we have developed an HPLC method to reliably detect both free and protein-bound BMAA in fly tissue extracts.

  17. Cancer in Drosophila

    DEFF Research Database (Denmark)

    Herranz, Héctor; Eichenlaub, Teresa; Cohen, Stephen M

    2016-01-01

    Cancer genomics has greatly increased our understanding of the complexity of the genetic and epigenetic changes found in human tumors. Understanding the functional relationships among these elements calls for the use of flexible genetic models. We discuss the use of Drosophila models to study...

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

    Science.gov (United States)

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

    2017-01-01

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

  19. 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

  20. Nucleocytoplasmic shuttling mediates the dynamic maintenance of nuclear Dorsal levels during Drosophila embryogenesis

    DEFF Research Database (Denmark)

    DeLotto, Robert; DeLotto, Yvonne; Steward, Ruth

    2007-01-01

    of Dorsal. We also find that diffusion of Dorsal is partially constrained to cytoplasmic islands surrounding individual syncitial nuclei. A model is proposed in which the generation and maintenance of the Dorsal gradient is a consequence of an active process involving both restricted long-range diffusion......In Drosophila, the NF-kappaB/REL family transcription factor, Dorsal, redistributes from the cytoplasm to nuclei, forming a concentration gradient across the dorsoventral axis of the embryo. Using live imaging techniques in conjunction with embryos expressing a chimeric Dorsal-GFP, we demonstrate...

  1. 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.

  2. 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.

  3. Oriented cell divisions in the extending germband of Drosophila.

    Science.gov (United States)

    da Silva, Sara Morais; Vincent, Jean-Paul

    2007-09-01

    Tissue elongation is a general feature of morphogenesis. One example is the extension of the germband, which occurs during early embryogenesis in Drosophila. In the anterior part of the embryo, elongation follows from a process of cell intercalation. In this study, we follow cell behaviour at the posterior of the extending germband. We find that, in this region, cell divisions are mostly oriented longitudinally during the fast phase of elongation. Inhibiting cell divisions prevents longitudinal deformation of the posterior region and leads to an overall reduction in the rate and extent of elongation. Thus, as in zebrafish embryos, cell intercalation and oriented cell division together contribute to tissue elongation. We also show that the proportion of longitudinal divisions is reduced when segmental patterning is compromised, as, for example, in even skipped (eve) mutants. Because polarised cell intercalation at the anterior germband also requires segmental patterning, a common polarising cue might be used for both processes. Even though, in fish embryos, both mechanisms require the classical planar cell polarity (PCP) pathway, germband extension and oriented cell divisions proceed normally in embryos lacking dishevelled (dsh), a key component of the PCP pathway. An alternative means of planar polarisation must therefore be at work in the embryonic epidermis.

  4. Ontogeny of rat chondrocyte proliferation: studies in embryo, adult and osteoarthritic (OA) cartilage

    Institute of Scientific and Technical Information of China (English)

    Madaí A GóMEZ-CAMARILLO; Juan B.KOURI

    2005-01-01

    The aim of this work was to study the ontogeny of chondrocyte cell division using embryo, adult and osteoarthritic (OA) cartilage. We searched for mitosis phases and performed a comparative evaluation of mitotic index, basic fibroblast growth factor b (FGFb), transforming growth factor β1 (TGF-β1) receptors, cyclin dependent kinase (CDK1)and Cyclin-B expression in fetal, neonate, 3, 5, 8 weeks old rats and experimental OA. Our results showed that mitosis phases were observed in all normal cartilage studied, although, we found a decrease in mitotic index in relation to tissue development. No mitosis was detected in OA cartilage. We also found a statistical significant reduction in cell number in OA cartilage, compared with the normal tissue. Furthermore, FGFb and TGF-β1 receptors diminished in relation to tissue development, and were very scarce in experimental OA. Western blot assays showed CDK-1 expression in all cases, including human-OA cartilage. Similar results were observed for Cyclin-B, except for 8 weeks, when it was not expressed. Our results suggest that cell division seems to be scarce, if not absent within the OA cartilage studied.Nevertheless, the existence of factors essential for cell division leaves open the question concerning chondrocyte proliferation in OA cartilage, which is likely to be present in the early stages of the disease.

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

    Science.gov (United States)

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

    2014-01-21

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-08-13

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

  7. windbeutel, a gene required for dorsoventral patterning in Drosophila, encodes a protein that has homologies to vertebrate proteins of the endoplasmic reticulum

    OpenAIRE

    Konsolaki, Mary; Schüpbach, Trudi

    1998-01-01

    The formation of the dorsoventral axis of the Drosophila embryo depends on cell–cell interactions that take place in the female ovary and involve the activation of transmembrane receptors by secreted ligands. The gene windbeutel functions in the somatic follicle cells of the ovary and is required for the generation of a signal that will determine the ventral side of the embryo. This signal originates in the follicle cells during oogenesis, but its actions are only manifested after fertilizati...

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

    Science.gov (United States)

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

    2011-01-01

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

  9. Mitosis Counting in Breast Cancer: Object-Level Interobserver Agreement and Comparison to an Automatic Method.

    Science.gov (United States)

    Veta, Mitko; van Diest, Paul J; Jiwa, Mehdi; Al-Janabi, Shaimaa; Pluim, Josien P W

    2016-01-01

    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 reproducibility, particularly among non-experts. Inter- and intraobserver reproducibility of mitosis counting can be improved when a strict protocol is defined and followed. Previous studies have examined only the agreement in terms of the mitotic count or the mitotic activity score. Studies of the observer agreement at the level of individual objects, which can provide more insight into the procedure, have not been performed thus far. The development of automatic mitosis detection methods has received large interest in recent years. Automatic image analysis is viewed as a solution for the problem of subjectivity of mitosis counting by pathologists. In this paper we describe the results from an interobserver agreement study between three human observers and an automatic method, and make two unique contributions. For the first time, we present an analysis of the object-level interobserver agreement on mitosis counting. Furthermore, we train an automatic mitosis detection method that is robust with respect to staining appearance variability and compare it with the performance of expert observers on an "external" dataset, i.e. on histopathology images that originate from pathology labs other than the pathology lab that provided the training data for the automatic method. The object-level interobserver study revealed that pathologists often do not agree on individual objects, even if this is not reflected in the mitotic count. The disagreement is larger for objects from smaller size, which suggests that adding a size constraint in the mitosis counting protocol can improve reproducibility. The automatic mitosis detection method can perform mitosis counting in an unbiased way, with substantial

  10. Stage-specific proteome signatures in early bovine embryo development.

    Science.gov (United States)

    Deutsch, Daniela R; Fröhlich, Thomas; Otte, Kathrin A; Beck, Andrea; Habermann, Felix A; Wolf, Eckhard; Arnold, Georg J

    2014-10-03

    Development of early embryonic stages before activation of the embryonic genome depends on sufficiently stored products of the maternal genome, adequate recruitment and degradation of mRNAs, as well as activation, deactivation, and relocation of proteins. By application of an isobaric tagging for relative and absolute quantification (iTRAQ)-based approach, the proteomes of bovine embryos at the zygote and 2-cell and 4-cell stage with MII oocytes as a reference were quantitatively analyzed. Of 1072 quantified proteins, 87 differed significantly in abundance between the four stages. The proteomes of 2-cell and 4-cell embryos differed most from the reference MII oocyte, and a considerable fraction of proteins continuously increased in abundance during the stages analyzed, despite a strongly attenuated rate of translation reported for this period. Bioinformatic analysis revealed particularly interesting proteins involved in the p53 pathway, lipid metabolism, and mitosis. Verification of iTRAQ results by targeted SRM (selected reaction monitoring) analysis revealed excellent agreement for all five proteins analyzed. By principal component analysis, SRM quantifications comprising a panel of only five proteins were shown to discriminate between all four developmental stages analyzed here. For future experiments, an expanded SRM protein panel will provide the potential to detect developmental disturbances with high sensitivity and enable first insights into the underlying molecular pathways.

  11. Inhibition of endocytic vesicle fusion by Plk1-mediated phosphorylation of vimentin during mitosis.

    Science.gov (United States)

    Ikawa, Keisuke; Satou, Ayaka; Fukuhara, Mitsuko; Matsumura, Shigeru; Sugiyama, Naoyuki; Goto, Hidemasa; Fukuda, Mitsunori; Inagaki, Masaki; Ishihama, Yasushi; Toyoshima, Fumiko

    2014-01-01

    Endocytic vesicle fusion is inhibited during mitosis, but the molecular pathways that mediate the inhibition remain unclear. Here we uncovered an essential role of Polo-like kinase 1 (Plk1) in this mechanism. Phosphoproteomic analysis revealed that Plk1 phosphorylates the intermediate filament protein vimentin on Ser459, which is dispensable for its filament formation but is necessary for the inhibition of endocytic vesicle fusion in mitosis. Furthermore, this mechanism is required for integrin trafficking toward the cleavage furrow during cytokinesis. Our results thus identify a novel mechanism for fusion inhibition in mitosis and implicate its role in vesicle trafficking after anaphase onset.

  12. Singling out Drosophila tendon cells: a dialogue between two distinct cell types.

    Science.gov (United States)

    Volk, T

    1999-11-01

    The precise match between somatic muscles and their epidermal attachment cells is achieved through a continuous dialogue between these two cell types. Whereas tendon cells direct myotube migration and final patterning, the muscles are essential for the maintenance of the fate of tendon cells. The Drosophila neuregulin-like ligand, Vein, and its receptor, the epidermal growth factor receptor (Egfr), are critical components in the inductive signaling process that takes place between muscles and tendon cells. Additional gene products that relay the Vein-Egfr effect in Drosophila are conserved in the vertebrate neuregulin-mediated cascade. This review describes genetic and molecular aspects of the muscle-tendon inductive processes in Drosophila, and compares them with the relevant mechanisms in the vertebrate embryo.

  13. Drosophila by the dozen

    Energy Technology Data Exchange (ETDEWEB)

    Celniker, Susan E.; Hoskins, Roger A.

    2007-07-13

    This year's conference on Drosophila research illustratedwell the current focus of Drosophila genomics on the comprehensiveidentification of functional elements in the genome sequence, includingmRNA transcripts arising from multiple alternative start sites and splicesites, a multiplicity of noncoding transcripts and small RNAs,identification of binding sites for transcription factors, sequenceconservation in related species and sequence variation within species.Resources and technologies for genetics and functional genomics aresteadily being improved, including the building of collections oftransposon insertion mutants and hairpin constructs for RNA interference(RNAi). The conference also highlighted progress in the use of genomicinformation by many laboratories to study diverse aspects of biology andmodels of human disease. Here we will review a few highlights of especialinterest to readers of Genome Biology.

  14. Gender determination of avian embryo

    Science.gov (United States)

    Daum, Keith A.; Atkinson, David A.

    2002-01-01

    Disclosed is a method for gender determination of avian embryos. During the embryo incubation process, the outer hard shells of eggs are drilled and samples of allantoic fluid are removed. The allantoic fluids are directly introduced into an ion mobility spectrometer (IMS) for analysis. The resulting spectra contain the relevant marker peaks in the positive or negative mode which correlate with unique mobilities which are sex-specific. This way, the gender of the embryo can be determined.

  15. Schistosoma mansoni polo-like kinases and their function in control of mitosis and parasite reproduction

    Directory of Open Access Journals (Sweden)

    Colette Dissous

    2011-06-01

    Full Text Available Polo-like kinases are important regulators of cell cycle progression and mitosis. They constitute a family of conserved serine/threonine kinases which are highly related in their catalytic domains and contain polo boxes involved in protein-protein interactions and subcellular localization. In mammals, five Plks (Plk 1-5 encompass diverse roles in centrosome dynamics, spindle formation, intra S-phase and G2/M checkpoints and DNA damage response. Plk1 is a key positive regulator of mitosis and is overexpressed in various types of cancers. Plk4 is a divergent member of the Plk family, with essential functions in centriole duplication. Homozygous disruption of Plk1 or Plk4 in mice is lethal in embryos. Two Plk members SmPlk1 and SmSak, homologous to Plk1 and Plk4 respectively, are present in the parasitic platyhelminth Schistosoma mansoni. Structural and functional analyses of SmPlk1 have demonstrated its conserved function in the regulation of cell cycle G2/M transition in Xenopus oocytes. The anti-cancer drug BI 2536 (the most potent and selective Plk1 inhibitor inhibits specifically the catalytic activity of SmPlk1 and induced profound alterations in schistosome gonads, indicating a role of SmPlk1 in parasite gametogenesis and its potential as a novel chemotherapeutic target against schistosomiasis. Functions of SmSak in cell cycle regulation and schistosome gonad development are currently investigatedQuinases do tipo Polo ("polo-like" são importantes reguladores da progressão do ciclo celular e da mitose. Elas constituem uma família de serina/treonina quinases que são altamente relacionadas entre si no seu domínio catalítico e contêm blocos "polo" envolvidos com interações proteína-proteína e com localização subcelular. Em mamíferos, cinco Plks (Plk 1-5 englobam diversos papéis na dinâmica do centrossomo, formação do fuso, "checkpoints" dentro da fase S e da transição G2/M, e na resposta aos danos do DNA. Plk1 é um regulador

  16. A Conserved Di-Basic Motif of Drosophila Crumbs Contributes to Efficient ER Export.

    Science.gov (United States)

    Kumichel, Alexandra; Kapp, Katja; Knust, Elisabeth

    2015-06-01

    The Drosophila type I transmembrane protein Crumbs is an apical determinant required for the maintenance of apico-basal epithelial cell polarity. The level of Crumbs at the plasma membrane is crucial, but how it is regulated is poorly understood. In a genetic screen for regulators of Crumbs protein trafficking we identified Sar1, the core component of the coat protein complex II transport vesicles. sar1 mutant embryos show a reduced plasma membrane localization of Crumbs, a defect similar to that observed in haunted and ghost mutant embryos, which lack Sec23 and Sec24CD, respectively. By pulse-chase assays in Drosophila Schneider cells and analysis of protein transport kinetics based on Endoglycosidase H resistance we identified an RNKR motif in Crumbs, which contributes to efficient ER export. The motif identified fits the highly conserved di-basic RxKR motif and mediates interaction with Sar1. The RNKR motif is also required for plasma membrane delivery of transgene-encoded Crumbs in epithelial cells of Drosophila embryos. Our data are the first to show that a di-basic motif acts as a signal for ER exit of a type I plasma membrane protein in a metazoan organism. © 2015 The Authors. Traffic published by John Wiley & Sons Ltd.

  17. The Arabidopsis thaliana F-box protein FBL17 is essential for progression through the second mitosis during pollen development.

    Directory of Open Access Journals (Sweden)

    Andi Gusti

    Full Text Available In fungi and metazoans, the SCF-type Ubiquitin protein ligases (E3s play a critical role in cell cycle regulation by degrading negative regulators, such as cell cycle-dependent kinase inhibitors (CKIs at the G1-to-S-phase checkpoint. Here we report that FBL17, an Arabidopsis thaliana F-box protein, is involved in cell cycle regulation during male gametogenesis. FBL17 expression is strongly enhanced in plants co-expressing E2Fa and DPa, transcription factors that promote S-phase entry. FBL17 loss-of-function mutants fail to undergo pollen mitosis II, which generates the two sperm cells in mature A. thaliana pollen. Nonetheless, the single sperm cell-like cell in fbl17 mutants is functional but will exclusively fertilize the egg cell of the female gametophyte, giving rise to an embryo that will later abort, most likely due to the lack of functional endosperm. Seed abortion can, however, be overcome by mutations in FIE, a component of the Polycomb group complex, overall resembling loss-of-function mutations in the A. thaliana cyclin-dependent kinase CDKA;1. Finally we identified ASK11, as an SKP1-like partner protein of FBL17 and discuss a possible mechanism how SCF(FBL17 may regulate cell division during male gametogenesis.

  18. Genome-wide analysis of promoter architecture in Drosophila melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Hoskins, Roger A.; Landolin, Jane M.; Brown, James B.; Sandler, Jeremy E.; Takahashi, Hazuki; Lassmann, Timo; Yu, Charles; Booth, Benjamin W.; Zhang, Dayu; Wan, Kenneth H.; Yang, Li; Boley, Nathan; Andrews, Justen; Kaufman, Thomas C.; Graveley, Brenton R.; Bickel, Peter J.; Carninci, Piero; Carlson, Joseph W.; Celniker, Susan E.

    2010-10-20

    Core promoters are critical regions for gene regulation in higher eukaryotes. However, the boundaries of promoter regions, the relative rates of initiation at the transcription start sites (TSSs) distributed within them, and the functional significance of promoter architecture remain poorly understood. We produced a high-resolution map of promoters active in the Drosophila melanogaster embryo by integrating data from three independent and complementary methods: 21 million cap analysis of gene expression (CAGE) tags, 1.2 million RNA ligase mediated rapid amplification of cDNA ends (RLMRACE) reads, and 50,000 cap-trapped expressed sequence tags (ESTs). We defined 12,454 promoters of 8037 genes. Our analysis indicates that, due to non-promoter-associated RNA background signal, previous studies have likely overestimated the number of promoter-associated CAGE clusters by fivefold. We show that TSS distributions form a complex continuum of shapes, and that promoters active in the embryo and adult have highly similar shapes in 95% of cases. This suggests that these distributions are generally determined by static elements such as local DNA sequence and are not modulated by dynamic signals such as histone modifications. Transcription factor binding motifs are differentially enriched as a function of promoter shape, and peaked promoter shape is correlated with both temporal and spatial regulation of gene expression. Our results contribute to the emerging view that core promoters are functionally diverse and control patterning of gene expression in Drosophila and mammals.

  19. Both Chromosome Decondensation and Condensation Are Dependent on DNA Replication in C. elegans Embryos

    Directory of Open Access Journals (Sweden)

    Remi Sonneville

    2015-07-01

    Full Text Available During cell division, chromatin alternates between a condensed state to facilitate chromosome segregation and a decondensed form when DNA replicates. In most tissues, S phase and mitosis are separated by defined G1 and G2 gap phases, but early embryogenesis involves rapid oscillations between replication and mitosis. Using Caenorhabditis elegans embryos as a model system, we show that chromosome condensation and condensin II concentration on chromosomal axes require replicated DNA. In addition, we found that, during late telophase, replication initiates on condensed chromosomes and promotes the rapid decondensation of the chromatin. Upon replication initiation, the CDC-45-MCM-GINS (CMG DNA helicase drives the release of condensin I complexes from chromatin and the activation or displacement of inactive MCM-2–7 complexes, which together with the nucleoporin MEL-28/ELYS tethers condensed chromatin to the nuclear envelope, thereby promoting chromatin decondensation. Our results show how, in an early embryo, the chromosome-condensation cycle is functionally linked with DNA replication.

  20. 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.

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

    Science.gov (United States)

    Liang, Kaiwei; Woodfin, Ashley R; Slaughter, Brian D; Unruh, Jay R; Box, Andrew C; Rickels, Ryan A; Gao, Xin; Haug, Jeffrey S; Jaspersen, Sue L; Shilatifard, Ali

    2015-11-05

    Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division.

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

    Science.gov (United States)

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

    2013-01-01

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

  3. 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.

  4. Release of chromosomes from the nuclear envelope: a universal mechanism for eukaryotic mitosis?

    Science.gov (United States)

    Kanoh, Junko

    2013-01-01

    Multiple domains of chromosomes are associated with the nuclear envelope (NE) in interphase. The association between chromosomes and the NE is involved in a variety of chromosomal reactions, such as gene expression and DNA repair. However, efficient chromosome movements are required for the fidelity of chromosome segregation in mitosis. Most higher eukaryotes perform open mitosis, in which the NE is broken down, enabling chromosomes to be released from the NE as well as spindle microtubules to access to kinetochores. By contrast, lower eukaryotes, such as Schizosaccharomyces pombe, perform closed mitosis, during which NE breakdown does not occur. In S. pombe, telomeres are tethered to the NE in interphase. Phosphorylation of the telomere-binding protein Rap1 at M phase promotes transient dissociation of telomeres from the NE, facilitating the faithful chromosome segregation. These findings imply a common mechanism for genome stability via the dissociation of chromosomes from the NE in eukaryotic mitosis.

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

    Science.gov (United States)

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

    2015-08-05

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

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

    Science.gov (United States)

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

    2002-08-01

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

  7. 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

  8. GLD-3 and Control of the Mitosis/Meiosis Decision in the Germline of Caenorhabditis elegans

    National Research Council Canada - National Science Library

    Eckmann, Christian R; Crittenden, Sarah L; Suh, Nayoung; Kimble, Judith

    2004-01-01

    .... Furthermore, FBF acts largely upstream of gld-3 in the mitosis/meiosis decision. By contrast, GLD-3 acts upstream of FBF in the sperm/oocyte decision, and GLD-3 protein can antagonize FBF binding to RNA regulatory elements...

  9. The Hydra FGFR, Kringelchen, partially replaces the Drosophila Heartless FGFR.

    Science.gov (United States)

    Rudolf, Anja; Hübinger, Christine; Hüsken, Katrin; Vogt, Angelika; Rebscher, Nicole; Onel, Susanne-Filiz; Renkawitz-Pohl, Renate; Hassel, Monika

    2013-05-01

    Fibroblast growth factor receptors (FGFR) are highly conserved receptor tyrosine kinases, and evolved early in metazoan evolution. In order to investigate their functional conservation, we asked whether the Kringelchen FGFR in the freshwater polyp Hydra vulgaris, is able to functionally replace FGFR in fly embryos. In Drosophila, two endogenous FGFR, Breathless (Btl) and Heartless (Htl), ensure formation of the tracheal system and mesodermal cell migration as well as formation of the heart. Using UAS-kringelchen-5xmyc transgenic flies and targeted expression, we show that Kringelchen is integrated correctly into the cell membrane of mesodermal and tracheal cells in Drosophila. Nevertheless, Kringelchen expression driven in tracheal cells failed to rescue the btl (LG19) mutant. The Hydra FGFR was able to substitute for Heartless in the htl (AB42) null mutant; however, this occurred only during early mesodermal cell migration. Our data provide evidence for functional conservation of this early-diverged FGFR across these distantly related phyla, but also selectivity for the Htl FGFR in the Drosophila system.

  10. Hybrid Lethal Systems in the Drosophila Melanogaster Species Complex. I. the Maternal Hybrid Rescue (Mhr) Gene of Drosophila Simulans

    Science.gov (United States)

    Sawamura, K.; Taira, T.; Watanabe, T. K.

    1993-01-01

    Hybrid females from Drosophila simulans females X Drosophila melanogaster males die as embryos while hybrid males from the reciprocal cross die as late larvae. The other two classes are sterile adults. Letting C, X, and Y designate egg cytoplasm, X, and Y chromosomes, respectively, and subscripts m and s stand for melanogaster and simulans, C(m)X(m)Y(s) males are lethal in the larval stage and are rescued by the previously reported genes, Lhr (Lethal hybrid rescue) in simulans or Hmr (Hybrid male rescue) in melanogaster. We report here another rescue gene located on the second chromosome of simulans, mhr (maternal hybrid rescue) that, when present in the mother, rescues C(s)X(m)X(s) females from embryonic lethality. It has been postulated that the hybrids not carrying the X(s) like C(m)X(m)Y(s) males are larval lethal and that the hybrids carrying both the C(s) and the X(m) like C(s)X(m)X(s) females are embryonic lethal. According to these postulates C(s)X(m)Y(s) males (obtained by mating attached-X simulans females to melanogaster males) should be doubly lethal, at both embryo and larval stages. When both rescuing genes are present, Hmr in the father and mhr in the mother, males of this genotype are fully viable, as predicted. PMID:8436276

  11. Who abandons embryos after IVF?

    LENUS (Irish Health Repository)

    Walsh, A P H

    2010-04-01

    This investigation describes features of in vitro fertilisation (IVF) patients who never returned to claim their embryos following cryopreservation. Frozen embryo data were reviewed to establish communication patterns between patient and clinic; embryos were considered abandoned when 1) an IVF patient with frozen embryo\\/s stored at our facility failed to make contact with our clinic for > 2 yrs and 2) the patient could not be located after a multi-modal outreach effort was undertaken. For these patients, telephone numbers had been disconnected and no forwarding address was available. Patient, spouse and emergency family contact\\/s all escaped detection efforts despite an exhaustive public database search including death records and Internet directory portals. From 3244 IVF cycles completed from 2000 to 2008, > or = 1 embryo was frozen in 1159 cases (35.7%). Those without correspondence for > 2 yrs accounted for 292 (25.2%) patients with frozen embryos; 281 were contacted by methods including registered (signature involving abandoned embryos did not differ substantially from other patients. The goal of having a baby was achieved by 10\\/11 patients either by spontaneous conception, adoption or IVF. One patient moved away with conception status unconfirmed. The overall rate of embryo abandonment was 11\\/1159 (< 1%) in this IVF population. Pre-IVF counselling minimises, but does not totally eliminate, the problem of abandoned embryos. As the number of abandoned embryos from IVF accumulates, their fate urgently requires clarification. We propose that clinicians develop a policy consistent with relevant Irish Constitutional provisions to address this medical dilemma.

  12. The Drosophila melanogaster host model

    Science.gov (United States)

    Igboin, Christina O.; Griffen, Ann L.; Leys, Eugene J.

    2012-01-01

    The deleterious and sometimes fatal outcomes of bacterial infectious diseases are the net result of the interactions between the pathogen and the host, and the genetically tractable fruit fly, Drosophila melanogaster, has emerged as a valuable tool for modeling the pathogen–host interactions of a wide variety of bacteria. These studies have revealed that there is a remarkable conservation of bacterial pathogenesis and host defence mechanisms between higher host organisms and Drosophila. This review presents an in-depth discussion of the Drosophila immune response, the Drosophila killing model, and the use of the model to examine bacterial–host interactions. The recent introduction of the Drosophila model into the oral microbiology field is discussed, specifically the use of the model to examine Porphyromonas gingivalis–host interactions, and finally the potential uses of this powerful model system to further elucidate oral bacterial-host interactions are addressed. PMID:22368770

  13. The Drosophila melanogaster host model

    Directory of Open Access Journals (Sweden)

    Christina O. Igboin

    2012-02-01

    Full Text Available The deleterious and sometimes fatal outcomes of bacterial infectious diseases are the net result of the interactions between the pathogen and the host, and the genetically tractable fruit fly, Drosophila melanogaster, has emerged as a valuable tool for modeling the pathogen–host interactions of a wide variety of bacteria. These studies have revealed that there is a remarkable conservation of bacterial pathogenesis and host defence mechanisms between higher host organisms and Drosophila. This review presents an in-depth discussion of the Drosophila immune response, the Drosophila killing model, and the use of the model to examine bacterial–host interactions. The recent introduction of the Drosophila model into the oral microbiology field is discussed, specifically the use of the model to examine Porphyromonas gingivalis–host interactions, and finally the potential uses of this powerful model system to further elucidate oral bacterial-host interactions are addressed.

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

    OpenAIRE

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

    2015-01-01

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

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

  16. 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....

  17. 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.

  18. 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.

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

    Science.gov (United States)

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

    2016-10-20

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

  20. 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.

  1. A conserved role for kinesin-5 in plant mitosis.

    Science.gov (United States)

    Bannigan, Alex; Scheible, Wolf-Rüdiger; Lukowitz, Wolfgang; Fagerstrom, Carey; Wadsworth, Patricia; Somerville, Chris; Baskin, Tobias I

    2007-08-15

    The mitotic spindle of vascular plants is assembled and maintained by processes that remain poorly explored at a molecular level. Here, we report that AtKRP125c, one of four kinesin-5 motor proteins in arabidopsis, decorates microtubules throughout the cell cycle and appears to function in both interphase and mitosis. In a temperature-sensitive mutant, interphase cortical microtubules are disorganized at the restrictive temperature and mitotic spindles are massively disrupted, consistent with a defect in the stabilization of anti-parallel microtubules in the spindle midzone, as previously described in kinesin-5 mutants from animals and yeast. AtKRP125c introduced into mammalian epithelial cells by transfection decorates microtubules throughout the cell cycle but is unable to complement the loss of the endogenous kinesin-5 motor (Eg5). These results are among the first reports of any motor with a major role in anastral spindle structure in plants and demonstrate that the conservation of kinesin-5 motor function throughout eukaryotes extends to vascular plants.

  2. Expression image data of Drosophila GAL4 enhancer trap lines - GETDB | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available [ Credits ] BLAST Search Image Search Home About Archive Update History Contact us GETDB Expression image... data of Drosophila GAL4 enhancer trap lines Data detail Data name Expression image da...ta of Drosophila GAL4 enhancer trap lines Description of data contents 3,075 expression image data by develo...pmental stages. - Expression image in imaginal disc - Expression image in embryo - Fluorescence expression image... in GFP-dyed larva - Expression image in adult The image file is provided in the JPEG format. The file n

  3. Viability of bovine demi embryo after splitting of fresh and frozen thawed embryo derived from in vitro embryo production

    Directory of Open Access Journals (Sweden)

    M Imron

    2007-06-01

    Full Text Available In vivo embryo production was limited by number of donor, wide variability respond due to superovulation program and also immunoactifity of superovulation hormone (FSH. Splitting technology could be an alternative to increase the number of transferrable embryos into recipien cows. Splitting is done with cutting embryo becoming two equal pieces (called demi embrio base on ICM orientation. The objective of this research was to determine the viability of demi embryo obtained from embryo splitting of fresh and frozen thawed embryo. The results showed that demi embryos which performed blastocoel reexpansion 3 hours after embryo splitting using fresh and frozen thawed embryos were 76.9 and 76.2% respectively. Base on existention of inner cell mass (ICM, the number of demi embryos developed with ICM from fresh and frozen thawed embryos were not significantly different (90.6 and 85.7% respectively. The cell number of demi embryo from fresh embryos splitting was not different compared with those from frozen thawed embryos (36.1 and 35.9 respectively. These finding indicated that embryo splitting can be applied to frozen thawed embryos with certain condition as well as fresh embryos.

  4. Exploring Autophagy in Drosophila

    Directory of Open Access Journals (Sweden)

    Péter Lőrincz

    2017-07-01

    Full Text Available Autophagy is a catabolic process in eukaryotic cells promoting bulk or selective degradation of cellular components within lysosomes. In recent decades, several model systems were utilized to dissect the molecular machinery of autophagy and to identify the impact of this cellular “self-eating” process on various physiological and pathological processes. Here we briefly discuss the advantages and limitations of using the fruit fly Drosophila melanogaster, a popular model in cell and developmental biology, to apprehend the main pathway of autophagy in a complete animal.

  5. Drosophila as a genetic and cellular model for studies on axonal growth

    Directory of Open Access Journals (Sweden)

    Whitington Paul

    2007-05-01

    Full Text Available Abstract One of the most fascinating processes during nervous system development is the establishment of stereotypic neuronal networks. An essential step in this process is the outgrowth and precise navigation (pathfinding of axons and dendrites towards their synaptic partner cells. This phenomenon was first described more than a century ago and, over the past decades, increasing insights have been gained into the cellular and molecular mechanisms regulating neuronal growth and navigation. Progress in this area has been greatly assisted by the use of simple and genetically tractable invertebrate model systems, such as the fruit fly Drosophila melanogaster. This review is dedicated to Drosophila as a genetic and cellular model to study axonal growth and demonstrates how it can and has been used for this research. We describe the various cellular systems of Drosophila used for such studies, insights into axonal growth cones and their cytoskeletal dynamics, and summarise identified molecular signalling pathways required for growth cone navigation, with particular focus on pathfinding decisions in the ventral nerve cord of Drosophila embryos. These Drosophila-specific aspects are viewed in the general context of our current knowledge about neuronal growth.

  6. Efficient and Specific Modifications of the Drosophila Genome by Means of an Easy TALEN Strategy

    Institute of Scientific and Technical Information of China (English)

    Jiyong Liu; Bo Zhang; Wu-Min Deng; Renjie Jiao; Changqing Li; Zhongsheng Yu; Peng Huang; Honggang Wu; Chuanxian Wei; Nannan Zhu; Yan Shen; Yixu Chen

    2012-01-01

    Technology development has always been one of the forces driving breakthroughs in biomedical research.Since the time of Thomas Morgan,Drosophilists have,step by step,developed powerful genetic tools for manipulating and functionally dissecting the Drosophila genome,but room for improving these technologies and developing new techniques is still large,especially today as biologists start to study systematically the functional genomics of different model organisms,including humans,in a high-throughput manner.Here,we report,for the first time in Drosophila,a rapid,easy,and highly specific method for modifying the Drosophila genome at a very high efficiency by means of an improved transcription activator-like effector nuclease (TALEN) strategy.We took advantage of the very recently developed "unit assembly" strategy to assemble two pairs of specific TALENs designed to modify the yellow gene (on the sex chromosome) and a novel autosomal gene.The mRNAs of TALENs were subsequently injected into Drosophila embryos.From 31.2% of the injected Fo fertile flies,we detected inheritable modification involving the yellow gene.The entire process from construction of specific TALENs to detection of inheritable modifications can be accomplished within one month.The potential applications of this TALEN-mediated genome modification method in Drosophila are discussed.

  7. Spindle checkpoint-independent inhibition of mitotic chromosome segregation by Drosophila Mps1.

    Science.gov (United States)

    Althoff, Friederike; Karess, Roger E; Lehner, Christian F

    2012-06-01

    Monopolar spindle 1 (Mps1) is essential for the spindle assembly checkpoint (SAC), which prevents anaphase onset in the presence of misaligned chromosomes. Moreover, Mps1 kinase contributes in a SAC-independent manner to the correction of erroneous initial attachments of chromosomes to the spindle. Our characterization of the Drosophila homologue reveals yet another SAC-independent role. As in yeast, modest overexpression of Drosophila Mps1 is sufficient to delay progression through mitosis during metaphase, even though chromosome congression and metaphase alignment do not appear to be affected. This delay in metaphase depends on the SAC component Mad2. Although Mps1 overexpression in mad2 mutants no longer causes a metaphase delay, it perturbs anaphase. Sister kinetochores barely move apart toward spindle poles. However, kinetochore movements can be restored experimentally by separase-independent resolution of sister chromatid cohesion. We propose therefore that Mps1 inhibits sister chromatid separation in a SAC-independent manner. Moreover, we report unexpected results concerning the requirement of Mps1 dimerization and kinase activity for its kinetochore localization in Drosophila. These findings further expand Mps1's significance for faithful mitotic chromosome segregation and emphasize the importance of its careful regulation.

  8. Initial neurogenesis in Drosophila

    OpenAIRE

    Hartenstein, Volker; Wodarz, Andreas

    2013-01-01

    Early neurogenesis comprises the phase of nervous system development during which neural progenitor cells are born. In early development, the embryonic ectoderm is subdivided by a conserved signaling mechanism into two main domains, the epidermal ectoderm and the neurectoderm. Subsequently, cells of the neurectoderm are internalized and form a cell layer of proliferating neural progenitors. In vertebrates, the entire neurectoderm folds into the embryo to give rise to the neural tube. In Droso...

  9. Identification of a novel Drosophila gene, beltless, using injectable embryonic and adult RNA interference (RNAi

    Directory of Open Access Journals (Sweden)

    Manev Hari

    2003-08-01

    Full Text Available Abstract Background RNA interference (RNAi is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly gene (corresponding to a putative gene CG5652/GM06434, that we named beltless based on an embryonic loss-of-function phenotype. Results Beltless mRNA is expressed in all developmental stages except in 0–6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless of the abdominal segments A2–A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts. Conclusions We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should

  10. SUMOylation in Drosophila Development

    Directory of Open Access Journals (Sweden)

    Albert J. Courey

    2012-07-01

    Full Text Available Small ubiquitin-related modifier (SUMO, an ~90 amino acid ubiquitin-like protein, is highly conserved throughout the eukaryotic domain. Like ubiquitin, SUMO is covalently attached to lysine side chains in a large number of target proteins. In contrast to ubiquitin, SUMO does not have a direct role in targeting proteins for proteasomal degradation. However, like ubiquitin, SUMO does modulate protein function in a variety of other ways. This includes effects on protein conformation, subcellular localization, and protein–protein interactions. Significant insight into the in vivo role of SUMOylation has been provided by studies in Drosophila that combine genetic manipulation, proteomic, and biochemical analysis. Such studies have revealed that the SUMO conjugation pathway regulates a wide variety of critical cellular and developmental processes, including chromatin/chromosome function, eggshell patterning, embryonic pattern formation, metamorphosis, larval and pupal development, neurogenesis, development of the innate immune system, and apoptosis. This review discusses our current understanding of the diverse roles for SUMO in Drosophila development.

  11. CELLULAR LOCALIZATION AND EXPRESSION OF pygo DURING DROSOPHILA DEVELOPMENT

    Institute of Scientific and Technical Information of China (English)

    LINXin-da; LINXin-hua; CHENGJia-an

    2003-01-01

    Wg/Wnt signaling is a key signaling pathway in Drosophila. Many genes involved in Wingless(wg) signal transduction pathway downstream of Wg, or it'' s vertebrate Wg homologue Wnt, have been identified.Transduction of the Wg signal downstream of Wg is mediated by nuclear TCF/LEF-1, through association with Ar-madillo (Arm)/β-catenin. Pygopus (pygo) is a new identified component in this pathway . Cellular localization experiment showed that pygo was expressed specifically in the nucleus. The expression profile of pygo in embryos was examined using in situ hybridization. Although pygo expressed ubiquitously in the embryos, it expressed at relatively high level in pre-blastoderm embryos which indicate a high degree of maternally provided message, fol-lowed by a low level of ubiquitous zygotic expression. This continues into larval tissues (including wing disc, eye disc and leg disc), where pygo appears to be expressed at low level. Comparison of pygo expression levels, in the wing disc, eye disc and leg disc, showed pygo expression level in the wing disc pouch and leg disc were rela-tive higher.

  12. Hyperphosphorylation regulates the activity of SREBP1 during mitosis.

    Science.gov (United States)

    Bengoechea-Alonso, Maria T; Punga, Tanel; Ericsson, Johan

    2005-08-16

    The sterol regulatory element-binding protein (SREBP) family of transcription factors controls the biosynthesis of cholesterol and other lipids, and lipid synthesis is critical for cell growth and proliferation. We were, therefore, interested in the expression and activity of SREBPs during the cell cycle. We found that the expression of a number of SREBP-responsive promoter-reporter genes were induced in a SREBP-dependent manner in cells arrested in G2/M. In addition, the mature forms of SREBP1a and SREBP1c were hyperphosphorylated in mitotic cells, giving rise to a phosphoepitope recognized by the mitotic protein monoclonal-2 (MPM-2) antibody. In contrast, SREBP2 was not hyperphosphorylated in mitotic cells and was not recognized by the MPM-2 antibody. The MPM-2 epitope was mapped to the C terminus of mature SREBP1, and the mitosis-specific hyperphosphorylation of SREBP1 depended on this domain of the protein. The transcriptional and DNA-binding activity of SREBP1 was enhanced in cells arrested in G2/M, and these effects depended on the C-terminal domain of the protein. In part, these effects could be explained by our observation that mature SREBP1 was stabilized in G2/M. In agreement with these observations, we found that the synthesis of cholesterol was increased in G2/M-arrested cells. Thus, our results demonstrate that the activity of mature SREBP1 is regulated by phosphorylation during the cell cycle, suggesting that SREBP1 may provide a link between lipid synthesis, proliferation, and cell growth.

  13. The planar cell polarity protein Strabismus promotes Pins anterior localization during asymmetric division of sensory organ precursor cells in Drosophila.

    Science.gov (United States)

    Bellaïche, Yohanns; Beaudoin-Massiani, Olivia; Stuttem, Isabella; Schweisguth, François

    2004-01-01

    Cell fate diversity is generated in part by the unequal segregation of cell-fate determinants during asymmetric cell division. In the Drosophila bristle lineage, the sensory organ precursor (pI) cell is polarized along the anteroposterior (AP) axis by Frizzled (Fz) receptor signaling. We show here that Fz localizes at the posterior apical cortex of the pI cell prior to mitosis, whereas Strabismus (Stbm) and Prickle (Pk), which are also required for AP polarization of the pI cell, co-localize at the anterior apical cortex. Thus, asymmetric localization of Fz, Stbm and Pk define two opposite cortical domains prior to mitosis of the pI cell. At mitosis, Stbm forms an anterior crescent that overlaps with the distribution of Partner of Inscuteable (Pins) and Discs-large (Dlg), two components of the anterior Dlg-Pins-Galphai complex that regulates the localization of cell-fate determinants. At prophase, Stbm promotes the anterior localization of Pins. By contrast, Dishevelled (Dsh) acts antagonistically to Stbm by excluding Pins from the posterior cortex. We propose that the Stbm-dependent recruitment of Pins at the anterior cortex of the pI cell is a novel read-out of planar cell polarity.

  14. Physical influences on embryo development.

    Science.gov (United States)

    Deeming, D C; Rowlett, K; Simkiss, K

    1987-01-01

    There is a critical period between 3 and 7 days of incubation when the absence of turning in eggs of the domestic fowl leads to increased mortality and decreased embryo growth. This critical period coincides with the time of subembryonic fluid formation, and it is suggested that the absence of turning leads to the presence of unstirred layer effects in fluid secretion. This fluid deficiency persists throughout the subsequent development of the embryo. Experiments on shell-less culture systems support this interpretation in preference to other explanations of embryo death in unturned eggs, which usually refer to chorion adhesion to shell membranes.

  15. New techniques on embryo manipulation.

    Science.gov (United States)

    Escribá, M J; Valbuena, D; Remohí, J; Pellicer, A; Simón, C

    2002-01-01

    For many years, experience has been accumulated on embryo and gamete manipulation in livestock animals. The present work is a review of these techniques and their possible application in human embryology in specific cases. It is possible to manipulate gametes at different levels, producing paternal or maternal haploid embryos (hemicloning), using different techniques including nuclear transfer. At the embryonic stage, considering practical, ethical and legal issues, techniques will be reviewed that include cloning and embryo splitting at the cleavage stage, morula, or blastocyst stage.

  16. ATRX contributes to epigenetic asymmetry and silencing of major satellite transcripts in the maternal genome of the mouse embryo.

    Science.gov (United States)

    De La Fuente, Rabindranath; Baumann, Claudia; Viveiros, Maria M

    2015-05-15

    A striking proportion of human cleavage-stage embryos exhibit chromosome instability (CIN). Notably, until now, no experimental model has been described to determine the origin and mechanisms of complex chromosomal rearrangements. Here, we examined mouse embryos deficient for the chromatin remodeling protein ATRX to determine the cellular mechanisms activated in response to CIN. We demonstrate that ATRX is required for silencing of major satellite transcripts in the maternal genome, where it confers epigenetic asymmetry to pericentric heterochromatin during the transition to the first mitosis. This stage is also characterized by a striking kinetochore size asymmetry established by differences in CENP-C protein between the parental genomes. Loss of ATRX results in increased centromeric mitotic recombination, a high frequency of sister chromatid exchanges and double strand DNA breaks, indicating the formation of mitotic recombination break points. ATRX-deficient embryos exhibit a twofold increase in transcripts for aurora kinase B, the centromeric cohesin ESCO2, DNMT1, the ubiquitin-ligase (DZIP3) and the histone methyl transferase (EHMT1). Thus, loss of ATRX activates a pathway that integrates epigenetic modifications and DNA repair in response to chromosome breaks. These results reveal the cellular response of the cleavage-stage embryo to CIN and uncover a mechanism by which centromeric fission induces the formation of large-scale chromosomal rearrangements. Our results have important implications to determine the epigenetic origins of CIN that lead to congenital birth defects and early pregnancy loss, as well as the mechanisms involved in the oocyte to embryo transition.

  17. Non-surgical embryo transfer in pigs

    NARCIS (Netherlands)

    Hazeleger, W.

    1999-01-01

    Embryo transfer in pigs has been performed surgically for a long time. However, a less invasive, non-surgical, procedure of embryo transfer could be a valuable tool for research (to study embryo survival and embryo-uterus interactions) and practical applications (export, prevention of

  18. Identification of novel Drosophila centromere-associated proteins.

    Science.gov (United States)

    Barth, Teresa K; Schade, Georg O M; Schmidt, Andreas; Vetter, Irene; Wirth, Marc; Heun, Patrick; Thomae, Andreas W; Imhof, Axel

    2014-10-01

    Centromeres are chromosomal regions crucial for correct chromosome segregation during mitosis and meiosis. They are epigenetically defined by centromeric proteins such as the centromere-specific histone H3-variant centromere protein A (CENP-A). In humans, 16 additional proteins have been described to be constitutively associated with centromeres throughout the cell cycle, known as the constitutive centromere-associated network (CCAN). In contrast, only one additional constitutive centromeric protein is known in Drosophila melanogaster (D.mel), the conserved CCAN member CENP-C. To gain further insights into D.mel centromere composition and biology, we analyzed affinity-purified chromatin prepared from D.mel cell lines expressing green fluorescent protein tagged histone three variants by MS. In addition to already-known centromeric proteins, we identified novel factors that were repeatedly enriched in affinity purification-MS experiments. We analyzed the cellular localization of selected candidates by immunocytochemistry and confirmed localization to the centromere and other genomic regions for ten factors. Furthermore, RNA interference mediated depletion of CG2051, CG14480, and hyperplastic discs, three of our strongest candidates, leads to elevated mitotic defects. Knockdowns of these candidates neither impair the localization of several known kinetochore proteins nor CENP-A(CID) loading, suggesting their involvement in alternative pathways that contribute to proper centromere function. In summary, we provide a comprehensive analysis of the proteomic composition of Drosophila centromeres. All MS data have been deposited in the ProteomeXchange with identifier PXD000758 (http://proteomecentral.proteomexchange.org/dataset/PXD000758).

  19. Centrosome and microtubule instability in aging Drosophila cells

    Science.gov (United States)

    Schatten, H.; Chakrabarti, A.; Hedrick, J.

    1999-01-01

    Several cytoskeletal changes are associated with aging which includes alterations in muscle structure leading to muscular atrophy, and weakening of the microtubule network which affects cellular secretion and maintenance of cell shape. Weakening of the microtubule network during meiosis in aging oocytes can result in aneuploidy or trisomic zygotes with increasing maternal age. Imbalances of cytoskeletal organization can lead to disease such as Alzheimer's, muscular disorders, and cancer. Because many cytoskeletal diseases are related to age we investigated the effects of aging on microtubule organization in cell cultures of the Drosophila cell model system (Schneider S-1 and Kc23 cell lines). This cell model is increasingly being used as an alternative system to mammalian cell cultures. Drosophila cells are amenable to genetic manipulations and can be used to identify and manipulate genes which are involved in the aging processes. Immunofluorescence, scanning, and transmission electron microscopy were employed for the analysis of microtubule organizing centers (centrosomes) and microtubules at various times after subculturing cells in fresh medium. Our results reveal that centrosomes and the microtubule network becomes significantly affected in aging cells after 5 days of subculture. At 5-14 days of subculture, 1% abnormal out of 3% mitoses were noted which were clearly distinguishable from freshly subcultured control cells in which 3% of cells undergo normal mitosis with bipolar configurations. Microtubules are also affected in the midbody during cell division. The midbody in aging cells becomes up to 10 times longer when compared with midbodies in freshly subcultured cells. During interphase, microtubules are often disrupted and disorganized, which may indicate improper function related to transport of cell organelles along microtubules. These results are likely to help explain some cytoskeletal disorders and diseases related to aging.

  20. Sterol requirements in Drosophila melanogaster

    OpenAIRE

    Almeida de Carvalho, Maria Joao

    2009-01-01

    Sterol is an abundant component of eukaryotic cell membranes and is thought to influence membrane properties such as permeability, fluidity and microdomain formation. Drosophila is an excellent model system in which to study functional requirements for membrane sterol because, although it does not synthesize sterol, it nevertheless requires sterols to complete development. Moreover, Drosophila normally incorporates sterols into cell membranes. Thus, dietary sterol depletion can be used to ...

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

    Science.gov (United States)

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

    2014-01-01

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

  2. Phosphorylation of p37 is important for Golgi disassembly at mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Kaneko, Yayoi [Department of Molecular Cell Biology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Mitsubishi Kagaku Institute of Life Sciences, Tokyo 194-8511 (Japan); Tamura, Kaori [Department of Molecular Cell Biology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Totsukawa, Go [Department of Molecular Cell Biology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Mitsubishi Kagaku Institute of Life Sciences, Tokyo 194-8511 (Japan); Kondo, Hisao, E-mail: hk228@med.kyushu-u.ac.jp [Department of Molecular Cell Biology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan)

    2010-11-05

    Research highlights: {yields} p37 is phosphorylated on Serine-56 and Threonine-59 by Cdc2 at mitosis. {yields} Phosphorylated p37 does not bind to Golgi membranes. {yields} p37 phosphorylation inhibits p97/p37-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled at early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 by Cdc2 results in mitotic inhibition of the p97/p47 pathway . In this study, we demonstrate that p37 is phosphorylated on Serine-56 and Threonine-59 by Cdc2 at mitosis, and this phosphorylated p37 does not bind to Golgi membranes. Using an in vitro Golgi reassembly assay, we show that mutated p37(S56D, T59D), which mimics mitotic phosphorylation, does not cause any cisternal regrowth, indicating that p37 phosphorylation inhibits the p97/p37 pathway. Our results demonstrate that p37 phosphorylation on Serine-56 and Threonine-59 is important for Golgi disassembly at mitosis.

  3. 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.

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

    Science.gov (United States)

    Hameroff, Stuart R

    2004-11-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2014-11-01

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

  7. twin of eyeless, a second Pax-6 gene of Drosophila, acts upstream of eyeless in the control of eye development.

    Science.gov (United States)

    Czerny, T; Halder, G; Kloter, U; Souabni, A; Gehring, W J; Busslinger, M

    1999-03-01

    The Drosophila Pax-6 gene eyeless (ey) plays a key role in eye development. Here we show tht Drosophila contains a second Pax-6 gene, twin of eyeless (toy), due to a duplication during insect evolution. Toy is more similar to vertebrate Pax-6 proteins than Ey with regard to overall sequence conservation, DNA-binding function, and early expression in the embryo, toy and ey share a similar expression pattern in the developing visual system, and targeted expression of Toy, like Ey, induces the formation of ectopic eyes. Genetic and biochemical evidence indicates, however, that Toy functions upstream of ey by directly regulating the eye-specific enhancer of ey. Toy is therefore required for initiation of ey expression in the embryo and acts through Ey to activate the eye developmental program.

  8. Phaseolus immature embryo rescue technology.

    Science.gov (United States)

    Geerts, Pascal; Toussaint, André; Mergeai, Guy; Baudoin, Jean-Pierre

    2011-01-01

    Predominant among the production constraints of the common bean Phaseolus vulgaris are infestation of Ascochyta blight, Bean Golden Mosaic virus (BGMV), and Bean Fly. Interbreeding with Phaseolus -coccineus L. and/or Phaseolus polyanthus Greenm has been shown to provide P. vulgaris with greater resistance to these diseases. For interspecific crosses to be successful, it is important to use P. coccineus and P. polyanthus as female parents; this prevents rapid reversal to the recurrent parent P. vulgaris. Although incompatibility barriers are post-zygotic, early hybrid embryo abortion limits the success of F1 crosses. While rescue techniques for globular and early heart-shaped embryos have improved in recent years, -success in hybridization remains very low. In this study, we describe six steps that allowed us to rescue 2-day-old P. vulgaris embryos using a pod culture technique. Our methods consisted of (i) pod culture, (ii) extraction and culture of immature embryos, (iii) dehydration of embryos, (iv) germination of embryos, (v) rooting of developed shoots, and (vi) hardening of plantlets.

  9. Cytokines in Drosophila immunity.

    Science.gov (United States)

    Vanha-Aho, Leena-Maija; Valanne, Susanna; Rämet, Mika

    2016-02-01

    Cytokines are a large and diverse group of small proteins that can affect many biological processes, but most commonly cytokines are known as mediators of the immune response. In the event of an infection, cytokines are produced in response to an immune stimulus, and they function as key regulators of the immune response. Cytokines come in many shapes and sizes, and although they vary greatly in structure, their functions have been well conserved in evolution. The immune signaling pathways that respond to cytokines are remarkably conserved from fly to man. Therefore, Drosophila melanogaster, provides an excellent platform for studying the biology and function of cytokines. In this review, we will describe the cytokines and cytokine-like molecules found in the fly and discuss their roles in host immunity.

  10. Optogenetics in Drosophila Neuroscience.

    Science.gov (United States)

    Riemensperger, Thomas; Kittel, Robert J; Fiala, André

    2016-01-01

    Optogenetic techniques enable one to target specific neurons with light-sensitive proteins, e.g., ion channels, ion pumps, or enzymes, and to manipulate their physiological state through illumination. Such artificial interference with selected elements of complex neuronal circuits can help to determine causal relationships between neuronal activity and the effect on the functioning of neuronal circuits controlling animal behavior. The advantages of optogenetics can best be exploited in genetically tractable animals whose nervous systems are, on the one hand, small enough in terms of cell numbers and to a certain degree stereotypically organized, such that distinct and identifiable neurons can be targeted reproducibly. On the other hand, the neuronal circuitry and the behavioral repertoire should be complex enough to enable one to address interesting questions. The fruit fly Drosophila melanogaster is a favorable model organism in this regard. However, the application of optogenetic tools to depolarize or hyperpolarize neurons through light-induced ionic currents has been difficult in adult flies. Only recently, several variants of Channelrhodopsin-2 (ChR2) have been introduced that provide sufficient light sensitivity, expression, and stability to depolarize central brain neurons efficiently in adult Drosophila. Here, we focus on the version currently providing highest photostimulation efficiency, ChR2-XXL. We exemplify the use of this optogenetic tool by applying it to a widely used aversive olfactory learning paradigm. Optogenetic activation of a population of dopamine-releasing neurons mimics the reinforcing properties of a punitive electric shock typically used as an unconditioned stimulus. In temporal coincidence with an odor stimulus this artificially induced neuronal activity causes learning of the odor signal, thereby creating a light-induced memory.

  11. Microtubules as key coordinators of nuclear envelope and endoplasmic reticulum dynamics during mitosis.

    Science.gov (United States)

    Schlaitz, Anne-Lore

    2014-07-01

    During mitosis, cells comprehensively restructure their interior to promote the faithful inheritance of DNA and cytoplasmic contents. In metazoans, this restructuring entails disassembly of the nuclear envelope, redistribution of its components into the endoplasmic reticulum (ER) and eventually nuclear envelope reassembly around the segregated chromosomes. The microtubule cytoskeleton has recently emerged as a critical regulator of mitotic nuclear envelope and ER dynamics. Microtubules and associated molecular motors tear open the nuclear envelope in prophase and remove nuclear envelope remnants from chromatin. Additionally, two distinct mechanisms of microtubule-based regulation of ER dynamics operate later in mitosis. First, association of the ER with microtubules is reduced, preventing invasion of ER into the spindle area, and second, organelle membrane is actively cleared from metaphase chromosomes. However, we are only beginning to understand the role of microtubules in shaping and distributing ER and other organelles during mitosis. © 2014 WILEY Periodicals, Inc.

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

    Science.gov (United States)

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

    2012-10-01

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

  13. 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.

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

    Science.gov (United States)

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

    2015-12-10

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

  15. 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...

  16. 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.

  17. Lessons from Embryos: Haeckel's Embryo Drawings, Evolution, and Secondary Biology Textbooks

    Science.gov (United States)

    Wellner, Karen L.

    2014-01-01

    In 1997, developmental biologist Michael Richardson compared his research team's embryo photographs to Ernst Haeckel's 1874 embryo drawings and called Haeckel's work "noncredible". "Science" soon published "Haeckel's Embryos: Fraud Rediscovered," and Richardson's comments further reinvigorated criticism of Haeckel by…

  18. Lessons from Embryos: Haeckel's Embryo Drawings, Evolution, and Secondary Biology Textbooks

    Science.gov (United States)

    Wellner, Karen L.

    2014-01-01

    In 1997, developmental biologist Michael Richardson compared his research team's embryo photographs to Ernst Haeckel's 1874 embryo drawings and called Haeckel's work "noncredible". "Science" soon published "Haeckel's Embryos: Fraud Rediscovered," and Richardson's comments further reinvigorated criticism of Haeckel by…

  19. Drosophila laminins act as key regulators of basement membrane assembly and morphogenesis.

    Science.gov (United States)

    Urbano, Jose M; Torgler, Catherine N; Molnar, Cristina; Tepass, Ulrich; López-Varea, Ana; Brown, Nicholas H; de Celis, Jose F; Martín-Bermudo, Maria D

    2009-12-01

    Laminins are heterotrimeric molecules found in all basement membranes. In mammals, they have been involved in diverse developmental processes, from gastrulation to tissue maintenance. The Drosophila genome encodes two laminin alpha chains, one beta and one Gamma, which form two distinct laminin trimers. So far, only mutations affecting one or other trimer have been analysed. In order to study embryonic development in the complete absence of laminins, we mutated the gene encoding the sole laminin beta chain in Drosophila, LanB1, so that no trimers can be made. We show that LanB1 mutant embryos develop until the end of embryogenesis. Electron microscopy analysis of mutant embryos reveals that the basement membranes are absent and the remaining extracellular material appears disorganised and diffuse. Accordingly, abnormal accumulation of major basement membrane components, such as Collagen IV and Perlecan, is observed in mutant tissues. In addition, we show that elimination of LanB1 prevents the normal morphogenesis of most organs and tissues, including the gut, trachea, muscles and nervous system. In spite of the above structural roles for laminins, our results unravel novel functions in cell adhesion, migration and rearrangement. We propose that while an early function of laminins in gastrulation is not conserved in Drosophila and mammals, their function in basement membrane assembly and organogenesis seems to be maintained throughout evolution.

  20. Microsatellite repeat instability fuels evolution of embryonic enhancers in Hawaiian Drosophila.

    Science.gov (United States)

    Brittain, Andrew; Stroebele, Elizabeth; Erives, Albert

    2014-01-01

    For ∼30 million years, the eggs of Hawaiian Drosophila were laid in ever-changing environments caused by high rates of island formation. The associated diversification of the size and developmental rate of the syncytial fly embryo would have altered morphogenic gradients, thus necessitating frequent evolutionary compensation of transcriptional responses. We investigate the consequences these radiations had on transcriptional enhancers patterning the embryo to see whether their pattern of molecular evolution is different from non-Hawaiian species. We identify and functionally assay in transgenic D. melanogaster the Neurogenic Ectoderm Enhancers from two different Hawaiian Drosophila groups: (i) the picture wing group, and (ii) the modified mouthparts group. We find that the binding sites in this set of well-characterized enhancers are footprinted by diverse microsatellite repeat (MSR) sequences. We further show that Hawaiian embryonic enhancers in general are enriched in MSR relative to both Hawaiian non-embryonic enhancers and non-Hawaiian embryonic enhancers. We propose embryonic enhancers are sensitive to Activator spacing because they often serve as assembly scaffolds for the aggregation of transcription factor activator complexes. Furthermore, as most indels are produced by microsatellite repeat slippage, enhancers from Hawaiian Drosophila lineages, which experience dynamic evolutionary pressures, would become grossly enriched in MSR content.

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

    Science.gov (United States)

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

    1999-08-09

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

  2. Tetrameric structure of centromeric nucleosomes in interphase Drosophila cells.

    Directory of Open Access Journals (Sweden)

    Yamini Dalal

    2007-08-01

    Full Text Available Centromeres, the specialized chromatin structures that are responsible for equal segregation of chromosomes at mitosis, are epigenetically maintained by a centromere-specific histone H3 variant (CenH3. However, the mechanistic basis for centromere maintenance is unknown. We investigated biochemical properties of CenH3 nucleosomes from Drosophila melanogaster cells. Cross-linking of CenH3 nucleosomes identifies heterotypic tetramers containing one copy of CenH3, H2A, H2B, and H4 each. Interphase CenH3 particles display a stable association of approximately 120 DNA base pairs. Purified centromeric nucleosomal arrays have typical "beads-on-a-string" appearance by electron microscopy but appear to resist condensation under physiological conditions. Atomic force microscopy reveals that native CenH3-containing nucleosomes are only half as high as canonical octameric nucleosomes are, confirming that the tetrameric structure detected by cross-linking comprises the entire interphase nucleosome particle. This demonstration of stable half-nucleosomes in vivo provides a possible basis for the instability of centromeric nucleosomes that are deposited in euchromatic regions, which might help maintain centromere identity.

  3. VISUALIZACIÓN DE LA MITOSIS CON EL MICROSCOPIO DE FUERZA ATÓMICA

    Directory of Open Access Journals (Sweden)

    María de Lourdes Segura-Valdez

    2008-01-01

    Full Text Available En eucariontes, la división celular generalmente ocurre por medio de la mitosis. En estudios previos hemos documentado la posibilidad de estudiar la estructura celular in situ con el microscopio de fuerza atómica, con énfasis en la estructura nuclear en interfase. En este trabajo mostramos que las diferentes etapas de la mitosis pueden ser visualizadas con este instrumento, lo que abre la posibilidad de estudiar este fenómeno en el rango nanométrico.

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

    OpenAIRE

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

    2016-01-01

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

  5. Demographic Aspects In The Use Of Music- Action Integration In Teaching Mitosis

    Directory of Open Access Journals (Sweden)

    Bryan Joseph E. Matillano

    2015-08-01

    Full Text Available Music and action integration had been used in a lot of pedagogical studies in the field of science education. This study sought to situatedemographic factors such as sex and educational background to use it as a tool in teaching mitosis. Using experimental- correlational design the level of significance was identified using t-test for the performance based on the pre and posttests. Results showed that music-action integration was an effective tool in teaching mitosis compared to the traditional lecture discussion. Factors such as sex and educational background did not affect students performance.

  6. 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...

  7. 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...

  8. 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...

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

    OpenAIRE

    Berry, Lynne D.; Gould, Kathleen L.

    1997-01-01

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

  10. VISUALIZACIÓN DE LA MITOSIS CON EL MICROSCOPIO DE FUERZA ATÓMICA

    OpenAIRE

    María de Lourdes Segura-Valdez; Sarai de Jesús Cruz-Gómez; Roberto López-Cruz; Guadalupe Zavala; Luis Felipe Jiménez-García

    2008-01-01

    En eucariontes, la división celular generalmente ocurre por medio de la mitosis. En estudios previos hemos documentado la posibilidad de estudiar la estructura celular in situ con el microscopio de fuerza atómica, con énfasis en la estructura nuclear en interfase. En este trabajo mostramos que las diferentes etapas de la mitosis pueden ser visualizadas con este instrumento, lo que abre la posibilidad de estudiar este fenómeno en el rango nanométrico.

  11. A vertex model of Drosophila ventral furrow formation.

    Directory of Open Access Journals (Sweden)

    Philipp Spahn

    Full Text Available Ventral furrow formation in Drosophila is an outstanding model system to study the mechanisms involved in large-scale tissue rearrangements. Ventral cells accumulate myosin at their apical sides and, while being tightly coupled to each other via apical adherens junctions, execute actomyosin contractions that lead to reduction of their apical cell surface. Thereby, a band of constricted cells along the ventral epithelium emerges which will form a tissue indentation along the ventral midline (the ventral furrow. Here we adopt a 2D vertex model to simulate ventral furrow formation in a surface view allowing easy comparison with confocal live-recordings. We show that in order to reproduce furrow morphology seen in vivo, a gradient of contractility must be assumed in the ventral epithelium which renders cells more contractile the closer they lie to the ventral midline. The model predicts previous experimental findings, such as the gain of eccentric morphology of constricting cells and an incremental fashion of apical cell area reduction. Analysis of the model suggests that this incremental area reduction is caused by the dynamical interplay of cell elasticity and stochastic contractility as well as by the opposing forces from contracting neighbour cells. We underpin results from the model through in vivo analysis of ventral furrow formation in wildtype and twi mutant embryos. Our results show that ventral furrow formation can be accomplished as a "tug-of-war" between stochastically contracting, mechanically coupled cells and may require less rigorous regulation than previously thought.For the developmental biologist it is a fascinating question how cells can coordinate major tissue movements during embryonic development. The so-called ventral furrow of the Drosophila embryo is a well-studied example of such a process when cells from a ventral band, spanning nearly the entire length of the embryo, undergo dramatic shape change by contracting their

  12. Passive cellular microrheology in developing fruit fly embryos

    Science.gov (United States)

    Crews, Sarah; Ma, Xiaoyan; Lawrence, Stacey; Hutson, M. Shane

    2012-02-01

    The development of fruit fly (Drosophila) embryos involves spatial and temporal regulation of cellular mechanical properties. These properties can be probed in vivo using laser hole drilling experiments; however, this technique only infers relative forces. Conversion to absolute forces requires measurement of cellular viscoelastic properties. Here, we use passive microrheology of fluorescently labeled cell membranes to measure the viscoelastic properties of amnioserosa cells. These dynamic epithelial cells play an important mechanical role during two developmental stages: germ band retraction and dorsal closure. Passive microrheology in this system is confounded by active contractions in the cytoskeleton. Thus, the fruit fly embryos are transiently anesthetized with CO2, halting active cellular movements, leaving only passive Brownian motion. The power spectra of these fluctuations are well fit by a Lorentzian -- as expected for Brownian motion -- and allow us to extract cellular viscoelastic parameters at different developmental stages. These measured parameters inform previous hole-drilling experiments and provide inputs for quantitative computational models of fruit fly embryonic development.

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

    OpenAIRE

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

    2006-01-01

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

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

    NARCIS (Netherlands)

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

    1998-01-01

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

  15. Embryo density may affect embryo quality during in vitro culture in a microwell group culture dish.

    Science.gov (United States)

    Lehner, Adam; Kaszas, Zita; Murber, Akos; Rigo, Janos; Urbancsek, Janos; Fancsovits, Peter

    2017-08-01

    Culturing embryos in groups is a common practice in mammalian embryology. Since the introduction of different microwell dishes, it is possible to identify oocytes or embryos individually. As embryo density (embryo-to-volume ratio) may affect the development and viability of the embryos, the purpose of this study was to assess the effect of different embryo densities on embryo quality. Data of 1337 embryos from 228 in vitro fertilization treatment cycles were retrospectively analyzed. Embryos were cultured in a 25 μl microdrop in a microwell group culture dish containing 9 microwells. Three density groups were defined: Group 1 with 2-4 (6.3-12.5 μl/embryo), Group 2 with 5-6 (4.2-5.0 μl/embryo), and Group 3 with 7-9 (2.8-3.6 μl/embryo) embryos. Proportion of good quality embryos was higher in Group 2 on both days (D2: 18.9 vs. 31.5 vs. 24.7%; p Culturing 5-6 embryos together in a culture volume of 25 μl may benefit embryo quality. As low egg number, position, and distance of the embryos may influence embryo quality, results should be interpreted with caution.

  16. Origin and dynamic lineage characteristics of the developing Drosophila midgut stem cells.

    Science.gov (United States)

    Takashima, Shigeo; Aghajanian, Patrick; Younossi-Hartenstein, Amelia; Hartenstein, Volker

    2016-08-15

    Proliferating intestinal stem cells (ISCs) generate all cell types of the Drosophila midgut, including enterocytes, endocrine cells, and gland cells (e.g., copper cells), throughout the lifetime of the animal. Among the signaling mechanisms controlling the balance between ISC self-renewal and the production of different cell types, Notch (N) plays a pivotal role. In this paper we investigated the emergence of ISCs during metamorphosis and the role of N in this process. Precursors of the Drosophila adult intestinal stem cells (pISCs) can be first detected within the pupal midgut during the first hours after onset of metamorphosis as motile mesenchymal cells. pISCs perform 2-3 rounds of parasynchronous divisions. The first mitosis yields only an increase in pISC number. During the following rounds of mitosis, dividing pISCs give rise to more pISCs, as well as the endocrine cells that populate the midgut of the eclosing fly. Enterocytes do not appear among the pISC progeny until around the time of eclosion. The "proendocrine" gene prospero (pros), expressed from mid-pupal stages onward in pISCs, is responsible to advance the endocrine fate in these cells; following removal of pros, pISCs continue to proliferate, but endocrine cells do not form. Conversely, the onset of N activity that occurs around the stage when pros comes on restricts pros expression among pISCs. Loss of N abrogates proliferation and switches on an endocrine fate among all pISCs. Our results suggest that a switch depending on the activity of N and pros acts at the level of the pISC to decide between continued proliferation and endocrine differentiation.

  17. The arrest gene is required for germline cyst formation during Drosophila oogenesis.

    Science.gov (United States)

    Parisi, M J; Deng, W; Wang, Z; Lin, H

    2001-04-01

    In Drosophila, oogenesis is initiated when a germline stem cell produces a differentiating daughter cell called the cystoblast. The cystoblast undergoes four rounds of synchronous divisions with incomplete cytokinesis to generate a syncytial cyst of 16 interconnected cystocytes, in which one cystocyte differentiates into an oocyte. Strong mutations of the arrest (aret) gene disrupt cyst formation and cause the production of clusters of ill-differentiated germline cells that retain cellular and molecular characteristics of cystoblasts. These mutant germ cells express high levels of BAM-C and SXL proteins in the cytoplasm but do not accumulate markers for advanced cystocytes or differentiating oocytes, such as the nuclear localization of SXL or the accumulation of osk mRNA, orb mRNA, and cytoplasmic dynein. However, the mutant germ cells do not contain spectrosomes, the cytoplasmic structure that objectifies the divisional asymmetry of the cystoblast. The aret mutant germ cells undergo active mitosis with complete cytokinesis. Their mitosis is accompanied by massive necrosis, so that the number of germ cells in a stem cell-derived cluster ranges from one to greater than 70. These defects of aret mutants reveal a novel function of aret as the first gene with a defined function in the cystoblast to cyst transition during early oogenesis.

  18. Asymmetric distribution of histones during Drosophila male germline stem cell asymmetric divisions.

    Science.gov (United States)

    Tran, Vuong; Feng, Lijuan; Chen, Xin

    2013-05-01

    It has long been known that epigenetic changes are inheritable. However, except for DNA methylation, little is known about the molecular mechanisms of epigenetic inheritance. Many types of stem cells undergo asymmetric cell divisions to generate self-renewed stem cells and daughter cells committed for differentiation. Still, whether and how stem cells retain their epigenetic memory remain questions to be elucidated. During the asymmetric division of Drosophila male germline stem cell (GSC), our recent studies revealed that the preexisting histone 3 (H3) are selectively segregated to the GSC, whereas newly synthesized H3 deposited during DNA replication are enriched in the differentiating daughter cell. We propose a two-step model to explain this asymmetric histone distribution. First, prior to mitosis, preexisting histones and newly synthesized histones are differentially distributed at two sets of sister chromatids. Next, during mitosis, the set of sister chromatids that mainly consist of preexisting histones are segregated to GSCs, while the other set of sister chromatids enriched with newly synthesized histones are partitioned to the daughter cell committed for differentiation. In this review, we apply current knowledge about epigenetic inheritance and asymmetric cell division to inform our discussion of potential molecular mechanisms and the cellular basis underlying this asymmetric histone distribution pattern. We will also discuss whether this phenomenon contributes to the maintenance of stem cell identity and resetting chromatin structure in the other daughter cell for differentiation.

  19. A cellular automaton model for neurogenesis in Drosophila

    Science.gov (United States)

    Luthi, Pascal O.; Chopard, Bastien; Preiss, Anette; Ramsden, Jeremy J.

    1998-07-01

    A cellular automaton (CA) is constructed for the formation of the central nervous system of the Drosophila embryo. This is an experimentally well-studied system in which complex interactions between neighbouring cells appear to drive their differentiation into different types. It appears that all the cells initially have the potential to become neuroblasts, and all strive to this end, but those which differentiate first block their as yet undifferentiated neighbours from doing so. The CA makes use of observational evidence for a lateral inhibition mechanism involving signalling products S of the ‘proneural’ or neuralizing genes. The key concept of the model is that cells are continuously producing S, but the production rate is lowered by inhibitory signals received from neighbouring cells which have advanced further along the developmental pathway. Comparison with experimental data shows that it well accounts for the observed proportion of neuroectodermal cells delaminating as neuroblasts.

  20. DeadEasy caspase: automatic counting of apoptotic cells in Drosophila.

    Directory of Open Access Journals (Sweden)

    Manuel G Forero

    Full Text Available Development, cancer, neurodegenerative and demyelinating diseases, injury, and stem cell manipulations are characterised by alterations in cell number. Research into development, disease, and the effects of drugs require cell number counts. These are generally indirect estimates, because counting cells in an animal or organ is paradoxically difficult, as well as being tedious and unmanageable. Drosophila is a powerful model organism used to investigate the genetic bases of development and disease. There are Drosophila models for multiple neurodegenerative diseases, characterised by an increase in cell death. However, a fast, reliable, and accurate way to count the number of dying cells in vivo is not available. Here, we present a method based on image filtering and mathematical morphology techniques, to count automatically the number of dying cells in intact fruit-fly embryos. We call the resulting programme DeadEasy Caspase. It has been validated for Drosophila and we present examples of its power to address biological questions. Quantification is automatic, accurate, objective, and very fast. DeadEasy Caspase will be freely available as an ImageJ plug-in, and it can be modified for use in other sample types. It is of interest to the Drosophila and wider biomedical communities. DeadEasy Caspase is a powerful tool for the analysis of cell survival and cell death in development and in disease, such as neurodegenerative diseases and ageing. Combined with the power of Drosophila genetics, DeadEasy expands the tools that enable the use of Drosophila to analyse gene function, model disease and test drugs in the intact nervous system and whole animal.

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

    Science.gov (United States)

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

    2016-08-17

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

  2. Nonperiodic activity of the human anaphase-promoting complex-Cdh1 ubiquitin ligase results in continuous DNA synthesis uncoupled from mitosis

    DEFF Research Database (Denmark)

    Lukas, C; Kramer, E R; Peters, J M

    2000-01-01

    Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccha......Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which......, in Saccharomyces cerevisiae and Drosophila spp., triggers exit from mitosis and during G(1) prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference...... ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells....

  3. What is the preimplantation embryo?

    Science.gov (United States)

    Krones, Tanja; Schlüter, Elmar; Neuwohner, Elke; El Ansari, Susan; Wissner, Thomas; Richter, Gerd

    2006-07-01

    We present results from our 'bioethical field studies', which explore and compare the views of experts, patients and the general public on the beginning of human life and the status of the preimplantation embryo in Germany. Using a qualitative and quantitative multi-method approach, we found crucial differences in the categorization of the beginning of human life within the expert group (representative samples of human geneticists n=104, ethicists n=168, midwives n=294, obstetricians n=147, paediatricians n=166), and between expert and lay samples (IVF couples n=108, high genetic risk couples n=324, general population n=1017). The majority of lay respondents as well as paediatricians and obstetricians chose nidation, the moment when the implantation of the fertilized egg into the uterus takes place, as the crucial boundary that marks the beginning of human life, whereas the majority of (female) human geneticists, ethicists and midwives voted for conception as the decisive point in time. The views of all groups on the status of the preimplantation embryo differed from the assumptions underlying German legislation (Embryo Protection Act). Religiousness and religious affiliation, gender, attitudes towards disabled people, post-material values and a present desire for a child were identified as independent factors influencing attitudes towards the preimplantation embryo in the population sample. The results are discussed within a broader philosophical and social science perspective of constructivism versus essentialism, proposing a truly interdisciplinary approach to such bioethical core issues as new reproductive technologies and the status of the preimplantation embryo.

  4. The TALE transcription factor homothorax functions to assemble heterochromatin during Drosophila embryogenesis.

    Directory of Open Access Journals (Sweden)

    Miguel Angel Zaballos

    Full Text Available We have previously identified Homothorax (Hth as an important factor for the correct assembly of the pericentromeric heterochromatin during the first fast syncytial divisions of the Drosophila embryo. Here we have extended our studies to later stages of embryonic development. We were able to show that hth mutants exhibit a drastic overall reduction in the tri-methylation of H3 in Lys9, with no reduction of the previous di-methylation. One phenotypic outcome of such a reduction is a genome instability visualized by the many DNA breaks observed in the mutant nuclei. Moreover, loss of Hth leads to the opening of closed heterochromatic regions, including the rDNA genomic region. Our data show that the satellite repeats get transcribed in wild type embryos and that this transcription depends on the presence of Hth, which binds to them as well as to the rDNA region. This work indicates that there is an important role of transcription of non-coding RNAs for constitutive heterochromatin assembly in the Drosophila embryo, and suggests that Hth plays an important role in this process.

  5. The TALE transcription factor homothorax functions to assemble heterochromatin during Drosophila embryogenesis.

    Science.gov (United States)

    Zaballos, Miguel Angel; Cantero, Walter; Azpiazu, Natalia

    2015-01-01

    We have previously identified Homothorax (Hth) as an important factor for the correct assembly of the pericentromeric heterochromatin during the first fast syncytial divisions of the Drosophila embryo. Here we have extended our studies to later stages of embryonic development. We were able to show that hth mutants exhibit a drastic overall reduction in the tri-methylation of H3 in Lys9, with no reduction of the previous di-methylation. One phenotypic outcome of such a reduction is a genome instability visualized by the many DNA breaks observed in the mutant nuclei. Moreover, loss of Hth leads to the opening of closed heterochromatic regions, including the rDNA genomic region. Our data show that the satellite repeats get transcribed in wild type embryos and that this transcription depends on the presence of Hth, which binds to them as well as to the rDNA region. This work indicates that there is an important role of transcription of non-coding RNAs for constitutive heterochromatin assembly in the Drosophila embryo, and suggests that Hth plays an important role in this process.

  6. 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.

  7. A Tribolium castaneum whole-embryo culture protocol for studying the molecular mechanisms and morphogenetic movements involved in insect development.

    Science.gov (United States)

    Macaya, Constanza C; Saavedra, Patricio E; Cepeda, Rodrigo E; Nuñez, Viviana A; Sarrazin, Andres F

    2016-01-01

    The development of the red flour beetle Tribolium castaneum is more representative of arthropods than the evolutionarily derived fly, Drosophila melanogaster. Thus, Tribolium is becoming an emerging organism model for studying the evolution of the mechanisms that control embryonic development in arthropods. In this regard, diverse genetic and molecular tools are currently available for Tribolium, as well as imaging and embryonic techniques. Recently, we developed a method for culturing embryos in order to study specific stages during Tribolium development. In this report, we present a detailed and "easy-to-follow" protocol for embryo handling and dissection, extending the use of whole-embryo culture to functional analysis by performing in vivo pharmacological manipulations. This experimental accessibility allowed us to study the relevance of microtubules in axis elongation, using nocodazole and taxol drugs to interfere with microtubule networks, followed by length measurement analysis. Additionally, we demonstrated that embryo handling had no effect on the development of Tribolium embryos, and we checked viability after dissection and bisection and during incubation using propidium iodide. The embryo culture protocol we describe here can be applied to study diverse developmental processes in Tribolium. We expect that this protocol can be adapted and applied to other arthropods.

  8. A simple TALEN-based protocol for efficient genome-editing in Drosophila.

    Science.gov (United States)

    Zhang, Xu; Ferreira, Irene R S; Schnorrer, Frank

    2014-08-15

    Drosophila is a well-established genetic model organism: thousands of point mutations, deficiencies or transposon insertions are available from stock centres. However, to date, it is still difficult to modify a specific gene locus in a defined manner. A potential solution is the application of transcription activator-like effector nucleases (TALENs), which have been used successfully to mutate genes in various model organisms. TALENs are constructed by fusion of TALE proteins to the endonuclease FokI, resulting in artificial, sequence-specific endonucleases. They induce double strand breaks, which are either repaired by error-prone non-homologous end joining (NHEJ) or homology directed repair (HDR). We developed a simple TALEN-based protocol to mutate any gene of interest in Drosophila within approximately 2 months. We inject mRNA coding for two TALEN pairs targeting the same gene into embryos, employ T7 endonuclease I screening of pooled F1 flies to identify mutations and generate a stable mutant stock in the F3 generation. We illustrate the efficacy of our strategy by mutating CG11617, a previously uncharacterized putative transcription factor with an unknown function in Drosophila. This demonstrates that TALENs are a reliable and efficient strategy to mutate any gene of interest in Drosophila.

  9. Drosophila X-Linked Genes Have Lower Translation Rates than Autosomal Genes.

    Science.gov (United States)

    Zhang, Zhenguo; Presgraves, Daven C

    2016-02-01

    In Drosophila, X-linked and autosomal genes achieve comparable expression at the mRNA level. Whether comparable X-autosome gene expression is realized at the translational and, ultimately, the protein levels is, however, unknown. Previous studies suggest the possibility of higher translation rates for X-linked genes owing to stronger usage of preferred codons. In this study, we use public ribosome profiling data from Drosophila melanogaster to infer translation rates on the X chromosome versus the autosomes. We find that X-linked genes have consistently lower ribosome densities than autosomal genes in S2 cells, early embryos, eggs, and mature oocytes. Surprisingly, the lower ribosome densities of X-linked genes are not consistent with faster translation elongation but instead imply slower translation initiation. In particular, X-linked genes have sequence features known to slow translation initiation such as stronger mRNA structure near start codons and longer 5'-UTRs. Comparison to outgroup species suggests that stronger mRNA structure is an evolved feature of Drosophila X chromosomes. Finally, we find that the magnitude of the X-autosome difference in ribosome densities is smaller for genes encoding members of protein complexes, suggesting that stoichiometry constrains the evolution of translation rates. In sum, our analyses suggest that Drosophila X-linked genes have evolved lower translation rates than autosomal genes despite stronger usage of preferred codons.

  10. Proteomics of early zebrafish embryos

    Directory of Open Access Journals (Sweden)

    Heisenberg Carl-Philipp

    2006-01-01

    Full Text Available Abstract Background Zebrafish (D. rerio has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development. While genetic methods are readily available in zebrafish, protocols for two dimensional (2D gel electrophoresis and proteomics have yet to be developed. Results As a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a method to efficiently remove the yolk from large batches of embryos. This method enabled high resolution 2D gel electrophoresis and improved Western blotting considerably. Here, we provide detailed protocols for proteomics in zebrafish from sample preparation to mass spectrometry (MS, including a comparison of databases for MS identification of zebrafish proteins. Conclusion The provided protocols for proteomic analysis of early embryos enable research to be taken in novel directions in embryogenesis.

  11. Cell adhesion in embryo morphogenesis.

    Science.gov (United States)

    Barone, Vanessa; Heisenberg, Carl-Philipp

    2012-02-01

    Visualizing and analyzing shape changes at various scales, ranging from single molecules to whole organisms, are essential for understanding complex morphogenetic processes, such as early embryonic development. Embryo morphogenesis relies on the interplay between different tissues, the properties of which are again determined by the interaction between their constituent cells. Cell interactions, on the other hand, are controlled by various molecules, such as signaling and adhesion molecules, which in order to exert their functions need to be spatiotemporally organized within and between the interacting cells. In this review, we will focus on the role of cell adhesion functioning at different scales to organize cell, tissue and embryo morphogenesis. We will specifically ask how the subcellular distribution of adhesion molecules controls the formation of cell-cell contacts, how cell-cell contacts determine tissue shape, and how tissue interactions regulate embryo morphogenesis.

  12. Control of dendritic morphogenesis by Trio in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Madhuri Shivalkar

    Full Text Available Abl tyrosine kinase and its effectors among the Rho family of GTPases each act to control dendritic morphogenesis in Drosophila. It has not been established, however, which of the many GTPase regulators in the cell link these signaling molecules in the dendrite. In axons, the bifunctional guanine exchange factor, Trio, is an essential link between the Abl tyrosine kinase signaling pathway and Rho GTPases, particularly Rac, allowing these systems to act coordinately to control actin organization. In dendritic morphogenesis, however, Abl and Rac have contrary rather than reinforcing effects, raising the question of whether Trio is involved, and if so, whether it acts through Rac, Rho or both. We now find that Trio is expressed in sensory neurons of the Drosophila embryo and regulates their dendritic arborization. trio mutants display a reduction in dendritic branching and increase in average branch length, whereas over-expression of trio has the opposite effect. We further show that it is the Rac GEF domain of Trio, and not its Rho GEF domain that is primarily responsible for the dendritic function of Trio. Thus, Trio shapes the complexity of dendritic arbors and does so in a way that mimics the effects of its target, Rac.

  13. Accelerated homologous recombination and subsequent genome modification in Drosophila

    Science.gov (United States)

    Baena-Lopez, Luis Alberto; Alexandre, Cyrille; Mitchell, Alice; Pasakarnis, Laurynas; Vincent, Jean-Paul

    2013-01-01

    Gene targeting by ‘ends-out’ homologous recombination enables the deletion of genomic sequences and concurrent introduction of exogenous DNA with base-pair precision without sequence constraint. In Drosophila, this powerful technique has remained laborious and hence seldom implemented. We describe a targeting vector and protocols that achieve this at high frequency and with very few false positives in Drosophila, either with a two-generation crossing scheme or by direct injection in embryos. The frequency of injection-mediated gene targeting can be further increased with CRISPR-induced double-strand breaks within the region to be deleted, thus making homologous recombination almost as easy as conventional transgenesis. Our targeting vector replaces genomic sequences with a multifunctional fragment comprising an easy-to-select genetic marker, a fluorescent reporter, as well as an attP site, which acts as a landing platform for reintegration vectors. These vectors allow the insertion of a variety of transcription reporters or cDNAs to express tagged or mutant isoforms at endogenous levels. In addition, they pave the way for difficult experiments such as tissue-specific allele switching and functional analysis in post-mitotic or polyploid cells. Therefore, our method retains the advantages of homologous recombination while capitalising on the mutagenic power of CRISPR. PMID:24154526

  14. In amnio MRI of mouse embryos.

    Directory of Open Access Journals (Sweden)

    Thomas A Roberts

    Full Text Available Mouse embryo imaging is conventionally carried out on ex vivo embryos excised from the amniotic sac, omitting vital structures and abnormalities external to the body. Here, we present an in amnio MR imaging methodology in which the mouse embryo is retained in the amniotic sac and demonstrate how important embryonic structures can be visualised in 3D with high spatial resolution (100 µm/px. To illustrate the utility of in amnio imaging, we subsequently apply the technique to examine abnormal mouse embryos with abdominal wall defects. Mouse embryos at E17.5 were imaged and compared, including three normal phenotype embryos, an abnormal embryo with a clear exomphalos defect, and one with a suspected gastroschisis phenotype. Embryos were excised from the mother ensuring the amnion remained intact and stereo microscopy was performed. Embryos were next embedded in agarose for 3D, high resolution MRI on a 9.4T scanner. Identification of the abnormal embryo phenotypes was not possible using stereo microscopy or conventional ex vivo MRI. Using in amnio MRI, we determined that the abnormal embryos had an exomphalos phenotype with varying severities. In amnio MRI is ideally suited to investigate the complex relationship between embryo and amnion, together with screening for other abnormalities located outside of the mouse embryo, providing a valuable complement to histology and existing imaging methods available to the phenotyping community.

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

    Science.gov (United States)

    Li, Yan; Butenko, Yana; Grafi, Gideon

    2005-02-01

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

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

    Science.gov (United States)

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

    2002-01-01

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

  17. Translocation of histone H1 subtypes between chromatin and cytoplasm during mitosis in normal human fibroblasts.

    Science.gov (United States)

    Gréen, Anna; Lönn, Anita; Peterson, Kajsa Holmgren; Ollinger, Karin; Rundquist, Ingemar

    2010-05-01

    Histone H1 is an important constituent of chromatin, which undergoes major structural rearrangements during mitosis. However, the role of H1, multiple H1 subtypes, and H1 phosphorylation is still unclear. In normal human fibroblasts, phosphorylated H1 was found located in nuclei during prophase and in both cytoplasm and condensed chromosomes during metaphase, anaphase, and telophase as detected by immunocytochemistry. Moreover, we detected remarkable differences in the distribution of the histone H1 subtypes H1.2, H1.3, and H1.5 during mitosis. H1.2 was found in chromatin during prophase and almost solely in the cytoplasm of metaphase and early anaphase cells. In late anaphase, it appeared in both chromatin and cytoplasm and again in chromatin during telophase. H1.5 distribution pattern resembled that of H1.2, but H1.5 was partitioned between chromatin and cytoplasm during metaphase and early anaphase. H1.3 was detected in chromatin in all cell cycle phases. We propose therefore, that H1 subtype translocation during mitosis is controlled by phosphorylation, in combination with H1 subtype inherent affinity. We conclude that H1 subtypes, or theirphosphorylated forms, may leave chromatin in a regulated way to give access for chromatin condensing factors or transcriptional regulators during mitosis.

  18. CDK-1 Inhibition in G2 Stabilizes Kinetochore-Microtubules in the following Mitosis.

    Science.gov (United States)

    Gayek, A Sophia; Ohi, Ryoma

    2016-01-01

    Cell proliferation is driven by cyclical activation of cyclin-dependent kinases (CDKs), which produce distinct biochemical cell cycle phases. Mitosis (M phase) is orchestrated by CDK-1, complexed with mitotic cyclins. During M phase, chromosomes are segregated by a bipolar array of microtubules called the mitotic spindle. The essential bipolarity of the mitotic spindle is established by the kinesin-5 Eg5, but factors influencing the maintenance of spindle bipolarity are not fully understood. Here, we describe an unexpected link between inhibiting CDK-1 before mitosis and bipolar spindle maintenance. Spindles in human RPE-1 cells normally collapse to monopolar structures when Eg5 is inhibited at metaphase. However, we found that inhibition of CDK-1 in the G2 phase of the cell cycle improved the ability of RPE-1 cells to maintain spindle bipolarity without Eg5 activity in the mitosis immediately after release from CDK-1 inhibition. This improved bipolarity maintenance correlated with an increase in the stability of kinetochore-microtubules, the subset of microtubules that link chromosomes to the spindle. The improvement in bipolarity maintenance after CDK-1 inhibition in G2 required both the kinesin-12 Kif15 and increased stability of kinetochore-microtubules. Consistent with increased kinetochore-microtubule stability, we find that inhibition of CDK-1 in G2 impairs mitotic fidelity by increasing the incidence of lagging chromosomes in anaphase. These results suggest that inhibition of CDK-1 in G2 causes unpredicted effects in mitosis, even after CDK-1 inhibition is relieved.

  19. 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…

  20. 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.

  1. 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.

  2. Nuclear envelope expansion is crucial for proper chromosomal segregation during a closed mitosis.

    Science.gov (United States)

    Takemoto, Ai; Kawashima, Shigehiro A; Li, Juan-Juan; Jeffery, Linda; Yamatsugu, Kenzo; Elemento, Olivier; Nurse, Paul

    2016-03-15

    Here, we screened a 10,371 library of diverse molecules using a drug-sensitive fission yeast strain to identify compounds which cause defects in chromosome segregation during mitosis. We identified a phosphorium-ylide-based compound Cutin-1 which inhibits nuclear envelope expansion and nuclear elongation during the closed mitosis of fission yeast, and showed that its target is the β-subunit of fatty acid synthase. A point mutation in the dehydratase domain of Fas1 conferred in vivo and in vitro resistance to Cutin-1. Time-lapse photomicrography showed that the bulk of the chromosomes were only transiently separated during mitosis, and nucleoli separation was defective. Subsequently sister chromatids re-associated leading to chromosomal mis-segregation. These segregation defects were reduced when the nuclear volume was increased and were increased when the nuclear volume was reduced. We propose that there needs to be sufficient nuclear volume to allow the nuclear elongation necessary during a closed mitosis to take place for proper chromosome segregation, and that inhibition of fatty acid synthase compromises nuclear elongation and leads to defects in chromosomal segregation.

  3. 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.

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

    Science.gov (United States)

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

    2014-09-15

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

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

    DEFF Research Database (Denmark)

    Ying, Songmin; Hickson, Ian D

    2011-01-01

    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...

  6. 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

  7. 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

  8. 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

  9. Assessment of algorithms for mitosis detection in breast cancer histopathology images

    NARCIS (Netherlands)

    Veta, Mitko; van Diest, Paul J.|info:eu-repo/dai/nl/075281775; Willems, Stefan M.; Madabhushi, Anant; Cruz-Roa, Angel; Gonzalez, Fabio; Larsen, Anders B L; Vestergaard, Jacob S.; Dahl, Anders B.; Cireşan, Dan C.; Schmidhuber, Jürgen; Giusti, Alessandro; Gambardella, Luca M.; Tek, F. Boray; Walter, Thomas; Wang, Ching Wei; Kondo, Satoshi; Matuszewski, Bogdan J.; Precioso, Frederic; Snell, Violet; Kittler, Josef; de Campos, Teofilo E.; Khan, Adnan M.; Rajpoot, Nasir M.; Arkoumani, Evdokia; Lacle, Miangela M.; Viergever, Max A.|info:eu-repo/dai/nl/108781828; Pluim, JPW|info:eu-repo/dai/nl/224274333

    2015-01-01

    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 low i

  10. 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

    inter-observer agreement. With the wider acceptance of whole slide images in pathology labs, automatic image analysis has been proposed as a potential solution for these issues.In this paper, the results from the Assessment of Mitosis Detection Algorithms 2013 (AMIDA13) challenge are described...

  11. Antagonistic effect of polyamines on ABA-induced suppression of mitosis in Allium cepa L.

    Science.gov (United States)

    Mahajan, Arpana; Sharma, Shashi

    2009-02-01

    Effect of abscisic acid (ABA) and polyamines (PAs) [putrescine (Put), spermidine (Spd) and spermine (Spm)] on mitosis in root tips of A. cepa was studied. Treatment with ABA (0.1 to 100 microM) for 24 hr suppressed the mitosis, measured as mitotic index (MI), in a concentration-dependent manner with approx. 50% suppression at 10 microM of ABA. Treatment with different PAs (1 to 100 microM) had differential mitosis suppression effect. Spm was most inhibitory followed by Spd and Put, respectively. The higher concentrations of PAs (1 mM Put; 0.1 and 1 mM Spd or Spm) caused cell distortion. Remarkably, a 24 hr pretreatment of root tips with PAs prior to ABA (100 microM) treatment resulted in a general concentration-dependent reversal of ABA-induced suppression of MI. Catalase (CAT) activity in the root tips, an indicator of redox metabolism, increased due to ABA treatment in a concentration-dependent manner, remained unaltered in response to Put and declined due to Spd and Spm (> or = 0.1 mM). However, all PAs, irrespective of their individual effects, generally antagonized the ABA-dependent increase in CAT activity. Data indicate the possibility of ABA-PA interaction in the regulation of mitosis.

  12. Assessment of algorithms for mitosis detection in breast cancer histopathology images

    NARCIS (Netherlands)

    Veta, Mitko; van Diest, Paul J.; Willems, Stefan M.; Madabhushi, Anant; Cruz-Roa, Angel; Gonzalez, Fabio; Larsen, Anders B L; Vestergaard, Jacob S.; Dahl, Anders B.; Cireşan, Dan C.; Schmidhuber, Jürgen; Giusti, Alessandro; Gambardella, Luca M.; Tek, F. Boray; Walter, Thomas; Wang, Ching Wei; Kondo, Satoshi; Matuszewski, Bogdan J.; Precioso, Frederic; Snell, Violet; Kittler, Josef; de Campos, Teofilo E.; Khan, Adnan M.; Rajpoot, Nasir M.; Arkoumani, Evdokia; Lacle, Miangela M.; Viergever, Max A.; Pluim, JPW

    2015-01-01

    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 low i

  13. 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…

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

    Science.gov (United States)

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

    2014-01-01

    Unless mitigated, external and physiological stresses are detrimental for cells, especially in mitosis, resulting in chromosomal missegregation, aneuploidy, or apoptosis. Heat shock proteins (Hsps) maintain protein homeostasis and promote cell survival. Hsps are transcriptionally regulated by heat shock factors (HSFs). Of these, HSF1 is the master regulator and HSF2 modulates Hsp expression by interacting with HSF1. Due to global inhibition of transcription in mitosis, including HSF1-mediated expression of Hsps, mitotic cells are highly vulnerable to stress. Here, we show that cells can counteract transcriptional silencing and protect themselves against proteotoxicity in mitosis. We found that the condensed chromatin of HSF2-deficient cells is accessible for HSF1 and RNA polymerase II, allowing stress-inducible Hsp expression. Consequently, HSF2-deficient cells exposed to acute stress display diminished mitotic errors and have a survival advantage. We also show that HSF2 expression declines during mitosis in several but not all human cell lines, which corresponds to the Hsp70 induction and protection against stress-induced mitotic abnormalities and apoptosis. PMID:25202032

  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. 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

  17. 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.

  18. Xenopus BTBD6 and its Drosophila homologue lute are required for neuronal development.

    Science.gov (United States)

    Bury, Frédéric J; Moers, Virginie; Yan, Jiekun; Souopgui, Jacob; Quan, Xiao-Jiang; De Geest, Natalie; Kricha, Sadia; Hassan, Bassem A; Bellefroid, Eric J

    2008-11-01

    BBP proteins constitute a subclass of CUL3 interacting BTB proteins whose in vivo function remains unknown. Here, we show that the Xenopus BBP gene BTBD6 and the single Drosophila homologue of mammalian BBP genes lute are strongly expressed in the developing nervous system. In Xenopus, BTBD6 expression responds positively to proneural and negatively to neurogenic gene overexpression. Knockdown of BTBD6 in Xenopus or loss of Drosophila lute result in embryos with strong defects in late neuronal markers and strongly reduced and disorganized axons while early neural development is unaffected. XBTBD6 knockdown in Xenopus also affects muscle development. Together, these data indicate that BTBD6/lute is required for proper embryogenesis and plays an essential evolutionary conserved role during neuronal development.

  19. From the Eye to the Brain: Development of the Drosophila Visual System.

    Science.gov (United States)

    Nériec, Nathalie; Desplan, Claude

    2016-01-01

    How stem cells produce the huge diversity of neurons that form the visual system, and how these cells are assembled in neural circuits are a critical question in developmental neurobiology. Investigations in Drosophila have led to the discovery of several basic principles of neural patterning. In this chapter, we provide an overview of the field by describing the development of the Drosophila visual system, from the embryo to the adult and from the gross anatomy to the cellular level. We then explore the general molecular mechanisms identified that might apply to other neural structures in flies or in vertebrates. Finally, we discuss the major challenges that remain to be addressed in the field. © 2016 Elsevier Inc. All rights reserved.

  20. Two-photon fluorescence imaging and femtosecond laser microsurgery to study drosophila dorsal closure

    Science.gov (United States)

    Thayil K. N., Anisha; Pereira, Andrea; Mathew, Manoj; Artigas, David; Martín Blanco, Enrique; Loza-Alvarez, Pablo

    2008-02-01

    Dorsal closure is a key morphogenic process that occurs at the last stages of Drosophila melanogaster embryogenesis. It involves a well coordinated rearrangement and movement of tissues that resemble epithelial wound healing in mammals. The cell dynamics and intracellular signaling pathways that accompany hole closure are expected to be similar during would healing providing a model system to study epithelial healing. Here we demonstrate the use of two-photon fluorescence microscope together with femtosecond laser ablation to examine the epithelial wound healing during embryonic dorsal closure. By using tightly focused NIR femtosecond pulses of subnanojoule energy we are able to produce highly confined microsurgery on the epithelial cells of a developing embryo. We observed that drosophila epidermis heals from the laser wounds with increased activity of actin near the wound edges.

  1. Highly Efficient Targeted Mutagenesis of Drosophila with the CRISPR/Cas9 System

    Directory of Open Access Journals (Sweden)

    Andrew R. Bassett

    2013-07-01

    Full Text Available Here, we present a simple and highly efficient method for generating and detecting mutations of any gene in Drosophila melanogaster through the use of the CRISPR/Cas9 system (clustered regularly interspaced palindromic repeats/CRISPR-associated. We show that injection of RNA into the Drosophila embryo can induce highly efficient mutagenesis of desired target genes in up to 88% of injected flies. These mutations can be transmitted through the germline to make stable lines. Our system provides at least a 10-fold improvement in efficiency over previously published reports, enabling wider application of this technique. We also describe a simple and highly sensitive method of detecting mutations in the target gene by high-resolution melt analysis and discuss how the new technology enables the study of gene function.

  2. Evolutionary Techniques for Image Processing a Large Dataset of Early Drosophila Gene Expression

    Directory of Open Access Journals (Sweden)

    David M. Holloway

    2003-07-01

    Full Text Available Understanding how genetic networks act in embryonic development requires a detailed and statistically significant dataset integrating diverse observational results. The fruit fly (Drosophila melanogaster is used as a model organism for studying developmental genetics. In recent years, several laboratories have systematically gathered confocal microscopy images of patterns of activity (expression for genes governing early Drosophila development. Due to both the high variability between fruit fly embryos and diverse sources of observational errors, some new nontrivial procedures for processing and integrating the raw observations are required. Here we describe processing techniques based on genetic algorithms and discuss their efficacy in decreasing observational errors and illuminating the natural variability in gene expression patterns. The specific developmental problem studied is anteroposterior specification of the body plan.

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

    Science.gov (United States)

    Markov, Alexander V; Kaznacheev, Ilya S

    2016-06-08

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

  4. Drosophila models for cancer research.

    Science.gov (United States)

    Vidal, Marcos; Cagan, Ross L

    2006-02-01

    Drosophila is a model system for cancer research. Investigation with fruit flies has facilitated a number of important recent discoveries in the field: the hippo signaling pathway, which coordinates cell proliferation and death to achieve normal tissue size; 'social' behaviors of cells, including cell competition and apoptosis-induced compensatory proliferation, that help ensure normal tissue size; and a growing understanding of how oncogenes and tumor suppressors cooperate to achieve tumor growth and metastasis in situ. In the future, Drosophila models can be extended beyond basic research in the search for human therapeutics.

  5. Future aspects of micromanipualtion with embryos for

    African Journals Online (AJOL)

    Embryo micromanipulation techniques and their potential genetic impact in dairy cattle ... bovine embryos and the subsequent transfer of halfembryos has reached a .... (e) to provide synchronously developing pronuclear srage ova for nuclear ...

  6. Embryo splitting: a role in infertility?

    Science.gov (United States)

    Wood, C

    2001-01-01

    Embryo splitting may be used to increase the potential fertility of couples requiring IVF. Using cattle as a model, it is possible to increase pregnancy rates from 70% per transfer of good quality in-vivo-produced embryos, to 110% by transferring the two demi-embryos resulting from the bisection of one embryo. The 30-40% greater chance of conception would reduce costs for the government, health authorities and patients, and reduce stress, time and complications for women having IVF treatment. Embryo splitting may also provide donor embryos for infertile couples that cannot conceive naturally or with IVF. The shortage of children for adoption and donor embryos may be overcome by the production of demi-embryos.

  7. Negative regulation of the endocytic adaptor disabled-2 (Dab2) in mitosis.

    Science.gov (United States)

    Chetrit, David; Barzilay, Lior; Horn, Galit; Bielik, Tom; Smorodinsky, Nechama I; Ehrlich, Marcelo

    2011-02-18

    Mitotic cells undergo extensive changes in shape and size through the altered regulation and function of their membrane trafficking machinery. Disabled 2 (Dab2), a multidomain cargo-specific endocytic adaptor and a mediator of signal transduction, is a potential integrator of trafficking and signaling. Dab2 binds effectors of signaling and trafficking that localize to different intracellular compartments. Thus, differential localization is a putative regulatory mechanism of Dab2 function. Furthermore, Dab2 is phosphorylated in mitosis and is thus regulated in the cell cycle. However, a detailed description of the intracellular localization of Dab2 in the different phases of mitosis and an understanding of the functional consequences of its phosphorylation are lacking. Here, we show that Dab2 is progressively displaced from the membrane in mitosis. This phenomenon is paralleled by a loss of co-localization with clathrin. Both phenomena culminate in metaphase/anaphase and undergo partial recovery in cytokinesis. Treatment with 2-methoxyestradiol, which arrests cells at the spindle assembly checkpoint, induces the same effects observed in metaphase cells. Moreover, 2-methoxyestradiol also induced Dab2 phosphorylation and reduced Dab2/clathrin interactions, endocytic vesicle motility, clathrin exchange dynamics, and the internalization of a receptor endowed with an NPXY endocytic signal. Serine/threonine to alanine mutations, of residues localized to the central region of Dab2, attenuated its phosphorylation, reduced its membrane displacement, and maintained its endocytic abilities in mitosis. We propose that the negative regulation of Dab2 is part of an accommodation of the cell to the altered physicochemical conditions prevalent in mitosis, aimed at allowing endocytic activity throughout the cell cycle.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  10. Cdc15 Phosphorylates the C-terminal Domain of RNA Polymerase II for Transcription during Mitosis.

    Science.gov (United States)

    Singh, Amit Kumar; Rastogi, Shivangi; Shukla, Harish; Asalam, Mohd; Rath, Srikanta Kumar; Akhtar, Md Sohail

    2017-03-31

    In eukaryotes, the basal transcription in interphase is orchestrated through the regulation by kinases (Kin28, Bur1, and Ctk1) and phosphatases (Ssu72, Rtr1, and Fcp1), which act through the post-translational modification of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. The CTD comprises the repeated Tyr-Ser-Pro-Thr-Ser-Pro-Ser motif with potential epigenetic modification sites. Despite the observation of transcription and periodic expression of genes during mitosis with entailing CTD phosphorylation and dephosphorylation, the associated CTD specific kinase(s) and its role in transcription remains unknown. Here we have identified Cdc15 as a potential kinase phosphorylating Ser-2 and Ser-5 of CTD for transcription during mitosis in the budding yeast. The phosphorylation of CTD by Cdc15 is independent of any prior Ser phosphorylation(s). The inactivation of Cdc15 causes reduction of global CTD phosphorylation during mitosis and affects the expression of genes whose transcript levels peak during mitosis. Cdc15 also influences the complete transcription of clb2 gene and phosphorylates Ser-5 at the promoter and Ser-2 toward the 3' end of the gene. The observation that Cdc15 could phosphorylate Ser-5, as well as Ser-2, during transcription in mitosis is in contrast to the phosphorylation marks put by the kinases in interphase (G1, S, and G2), where Cdck7/Kin28 phosphorylates Ser-5 at promoter and Bur1/Ctk1 phosphorylates Ser-2 at the 3' end of the genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. A conserved developmental patterning network produces quantitatively different output in multiple species of Drosophila.

    Science.gov (United States)

    Fowlkes, Charless C; Eckenrode, Kelly B; Bragdon, Meghan D; Meyer, Miriah; Wunderlich, Zeba; Simirenko, Lisa; Luengo Hendriks, Cris L; Keränen, Soile V E; Henriquez, Clara; Knowles, David W; Biggin, Mark D; Eisen, Michael B; DePace, Angela H

    2011-10-01

    Differences in the level, timing, or location of gene expression can contribute to alternative phenotypes at the molecular and organismal level. Understanding the origins of expression differences is complicated by the fact that organismal morphology and gene regulatory networks could potentially vary even between closely related species. To assess the scope of such changes, we used high-resolution imaging methods to measure mRNA expression in blastoderm embryos of Drosophila yakuba and Drosophila pseudoobscura and assembled these data into cellular resolution atlases, where expression levels for 13 genes in the segmentation network are averaged into species-specific, cellular resolution morphological frameworks. We demonstrate that the blastoderm embryos of these species differ in their morphology in terms of size, shape, and number of nuclei. We present an approach to compare cellular gene expression patterns between species, while accounting for varying embryo morphology, and apply it to our data and an equivalent dataset for Drosophila melanogaster. Our analysis reveals that all individual genes differ quantitatively in their spatio-temporal expression patterns between these species, primarily in terms of their relative position and dynamics. Despite many small quantitative differences, cellular gene expression profiles for the whole set of genes examined are largely similar. This suggests that cell types at this stage of development are conserved, though they can differ in their relative position by up to 3-4 cell widths and in their relative proportion between species by as much as 5-fold. Quantitative differences in the dynamics and relative level of a subset of genes between corresponding cell types may reflect altered regulatory functions between species. Our results emphasize that transcriptional networks can diverge over short evolutionary timescales and that even small changes can lead to distinct output in terms of the placement and number of

  12. A conserved developmental patterning network produces quantitatively different output in multiple species of Drosophila.

    Directory of Open Access Journals (Sweden)

    Charless C Fowlkes

    2011-10-01

    Full Text Available Differences in the level, timing, or location of gene expression can contribute to alternative phenotypes at the molecular and organismal level. Understanding the origins of expression differences is complicated by the fact that organismal morphology and gene regulatory networks could potentially vary even between closely related species. To assess the scope of such changes, we used high-resolution imaging methods to measure mRNA expression in blastoderm embryos of Drosophila yakuba and Drosophila pseudoobscura and assembled these data into cellular resolution atlases, where expression levels for 13 genes in the segmentation network are averaged into species-specific, cellular resolution morphological frameworks. We demonstrate that the blastoderm embryos of these species differ in their morphology in terms of size, shape, and number of nuclei. We present an approach to compare cellular gene expression patterns between species, while accounting for varying embryo morphology, and apply it to our data and an equivalent dataset for Drosophila melanogaster. Our analysis reveals that all individual genes differ quantitatively in their spatio-temporal expression patterns between these species, primarily in terms of their relative position and dynamics. Despite many small quantitative differences, cellular gene expression profiles for the whole set of genes examined are largely similar. This suggests that cell types at this stage of development are conserved, though they can differ in their relative position by up to 3-4 cell widths and in their relative proportion between species by as much as 5-fold. Quantitative differences in the dynamics and relative level of a subset of genes between corresponding cell types may reflect altered regulatory functions between species. Our results emphasize that transcriptional networks can diverge over short evolutionary timescales and that even small changes can lead to distinct output in terms of the placement and

  13. Comprehensive transcriptome analysis of early male and female Bactrocera jarvisi embryos.

    Science.gov (United States)

    Morrow, Jennifer L; Riegler, Markus; Gilchrist, A Stuart; Shearman, Deborah C A; Frommer, Marianne

    2014-01-01

    Developing embryos are provided with maternal RNA transcripts and proteins, but transcription from the zygotic nuclei must be activated to control continuing embryonic development. Transcripts are generated at different stages of early development, and those involved in sex determination and cellularisation are some of the earliest to be activated. The male sex in tephritid fruit flies is determined by the presence of a Y chromosome, and it is believed that a transcript from the Y-chromosome sets in motion a cascade that determines male development, as part of the greater maternal to zygotic transition (MTZ). Here we investigate the poly(A+) transcriptome in early male and female embryos of the horticultural pest Bactrocera jarvisi (Diptera: Tephritidae). Bactrocera jarvisi embryos were collected over two pre-blastoderm time periods, 2-3h and 3-5h after egg laying. Embryos were individually sexed using a Y-chromosome marker, allowing the sex-specific poly(A+) transcriptome of single-sex embryo pools to be deep-sequenced and assembled de novo. Transcripts for sixteen sex-determination and two cellularisation gene homologues of Drosophila melanogaster (Diptera: Drosophilidae) were identified in early embryos of B. jarvisi, including transcripts highly upregulated prior to cellularisation. No strong candidates for transcripts derived solely from the Y chromosome were recovered from the poly(A+) fraction. Bactrocera jarvisi provides an excellent model for embryonic studies due to available Y-chromosome markers and the compact time frame for zygotic transcription and the sex-determined state. Our data contribute fundamental information to sex-determination research, and provide candidates for the sourcing of gene promoters for transgenic pest-management strategies of tephritid fruit flies.

  14. 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.

  15. Human Cdc14B promotes progression through mitosis by dephosphorylating Cdc25 and regulating Cdk1/cyclin B activity.

    Directory of Open Access Journals (Sweden)

    Indra Tumurbaatar

    Full Text Available Entry into and progression through mitosis depends on phosphorylation and dephosphorylation of key substrates. In yeast, the nucleolar phosphatase Cdc14 is pivotal for exit from mitosis counteracting Cdk1-dependent phosphorylations. Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known. Here we show that hCdc14B serves a critical role in regulating progression through mitosis, which is distinct from hCdc14A. Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis. Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation. The results demonstrate that hCdc14B-dependent modulation of Cdc25 phosphatase and Cdk1/cyclin B activity is tightly linked to correct chromosome segregation and bipolar spindle formation, processes that are required for proper progression through mitosis and maintenance of genomic stability.

  16. Embryo growth in mature celery seeds

    NARCIS (Netherlands)

    Toorn, van der P.

    1989-01-01

    Germination of celery seeds is slow, due to the need for embryo growth before radicle protrusion can occur. Germination rate was correlated with embryo growth rate. Celery seeds with different embryo growth rates were obtained with fluid density separation of a seed lot. Low density seeds g

  17. Embryo temperature during incubation: practice and theory

    NARCIS (Netherlands)

    Lourens, A.

    2008-01-01

    (Key words: incubation, embryo temperature, embryonic development, heat production, heat loss) Until recently, all incubator studies were performed using a constant machine temperature (MT). But it is embryo temperature (ET) that is of importance to the embryo, and not MT. In practice, MT is often

  18. Improving embryo quality in assisted reproduction

    NARCIS (Netherlands)

    Mantikou, E.

    2013-01-01

    The goal of this thesis was to improve embryo quality in assisted reproductive technologies by gaining more insight into human preimplantation embryo development and by improving in vitro culture conditions. To do so, we investigated an intriguing feature of the human preimplantation embryo, i.e. it

  19. Embryo growth in mature celery seeds

    NARCIS (Netherlands)

    Toorn, van der P.

    1989-01-01

    Germination of celery seeds is slow, due to the need for embryo growth before radicle protrusion can occur. Germination rate was correlated with embryo growth rate. Celery seeds with different embryo growth rates were obtained with fluid density separation of a seed lot. Low density seeds

  20. Single-Cell Dynamic Analysis of Mitosis in Haploid Embryonic Stem Cells Shows the Prolonged Metaphase and Its Association with Self-diploidization

    Directory of Open Access Journals (Sweden)

    Ao Guo

    2017-05-01

    Full Text Available The recent establishment of mammalian haploid embryonic stem cells (ESCs provides new possibilities for genetic screening and for understanding genome evolution and function. However, the dynamics of mitosis in haploid ESCs is still unclear. Here, we report that the duration of mitosis in haploid ESCs, especially the metaphase, is significantly longer than that in diploid ESCs. Delaying mitosis by chemicals increased self-diploidization of haploid ESCs, while shortening mitosis stabilized haploid ESCs. Taken together, our study suggests that the delayed mitosis of haploid ESCs is associated with self-diploidization.

  1. Myc localizes to histone locus bodies during replication in Drosophila.

    Directory of Open Access Journals (Sweden)

    Kaveh Daneshvar

    Full Text Available Myc is an important protein at the center of multiple pathways required for growth and proliferation in animals. The absence of Myc is lethal in flies and mice, and its over-production is a potent inducer of over-proliferation and cancer. Myc protein is localized to the nucleus where it executes its many functions, however the specific sub-nuclear localization of Myc has rarely been reported. The work we describe here began with an observation of unexpected, punctate spots of Myc protein in certain regions of Drosophila embryos. We investigated the identity of these puncta and demonstrate that Myc is co-localized with coilin, a marker for sub-nuclear organelles known as Cajal Bodies (CBs, in embryos, larvae and ovaries. Using antibodies specific for U7 snRNP component Lsm11, we show that the majority of Myc and coilin co-localization occurs in Histone Locus Bodies (HLBs, the sites of histone mRNA transcription and processing. Furthermore, Myc localizes to HLBs only during replication in mitotic and endocycling cells, suggesting that its role there relates to replication-dependent canonical histone gene transcription. These results provide evidence that sub-nuclear localization of Myc is cell-cycle dependent and potentially important for histone mRNA production and processing.

  2. zen and the art of phenotypic maintenance: canalization of embryonic dorsal-ventral patterning in Drosophila.

    Science.gov (United States)

    Gavin-Smyth, Jackie; Ferguson, Edwin L

    2014-01-01

    We recently uncovered a novel genetic mechanism that generates the phenotypic uniformity, or canalization, of BMP signaling and cell fate specification during patterning of the dorsal-ventral (D/V) axis in D. melanogaster embryos. We went on to show that other wild-type Drosophila species lack this canalizing genetic circuitry and, consequently, have non-robust D/V patterning. In this review, we propose molecular mechanisms that may give rise to stereotyped BMP signaling, and we identify an additional species that could have decanalized D/V patterning. Extension of these analyses could in turn help explain why canalization is not a universal necessity for species survival.

  3. Cloning and expression of Xenopus Prickle, an orthologue of a Drosophila planar cell polarity gene.

    Science.gov (United States)

    Wallingford, John B; Goto, Toshiyasu; Keller, Ray; Harland, Richard M

    2002-08-01

    We have cloned Xenopus orthologues of the Drosophila planar cell polarity (PCP) gene Prickle. Xenopus Prickle (XPk) is expressed in tissues at the dorsal midline during gastrulation and early neurulation. XPk is later expressed in a segmental pattern in the presomitic mesoderm and then in recently formed somites. XPk is also expressed in the tailbud, pronephric duct, retina, and the otic vesicle. The complex expression pattern of XPk suggests that PCP signaling is used in a diverse array of developmental processes in vertebrate embryos.

  4. An epidermal barrier wound repair pathway in Drosophila is mediated by grainy head.

    Science.gov (United States)

    Mace, Kimberly A; Pearson, Joseph C; McGinnis, William

    2005-04-15

    We used wounded Drosophila embryos to define an evolutionarily conserved pathway for repairing the epidermal surface barrier. This pathway includes a wound response enhancer from the Ddc gene that requires grainy head (grh) function and binding sites for the Grh transcription factor. At the signaling level, tyrosine kinase and extracellular signal-regulated kinase (ERK) activities are induced in epidermal cells near wounds, and activated ERK is required for a robust wound response. The conservation of this Grh-dependent pathway suggests that the repair of insect cuticle and mammal skin is controlled by an ancient, shared control system for constructing and healing the animal body surface barrier.

  5. Establishment and mitotic characterization of new Drosophila acentriolar cell lines from DSas-4 mutant

    Directory of Open Access Journals (Sweden)

    Nicolas Lecland

    2013-01-01

    In animal cells the centrosome is commonly viewed as the main cellular structure driving microtubule (MT assembly into the mitotic spindle apparatus. However, additional pathways, such as those mediated by chromatin and augmin, are involved in the establishment of functional spindles. The molecular mechanisms involved in these pathways remain poorly understood, mostly due to limitations inherent to current experimental systems available. To overcome these limitations we have developed six new Drosophila cell lines derived from Drosophila homozygous mutants for DSas-4, a protein essential for centriole biogenesis. These cells lack detectable centrosomal structures, astral MT, with dispersed pericentriolar proteins D-PLP, Centrosomin and γ-tubulin. They show poorly focused spindle poles that reach the plasma membrane. Despite being compromised for functional centrosome, these cells could successfully undergo mitosis. Live-cell imaging analysis of acentriolar spindle assembly revealed that nascent MTs are nucleated from multiple points in the vicinity of chromosomes. These nascent MTs then grow away from kinetochores allowing the expansion of fibers that will be part of the future acentriolar spindle. MT repolymerization assays illustrate that acentriolar spindle assembly occurs “inside-out” from the chromosomes. Colchicine-mediated depolymerization of MTs further revealed the presence of a functional Spindle Assembly Checkpoint (SAC in the acentriolar cells. Finally, pilot RNAi experiments open the potential use of these cell lines for the molecular dissection of anastral pathways in spindle and centrosome assembly.

  6. A genome-wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila.

    Directory of Open Access Journals (Sweden)

    Jeroen Dobbelaere

    2008-09-01

    Full Text Available Centrosomes comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM. Here, we have performed a microscopy-based genome-wide RNA interference (RNAi screen in Drosophila cells to identify proteins required for centriole duplication and mitotic PCM recruitment. We analysed 92% of the Drosophila genome (13,059 genes and identified 32 genes involved in centrosome function. An extensive series of secondary screens classified these genes into four categories: (1 nine are required for centriole duplication, (2 11 are required for centrosome maturation, (3 nine are required for both functions, and (4 three genes regulate centrosome separation. These 32 hits include several new centrosomal components, some of which have human homologs. In addition, we find that the individual depletion of only two proteins, Polo and Centrosomin (Cnn can completely block centrosome maturation. Cnn is phosphorylated during mitosis in a Polo-dependent manner, suggesting that the Polo-dependent phosphorylation of Cnn initiates centrosome maturation in flies.

  7. Iron Absorption in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Fanis Missirlis

    2013-05-01

    Full Text Available The way in which Drosophila melanogaster acquires iron from the diet remains poorly understood despite iron absorption being of vital significance for larval growth. To describe the process of organismal iron absorption, consideration needs to be given to cellular iron import, storage, export and how intestinal epithelial cells sense and respond to iron availability. Here we review studies on the Divalent Metal Transporter-1 homolog Malvolio (iron import, the recent discovery that Multicopper Oxidase-1 has ferroxidase activity (iron export and the role of ferritin in the process of iron acquisition (iron storage. We also describe what is known about iron regulation in insect cells. We then draw upon knowledge from mammalian iron homeostasis to identify candidate genes in flies. Questions arise from the lack of conservation in Drosophila for key mammalian players, such as ferroportin, hepcidin and all the components of the hemochromatosis-related pathway. Drosophila and other insects also lack erythropoiesis. Thus, systemic iron regulation is likely to be conveyed by different signaling pathways and tissue requirements. The significance of regulating intestinal iron uptake is inferred from reports linking Drosophila developmental, immune, heat-shock and behavioral responses to iron sequestration.

  8. Iron Absorption in Drosophila melanogaster

    Science.gov (United States)

    Mandilaras, Konstantinos; Pathmanathan, Tharse; Missirlis, Fanis

    2013-01-01

    The way in which Drosophila melanogaster acquires iron from the diet remains poorly understood despite iron absorption being of vital significance for larval growth. To describe the process of organismal iron absorption, consideration needs to be given to cellular iron import, storage, export and how intestinal epithelial cells sense and respond to iron availability. Here we review studies on the Divalent Metal Transporter-1 homolog Malvolio (iron import), the recent discovery that Multicopper Oxidase-1 has ferroxidase activity (iron export) and the role of ferritin in the process of iron acquisition (iron storage). We also describe what is known about iron regulation in insect cells. We then draw upon knowledge from mammalian iron homeostasis to identify candidate genes in flies. Questions arise from the lack of conservation in Drosophila for key mammalian players, such as ferroportin, hepcidin and all the components of the hemochromatosis-related pathway. Drosophila and other insects also lack erythropoiesis. Thus, systemic iron regulation is likely to be conveyed by different signaling pathways and tissue requirements. The significance of regulating intestinal iron uptake is inferred from reports linking Drosophila developmental, immune, heat-shock and behavioral responses to iron sequestration. PMID:23686013

  9. Effects of mutations at the stambh A locus of Drosophila melanogaster

    Indian Academy of Sciences (India)

    M. Kumar; Minu Joseph; Shanti Chandrashekaran

    2001-08-01

    We report novel findings on the cytogenetic location, functional complexity and maternal and germline roles of the stambh A locus of Drosophila melanogaster. stmA is localized to polytene bands 44D1.2 on 2R. stmA mutations are of two types: temperature-sensitive (ts) adult and larval paralytic or unconditional embryonic or larval lethal. Twelve alleles reported in this study fall into two intragenic complementing groups suggesting that stmA is a complex locus with more than one functional domain. Some unconditional embryonic lethal alleles show a ‘neurogenic’ phenotype of cuticle loss accompanied by neural hypertrophy. It is shown that embryos of ts paralytic alleles also show mild neural hypertrophy at permissive temperatures while short exposure to heat induces severe cuticle loss in these embryos. stmA exerts a maternal influence over heat-induced cuticle loss. Unconditional embryonic lethal alleles of stmA are also germline lethal.

  10. centrosomin's beautiful sister (cbs) encodes a GRIP-domain protein that marks Golgi inheritance and functions in the centrosome cycle in Drosophila.

    Science.gov (United States)

    Eisman, Robert C; Stewart, Natasha; Miller, David; Kaufman, Thomas C

    2006-08-15

    The mechanism of inheritance of the Golgi complex is an important problem in cell biology. In this study, we examine the localization and function of a Golgi protein encoded by centrosomin's beautiful sister (cbs) during cleavage in Drosophila melanogaster. Cbs contains a GRIP domain that is 57% identical to vertebrate Golgin-97. Cbs undergoes a dramatic relocalization during mitosis from the cytoplasm to an association with chromosomes from late prometaphase to early telophase, by a transport mechanism that requires the GRIP domain and Arl1, the product of the Arf72A locus. Additionally, Cbs remains independent of the endoplasmic reticulum throughout cleavage. The use of RNAi, Arf72A mutant analysis and ectopic expression of the GRIP domain, shows that cycling of Cbs during mitosis is required for the centrosome cycle. The effects on the centrosome cycle depend on Cbs concentration and Cbs transport from the cytoplasm to DNA. When Cbs levels are reduced centrosomes fail to mature, and when Cbs transport is impeded by ectopic expression of the GRIP domain, centrosomes undergo hypertrophy. We propose that, Cbs is a trans-Golgi protein that links Golgi inheritance to the cell cycle and the Drosophila Golgi is more vertebrate-like than previously recognized.

  11. Transcriptional control in embryonic Drosophila midline guidance assessed through a whole genome approach

    Directory of Open Access Journals (Sweden)

    Tomancak Pavel

    2007-07-01

    Full Text Available Abstract Background During the development of the Drosophila central nervous system the process of midline crossing is orchestrated by a number of guidance receptors and ligands. Many key axon guidance molecules have been identified in both invertebrates and vertebrates, but the transcriptional regulation of growth cone guidance remains largely unknown. It is established that translational regulation plays a role in midline crossing, and there are indications that transcriptional regulation is also involved. To investigate this issue, we conducted a genome-wide study of transcription in Drosophila embryos using wild type and a number of well-characterized Drosophila guidance mutants and transgenics. We also analyzed a previously published microarray time course of Drosophila embryonic development with an axon guidance focus. Results Using hopach, a novel clustering method which is well suited to microarray data analysis, we identified groups of genes with similar expression patterns across guidance mutants and transgenics. We then systematically characterized the resulting clusters with respect to their relevance to axon guidance using two complementary controlled vocabularies: the Gene Ontology (GO and anatomical annotations of the Atlas of Pattern of Gene Expression (APoGE in situ hybridization database. The analysis indicates that regulation of gene expression does play a role in the process of axon guidance in Drosophila. We also find a strong link between axon guidance and hemocyte migration, a result that agrees with mounting evidence that axon guidance molecules are co-opted in vertebrate vascularization. Cell cyclin activity in the context of axon guidance is also suggested from our array data. RNA and protein expression patterns of cell cyclins in axon guidance mutants and transgenics support this possible link. Conclusion This study provides important insights into the regulation of axon guidance in vivo.

  12. Identification of a TPX2-like microtubule-associated protein in Drosophila.

    Directory of Open Access Journals (Sweden)

    Gohta Goshima

    Full Text Available Chromosome segregation during mitosis and meiosis relies on the spindle and the functions of numerous microtubule-associated proteins (MAPs. One of the best-studied spindle MAPs is the highly conserved TPX2, which has been reported to have characteristic intracellular dynamics and molecular activities, such as nuclear localisation in interphase, poleward movement in the metaphase spindle, microtubule nucleation, microtubule stabilisation, microtubule bundling, Aurora A kinase activation, kinesin-5 binding, and kinesin-12 recruitment. This protein has been shown to be essential for spindle formation in every cell type analysed so far. However, as yet, TPX2 homologues have not been found in the Drosophila genome. In this study, I found that the Drosophila protein Ssp1/Mei-38 has significant homology to TPX2. Sequence conservation was limited to the putative spindle microtubule-associated region of TPX2, and intriguingly, D-TPX2 (Ssp1/Mei-38 lacks Aurora A- and kinesin-5-binding domains, which are highly conserved in other animal and plant species, including many insects such as ants and bees. D-TPX2 uniformly localised to kinetochore microtubule-enriched regions of the metaphase spindle in the S2 cell line, and it had microtubule binding and bundling activities in vitro. In comparison with other systems, the contribution of D-TPX2 to cell division seems to be minor; live cell imaging of microtubules and chromosomes after RNAi knockdown identified significant delay in chromosome congression in only 18% of the cells. Thus, while this conserved spindle protein is present in Drosophila, other mechanisms may largely compensate for its spindle assembly and chromosome segregation functions.

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

    Science.gov (United States)

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

    2012-01-01

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

  14. 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 with unreso......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...... complemented by immunofluorescence (IF) analysis in fixed cells, we were able to correlate the impact of mitotic perturbations with the occurrence of DNA damage. Surprisingly, we saw that siRNA-mediated knockdown of only a subset of mitotic genes was accompanied by an increase in DNA damage, showing that even...

  15. How-to-Do-It: Hands-on Activity for Mitosis, Meiosis and the Fundamentals of Heredity.

    Science.gov (United States)

    Taylor, Mark F.

    1988-01-01

    Described is an exercise which uses inexpensive and easy-to-make materials to demonstrate the basic fundamentals of heredity. Discusses two approaches using a hypothetical insert to demonstrate inheritance, mitosis, meiosis, and genotypic and phenotypic frequencies. (CW)

  16. Oocyte and embryonic cytoskeletal defects caused by mutations in the Drosophila swallow gene.

    Science.gov (United States)

    Meng, Jing; Stephenson, Edwin C

    2002-06-01

    The maternal effect gene swallow ( swa) of Drosophila is required for bicoid and htsN4 mRNA localization during oogenesis. Swallow is also required for additional, poorly understood, functions in early embryogenesis. We have examined the cytoskeleton in swa mutant oocytes and embryos by immunocytochemistry and confocal microscopy. Mid- and late-stage swaoocytes have defective cytoplasmic actin networks. Stage-10 oocytes have solid actin clumps and hollow actin spheres in the subcortical layer, and late-stage oocytes have uniformly distributed hollow actin spheres in the subcortical layer and in deeper cytoplasm. Swa preblastoderm embryos have uneven and irregularly distributed actin at the cortex, and defective subcortical actin networks that contain hollow and solid spheres. In swa syncytial blastoderm embryos, the abnormal actin cytoskeleton is associated with defects in nuclear distribution, migration and cleavage. Actin cytoskeletal defects correlate with spindle defects, suggesting that the abnormal organization of the actin cytoskeleton allows interaction of mitotic spindles, which induces defective nuclear divisions and loss of nuclei from the surface of the embryo.

  17. Electroporation into Cultured Mammalian Embryos

    Science.gov (United States)

    Nomura, Tadashi; Takahashi, Masanori; Osumi, Noriko

    Over the last century, mammalian embryos have been used extensively as a common animal model to investigate fundamental questions in the field of developmental biology. More recently, the establishment of transgenic and gene-targeting systems in laboratory mice has enabled researchers to unveil the genetic mechanisms under lying complex developmental processes (Mak, 2007). However, our understanding of cell—cell interactions and their molecular basis in the early stages of mammalian embryogenesis is still very fragmentary. One of the major problems is the difficulty of precise manipulation and limited accessibility to mammalian embryos via uterus wall. Unfortunately, existing tissue and organotypic culture systems per se do not fully recapitulate three-dimensional, dynamic processes of organogenesis observed in vivo. Although transgenic animal technology and virus-mediated gene delivery are useful to manipulate gene expression, these techniques take much time and financial costs, which limit their use.

  18. The ABNORMAL GAMETOPHYTES (AGM) gene product of Arabidopsis demonstrates a role in mitosis during gamete development.

    Science.gov (United States)

    Sorensen, Anna-Marie; Kroeber, Sandra; Saedler, Heinz

    2004-07-01

    Screening a T-DNA mutagenized population of Arabidopsis thaliana for reduced seed set and segregation distortion led to the isolation of the ABNORMAL GAMETOPHYTES (AGM) mutant. Homozygous plants were never recovered, but heterozygous plants showed mitotic defects during gametogenesis resulting in approximately 50% abortion of both the male and female gametes. Isolation of the genomic sequence flanking the co-segregating T-DNA element led to the identification of a gene located on chromosome 5, predicted to encode a transmembrane protein. BLAST homology searches identified two homologous proteins that are not redundant, as is clear from the existence of the agm mutant. Unexpectedly, expression studies using the beta-glucuronidase reporter gene suggest that AGM and its closest Arabidopsis homolog are mostly expressed in cells undergoing mitosis. Thus, AGM is not a gametophytic gene as originally speculated on the basis of segregation distortion, but rather classified as an essential gene crucial to the process of mitosis in plants.

  19. 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.

  20. 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.

  1. The roles of cohesins in mitosis, meiosis, and human health and disease

    Science.gov (United States)

    Brooker, Amanda S.; Berkowitz, Karen M.

    2015-01-01

    Summary Mitosis and meiosis are essential processes that occur during development. Throughout these processes, cohesion is required to keep the sister chromatids together until their separation at anaphase. Cohesion is created by multi-protein subunit complexes called cohesins. Although the subunits differ slightly in mitosis and meiosis, the canonical cohesin complex is composed of four subunits that are quite diverse. The cohesin complexes are also important for DNA repair, gene expression, development, and genome integrity. Here we provide an overview of the roles of cohesins during these different events, as well as their roles in human health and disease, including the cohesinopathies. Although the exact roles and mechanisms of these proteins are still being elucidated, this review will serve as a guide for the current knowledge of cohesins. PMID:24906316

  2. MicroRNAs and DNA methylation as epigenetic regulators of mitosis, meiosis and spermiogenesis.

    Science.gov (United States)

    Yao, Chencheng; Liu, Yun; Sun, Min; Niu, Minghui; Yuan, Qingqing; Hai, Yanan; Guo, Ying; Chen, Zheng; Hou, Jingmei; Liu, Yang; He, Zuping

    2015-07-01

    Spermatogenesis is composed of three distinctive phases, which include self-renewal of spermatogonia via mitosis, spermatocytes undergoing meiosis I/II and post-meiotic development of haploid spermatids via spermiogenesis. Spermatogenesis also involves condensation of chromatin in the spermatid head before transformation of spermatids to spermatozoa. Epigenetic regulation refers to changes of heritably cellular and physiological traits not caused by modifications in the DNA sequences of the chromatin such as mutations. Major advances have been made in the epigenetic regulation of spermatogenesis. In this review, we address the roles and mechanisms of epigenetic regulators, with a focus on the role of microRNAs and DNA methylation during mitosis, meiosis and spermiogenesis. We also highlight issues that deserve attention for further investigation on the epigenetic regulation of spermatogenesis. More importantly, a thorough understanding of the epigenetic regulation in spermatogenesis will provide insightful information into the etiology of some unexplained infertility, offering new approaches for the treatment of male infertility.

  3. The roles of cohesins in mitosis, meiosis, and human health and disease.

    Science.gov (United States)

    Brooker, Amanda S; Berkowitz, Karen M

    2014-01-01

    Mitosis and meiosis are essential processes that occur during development. Throughout these processes, cohesion is required to keep the sister chromatids together until their separation at anaphase. Cohesion is created by multiprotein subunit complexes called cohesins. Although the subunits differ slightly in mitosis and meiosis, the canonical cohesin complex is composed of four subunits that are quite diverse. The cohesin complexes are also important for DNA repair, gene expression, development, and genome integrity. Here we provide an overview of the roles of cohesins during these different events as well as their roles in human health and disease, including the cohesinopathies. Although the exact roles and mechanisms of these proteins are still being elucidated, this review serves as a guide for the current knowledge of cohesins.

  4. Mitosis gives a brief window of opportunity for a change in gene transcription.

    Directory of Open Access Journals (Sweden)

    Richard P Halley-Stott

    2014-07-01

    Full Text Available Cell differentiation is remarkably stable but can be reversed by somatic cell nuclear transfer, cell fusion, and iPS. Nuclear transfer to amphibian oocytes provides a special opportunity to test transcriptional reprogramming without cell division. We show here that, after nuclear transfer to amphibian oocytes, mitotic chromatin is reprogrammed up to 100 times faster than interphase nuclei. We find that, as cells traverse mitosis, their genes pass through a temporary phase of unusually high responsiveness to oocyte reprogramming factors (mitotic advantage. Mitotic advantage is not explained by nuclear penetration, DNA modifications, histone acetylation, phosphorylation, methylation, nor by salt soluble chromosomal proteins. Our results suggest that histone H2A deubiquitination may account, at least in part, for the acquisition of mitotic advantage. They support the general principle that a temporary access of cytoplasmic factors to genes during mitosis may facilitate somatic cell nuclear reprogramming and the acquisition of new cell fates in normal development.

  5. Assessment of algorithms for mitosis detection in breast cancer histopathology images.

    Science.gov (United States)

    Veta, Mitko; van Diest, Paul J; Willems, Stefan M; Wang, Haibo; Madabhushi, Anant; Cruz-Roa, Angel; Gonzalez, Fabio; Larsen, Anders B L; Vestergaard, Jacob S; Dahl, Anders B; Cireşan, Dan C; Schmidhuber, Jürgen; Giusti, Alessandro; Gambardella, Luca M; Tek, F Boray; Walter, Thomas; Wang, Ching-Wei; Kondo, Satoshi; Matuszewski, Bogdan J; Precioso, Frederic; Snell, Violet; Kittler, Josef; de Campos, Teofilo E; Khan, Adnan M; Rajpoot, Nasir M; Arkoumani, Evdokia; Lacle, Miangela M; Viergever, Max A; Pluim, Josien P W

    2015-02-01

    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 low inter-observer agreement. With the wider acceptance of whole slide images in pathology labs, automatic image analysis has been proposed as a potential solution for these issues. In this paper, the results from the Assessment of Mitosis Detection Algorithms 2013 (AMIDA13) challenge are described. The challenge was based on a data set consisting of 12 training and 11 testing subjects, with more than one thousand annotated mitotic figures by multiple observers. Short descriptions and results from the evaluation of eleven methods are presented. The top performing method has an error rate that is comparable to the inter-observer agreement among pathologists.

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    I.J. Udo

    2014-03-01

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

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

    Science.gov (United States)

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

    1978-02-01

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

  9. Electron-microscope observations of mitosis and cytokinesis in multinucleate protoplasts of soybean.

    Science.gov (United States)

    Fowke, L C; Bech-Hansen, C W; Gamborg, O L; Constabel, F

    1975-08-01

    Multinucleate soybean protoplasts produced by spontaneous fusion during enzyme digestion of the cell wall initiated cell division after approximately 40 h in culture. The structure of these protoplasts during mitosis and cytokinesis was studied with both light and electron microscopes. Most nuclei did not fuse but divided synchronously. Interphase nuclei was commonly connected by short narrow nuclear bridges. At prophase and metaphase the nuclei appeared typical of those in most higher plants; technical difficulties prevented an adequate examination of protoplasts at anaphase. Telophase was characterized by cytokinesis involving phragmoplast and cell plate formation; however, complete partitioning of the cytoplasm by cell plants was not observed. Numerous coated vesicles were present near to or continuous with the cell plate and plasmalemma. The presence of a few dividing protoplasts with at least double the normal chromosome number suggests that some nuclear fusion occurred prior to mitosis. Very little cell wall material was detected at the margin of the dividing protoplasts.

  10. Molecular Regulation of the Mitosis/Meiosis Decision in Multicellular Organisms

    Science.gov (United States)

    Kimble, Judith

    2011-01-01

    A major step in the journey from germline stem cell to differentiated gamete is the decision to leave the mitotic cell cycle and begin progression through the meiotic cell cycle. Over the past decade, molecular regulators of the mitosis/meiosis decision have been discovered in most of the major model multicellular organisms. Historically, the mitosis/meiosis decision has been closely linked with controls of germline self-renewal and the sperm/egg decision, especially in nematodes and mice. Molecular explanations of those linkages clarify our understanding of this fundamental germ cell decision, and unifying themes have begun to emerge. Although the complete circuitry of the decision is not known in any organism, the recent advances promise to impact key issues in human reproduction and agriculture. PMID:21646377

  11. Drosophila as a model for antiviral immunity

    Institute of Scientific and Technical Information of China (English)

    Susanna; Valanne; Mika; Rmet

    2010-01-01

    The fruit fly Drosophila melanogaster has been successfully used to study numerous biological processes including immune response.Flies are naturally infected with more than twenty RNA viruses making it a valid model organism to study host-pathogen interactions during viral infections.The Drosophila antiviral immunity includes RNA interference,activation of the JAK/STAT and other signaling cascades and other mechanisms such as autophagy and interactions with other microorganisms.Here we review Drosophila as an immunological research model as well as recent advances in the field ofDrosophila antiviral immunity.

  12. Non-invasive long-term fluorescence live imaging of Tribolium castaneum embryos.

    Science.gov (United States)

    Strobl, Frederic; Stelzer, Ernst H K

    2014-06-01

    Insect development has contributed significantly to our understanding of metazoan development. However, most information has been obtained by analyzing a single species, the fruit fly Drosophila melanogaster. Embryonic development of the red flour beetle Tribolium castaneum differs fundamentally from that of Drosophila in aspects such as short-germ development, embryonic leg development, extensive extra-embryonic membrane formation and non-involuted head development. Although Tribolium has become the second most important insect model organism, previous live imaging attempts have addressed only specific questions and no long-term live imaging data of Tribolium embryogenesis have been available. By combining light sheet-based fluorescence microscopy with a novel mounting method, we achieved complete, continuous and non-invasive fluorescence live imaging of Tribolium embryogenesis at high spatiotemporal resolution. The embryos survived the 2-day or longer imaging process, developed into adults and produced fertile progeny. Our data document all morphogenetic processes from the rearrangement of the uniform blastoderm to the onset of regular muscular movement in the same embryo and in four orientations, contributing significantly to the understanding of Tribolium development. Furthermore, we created a comprehensive chronological table of Tribolium embryogenesis, integrating most previous work and providing a reference for future studies. Based on our observations, we provide evidence that serosa window closure and serosa opening, although deferred by more than 1 day, are linked. All our long-term imaging datasets are available as a resource for the community. Tribolium is only the second insect species, after Drosophila, for which non-invasive long-term fluorescence live imaging has been achieved.

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

    Directory of Open Access Journals (Sweden)

    Judit Pampalona

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

  14. 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.

  15. Phospho-Bcl-xL(Ser62) influences spindle assembly and chromosome segregation during mitosis.

    Science.gov (United States)

    Wang, Jianfang; Beauchemin, Myriam; Bertrand, Richard

    2014-01-01

    Functional analysis of a series of phosphorylation mutants reveals that Bcl-xL(Ser62Ala) influences cell entry into anaphase and mitotic exit in taxol-exposed cells compared with cells expressing wild-type Bcl-xL or a series of other phosphorylation mutants, an effect that appears to be independent of its anti-apoptotic activity. During normal mitosis progression, Bcl-xL(Ser62) is strongly phosphorylated by PLK1 and MAPK14/SAPKp38α at the prometaphase, metaphase, and the anaphase boundaries, while it is de-phosphorylated at telophase and cytokinesis. Phospho-Bcl-xL(Ser62) localizes in centrosomes with γ-tubulin and in the mitotic cytosol with some spindle-assembly checkpoint signaling components, including PLK1, BubR1, and Mad2. In taxol- and nocodazole-exposed cells, phospho-Bcl-xL(Ser62) also binds to Cdc20- Mad2-, BubR1-, and Bub3-bound complexes, while Bcl-xL(Ser62Ala) does not. Silencing Bcl-xL expression and expressing the phosphorylation mutant Bcl-xL(Ser62Ala) lead to an increased number of cells harboring mitotic spindle defects including multipolar spindle, chromosome lagging and bridging, aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h. Together, the data indicate that during mitosis, Bcl-xL(Ser62) phosphorylation impacts on spindle assembly and chromosome segregation, influencing chromosome stability. Observations of mitotic cells harboring aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h were also made with cells expressing the phosphorylation mutant Bcl-xL(Ser49Ala) and dual mutant Bcl-xL(Ser49/62Ala).

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

    Science.gov (United States)

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

    2013-08-21

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

  17. Subcellular localization of the APOBEC3 proteins during mitosis and implications for genomic DNA deamination.

    Science.gov (United States)

    Lackey, Lela; Law, Emily K; Brown, William L; Harris, Reuben S

    2013-03-01

    Humans have seven APOBEC3 DNA cytosine deaminases. The activity of these enzymes allows them to restrict a variety of retroviruses and retrotransposons, but may also cause pro-mutagenic genomic uracil lesions. During interphase the APOBEC3 proteins have different subcellular localizations: cell-wide, cytoplasmic or nuclear. This implies that only a subset of APOBEC3s have contact with nuclear DNA. However, during mitosis, the nuclear envelope breaks down and cytoplasmic proteins may enter what was formerly a privileged zone. To address the hypothesis that all APOBEC3 proteins have access to genomic DNA, we analyzed the localization of the APOBEC3 proteins during mitosis. We show that APOBEC3A, APOBEC3C and APOBEC3H are excluded from condensed chromosomes, but become cell-wide during telophase. However, APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3G are excluded from chromatin throughout mitosis. After mitosis, APOBEC3B becomes nuclear, and APOBEC3D, APOBEC3F and APOBEC3G become cytoplasmic. Both structural motifs as well as size may be factors in regulating chromatin exclusion. Deaminase activity was not dependent on cell cycle phase. We also analyzed APOBEC3-induced cell cycle perturbations as a measure of each enzyme's capacity to inflict genomic DNA damage. AID, APOBEC3A and APOBEC3B altered the cell cycle profile, and, unexpectedly, APOBEC3D also caused changes. We conclude that several APOBEC3 family members have access to the nuclear compartment and can impede the cell cycle, most likely through DNA deamination and the ensuing DNA damage response. Such genomic damage may contribute to carcinogenesis, as demonstrated by AID in B cell cancers and, recently, APOBEC3B in breast cancers.

  18. 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.

  19. Seasonal temperature variations influence tapetum mitosis patterns associated with reproductive fitness.

    Science.gov (United States)

    Lavania, Umesh C; Basu, Surochita; Kushwaha, Jyotsana Singh; Lavania, Seshu

    2014-09-01

    Environmental stress in plants impacts many biological processes, including male gametogenesis, and affects several cytological mechanisms that are strongly interrelated. To understand the likely impact of rising temperature on reproductive fitness in the climate change regime, a study of tapetal mitosis and its accompanying meiosis over seasons was made to elucidate the influence of temperature change on the cytological events occurring during microsporogenesis. For this we used two species of an environmentally sensitive plant system, i.e., genus Cymbopogon Sprengel (Poaceae), namely Cymbopogon nardus (L.) Rendle var. confertiflorus (Steud.) Bor (2n = 20) and Cymbopogon jwaruncusha (Jones) Schult. (2n = 20). Both species flower profusely during extreme summer (48 °C) and mild winter (15 °C) but support low and high seed fertility, respectively, in the two seasons. We have shown that tapetal mitotic patterns over seasons entail differential behavior for tapetal mitosis. During the process of tapetum development there are episodes of endomitosis that form either (i) an endopolyploid genomically imbalanced uninucleate and multinucleate tapetum, and (or) (ii) an acytokinetic multinucleate genomically balanced tapetum, with the progression of meiosis in the accompanying sporogenous tissue. The relative frequency of occurrence of the two types of tapetum mitosis patterns is significantly different in the two seasons, and it is found to be correlated with the temperature conditions. Whereas, the former (genomically imbalanced tapetum) are prevalent during the hot summer, the latter (genomically balanced tapetum) are frequent under optimal conditions. Such a differential behaviour in tapetal mitosis vis-à-vis temperature change is also correspondingly accompanied by substantial disturbances or regularity in meiotic anaphase disjunction. Both species show similar patterns. The study underpins that tapetal mitotic behaviour per se could be a reasonable indicator to

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

    Science.gov (United States)

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

    2016-07-01

    To explore the role of p21 in ionizing radiation-induced changes in protein levels during the G2/M transition and long-term G2 arrest. Protein expression levels were assessed by western blot in the human uveal melanoma 92-1 cells after treatment with ionizing radiation. Depletion of p21 was carried out by employing the siRNA technique. Cell cycle distribution was determined by flow cytometry combined with histone H3 phosphorylation at Ser28, an M-phase marker. Senescence was assessed by senescence- associated-β-galactosidase (SA-β-gal) staining combined with Ki67 staining, a cell proliferation marker. Accompanying increased p21, the protein levels of G2/M transition genes declined significantly in 92-1 cells irradiated with 5 Gy of X-rays. Furthermore, these irradiated cells were blocked at the G2 phase followed by cellular senescence. Depletion of p21 rescued radiation-induced G2 arrest as demonstrated by the upregulation of G2/M transition kinases, as well as the high expression of histone H3 phosphorylated at Ser28. Knockdown of p21 resulted in entry into mitosis of irradiated 92-1 cells. However, cells with serious DNA damage failed to undergo cytokinesis, leading to the accumulation of multinucleated cells. Our results indicated that p21 was responsible for the downregulation of G2/M transition regulatory proteins and the bypass of mitosis induced by irradiation. Downregulation of p21 by siRNA resulted in G2-arrested cells entering into mitosis with serious DNA damage. This is the first report on elucidating the role of p21 in the bypass of mitosis. Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.