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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

    Science.gov (United States)

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

    2015-01-01

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

  2. Heterologous expression of mammalian Plk1 in Drosophila reveals divergence from Polo during late mitosis

    International Nuclear Information System (INIS)

    Pearson, John; Godinho, Susana A.; Tavares, Alvaro; Glover, David M.

    2006-01-01

    Drosophila Polo kinase is the founder member of a conserved kinase family required for multiple stages of mitosis. We assessed the ability of mouse Polo-like kinase 1 (Plk1) to perform the multiple mitotic functions of Polo kinase, by expressing a Plk1-GFP fusion in Drosophila. Consistent with the previously reported localization of Polo kinase, Plk1-GFP was strongly localized to centrosomes and recruited to the centromeric regions of condensing chromosomes during early mitosis. However, in contrast to a functional Polo-GFP fusion, Plk1-GFP failed to localize to the central spindle midzone in both syncytial embryo mitosis and the conventional mitoses of cellularized embryos and S2 cells. Moreover, unlike endogenous Polo kinase and Polo-GFP, Plk1-GFP failed to associate with the contractile ring. Expression of Plk1-GFP enhanced the lethality of hypomorphic polo mutants and disrupted the organization of the actinomyosin cytoskeleton in a dominant-negative manner. Taken together, our results suggest that endogenous Polo kinase has specific roles in regulating actinomyosin rearrangements during Drosophila mitoses that its mammalian counterpart, Plk1, cannot fulfill. Consistent with this hypothesis, we observed defects in the cortical recruitment of myosin and myosin regulatory light chain in Polo deficient cells

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

    National Research Council Canada - National Science Library

    Chang, Long-Sheng

    2007-01-01

    To better understand the mechanism by which Merlin functions as a tumor suppressor we have shown that mutations in the Drosophila Merlin gene lead to increased mitosis and alter the duration of the G2...

  4. 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......(S): Forty IVF and/or intracytoplasmic sperm injection patients. INTERVENTION(S): Embryo thawing, morphological evaluation, and fluorescence in situ hybridization analysis for aneuploidy screening. MAIN OUTCOME MEASURE(S): Resumption of mitosis, blastomere loss, multinucleation, and chromosome enumeration....... 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...

  5. Cdk1 and Okadaic Acid-sensitive Phosphatases Control Assembly of Nuclear Pore Complexes in Drosophila EmbryosV⃞

    OpenAIRE

    Onischenko, Evgeny A.; Gubanova, Natalia V.; Kiseleva, Elena V.; Hallberg, Einar

    2005-01-01

    Disassembly and reassembly of the nuclear pore complexes (NPCs) is one of the major events during open mitosis in higher eukaryotes. However, how this process is controlled by the mitotic machinery is not clear. To investigate this we developed a novel in vivo model system based on syncytial Drosophila embryos. We microinjected different mitotic effectors into the embryonic cytoplasm and monitored the dynamics of disassembly/reassembly of NPCs in live embryos using fluorescently labeled wheat...

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

    International Nuclear Information System (INIS)

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

    1991-01-01

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

  7. Mitosis.

    Science.gov (United States)

    Henderson, Paula

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

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

    Science.gov (United States)

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

    2015-01-01

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

  9. Effect of localized hypoxia on Drosophila embryo development.

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    Zhinan Wang

    Full Text Available Environmental stress, such as oxygen deprivation, affects various cellular activities and developmental processes. In this study, we directly investigated Drosophila embryo development in vivo while cultured on a microfluidic device, which imposed an oxygen gradient on the developing embryos. The designed microfluidic device enabled both temporal and spatial control of the local oxygen gradient applied to the live embryos. Time-lapse live cell imaging was used to monitor the morphology and cellular migration patterns as embryos were placed in various geometries relative to the oxygen gradient. Results show that pole cell movement and tail retraction during Drosophila embryogenesis are highly sensitive to oxygen concentrations. Through modeling, we also estimated the oxygen permeability across the Drosophila embryonic layers for the first time using parameters measured on our oxygen control device.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. Cdk1 and okadaic acid-sensitive phosphatases control assembly of nuclear pore complexes in Drosophila embryos.

    Science.gov (United States)

    Onischenko, Evgeny A; Gubanova, Natalia V; Kiseleva, Elena V; Hallberg, Einar

    2005-11-01

    Disassembly and reassembly of the nuclear pore complexes (NPCs) is one of the major events during open mitosis in higher eukaryotes. However, how this process is controlled by the mitotic machinery is not clear. To investigate this we developed a novel in vivo model system based on syncytial Drosophila embryos. We microinjected different mitotic effectors into the embryonic cytoplasm and monitored the dynamics of disassembly/reassembly of NPCs in live embryos using fluorescently labeled wheat germ agglutinin (WGA) or in fixed embryos using electron microscopy and immunostaining techniques. We found that in live embryos Cdk1 activity was necessary and sufficient to induce disassembly of NPCs as well as their cytoplasmic mimics: annulate lamellae pore complexes (ALPCs). Cdk1 activity was also required for keeping NPCs and ALPCs disassembled during mitosis. In agreement recombinant Cdk1/cyclin B was able to induce phosphorylation and dissociation of nucleoporins from the NPCs in vitro. Conversely, reassembly of NPCs and ALPCs was dependent on the activity of protein phosphatases, sensitive to okadaic acid (OA). Our findings suggest a model where mitotic disassembly/reassembly of the NPCs is regulated by a dynamic equilibrium of Cdk1 and OA-sensitive phosphatase activities and provide evidence that mitotic phosphorylation mediates disassembly of the NPC.

  12. Gene expression profiling of brakeless mutant Drosophila embryos.

    Science.gov (United States)

    Crona, Filip; Singla, Bhumica; Mannervik, Mattias

    2015-12-01

    The transcriptional co-regulator Brakeless performs many important functions during Drosophila development, but few target genes have been identified. Here we use Affymetrix microarrays to identify Brakeless-regulated genes in 2-4 h old Drosophila embryos. Robust multi-array analysis (RMA) and statistical tests revealed 240 genes that changed their expression more than 1.5 fold. We find that up- and down-regulated genes fall into distinct gene ontology categories. In our associated study [2] we demonstrate that both up- and down-regulated genes can be direct Brakeless targets. Our results indicate that the co-repressor and co-activator activities of Brakeless may result in distinct biological responses. The microarray data complies with MIAME guidelines and is deposited in GEO under accession number GSE60048.

  13. Single molecule transcription factor dynamics in the syncytial Drosophila embryo

    Science.gov (United States)

    Darzacq, Xavier

    During early development in the Drosophila embryo, cell fates are determined over the course of just 2 hours with exquisite spatio-temoral precision. One of the key regulators of this process is the transcription factor Bicoid which forms a concentration gradient across the long axis of the embryo. Although Bicoids' primary role is activation at the anterior, where concentrations are highest, it is also known to play a role in the posterior where there are only 100s of molecules per nucleus. Understanding how Bicoid can find its target at such low concentrations has remained intractable, largely due to the inability to perform single molecule imaging in the context of the developing embryo. Here we use lattice light sheet microscopy to overcome the technical barriers of sample thickness and auto-fluorescence to characterize the single molecule dynamics of Bicoid. We find that off-rates do not vary across the embryo and that instead the on-rates are modulated through the formation of clusters that enrich local concentration. This data is contrary to the current concentration dependent model of Bicoid function since local concentration within the nucleus is now a regulated parameter and suggests a previously unknown mechanism for regulation at extremely low concentrations.

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

    International Nuclear Information System (INIS)

    Weissenborn, U.; Streffer, C.

    1989-01-01

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

  15. Sample Preparation and Mounting of Drosophila Embryos for Multiview Light Sheet Microscopy.

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    Schmied, Christopher; Tomancak, Pavel

    2016-01-01

    Light sheet fluorescent microscopy (LSFM), and in particular its most widespread flavor Selective Plane Illumination Microscopy (SPIM), promises to provide unprecedented insights into developmental dynamics of entire living systems. By combining minimal photo-damage with high imaging speed and sample mounting tailored toward the needs of the specimen, it enables in toto imaging of embryogenesis with high spatial and temporal resolution. Drosophila embryos are particularly well suited for SPIM imaging because the volume of the embryo does not change from the single cell embryo to the hatching larva. SPIM microscopes can therefore image Drosophila embryos embedded in rigid media, such as agarose, from multiple angles every few minutes from the blastoderm stage until hatching. Here, we describe sample mounting strategies to achieve such a recording. We also provide detailed protocols to realize multiview, long-term, time-lapse recording of Drosophila embryos expressing fluorescent markers on the commercially available Zeiss Lightsheet Z.1 microscope and the OpenSPIM.

  16. The preparation of Drosophila embryos for live-imaging using the hanging drop protocol.

    Science.gov (United States)

    Reed, Bruce H; McMillan, Stephanie C; Chaudhary, Roopali

    2009-03-13

    Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

  17. 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.

  18. 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

  19. PSK, a biological response modifier, modifies p53 expression, mitosis and apoptosis in X-ray irradiated mouse embryos. Possible cellular mechanism of the anti-teratogenic effect

    International Nuclear Information System (INIS)

    Kagohashi, Yukiko; Naora, Hiroyuki; Otani, Hiroki

    2002-01-01

    We previously showed that PSK, a biological response modifier, suppressed X-ray irradiation induced ocular anomalies in mouse embryos. In the present study, in mouse embryos irradiated at E7.5, PSK, when administered immediately after irradiation, suppressed mitosis and increased apoptosis as compared with embryos not treated with PSK at 12 hrs after irradiation. In the irradiated embryos, p53, which is normally expressed at a high level in early embryos, increased at 6 hrs and decreased at 12 hrs after irradiation. In the irradiated and PSK-treated embryos, the p53 level did not change at 6 hrs, increased at 12 hrs and decreased at 24 hrs after irradiation. This timing of PSK-induced delayed increase of p53 coincided with that of the PSK-induced decrease in mitosis and increase in apoptosis. These results suggested that PSK modified the p53 level and affected cell proliferation and apoptosis, which might contribute to the suppression of teratogenesis. (author)

  20. 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

  1. 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.

  2. 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.

  3. 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....

  4. Repair of lesions provoked by X-rays in embryos of Drosophila (D. melanogaster Meig)

    International Nuclear Information System (INIS)

    Ghelelovitch, S.

    1975-01-01

    The sensitivity of Drosophila eggs to the lethal action of X-rays did not remain constant during embryogenesis. The X-ray doses used in the present investigation may have retarded the hatching of the larvae but did not block development immediately after irradiation. A fraction of the damage induced in young embryos was repaired during gastrulation. The amount of repair was independent of the X-ray dose but was influenced by the temperature. The damage could be repaired even after the cells of the embryo had undergone many mitotic cycles. (author)

  5. 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.

  6. 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.

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2015-04-20

    During development, cell-fate diversity can result from the unequal segregation of fate determinants at mitosis. Polarization of the mother cell is essential for asymmetric cell division (ACD). It often involves the formation of a cortical domain containing the PAR complex proteins Par3, Par6, and atypical protein kinase C (aPKC). In the fly notum, sensory organ precursor cells (SOPs) divide asymmetrically within the plane of the epithelium and along the body axis to generate two distinct cells. Fate asymmetry depends on the asymmetric localization of the PAR complex. In the absence of planar cell polarity (PCP), SOPs divide with a random planar orientation but still asymmetrically, showing that PCP is dispensable for PAR asymmetry at mitosis. To study when and how the PAR complex localizes asymmetrically, we have used a quantitative imaging approach to measure the planar polarization of the proteins Bazooka (Baz, fly Par3), Par6, and aPKC in living pupae. By using imaging of functional GFP-tagged proteins with image processing and computational modeling, we find that Baz, Par6, and aPKC become planar polarized prior to mitosis in a manner independent of the AuroraA kinase and that PCP is required for the planar polarization of Baz, Par6, and aPKC during interphase. This indicates that a "mitosis rescue" mechanism establishes asymmetry at mitosis in PCP mutants. This study therefore identifies PCP as the initial symmetry-breaking signal for the planar polarization of PAR proteins in asymmetrically dividing SOPs. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. A modifier screen for Bazooka/PAR-3 interacting genes in the Drosophila embryo epithelium.

    Directory of Open Access Journals (Sweden)

    Wei Shao

    2010-04-01

    Full Text Available The development and homeostasis of multicellular organisms depends on sheets of epithelial cells. Bazooka (Baz; PAR-3 localizes to the apical circumference of epithelial cells and is a key hub in the protein interaction network regulating epithelial structure. We sought to identify additional proteins that function with Baz to regulate epithelial structure in the Drosophila embryo.The baz zygotic mutant cuticle phenotype could be dominantly enhanced by loss of known interaction partners. To identify additional enhancers, we screened molecularly defined chromosome 2 and 3 deficiencies. 37 deficiencies acted as strong dominant enhancers. Using deficiency mapping, bioinformatics, and available single gene mutations, we identified 17 interacting genes encoding known and predicted polarity, cytoskeletal, transmembrane, trafficking and signaling proteins. For each gene, their loss of function enhanced adherens junction defects in zygotic baz mutants during early embryogenesis. To further evaluate involvement in epithelial polarity, we generated GFP fusion proteins for 15 of the genes which had not been found to localize to the apical domain previously. We found that GFP fusion proteins for Drosophila ASAP, Arf79F, CG11210, Septin 5 and Sds22 could be recruited to the apical circumference of epithelial cells. Nine of the other proteins showed various intracellular distributions, and one was not detected.Our enhancer screen identified 17 genes that function with Baz to regulate epithelial structure in the Drosophila embryo. Our secondary localization screen indicated that some of the proteins may affect epithelial cell polarity by acting at the apical cell cortex while others may act through intracellular processes. For 13 of the 17 genes, this is the first report of a link to baz or the regulation of epithelial structure.

  11. The Brakeless co-regulator can directly activate and repress transcription in early Drosophila embryos.

    Science.gov (United States)

    Crona, Filip; Holmqvist, Per-Henrik; Tang, Min; Singla, Bhumica; Vakifahmetoglu-Norberg, Helin; Fantur, Katrin; Mannervik, Mattias

    2015-11-01

    The Brakeless protein performs many important functions during Drosophila development, but how it controls gene expression is poorly understood. We previously showed that Brakeless can function as a transcriptional co-repressor. In this work, we perform transcriptional profiling of brakeless mutant embryos. Unexpectedly, the majority of affected genes are down-regulated in brakeless mutants. We demonstrate that genomic regions in close proximity to some of these genes are occupied by Brakeless, that over-expression of Brakeless causes a reciprocal effect on expression of these genes, and that Brakeless remains an activator of the genes upon fusion to an activation domain. Together, our results show that Brakeless can both repress and activate gene expression. A yeast two-hybrid screen identified the Mediator complex subunit Med19 as interacting with an evolutionarily conserved part of Brakeless. Both down- and up-regulated Brakeless target genes are also affected in Med19-depleted embryos, but only down-regulated targets are influenced in embryos depleted of both Brakeless and Med19. Our data provide support for a Brakeless activator function that regulates transcription by interacting with Med19. We conclude that the transcriptional co-regulator Brakeless can either activate or repress transcription depending on context. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2012-01-01

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

  14. Origin and specification of type II neuroblasts in the Drosophila embryo.

    Science.gov (United States)

    Álvarez, José-Andrés; Díaz-Benjumea, Fernando J

    2018-04-05

    In Drosophila , neural stem cells or neuroblasts (NBs) acquire different identities according to their site of origin in the embryonic neuroectoderm. Their identity determines the number of times they will divide and the types of daughter cells they will generate. All NBs divide asymmetrically, with type I NBs undergoing self-renewal and generating another cell that will divide only once more. By contrast, a small set of NBs in the larval brain, type II NBs, divides differently, undergoing self-renewal and generating an intermediate neural progenitor (INP) that continues to divide asymmetrically several more times, generating larger lineages. In this study, we have analysed the origin of type II NBs and how they are specified. Our results indicate that these cells originate in three distinct clusters in the dorsal protocerebrum during stage 12 of embryonic development. Moreover, it appears that their specification requires the combined action of EGFR signalling and the activity of the related genes buttonhead and Drosophila Sp1 In addition, we also show that the INPs generated in the embryo enter quiescence at the end of embryogenesis, resuming proliferation during the larval stage. © 2018. Published by The Company of Biologists Ltd.

  15. Mitochondrial DNA polymerase from embryos of Drosophila melanogaster: purification, subunit structure, and partial characterization

    International Nuclear Information System (INIS)

    Wernette, C.M.; Kaguni, L.S.

    1986-01-01

    The mitochondrial DNA polymerase has been purified to near-homogeneity from early embryos of Drosophila melanogaster. Sodium dodecyl sulfate gel electrophoresis of the highly purified enzyme reveals two polypeptides with molecular masses of 125,000 and 35,000 daltons, in a ratio of 1:1. The enzyme has a sedimentation coefficient of 7.6 S and a stokes radius of 51 A. Taken together, the data suggest that the D. melanogaster DNA polymerase γ is a heterodimer. DNA polymerase activity gel analysis has allowed the assignment of the DNA polymerization function to the large subunit. The DNA polymerase exhibits a remarkable ability to utilize efficiently a variety of template-primers including gapped DNA, poly(rA).oligo(dT) and singly primed phiX174 DNA. Both the crude and the highly purified enzymes are stimulated by KCl, and inhibited by dideoxythymidine triphosphate and by N-ethylmaleimide. Thus, the catalytic properties of the near-homogeneous Drosophila enzyme are consistent with those of DNA polymerase γ as partially purified from several vertebrates

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

    Directory of Open Access Journals (Sweden)

    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.

  17. A biomechanical analysis of ventral furrow formation in the Drosophila melanogaster embryo.

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    Vito Conte

    Full Text Available The article provides a biomechanical analysis of ventral furrow formation in the Drosophila melanogaster embryo. Ventral furrow formation is the first large-scale morphogenetic movement in the fly embryo. It involves deformation of a uniform cellular monolayer formed following cellularisation, and has therefore long been used as a simple system in which to explore the role of mechanics in force generation. Here we use a quantitative framework to carry out a systematic perturbation analysis to determine the role of each of the active forces observed. The analysis confirms that ventral furrow invagination arises from a combination of apical constriction and apical-basal shortening forces in the mesoderm, together with a combination of ectodermal forces. We show that the mesodermal forces are crucial for invagination: the loss of apical constriction leads to a loss of the furrow, while the mesodermal radial shortening forces are the primary cause of the internalisation of the future mesoderm as the furrow rises. Ectodermal forces play a minor but significant role in furrow formation: without ectodermal forces the furrow is slower to form, does not close properly and has an aberrant morphology. Nevertheless, despite changes in the active mesodermal and ectodermal forces lead to changes in the timing and extent of furrow, invagination is eventually achieved in most cases, implying that the system is robust to perturbation and therefore over-determined.

  18. 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.

  19. Notch and PKC Are Involved in Formation of the Lateral Region of the Dorso-Ventral Axis in Drosophila Embryos

    OpenAIRE

    Tremmel, Daniel M.; Resad, Sedat; Little, Christopher J.; Wesley, Cedric S.

    2013-01-01

    The Notch gene encodes an evolutionarily conserved cell surface receptor that generates regulatory signals based on interactions between neighboring cells. In Drosophila embryos it is normally expressed at a low level due to strong negative regulation. When this negative regulation is abrogated neurogenesis in the ventral region is suppressed, the development of lateral epidermis is severely disrupted, and the dorsal aminoserosa is expanded. Of these phenotypes only the anti-neurogenic phenot...

  20. 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…

  1. 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…

  2. Global sensitivity analysis of a dynamic model for gene expression in Drosophila embryos

    Science.gov (United States)

    McCarthy, Gregory D.; Drewell, Robert A.

    2015-01-01

    It is well known that gene regulation is a tightly controlled process in early organismal development. However, the roles of key processes involved in this regulation, such as transcription and translation, are less well understood, and mathematical modeling approaches in this field are still in their infancy. In recent studies, biologists have taken precise measurements of protein and mRNA abundance to determine the relative contributions of key factors involved in regulating protein levels in mammalian cells. We now approach this question from a mathematical modeling perspective. In this study, we use a simple dynamic mathematical model that incorporates terms representing transcription, translation, mRNA and protein decay, and diffusion in an early Drosophila embryo. We perform global sensitivity analyses on this model using various different initial conditions and spatial and temporal outputs. Our results indicate that transcription and translation are often the key parameters to determine protein abundance. This observation is in close agreement with the experimental results from mammalian cells for various initial conditions at particular time points, suggesting that a simple dynamic model can capture the qualitative behavior of a gene. Additionally, we find that parameter sensitivites are temporally dynamic, illustrating the importance of conducting a thorough global sensitivity analysis across multiple time points when analyzing mathematical models of gene regulation. PMID:26157608

  3. Interdependence of macrophage migration and ventral nerve cord development in Drosophila embryos.

    Science.gov (United States)

    Evans, Iwan R; Hu, Nan; Skaer, Helen; Wood, Will

    2010-05-01

    During embryonic development, Drosophila macrophages (haemocytes) undergo a series of stereotypical migrations to disperse throughout the embryo. One major migratory route is along the ventral nerve cord (VNC), where haemocytes are required for the correct development of this tissue. We show, for the first time, that a reciprocal relationship exists between haemocytes and the VNC and that defects in nerve cord development prevent haemocyte migration along this structure. Using live imaging, we demonstrate that the axonal guidance cue Slit and its receptor Robo are both required for haemocyte migration, but signalling is not autonomously required in haemocytes. We show that the failure of haemocyte migration along the VNC in slit mutants is not due to a lack of chemotactic signals within this structure, but rather to a failure in its detachment from the overlying epithelium, creating a physical barrier to haemocyte migration. This block of haemocyte migration in turn disrupts the formation of the dorsoventral channels within the VNC, further highlighting the importance of haemocyte migration for correct neural development. This study illustrates the important role played by the three-dimensional environment in directing cell migration in vivo and reveals an intriguing interplay between the developing nervous system and the blood cells within the fly, demonstrating that their development is both closely coupled and interdependent.

  4. Maternal Nanos-Dependent RNA Stabilization in the Primordial Germ Cells of Drosophila Embryos.

    Science.gov (United States)

    Sugimori, Seiko; Kumata, Yuji; Kobayashi, Satoru

    2018-01-01

    Nanos (Nos) is an evolutionary conserved protein expressed in the germline of various animal species. In Drosophila, maternal Nos protein is essential for germline development. In the germline progenitors, or the primordial germ cells (PGCs), Nos binds to the 3' UTR of target mRNAs to repress their translation. In contrast to this prevailing role of Nos, here we report that the 3' UTR of CG32425 mRNA mediates Nos-dependent RNA stabilization in PGCs. We found that the level of mRNA expressed from a reporter gene fused to the CG32425 3' UTR was significantly reduced in PGCs lacking maternal Nos (nos PGCs) as compared with normal PGCs. By deleting the CG32425 3' UTR, we identified the region required for mRNA stabilization, which includes Nos-binding sites. In normal embryos, CG32425 mRNA was maternally supplied into PGCs and remained in this cell type during embryogenesis. However, as expected from our reporter assay, the levels of CG32425 mRNA and its protein product expressed in nos PGCs were lower than in normal PGCs. Thus, we propose that Nos protein has dual functions in translational repression and stabilization of specific RNAs to ensure proper germline development. © 2017 Japanese Society of Developmental Biologists.

  5. Mid-embryo patterning and precision in Drosophila segmentation: Krüppel 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

  6. Protein O-Mannosyltransferases Affect Sensory Axon Wiring and Dynamic Chirality of Body Posture in the Drosophila Embryo.

    Science.gov (United States)

    Baker, Ryan; Nakamura, Naosuke; Chandel, Ishita; Howell, Brooke; Lyalin, Dmitry; Panin, Vladislav M

    2018-02-14

    Genetic defects in protein O-mannosyltransferase 1 (POMT1) and POMT2 underlie severe muscular dystrophies. POMT genes are evolutionarily conserved in metazoan organisms. In Drosophila , both male and female POMT mutants show a clockwise rotation of adult abdominal segments, suggesting a chirality of underlying pathogenic mechanisms. Here we described and analyzed a similar phenotype in POMT mutant embryos that shows left-handed body torsion. Our experiments demonstrated that coordinated muscle contraction waves are associated with asymmetric embryo rolling, unveiling a new chirality marker in Drosophila development. Using genetic and live-imaging approaches, we revealed that the torsion phenotype results from differential rolling and aberrant patterning of peristaltic waves of muscle contractions. Our results demonstrated that peripheral sensory neurons are required for normal contractions that prevent the accumulation of torsion. We found that POMT mutants show abnormal axonal connections of sensory neurons. POMT transgenic expression limited to sensory neurons significantly rescued the torsion phenotype, axonal connectivity defects, and abnormal contractions in POMT mutant embryos. Together, our data suggested that protein O-mannosylation is required for normal sensory feedback to control coordinated muscle contractions and body posture. This mechanism may shed light on analogous functions of POMT genes in mammals and help to elucidate the etiology of neurological defects in muscular dystrophies. SIGNIFICANCE STATEMENT Protein O-mannosyltransferases (POMTs) are evolutionarily conserved in metazoans. Mutations in POMTs cause severe muscular dystrophies associated with pronounced neurological defects. However, neurological functions of POMTs remain poorly understood. We demonstrated that POMT mutations in Drosophila result in abnormal muscle contractions and cause embryo torsion. Our experiments uncovered a chirality of embryo movements and a unique POMT -dependent

  7. Changes in protein synthetic activity in early Drosophila embryos mutant for the segmentation gene Krueppel

    International Nuclear Information System (INIS)

    Bedian, V.; Summers, M.C.; Kauffman, S.A.

    1988-01-01

    We have identified early embryo proteins related to the segmentation gene Krueppel by [35S]methionine pulse labelling and two-dimensional gel electrophoresis. Protein synthesis differences shared by homozygous embryos of two Krueppel alleles when compared to heterozygous and wild-type embryos are reported. The study was extended to syncytial blastoderm stages by pulse labelling and gel analysis of single embryos, using Krueppel-specific proteins from gastrula stages as molecular markers for identifying homozygous Krueppel embryos. Localized expression of interesting proteins was examined in embryo fragments. The earliest differences detected at nuclear migration stages showed unregulated synthesis in mutant embryos of two proteins that have stage specific synthesis in normal embryos. At the cellular blastoderm stage one protein was not synthesized and two proteins showed apparent shifts in isoelectric point in mutant embryos. Differences observed in older embryos included additional proteins with shifted isoelectric points and a number of qualitative and quantitative changes in protein synthesis. Five of the proteins with altered rates of synthesis in mutant embryos showed localized synthesis in normal embryos. The early effects observed are consistent with the hypothesis that the Krueppel product can be a negative or positive regulator of expression of other loci, while blastoderm and gastrula stage shifts in isoelectric point indicate that a secondary effect of Krueppel function may involve post-translational modification of proteins

  8. Notch and PKC are involved in formation of the lateral region of the dorso-ventral axis in Drosophila embryos.

    Science.gov (United States)

    Tremmel, Daniel M; Resad, Sedat; Little, Christopher J; Wesley, Cedric S

    2013-01-01

    The Notch gene encodes an evolutionarily conserved cell surface receptor that generates regulatory signals based on interactions between neighboring cells. In Drosophila embryos it is normally expressed at a low level due to strong negative regulation. When this negative regulation is abrogated neurogenesis in the ventral region is suppressed, the development of lateral epidermis is severely disrupted, and the dorsal aminoserosa is expanded. Of these phenotypes only the anti-neurogenic phenotype could be linked to excess canonical Notch signaling. The other phenotypes were linked to high levels of Notch protein expression at the surface of cells in the lateral regions indicating that a non-canonical Notch signaling activity normally functions in these regions. Results of our studies reported here provide evidence. They show that Notch activities are inextricably linked to that of Pkc98E, the homolog of mammalian PKCδ. Notch and Pkc98E up-regulate the levels of the phosphorylated form of IκBCactus, a negative regulator of Toll signaling, and Mothers against dpp (MAD), an effector of Dpp signaling. Our data suggest that in the lateral regions of the Drosophila embryos Notch activity, in conjunction with Pkc98E activity, is used to form the slopes of the opposing gradients of Toll and Dpp signaling that specify cell fates along the dorso-ventral axis.

  9. Notch and PKC are involved in formation of the lateral region of the dorso-ventral axis in Drosophila embryos.

    Directory of Open Access Journals (Sweden)

    Daniel M Tremmel

    Full Text Available The Notch gene encodes an evolutionarily conserved cell surface receptor that generates regulatory signals based on interactions between neighboring cells. In Drosophila embryos it is normally expressed at a low level due to strong negative regulation. When this negative regulation is abrogated neurogenesis in the ventral region is suppressed, the development of lateral epidermis is severely disrupted, and the dorsal aminoserosa is expanded. Of these phenotypes only the anti-neurogenic phenotype could be linked to excess canonical Notch signaling. The other phenotypes were linked to high levels of Notch protein expression at the surface of cells in the lateral regions indicating that a non-canonical Notch signaling activity normally functions in these regions. Results of our studies reported here provide evidence. They show that Notch activities are inextricably linked to that of Pkc98E, the homolog of mammalian PKCδ. Notch and Pkc98E up-regulate the levels of the phosphorylated form of IκBCactus, a negative regulator of Toll signaling, and Mothers against dpp (MAD, an effector of Dpp signaling. Our data suggest that in the lateral regions of the Drosophila embryos Notch activity, in conjunction with Pkc98E activity, is used to form the slopes of the opposing gradients of Toll and Dpp signaling that specify cell fates along the dorso-ventral axis.

  10. Drosophila brakeless interacts with atrophin and is required for tailless-mediated transcriptional repression in early embryos.

    Science.gov (United States)

    Haecker, Achim; Qi, Dai; Lilja, Tobias; Moussian, Bernard; Andrioli, Luiz Paulo; Luschnig, Stefan; Mannervik, Mattias

    2007-06-01

    Complex gene expression patterns in animal development are generated by the interplay of transcriptional activators and repressors at cis-regulatory DNA modules (CRMs). How repressors work is not well understood, but often involves interactions with co-repressors. We isolated mutations in the brakeless gene in a screen for maternal factors affecting segmentation of the Drosophila embryo. Brakeless, also known as Scribbler, or Master of thickveins, is a nuclear protein of unknown function. In brakeless embryos, we noted an expanded expression pattern of the Krüppel (Kr) and knirps (kni) genes. We found that Tailless-mediated repression of kni expression is impaired in brakeless mutants. Tailless and Brakeless bind each other in vitro and interact genetically. Brakeless is recruited to the Kr and kni CRMs, and represses transcription when tethered to DNA. This suggests that Brakeless is a novel co-repressor. Orphan nuclear receptors of the Tailless type also interact with Atrophin co-repressors. We show that both Drosophila and human Brakeless and Atrophin interact in vitro, and propose that they act together as a co-repressor complex in many developmental contexts. We discuss the possibility that human Brakeless homologs may influence the toxicity of polyglutamine-expanded Atrophin-1, which causes the human neurodegenerative disease dentatorubral-pallidoluysian atrophy (DRPLA).

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

    Directory of Open Access Journals (Sweden)

    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.

  12. 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.

  13. No significant regulation of bicoid mRNA by Pumilio or Nanos in the early Drosophila embryo.

    Science.gov (United States)

    Wharton, Tammy H; Nomie, Krystle J; Wharton, Robin P

    2018-01-01

    Drosophila Pumilio (Pum) is a founding member of the conserved Puf domain class of RNA-binding translational regulators. Pum binds with high specificity, contacting eight nucleotides, one with each of the repeats in its RNA-binding domain. In general, Pum is thought to block translation in collaboration with Nanos (Nos), which exhibits no binding specificity in isolation but is recruited jointly to regulatory sequences containing a Pum binding site in the 3'-UTRs of target mRNAs. Unlike Pum, which is ubiquitous in the early embryo, Nos is tightly restricted to the posterior, ensuring that repression of its best-characterized target, maternal hunchback (hb) mRNA, takes place exclusively in the posterior. An exceptional case of Nos-independent regulation by Pum has been described-repression of maternal bicoid (bcd) mRNA at the anterior pole of the early embryo, dependent on both Pum and conserved Pum binding sites in the 3'-UTR of the mRNA. We have re-investigated regulation of bcd in the early embryo; our experiments reveal no evidence of a role for Pum or its conserved binding sites in regulation of the perdurance of bcd mRNA or protein. Instead, we find that Pum and Nos control the accumulation of bcd mRNA in testes.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Loss of PTB or Negative Regulation of Notch mRNA Reveals Distinct Zones of Notch and Actin Protein Accumulation in Drosophila Embryo

    Science.gov (United States)

    Wesley, Cedric S.; Guo, Heng; Chaudhry, Kanita A.; Thali, Markus J.; Yin, Jerry C.; Clason, Todd; Wesley, Umadevi V.

    2011-01-01

    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. PMID:21750738

  16. Pipe-dependent ventral processing of Easter by Snake is the defining step in Drosophila embryo DV axis formation.

    Science.gov (United States)

    Cho, Yong Suk; Stevens, Leslie M; Stein, David

    2010-06-22

    The establishment of Drosophila embryonic dorsal-ventral (DV) polarity relies on serine proteolytic activity in the perivitelline space between the embryonic membrane and the eggshell. Gastrulation Defective cleaves and activates Snake, which processes and activates Easter, which cleaves Spätzle to form the activating ligand for the Toll receptor. Ventral restriction of ligand formation depends on the Pipe sulfotransferase, which is expressed in ventral cells of the follicular epithelium surrounding the developing oocyte. Pipe modifies components of the developing eggshell to produce a ventral cue embedded in the vitelline membrane. This ventral cue is believed to promote one or more of the proteolysis steps in the perivitelline space. By examining the processing of transgenic, tagged versions of the perivitelline proteins during DV patterning, we find that the proteolysis of Easter by Snake is the first Pipe-dependent step and therefore the key ventrally restricted event in the protease cascade. We also find that Snake and Easter associate together in a complex in both wild-type and pipe mutant-derived embryos. This observation suggests a mechanism in which the sulfated target of Pipe promotes a productive interaction between Snake and Easter, perhaps by facilitating conformational changes in a complex containing the two proteins. Copyright 2010 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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

  18. Asterless licenses daughter centrioles to duplicate for the first time in Drosophila embryos.

    Science.gov (United States)

    Novak, Zsofia A; Conduit, Paul T; Wainman, Alan; Raff, Jordan W

    2014-06-02

    Centrioles form centrosomes and cilia, and defects in any of these three organelles are associated with human disease [1]. Centrioles duplicate once per cell cycle, when a mother centriole assembles an adjacent daughter during S phase. Daughter centrioles cannot support the assembly of another daughter until they mature into mothers during the next cell cycle [2-5]. The molecular nature of this daughter-to-mother transition remains mysterious. Pioneering studies in C. elegans identified a set of core proteins essential for centriole duplication [6-12], and a similar set have now been identified in other species [10, 13-18]. The protein kinase ZYG-1/Sak/Plk4 recruits the inner centriole cartwheel components SAS-6 and SAS-5/Ana2/STIL, which then recruit SAS-4/CPAP, which in turn helps assemble the outer centriole microtubules [19, 20]. In flies and humans, the Asterless/Cep152 protein interacts with Sak/Plk4 and Sas-4/CPAP and is required for centriole duplication, although its precise role in the assembly pathway is unclear [21-24]. Here, we show that Asl is not incorporated into daughter centrioles as they assemble during S phase but is only incorporated once mother and daughter separate at the end of mitosis. The initial incorporation of Asterless (Asl) is irreversible, requires DSas-4, and, crucially, is essential for daughter centrioles to mature into mothers that can support centriole duplication. We therefore propose a "dual-licensing" model of centriole duplication, in which Asl incorporation provides a permanent primary license to allow new centrioles to duplicate for the first time, while centriole disengagement provides a reduplication license to allow mother centrioles to duplicate again. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Cortical movement of Bicoid in early Drosophila embryos is actin- and microtubule-dependent and disagrees with the SDD diffusion model.

    Directory of Open Access Journals (Sweden)

    Xiaoli Cai

    Full Text Available The Bicoid (Bcd protein gradient in Drosophila serves as a paradigm for gradient formation in textbooks. The SDD model (synthesis, diffusion, degradation was proposed to explain the formation of the gradient. The SDD model states that the bcd mRNA is located at the anterior pole of the embryo at all times and serves a source for translation of the Bicoid protein, coupled with diffusion and uniform degradation throughout the embryo. Recently, the ARTS model (active RNA transport, synthesis challenged the SDD model. In this model, the mRNA is transported at the cortex along microtubules to form a mRNA gradient which serves as template for the production of Bcd, hence little Bcd movement is involved. To test the validity of the SDD model, we developed a sensitive assay to monitor the movement of Bcd during early nuclear cycles. We observed that Bcd moved along the cortex and not in a broad front towards the posterior as the SDD model would have predicted. We subjected embryos to hypoxia where the mRNA remained strictly located at the tip at all times, while the protein was allowed to move freely, thus conforming to an ideal experimental setup to test the SDD model. Unexpectedly, Bcd still moved along the cortex. Moreover, cortical Bcd movement was sparse, even under longer hypoxic conditions. Hypoxic embryos treated with drugs compromising microtubule and actin function affected Bcd cortical movement and stability. Vinblastine treatment allowed the simulation of an ideal SDD model whereby the protein moved throughout the embryo in a broad front. In unfertilized embryos, the Bcd protein followed the mRNA which itself was transported into the interior of the embryo utilizing a hitherto undiscovered microtubular network. Our data suggest that the Bcd gradient formation is probably more complex than previously anticipated.

  20. 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.

  1. Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein.

    Science.gov (United States)

    Chen, Linan; Dumelie, Jason G; Li, Xiao; Cheng, Matthew Hk; Yang, Zhiyong; Laver, John D; Siddiqui, Najeeb U; Westwood, J Timothy; Morris, Quaid; Lipshitz, Howard D; Smibert, Craig A

    2014-01-07

    Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown. To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug's target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes. Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism.

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    L. F. Kurilo

    2016-01-01

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

  4. Role-Playing Mitosis.

    Science.gov (United States)

    Wyn, Mark A.; Stegink, Steven J.

    2000-01-01

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

  5. A novel function for the IκB inhibitor Cactus in promoting Dorsal nuclear localization and activity in the Drosophila embryo.

    Science.gov (United States)

    Cardoso, Maira Arruda; Fontenele, Marcio; Lim, Bomyi; Bisch, Paulo Mascarello; Shvartsman, Stanislav Y; Araujo, Helena Marcolla

    2017-08-15

    The evolutionarily conserved Toll signaling pathway controls innate immunity across phyla and embryonic patterning in insects. In the Drosophila embryo, Toll is required to establish gene expression domains along the dorsal-ventral axis. Pathway activation induces degradation of the IκB inhibitor Cactus, resulting in a ventral-to-dorsal nuclear gradient of the NFκB effector Dorsal. Here, we investigate how cactus modulates Toll signals through its effects on the Dorsal gradient and on Dorsal target genes. Quantitative analysis using a series of loss- and gain-of-function conditions shows that the ventral and lateral aspects of the Dorsal gradient can behave differently with respect to Cactus fluctuations. In lateral and dorsal embryo domains, loss of Cactus allows more Dorsal to translocate to the nucleus. Unexpectedly, cactus loss-of-function alleles decrease Dorsal nuclear localization ventrally, where Toll signals are high. Overexpression analysis suggests that this ability of Cactus to enhance Toll stems from the mobilization of a free Cactus pool induced by the Calpain A protease. These results indicate that Cactus acts to bolster Dorsal activation, in addition to its role as a NFκB inhibitor, ensuring a correct response to Toll signals. © 2017. Published by The Company of Biologists Ltd.

  6. 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…

  7. Effects of copper on mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Kostal, L

    1971-01-01

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

  8. From stem cell to embryo without centrioles.

    Science.gov (United States)

    Stevens, Naomi R; Raposo, Alexandre A S F; Basto, Renata; St Johnston, Daniel; Raff, Jordan W

    2007-09-04

    Centrosome asymmetry plays a key role in ensuring the asymmetric division of Drosophila neural stem cells (neuroblasts [NBs]) and male germline stem cells (GSCs) [1-3]. In both cases, one centrosome is anchored close to a specific cortical region during interphase, thus defining the orientation of the spindle during the ensuing mitosis. To test whether asymmetric centrosome behavior is a general feature of stem cells, we have studied female GSCs, which divide asymmetrically, producing another GSC and a cystoblast. The cystoblast then divides and matures into an oocyte, a process in which centrosomes exhibit a series of complex behaviors proposed to play a crucial role in oogenesis [4-6]. We show that the interphase centrosome does not define spindle orientation in female GSCs and that DSas-4 mutant GSCs [7], lacking centrioles and centrosomes, invariably divide asymmetrically to produce cystoblasts that proceed normally through oogenesis-remarkably, oocyte specification, microtubule organization, and mRNA localization are all unperturbed. Mature oocytes can be fertilized, but embryos that cannot support centriole replication arrest very early in development. Thus, centrosomes are dispensable for oogenesis but essential for early embryogenesis. These results reveal that asymmetric centrosome behavior is not an essential feature of stem cell divisions.

  9. The Drosophila Pericentrin-like-protein (PLP cooperates with Cnn to maintain the integrity of the outer PCM

    Directory of Open Access Journals (Sweden)

    Jennifer H. Richens

    2015-08-01

    Full Text Available Centrosomes comprise a pair of centrioles surrounded by a matrix of pericentriolar material (PCM. In vertebrate cells, Pericentrin plays an important part in mitotic PCM assembly, but the Drosophila Pericentrin-like protein (PLP appears to have a more minor role in mitotic fly cells. Here we investigate the function of PLP during the rapid mitotic cycles of the early Drosophila embryo. Unexpectedly, we find that PLP is specifically enriched in the outer-most regions of the PCM, where it largely co-localizes with the PCM scaffold protein Cnn. In the absence of PLP the outer PCM appears to be structurally weakened, and it rapidly disperses along the centrosomal microtubules (MTs. As a result, centrosomal MTs are subtly disorganized in embryos lacking PLP, although mitosis is largely unperturbed and these embryos develop and hatch at near-normal rates. Y2H analysis reveals that PLP can potentially form multiple interactions with itself and with the PCM recruiting proteins Asl, Spd-2 and Cnn. A deletion analysis suggests that PLP participates in a complex network of interactions that ultimately help to strengthen the PCM.

  10. Determination of Relative Biological Efficacy (RBE) and Oxygen Enhancement Ratio (OER) for the entire negative and positive pion beam profile using Vicia faba roots and Drosophila embryos as biological model systems

    International Nuclear Information System (INIS)

    Baarli, J.; Bianchi, M.; Keusch, F.; Mindek, G.; Sullivan, A.H.

    As an introduction to preclinical studies, pilot studies of pion beams are planned with relatively simple biological model systems that can be quickly evaluated and that yield indicative data for further action. Inhibition of growth was studied in Vicia faba roots, a biological system excellently suited for RBE and OER studies. For comparison there are already results from a low-intensity pion irradiation. A second system used Drosophila embryos 1 and 4 hours old, which are especially well suited for LET studies. The unambiguous criterion will be failure to slip out of the oolemma. The smallness of the objects (their beam sensitivity) will make it possible to determine empirically the peak region and to determine Gain factors; furthermore, the known dependency of RBE on the development stage promises highly informative results

  11. 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.

  12. Maintaining the proper connection between the centrioles and the pericentriolar matrix requires Drosophila centrosomin.

    Science.gov (United States)

    Lucas, Eliana P; Raff, Jordan W

    2007-08-27

    Centrosomes consist of two centrioles surrounded by an amorphous pericentriolar matrix (PCM), but it is unknown how centrioles and PCM are connected. We show that the centrioles in Drosophila embryos that lack the centrosomal protein Centrosomin (Cnn) can recruit PCM components but cannot maintain a proper attachment to the PCM. As a result, the centrioles "rocket" around in the embryo and often lose their connection to the nucleus in interphase and to the spindle poles in mitosis. This leads to severe mitotic defects in embryos and to errors in centriole segregation in somatic cells. The Cnn-related protein CDK5RAP2 is linked to microcephaly in humans, but cnn mutant brains are of normal size, and we observe only subtle defects in the asymmetric divisions of mutant neuroblasts. We conclude that Cnn maintains the proper connection between the centrioles and the PCM; this connection is required for accurate centriole segregation in somatic cells but is not essential for the asymmetric division of neuroblasts.

  13. Metabolome analysis of Drosophila melanogaster during embryogenesis.

    Science.gov (United States)

    An, Phan Nguyen Thuy; Yamaguchi, Masamitsu; Bamba, Takeshi; Fukusaki, Eiichiro

    2014-01-01

    The Drosophila melanogaster embryo has been widely utilized as a model for genetics and developmental biology due to its small size, short generation time, and large brood size. Information on embryonic metabolism during developmental progression is important for further understanding the mechanisms of Drosophila embryogenesis. Therefore, the aim of this study is to assess the changes in embryos' metabolome that occur at different stages of the Drosophila embryonic development. Time course samples of Drosophila embryos were subjected to GC/MS-based metabolome analysis for profiling of low molecular weight hydrophilic metabolites, including sugars, amino acids, and organic acids. The results showed that the metabolic profiles of Drosophila embryo varied during the course of development and there was a strong correlation between the metabolome and different embryonic stages. Using the metabolome information, we were able to establish a prediction model for developmental stages of embryos starting from their high-resolution quantitative metabolite composition. Among the important metabolites revealed from our model, we suggest that different amino acids appear to play distinct roles in different developmental stages and an appropriate balance in trehalose-glucose ratio is crucial to supply the carbohydrate source for the development of Drosophila embryo.

  14. 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

  15. Embryo splitting

    Directory of Open Access Journals (Sweden)

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

    Science.gov (United States)

    Bougé, Anne-Laure; Parmentier, Marie-Laure

    2016-03-01

    In neurodegenerative diseases such as Alzheimer's disease (AD), cell cycle defects and associated aneuploidy have been described. However, the importance of these defects in the physiopathology of AD and the underlying mechanistic processes are largely unknown, in particular with respect to the microtubule (MT)-binding protein Tau, which is found in excess in the brain and cerebrospinal fluid of affected individuals. Although it has long been known that Tau is phosphorylated during mitosis to generate a lower affinity for MTs, there is, to our knowledge, no indication that an excess of this protein could affect mitosis. Here, we studied the effect of an excess of human Tau (hTau) protein on cell mitosis in vivo. Using the Drosophila developing wing disc epithelium as a model, we show that an excess of hTau induces a mitotic arrest, with the presence of monopolar spindles. This mitotic defect leads to aneuploidy and apoptotic cell death. We studied the mechanism of action of hTau and found that the MT-binding domain of hTau is responsible for these defects. We also demonstrate that the effects of hTau occur via the inhibition of the function of the kinesin Klp61F, the Drosophila homologue of kinesin-5 (also called Eg5 or KIF11). We finally show that this deleterious effect of hTau is also found in other Drosophila cell types (neuroblasts) and tissues (the developing eye disc), as well as in human HeLa cells. By demonstrating that MT-bound Tau inhibits the Eg5 kinesin and cell mitosis, our work provides a new framework to consider the role of Tau in neurodegenerative diseases. © 2016. Published by The Company of Biologists Ltd.

  17. 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. © 2014 The Authors.

  18. 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

    , 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...

  19. Non-coding RNAs enter mitosis: functions, conservation and implications

    Directory of Open Access Journals (Sweden)

    Kai Toshie

    2011-02-01

    Full Text Available Abstract Nuage (or commonly known as chromatoid body in mammals is a conserved germline-specific organelle that has been linked to the Piwi-interacting RNA (piRNA pathway. piRNAs are a class of gonadal-specific RNAs that are ~23-29 nucleotides in length and protect genome stability by repressing the expression of deleterious retrotransposons. More recent studies in Drosophila have implicated the piRNA pathway in other functions including canalization of embryonic development, regulation of maternal gene expression and telomere protection. We have recently shown that Vasa (known as Mouse Vasa Homolog in mouse, a nuage component, plays a mitotic role in promoting chromosome condensation and segregation by facilitating robust chromosomal localization of condensin I in the Drosophila germline. Vasa functions together with Aubergine (a PIWI family protein and Spindle-E/mouse TDRD-9, two other nuage components that are involved in the piRNA pathway, therefore providing a link between the piRNA pathway and mitotic chromosome condensation. Here, we propose and discuss possible models for the role of Vasa and the piRNA pathway during mitosis. We also highlight relevant studies implicating mitotic roles for RNAs and/or nuage in other model systems and their implications for cancer development.

  20. 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.

  1. 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.

  2. Myoblast fusion in Drosophila

    International Nuclear Information System (INIS)

    Haralalka, Shruti; Abmayr, Susan M.

    2010-01-01

    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.

  3. 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.

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

    Science.gov (United States)

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

    2018-04-01

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

  5. Imaging Mitosis in the Moss Physcomitrella patens.

    Science.gov (United States)

    Yamada, Moé; Miki, Tomohiro; Goshima, Gohta

    2016-01-01

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

  6. 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.

  7. Genetic and proteomic evidence for roles of Drosophila SUMO in cell cycle control, Ras signaling, and early pattern formation.

    Directory of Open Access Journals (Sweden)

    Minghua Nie

    2009-06-01

    Full Text Available SUMO is a protein modifier that is vital for multicellular development. Here we present the first system-wide analysis, combining multiple approaches, to correlate the sumoylated proteome (SUMO-ome in a multicellular organism with the developmental roles of SUMO. Using mass-spectrometry-based protein identification, we found over 140 largely novel SUMO conjugates in the early Drosophila embryo. Enriched functional groups include proteins involved in Ras signaling, cell cycle, and pattern formation. In support of the functional significance of these findings, sumo germline clone embryos exhibited phenotypes indicative of defects in these same three processes. Our cell culture and immunolocalization studies further substantiate roles for SUMO in Ras signaling and cell cycle regulation. For example, we found that SUMO is required for efficient Ras-mediated MAP kinase activation upstream or at the level of Ras activation. We further found that SUMO is dynamically localized during mitosis to the condensed chromosomes, and later also to the midbody. Polo kinase, a SUMO substrate found in our screen, partially colocalizes with SUMO at both sites. These studies show that SUMO coordinates multiple regulatory processes during oogenesis and early embryogenesis. In addition, our database of sumoylated proteins provides a valuable resource for those studying the roles of SUMO in development.

  8. The DNA damage response during mitosis

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  9. 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.

  10. The DNA damage response during mitosis.

    Science.gov (United States)

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

    2013-10-01

    Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Kinases Involved in Both Autophagy and Mitosis.

    Science.gov (United States)

    Li, Zhiyuan; Zhang, Xin

    2017-08-31

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

  12. Kinases Involved in Both Autophagy and Mitosis

    Directory of Open Access Journals (Sweden)

    Zhiyuan Li

    2017-08-01

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

  13. Rescue from replication stress during mitosis.

    Science.gov (United States)

    Fragkos, Michalis; Naim, Valeria

    2017-04-03

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

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

    Science.gov (United States)

    Dienemann, Axel; Sprenger, Frank

    2004-06-22

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

  15. 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.

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

    OpenAIRE

    Pilaz, Louis-Jan; Silver, Debra L.

    2014-01-01

    Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixe...

  17. 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

    The database of the Drosophila Genome Project contains the sequences of two genes, CG8784 and CG8795, predicted to code for two structurally related G protein-coupled receptors. We have cloned these genes and expressed their coding parts in Chinese hamster ovary cells. We found that both receptors...... can be activated by low concentrations of the Drosophila neuropeptide pyrokinin-2 (CG8784, EC(50) for pyrokinin-2, 1x10(-9)M; CG8795, EC(50) for pyrokinin-2, 5 x 10(-10)M). The precise role of Drosophila pyrokinin-2 (SVPFKPRLamide) in Drosophila is unknown, but in other insects, pyrokinins have...... 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...

  18. Cultures of preimplantation mouse embryos

    International Nuclear Information System (INIS)

    Streffer, C.; Molls, M.

    1987-01-01

    In the preimplantation mouse embryos the chromosomal damage develops through several postradiation cell cycles and mitoses. New chromosome aberrations are seen during the second and third postradiation mitoses. Also, more micronuclei appear during later postradiation interphases. This is in agreement with the assumption that unrepaired chromosomal radiation damage develops during the cell generation cycle to such a form (i.e. double-strand breaks in DNA) that chromosomal breaks occur. This proposition is strengthened by the observation that radiation-induced damage is more rapidly expressed after neutron exposure (first or second postradiation mitosis) than after exposure to X rays at the one- or two-cell stage. The preimplantation mouse embryo culture is an inviting system for additional studies at the molecular level, especially now that within the last few years more sensitive methods have been developed for study of DNA and protein structure, regulation, and synthesis. The results from these studies of cultures of preimplantation mouse embryos present a favorable case for the study of complex biological systems under very defined conditions in vitro for extrapolation to effects in vivo

  19. Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions.

    Science.gov (United States)

    Koyle, Melinda L; Veloz, Madeline; Judd, Alec M; Wong, Adam C-N; Newell, Peter D; Douglas, Angela E; Chaston, John M

    2016-07-30

    The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

  20. Rapid DNA Synthesis During Early Drosophila Embryogenesis Is Sensitive to Maternal Humpty Dumpty Protein Function.

    Science.gov (United States)

    Lesly, Shera; Bandura, Jennifer L; Calvi, Brian R

    2017-11-01

    Problems with DNA replication cause cancer and developmental malformations. It is not fully understood how DNA replication is coordinated with development and perturbed in disease. We had previously identified the Drosophila gene humpty dumpty ( hd ), and showed that null alleles cause incomplete DNA replication, tissue undergrowth, and lethality. Animals homozygous for the missense allele, hd 272-9 , were viable, but adult females had impaired amplification of eggshell protein genes in the ovary, resulting in the maternal effects of thin eggshells and embryonic lethality. Here, we show that expression of an hd transgene in somatic cells of the ovary rescues amplification and eggshell synthesis but not embryo viability. The germline of these mothers remain mutant for the hd 272-9 allele, resulting in reduced maternal Hd protein and embryonic arrest during mitosis of the first few S/M nuclear cleavage cycles with chromosome instability and chromosome bridges. Epistasis analysis of hd with the rereplication mutation plutonium indicates that the chromosome bridges of hd embryos are the result of a failed attempt to segregate incompletely replicated sister chromatids. This study reveals that maternally encoded Humpty dumpty protein is essential for DNA replication and genome integrity during the little-understood embryonic S/M cycles. Moreover, the two hd 272-9 maternal-effect phenotypes suggest that ovarian gene amplification and embryonic cleavage are two time periods in development that are particularly sensitive to mild deficits in DNA replication function. This last observation has broader relevance for interpreting why mild mutations in the human ortholog of humpty dumpty and other DNA replication genes cause tissue-specific malformations of microcephalic dwarfisms. Copyright © 2017 by the Genetics Society of America.

  1. Meiosis: An Overview of Key Differences from Mitosis

    Science.gov (United States)

    Ohkura, Hiroyuki

    2015-01-01

    Meiosis is the specialized cell division that generates gametes. In contrast to mitosis, molecular mechanisms and regulation of meiosis are much less understood. Meiosis shares mechanisms and regulation with mitosis in many aspects, but also has critical differences from mitosis. This review highlights these differences between meiosis and mitosis. Recent studies using various model systems revealed differences in a surprisingly wide range of aspects, including cell-cycle regulation, recombination, postrecombination events, spindle assembly, chromosome–spindle interaction, and chromosome segregation. Although a great degree of diversity can be found among organisms, meiosis-specific processes, and regulation are generally conserved. PMID:25605710

  2. 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.

  3. 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.

  4. 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.

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

    Science.gov (United States)

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

    2017-02-20

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

  6. Unsuccessful mitosis in multicellular tumour spheroids.

    Science.gov (United States)

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

    2017-04-25

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

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

    Science.gov (United States)

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

    2012-01-01

    We analyzed modification of chromatin by ubiquitination in human cells and whether this mark changes through the cell cycle. HeLa cells were synchronized at different stages and regions of the genome with ubiquitinated chromatin were identified by affinity purification coupled with next-generation sequencing. During interphase, ubiquitin marked the chromatin on the transcribed regions of ∼70% of highly active genes and deposition of this mark was sensitive to transcriptional inhibition. Promoters of nearly half of the active genes were highly ubiquitinated specifically during mitosis. The ubiquitination at the coding regions in interphase but not at promoters during mitosis was enriched for ubH2B and dependent on the presence of RNF20. Ubiquitin labeling of both promoters during mitosis and transcribed regions during interphase, correlated with active histone marks H3K4me3 and H3K36me3 but not a repressive histone modification, H3K27me3. The high level of ubiquitination at the promoter chromatin during mitosis was transient and was removed within 2 h after the cells exited mitosis and entered the next cell cycle. These results reveal that the ubiquitination of promoter chromatin during mitosis is a bookmark identifying active genes during chromosomal condensation in mitosis, and we suggest that this process facilitates transcriptional reactivation post-mitosis. PMID:22941662

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

    Science.gov (United States)

    Van Hoewyk, Doug

    2007-01-01

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

  9. 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.

  10. 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...

  11. Centrioles: active players or passengers during mitosis?

    Science.gov (United States)

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

    2010-07-01

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

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

    Science.gov (United States)

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

    2015-02-01

    Mitosis entails global alterations to chromosome structure and nuclear architecture, concomitant with transient silencing of transcription. How cells transmit transcriptional states through mitosis remains incompletely understood. While many nuclear factors dissociate from mitotic chromosomes, the observation that certain nuclear factors and chromatin features remain associated with individual loci during mitosis originated the hypothesis that such mitotically retained molecular signatures could provide transcriptional memory through mitosis. To understand the role of chromatin structure in mitotic memory, we performed the first genome-wide comparison of DNase I sensitivity of chromatin in mitosis and interphase, using a murine erythroblast model. Despite chromosome condensation during mitosis visible by microscopy, the landscape of chromatin accessibility at the macromolecular level is largely unaltered. However, mitotic chromatin accessibility is locally dynamic, with individual loci maintaining none, some, or all of their interphase accessibility. Mitotic reduction in accessibility occurs primarily within narrow, highly DNase hypersensitive sites that frequently coincide with transcription factor binding sites, whereas broader domains of moderate accessibility tend to be more stable. In mitosis, proximal promoters generally maintain their accessibility more strongly, whereas distal regulatory elements tend to lose accessibility. Large domains of DNA hypomethylation mark a subset of promoters that retain accessibility during mitosis and across many cell types in interphase. Erythroid transcription factor GATA1 exerts site-specific changes in interphase accessibility that are most pronounced at distal regulatory elements, but has little influence on mitotic accessibility. We conclude that features of open chromatin are remarkably stable through mitosis, but are modulated at the level of individual genes and regulatory elements. © 2015 Hsiung et al.; Published by

  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. Deciphering the evolutionary history of open and closed mitosis.

    Science.gov (United States)

    Sazer, Shelley; Lynch, Michael; Needleman, Daniel

    2014-11-17

    The origin of the nucleus at the prokaryote-to-eukaryote transition represents one of the most important events in the evolution of cellular organization. The nuclear envelope encircles the chromosomes in interphase and is a selectively permeable barrier between the nucleoplasm and cytoplasm and an organizational scaffold for the nucleus. It remains intact in the 'closed' mitosis of some yeasts, but loses its integrity in the 'open' mitosis of mammals. Instances of both types of mitosis within two evolutionary clades indicate multiple evolutionary transitions between open and closed mitosis, although the underlying genetic changes that influenced these transitions remain unknown. A survey of the diversity of mitotic nuclei that fall between these extremes is the starting point from which to determine the physiologically relevant characteristics distinguishing open from closed mitosis and to understand how they evolved and why they are retained in present-day organisms. The field is now poised to begin addressing these issues by defining and documenting patterns of mitotic nuclear variation within and among species and mapping them onto a phylogenic tree. Deciphering the evolutionary history of open and closed mitosis will complement cell biological and genetic approaches aimed at deciphering the fundamental organizational principles of the nucleus. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. Lethals induced by γ-radiation in drosophila somatic cells

    International Nuclear Information System (INIS)

    Ivanov, A.I.

    1989-01-01

    Exposure of 3-hour drosophila male embryos to γ-radiation during the topographic segregation of the germ anlage nuclei caused recessive sex-linked lethals in somatic cells only. The selectivity of the screening was determined by the ratio of mutation frequencies induced in embryos and adult males. Analysis of lethal mutations shows that a minimal rate of the divergence between germinal and somatic patterns of the cell development is observed in the embryogenesis, the 3d instar larva and prepupa, and maximal in the 1st and 2nd larva and pupa

  1. Rab5 GTPase controls chromosome alignment through Lamin disassembly and relocation of the NuMA-like protein Mud to the poles during mitosis

    Science.gov (United States)

    Capalbo, Luisa; D'Avino, Pier Paolo; Archambault, Vincent; Glover, David M.

    2011-01-01

    The small GTPase Rab5 is a conserved regulator of membrane trafficking; it regulates the formation of early endosomes, their transport along microtubules, and the fusion to the target organelles. Although several members of the endocytic pathway were recently implicated in spindle organization, it is unclear whether Rab5 has any role during mitosis. Here, we describe that Rab5 is required for proper chromosome alignment during Drosophila mitoses. We also found that Rab5 associated in vivo with nuclear Lamin and mushroom body defect (Mud), the Drosophila counterpart of nuclear mitotic apparatus protein (NuMA). Consistent with this finding, Rab5 was required for the disassembly of the nuclear envelope at mitotic entry and the accumulation of Mud at the spindle poles. Furthermore, Mud depletion caused chromosome misalignment defects that resembled the defects of Rab5 RNAi cells, and double-knockdown experiments indicated that the two proteins function in a linear pathway. Our results indicate a role for Rab5 in mitosis and reinforce the emerging view of the contributions made by cell membrane dynamics to spindle function. PMID:21987826

  2. Phosphorylation of CPAP by Aurora-A Maintains Spindle Pole Integrity during Mitosis

    OpenAIRE

    En-Ju Chou; Liang-Yi Hung; Chieh-Ju C. Tang; Wen-Bin Hsu; Hsin-Yi Wu; Pao-Chi Liao; Tang K. Tang

    2016-01-01

    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. In...

  3. 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

  4. 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.

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

    National Research Council Canada - National Science Library

    Chang, Long-Sheng

    2008-01-01

    ... determination of dorsal/ventral compartment border during wing imaginal disc development. We show that the Merlin protein is dynamically redistributed during meiosis and demonstrate, for the first time, Merlin immunoreactivity in mitochondria...

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

    Science.gov (United States)

    2007-07-01

    axoneme, or with two axonemes, were found. Also, cytoplasmic fragmentation together with condensed cytoplasmic remnants and gigantic cytoplasmic... Endocrine - Related Cancer 2001; 8: 249-258. 33. Faivre S, Kroemer G and Raymond E. Current development of mTOR inhibitors as anticancer agents

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

    Science.gov (United States)

    2008-07-01

    chrysosporium --- http://genome.jgi-p Aspergillus flavus --- http://www.tigr.or Arabidopsis thaliana --- http://www.tigr.or Oryza sativa --- http...distinguish colon and ovarian adenocarcinomas: identi- fication, genomic, proteomic , and tissue array profiling. Cancer Res 2003;63:5243–50. 108

  8. 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

    2017-10-15

    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 about 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. Altogether, we propose that Dynein, Mud and Asp operate sequentially during the cell cycle to ensure efficient centrosome-spindle coupling in mitosis, thereby preventing centrosome mis-segregation to maintain centrosome number. © 2017. Published by The Company of Biologists Ltd.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. Raptor is phosphorylated by cdc2 during mitosis.

    Directory of Open Access Journals (Sweden)

    Dana M Gwinn

    2010-02-01

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

  14. Tet protein function during Drosophila development.

    Directory of Open Access Journals (Sweden)

    Fei Wang

    Full Text Available The TET (Ten-eleven translocation 1, 2 and 3 proteins have been shown to function as DNA hydroxymethylases in vertebrates and their requirements have been documented extensively. Recently, the Tet proteins have been shown to also hydroxylate 5-methylcytosine in RNA. 5-hydroxymethylcytosine (5hmrC is enriched in messenger RNA but the function of this modification has yet to be elucidated. Because Cytosine methylation in DNA is barely detectable in Drosophila, it serves as an ideal model to study the biological function of 5hmrC. Here, we characterized the temporal and spatial expression and requirement of Tet throughout Drosophila development. We show that Tet is essential for viability as Tet complete loss-of-function animals die at the late pupal stage. Tet is highly expressed in neuronal tissues and at more moderate levels in somatic muscle precursors in embryos and larvae. Depletion of Tet in muscle precursors at early embryonic stages leads to defects in larval locomotion and late pupal lethality. Although Tet knock-down in neuronal tissue does not cause lethality, it is essential for neuronal function during development through its affects upon locomotion in larvae and the circadian rhythm of adult flies. Further, we report the function of Tet in ovarian morphogenesis. Together, our findings provide basic insights into the biological function of Tet in Drosophila, and may illuminate observed neuronal and muscle phenotypes observed in vertebrates.

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

    Science.gov (United States)

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

    2016-06-09

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

  16. Regulation of cAMP on the first mitotic cell cycle of mouse embryos.

    Science.gov (United States)

    Yu, Aiming; Zhang, Zhe; Bi, Qiang; Sun, Bingqi; Su, Wenhui; Guan, Yifu; Mu, Runqing; Miao, Changsheng; Zhang, Jie; Yu, Bingzhi

    2008-03-01

    Mitosis promoting factor (MPF) plays a central role during the first mitosis of mouse embryo. We demonstrated that MPF activity increased when one-cell stage mouse embryo initiated G2/M transition following the decrease of cyclic adenosine 3', 5'-monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) activity. When cAMP and PKA activity increases again, MPF activity decreases and mouse embryo starts metaphase-anaphase transition. In the downstream of cAMP/PKA, there are some effectors such as polo-like kinase 1 (Plk1), Cdc25, Mos (mitogen-activated protein kinase kinase kinase), MEK (mitogen-activated protein kinase kinase), mitogen-activated protein kinase (MAPK), Wee1, anaphase-promoting complex (APC), and phosphoprotein phosphatase that are involved in the regulation of MPF activity. Here, we demonstrated that following activation of MPF, MAPK activity was steady, whereas Plk1 activity fluctuated during the first cell cycle. Plk1 activity was the highest at metaphase and decreased at metaphase-anaphase transition. Further, we established a mathematical model using Gepasi algorithm and the simulation was in agreement with the experimental data. Above all the evidences, we suggested that cAMP and PKA might be the upstream factors which were included in the regulation of the first cell cycle development of mouse embryo. Copyright 2007 Wiley-Liss, Inc.

  17. Metabolomic Studies in Drosophila.

    Science.gov (United States)

    Cox, James E; Thummel, Carl S; Tennessen, Jason M

    2017-07-01

    Metabolomic analysis provides a powerful new tool for studies of Drosophila physiology. This approach allows investigators to detect thousands of chemical compounds in a single sample, representing the combined contributions of gene expression, enzyme activity, and environmental context. Metabolomics has been used for a wide range of studies in Drosophila , often providing new insights into gene function and metabolic state that could not be obtained using any other approach. In this review, we survey the uses of metabolomic analysis since its entry into the field. We also cover the major methods used for metabolomic studies in Drosophila and highlight new directions for future research. Copyright © 2017 by the Genetics Society of America.

  18. Cycling with BRCA2 from DNA repair to mitosis

    International Nuclear Information System (INIS)

    Lee, Hyunsook

    2014-01-01

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

  19. Cycling with BRCA2 from DNA repair to mitosis

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-11-15

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

  20. Relocalization of human chromatin remodeling cofactor TIP48 in mitosis

    International Nuclear Information System (INIS)

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

    2005-01-01

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

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

  2. 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.

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

    Science.gov (United States)

    Ma, Hoi Tang; Poon, Randy Y C

    2011-04-01

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

  4. 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 dire...... 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.......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...

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

    Science.gov (United States)

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

    2015-05-29

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

  6. Active forgetting of olfactory memories in Drosophila.

    Science.gov (United States)

    Berry, Jacob A; Davis, Ronald L

    2014-01-01

    Failure to remember, or forgetting, is a phenomenon familiar to everyone and despite more than a century of scientific inquiry, why we forget what we once knew remains unclear. If the brain marshals significant resources to form and store memories, why is it that these memories become lost? In the last century, psychological studies have divided forgetting into decay theory, in which memory simply dissipates with time, and interference theory, in which additional learning or mental activity hinders memory by reducing its stability or retrieval (for review, Dewar et al., 2007; Wixted, 2004). Importantly, these psychological models of forgetting posit that forgetting is a passive property of the brain and thus a failure of the brain to retain memories. However, recent neuroscience research on olfactory memory in Drosophila has offered evidence for an alternative conclusion that forgetting is an "active" process, with specific, biologically regulated mechanisms that remove existing memories (Berry et al., 2012; Shuai et al., 2010). Similar to the bidirectional regulation of cell number by mitosis and apoptosis, protein concentration by translation and lysosomal or proteomal degradation, and protein phosphate modification by kinases and phosphatases, biologically regulated memory formation and removal would be yet another example in biological systems where distinct and separate pathways regulate the creation and destruction of biological substrates. © 2014 Elsevier B.V. All rights reserved.

  7. 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

    2016-06-28

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

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

    Science.gov (United States)

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

    2016-06-15

    During mitosis, RNA polymerase II (Pol II) and many transcription factors dissociate from chromatin, and transcription ceases globally. Transcription is known to restart in bulk by telophase, but whether de novo transcription at the mitosis-G1 transition is in any way distinct from later in interphase remains unknown. We tracked Pol II occupancy genome-wide in mammalian cells progressing from mitosis through late G1. Unexpectedly, during the earliest rounds of transcription at the mitosis-G1 transition, ∼50% of active genes and distal enhancers exhibit a spike in transcription, exceeding levels observed later in G1 phase. Enhancer-promoter chromatin contacts are depleted during mitosis and restored rapidly upon G1 entry but do not spike. Of the chromatin-associated features examined, histone H3 Lys27 acetylation levels at individual loci in mitosis best predict the mitosis-G1 transcriptional spike. Single-molecule RNA imaging supports that the mitosis-G1 transcriptional spike can constitute the maximum transcriptional activity per DNA copy throughout the cell division cycle. The transcriptional spike occurs heterogeneously and propagates to cell-to-cell differences in mature mRNA expression. Our results raise the possibility that passage through the mitosis-G1 transition might predispose cells to diverge in gene expression states. © 2016 Hsiung et al.; Published by Cold Spring Harbor Laboratory Press.

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

    Science.gov (United States)

    O'Day, Danton H; Budniak, Aldona

    2015-02-01

    Mitosis is a fundamental and essential life process. It underlies the duplication and survival of all cells and, as a result, all eukaryotic organisms. Since uncontrolled mitosis is a dreaded component of many cancers, a full understanding of the process is critical. Evolution has led to the existence of three types of mitosis: closed, open, and semi-open. The significance of these different mitotic species, how they can lead to a full understanding of the critical events that underlie the asexual duplication of all cells, and how they may generate new insights into controlling unregulated cell division remains to be determined. The eukaryotic microbe Dictyostelium discoideum has proved to be a valuable biomedical model organism. While it appears to utilize closed mitosis, a review of the literature suggests that it possesses a form of mitosis that lies in the middle between truly open and fully closed mitosis-it utilizes a form of semi-open mitosis. Here, the nucleocytoplasmic translocation patterns of the proteins that have been studied during mitosis in the social amoebozoan D. discoideum are detailed followed by a discussion of how some of them provide support for the hypothesis of semi-open mitosis. © 2014 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.

  10. JAK/Stat signaling regulates heart precursor diversification in Drosophila

    Science.gov (United States)

    Johnson, Aaron N.; Mokalled, Mayssa H.; Haden, Tom N.; Olson, Eric N.

    2011-01-01

    Intercellular signal transduction pathways regulate the NK-2 family of transcription factors in a conserved gene regulatory network that directs cardiogenesis in both flies and mammals. The Drosophila NK-2 protein Tinman (Tin) was recently shown to regulate Stat92E, the Janus kinase (JAK) and Signal transducer and activator of transcription (Stat) pathway effector, in the developing mesoderm. To understand whether the JAK/Stat pathway also regulates cardiogenesis, we performed a systematic characterization of JAK/Stat signaling during mesoderm development. Drosophila embryos with mutations in the JAK/Stat ligand upd or in Stat92E have non-functional hearts with luminal defects and inappropriate cell aggregations. Using strong Stat92E loss-of-function alleles, we show that the JAK/Stat pathway regulates tin expression prior to heart precursor cell diversification. tin expression can be subdivided into four phases and, in Stat92E mutant embryos, the broad phase 2 expression pattern in the dorsal mesoderm does not restrict to the constrained phase 3 pattern. These embryos also have an expanded pericardial cell domain. We show the E(spl)-C gene HLHm5 is expressed in a pattern complementary to tin during phase 3 and that this expression is JAK/Stat dependent. In addition, E(spl)-C mutant embryos phenocopy the cardiac defects of Stat92E embryos. Mechanistically, JAK/Stat signals activate E(spl)-C genes to restrict Tin expression and the subsequent expression of the T-box transcription factor H15 to direct heart precursor diversification. This study is the first to characterize a role for the JAK/Stat pathway during cardiogenesis and identifies an autoregulatory circuit in which tin limits its own expression domain. PMID:21965617

  11. Mapping organism expression levels at cellular resolution in developing Drosophila

    Science.gov (United States)

    Knowles, David W.; Keranen, Soile; Biggin, Mark D.; Sudar, Damir

    2002-05-01

    The development of an animal embryo is orchestrated by a network of genetically determined, temporal and spatial gene expression patterns that determine the animals final form. To understand such networks, we are developing novel quantitative optical imaging techniques to map gene expression levels at cellular and sub-cellular resolution within pregastrula Drosophila. Embryos at different stages of development are labeled for total DNA and specific gene products using different fluorophors and imaged in 3D with confocal microscopy. Innovative steps have been made which allow the DNA-image to be automatically segmented to produce a morphological mask of the individual nuclear boundaries. For each stage of development an average morphology is chosen to which images from different embryo are compared. The morphological mask is then used to quantify gene-product on a per nuclei basis. What results is an atlas of the relative amount of the specific gene product expressed within the nucleus of every cell in the embryo at the various stages of development. We are creating a quantitative database of transcription factor and target gene expression patterns in wild-type and factor mutant embryos with single cell resolution. Our goal is to uncover the rules determining how patterns of gene expression are generated.

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

    Science.gov (United States)

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

    2015-11-15

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

  13. Asymmetrical distribution of the transcriptionally competent NORs in mitosis

    Czech Academy of Sciences Publication Activity Database

    Kalmárová, Markéta; Kováčik, Lubomír; Popov, Alexey; Testillano, P. S.; Smirnov, Evgeny

    2008-01-01

    Roč. 163, č. 1 (2008), s. 40-44 ISSN 1047-8477 R&D Projects: GA ČR(CZ) GA304/06/1691 Grant - others:Wellcome Trust(XE) 075834/04/Z; GA MŠk(CZ) LC535; GA ČR(CZ) GA304/06/1662; C.S.I.C.(ES) CS-ES2007-8/16 Program:LC Institutional research plan: CEZ:AV0Z50110509 Keywords : mitosis * NORs * asymmetry Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.059, year: 2008

  14. 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

  15. 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

  16. 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

  17. Manipulating early pig embryos.

    Science.gov (United States)

    Niemann, H; Reichelt, B

    1993-01-01

    On the basis of established surgical procedures for embryo recovery and transfer, the early pig embryo can be subjected to various manipulations aimed at a long-term preservation of genetic material, the generation of identical multiplets, the early determination of sex or the alteration of the genetic make-up. Most of these procedures are still at an experimental stage and despite recent considerable progress are far from practical application. Normal piglets have been obtained after cryopreservation of pig blastocysts hatched in vitro, whereas all attempts to freeze embryos with intact zona pellucida have been unsuccessful. Pig embryos at the morula and blastocyst stage can be bisected microsurgically and the resulting demi-embryos possess a high developmental potential in vitro, whereas their development in vivo is impaired. Pregnancy rates are similar (80%) but litter size is reduced compared with intact embryos and twinning rate is approximately 2%. Pig blastomeres isolated from embryos up to the 16-cell stage can be grown in culture and result in normal blastocysts. Normal piglets have been born upon transfer of blastocysts derived from isolated eight-cell blastomeres, clearly underlining the totipotency of this developmental stage. Upon nuclear transfer the developmental capacity of reconstituted pig embryos is low and culture. Sex determination can be achieved either by separation of X and Y chromosome bearing spermatozoa by flow cytometry or by analysing the expression of the HY antigen in pig embryos from the eight-cell to morula stage. Microinjection of foreign DNA has been successfully used to alter growth and development of transgenic pigs, and to produce foreign proteins in the mammary gland or in the bloodstream, indicating that pigs can be used as donors for valuable human pharmaceutical proteins. Another promising area of gene transfer is the increase of disease resistance in transgenic lines of pigs. Approximately 30% of pig spermatozoa bind

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    Humayun Irshad

    2013-01-01

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

  20. Mouse Embryo Compaction.

    Science.gov (United States)

    White, M D; Bissiere, S; Alvarez, Y D; Plachta, N

    2016-01-01

    Compaction is a critical first morphological event in the preimplantation development of the mammalian embryo. Characterized by the transformation of the embryo from a loose cluster of spherical cells into a tightly packed mass, compaction is a key step in the establishment of the first tissue-like structures of the embryo. Although early investigation of the mechanisms driving compaction implicated changes in cell-cell adhesion, recent work has identified essential roles for cortical tension and a compaction-specific class of filopodia. During the transition from 8 to 16 cells, as the embryo is compacting, it must also make fundamental decisions regarding cell position, polarity, and fate. Understanding how these and other processes are integrated with compaction requires further investigation. Emerging imaging-based techniques that enable quantitative analysis from the level of cell-cell interactions down to the level of individual regulatory molecules will provide a greater understanding of how compaction shapes the early mammalian embryo. © 2016 Elsevier Inc. All rights reserved.

  1. 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.

  2. Hearing regulates Drosophila aggression.

    Science.gov (United States)

    Versteven, Marijke; Vanden Broeck, Lies; Geurten, Bart; Zwarts, Liesbeth; Decraecker, Lisse; Beelen, Melissa; Göpfert, Martin C; Heinrich, Ralf; Callaerts, Patrick

    2017-02-21

    Aggression is a universal social behavior important for the acquisition of food, mates, territory, and social status. Aggression in Drosophila is context-dependent and can thus be expected to involve inputs from multiple sensory modalities. Here, we use mechanical disruption and genetic approaches in Drosophila melanogaster to identify hearing as an important sensory modality in the context of intermale aggressive behavior. We demonstrate that neuronal silencing and targeted knockdown of hearing genes in the fly's auditory organ elicit abnormal aggression. Further, we show that exposure to courtship or aggression song has opposite effects on aggression. Our data define the importance of hearing in the control of Drosophila intermale aggression and open perspectives to decipher how hearing and other sensory modalities are integrated at the neural circuit level.

  3. 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...

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

    Science.gov (United States)

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

    2016-11-15

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

  5. INPP5E Preserves Genomic Stability through Regulation of Mitosis.

    Science.gov (United States)

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

    2017-03-15

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

  6. Kindlin1 regulates microtubule function to ensure normal mitosis.

    Science.gov (United States)

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

    2016-08-01

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

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

    Science.gov (United States)

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

    2016-09-02

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

  8. 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)

  9. 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...

  10. 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.

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

    Science.gov (United States)

    Yanagida, Mitsuhiro

    2014-09-02

    Mitosis is a cell-cycle stage during which condensed chromosomes migrate to the middle of the cell and segregate into two daughter nuclei before cytokinesis (cell division) with the aid of a dynamic mitotic spindle. The history of mitosis research is quite long, commencing well before the discovery of DNA as the repository of genetic information. However, great and rapid progress has been made since the introduction of recombinant DNA technology and discovery of universal cell-cycle control. A large number of conserved eukaryotic genes required for the progression from early to late mitotic stages have been discovered, confirming that DNA replication and mitosis are the two main events in the cell-division cycle. In this article, a historical overview of mitosis is given, emphasizing the importance of diverse model organisms that have been used to solve fundamental questions about mitosis. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

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

    Science.gov (United States)

    Jin, J; Liu, J

    2016-08-31

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

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

    Science.gov (United States)

    Leitao, Ricardo M; Kellogg, Douglas R

    2017-11-06

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Ma, T H

    1982-01-01

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

  15. 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.

  16. Analysis of a new morphogenetic mutation in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Mglinets, V.A.

    1987-01-01

    Somatic mosaicism for mutations monster and yellow was induced by gamma-irradiation of Drosophila melanogaster y/y; Dp(1; 2)sc 19 M(2)z/mn d embryos and larvae. Frequencies of mosaicism increased with the age of treated larvae, especially in the end of the 2nd larval instar. Autonomous expression of mn was observed throughout the whole range of larval age studied, though neither for all y/y spots nor for all parts of the spots. Dissimilarities in dynamics of mosaic spots and duplication induction suggest that the latter are not due to mn expression in somatic clones

  17. Ovarian stimulation and embryo quality

    NARCIS (Netherlands)

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

    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

  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. impact on embryo quality

    Directory of Open Access Journals (Sweden)

    Marijan Tandara

    2013-05-01

    Conclusions: In men with poorer semen quality, evaluated by standard semen parameters, a higher proportion of sperm with damaged DNA can also be expected. Higher sperm DNA damage, established by Halosperm test, also had an impact on embryo quality in this group of patients.

  20. Non-coding RNAs enter mitosis: functions, conservation and implications

    OpenAIRE

    Pek, Jun Wei; Kai, Toshie

    2011-01-01

    Abstract Nuage (or commonly known as chromatoid body in mammals) is a conserved germline-specific organelle that has been linked to the Piwi-interacting RNA (piRNA) pathway. piRNAs are a class of gonadal-specific RNAs that are ~23-29 nucleotides in length and protect genome stability by repressing the expression of deleterious retrotransposons. More recent studies in Drosophila have implicated the piRNA pathway in other functions including canalization of embryonic development, regulation of ...

  1. 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.

  2. 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...

  3. Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

    Science.gov (United States)

    Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

    2016-01-01

    In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

  4. Clathrin is spindle-associated but not essential for mitosis.

    Directory of Open Access Journals (Sweden)

    Joana Borlido

    Full Text Available Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis.Previously a chicken pre-B lymphoma cell line (DKO-R was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression.We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the

  5. Abnormal early cleavage events predict early embryo demise: sperm oxidative stress and early abnormal cleavage.

    Science.gov (United States)

    Burruel, Victoria; Klooster, Katie; Barker, Christopher M; Pera, Renee Reijo; Meyers, Stuart

    2014-10-13

    Human embryos resulting from abnormal early cleavage can result in aneuploidy and failure to develop normally to the blastocyst stage. The nature of paternal influence on early embryo development has not been directly demonstrated although many studies have suggested effects from spermatozoal chromatin packaging, DNA damage, centriolar and mitotic spindle integrity, and plasma membrane integrity. The goal of this study was to determine whether early developmental events were affected by oxidative damage to the fertilizing sperm. Survival analysis was used to compare patterns of blastocyst formation based on P2 duration. Kaplan-Meier survival curves demonstrate that relatively few embryos with short (P2 times reached blastocysts, and the two curves diverged beginning on day 4, with nearly all of the embryos with longer P2 times reaching blastocysts by day 6 (p < .01). We determined that duration of the 2nd to 3rd mitoses were sensitive periods in the presence of spermatozoal oxidative stress. Embryos that displayed either too long or too short cytokineses demonstrated an increased failure to reach blastocyst stage and therefore survive for further development. Although paternal-derived gene expression occurs later in development, this study suggests a specific role in early mitosis that is highly influenced by paternal factors.

  6. Effects of hypo-O-GlcNAcylation on Drosophila development.

    Science.gov (United States)

    Mariappa, Daniel; Ferenbach, Andrew T; van Aalten, Daan M F

    2018-05-11

    Post-translational modification of serine/threonine residues in nucleocytoplasmic proteins with GlcNAc ( O -GlcNAcylation) is an essential regulatory mechanism in many cellular processes. In Drosophila , null mutants of the Polycomb gene O -GlcNAc transferase ( OGT ; also known as super sex combs ( sxc )) display homeotic phenotypes. To dissect the requirement for O -GlcNAc signaling in Drosophila development, we used CRISPR/Cas9 gene editing to generate rationally designed sxc catalytically hypomorphic or null point mutants. Of the fertile males derived from embryos injected with the CRISPR/Cas9 reagents, 25% produced progeny carrying precise point mutations with no detectable off-target effects. One of these mutants, the catalytically inactive sxc K872M , was recessive lethal, whereas a second mutant, the hypomorphic sxc H537A , was homozygous viable. We observed that reduced total protein O -GlcNAcylation in the sxc H537A mutant is associated with a wing vein phenotype and temperature-dependent lethality. Genetic interaction between sxc H537A and a null allele of Drosophila host cell factor ( dHcf ), encoding an extensively O -GlcNAcylated transcriptional coactivator, resulted in abnormal scutellar bristle numbers. A similar phenotype was also observed in sxc H537A flies lacking a copy of skuld ( skd ), a Mediator complex gene known to affect scutellar bristle formation. Interestingly, this phenotype was independent of OGT Polycomb function or dHcf downstream targets. In conclusion, the generation of the endogenous OGT hypomorphic mutant sxc H537A enabled us to identify pleiotropic effects of globally reduced protein O -GlcNAc during Drosophila development. The mutants generated and phenotypes observed in this study provide a platform for discovery of OGT substrates that are critical for Drosophila development. © 2018 Mariappa et al.

  7. Modeling Human Cancers in Drosophila.

    Science.gov (United States)

    Sonoshita, M; Cagan, R L

    2017-01-01

    Cancer is a complex disease that affects multiple organs. Whole-body animal models provide important insights into oncology that can lead to clinical impact. Here, we review novel concepts that Drosophila studies have established for cancer biology, drug discovery, and patient therapy. Genetic studies using Drosophila have explored the roles of oncogenes and tumor-suppressor genes that when dysregulated promote cancer formation, making Drosophila a useful model to study multiple aspects of transformation. Not limited to mechanism analyses, Drosophila has recently been showing its value in facilitating drug development. Flies offer rapid, efficient platforms by which novel classes of drugs can be identified as candidate anticancer leads. Further, we discuss the use of Drosophila as a platform to develop therapies for individual patients by modeling the tumor's genetic complexity. Drosophila provides both a classical and a novel tool to identify new therapeutics, complementing other more traditional cancer tools. © 2017 Elsevier Inc. All rights reserved.

  8. Replication stress activates DNA repair synthesis in mitosis

    DEFF Research Database (Denmark)

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

    2015-01-01

    Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis. Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs). CFSs are difficult-to-replicate loci that manifest as gaps...... into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early...... mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest...

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

    Science.gov (United States)

    1984-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-08-13

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

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

    Science.gov (United States)

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

    2014-01-21

    Nutrients from living environment are vital for the survival and growth of any organism. Budding yeast diploid cells decide to grow by mitosis type cell division or decide to create unique, stress resistant spores by meiosis type cell division depending on the available nutrient conditions. To gain a molecular systems level understanding of the nutrient dependant switching between meiosis and mitosis initiation in diploid cells of budding yeast, we develop a theoretical model based on ordinary differential equations (ODEs) including the mitosis initiator and its relations to budding yeast meiosis initiation network. Our model accurately and qualitatively predicts the experimentally revealed temporal variations of related proteins under different nutrient conditions as well as the diverse mutant studies related to meiosis and mitosis initiation. Using this model, we show how the meiosis and mitosis initiators form an all-or-none type bistable switch in response to available nutrient level (mainly nitrogen). The transitions to and from meiosis or mitosis initiation states occur via saddle node bifurcation. This bidirectional switch helps the optimal usage of available nutrients and explains the mutually exclusive existence of meiosis and mitosis pathways. © 2013 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

    Research highlights: → hDlg is phosphorylated during mitosis in multiple residues. → Prospho-hDlg is excluded from the midbody during mitosis. → hDlg is not phosphorylated by p38γ or JNK1/2 during mitosis. → ERK5 pathway mediates hDlg phosphorylation in mitosis. -- Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

  13. 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

  14. 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

  15. Immunoelectron microscopy in embryos.

    Science.gov (United States)

    Sierralta, W D

    2001-05-01

    Immunogold labeling of proteins in sections of embryos embedded in acrylate media provides an important analytical tool when the resolving power of the electron microscope is required to define sites of protein function. The protocol presented here was established to analyze the role and dynamics of the activated protein kinase C/Rack1 regulatory system in the patterning and outgrowth of limb bud mesenchyme. With minor changes, especially in the composition of the fixative solution, the protocol should be easily adaptable for the postembedding immunogold labeling of any other antigen in tissues of embryos of diverse species. Quantification of the labeling can be achieved by using electron microscope systems capable of supporting digital image analysis. Copyright 2001 Academic Press.

  16. Tolerance in Drosophila

    OpenAIRE

    Atkinson, Nigel S.

    2009-01-01

    The set of genes that underlie ethanol tolerance (inducible resistance) are likely to overlap with the set of genes responsible for ethanol addiction. Whereas addiction is difficult to recognize in simple model systems, behavioral tolerance is readily identifiable and can be induced in large populations of animals. Thus, tolerance lends itself to analysis in model systems with powerful genetics. Drosophila melanogaster has been used by a variety of laboratories for the identification of genes...

  17. 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.

  18. Behavioral Teratogenesis in Drosophila melanogaster.

    Science.gov (United States)

    Mishra, Monalisa; Barik, Bedanta Kumar

    2018-01-01

    Developmental biology is a fascinating branch of science which helps us to understand the mechanism of development, thus the findings are used in various therapeutic approach. Drosophila melanogaster served as a model to find the key molecules that initiate and regulate the mechanism of development. Various genes, transcription factors, and signaling pathways helping in development are identified in Drosophila. Many toxic compounds, which can affect the development, are also recognized using Drosophila model. These compounds, which can affect the development, are named as a teratogen. Many teratogens identified using Drosophila may also act as a teratogen for a human being since 75% of conservation exist between the disease genes present in Drosophila and human. There are certain teratogens, which do not cause developmental defect if exposed during pregnancy, however; behavioral defect appears in later part of development. Such compounds are named as a behavioral teratogen. Thus, it is worthy to identify the potential behavioral teratogen using Drosophila model. Drosophila behavior is well studied in various developmental stages. This chapter describes various methods which can be employed to test behavioral teratogenesis in Drosophila.

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

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

    Science.gov (United States)

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

    2015-11-05

    Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    1996-01-01

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

  2. Mitosis counting in breast cancer : object-level interobserver agreement and comparison to an automatic method

    NARCIS (Netherlands)

    Veta, M.; van Diest, P.J.; Jiwa, M.; Al-Janabi, S.; Pluim, J.P.W.

    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

  3. 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

  4. 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

  5. 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.

  6. 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.

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

    OpenAIRE

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

    2005-01-01

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

  8. 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....

  9. Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein

    OpenAIRE

    Hong, Kyung Uk; Kim, Hyun-Jun; Bae, Chang-Dae; Park, Joobae

    2009-01-01

    Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one o...

  10. Mechanisms of gap gene expression canalization in the Drosophila blastoderm

    Directory of Open Access Journals (Sweden)

    Samsonova Maria G

    2011-07-01

    Full Text Available Abstract Background Extensive variation in early gap gene expression in the Drosophila blastoderm is reduced over time because of gap gene cross regulation. This phenomenon is a manifestation of canalization, the ability of an organism to produce a consistent phenotype despite variations in genotype or environment. The canalization of gap gene expression can be understood as arising from the actions of attractors in the gap gene dynamical system. Results In order to better understand the processes of developmental robustness and canalization in the early Drosophila embryo, we investigated the dynamical effects of varying spatial profiles of Bicoid protein concentration on the formation of the expression border of the gap gene hunchback. At several positions on the anterior-posterior axis of the embryo, we analyzed attractors and their basins of attraction in a dynamical model describing expression of four gap genes with the Bicoid concentration profile accounted as a given input in the model equations. This model was tested against a family of Bicoid gradients obtained from individual embryos. These gradients were normalized by two independent methods, which are based on distinct biological hypotheses and provide different magnitudes for Bicoid spatial variability. We showed how the border formation is dictated by the biological initial conditions (the concentration gradient of maternal Hunchback protein being attracted to specific attracting sets in a local vicinity of the border. Different types of these attracting sets (point attractors or one dimensional attracting manifolds define several possible mechanisms of border formation. The hunchback border formation is associated with intersection of the spatial gradient of the maternal Hunchback protein and a boundary between the attraction basins of two different point attractors. We demonstrated how the positional variability for hunchback is related to the corresponding variability of the

  11. 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.

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

    Science.gov (United States)

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

    2017-06-06

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

  13. 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.

  14. Compartmentalizing the embryo

    Science.gov (United States)

    Ile, Kristina E.; Renault, Andrew D.

    2013-01-01

    Lipid phosphate phosphatases (LPPs) are a class of enzymes that can dephosphorylate a number of lysophopholipids in vitro. Analysis of knockouts of LPP family members has demonstrated striking but diverse developmental roles for these enzymes. LPP3 is required for mouse vascular development while the Drosophila LPPs Wunen (Wun) and Wunen2 (Wun2) are required during embryogenesis for germ cell migration and survival. In a recent publication we examined if these fly LPPs have further developmental roles and found that Wun is required for proper tracheal formation. In particular we highlight a role for Wun in septate junction mediated barrier function in the tracheal system. In this paper we discuss further the possible mechanisms by which LPPs may influence barrier activity. PMID:23221483

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

    Science.gov (United States)

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

    2010-07-01

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

  16. Unconventional actin conformations localize on intermediate filaments in mitosis

    International Nuclear Information System (INIS)

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan

    2011-01-01

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

  17. Self-organization of intracellular gradients during mitosis

    Directory of Open Access Journals (Sweden)

    Fuller Brian G

    2010-01-01

    Full Text Available Abstract Gradients are used in a number of biological systems to transmit spatial information over a range of distances. The best studied are morphogen gradients where information is transmitted over many cell lengths. Smaller mitotic gradients reflect the need to organize several distinct events along the length of the mitotic spindle. The intracellular gradients that characterize mitosis are emerging as important regulatory paradigms. Intracellular gradients utilize intrinsic auto-regulatory feedback loops and diffusion to establish stable regions of activity within the mitotic cytosol. We review three recently described intracellular mitotic gradients. The Ran GTP gradient with its elaborate cascade of nuclear transport receptors and cargoes is the best characterized, yet the dynamics underlying the robust gradient of Ran-GTP have received little attention. Gradients of phosphorylation have been observed on Aurora B kinase substrates both before and after anaphase onset. In both instances the phosphorylation gradient appears to result from a soluble gradient of Aurora B kinase activity. Regulatory properties that support gradient formation are highlighted. Intracellular activity gradients that regulate localized mitotic events bare several hallmarks of self-organizing biologic systems that designate spatial information during pattern formation. Intracellular pattern formation represents a new paradigm in mitotic regulation.

  18. 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.

  19. Embryos, genes, and birth defects

    National Research Council Canada - National Science Library

    Ferretti, Patrizia

    2006-01-01

    ... Structural anomalies The genesis of chromosome abnormalities Embryo survival The cause of high levels of chromosome abnormality in human embryos Relative parental risks - age, translocations, inversions, gonadal and germinal mosaics 33 33 34 35 36 44 44 45 4 Identification and Analysis of Genes Involved in Congenital Malformation Syndromes Peter J. Scambler Ge...

  20. Laboratory techniques for human embryos.

    Science.gov (United States)

    Geber, Selmo; Sales, Liana; Sampaio, Marcos A C

    2002-01-01

    This review is concerned with laboratory techniques needed for assisted conception, particularly the handling of gametes and embryos. Such methods are being increasingly refined. Successive stages of fertilization and embryogenesis require especial care, and often involve the use of micromanipulative methods for intracytoplasmic sperm injection (ICSI) or preimplantation genetic diagnosis. Embryologists must take responsibility for gamete collection and preparation, and for deciding on the means of insemination or ICSI. Embryos must be assessed in culture, during the 1-cell, cleaving and morula/blastocyst stages, and classified according to quality. Co-culture methods may be necessary. The best embryos for transfer must be selected and loaded into the transfer catheter. Embryos not transferred must be cryopreserved, which demands the correct application of current methods of media preparation, seeding and the correct speed for cooling and warming. Before too long, methods of detecting abnormal embryos and avoiding their transfer may become widespread.

  1. Cell-stage-specific enhancement by caffeine of the frequency of chromatid aberrations induced by X-rays in neutral ganglia of Drosophila melanogaster

    International Nuclear Information System (INIS)

    De Marco, A.; Polani, S.

    1981-01-01

    Caffeine (10 -2 M) induced a high level of chromatid aberrations in neural ganglia of third-instar larvae of Drosophila melanogaster only when it was added to cells in late G 2 and mitotic prophase. No aberrations were observed after treatment in late S-middle G 2 or C-mitosis. We observed that, in these stages, caffeine strongly increased X-ray-induced damage (500 R). This potentiation was quantitatively similar. But it involved all types of aberration after treatment in C-mitosis, and essentially isochromatid deletions and chromatid exhanges after treatment in S-G 2 . Some hypotheses are put forth to explain the possible mechanism of action of caffeine in the potentiation of X-ray-induced damage. (orig.)

  2. 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

  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. Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein.

    Science.gov (United States)

    Hong, Kyung Uk; Kim, Hyun-Jun; Bae, Chang-Dae; Park, Joobae

    2009-11-30

    Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one of the mitosis-specific phosphorylation residues (i.e., Thr-622). However, the phosphorylation events at the remaining mitotic phosphorylation sites of TMAP have not been fully characterized in detail. Here, we report on generation and characterization of phosphorylated Thr-578- and phosphorylated Thr-596-specific antibodies. Using the antibodies, we show that phosphorylation of TMAP at Thr-578 and Thr-596 indeed occurs specifically during mitosis. Immunofluorescent staining using the antibodies shows that these residues become phosphorylated starting at prophase and then become rapidly dephosphorylated soon after initiation of anaphase. Subtle differences in the kinetics of phosphorylation between Thr-578 and Thr-596 imply that they may be under different mechanisms of phosphorylation during mitosis. Unlike the phosphorylation-deficient mutant form for Thr-622, the mutant in which both Thr-578 and Thr-596 had been mutated to alanines did not induce significant delay in progression of mitosis. These results show that the majority of mitosis-specific phosphorylation of TMAP is limited to pre-anaphase stages and suggest that the multiple phosphorylation may not act in concert but serve diverse functions.

  5. 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.

  6. 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.

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

    Science.gov (United States)

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

    2017-08-14

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

  8. Effects of Spaceflight on Drosophila Neural Development

    Science.gov (United States)

    Keshishian, Haig S.

    1997-01-01

    The major goal from the animal side, however, has been achieved, namely to develop Drosophila lines where we can assay individual neuromuscular endings directly without dissection. This was achieved by means of using the GAL4-UAS system, where we have succeeded in establishing stocks of flies where the key neuromuscular connections can be assayed directly in undissected larvae by means of the expression of endogenously fluorescent reporters in the specific motor endings. The green fluorescent protein (GFP) as a reporter allows scoring of neural anatomy en-masse in whole mount using fluorescent microscopy without the need for either dissection or specific labeling. Two stocks have been developed. The first, which we developed first, uses the S65T mutant form, which has a dramatically brighter expression than the native protein. This animal will use GAL4 drivers with expression under the control of the elav gene, and which will ensure expression in all neurons of the embryo and larva. The second transgenic animal we have developed is of a novel kind, and makes use of dicistronic design, so that two copies of the protein will be expressed per insert. We have also developed a tricistronic form, but this has not yet been transformed into flies, and we do not imagine that this third line will be ready in time for the flight.

  9. 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.

  10. Evolutionary genetics: the Drosophila model

    Indian Academy of Sciences (India)

    Unknown

    Evolutionary genetics straddles the two fundamental processes of life, ... of the genus Drosophila have been used extensively as model systems in experimental ... issue will prove interesting, informative and thought-provoking for both estab-.

  11. 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.

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

    Science.gov (United States)

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

    2016-01-06

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

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

    International Nuclear Information System (INIS)

    Kaneko, Yayoi; Tamura, Kaori; Totsukawa, Go; Kondo, Hisao

    2010-01-01

    Research highlights: → p37 is phosphorylated on Serine-56 and Threonine-59 by Cdc2 at mitosis. → Phosphorylated p37 does not bind to Golgi membranes. → 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.

  14. 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.

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

    Science.gov (United States)

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

    2017-02-15

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

  16. 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

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

    Science.gov (United States)

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

    2017-09-15

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

  18. 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.

  19. Surgical manipulation of mammalian embryos in vitro.

    Science.gov (United States)

    Naruse, I; Keino, H; Taniguchi, M

    1997-04-01

    Whole-embryo culture systems are useful in the fields of not only embryology but also teratology, toxicology, pharmacology, and physiology. Of the many advantages of whole-embryo culture, we focus here on the surgical manipulation of mammalian embryos. Whole-embryo culture allows us to manipulate mammalian embryos, similarly to fish, amphibian and avian embryos. Many surgical experiments have been performed in mammalian embryos in vitro. Such surgical manipulation alters the destiny of morphogenesis of the embryos and can answer many questions concerning developmental issues. As an example of surgical manipulation using whole-embryo culture systems, one of our experiments is described. Microsurgical electrocauterization of the deep preaxial mesodermal programmed cell death zone (fpp) in the footplate prevented the manifestation of polydactyly in genetic polydactyly mouse embryos (Pdn/Pdn), in which fpp was abolished.

  20. 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...

  1. 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

  2. Humidity Sensing in Drosophila.

    Science.gov (United States)

    Enjin, Anders; Zaharieva, Emanuela E; Frank, Dominic D; Mansourian, Suzan; Suh, Greg S B; Gallio, Marco; Stensmyr, Marcus C

    2016-05-23

    Environmental humidity influences the fitness and geographic distribution of all animals [1]. Insects in particular use humidity cues to navigate the environment, and previous work suggests the existence of specific sensory mechanisms to detect favorable humidity ranges [2-5]. Yet, the molecular and cellular basis of humidity sensing (hygrosensation) remains poorly understood. Here we describe genes and neurons necessary for hygrosensation in the vinegar fly Drosophila melanogaster. We find that members of the Drosophila genus display species-specific humidity preferences related to conditions in their native habitats. Using a simple behavioral assay, we find that the ionotropic receptors IR40a, IR93a, and IR25a are all required for humidity preference in D. melanogaster. Yet, whereas IR40a is selectively required for hygrosensory responses, IR93a and IR25a mediate both humidity and temperature preference. Consistent with this, the expression of IR93a and IR25a includes thermosensory neurons of the arista. In contrast, IR40a is excluded from the arista but is expressed (and required) in specialized neurons innervating pore-less sensilla of the sacculus, a unique invagination of the third antennal segment. Indeed, calcium imaging showed that IR40a neurons directly respond to changes in humidity, and IR40a knockdown or IR93a mutation reduced their responses to stimuli. Taken together, our results suggest that the preference for a specific humidity range depends on specialized sacculus neurons, and that the processing of environmental humidity can happen largely in parallel to that of temperature. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. [The destiny of cryopreserved embryos].

    Science.gov (United States)

    Karpel, L; Achour-Frydman, N; Frydman, R; Flis-Trèves, M

    2007-12-01

    To know the psychological motivations of couples who keep their embryos so long (five years and more) and do not make a decision about them. We studied 84 couples refrained from making a decision on their cryopreserved embryos for at least five years. They were invited to fill out a questionnaire focusing on three points: the reasons of the indecision, their own representation of the cryopreserved embryos and their choice for the future: donation to another couple, to research, pregnancy or no solution for the moment. Mean (S.D.) women's and men's age were respectively, 38.8 (2.5)- and 41.3 (2.5)-years old. On average, three (1-9) embryos are preserved since 7.5 (5-12) years. Most of couples are parents. Four major reasons explain their attitudes: feeling of being too aged (25%), fear of a multiple pregnancy (45%), disagreement between members of couple (20%) and fear of failure (42.5%). Multiple choices were given to the future of the embryos: 25% wanted a pregnancy, 8% wanted to give them to infertile couples, 20% to research and 27.5% did not find any solution. Twenty percent were hesitating. The representation of those embryos is more symbolic than material. Most of the time, they see them like a potential child, a hope for the future or a brother or sister of their alive children. Those embryos are symbolized. They are a proof of fertility, a hope for another child. So, whatever the legal statement, couples will be in a dilemma because it is never easy for an infertile person to renounce to embryos, and the hope for children.

  4. 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...

  5. 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...

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

    Directory of Open Access Journals (Sweden)

    Tatyana Eugenivna KOPYTCHUK

    2012-05-01

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

  7. 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.

  8. Hermann Muller and Mutations in Drosophila

    Science.gov (United States)

    dropdown arrow Site Map A-Z Index Menu Synopsis Hermann Muller and Mutations in Drosophila Resources with University of Texas. In Austin his experiments on fruit flies (Drosophila) first showed that exposure to September to spend a year at the only Drosophila laboratory in Europe which was doing parallel work

  9. Use of Drosophila to study DNA repair

    International Nuclear Information System (INIS)

    Boyd, J.B.; Harris, P.V.; Sakaguchi, K.

    1988-01-01

    This paper discusses Drosophila, the premier metazoan organism for analyzing many fundamental features of eukaryotic gene regulation. The authors present adaptations of several approaches for studying DNA repair to an analysis of repair-defective mutants in Drosophila. A current understanding of Drosophila DNA repair is described

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

    NARCIS (Netherlands)

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

    1998-01-01

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

  11. Teratogenic effects of retinoic acid on neurulation in mice embryos.

    Science.gov (United States)

    Nobakht, M; Zirak, A; Mehdizadeh, M; Tabatabaeei, P

    2006-02-21

    Retinoic acids (RA) are natural chemicals that exert a hormone-like activity and a variety of biological effects on early development of mouse. In this study, the probable teratogenic effects of RA on CNS have been investigated in pregnant mice (n = 20) divided into four groups: (1) untreated controls, (2) controls which received a single dose of DMSO, (3) a group that received 40 mg/kg, and (4) a group that received 60 mg/kg of all-trans RA in DMSO, respectively on the eighth day of gestation. Embryos whose dams had received 40 and 60 mg/kg doses of RA, showed malformations and decreased size. At 40 mg/kg dosage level, 50% of the embryos had closed neural tubes while at 60 mg/kg dosage level the neural tube failed to close. The neuroblast mantle layers were disorganized in the 40 mg/kg and even more in the 60 mg/kg exposed group compared to the controls. In mitosis, the density of chromatin was increased in the 60 mg/kg dose group. Compared to controls the 40 and 60 mg/kg dose groups of RA treated dams decreases in the luminal longitudinal and internal measures were observed. Also the thickness of ventricular, mantle and marginal layers was smaller. Wide intercellular spaces due to the degenerated cells at high doses of RA as well as an accumulation of intercellular fluid were observed. Therefore, the wedge shape of neuroepithelium was abolished, preventing the elevation of the neural wall.

  12. 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…

  13. 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...... transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27(Kip1) cyclin-dependent kinase inhibitor. Consequently...

  14. 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.

  15. Mitosis, double strand break repair, and telomeres: a view from the end: how telomeres and the DNA damage response cooperate during mitosis to maintain genome stability.

    Science.gov (United States)

    Cesare, Anthony J

    2014-11-01

    Double strand break (DSB) repair is suppressed during mitosis because RNF8 and downstream DNA damage response (DDR) factors, including 53BP1, do not localize to mitotic chromatin. Discovery of the mitotic kinase-dependent mechanism that inhibits DSB repair during cell division was recently reported. It was shown that restoring mitotic DSB repair was detrimental, resulting in repair dependent genome instability and covalent telomere fusions. The telomere DDR that occurs naturally during cellular aging and in cancer is known to be refractory to G2/M checkpoint activation. Such DDR-positive telomeres, and those that occur as part of the telomere-dependent prolonged mitotic arrest checkpoint, normally pass through mitosis without covalent ligation, but result in cell growth arrest in G1 phase. The discovery that suppressing DSB repair during mitosis may function primarily to protect DDR-positive telomeres from fusing during cell division reinforces the unique cooperation between telomeres and the DDR to mediate tumor suppression. © 2014 The Author. Bioessays published by WILEY Periodicals, Inc.

  16. Disparate patterns of thermal adaptation between life stages in temperate vs. tropical Drosophila melanogaster.

    Science.gov (United States)

    Lockwood, B L; Gupta, T; Scavotto, R

    2018-02-01

    Many terrestrial ectothermic species exhibit limited variation in upper thermal tolerance across latitude. However, these trends may not signify limited adaptive capacity to increase thermal tolerance in the face of climate change. Instead, thermal tolerance may be similar among populations because behavioural thermoregulation by mobile organisms or life stages may buffer natural selection for thermal tolerance. We compared thermal tolerance of adults and embryos among natural populations of Drosophila melanogaster from a broad range of thermal habitats around the globe to assess natural variation of thermal tolerance in mobile vs. immobile life stages. We found no variation among populations in adult thermal tolerance, but embryonic thermal tolerance was higher in tropical strains than in temperate strains. We further report that embryos live closer to their upper thermal limits than adults - that is, thermal safety margins are smaller for embryos than adults. F1 hybrid embryos from crosses between temperate and tropical populations had thermal tolerance that matched that of tropical embryos, suggesting the dominance of heat-tolerant alleles. Together, our findings suggest that thermal selection has led to divergence in embryonic thermal tolerance but that selection for divergent thermal tolerance may be limited in adults. Further, our results suggest that thermal traits should be measured across life stages to better predict adaptive limits. © 2017 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2017 European Society For Evolutionary Biology.

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

    Science.gov (United States)

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

    2015-03-01

    Mitosis and epidermal growth factor (EGF) receptor (EGFR) are both targets for cancer therapy. The role of EGFR signaling in mitosis has been rarely studied and poorly understood. The limited studies indicate that the activation of EGFR and downstream signaling pathways is mostly inhibited during mitosis. However, we recently showed that EGFR is phosphorylated in response to EGF stimulation in mitosis. Here we studied EGF-induced EGFR activation and the activation of major signaling pathways downstream of EGFR during mitosis. We showed that EGFR was strongly activated by EGF during mitosis as all the five major tyrosine residues including Y992, Y1045, Y1068, Y1086, and Y1173 were phosphorylated to a level similar to that in the interphase. We further showed that the activated EGFR is able to selectively activate some downstream signaling pathways while avoiding others. Activated EGFR is able to activate PI3K and AKT2, but not AKT1, which may be responsible for the observed effects of EGF against nocodazole-induced cell death. Activated EGFR is also able to activate c-Src, c-Cbl and PLC-γ1 during mitosis. However, activated EGFR is unable to activate ERK1/2 and their downstream substrates RSK and Elk-1. While it activated Ras, EGFR failed to fully activate Raf-1 in mitosis due to the lack of phosphorylation at Y341 and the lack of dephosphorylation at pS259. We conclude that contrary to the dogma, EGFR is activated by EGF during mitosis. Moreover, EGFR-mediated cell signaling is regulated differently from the interphase to specifically serve the needs of the cell in mitosis. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. A damped oscillator imposes temporal order on posterior gap gene expression in Drosophila

    Science.gov (United States)

    Verd, Berta; Clark, Erik; Wotton, Karl R.; Janssens, Hilde; Jiménez-Guri, Eva; Crombach, Anton

    2018-01-01

    Insects determine their body segments in two different ways. Short-germband insects, such as the flour beetle Tribolium castaneum, use a molecular clock to establish segments sequentially. In contrast, long-germband insects, such as the vinegar fly Drosophila melanogaster, determine all segments simultaneously through a hierarchical cascade of gene regulation. Gap genes constitute the first layer of the Drosophila segmentation gene hierarchy, downstream of maternal gradients such as that of Caudal (Cad). We use data-driven mathematical modelling and phase space analysis to show that shifting gap domains in the posterior half of the Drosophila embryo are an emergent property of a robust damped oscillator mechanism, suggesting that the regulatory dynamics underlying long- and short-germband segmentation are much more similar than previously thought. In Tribolium, Cad has been proposed to modulate the frequency of the segmentation oscillator. Surprisingly, our simulations and experiments show that the shift rate of posterior gap domains is independent of maternal Cad levels in Drosophila. Our results suggest a novel evolutionary scenario for the short- to long-germband transition and help explain why this transition occurred convergently multiple times during the radiation of the holometabolan insects. PMID:29451884

  19. 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.

  20. The Centrioles, Centrosomes, Basal Bodies, and Cilia of Drosophila melanogaster.

    Science.gov (United States)

    Lattao, Ramona; Kovács, Levente; Glover, David M

    2017-05-01

    Centrioles play a key role in the development of the fly. They are needed for the correct formation of centrosomes, the organelles at the poles of the spindle that can persist as microtubule organizing centers (MTOCs) into interphase. The ability to nucleate cytoplasmic microtubules (MTs) is a property of the surrounding pericentriolar material (PCM). The centriole has a dual life, existing not only as the core of the centrosome but also as the basal body, the structure that templates the formation of cilia and flagellae. Thus the structure and functions of the centriole, the centrosome, and the basal body have an impact upon many aspects of development and physiology that can readily be modeled in Drosophila Centrosomes are essential to give organization to the rapidly increasing numbers of nuclei in the syncytial embryo and for the spatially precise execution of cell division in numerous tissues, particularly during male meiosis. Although mitotic cell cycles can take place in the absence of centrosomes, this is an error-prone process that opens up the fly to developmental defects and the potential of tumor formation. Here, we review the structure and functions of the centriole, the centrosome, and the basal body in different tissues and cultured cells of Drosophila melanogaster , highlighting their contributions to different aspects of development and cell division. Copyright © 2017 Lattao et al.

  1. The Origin of the Second Centriole in the Zygote of Drosophila melanogaster

    Science.gov (United States)

    Blachon, Stephanie; Khire, Atul; Avidor-Reiss, Tomer

    2014-01-01

    Centrosomes are composed of two centrioles surrounded by pericentriolar material (PCM). However, the sperm and the oocyte modify or lose their centrosomes. Consequently, how the zygote establishes its first centrosome, and in particular, the origin of the second zygotic centriole, is uncertain. Drosophila melanogaster spermatids contain a single centriole called the Giant Centriole (GC) and a Proximal centriole-like (PCL) structure whose function is unknown. We found that, like the centriole, the PCL loses its protein markers at the end of spermiogenesis. After fertilization, the first two centrioles are observed via the recruitment of the zygotic PCM proteins and are seen in asterless mutant embryos that cannot form centrioles. The zygote’s centriolar proteins label only the daughter centrioles of the first two centrioles. These observations demonstrate that the PCL is the origin for the second centriole in the Drosophila zygote and that a paternal centriole precursor, without centriolar proteins, is transmitted to the egg during fertilization. PMID:24532732

  2. Functional conservation of the Drosophila gooseberry gene and its evolutionary alleles.

    Directory of Open Access Journals (Sweden)

    Wei Liu

    Full Text Available The Drosophila Pax gene gooseberry (gsb is required for development of the larval cuticle and CNS, survival to adulthood, and male fertility. These functions can be rescued in gsb mutants by two gsb evolutionary alleles, gsb-Prd and gsb-Pax3, which express the Drosophila Paired and mouse Pax3 proteins under the control of gooseberry cis-regulatory region. Therefore, both Paired and Pax3 proteins have conserved all the Gsb functions that are required for survival of embryos to fertile adults, despite the divergent primary sequences in their C-terminal halves. As gsb-Prd and gsb-Pax3 uncover a gsb function involved in male fertility, construction of evolutionary alleles may provide a powerful strategy to dissect hitherto unknown gene functions. Our results provide further evidence for the essential role of cis-regulatory regions in the functional diversification of duplicated genes during evolution.

  3. Characterization of a Smad motif similar to Drosophila mad in the mouse Msx 1 promoter.

    Science.gov (United States)

    Alvarez Martinez, Cristina E; Binato, Renata; Gonzalez, Sayonara; Pereira, Monica; Robert, Benoit; Abdelhay, Eliana

    2002-03-01

    Mouse Msx 1 gene, orthologous of the Drosophila msh, is involved in several developmental processes. BMP family members are major proteins in the regulation of Msx 1 expression. BMP signaling activates Smad 1/5/8 proteins, which associate to Smad 4 before translocating to the nucleus. Analysis of Msx 1 promoter revealed the presence of three elements similar to the consensus established for Mad, the Smad 1 Drosophila counterpart. Notably, such an element was identified in an enhancer important for Msx 1 regulation. Gel shift analysis demonstrated that proteins from 13.5 dpc embryo associate to this enhancer. Remarkably, supershift assays showed that Smad proteins are present in the complex. Purified Smad 1 and 4 also bind to this fragment. We demonstrate that functional binding sites in this enhancer are confined to the Mad motif and flanking region. Our data suggest that this Mad motif may be functional in response to BMP signaling. ©2002 Elsevier Science (USA).

  4. Efficient gene knock-out and knock-in with transgenic Cas9 in Drosophila.

    Science.gov (United States)

    Xue, Zhaoyu; Ren, Mengda; Wu, Menghua; Dai, Junbiao; Rong, Yikang S; Gao, Guanjun

    2014-03-21

    Bacterial Cas9 nuclease induces site-specific DNA breaks using small gRNA as guides. Cas9 has been successfully introduced into Drosophila for genome editing. Here, we improve the versatility of this method by developing a transgenic system that expresses Cas9 in the Drosophila germline. Using this system, we induced inheritable knock-out mutations by injecting only the gRNA into embryos, achieved highly efficient mutagenesis by expressing gRNA from the promoter of a novel non-coding RNA gene, and recovered homologous recombination-based knock-in of a fluorescent marker at a rate of 4.5% by co-injecting gRNA with a circular DNA donor. Copyright © 2014 Xue et al.

  5. Phylogeny of the Genus Drosophila

    Science.gov (United States)

    O’Grady, Patrick M.; DeSalle, Rob

    2018-01-01

    Understanding phylogenetic relationships among taxa is key to designing and implementing comparative analyses. The genus Drosophila, which contains over 1600 species, is one of the most important model systems in the biological sciences. For over a century, one species in this group, Drosophila melanogaster, has been key to studies of animal development and genetics, genome organization and evolution, and human disease. As whole-genome sequencing becomes more cost-effective, there is increasing interest in other members of this morphologically, ecologically, and behaviorally diverse genus. Phylogenetic relationships within Drosophila are complicated, and the goal of this paper is to provide a review of the recent taxonomic changes and phylogenetic relationships in this genus to aid in further comparative studies. PMID:29716983

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1977-12-01

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

  7. 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. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  8. A mitosis-specific and R loop-driven ATR pathway promotes faithful chromosome segregation.

    Science.gov (United States)

    Kabeche, Lilian; Nguyen, Hai Dang; Buisson, Rémi; Zou, Lee

    2018-01-05

    The ataxia telangiectasia mutated and Rad3-related (ATR) kinase is crucial for DNA damage and replication stress responses. Here, we describe an unexpected role of ATR in mitosis. Acute inhibition or degradation of ATR in mitosis induces whole-chromosome missegregation. The effect of ATR ablation is not due to altered cyclin-dependent kinase 1 (CDK1) activity, DNA damage responses, or unscheduled DNA synthesis but to loss of an ATR function at centromeres. In mitosis, ATR localizes to centromeres through Aurora A-regulated association with centromere protein F (CENP-F), allowing ATR to engage replication protein A (RPA)-coated centromeric R loops. As ATR is activated at centromeres, it stimulates Aurora B through Chk1, preventing formation of lagging chromosomes. Thus, a mitosis-specific and R loop-driven ATR pathway acts at centromeres to promote faithful chromosome segregation, revealing functions of R loops and ATR in suppressing chromosome instability. Copyright © 2018, American Association for the Advancement of Science.

  9. 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…

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

    Science.gov (United States)

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

    2016-01-01

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

  11. 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

  12. Novel functions for the endocytic regulatory proteins MICAL-L1 and EHD1 in mitosis.

    Science.gov (United States)

    Reinecke, James B; Katafiasz, Dawn; Naslavsky, Naava; Caplan, Steve

    2015-01-01

    During interphase, recycling endosomes mediate the transport of internalized cargo back to the plasma membrane. However, in mitotic cells, recycling endosomes are essential for the completion of cytokinesis, the last phase of mitosis that promotes the physical separation the two daughter cells. Despite recent advances, our understanding of the molecular determinants that regulate recycling endosome dynamics during cytokinesis remains incomplete. We have previously demonstrated that Molecule Interacting with CasL Like-1 (MICAL-L1) and C-terminal Eps15 Homology Domain protein 1 (EHD1) coordinately regulate receptor transport from tubular recycling endosomes during interphase. However, their potential roles in controlling cytokinesis had not been addressed. In this study, we show that MICAL-L1 and EHD1 regulate mitosis. Depletion of either protein resulted in increased numbers of bi-nucleated cells. We provide evidence that bi-nucleation in MICAL-L1- and EHD1-depleted cells is a consequence of impaired recycling endosome transport during late cytokinesis. However, depletion of MICAL-L1, but not EHD1, resulted in aberrant chromosome alignment and lagging chromosomes, suggesting an EHD1-independent function for MICAL-L1 earlier in mitosis. Moreover, we provide evidence that MICAL-L1 and EHD1 differentially influence microtubule dynamics during early and late mitosis. Collectively, our new data suggest several unanticipated roles for MICAL-L1 and EHD1 during the cell cycle. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2017-06-01

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

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

    International Nuclear Information System (INIS)

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

    1977-01-01

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

  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, J.; 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. 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. © 2016. Published by The Company of Biologists Ltd.

  18. 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…

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

    Science.gov (United States)

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

    2013-01-07

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

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

    NARCIS (Netherlands)

    Veta, M.; Diest, van P.J.; Willems, S.M.; Wang, Haibo; Madabhushi, A. (Anant); Cruz-Roa, A. (Angel); González, F.; Larsen, A.B.L. (Anders); Vestergaard, J.S. (Jacob); Dahl, A.B. (Anders); Ciresan, D.C. (Dan); Schmidhuber, J.; Giusti, A. (Alessandro); Gambardella, L.M. (Luca); Tek, F. Boray; Walter, Th. (Thomas); Wang, Ching-Wei; Kondo, Satoshi; Matuszewski, B.J. (Bogdan); Precioso, F. (Frederic); Snell, V. (Violet); Kittler, Josef; de Campos, Teofilo E.; Khan, Adnan M.; Rajpoot, Nasir M.; Arkoumani, Evdokia; Lacle, Miangela M.; Viergever, M.A.; Pluim, J.P.W.

    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

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

    DEFF Research Database (Denmark)

    Veta, Mitko; van Diest, Paul J.; Willems, Stefan M.

    2014-01-01

    The proliferative activity of breast tumors, which is routinely estimated by counting of mitotic figures in hematoxylin and eosin stained histology sections, is considered to be one of the most important prognostic markers. However, mitosis counting is laborious, subjective and may suffer from lo...

  2. 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...

  3. 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.

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

    Science.gov (United States)

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

    2018-06-01

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

  5. Feminists on the inalienability of human embryos.

    Science.gov (United States)

    McLeod, Carolyn; Baylis, Francoise

    2006-01-01

    The feminist literature against the commodification of embryos in human embryo research includes an argument to the effect that embryos are "intimately connected" to persons, or morally inalienable from them. We explore why embryos might be inalienable to persons and why feminists might find this view appealing. But, ultimately, as feminists, we reject this view because it is inconsistent with full respect for women's reproductive autonomy and with a feminist conception of persons as relational, embodied beings. Overall, feminists should avoid claims about embryos' being inalienable to persons in arguments for or against the commodification of human embryos.

  6. 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

  7. Localization and activation of the Drosophila protease easter require the ER-resident saposin-like protein seele.

    Science.gov (United States)

    Stein, David; Charatsi, Iphigenie; Cho, Yong Suk; Zhang, Zhenyu; Nguyen, Jesse; DeLotto, Robert; Luschnig, Stefan; Moussian, Bernard

    2010-11-09

    Drosophila embryonic dorsal-ventral polarity is generated by a series of serine protease processing events in the egg perivitelline space. Gastrulation Defective processes Snake, which then cleaves Easter, which then processes Spätzle into the activating ligand for the Toll receptor. seele was identified in a screen for mutations that, when homozygous in ovarian germline clones, lead to the formation of progeny embryos with altered embryonic patterning; maternal loss of seele function leads to the production of moderately dorsalized embryos. By combining constitutively active versions of Gastrulation Defective, Snake, Easter, and Spätzle with loss-of-function alleles of seele, we find that Seele activity is dispensable for Spätzle-mediated activation of Toll but is required for Easter, Snake, and Gastrulation Defective to exert their effects on dorsal-ventral patterning. Moreover, Seele function is required specifically for secretion of Easter from the developing embryo into the perivitelline space and for Easter processing. Seele protein resides in the endoplasmic reticulum of blastoderm embryos, suggesting a role in the trafficking of Easter to the perivitelline space, prerequisite to its processing and function. Easter transport to the perivitelline space represents a previously unappreciated control point in the signal transduction pathway that controls Drosophila embryonic dorsal-ventral polarity. Copyright © 2010 Elsevier Ltd. All rights reserved.

  8. Modelling Cooperative Tumorigenesis in Drosophila

    Science.gov (United States)

    2018-01-01

    The development of human metastatic cancer is a multistep process, involving the acquisition of several genetic mutations, tumour heterogeneity, and interactions with the surrounding microenvironment. Due to the complexity of cancer development in mammals, simpler model organisms, such as the vinegar fly, Drosophila melanogaster, are being utilized to provide novel insights into the molecular mechanisms involved. In this review, we highlight recent advances in modelling tumorigenesis using the Drosophila model, focusing on the cooperation of oncogenes or tumour suppressors, and the interaction of mutant cells with the surrounding tissue in epithelial tumour initiation and progression. PMID:29693007

  9. Modelling Cooperative Tumorigenesis in Drosophila

    Directory of Open Access Journals (Sweden)

    Helena E. Richardson

    2018-01-01

    Full Text Available The development of human metastatic cancer is a multistep process, involving the acquisition of several genetic mutations, tumour heterogeneity, and interactions with the surrounding microenvironment. Due to the complexity of cancer development in mammals, simpler model organisms, such as the vinegar fly, Drosophila melanogaster, are being utilized to provide novel insights into the molecular mechanisms involved. In this review, we highlight recent advances in modelling tumorigenesis using the Drosophila model, focusing on the cooperation of oncogenes or tumour suppressors, and the interaction of mutant cells with the surrounding tissue in epithelial tumour initiation and progression.

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

    Science.gov (United States)

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

    2012-01-01

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

  11. 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 (G 1 , S, and G 2 ), 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.

  12. 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

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

    Science.gov (United States)

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

    2015-08-01

    Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. Single-embryo transfer versus multiple-embryo transfer.

    Science.gov (United States)

    Gerris, Jan

    2009-01-01

    Despite the progress made in assisted reproductive technology, live birth rates remain disappointingly low. Multiple-embryo transfer has been an accepted practice with which to increase the success rate. This has led to a higher incidence of multiple-order births compared with natural conception, which not only increase the risk of mortality and morbidity to both mother and children but are also associated with social and economic consequences. Elective single-embryo transfer (eSET) was developed in an effort to increase singleton pregnancies in assisted reproduction. Studies comparing eSET with multiple-embryo transfer highlight the benefit of this approach and suggest that, with careful patient selection and the transfer of good-quality embryos, the risk of a multiple-order pregnancy can be reduced without significantly decreasing live birth rates. Although the use of eSET has gradually increased in clinical practice, its acceptance has been limited by factors such as availability of funding and awareness of the procedure. An open discussion of eSET is warranted in an effort to enable a broader understanding by physicians and patients of the merits of this approach. Ultimately, eSET may provide a more cost-effective, potentially safer approach to patients undergoing assisted reproduction technology.

  15. 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.

  16. 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.

  17. A regulatory transcriptional loop controls proliferation and differentiation in Drosophila neural stem cells.

    Directory of Open Access Journals (Sweden)

    Tetsuo Yasugi

    Full Text Available Neurogenesis is initiated by a set of basic Helix-Loop-Helix (bHLH transcription factors that specify neural progenitors and allow them to generate neurons in multiple rounds of asymmetric cell division. The Drosophila Daughterless (Da protein and its mammalian counterparts (E12/E47 act as heterodimerization factors for proneural genes and are therefore critically required for neurogenesis. Here, we demonstrate that Da can also be an inhibitor of the neural progenitor fate whose absence leads to stem cell overproliferation and tumor formation. We explain this paradox by demonstrating that Da induces the differentiation factor Prospero (Pros whose asymmetric segregation is essential for differentiation in one of the two daughter cells. Da co-operates with the bHLH transcription factor Asense, whereas the other proneural genes are dispensible. After mitosis, Pros terminates Asense expression in one of the two daughter cells. In da mutants, pros is not expressed, leading to the formation of lethal transplantable brain tumors. Our results define a transcriptional feedback loop that regulates the balance between self-renewal and differentiation in Drosophila optic lobe neuroblasts. They indicate that initiation of a neural differentiation program in stem cells is essential to prevent tumorigenesis.

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

    DEFF Research Database (Denmark)

    Pedersen, Ronni Sølvhøi

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

  19. 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.

  20. Mechanistic dissection of plant embryo initiation

    NARCIS (Netherlands)

    Radoeva, T.M.

    2016-01-01

    Land plants can reproduce sexually by developing an embryo from a fertilized egg cell, the zygote. After fertilization, the zygote undergoes several rounds of controlled cell divisions to generate a mature embryo. However, embryo formation can also be induced in a variety of other cell types in

  1. Untwisting the Caenorhabditis elegans embryo

    Science.gov (United States)

    Christensen, Ryan Patrick; Bokinsky, Alexandra; Santella, Anthony; Wu, Yicong; Marquina-Solis, Javier; Guo, Min; Kovacevic, Ismar; Kumar, Abhishek; Winter, Peter W; Tashakkori, Nicole; McCreedy, Evan; Liu, Huafeng; McAuliffe, Matthew; Mohler, William; Colón-Ramos, Daniel A; Bao, Zhirong; Shroff, Hari

    2015-01-01

    The nematode Caenorhabditis elegans possesses a simple embryonic nervous system with few enough neurons that the growth of each cell could be followed to provide a systems-level view of development. However, studies of single cell development have largely been conducted in fixed or pre-twitching live embryos, because of technical difficulties associated with embryo movement in late embryogenesis. We present open-source untwisting and annotation software (http://mipav.cit.nih.gov/plugin_jws/mipav_worm_plugin.php) that allows the investigation of neurodevelopmental events in late embryogenesis and apply it to track the 3D positions of seam cell nuclei, neurons, and neurites in multiple elongating embryos. We also provide a tutorial describing how to use the software (Supplementary file 1) and a detailed description of the untwisting algorithm (Appendix). The detailed positional information we obtained enabled us to develop a composite model showing movement of these cells and neurites in an 'average' worm embryo. The untwisting and cell tracking capabilities of our method provide a foundation on which to catalog C. elegans neurodevelopment, allowing interrogation of developmental events in previously inaccessible periods of embryogenesis. DOI: http://dx.doi.org/10.7554/eLife.10070.001 PMID:26633880

  2. Untwisting the Caenorhabditis elegans embryo.

    Science.gov (United States)

    Christensen, Ryan Patrick; Bokinsky, Alexandra; Santella, Anthony; Wu, Yicong; Marquina-Solis, Javier; Guo, Min; Kovacevic, Ismar; Kumar, Abhishek; Winter, Peter W; Tashakkori, Nicole; McCreedy, Evan; Liu, Huafeng; McAuliffe, Matthew; Mohler, William; Colón-Ramos, Daniel A; Bao, Zhirong; Shroff, Hari

    2015-12-03

    The nematode Caenorhabditis elegans possesses a simple embryonic nervous system with few enough neurons that the growth of each cell could be followed to provide a systems-level view of development. However, studies of single cell development have largely been conducted in fixed or pre-twitching live embryos, because of technical difficulties associated with embryo movement in late embryogenesis. We present open-source untwisting and annotation software (http://mipav.cit.nih.gov/plugin_jws/mipav_worm_plugin.php) that allows the investigation of neurodevelopmental events in late embryogenesis and apply it to track the 3D positions of seam cell nuclei, neurons, and neurites in multiple elongating embryos. We also provide a tutorial describing how to use the software (Supplementary file 1) and a detailed description of the untwisting algorithm (Appendix). The detailed positional information we obtained enabled us to develop a composite model showing movement of these cells and neurites in an 'average' worm embryo. The untwisting and cell tracking capabilities of our method provide a foundation on which to catalog C. elegans neurodevelopment, allowing interrogation of developmental events in previously inaccessible periods of embryogenesis.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1975-10-01

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

  4. Functional evolution of cis-regulatory modules at a homeotic gene in Drosophila.

    Directory of Open Access Journals (Sweden)

    Margaret C W Ho

    2009-11-01

    Full Text Available It is a long-held belief in evolutionary biology that the rate of molecular evolution for a given DNA sequence is inversely related to the level of functional constraint. This belief holds true for the protein-coding homeotic (Hox genes originally discovered in Drosophila melanogaster. Expression of the Hox genes in Drosophila embryos is essential for body patterning and is controlled by an extensive array of cis-regulatory modules (CRMs. How the regulatory modules functionally evolve in different species is not clear. A comparison of the CRMs for the Abdominal-B gene from different Drosophila species reveals relatively low levels of overall sequence conservation. However, embryonic enhancer CRMs from other Drosophila species direct transgenic reporter gene expression in the same spatial and temporal patterns during development as their D. melanogaster orthologs. Bioinformatic analysis reveals the presence of short conserved sequences within defined CRMs, representing gap and pair-rule transcription factor binding sites. One predicted binding site for the gap transcription factor KRUPPEL in the IAB5 CRM was found to be altered in Superabdominal (Sab mutations. In Sab mutant flies, the third abdominal segment is transformed into a copy of the fifth abdominal segment. A model for KRUPPEL-mediated repression at this binding site is presented. These findings challenge our current understanding of the relationship between sequence evolution at the molecular level and functional activity of a CRM. While the overall sequence conservation at Drosophila CRMs is not distinctive from neighboring genomic regions, functionally critical transcription factor binding sites within embryonic enhancer CRMs are highly conserved. These results have implications for understanding mechanisms of gene expression during embryonic development, enhancer function, and the molecular evolution of eukaryotic regulatory modules.

  5. Embryo density and medium volume effects on early murine embryo development.

    Science.gov (United States)

    Canseco, R S; Sparks, A E; Pearson, R E; Gwazdauskas, F C

    1992-10-01

    One-cell mouse embryos were used to determine the effects of drop size and number of embryos per drop for optimum development in vitro. Embryos were collected from immature C57BL6 female mice superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin and mated by CD1 males. Groups of 1, 5, 10, or 20 embryos were cultured in 5-, 10-, 20-, or 40-microliters drops of CZB under silicon oil at 37.5 degrees C in a humidified atmosphere of 5% CO2 and 95% air. Development score for embryos cultured in 10 microliters was higher than that of embryos cultured in 20 or 40 microliters. Embryos cultured in groups of 5, 10, or 20 had higher development scores than embryos cultured singly. The highest development score was obtained by the combination of 5 embryos per 10-microliters drop. The percentage of live embryos in 20 or 40 microliters was lower than that of embryos cultured in 10 microliters. Additionally, the percentage of live embryos cultured singly was lower than that of embryos cultured in groups. Our results suggest that a stimulatory interaction occurs among embryos possibly exerted through the secretion of growth factors. This effect can be diluted if the embryos are cultured in large drops or singly.

  6. [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.

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

    Science.gov (United States)

    Yasuhara, Hiroki; Oe, Yuki

    2011-07-01

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

  8. 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)

  9. Blocking Internalization of Phosphatidylethanolamine at Cleavage Furrow of Mitosis as a Novel Mechanism of Anti-Breast Cancer Strategy

    National Research Council Canada - National Science Library

    Cui, Zhen

    2002-01-01

    During the formation of cleavage furrow of mitosis, phosphatidylethanolamine (PE) flips from inner leaflet of the plasma membrane to the outer leaflet specifically in the furrow region near the contractile ring...

  10. Blocking Internalization of Phosphatidylethanolamine at Cleavage Furrow of Mitosis as a Novel Mechanism of Anti-Breast-Cancer Strategy

    National Research Council Canada - National Science Library

    Cui, Zheng

    2003-01-01

    During the formation of cleavage furrow of mitosis, phosphatidylethanolamine (PE) flips from inner leaflet of the plasma membrane to the outer leaflet specifically in the furrow region near the contractile ring...

  11. Semi-automated quantitative Drosophila wings measurements.

    Science.gov (United States)

    Loh, Sheng Yang Michael; Ogawa, Yoshitaka; Kawana, Sara; Tamura, Koichiro; Lee, Hwee Kuan

    2017-06-28

    Drosophila melanogaster is an important organism used in many fields of biological research such as genetics and developmental biology. Drosophila wings have been widely used to study the genetics of development, morphometrics and evolution. Therefore there is much interest in quantifying wing structures of Drosophila. Advancement in technology has increased the ease in which images of Drosophila can be acquired. However such studies have been limited by the slow and tedious process of acquiring phenotypic data. We have developed a system that automatically detects and measures key points and vein segments on a Drosophila wing. Key points are detected by performing image transformations and template matching on Drosophila wing images while vein segments are detected using an Active Contour algorithm. The accuracy of our key point detection was compared against key point annotations of users. We also performed key point detection using different training data sets of Drosophila wing images. We compared our software with an existing automated image analysis system for Drosophila wings and showed that our system performs better than the state of the art. Vein segments were manually measured and compared against the measurements obtained from our system. Our system was able to detect specific key points and vein segments from Drosophila wing images with high accuracy.

  12. 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

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

    Directory of Open Access Journals (Sweden)

    B. S. Kirbik

    2015-01-01

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

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

    International Nuclear Information System (INIS)

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

    1976-01-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    OpenAIRE

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

    2011-01-01

    The efficient and timely resolution of DNA recombination intermediates is essential for bipolar chromosome segregation. Here, we show that the specialized chromosome segregation patterns of meiosis and mitosis, which require the coordination of recombination with cell-cycle progression, are achieved by regulating the timing of activation of two crossover-promoting endonucleases. In yeast meiosis, Mus81-Mms4 and Yen1 are controlled by phosphorylation events that lead to their sequential activa...

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

    Czech Academy of Sciences Publication Activity Database

    Bruinsma, W.; Macůrek, Libor; Freire, R.; Lindqvist, A.; Medema, R.H.

    2014-01-01

    Roč. 127, č. 4 (2014), s. 801-811 ISSN 0021-9533 R&D Projects: GA ČR GA13-18392S Grant - others:Ministerio de Economía y Competitividad(ES) SAF2010-22357; CONSOLIDER-Ingenio(NL) CDS2007-0015 Keywords : Aurora-A * Bora * Mitosis * Plk1 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.432, year: 2014

  18. 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.

  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.

  1. 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).

  2. 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. © 2014. Published by The Company of Biologists Ltd.

  3. Drosophila homologues of adenomatous polyposis coli (APC) and the formin diaphanous collaborate by a conserved mechanism to stimulate actin filament assembly.

    Science.gov (United States)

    Jaiswal, Richa; Stepanik, Vince; Rankova, Aneliya; Molinar, Olivia; Goode, Bruce L; McCartney, Brooke M

    2013-05-10

    Vertebrate APC collaborates with Dia through its Basic domain to assemble actin filaments. Despite limited sequence homology between the vertebrate and Drosophila APC Basic domains, Drosophila APC1 collaborates with Dia to stimulate actin assembly in vitro. The mechanism of actin assembly is highly conserved over evolution. APC-Dia collaborations may be crucial in a wide range of animal cells. Adenomatous polyposis coli (APC) is a large multidomain protein that regulates the cytoskeleton. Recently, it was shown that vertebrate APC through its Basic domain directly collaborates with the formin mDia1 to stimulate actin filament assembly in the presence of nucleation barriers. However, it has been unclear whether these activities extend to homologues of APC and Dia in other organisms. Drosophila APC and Dia are each required to promote actin furrow formation in the syncytial embryo, suggesting a potential collaboration in actin assembly, but low sequence homology between the Basic domains of Drosophila and vertebrate APC has left their functional and mechanistic parallels uncertain. To address this question, we purified Drosophila APC1 and Dia and determined their individual and combined effects on actin assembly using both bulk fluorescence assays and total internal reflection fluorescence microscopy. Our data show that APC1, similar to its vertebrate homologue, bound to actin monomers and nucleated and bundled filaments. Further, Drosophila Dia nucleated actin assembly and protected growing filament barbed ends from capping protein. Drosophila APC1 and Dia directly interacted and collaborated to promote actin assembly in the combined presence of profilin and capping protein. Thus, despite limited sequence homology, Drosophila and vertebrate APCs exhibit highly related activities and mechanisms and directly collaborate with formins. These results suggest that APC-Dia interactions in actin assembly are conserved and may underlie important in vivo functions in a broad

  4. Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver

    International Nuclear Information System (INIS)

    Reaiche-Miller, Georget Y.; Thorpe, Michael; Low, Huey Chi; Qiao, Qiao; Scougall, Catherine A.; Mason, William S.; Litwin, Samuel; Jilbert, Allison R.

    2013-01-01

    Nucleos(t)ide analogues that inhibit hepatitis B virus (HBV) DNA replication are typically used as monotherapy for chronically infected patients. Treatment with a nucleos(t)ide analogue eliminates most HBV DNA replication intermediates and produces a gradual decline in levels of covalently closed circular DNA (cccDNA), the template for viral RNA synthesis. It remains uncertain if levels of cccDNA decline primarily through hepatocyte death, or if loss also occurs during hepatocyte mitosis. To determine if cccDNA survives mitosis, growing ducklings infected with duck hepatitis B virus (DHBV) were treated with the nucleoside analogue, Entecavir. Viremia was suppressed at least 10 5 -fold, during a period when average liver mass increased 23-fold. Analysis of the data suggested that if cccDNA synthesis was completely inhibited, at least 49% of cccDNA survived hepatocyte mitosis. However, there was a large duck-to-duck variation in cccDNA levels, suggesting that low level cccDNA synthesis may contribute to this apparent survival through mitosis. - Highlights: • The hepatitis B virus nuclear template is covalently closed circular DNA (cccDNA). • cccDNA was studied during liver growth in duck hepatitis B virus infected ducks. • Virus DNA replication and new cccDNA synthesis were inhibited with Entecavir. • At least 49% of cccDNA appeared to survive hepatocyte mitosis. • Low level virus DNA synthesis may contribute to survival of cccDNA through mitosis

  5. The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation.

    Science.gov (United States)

    Chiu, Shao-Chih; Chen, Jo-Mei Maureen; Wei, Tong-You Wade; Cheng, Tai-Shan; Wang, Ya-Hui Candice; Ku, Chia-Feng; Lian, Chiao-Hsuan; Liu, Chun-Chih Jared; Kuo, Yi-Chun; Yu, Chang-Tze Ricky

    2014-09-01

    Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser(115). The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis. Copyright © 2014 the American Physiological Society.

  6. Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver

    Energy Technology Data Exchange (ETDEWEB)

    Reaiche-Miller, Georget Y.; Thorpe, Michael; Low, Huey Chi; Qiao, Qiao; Scougall, Catherine A. [School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005 (Australia); Mason, William S.; Litwin, Samuel [Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111 (United States); Jilbert, Allison R., E-mail: allison.jilbert@adelaide.edu.au [School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005 (Australia)

    2013-11-15

    Nucleos(t)ide analogues that inhibit hepatitis B virus (HBV) DNA replication are typically used as monotherapy for chronically infected patients. Treatment with a nucleos(t)ide analogue eliminates most HBV DNA replication intermediates and produces a gradual decline in levels of covalently closed circular DNA (cccDNA), the template for viral RNA synthesis. It remains uncertain if levels of cccDNA decline primarily through hepatocyte death, or if loss also occurs during hepatocyte mitosis. To determine if cccDNA survives mitosis, growing ducklings infected with duck hepatitis B virus (DHBV) were treated with the nucleoside analogue, Entecavir. Viremia was suppressed at least 10{sup 5}-fold, during a period when average liver mass increased 23-fold. Analysis of the data suggested that if cccDNA synthesis was completely inhibited, at least 49% of cccDNA survived hepatocyte mitosis. However, there was a large duck-to-duck variation in cccDNA levels, suggesting that low level cccDNA synthesis may contribute to this apparent survival through mitosis. - Highlights: • The hepatitis B virus nuclear template is covalently closed circular DNA (cccDNA). • cccDNA was studied during liver growth in duck hepatitis B virus infected ducks. • Virus DNA replication and new cccDNA synthesis were inhibited with Entecavir. • At least 49% of cccDNA appeared to survive hepatocyte mitosis. • Low level virus DNA synthesis may contribute to survival of cccDNA through mitosis.

  7. Noninvasive embryo assessment technique based on buoyancy and its association with embryo survival after cryopreservation.

    Science.gov (United States)

    Wessels, Cara; Penrose, Lindsay; Ahmad, Khaliq; Prien, Samuel

    2017-11-01

    Embryo cryopreservation offers many benefits by allowing genetic preservation, genetic screening, cost reduction, global embryo transport and single embryo transfer. However, freezing of embryos decreases embryo viability, as intracellular ice crystal formation often damages embryos. Success rates of frozen embryo transfer are expected to be 15-20% less than fresh embryo transfer. We have developed a noninvasive embryo assessment technique (NEAT) which enables us to predict embryo viability based on buoyancy. The purpose of this research was twofold. First was to determine if a NEAT, through a specific gravity device can detect embryo survival of cryopreservation. Second, it was to relate embryo buoyancy to embryo viability for establishing pregnancies in sheep. Blastocysts descent times were measured on one-hundred sixty-nine mice blastocysts before cryopreservation, according to standard protocol and post-thawing blastocysts descent times were measured again. There was a significant difference in blastocyst post-thaw descent times with NEAT in those blastocysts which demonstrated viability from those that did not (P embryos. Further studies on a larger scale commercial setting will evaluate the efficacy of NEAT. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. PLK1 Activation in Late G2 Sets Up Commitment to Mitosis.

    Science.gov (United States)

    Gheghiani, Lilia; Loew, Damarys; Lombard, Bérangère; Mansfeld, Jörg; Gavet, Olivier

    2017-06-06

    Commitment to mitosis must be tightly coordinated with DNA replication to preserve genome integrity. While we have previously established that the timely activation of CyclinB1-Cdk1 in late G2 triggers mitotic entry, the upstream regulatory mechanisms remain unclear. Here, we report that Polo-like kinase 1 (Plk1) is required for entry into mitosis during an unperturbed cell cycle and is rapidly activated shortly before CyclinB1-Cdk1. We determine that Plk1 associates with the Cdc25C1 phosphatase and induces its phosphorylation before mitotic entry. Plk1-dependent Cdc25C1 phosphosites are sufficient to promote mitotic entry, even when Plk1 activity is inhibited. Furthermore, we find that activation of Plk1 during G2 relies on CyclinA2-Cdk activity levels. Our findings thus elucidate a critical role for Plk1 in CyclinB1-Cdk1 activation and mitotic entry and outline how CyclinA2-Cdk, an S-promoting factor, poises cells for commitment to mitosis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  9. INDEKS MITOSIS UJUNG AKAR KECAMBAH CABE BESAR (Capsicum annuum L. SETELAH PERLAKUAN SUSPENSI Trichoderma sp.

    Directory of Open Access Journals (Sweden)

    PetroneLa Deno Raja

    2016-06-01

    Full Text Available Penelitian ini bertujuan untuk mengetahui indeks mitosis ujung akar kecambah cabe besar (Capsicum annuum L. setelah perlakuan suspensi Trichoderma sp. Penelitian ini dilakukan di laboratorium Struktur Perkembangan Tumbuhan Jurusan Biologi FMIPA, Universitas Udayana dari Oktober 2013-November 2013. Metode yang digunakan adalah metode squash, biji cabe untuk kontrol direndam dalam air ± 6 jam, untuk perlakuan biji setelah direndam air, direndam lagi dalam suspensi Trichoderma sp. 10-7 selama ± 6 jam, selanjutnya dikecambahkan. Ujung akar kecambah 2 mm dipotong, difiksasi dalam larutan farmer ± 2-24 jam, dihidrolisis dalam larutan 3N HCL ± 2-5 menit dan kemudian pewarnaan dengan aceto orcein ± 5 menit. Pengamatan dilakukan dengan mikroskop binokuler, data pembelahan tiap fase mitosis dihitung (%, dicatat dan difoto, dan dianalisis dengan menggunakan uji paired T tes.Hasil penelitian menunjukkan bahwa Trichoderma sp. berpengaruh terhadap indeks mitosis sel ujung akar Capsicum annuum L.,  pada fase metafase berbeda nyata antara kontrol dan perlakuan, sedangkan pada fase profase, anafase dan telofase berbeda tidak nyata.  Pada perlakuan persentase fase profase, metafase, anafase dan telofase (77,14%; 12,96 %; 5,88 % dan 5,23 % lebih tinggi dari kontrol (66,40 %; 5,44 %; 4,96 % dan 4,66 %.

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

    Science.gov (United States)

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

    2016-05-31

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

  11. Polo-like kinase 1 inhibits DNA damage response during mitosis.

    Science.gov (United States)

    Benada, Jan; Burdová, Kamila; Lidak, Tomáš; von Morgen, Patrick; Macurek, Libor

    2015-01-01

    In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.

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

    Science.gov (United States)

    Govindaraghavan, Meera; Lad, Alisha A.

    2014-01-01

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

  13. Proteomic analysis of polyribosomes identifies splicing factors as potential regulators of translation during mitosis.

    Science.gov (United States)

    Aviner, Ranen; Hofmann, Sarah; Elman, Tamar; Shenoy, Anjana; Geiger, Tamar; Elkon, Ran; Ehrlich, Marcelo; Elroy-Stein, Orna

    2017-06-02

    Precise regulation of mRNA translation is critical for proper cell division, but little is known about the factors that mediate it. To identify mRNA-binding proteins that regulate translation during mitosis, we analyzed the composition of polysomes from interphase and mitotic cells using unbiased quantitative mass-spectrometry (LC-MS/MS). We found that mitotic polysomes are enriched with a subset of proteins involved in RNA processing, including alternative splicing and RNA export. To demonstrate that these may indeed be regulators of translation, we focused on heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a test case and confirmed that it is recruited to elongating ribosomes during mitosis. Then, using a combination of pulsed SILAC, metabolic labeling and ribosome profiling, we showed that knockdown of hnRNP C affects both global and transcript-specific translation rates and found that hnRNP C is specifically important for translation of mRNAs that encode ribosomal proteins and translation factors. Taken together, our results demonstrate how proteomic analysis of polysomes can provide insight into translation regulation under various cellular conditions of interest and suggest that hnRNP C facilitates production of translation machinery components during mitosis to provide daughter cells with the ability to efficiently synthesize proteins as they enter G1 phase. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    Science.gov (United States)

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

    2015-04-01

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

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

    International Nuclear Information System (INIS)

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

    1991-01-01

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

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

    Science.gov (United States)

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

    2009-01-01

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

  17. Manipulation of gene expression by infrared laser heat shock and its application to the study of tracheal development in Drosophila.

    Science.gov (United States)

    Miao, Guangxia; Hayashi, Shigeo

    2015-03-01

    Induction of gene expression in a specific cell and a defined time window is desirable to investigate gene function at the cellular level during morphogenesis. To achieve this, we attempted to introduce the infrared laser-evoked gene operator system (IR-LEGO, Kamei et al., 2009) in the Drosophila embryo. In this technique, infrared laser light illumination induces genes to be expressed under the control of heat shock promoters at the single cell level. We applied IR-LEGO to a transgenic fly stock, HS-eGFP, in which the enhanced green fluorescent protein (eGFP) gene is placed under the control of heat shock protein 70 promoter, and showed that eGFP expression can be induced in single cells within 1-2 hr after IR illumination. Furthermore, induction of HS-Branchless transgene encoding the Drosophila fibroblast growth factor (FGF) effectively altered the migration and branching patterns of the tracheal system. Our results indicated that IR-LEGO is a promising choice for the timely control of gene expression in a small group of cells in the Drosophila embryo. By using IR-LEGO, we further demonstrated that the tracheal terminal branching program is sensitive to localized expression of exogenous FGF. © 2014 Wiley Periodicals, Inc.

  18. A Drosophila wing spot test

    International Nuclear Information System (INIS)

    Ayaki, Toshikazu; Yoshikawa, Isao; Niikawa, Norio; Hoshi, Masaharu.

    1986-01-01

    A Drosophila wing spot test system was used to investigate the effects of low doses of X-rays, gamma rays, and both 2.3 and 14.1 MeV neutrons on somatic chromosome mutation (SCM) induction. The incidence of SCM was significantly increased with any type of radiation, with evident linear dose-response relationship within the range of 3 to 20 cGy. It was estimated that relative biological effectiveness value for SCM induction of 2.3 MeV neutrons to X-rays and gamma rays is much higher than that of 14.1 MeV neutrons to those photons (2.4 vs 8.0). The Drosophila wing spot test system seems to become a promising in vivo experimental method for higher animals in terms of the lack of necessity for a marvelously large number of materials required in conventional test system. (Namekawa, K.)

  19. Limited taste discrimination in Drosophila.

    Science.gov (United States)

    Masek, Pavel; Scott, Kristin

    2010-08-17

    In the gustatory systems of mammals and flies, different populations of sensory cells recognize different taste modalities, such that there are cells that respond selectively to sugars and others to bitter compounds. This organization readily allows animals to distinguish compounds of different modalities but may limit the ability to distinguish compounds within one taste modality. Here, we developed a behavioral paradigm in Drosophila melanogaster to evaluate directly the tastes that a fly distinguishes. These studies reveal that flies do not discriminate among different sugars, or among different bitter compounds, based on chemical identity. Instead, flies show a limited ability to distinguish compounds within a modality based on intensity or palatability. Taste associative learning, similar to olfactory learning, requires the mushroom bodies, suggesting fundamental similarities in brain mechanisms underlying behavioral plasticity. Overall, these studies provide insight into the discriminative capacity of the Drosophila gustatory system and the modulation of taste behavior.

  20. A software tool to model genetic regulatory networks. Applications to the modeling of threshold phenomena and of spatial patterning in Drosophila.

    Directory of Open Access Journals (Sweden)

    Rui Dilão

    Full Text Available We present a general methodology in order to build mathematical models of genetic regulatory networks. This approach is based on the mass action law and on the Jacob and Monod operon model. The mathematical models are built symbolically by the Mathematica software package GeneticNetworks. This package accepts as input the interaction graphs of the transcriptional activators and repressors of a biological process and, as output, gives the mathematical model in the form of a system of ordinary differential equations. All the relevant biological parameters are chosen automatically by the software. Within this framework, we show that concentration dependent threshold effects in biology emerge from the catalytic properties of genes and its associated conservation laws. We apply this methodology to the segment patterning in Drosophila early development and we calibrate the genetic transcriptional network responsible for the patterning of the gap gene proteins Hunchback and Knirps, along the antero-posterior axis of the Drosophila embryo. In this approach, the zygotically produced proteins Hunchback and Knirps do not diffuse along the antero-posterior axis of the embryo of Drosophila, developing a spatial pattern due to concentration dependent thresholds. This shows that patterning at the gap genes stage can be explained by the concentration gradients along the embryo of the transcriptional regulators.

  1. Studies on Drosophila radiosensitive strains

    International Nuclear Information System (INIS)

    Varentsova, E.P.; Zakharov, I.A.

    1976-01-01

    45 of radiosensitive strains of Drosophila melanogaster were isolated by Curly/Lobe technique after EMS treatment of Livadia population males. The lethality of non-Curly late larvae after gamma-irradiation (4000r) characterized radiosensitivity strains. Most of them exhibited higher frequency of the spontaneous dominant lethals (up to 69%). The males of 6 strains were semi-sterile. 5 of these strains exhibited higher frequency of X-chromosome non-disjunction

  2. Olfactory memory traces in Drosophila

    OpenAIRE

    Berry, Jacob; Krause, William C.; Davis, Ronald L.

    2008-01-01

    In Drosophila the fruit fly, coincident exposure to an odor and an aversive electric shock can produce robust behavioral memory. This behavioral memory is thought to be regulated by cellular memory traces within the central nervous system of the fly. These molecular, physiological or structural changes in neurons, induced by pairing odor and shock, regulate behavior by altering the neurons’ response to the learned environment. Recently, novel in vivo functional imaging techniques have allowed...

  3. Human embryo culture media comparisons.

    Science.gov (United States)

    Pool, Thomas B; Schoolfield, John; Han, David

    2012-01-01

    Every program of assisted reproduction strives to maximize pregnancy outcomes from in vitro fertilization and selecting an embryo culture medium, or medium pair, consistent with high success rates is key to this process. The common approach is to replace an existing medium with a new one of interest in the overall culture system and then perform enough cycles of IVF to see if a difference is noted both in laboratory measures of embryo quality and in pregnancy. This approach may allow a laboratory to select one medium over another but the outcomes are only relevant to that program, given that there are well over 200 other variables that may influence the results in an IVF cycle. A study design that will allow for a more global application of IVF results, ones due to culture medium composition as the single variable, is suggested. To perform a study of this design, the center must have a patient caseload appropriate to meet study entrance criteria, success rates high enough to reveal a difference if one exists and a strong program of quality assurance and control in both the laboratory and clinic. Sibling oocytes are randomized to two study arms and embryos are evaluated on day 3 for quality grades. Inter and intra-observer variability are evaluated by kappa statistics and statistical power and study size estimates are performed to bring discriminatory capability to the study. Finally, the complications associated with extending such a study to include blastocyst production on day 5 or 6 are enumerated.

  4. Drosophila: Retrotransposons Making up Telomeres.

    Science.gov (United States)

    Casacuberta, Elena

    2017-07-19

    Drosophila and extant species are the best-studied telomerase exception. In this organism, telomere elongation is coupled with targeted retrotransposition of Healing Transposon (HeT-A) and Telomere Associated Retrotransposon (TART) with sporadic additions of Telomere Associated and HeT-A Related (TAHRE), all three specialized non-Long Terminal Repeat (non-LTR) retrotransposons. These three very special retroelements transpose in head to tail arrays, always in the same orientation at the end of the chromosomes but never in interior locations. Apparently, retrotransposon and telomerase telomeres might seem very different, but a detailed view of their mechanisms reveals similarities explaining how the loss of telomerase in a Drosophila ancestor could successfully have been replaced by the telomere retrotransposons. In this review, we will discover that although HeT-A, TART, and TAHRE are still the only examples to date where their targeted transposition is perfectly tamed into the telomere biology of Drosophila, there are other examples of retrotransposons that manage to successfully integrate inside and at the end of telomeres. Because the aim of this special issue is viral integration at telomeres, understanding the base of the telomerase exceptions will help to obtain clues on similar strategies that mobile elements and viruses could have acquired in order to ensure their survival in the host genome.

  5. Optogenetic pacing in Drosophila melanogaster

    Science.gov (United States)

    Alex, Aneesh; Li, Airong; Tanzi, Rudolph E.; Zhou, Chao

    2015-01-01

    Electrical stimulation is currently the gold standard for cardiac pacing. However, it is invasive and nonspecific for cardiac tissues. We recently developed a noninvasive cardiac pacing technique using optogenetic tools, which are widely used in neuroscience. Optogenetic pacing of the heart provides high spatial and temporal precisions, is specific for cardiac tissues, avoids artifacts associated with electrical stimulation, and therefore promises to be a powerful tool in basic cardiac research. We demonstrated optogenetic control of heart rhythm in a well-established model organism, Drosophila melanogaster. We developed transgenic flies expressing a light-gated cation channel, channelrhodopsin-2 (ChR2), specifically in their hearts and demonstrated successful optogenetic pacing of ChR2-expressing Drosophila at different developmental stages, including the larva, pupa, and adult stages. A high-speed and ultrahigh-resolution optical coherence microscopy imaging system that is capable of providing images at a rate of 130 frames/s with axial and transverse resolutions of 1.5 and 3.9 μm, respectively, was used to noninvasively monitor Drosophila cardiac function and its response to pacing stimulation. The development of a noninvasive integrated optical pacing and imaging system provides a novel platform for performing research studies in developmental cardiology. PMID:26601299

  6. 'Peer pressure' in larval Drosophila?

    Science.gov (United States)

    Niewalda, Thomas; Jeske, Ines; Michels, Birgit; Gerber, Bertram

    2014-06-06

    Understanding social behaviour requires a study case that is simple enough to be tractable, yet complex enough to remain interesting. Do larval Drosophila meet these requirements? In a broad sense, this question can refer to effects of the mere presence of other larvae on the behaviour of a target individual. Here we focused in a more strict sense on 'peer pressure', that is on the question of whether the behaviour of a target individual larva is affected by what a surrounding group of larvae is doing. We found that innate olfactory preference of a target individual was neither affected (i) by the level of innate olfactory preference in the surrounding group nor (ii) by the expression of learned olfactory preference in the group. Likewise, learned olfactory preference of a target individual was neither affected (iii) by the level of innate olfactory preference of the surrounding group nor (iv) by the learned olfactory preference the group was expressing. We conclude that larval Drosophila thus do not take note of specifically what surrounding larvae are doing. This implies that in a strict sense, and to the extent tested, there is no social interaction between larvae. These results validate widely used en mass approaches to the behaviour of larval Drosophila. © 2014. Published by The Company of Biologists Ltd.

  7. Quantification of Drosophila Grooming Behavior.

    Science.gov (United States)

    Barradale, Francesca; Sinha, Kairav; Lebestky, Tim

    2017-07-19

    Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

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

    Science.gov (United States)

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

    2018-04-24

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

  9. Down-regulation of tricarboxylic acid (TCA) cycle genes blocks progression through the first mitotic division in Caenorhabditis elegans embryos.

    Science.gov (United States)

    Rahman, Mohammad M; Rosu, Simona; Joseph-Strauss, Daphna; Cohen-Fix, Orna

    2014-02-18

    The cell cycle is a highly regulated process that enables the accurate transmission of chromosomes to daughter cells. Here we uncover a previously unknown link between the tricarboxylic acid (TCA) cycle and cell cycle progression in the Caenorhabditis elegans early embryo. We found that down-regulation of TCA cycle components, including citrate synthase, malate dehydrogenase, and aconitase, resulted in a one-cell stage arrest before entry into mitosis: pronuclear meeting occurred normally, but nuclear envelope breakdown, centrosome separation, and chromosome condensation did not take place. Mitotic entry is controlled by the cyclin B-cyclin-dependent kinase 1 (Cdk1) complex, and the inhibitory phosphorylation of Cdk1 must be removed in order for the complex to be active. We found that following down-regulation of the TCA cycle, cyclin B levels were normal but CDK-1 remained inhibitory-phosphorylated in one-cell stage-arrested embryos, indicative of a G2-like arrest. Moreover, this was not due to an indirect effect caused by checkpoint activation by DNA damage or replication defects. These observations suggest that CDK-1 activation in the C. elegans one-cell embryo is sensitive to the metabolic state of the cell, and that down-regulation of the TCA cycle prevents the removal of CDK-1 inhibitory phosphorylation. The TCA cycle was previously shown to be necessary for the development of the early embryo in mammals, but the molecular processes affected were not known. Our study demonstrates a link between the TCA cycle and a specific cell cycle transition in the one-cell stage embryo.

  10. Germline progenitors escape the widespread phenomenon of homolog pairing during Drosophila development.

    Directory of Open Access Journals (Sweden)

    Eric F Joyce

    Full Text Available Homolog pairing, which plays a critical role in meiosis, poses a potential risk if it occurs in inappropriate tissues or between nonallelic sites, as it can lead to changes in gene expression, chromosome entanglements, and loss-of-heterozygosity due to mitotic recombination. This is particularly true in Drosophila, which supports organismal-wide pairing throughout development. Discovered over a century ago, such extensive pairing has led to the perception that germline pairing in the adult gonad is an extension of the pairing established during embryogenesis and, therefore, differs from the mechanism utilized in most species to initiate pairing specifically in the germline. Here, we show that, contrary to long-standing assumptions, Drosophila meiotic pairing in the gonad is not an extension of pairing established during embryogenesis. Instead, we find that homologous chromosomes are unpaired in primordial germ cells from the moment the germline can be distinguished from the soma in the embryo and remain unpaired even in the germline stem cells of the adult gonad. We further establish that pairing originates immediately after the stem cell stage. This pairing occurs well before the initiation of meiosis and, strikingly, continues through the several mitotic divisions preceding meiosis. These discoveries indicate that the spatial organization of the Drosophila genome differs between the germline and the soma from the earliest moments of development and thus argue that homolog pairing in the germline is an active process as versus a passive continuation of pairing established during embryogenesis.

  11. The Drosophila melanogaster methuselah gene: a novel gene with ancient functions.

    Directory of Open Access Journals (Sweden)

    Ana Rita Araújo

    Full Text Available The Drosophila melanogaster G protein-coupled receptor gene, methuselah (mth, has been described as a novel gene that is less than 10 million years old. Nevertheless, it shows a highly specific expression pattern in embryos, larvae, and adults, and has been implicated in larval development, stress resistance, and in the setting of adult lifespan, among others. Although mth belongs to a gene subfamily with 16 members in D. melanogaster, there is no evidence for functional redundancy in this subfamily. Therefore, it is surprising that a novel gene influences so many traits. Here, we explore the alternative hypothesis that mth is an old gene. Under this hypothesis, in species distantly related to D. melanogaster, there should be a gene with features similar to those of mth. By performing detailed phylogenetic, synteny, protein structure, and gene expression analyses we show that the D. virilis GJ12490 gene is the orthologous of mth in species distantly related to D. melanogaster. We also show that, in D. americana (a species of the virilis group of Drosophila, a common amino acid polymorphism at the GJ12490 orthologous gene is significantly associated with developmental time, size, and lifespan differences. Our results imply that GJ12490 orthologous genes are candidates for developmental time and lifespan differences in Drosophila in general.

  12. Neurl4 contributes to germ cell formation and integrity in Drosophila

    Directory of Open Access Journals (Sweden)

    Jennifer Jones

    2015-08-01

    Full Text Available Primordial germ cells (PGCs form at the posterior pole of the Drosophila embryo, and then migrate to their final destination in the gonad where they will produce eggs or sperm. Studies of the different stages in this process, including assembly of germ plasm in the oocyte during oogenesis, specification of a subset of syncytial embryonic nuclei as PGCs, and migration, have been informed by genetic analyses. Mutants have defined steps in the process, and the identities of the affected genes have suggested biochemical mechanisms. Here we describe a novel PGC phenotype. When Neurl4 activity is reduced, newly formed PGCs frequently adopt irregular shapes and appear to bud off vesicles. PGC number is also reduced, an effect exacerbated by a separate role for Neurl4 in germ plasm formation during oogenesis. Like its mammalian homolog, Drosophila Neurl4 protein is concentrated in centrosomes and downregulates centrosomal protein CP110. Reducing CP110 activity suppresses the abnormal PGC morphology of Neurl4 mutants. These results extend prior analyses of Neurl4 in cultured cells, revealing a heightened requirement for Neurl4 in germ-line cells in Drosophila.

  13. Radioresistance and radiosensitivity in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Reguly, M.L.

    1983-01-01

    Studying the mechanisms controlling radioresistant in Drosophila the sensibility of four strains of Drosophila melanogaster to sex-linked recessive lethal mutations induced by 5kR Cobalt-60 gamma radiation and 0,006 M EMS or 0,25% of caffeine was determined. (M.A.C.) [pt

  14. The Drosophila melanogaster circadian pacemaker circuit

    Indian Academy of Sciences (India)

    2016-08-26

    Aug 26, 2016 ... Keywords. circadian rhythm; neuronal network; ion channel; behaviour; neurotransmitter; electrophysiology; Drosophila. Abstract. As an experimental model system, the fruit fly Drosophila melanogaster has been seminal in shaping our understanding of the circadian clockwork. The wealth of genetic tools ...

  15. Radionuclide transfer from mother to embryo

    International Nuclear Information System (INIS)

    Toader, M.; Vasilache, R.A.; Scridon, R.; Toader, M.L.

    1998-01-01

    The transfer of radionuclides from mother to embryo is still a matter of high interest. Therefore, the relation was investigated between the amount of radionuclides in the embryo and the dietary intake of the mother, this for two scenarios: a recurrent intake of variable amounts of radionuclides, and a long-term intake of a relatively constant amount of radionuclides, the radionuclide being 137 Cs. In the first case, the amount of radionuclides present in the embryo increases with the age of the embryo and with the intake of the mother. In the second case, no correlation could be found between the age of the embryo and its radioactive content; only the correlation between the intake of the mother and the radionuclide content of the embryo remained. (A.K.)

  16. Large-scale discovery of promoter motifs in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Thomas A Down

    2007-01-01

    Full Text Available A key step in understanding gene regulation is to identify the repertoire of transcription factor binding motifs (TFBMs that form the building blocks of promoters and other regulatory elements. Identifying these experimentally is very laborious, and the number of TFBMs discovered remains relatively small, especially when compared with the hundreds of transcription factor genes predicted in metazoan genomes. We have used a recently developed statistical motif discovery approach, NestedMICA, to detect candidate TFBMs from a large set of Drosophila melanogaster promoter regions. Of the 120 motifs inferred in our initial analysis, 25 were statistically significant matches to previously reported motifs, while 87 appeared to be novel. Analysis of sequence conservation and motif positioning suggested that the great majority of these discovered motifs are predictive of functional elements in the genome. Many motifs showed associations with specific patterns of gene expression in the D. melanogaster embryo, and we were able to obtain confident annotation of expression patterns for 25 of our motifs, including eight of the novel motifs. The motifs are available through Tiffin, a new database of DNA sequence motifs. We have discovered many new motifs that are overrepresented in D. melanogaster promoter regions, and offer several independent lines of evidence that these are novel TFBMs. Our motif dictionary provides a solid foundation for further investigation of regulatory elements in Drosophila, and demonstrates techniques that should be applicable in other species. We suggest that further improvements in computational motif discovery should narrow the gap between the set of known motifs and the total number of transcription factors in metazoan genomes.

  17. Methanol as a cryoprotectant for equine embryos.

    Science.gov (United States)

    Bass, L D; Denniston, D J; Maclellan, L J; McCue, P M; Seidel, G E; Squires, E L

    2004-09-15

    Equine embryos (n=43) were recovered nonsurgically 7-8 days after ovulation and randomly assigned to be cryopreserved in one of two cryoprotectants: 48% (15M) methanol (n=22) or 10% (136 M) glycerol (n=21). Embryos (300-1000 microm) were measured at five intervals after exposure to glycerol (0, 2, 5, 10 and 15 min) or methanol (0, 15, 35, 75 and 10 min) to determine changes (%) in diameter over time (+/-S.D.). Embryos were loaded into 0.25-ml plastic straws, sealed, placed in a programmable cell freezer and cooled from room temperature (22 degrees C) to -6 degrees C. Straws were then seeded, held at -6 degrees C for 10 min and then cooled to -33 degrees C before being plunged into liquid nitrogen. Two or three embryos within a treatment group were thawed and assigned to be either cultured for 12 h prior to transfer or immediately nonsurgically transferred to a single mare. Embryo diameter decreased in all embryos upon initial exposure to cryoprotectant. Embryos in methanol shrank and recovered slightly to 76+/-8 % of their original diameter; however, embryos in glycerol continued to shrink, reaching 57+/-6 % of their original diameter prior to cryopreservation. Survival rates of embryos through Day 16 of pregnancy were 38 and 23%, respectively (P>0.05) for embryos cryopreserved in the presence of glycerol or methanol. There was no difference in pregnancy rates of mares receiving embryos that were cultured prior to transfer or not cultured (P>0.05). Preliminary experiments indicated that 48% methanol was not toxic to fresh equine embryos but methanol provided no advantage over glycerol as a cryoprotectant for equine blastocysts.

  18. Lethality of radioisotopes in early mouse embryos

    International Nuclear Information System (INIS)

    Macqueen, H.A.

    1979-01-01

    The development of pre-implantation mouse embryos was found to be prevented by exposure of the embryos to [ 35 S]methionine, but not to [ 3 H]methionine. Such embryos have also been shown to be highly sensitive to [ 3 H]thymidine. These observations are discussed with reference to the path lengths and energies of electrons emitted from the different radioisotopes. (author)

  19. Theory about the Embryo Cryo-Treatment.

    Science.gov (United States)

    Vladimirov, Iavor K; Tacheva, Desislava; Diez, Antonio

    2017-04-01

    To create hypothesis, which can give a logical explanation related to the benefits of freezing/thawing embryos. Cryopreservation is not only a technology used for storing embryos, but also a method of embryo treatment that can potentially improve the success rate in infertile couples. From the analysis of multiple results in assisted reproductive technology, which have no satisfactory explanation to date, we found evidence to support a 'therapeutic' effect of the freezing/thawing of embryos on the process of recovery of the embryo and its subsequent implantation. Freezing/thawing is a way to activate the endogenous survival and repair responses in preimplantation embryos. Several molecular mechanisms can explain the higher success rate of ET using thawed embryos compared to fresh ET in women of advanced reproductive age, the higher miscarriage rate in cases of thawed blastocyst ET compared to thawed ET at early cleavage embryo, and the higher perinatal parameters of born children after thawed ET. Embryo thawing induces a stress. Controlled stress is not necessarily detrimental, because it generates a phenomenon that is counteracted by several known biological responses aimed to repair mitochondrial damage of membrane and protein misfolding. The term for favorable biological responses to low exposures to stress is called hormesis. This thesis will summarize the role of cryopreservation in the activation of a hormetic response, preserving the mitochondrial function, improving survival, and having an impact on the process of implantation, miscarriage, and the development of pregnancy.

  20. Nano-nutrition of chicken embryos

    DEFF Research Database (Denmark)

    Sawosz, Filip; Pineda, Lane Manalili; Hotowy, Anna

    2013-01-01

    It has been suggested that the quantity and quality of nutrients stored in the egg might not be optimal for the fast rate of chicken embryo development in modern broilers, and embryos could be supplemented with nutrients by in ovo injection. Recent experiments showed that in ovo feeding reduces...... broiler eggs was randomly divided into a Control group without injection and injected groups with hydrocolloids of Nano-Ag, ATP or a complex of Nano-Ag and ATP (Nano-Ag/ATP). The embryos were evaluated on day 20 of incubation. The results indicate that the application of ATP to chicken embryos increases...

  1. Die Behandlung menschliches Embryos und Menschenwurde

    OpenAIRE

    Matsui, Fumio

    2002-01-01

    We are confronted with an old and new problem, which has come up with the progress of modern biotechnologies: what is a life or when does a life begin? The expectation of order-made medicine has build up since the discovery of Embryo Stem cell called "a dream master cell", while there is any condemnation against the destruction of human embryo in order to gain it. It is a question whether a human embryo is a human being in the world. Human dignity(=HD) is a principle that keeps human embryos ...

  2. Double-strand break repair-adox: Restoration of suppressed double-strand break repair during mitosis induces genomic instability.

    Science.gov (United States)

    Terasawa, Masahiro; Shinohara, Akira; Shinohara, Miki

    2014-12-01

    Double-strand breaks (DSBs) are one of the severest types of DNA damage. Unrepaired DSBs easily induce cell death and chromosome aberrations. To maintain genomic stability, cells have checkpoint and DSB repair systems to respond to DNA damage throughout most of the cell cycle. The failure of this process often results in apoptosis or genomic instability, such as aneuploidy, deletion, or translocation. Therefore, DSB repair is essential for maintenance of genomic stability. During mitosis, however, cells seem to suppress the DNA damage response and proceed to the next G1 phase, even if there are unrepaired DSBs. The biological significance of this suppression is not known. In this review, we summarize recent studies of mitotic DSB repair and discuss the mechanisms of suppression of DSB repair during mitosis. DSB repair, which maintains genomic integrity in other phases of the cell cycle, is rather toxic to cells during mitosis, often resulting in chromosome missegregation and aberration. Cells have multiple safeguards to prevent genomic instability during mitosis: inhibition of 53BP1 or BRCA1 localization to DSB sites, which is important to promote non-homologous end joining or homologous recombination, respectively, and also modulation of the non-homologous end joining core complex to inhibit DSB repair. We discuss how DSBs during mitosis are toxic and the multiple safeguard systems that suppress genomic instability. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

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

    Science.gov (United States)

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

    2010-05-15

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

  4. Identification and characterization of pyrokinin and CAPA peptides, and corresponding GPCRs from spotted wing drosophila, Drosophila suzukii.

    Science.gov (United States)

    Choi, Man-Yeon; Ahn, Seung-Joon; Kim, A Young; Koh, Youngho

    2017-05-15

    The family of FXPRLamide peptides serves as a major insect hormone. It is characterized by a core active amino acid sequence conserved at the C-terminal ends, and provides various physiological roles across the Insecta. In this study we identified and characterized pyrokinin (PK) and CAPA cDNAs encoding two FXPRLamide peptides, pyrokinin and CAPA-DH (diapause hormone), and two corresponding G protein-coupled receptors (GPCRs) from spotted wing drosophila (SWD), Drosophila suzukii. Expressions of PK and CAPA mRNAs were differentially observed during all life stages except the embryo, and the detection of CAPA transcription was relatively strong compared with the PK gene in SWD. Both D. suzukii pyrokinin receptor (DrosuPKr) and CAPA-DH receptor (DrosuCAPA-DHr) were functionally expressed and confirmed through binding to PK and DH peptides. Differential expression of two GPCRs occurred during all life stages; a strong transcription of DrosuPKr was observed in the 3rd instar. DrosuCAPA-DHr was clearly expressed from the embryo to the larva, but not detected in the adult. Gene regulation during the life stages was not synchronized between ligand and receptor. For example, SWD CAPA mRNA has been up-regulated in the adult while CAPA-DHr was down-regulated. The difference could be from the CAPA mRNA translating multiple peptides including CAPA-DH and two CAPA-PVK (periviscerokinin) peptides to act on different receptors. Comparing the genes of SWD PK, CAPA, PKr and CAPA-DHr to four corresponding genes of D. melanogaster, SWD CAPA and the receptor are more similar to D. melanogaster than PK and the receptor. These data suggest that the CAPA gene could be evolutionally more conserved to have a common biological role in insects. In addition, the effect of Kozak sequences was investigated by the expression of the GPCRs with or without Kozak sequences in Sf9 insect cells. The Kozak sequenced PK receptor was significantly less active than the original (= no Kozak sequenced

  5. Effects of caffeine on protein phosphorylation and cell cycle progression in X-irradiated two-cell mouse embryos

    International Nuclear Information System (INIS)

    Jung, Th.; Streffer, C.

    1992-01-01

    To understand the mechanism of the caffeine-induced uncoupling of mitosis and the cellular reactions to DNA-damaging agents, the authors studied the effects of caffeine treatment on cell cycle progression and protein phosphorylation in two-cell mouse embryos after X-irradiation. Caffeine alone had no effect on timing of and changes in phosphorylation associated with the embryonic cell cycle. In combination with X-rays, caffeine was able to override the radiation induced G 2 block and restored normal timing of these phosphorylation changes after X-irradiation. New additional changes in protein phosphorylation appeared after the combined treatment. Isobutyl-methylxanthine (IBMX), a substance chemically related to caffeine but a more specific inhibitor of the phosphodiesterase that breaks down cyclic AMP, reduced radiation induced G 2 block from 4 to 5 h to about 1 h and restored the cell cycle associated changes in protein phosphorylation. (author)

  6. Effects of caffeine on protein phosphorylation and cell cycle progression in X-irradiated two-cell mouse embryos

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Th. (AFRC Institute of Animal Physiology and Genetics Research, Babraham (United Kingdom)); Streffer, C. (Essen Univ (Germany). Inst. fuer Medizinische Strahlenbiolgie)

    1992-08-01

    To understand the mechanism of the caffeine-induced uncoupling of mitosis and the cellular reactions to DNA-damaging agents, the authors studied the effects of caffeine treatment on cell cycle progression and protein phosphorylation in two-cell mouse embryos after X-irradiation. Caffeine alone had no effect on timing of and changes in phosphorylation associated with the embryonic cell cycle. In combination with X-rays, caffeine was able to override the radiation induced G[sub 2] block and restored normal timing of these phosphorylation changes after X-irradiation. New additional changes in protein phosphorylation appeared after the combined treatment. Isobutyl-methylxanthine (IBMX), a substance chemically related to caffeine but a more specific inhibitor of the phosphodiesterase that breaks down cyclic AMP, reduced radiation induced G[sub 2] block from 4 to 5 h to about 1 h and restored the cell cycle associated changes in protein phosphorylation. (author).

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

    Science.gov (United States)

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

    2016-08-02

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

  8. Water droplet excess free energy determined by cluster mitosis using guided molecular dynamics

    Science.gov (United States)

    Lau, Gabriel V.; Hunt, Patricia A.; Müller, Erich A.; Jackson, George; Ford, Ian J.

    2015-12-01

    Atmospheric aerosols play a vital role in affecting climate by influencing the properties and lifetimes of clouds and precipitation. Understanding the underlying microscopic mechanisms involved in the nucleation of aerosol droplets from the vapour phase is therefore of great interest. One key thermodynamic quantity in nucleation is the excess free energy of cluster formation relative to that of the saturated vapour. In our current study, the excess free energy is extracted for clusters of pure water modelled with the TIP4P/2005 intermolecular potential using a method based on nonequilibrium molecular dynamics and the Jarzynski relation. The change in free energy associated with the "mitosis" or division of a cluster of N water molecules into two N/2 sub-clusters is evaluated. This methodology is an extension of the disassembly procedure used recently to calculate the excess free energy of argon clusters [H. Y. Tang and I. J. Ford, Phys. Rev. E 91, 023308 (2015)]. Our findings are compared to the corresponding excess free energies obtained from classical nucleation theory (CNT) as well as internally consistent classical theory (ICCT). The values of the excess free energy that we obtain with the mitosis method are consistent with CNT for large cluster sizes but for the smallest clusters, the results tend towards ICCT; for intermediate sized clusters, we obtain values between the ICCT and CNT predictions. Furthermore, the curvature-dependent surface tension which can be obtained by regarding the clusters as spherical droplets of bulk density is found to be a monotonically increasing function of cluster size for the studied range. The data are compared to other values reported in the literature, agreeing qualitatively with some but disagreeing with the values determined by Joswiak et al. [J. Phys. Chem. Lett. 4, 4267 (2013)] using a biased mitosis approach; an assessment of the differences is the main motivation for our current study.

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

    Science.gov (United States)

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

    2016-01-01

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

  10. [Relationship between mitochondrial DNA copy number, membrane potential of human embryo and embryo morphology].

    Science.gov (United States)

    Zhao, H; Teng, X M; Li, Y F

    2017-11-25

    Objective: To explore the relationship between the embryo with the different morphological types in the third day and its mitochondrial copy number, the membrane potential. Methods: Totally 117 embryos with poor development after normal fertilization and were not suitable transferred in the fresh cycle and 106 frozen embryos that were discarded voluntarily by infertility patients with in vitro fertilization-embryo transfer after successful pregnancy were selected. According to evaluation of international standard in embryos, all cleavage stage embryos were divided into class Ⅰ frozen embryo group ( n= 64), class Ⅱ frozen embryo group ( n= 42) and class Ⅲ fresh embryonic group (not transplanted embryos; n= 117). Real-time PCR and confocal microscopy methods were used to detect mitochondrial DNA (mtDNA) copy number and the mitochondrial membrane potential of a single embryo. The differences between embryo quality and mtDNA copy number and membrane potential of each group were compared. Results: The copy number of mtDNA and the mitochondrial membrane potential in class Ⅲ fresh embryonic group [(1.7±1.0)×10(5) copy/μl, 1.56±0.32] were significantly lower than those in class Ⅰ frozen embryo group [(3.4±1.7)×10(5) copy/μl, 2.66±0.21] and class Ⅱ frozen embryo group [(2.6±1.2)×10(5) copy/μl, 1.80±0.32; all Pembryo group were significantly higher than those in classⅡ frozen embryo group (both Pembryos of the better quality embryo are higher.

  11. Mouse one-cell embryos undergoing a radiation-induced G2 arrest may re-enter S-phase in the absence of cytokinesis

    International Nuclear Information System (INIS)

    Jacquet, P.; Buset, J.; Vankerkom, J.; Baatout, S.; De Saint-Georges, L.; Schoonjans, W.; Desaintes, C.

    2002-01-01

    PCC (premature chromosome condensation) can be used for visualizing and scoring damage induced by radiation in the chromatin of cells undergoing a G1 or G2 arrest. A method involving the fusion of irradiated single embryonic cells with single MI oocytes was used to induce PCC in mouse zygotes of the BALB/c strain, which suffer a drastic G2 arrest after X-irradiation (dose used 2.5 Gy). Other G2-arrested embryos were exposed in vitro to the phosphatase inhibitor calyculin A. Both methods furnished excellent chromosome preparations of the G2-arrested embryos. The mean number of chromosome fragments did not change significantly during G2 arrest, suggesting that zygotes of this strain are unable to repair DNA damage leading to such aberrations. Forty to fifty percent of the irradiated embryos were unable to cleave after G2 arrest and remained blocked at the one-cell stage for a few days before dying. PCC preparations obtained from such embryos suggested that about 30% of them had undergone a late mitosis not followed by cytokinesis and had entered a new DNA synthesis. These results are discussed in the light of recent observations in irradiated human cells deficient in the p53/14-3-3σ pathway. (author)

  12. A thermodynamic approach to the 'mitosis/apoptosis' ratio in cancer

    Science.gov (United States)

    Lucia, Umberto; Ponzetto, Antonio; Deisboeck, Thomas S.

    2015-10-01

    Cancer can be considered as an open, complex, (bio-thermo)dynamic and self-organizing system. Consequently, an entropy generation approach has been employed to analyze its mitosis/apoptosis ratio. Specifically, a novel thermodynamic anticancer strategy is suggested, based on the variation of entropy generation caused by the application of external fields, for example electro-magnetic fields, for therapeutic purposes. Eventually, this innovative approach could support conventional therapies, particularly for inoperable tumors or advanced stages of cancer, when larger tumor burden is diagnosed, and therapeutic options are often limited.

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

    Science.gov (United States)

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

    2014-03-01

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

  14. Regulation of Centromere Localization of the Drosophila Shugoshin MEI-S332 and Sister-Chromatid Cohesion in Meiosis

    Science.gov (United States)

    Nogueira, Cristina; Kashevsky, Helena; Pinto, Belinda; Clarke, Astrid; Orr-Weaver, Terry L.

    2014-01-01

    The Shugoshin (Sgo) protein family helps to ensure proper chromosome segregation by protecting cohesion at the centromere by preventing cleavage of the cohesin complex. Some Sgo proteins also influence other aspects of kinetochore-microtubule attachments. Although many Sgo members require Aurora B kinase to localize to the centromere, factors controlling delocalization are poorly understood and diverse. Moreover, it is not clear how Sgo function is inactivated and whether this is distinct from delocalization. We investigated these questions in Drosophila melanogaster, an organism with superb chromosome cytology to monitor Sgo localization and quantitative assays to test its function in sister-chromatid segregation in meiosis. Previous research showed that in mitosis in cell culture, phosphorylation of the Drosophila Sgo, MEI-S332, by Aurora B promotes centromere localization, whereas Polo phosphorylation promotes delocalization. These studies also suggested that MEI-S332 can be inactivated independently of delocalization, a conclusion supported here by localization and function studies in meiosis. Phosphoresistant and phosphomimetic mutants for the Aurora B and Polo phosphorylation sites were examined for effects on MEI-S332 localization and chromosome segregation in meiosis. Strikingly, MEI-S332 with a phosphomimetic mutation in the Aurora B phosphorylation site prematurely dissociates from the centromeres in meiosis I. Despite the absence of MEI-S332 on meiosis II centromeres in male meiosis, sister chromatids segregate normally, demonstrating that detectable levels of this Sgo are not essential for chromosome congression, kinetochore biorientation, or spindle assembly. PMID:25081981

  15. WDR81 mutations cause extreme microcephaly and impair mitotic progression in human fibroblasts and Drosophila neural stem cells.

    Science.gov (United States)

    Cavallin, Mara; Rujano, Maria A; Bednarek, Nathalie; Medina-Cano, Daniel; Bernabe Gelot, Antoinette; Drunat, Severine; Maillard, Camille; Garfa-Traore, Meriem; Bole, Christine; Nitschké, Patrick; Beneteau, Claire; Besnard, Thomas; Cogné, Benjamin; Eveillard, Marion; Kuster, Alice; Poirier, Karine; Verloes, Alain; Martinovic, Jelena; Bidat, Laurent; Rio, Marlene; Lyonnet, Stanislas; Reilly, M Louise; Boddaert, Nathalie; Jenneson-Liver, Melanie; Motte, Jacques; Doco-Fenzy, Martine; Chelly, Jamel; Attie-Bitach, Tania; Simons, Matias; Cantagrel, Vincent; Passemard, Sandrine; Baffet, Alexandre; Thomas, Sophie; Bahi-Buisson, Nadia

    2017-10-01

    Microlissencephaly is a rare brain malformation characterized by congenital microcephaly and lissencephaly. Microlissencephaly is suspected to result from abnormalities in the proliferation or survival of neural progenitors. Despite the recent identification of six genes involved in microlissencephaly, the pathophysiological basis of this condition remains poorly understood. We performed trio-based whole exome sequencing in seven subjects from five non-consanguineous families who presented with either microcephaly or microlissencephaly. This led to the identification of compound heterozygous mutations in WDR81, a gene previously associated with cerebellar ataxia, intellectual disability and quadrupedal locomotion. Patient phenotypes ranged from severe microcephaly with extremely reduced gyration with pontocerebellar hypoplasia to moderate microcephaly with cerebellar atrophy. In patient fibroblast cells, WDR81 mutations were associated with increased mitotic index and delayed prometaphase/metaphase transition. Similarly, in vivo, we showed that knockdown of the WDR81 orthologue in Drosophila led to increased mitotic index of neural stem cells with delayed mitotic progression. In summary, we highlight the broad phenotypic spectrum of WDR81-related brain malformations, which include microcephaly with moderate to extremely reduced gyration and cerebellar anomalies. Our results suggest that WDR81 might have a role in mitosis that is conserved between Drosophila and humans. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. Neural network classification of sweet potato embryos

    Science.gov (United States)

    Molto, Enrique; Harrell, Roy C.

    1993-05-01

    Somatic embryogenesis is a process that allows for the in vitro propagation of thousands of plants in sub-liter size vessels and has been successfully applied to many significant species. The heterogeneity of maturity and quality of embryos produced with this technique requires sorting to obtain a uniform product. An automated harvester is being developed at the University of Florida to sort embryos in vitro at different stages of maturation in a suspension culture. The system utilizes machine vision to characterize embryo morphology and a fluidic based separation device to isolate embryos associated with a pre-defined, targeted morphology. Two different backpropagation neural networks (BNN) were used to classify embryos based on information extracted from the vision system. One network utilized geometric features such as embryo area, length, and symmetry as inputs. The alternative network utilized polar coordinates of an embryo's perimeter with respect to its centroid as inputs. The performances of both techniques were compared with each other and with an embryo classification method based on linear discriminant analysis (LDA). Similar results were obtained with all three techniques. Classification efficiency was improved by reducing the dimension of the feature vector trough a forward stepwise analysis by LDA. In order to enhance the purity of the sample selected as harvestable, a reject to classify option was introduced in the model and analyzed. The best classifier performances (76% overall correct classifications, 75% harvestable objects properly classified, homogeneity improvement ratio 1.5) were obtained using 8 features in a BNN.

  17. Embryo transfer using cryopreserved Boer goat blastocysts ...

    African Journals Online (AJOL)

    The aim of this trial was to evaluate the effect of embryo cryopreservation techniques on the survivability of embryos and fertility following transfer to Boer goat does. The oestrous cycles of 27 mature recipients Boer goat does were synchronised using controlled internal drug release dispensers (CIDR's) for 16 days. At CIDR ...

  18. Novel embryo selection techniques to increase embryo implantation in IVF attempts.

    Science.gov (United States)

    Sigalos, George Α; Triantafyllidou, Olga; Vlahos, Nikos F

    2016-11-01

    The final success of an IVF attempt depends on several steps and decisions taken during the ovarian stimulation, the oocyte retrieval, the embryo culture and the embryo transfer. The final selection of the embryos most likely to implant is the final step in this process and the responsibility of the lab. Apart from strict morphologic criteria that historically have been used in embryo selection, additional information on genetic, metabolomic and morphokinetic characteristics of the embryo is recently combined to morphology to select the embryo most likely to produce a pregnancy. In this manuscript, we review the most recent information on the current methods used for embryo selection presenting the predictive capability of each one. A literature search was performed on Pubmed, Medline and Cochrane Database of Systematic Reviews for published studies using appropriate key words and phrases with no limits placed on time. It seems that the combination of morphologic criteria in conjunction to embryo kinetics as documented by time-lapse technology provides the most reliable information on embryo quality. Blastocyst biopsy with subsequent comprehensive chromosome analysis allows the selection of the euploid embryos with the higher implantation potential. Embryo time-lapse imaging and blastocyst biopsy combined to comprehensive chromosome analysis are the most promising technologies to increase pregnancy rates and reduce the possibility of multiple pregnancies. However, further studies will demonstrate the capability of routinely using these technologies to significantly improve IVF outcomes.

  19. Adaptive genic evolution in the Drosophila genomes

    DEFF Research Database (Denmark)

    Shapiro, Joshua A; Huang, Wei; Zhang, Chenhui

    2007-01-01

    and stable population. In this study, we sequenced 419 genes from 24 lines of Drosophila melanogaster and its close relatives. Together with data from Drosophila simulans, these data reveal the following. (i) Approximately 10% of the loci in regions of normal recombination are much less polymorphic at silent...... sites than expected, hinting at the action of selective sweeps. (ii) The level of polymorphism is negatively correlated with the rate of nonsynonymous divergence across loci. Thus, even under strict neutrality, the ratio of amino acid to silent nucleotide changes (A:S) between Drosophila species...

  20. Noninvasive metabolomic profiling as an adjunct to morphology for noninvasive embryo assessment in women undergoing single embryo transfer

    NARCIS (Netherlands)

    Seli, E.; Vergouw, C.G.; Morita, H.; Botros, L.; Roos, P.; Lambalk, C.B.; Yamashita, N.; Kato, O.; Sakkas, D.

    2010-01-01

    Objective: To determine whether metabolomic profiling of spent embryo culture media correlates with reproductive potential of human embryos. Design: Retrospective study. Setting: Academic and a private assisted reproductive technology (ART) programs. Patient(s): Women undergoing single embryo

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  2. Transcriptional intermediary factor 1γ binds to the anaphase-promoting complex/cyclosome and promotes mitosis

    DEFF Research Database (Denmark)

    Sedgwick, G.G.; Townsend, K.; Martin, A.

    2013-01-01

    The anaphase-promoting complex/cyclosome (APC/C) is an ubiquitin ligase that functions during mitosis. Here we identify the transcriptional regulator, transcriptional intermediary factor 1γ, TIF1γ, as an APC/C-interacting protein that regulates APC/C function. TIF1γ is not a substrate for APC....../C-dependent ubiquitylation but instead, associates specifically with the APC/C holoenzyme and Cdc20 to affect APC/C activity and progression through mitosis. RNA interference studies indicate that TIF1γ knockdown results in a specific reduction in APC/C ubiquitin ligase activity, the stabilization of APC/C substrates......, and an increase in the time taken for cells to progress through mitosis from nuclear envelope breakdown to anaphase. TIF1γ knockdown cells are also characterized by the inappropriate presence of cyclin A at metaphase, and an increase in the number of cells that fail to undergo metaphase-to-anaphase transition...

  3. Rape embryogenesis. III. Embryo development in time

    Directory of Open Access Journals (Sweden)

    Teresa Tykarska

    2014-01-01

    Full Text Available It was found that the growth curve of the rape embryo axis is of triple sigmoid type. Embryo growth occurs in 3 phases corresponding to 3 different periods of development. Phase I includes growth of the apical cell up to it's division into two layers of octants. Phase II comprises the increase of the spherical proembryo to the change of its symmetry from radial to bilateral. Phase III includes, growth of the embryo from the heart stage up to the end of embryogenesis. In each phase the relative growth rate increases drastically and then diminishes. The differences in growth intensity during the same phase are several-fold. The growth intensity maximum of the embryo axis occurs in phase II. The phasic growth intensity maxima occur: in phase I during apical cell elongation, :before its division, and in phases II and III in the periods of cell division ;growth in globular and torpedo-shaped -shaped embryos.

  4. Potential of human twin embryos generated by embryo splitting in assisted reproduction and research.

    Science.gov (United States)

    Noli, Laila; Ogilvie, Caroline; Khalaf, Yacoub; Ilic, Dusko

    2017-03-01

    Embryo splitting or twinning has been widely used in veterinary medicine over 20 years to generate monozygotic twins with desirable genetic characteristics. The first human embryo splitting, reported in 1993, triggered fierce ethical debate on human embryo cloning. Since Dolly the sheep was born in 1997, the international community has acknowledged the complexity of the moral arguments related to this research and has expressed concerns about the potential for reproductive cloning in humans. A number of countries have formulated bans either through laws, decrees or official statements. However, in general, these laws specifically define cloning as an embryo that is generated via nuclear transfer (NT) and do not mention embryo splitting. Only the UK includes under cloning both embryo splitting and NT in the same legislation. On the contrary, the Ethics Committee of the American Society for Reproductive Medicine does not have a major ethical objection to transferring two or more artificially created embryos with the same genome with the aim of producing a single pregnancy, stating that 'since embryo splitting has the potential to improve the efficacy of IVF treatments for infertility, research to investigate the technique is ethically acceptable'. Embryo splitting has been introduced successfully to the veterinary medicine several decades ago and today is a part of standard practice. We present here an overview of embryo splitting experiments in humans and non-human primates and discuss the potential of this technology in assisted reproduction and research. A comprehensive literature search was carried out using PUBMED and Google Scholar databases to identify studies on embryo splitting in humans and non-human primates. 'Embryo splitting' and 'embryo twinning' were used as the keywords, alone or in combination with other search phrases relevant to the topics of biology of preimplantation embryos. A very limited number of studies have been conducted in humans and non

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

    Science.gov (United States)

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

    2017-06-01

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

  6. Studies on Drosophila radiosensitivity strains

    International Nuclear Information System (INIS)

    Varentsova, E.R.; Sharygin, V.I.; Khromykh, Yu.U.

    1985-01-01

    Fertility of radiosensitive mutant drosophila female strain rad (2) 201 61 after irradiation and frequency of dominant lethal mutations (DLM), induced by γ-radiation for 0-5 h and 5-7 days, are investigated. It is shown, that oocytes of the mutant strain are more radiosensitive as compared with cells of mongrel flies as to criterion of DLM appearance over the period of maturing. Early oocytes of stages 2-7 are the most sensitive, i.e. at the stages, corresponding to the manifestation of previously established recombination-defective properties of mutations rad (2) 201 61 . It is also sown, that doses of γ-rays, exceeding 10 Gy produce a strong sterilizing effect on mutant females due to destruction and resorption of egg chambers, irradiated at the stages of previtellogenetic growth of oocytes. In females, carrying mutation of radiosensitivity there is no direct correlation betwen sensitivity of oocytes proper to DLM induction and sensitivity of egg folleicles to resorbing effect of γ-rays. The ways of possible involvement of mutant locus studied into genetic processes in various specialized cells of drosophila

  7. Isoform-specific functions of Mud/NuMA mediate binucleation of Drosophila male accessory gland cells.

    Science.gov (United States)

    Taniguchi, Kiichiro; Kokuryo, Akihiko; Imano, Takao; Minami, Ryunosuke; Nakagoshi, Hideki; Adachi-Yamada, Takashi

    2014-12-20

    In standard cell division, the cells undergo karyokinesis and then cytokinesis. Some cells, however, such as cardiomyocytes and hepatocytes, can produce binucleate cells by going through mitosis without cytokinesis. This cytokinesis skipping is thought to be due to the inhibition of cytokinesis machinery such as the central spindle or the contractile ring, but the mechanisms regulating it are unclear. We investigated them by characterizing the binucleation event during development of the Drosophila male accessory gland, in which all cells are binucleate. The accessory gland cells arrested the cell cycle at 50 hours after puparium formation (APF) and in the middle of the pupal stage stopped proliferating for 5 hours. They then restarted the cell cycle and at 55 hours APF entered the M-phase synchronously. At this stage, accessory gland cells binucleated by mitosis without cytokinesis. Binucleating cells displayed the standard karyokinesis progression but also showed unusual features such as a non-round shape, spindle orientation along the apico-basal axis, and poor assembly of the central spindle. Mud, a Drosophila homolog of NuMA, regulated the processes responsible for these three features, the classical isoform Mud(PBD) and the two newly characterized isoforms Mud(L) and Mud(S) regulated them differently: Mud(L) repressed cell rounding, Mud(PBD) and Mud(S) oriented the spindle along the apico-basal axis, and Mud(S) and Mud(L) repressed central spindle assembly. Importantly, overexpression of Mud(S) induced binucleation even in standard proliferating cells such as those in imaginal discs. We characterized the binucleation in the Drosophila male accessory gland and examined mechanisms that regulated unusual morphologies of binucleating cells. We demonstrated that Mud, a microtubule binding protein regulating spindle orientation, was involved in this binucleation. We suggest that atypical functions exerted by three structurally different isoforms of Mud regulate

  8. Bacterial mitosis

    DEFF Research Database (Denmark)

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

    2003-01-01

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

  9. The importance of G1/S-border and mitosis in the fixation

    International Nuclear Information System (INIS)

    Iliakis, G.; Nuesse, M.

    1983-01-01

    The ability of synchronized Ehrlich ascites tumour cells to repair PLD was measured by introducing delays in their progression through the cell cycle either in the same phase as that where the irradiation was given or in a subsequent phase. Cells were incubated for this purpose either in balanced salt solution which nonspecifically delayed progression in all cell cycle phases or with 0.5 μg/ml aphidicolin which delayed cells in S-phase. Cells which had been delayed in their progression through the cell cycle were able to repair PLD irrespective of the phase at which they were held. In cases where the delay in the progression through the cell cycle was introduced in a phase subsequent to that of the exposure to irradiation, repair of PLD was observed only if the cells had not passed the G1/S-border or mitosis. Based on these results, the importance of G1/S-border and mitosis in the fixation of PLD is suggested. (orig.)

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

    Science.gov (United States)

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

    2016-05-17

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

  11. CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast.

    Science.gov (United States)

    Převorovský, Martin; Oravcová, Martina; Zach, Róbert; Jordáková, Anna; Bähler, Jürg; Půta, František; Folk, Petr

    2016-11-16

    For every eukaryotic cell to grow and divide, intricately coordinated action of numerous proteins is required to ensure proper cell-cycle progression. The fission yeast Schizosaccharomyces pombe has been instrumental in elucidating the fundamental principles of cell-cycle control. Mutations in S. pombe 'cut' (cell untimely torn) genes cause failed coordination between cell and nuclear division, resulting in catastrophic mitosis. Deletion of cbf11, a fission yeast CSL transcription factor gene, triggers a 'cut' phenotype, but the precise role of Cbf11 in promoting mitotic fidelity is not known. We report that Cbf11 directly activates the transcription of the acetyl-coenzyme A carboxylase gene cut6, and the biotin uptake/biosynthesis genes vht1 and bio2, with the former 2 implicated in mitotic fidelity. Cbf11 binds to a canonical, metazoan-like CSL response element (GTGGGAA) in the cut6 promoter. Expression of Cbf11 target genes shows apparent oscillations during the cell cycle using temperature-sensitive cdc25-22 and cdc10-M17 block-release experiments, but not with other synchronization methods. The penetrance of catastrophic mitosis in cbf11 and cut6 mutants is nutrient-dependent. We also show that drastic decrease in biotin availability arrests cell proliferation but does not cause mitotic defects. Taken together, our results raise the possibility that CSL proteins play conserved roles in regulating cell-cycle progression, and they could guide experiments into mitotic CSL functions in mammals.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

    Albayrak, Abdülkadir; Bilgin, Gökhan

    2013-12-01

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

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

    Science.gov (United States)

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

    2014-10-15

    PCTAIRE1 is distant relative of the cyclin-dependent kinase family that has been implicated in spermatogenesis and neuronal development, but it has not been studied in cancer. Here, we report that PCTAIRE1 is expressed in prostate, breast, and cervical cancer cells, where its RNAi-mediated silencing causes growth inhibition with aberrant mitosis due to defects in centrosome dynamics. PCTAIRE1 was not similarly involved in proliferation of nontransformed cells, including diploid human IMR-90 fibroblasts. Through yeast two-hybrid screening, we identified tumor suppressor p27 as a PCTAIRE1 interactor. In vitro kinase assays showed PCTAIRE1 phosphorylates p27 at Ser10. PCTAIRE1 silencing modulated Ser10 phosphorylation on p27 and led to its accumulation in cancer cells but not in nontransformed cells. In a mouse xenograft model of PPC1 prostate cancer, conditional silencing of PCTAIRE1 restored p27 protein expression and suppressed tumor growth. Mechanistic studies in HeLa cells showed that PCTAIRE1 phosphorylates p27 during the S and M phases of the cell cycle. Notably, p27 silencing was sufficient to rescue cells from mitotic arrest caused by PCTAIRE1 silencing. Clinically, PCTAIRE1 was highly expressed in primary breast and prostate tumors compared with adjacent normal epithelial tissues. Together our findings reveal an unexpected role for PCTAIRE1 in regulating p27 stability, mitosis, and tumor growth, suggesting PCTAIRE1 as a candidate cancer therapeutic target. ©2014 American Association for Cancer Research.

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

    Science.gov (United States)

    Wang, Yingmei; Wen, Jing; Zhang, Wei

    2011-02-01

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

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

    Science.gov (United States)

    Wirenfeldt, Martin; Dissing-Olesen, Lasse; Anne Babcock, Alicia; Nielsen, Marianne; Meldgaard, Michael; Zimmer, Jens; Azcoitia, Iñigo; Leslie, Robert Graham Quinton; Dagnaes-Hansen, Frederik; Finsen, Bente

    2007-08-01

    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 investigated the mechanisms of microglial population control in a well-defined model of reactive microgliosis in the mouse dentate gyrus after perforant pathway axonal lesion. Unbiased stereological methods and flow cytometry demonstrate significant lesion-induced increases in microglial numbers. Reactive microglia often occurred in clusters, some having recently incorporated bromodeoxyuridine, showing that proliferation had occurred. Annexin V labeling and staining for activated caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling showed that apoptotic mechanisms participate in dissolution of the microglial response. Using bone marrow chimeric mice, we found that the lesion-induced proliferative capacity of resident microglia superseded that of immigrant microglia, whereas lesion-induced kinetics of apoptosis were comparable. Microglial numbers and responses were severely reduced 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.

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

    Directory of Open Access Journals (Sweden)

    Viktória Fisi

    2018-05-01

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

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

    Science.gov (United States)

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

    2008-11-01

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

  19. The FANC pathway and BLM collaborate during mitosis to prevent micro-nucleation and chromosome abnormalities.

    Science.gov (United States)

    Naim, Valeria; Rosselli, Filippo

    2009-06-01

    Loss-of-function of caretaker genes characterizes a group of cancer predisposition diseases that feature cellular hypersensitivity to DNA damage and chromosome fragility; this group includes Fanconi anaemia and Bloom syndrome. The products of the 13 FANC genes (mutated in Fanconi anaemia), which constitute the 'FANC' pathway, and BLM (the RecQ helicase mutated in Bloom syndrome) are thought to collaborate during the S phase of the cell cycle, preventing chromosome instability. Recently, BLM has been implicated in the completion of sister chromatid separation during mitosis, a complex process in which precise regulation and execution is crucial to preserve genomic stability. Here we show for the first time a role for the FANC pathway in chromosome segregation during mitotic cell division. FANCD2, a key component of the pathway, localizes to discrete spots on mitotic chromosomes. FANCD2 chromosomal localization is responsive to replicative stress and specifically targets aphidicolin (APH)-induced chromatid gaps and breaks. Our data indicate that the FANC pathway is involved in rescuing abnormal anaphase and telophase (ana-telophase) cells, limiting aneuploidy and reducing chromosome instability in daughter cells. We further address a cooperative role for the FANC pathway and BLM in preventing micronucleation, through FANC-dependent targeting of BLM to non-centromeric abnormal structures induced by replicative stress. We reveal new crosstalk between FANC and BLM proteins, extending their interaction beyond the S-phase rescue of damaged DNA to the safeguarding of chromosome stability during mitosis.

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

    International Nuclear Information System (INIS)

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

    2004-01-01

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

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

    International Nuclear Information System (INIS)

    Iliakis, G.; Nuesse, M.

    1984-01-01

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

  2. RECQ5 Helicase Cooperates with MUS81 Endonuclease in Processing Stalled Replication Forks at Common Fragile Sites during Mitosis

    DEFF Research Database (Denmark)

    Di Marco, Stefano; Hasanova, Zdenka; Kanagaraj, Radhakrishnan

    2017-01-01

    The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent...... on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain...

  3. Regulation of nuclear envelope dynamics via APC/C is necessary for the progression of semi-open mitosis in Schizosaccharomyces japonicus.

    Science.gov (United States)

    Aoki, Keita; Shiwa, Yuh; Takada, Hiraku; Yoshikawa, Hirofumi; Niki, Hironori

    2013-09-01

    Three types of mitosis, which are open, closed or semi-open mitosis, function in eukaryotic cells, respectively. The open mitosis involves breakage of the nuclear envelope before nuclear division, whereas the closed mitosis proceeds with an intact nuclear envelope. To understand the mechanism and significance of three types of mitotic division in eukaryotes, we investigated the process of semi-open mitosis, in which the nuclear envelope is only partially broken, in the fission yeast Schizosaccharomyces japonicus. In anaphase-promoting complex/cyclosome (APC/C) mutants of Sz. japonicus, the nuclear envelope remained relatively intact during anaphase, resulting in impaired semi-open mitosis. As a suppressor of apc2 mutant, a mutation of Oar2, which was a 3-oxoacyl-[acyl carrier protein] reductase, was obtained. The level of the Oar2, which had two destruction-box motifs recognized by APC/C, was increased in APC/C mutants. Furthermore, the defective semi-open mitosis observed in an apc2 mutant was restored by mutated oar2+. Based on these findings, we propose that APC/C regulates the dynamics of the nuclear envelope through degradation of Oar2 dependent on APC/C during the metaphase-to-anaphase transition of semi-open mitosis in Sz. japonicus. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

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

    Directory of Open Access Journals (Sweden)

    Ludovic Roux

    2013-01-01

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

  5. Emergent properties during dorsal closure in Drosophila morphogenesis

    International Nuclear Information System (INIS)

    Peralta, X G; Toyama, Y; Edwards, G S; Kiehart, D P

    2008-01-01

    Dorsal closure is an essential stage of Drosophila development that is a model system for research in morphogenesis and biological physics. Dorsal closure involves an orchestrated interplay between gene expression and cell activities that produce shape changes, exert forces and mediate tissue dynamics. We investigate the dynamics of dorsal closure based on confocal microscopic measurements of cell shortening in living embryos. During the mid-stages of dorsal closure we find that there are fluctuations in the width of the leading edge cells but the time-averaged analysis of measurements indicate that there is essentially no net shortening of cells in the bulk of the leading edge, that contraction predominantly occurs at the canthi as part of the process for zipping together the two leading edges of epidermis and that the rate constant for zipping correlates with the rate of movement of the leading edges. We characterize emergent properties that regulate dorsal closure, i.e., a velocity governor and the coordination and synchronization of tissue dynamics

  6. PH Domain-Arf G Protein Interactions Localize the Arf-GEF Steppke for Cleavage Furrow Regulation in Drosophila.

    Directory of Open Access Journals (Sweden)

    Donghoon M Lee

    Full Text Available The recruitment of GDP/GTP exchange factors (GEFs to specific subcellular sites dictates where they activate small G proteins for the regulation of various cellular processes. Cytohesins are a conserved family of plasma membrane GEFs for Arf small G proteins that regulate endocytosis. Analyses of mammalian cytohesins have identified a number of recruitment mechanisms for these multi-domain proteins, but the conservation and developmental roles for these mechanisms are unclear. Here, we report how the pleckstrin homology (PH domain of the Drosophila cytohesin Steppke affects its localization and activity at cleavage furrows of the early embryo. We found that the PH domain is necessary for Steppke furrow localization, and for it to regulate furrow structure. However, the PH domain was not sufficient for the localization. Next, we examined the role of conserved PH domain amino acid residues that are required for mammalian cytohesins to bind PIP3 or GTP-bound Arf G proteins. We confirmed that the Steppke PH domain preferentially binds PIP3 in vitro through a conserved mechanism. However, disruption of residues for PIP3 binding had no apparent effect on GFP-Steppke localization and effects. Rather, residues for binding to GTP-bound Arf G proteins made major contributions to this Steppke localization and activity. By analyzing GFP-tagged Arf and Arf-like small G proteins, we found that Arf1-GFP, Arf6-GFP and Arl4-GFP, but not Arf4-GFP, localized to furrows. However, analyses of embryos depleted of Arf1, Arf6 or Arl4 revealed either earlier defects than occur in embryos depleted of Steppke, or no detectable furrow defects, possibly because of redundancies, and thus it was difficult to assess how individual Arf small G proteins affect Steppke. Nonetheless, our data show that the Steppke PH domain and its conserved residues for binding to GTP-bound Arf G proteins have substantial effects on Steppke localization and activity in early Drosophila embryos.

  7. Functional Analysis of Drosophila NF1

    National Research Council Canada - National Science Library

    Bernards, Andre

    2005-01-01

    ...) for Ras, yet homozygous loss of a highly conserved Drosophila NF1 ortholog results in several phenotypes that are insensitive to manipulating Ras signal transduction, but rescued by increasing...

  8. The Drosophila bipectinata species complex: phylogenetic ...

    Indian Academy of Sciences (India)

    PARUL BANERJEE

    c Indian Academy of Sciences. RESEARCH ARTICLE. The Drosophila bipectinata species complex: phylogenetic relationship among different members based on chromosomal variations. PARUL BANERJEE and BASHISTH N. SINGH. ∗. Genetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi ...

  9. Drosophila melanogaster gene expression changes after spaceflight.

    Data.gov (United States)

    National Aeronautics and Space Administration — Gene expression levels were determined in 3rd instar and adult Drosophila melanogaster reared during spaceflight to elucidate the genetic and molecular mechanisms...

  10. Ecdysteroid receptors in Drosophila melanogaster adult females

    Science.gov (United States)

    Ecdysteroid receptors were identified and partially characterized from total cell extracts of whole animals and dissected tissues from Drosophila melanogaster adult females. Binding studies indicated the presence of two ecdysteroid binding components having high affinity and specificity consistent w...

  11. Urochordate ascidians possess a single isoform of Aurora kinase that localizes to the midbody via TPX2 in eggs and cleavage stage embryos.

    Directory of Open Access Journals (Sweden)

    Celine Hebras

    Full Text Available Aurora kinases are key proteins found throughout the eukaryotes that control mitotic progression. Vertebrate Aurora-A and B kinases are thought to have evolved from a single Aurora-kinase isoform closest to that found in present day urochordates. In urochordate ascidians Aurora binds both TPX2 (a vertebrate AURKA partner and INCENP (a vertebrate AURKB partner and localizes to centrosomes and spindle microtubules as well as chromosomes and midbody during both meiosis and mitosis. Ascidian Aurora also displays this localization pattern during mitosis in echinoderms, strengthening the idea that non-vertebrate deuterostomes such as the urochordates and echinoderms possess a single form of Aurora kinase that has properties of vertebrate Aurora-kinase A and B. In the ascidian, TPX2 localizes to the centrosome and the spindle poles also as in vertebrates. However, we were surprised to find that TPX2 also localized strongly to the midbody in ascidian eggs and embryos. We thus examined more closely Aurora localization to the midbody by creating two separate point mutations of ascidian Aurora predicted to perturb binding to TPX2. Both forms of mutated Aurora behaved as predicted: neither localized to spindle poles where TPX2 is enriched. Interestingly, neither form of mutated Aurora localized to the midbody where TPX2 is also enriched, suggesting that ascidian Aurora midbody localization required TPX2 binding in ascidians. Functional analysis revealed that inhibition of Aurora kinase with a pharmacological inhibitor or with a dominant negative kinase dead form of Aurora caused cytokinesis failure and perturbed midbody formation during polar body extrusion. Our data support the view that vertebrate Aurora-A and B kinases evolved from a single non-vertebrate deuterostome ancestor. Moreover, since TPX2 localizes to the midbody in ascidian eggs and cleavage stage embryos it may be worthwhile re-assessing whether Aurora A kinase or TPX2 localize to the midbody

  12. Embryo cryopreservation and preeclampsia risk.

    Science.gov (United States)

    Sites, Cynthia K; Wilson, Donna; Barsky, Maya; Bernson, Dana; Bernstein, Ira M; Boulet, Sheree; Zhang, Yujia

    2017-11-01

    To determine whether assisted reproductive technology (ART) cycles involving cryopreserved-warmed embryos are associated with the development of preeclampsia. Retrospective cohort study. IVF clinics and hospitals. A total of 15,937 births from ART: 9,417 singleton and 6,520 twin. We used linked ART surveillance, birth certificate, and maternal hospitalization discharge data, considering resident singleton and twin births from autologous or donor eggs from 2005-2010. We compared the frequency of preeclampsia diagnosis for cryopreserved-warmed versus fresh ET and used multivariable logistic regression to adjust for confounders. Among pregnancies conceived with autologous eggs resulting in singletons, preeclampsia was greater after cryopreserved-warmed versus fresh ET (7.51% vs. 4.29%, adjusted odds ratio = 2.17 [95% CI 1.67-2.82]). Preeclampsia without and with severe features, preeclampsia with preterm delivery, and chronic hypertension with superimposed preeclampsia were more frequent after cryopreserved-warmed versus fresh ET (3.99% vs. 2.55%; 2.95% vs. 1.41%; 2.76 vs. 1.48%; and 0.95% vs. 0.43%, respectively). Among pregnancies from autologous eggs resulting in twins, the frequency of preeclampsia with severe features (9.26% vs. 5.70%) and preeclampsia with preterm delivery (14.81% vs. 11.74%) was higher after cryopreserved versus fresh transfers. Among donor egg pregnancies, rates of preeclampsia did not differ significantly between cryopreserved-warmed and fresh ET (10.78% vs. 12.13% for singletons and 28.0% vs. 25.15% for twins). Among ART pregnancies conceived using autologous eggs resulting in live births, those involving transfer of cryopreserved-warmed embryos, as compared with fresh ETs, had increased risk for preeclampsia with severe features and preeclampsia with preterm delivery. Copyright © 2017 American Society for Reproductive Medicine. All rights reserved.

  13. Mapping of gene mutations in drosophila melanogaster

    OpenAIRE

    Halvorsen, Charlotte Marie

    2004-01-01

    In this experiment, mutant genes of a given unknown mutant strain of Drosophila melanogaster were mapped to specific chromosomes. Drosophila melanogaster, commonly known as the fruit fly, was the appropriate choice for the organism to use in this specific experiment because of its relatively rapid life cycle of 10-14 days and because of the small amount of space and food neccessary for maintaining thousands of flies. The D. Melanogaster unknown strain specifically used in this experiment wa...

  14. [Association of human chorionic gonadotropin level in embryo culture media with early embryo development].

    Science.gov (United States)

    Wang, Haiying; Zhang, Renli; Han, Dong; Liu, Caixia; Cai, Jiajie; Bi, Yanling; Wen, Anmin; Quan, Song

    2014-06-01

    To investigate the association of human chorionic gonadotropin (HCG) level on day 3 of embryo culture with embryo development. Spent culture media were collected from individually cultured embryos on day 3 of in vitro fertilization and embryo transfer (IVF-ET) cycles. HCG concentration in the culture media was measured using an ELISA kit and its association with embryo development was assessed. In the 163 samples of embryo culture media from 60 patients, HCG was positive in 153 sample (93.8%) with a mean level of 0.85 ± 0.43 mIU/ml. The concentration of hCG in the culture media increased gradually as the number of blastomeres increased (F=2.273, P=0.03), and decreased as the morphological grade of the embryo was lowered (F=3.900, P=0.02). ELISA is capable of detecting HCG levels in spent culture media of embryos on day 3 of in vitro culture. The concentration of HCG in spent culture media is positively correlated with the status of early embryo development and implantation rate and thus serves as a useful marker for embryo selection in IVF-ET procedure.

  15. Causality analysis detects the regulatory role of maternal effect genes in the early Drosophila embryo

    Directory of Open Access Journals (Sweden)

    Zara Ghodsi

    2017-03-01

    Full Text Available In developmental studies, inferring regulatory interactions of segmentation genetic network play a vital role in unveiling the mechanism of pattern formation. As such, there exists an opportune demand for theoretical developments and new mathematical models which can result in a more accurate illustration of this genetic network. Accordingly, this paper seeks to extract the meaningful regulatory role of the maternal effect genes using a variety of causality detection techniques and to explore whether these methods can suggest a new analytical view to the gene regulatory networks. We evaluate the use of three different powerful and widely-used models representing time and frequency domain Granger causality and convergent cross mapping technique with the results being thoroughly evaluated for statistical significance. Our findings show that the regulatory role of maternal effect genes is detectable in different time classes and thereby the method is applicable to infer the possible regulatory interactions present among the other genes of this network.

  16. Glassfrog embryos hatch early after parental desertion.

    Science.gov (United States)

    Delia, Jesse R J; Ramírez-Bautista, Aurelio; Summers, Kyle

    2014-06-22

    Both parental care and hatching plasticity can improve embryo survival. Research has found that parents can alter hatching time owing to a direct effect of care on embryogenesis or via forms of care that cue the hatching process. Because parental care alters conditions critical for offspring development, hatching plasticity could allow embryos to exploit variation in parental behaviour. However, this interaction of parental care and hatching plasticity remains largely unexplored. We tested the hypothesis that embryos hatch early to cope with paternal abandonment in the glassfrog Hyalinobatrachium fleischmanni (Centrolenidae). We conducted male-removal experiments in a wild population, and examined embryos' response to conditions with and without fathers. Embryos hatched early when abandoned, but extended development in the egg stage when fathers continued care. Paternal care had no effect on developmental rate. Rather, hatching plasticity was due to embryos actively hatching at different developmental stages, probably in response to deteriorating conditions without fathers. Our experimental results are supported by a significant correlation between the natural timing of abandonment and hatching in an unmanipulated population. This study demonstrates that embryos can respond to conditions resulting from parental abandonment, and provides insights into how variation in care can affect selection on egg-stage adaptations.

  17. Glassfrog embryos hatch early after parental desertion

    Science.gov (United States)

    Delia, Jesse R. J.; Ramírez-Bautista, Aurelio; Summers, Kyle

    2014-01-01

    Both parental care and hatching plasticity can improve embryo survival. Research has found that parents can alter hatching time owing to a direct effect of care on embryogenesis or via forms of care that cue the hatching process. Because parental care alters conditions critical for offspring development, hatching plasticity could allow embryos to exploit variation in parental behaviour. However, this interaction of parental care and hatching plasticity remains largely unexplored. We tested the hypothesis that embryos hatch early to cope with paternal abandonment in the glassfrog Hyalinobatrachium fleischmanni (Centrolenidae). We conducted male-removal experiments in a wild population, and examined embryos' response to conditions with and without fathers. Embryos hatched early when abandoned, but extended development in the egg stage when fathers continued care. Paternal care had no effect on developmental rate. Rather, hatching plasticity was due to embryos actively hatching at different developmental stages, probably in response to deteriorating conditions without fathers. Our experimental results are supported by a significant correlation between the natural timing of abandonment and hatching in an unmanipulated population. This study demonstrates that embryos can respond to conditions resulting from parental abandonment, and provides insights into how variation in care can affect selection on egg-stage adaptations. PMID:24789892

  18. Protein phosphorylation during coconut zygotic embryo development

    International Nuclear Information System (INIS)

    Islas-Flores, I.; Oropeza, C.; Hernandez-Sotomayor, S.M.T.

    1998-01-01

    Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [gamma-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species

  19. Characterization of Autophagic Responses in Drosophila melanogaster.

    Science.gov (United States)

    Xu, T; Kumar, S; Denton, D

    2017-01-01

    Drosophila is an excellent model system for studying autophagy during animal development due to the availability of genetic reagents and opportunity for in vivo cell biological analysis. The regulation and mechanism of autophagy are highly evolutionarily conserved and the role of autophagy has been characterized during various stages of Drosophila development as well as following starvation. Studies in Drosophila have revealed novel insights into the role of distinct components of the autophagy machinery. This chapter describes protocols for examining autophagy during Drosophila development. A crucial step in the induction of autophagy is the incorporation of Atg8a into the autophagosome. This can be measured as autophagic puncta using live fluorescent imaging, immunostaining, or immunoblot analysis of LC3/Atg8a processing. The level of autophagy can also be examined using other specific components of the autophagy pathway as markers detected by immunofluorescent imaging. Based on the distinct morphology of autophagy, it can also be examined by transmission electron microscopy. In addition, one of the advantages of using Drosophila as a model is the ability to undertake genetic analysis of individual components of the autophagy machinery. Current approaches that can be used to monitor autophagy, including the overall flux and individual steps in Drosophila melanogaster, will be discussed. © 2017 Elsevier Inc. All rights reserved.

  20. Effect of non-nutritive sugars to decrease the survivorship of spotted wing drosophila, Drosophila suzukii

    Science.gov (United States)

    In this study, we investigated the effects of non-nutritive sugars and sugar alcohols on the survivorship of spotted wing drosophila, Drosophila suzukii, and found erythritol and erythrose as potentially toxic to the fly. In a dose-dependent study, erythritol and erythrose significantly reduced fly ...

  1. New record for the invasive Spotted Wing Drosophila, Drosophila suzukii Matsumura (Diptera: Drosophilidae) in Anillaco, Argentina

    Science.gov (United States)

    The invasive Spotted Wing Drosophila (SWD), Drosophila suzukii Matsumura, is reported for the first time in La Rioja, Argentina. This represents a major range expansion for this species. The natural enemies of SWD, Leptopilina clavipes and Ganaspis hookeri were also collected with the SWD at the s...

  2. To transfer fresh or thawed embryos?

    DEFF Research Database (Denmark)

    Pinborg, Anja

    2012-01-01

    Worldwide freezing and thawing of embryos has been increasingly used since the first infant was born as a result of this technique in 1984. The use of frozen embryo replacement (FER) currently even exceeds the number of fresh cycles performed in some countries. This article discusses the pros...... and multiple pregnancies, thereby increasing the safety for mother and child. Finally the article describes the accumulating literature on perinatal and long-term child outcome after transfer of frozen/thawed embryos, including a discussion on the concerns regarding cryo techniques and their possible roles...

  3. Identification of four Drosophila allatostatins as the cognate ligands for the Drosophila orphan receptor DAR-2

    DEFF Research Database (Denmark)

    Lenz, C; Williamson, M; Hansen, G N

    2001-01-01

    The allatostatins are generally inhibitory insect neuropeptides. The Drosophila orphan receptor DAR-2 is a G-protein-coupled receptor, having 47% amino acid residue identity with another Drosophila receptor, DAR-1 (which is also called dros. GPCR, or DGR) that was previously shown...... to be the receptor for an intrinsic Drosophila A-type (cockroach-type) allatostatin. Here, we have permanently expressed DAR-2 in CHO cells and found that it is the cognate receptor for four Drosophila A-type allatostatins, the drostatins-A1 to -A4. Of all the drostatins, drostatin-A4 (Thr...... weakly in the brain. The Drosophila larval gut also contains about 20-30 endocrine cells, expressing the gene for the drostatins-A1 to -A4. We suggest, therefore, that DAR-2 mediates an allatostatin (drostatin)-induced inhibition of gut motility. This is the first report on the permanent and functional...

  4. Effect of Hawthorn on Drosophila Melanogaster Antioxidant-Related ...

    African Journals Online (AJOL)

    Results: The results indicate that hawthorn extract prolonged the life span of Drosophila, with 50 % survival time of 0.8 ... Drosophila's aging gene is highly similar to humans [4,5]. ..... reduces lipid peroxidation in senescence-accelerated mice .

  5. Gustatory Processing in Drosophila melanogaster.

    Science.gov (United States)

    Scott, Kristin

    2018-01-07

    The ability to identify nutrient-rich food and avoid toxic substances is essential for an animal's survival. Although olfaction and vision contribute to food detection, the gustatory system acts as a final checkpoint control for food acceptance or rejection. The vinegar fly Drosophila melanogaster tastes many of the same stimuli as mammals and provides an excellent model system for comparative studies of taste detection. The relative simplicity of the fly brain and behaviors, along with the molecular genetic and functional approaches available in this system, allow the examination of gustatory neural circuits from sensory input to motor output. This review discusses the molecules and cells that detect taste compounds in the periphery and the circuits that process taste information in the brain. These studies are providing insight into how the detection of taste compounds regulates feeding decisions.

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

    Science.gov (United States)

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

    2016-07-15

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

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

    Science.gov (United States)

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

    2016-02-26

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

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

    DEFF Research Database (Denmark)

    Czuchra, Aleksandra; Wu, Xunwei; Meyer, Hannelore

    2005-01-01

    of Cdc42 did not affect filopodium or lamellipodium formation and had no significant influence on the speed of directed migration nor on mitosis. Cdc42-deficient cells displayed a more elongated cell shape and had a reduced area. Furthermore, directionality during migration and reorientation of the Golgi...

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

    Science.gov (United States)

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

    2016-12-01

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

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

    NARCIS (Netherlands)

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

    2006-01-01

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

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

  12. Dissecting the function and assembly of acentriolar microtubule organizing centers in Drosophila cells in vivo.

    Directory of Open Access Journals (Sweden)

    Janina Baumbach

    2015-05-01

    Full Text Available Acentriolar microtubule organizing centers (aMTOCs are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. Here we show that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2--the same proteins that appear to drive mitotic centrosome assembly in flies. This finding enabled us to ablate aMTOC formation in acentriolar cells, and so perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. Here we show that although aMTOCs can nucleate microtubules, they do not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. We find that they are required, however, for robust microtubule array assembly in cells without centrioles that also lack microtubule nucleation from around the chromatin. Importantly, aMTOCs are also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems. We propose an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs.

  13. E-cadherin is required for centrosome and spindle orientation in Drosophila male germline stem cells.

    Directory of Open Access Journals (Sweden)

    Mayu Inaba

    2010-08-01

    Full Text Available Many adult stem cells reside in a special microenvironment known as the niche, where they receive essential signals that specify stem cell identity. Cell-cell adhesion mediated by cadherin and integrin plays a crucial role in maintaining stem cells within the niche. In Drosophila melanogaster, male germline stem cells (GSCs are attached to niche component cells (i.e., the hub via adherens junctions. The GSC centrosomes and spindle are oriented toward the hub-GSC junction, where E-cadherin-based adherens junctions are highly concentrated. For this reason, adherens junctions are thought to provide a polarity cue for GSCs to enable proper orientation of centrosomes and spindles, a critical step toward asymmetric stem cell division. However, understanding the role of E-cadherin in GSC polarity has been challenging, since GSCs carrying E-cadherin mutations are not maintained in the niche. Here, we tested whether E-cadherin is required for GSC polarity by expressing a dominant-negative form of E-cadherin. We found that E-cadherin is indeed required for polarizing GSCs toward the hub cells, an effect that may be mediated by Apc2. We also demonstrated that E-cadherin is required for the GSC centrosome orientation checkpoint, which prevents mitosis when centrosomes are not correctly oriented. We propose that E-cadherin orchestrates multiple aspects of stem cell behavior, including polarization of stem cells toward the stem cell-niche interface and adhesion of stem cells to the niche supporting cells.

  14. Histone H3 Serine 28 Is Essential for Efficient Polycomb-Mediated Gene Repression in Drosophila

    Directory of Open Access Journals (Sweden)

    Philip Yuk Kwong Yung

    2015-06-01

    Full Text Available Trimethylation at histone H3K27 is central to the polycomb repression system. Juxtaposed to H3K27 is a widely conserved phosphorylatable serine residue (H3S28 whose function is unclear. To assess the importance of H3S28, we generated a Drosophila H3 histone mutant with a serine-to-alanine mutation at position 28. H3S28A mutant cells lack H3S28ph on mitotic chromosomes but support normal mitosis. Strikingly, all methylation states of H3K27 drop in H3S28A cells, leading to Hox gene derepression and to homeotic transformations in adult tissues. These defects are not caused by active H3K27 demethylation nor by the loss of H3S28ph. Biochemical assays show that H3S28A nucleosomes are a suboptimal substrate for PRC2, suggesting that the unphosphorylated state of serine 28 is important for assisting in the function of polycomb complexes. Collectively, our data indicate that the conserved H3S28 residue in metazoans has a role in supporting PRC2 catalysis.

  15. Transgenic Drosophila simulans strains prove the identity of the speciation gene Lethal hybrid rescue.

    Science.gov (United States)

    Prigent, Stéphane R; Matsubayashi, Hiroshi; Yamamoto, Masa-Toshi

    2009-10-01

    Speciation genes are responsible for genetic incompatibilities in hybrids of incipient species and therefore participate in reproductive isolation leading to complete speciation. Hybrid males between Drosophila melanogaster females and D. simulans males die at late larval or prepupal stages due to a failure in chromosome condensation during mitosis. However a mutant male of D. simulans, named Lethal hybrid rescue (Lhr), produces viable hybrid males when crossed to females of D. melanogaster. Recently the Lhr gene has been proposed as corresponding to the CG18468 gene in D. melanogaster. However this identification relied on sequence characteristics more than on a precise mapping and the use of the GAL4/UAS system to drive the transgene in D. melanogaster might have increased the complexity of interaction. Thus here we propose an independent identification of the Lhr gene based on a more precise mapping and transgenic experiments in D. simulans. We have mapped the Lhr gene by using Single Nucleotide Polymorphisms (SNPs) and identified within the candidate region the gene homologous to CG18468 as the Lhr gene as it was previously reported. Transgenic experiments in D. simulans with the native promoter of CG18468 prove that it is the Lhr gene of D. simulans by inducing the lethality of the hybrid males.

  16. Single Nucleotide Polymorphism Markers for Genetic Mapping in Drosophila melanogaster

    OpenAIRE

    Hoskins, Roger A.; Phan, Alexander C.; Naeemuddin, Mohammed; Mapa, Felipa A.; Ruddy, David A.; Ryan, Jessica J.; Young, Lynn M.; Wells, Trent; Kopczynski, Casey; Ellis, Michael C.

    2001-01-01

    For nearly a century, genetic analysis in Drosophila melanogaster has been a powerful tool for analyzing gene function, yet Drosophila lacks the molecular genetic mapping tools that recently have revolutionized human, mouse, and plant genetics. Here, we describe the systematic characterization of a dense set of molecular markers in Drosophila by using a sequence tagged site-based physical map of the genome. We identify 474 biallelic markers in standard laboratory strains of Drosophila that sp...

  17. Nucleolar remodeling in nuclear transfer embryos

    DEFF Research Database (Denmark)

    Laurincik, Jozef; Maddox-Hyttel, Poul

    2007-01-01

    Transcription of the ribosomal RNA (rRNA) genes occurs in the nucleolus and results in ribosome biogenesis. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus serve to evaluate the devel......Transcription of the ribosomal RNA (rRNA) genes occurs in the nucleolus and results in ribosome biogenesis. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus serve to evaluate...... nucleoli are not apparent until the 5th cell cycle, whereas in somatic cell nuclear transfer embryos the functional nucleoli emerge already during the 3rd cell cycle. Intergeneric reconstructed embryos produced by the fusion of bovine differentiated somatic cell to a nonactivated ovine cytoplast fail...

  18. Testing the embryo, testing the fetus.

    Science.gov (United States)

    Ehrich, K; Farsides, B; Williams, C; Scott, Rosamund

    2007-12-01

    This paper stems from an ethnographic, multidisciplinary study that explored the views and experiences of practitioners and scientists on social, ethical and clinical dilemmas encountered when working in the area of PGD for serious genetic disorders. We focus here on staff perceptions and experiences of working with embryos and helping women/couples to make choices that will result in selecting embryos for transfer and disposal of 'affected' embryos, compared to the termination of affected pregnancies following PND. Analysis and discussion of our data led us to consider the possible advantages of PGD and whether a gradualist account of the embryo's and fetus's moral status can account for all of these, particularly since a gradualist account concentrates on the significance of time (developmental stage) and makes no comment as to the significance of place (in-vitro, in-utero).

  19. Bovine in vitro embryo production : An overview

    Directory of Open Access Journals (Sweden)

    V. S. Suthar

    Full Text Available Dairy industry perfected the application of the first reproductive biotechnology, i.e. artificial insemination (AI - a great success story and also remains the user of embryo transfer technology (ETT. In addition, recently the researchers taking interest to embraced the field of Transvaginal OocyteRecovery (TVOR and in vitro production (IVEP of embryos. IVF provides the starting point for the generation of reproductive material for a number of advanced reproduction techniques including sperm microinjection and nuclear transfer (cloning. In several countries commercial IVF facilities are already being employed by cattle ET operators. Various research groups have reported on modification of TVOR technique to give greater efficiency. Much research is still needed in domestic animal (Especially Indian species on mechanisms controlling embryo development and on development of totally in vitro system for embryo culture. [Vet World 2009; 2(12.000: 478-479`

  20. Nucleolar ultrastructure in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Kanka, J; Smith, S D; Soloy, E

    1999-01-01

    in nuclear morphology as a transformation of the nucleolus precursor body into a functional rRNA synthesising nucleolus with a characteristic ultrastructure. We examined nucleolar ultrastructure in bovine in vitro produced (control) embryos and in nuclear transfer embryos reconstructed from a MII phase...... at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary...... time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear...

  1. Predator recognition in rainbowfish, Melanotaenia duboulayi, embryos.

    Directory of Open Access Journals (Sweden)

    Lois Jane Oulton

    Full Text Available Exposure to olfactory cues during embryonic development can have long term impacts on birds and amphibians behaviour. Despite the vast literature on predator recognition and responses in fishes, few researchers have determined how fish embryos respond to predator cues. Here we exposed four-day-old rainbowfish (Melanotaenia duboulayi embryos to cues emanating from a novel predator, a native predator and injured conspecifics. Their response was assessed by monitoring heart rate and hatch time. Results showed that embryos have an innate capacity to differentiate between cues as illustrated by faster heart rates relative to controls. The greatest increase in heart rate occurred in response to native predator odour. While we found no significant change in the time taken for eggs to hatch, all treatments experienced slight delays as expected if embryos are attempting to reduce exposure to larval predators.

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    microglia often occurred in clusters, some having recently incorporated bromodeoxyuridine, showing that proliferation had occurred. Annexin V labeling and staining for activated caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling showed that apoptotic mechanisms participate...... in dissolution of the microglial response. Using bone marrow chimeric mice, we found that the lesion-induced proliferative capacity of resident microglia superseded that of immigrant microglia, whereas lesion-induced kinetics of apoptosis were comparable. Microglial numbers and responses were severely reduced...... 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....

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

    Energy Technology Data Exchange (ETDEWEB)

    Kostoff, D

    1940-01-01

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

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

    International Nuclear Information System (INIS)

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

    1980-01-01

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

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

    Science.gov (United States)

    Griffin, Brian; Kilfoil, Maria L.

    2013-03-01

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

  6. Mutations in genes encoding condensin complex proteins cause microcephaly through decatenation failure at mitosis.

    Science.gov (United States)

    Martin, Carol-Anne; Murray, Jennie E; Carroll, Paula; Leitch, Andrea; Mackenzie, Karen J; Halachev, Mihail; Fetit, Ahmed E; Keith, Charlotte; Bicknell, Louise S; Fluteau, Adeline; Gautier, Philippe; Hall, Emma A; Joss, Shelagh; Soares, Gabriela; Silva, João; Bober, Michael B; Duker, Angela; Wise, Carol A; Quigley, Alan J; Phadke, Shubha R; Wood, Andrew J; Vagnarelli, Paola; Jackson, Andrew P

    2016-10-01

    Compaction of chromosomes is essential for accurate segregation of the genome during mitosis. In vertebrates, two condensin complexes ensure timely chromosome condensation, sister chromatid disentanglement, and maintenance of mitotic chromosome structure. Here, we report that biallelic mutations in NCAPD2, NCAPH, or NCAPD3, encoding subunits of these complexes, cause microcephaly. In addition, hypomorphic Ncaph2 mice have significantly reduced brain size, with frequent anaphase chromatin bridge formation observed in apical neural progenitors during neurogenesis. Such DNA bridges also arise in condensin-deficient patient cells, where they are the consequence of failed sister chromatid disentanglement during chromosome compaction. This results in chromosome segregation errors, leading to micronucleus formation and increased aneuploidy in daughter cells. These findings establish "condensinopathies" as microcephalic disorders, with decatenation failure as an additional disease mechanism for microcephaly, implicating mitotic chromosome condensation as a key process ensuring mammalian cerebral cortex size. © 2016 Martin et al.; Published by Cold Spring Harbor Laboratory Press.

  7. Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis.

    Science.gov (United States)

    Luckner, Manja; Wanner, Gerhard

    2018-05-23

    A portfolio is presented documenting economic, high-resolution correlative focused ion beam scanning electron microscopy (FIB/SEM) in routine, comprising: (i) the use of custom-labeled slides and coverslips, (ii) embedding of cells in thin, or ultra-thin resin layers for correlative light and electron microscopy (CLEM) and (iii) the claim to reach the highest resolution possible with FIB/SEM in xyz. Regions of interest (ROIs) defined in light microscope (LM), can be relocated quickly and precisely in SEM. As proof of principle, HeLa cells were investigated in 3D context at all stages of the cell cycle, documenting ultrastructural changes during mitosis: nuclear envelope breakdown and reassembly, Golgi degradation and reconstitution and the formation of the midzone and midbody.

  8. Glassfrog embryos hatch early after parental desertion

    OpenAIRE

    Delia, Jesse R. J.; Ramírez-Bautista, Aurelio; Summers, Kyle

    2014-01-01

    Both parental care and hatching plasticity can improve embryo survival. Research has found that parents can alter hatching time owing to a direct effect of care on embryogenesis or via forms of care that cue the hatching process. Because parental care alters conditions critical for offspring development, hatching plasticity could allow embryos to exploit variation in parental behaviour. However, this interaction of parental care and hatching plasticity remains largely unexplored. We tested th...

  9. Embryo disposition and the new death scene

    Directory of Open Access Journals (Sweden)

    Ellison, David

    2011-01-01

    Full Text Available In the IVF clinic - a place designed principally for the production and implantation of embryos - scientists and IVF recipients are faced with decisions regarding the disposition of frozen embryos. At this time there are hundred of thousands of cryopreserved embryos awaiting such determinations. They may be thawed for transfer to the woman herself, they may be donated for research or for use by other infertile couples, they may remain in frozen storage, or they may variously be discarded by being allowed to 'succumb', or 'perish'. Where the choice is discard, some IVF clients have chosen to formalise the process through ceremony. A new language is emerging in response to the desires of the would-be-parents who might wish to characterise the discard experience as a ‘good death’. This article examines the procedure known as ‘compassionate transfer’ where the embryo to be discarded is placed in the woman’s vagina where it is clear that it will not develop further. An alternate method has the embryo transferred in the usual manner but without the benefit of fertility-enhancing hormones at a point in the cycle unreceptive to implantation. The embryo destined for disposal is thus removed from the realm of technological possibility and ‘returned’ to the female body for a homely death. While debates continue about whether or not embryos constitute life, new practices are developing in response to the emotional experience of embryo discard. We argue that compassionate transfer is a death scene taking shape. In this article, we take the measure of this new death scene’s fabrication, and consider the form, significance, and legal complexity of its ceremonies.

  10. Role of melatonin in embryo fetal development

    OpenAIRE

    Voiculescu, SE; Zygouropoulos, N; Zahiu, CD; Zagrean, AM

    2014-01-01

    Melatonin is an indoleamine produced by the pineal gland and secreted in a circadian manner. In the past few decades, research over this topic has been enhanced. Melatonin has many important roles in the human physiology: regulator of the circadian rhythms, sleep inducer, antioxidant, anticarcinogenic. This paper reviews the involvement of melatonin in embryo fetal development. The pineal gland develops completely postpartum, so both the embryo and the fetus are dependent on the maternal mela...

  11. [Assisted reproductive technologies and the embryo status].

    Science.gov (United States)

    Englert, Y

    The status of the human embryo has always be a subject of philosophical and theological thoughts with major social consequences, but, until the 19th century, it has been mainly an abstraction. The arrival of the human embryo in vitro, materialized by Louise Brown's birth in 1978 and above all by the supernumerary embryos produced by the Australian team of Trounson and Wood following the introduction of ovarian stimulation, will turn theoretical thoughts into a reality. Nobody may ignore the hidden intentions behind the debate, as to recognise a status to a few days old embryo will immediately have a major impact on the status of a few weeks old foetus and therefore on the abortion rights. We will see that the embryo status, essentially based as well on a vision on the good and evil as on social order, cannot be based on a scientific analysis of the reproduction process but comes from a society's choice, by essence " arbitrary " and always disputable. This does not preclude the collectivity right and legitimacy to give a precise status and it is remarkable to observe the law is careful not to specify which status to give to the human embryo. It is more thru handling procedures and functioning rules that the law designed the embryo position, neither with a status of a person, nor of a thing. It nevertheless remains true that there is a constant risk that the legislation gives the embryo a status that would call into question it's unique characteristic of early reproductive stage, jeopardizing at once the hard-won reproductive freedom (reproductive choice) as well as freedom of research on embryonic stem cells, one of the most promising field of medical research.

  12. Induction of mitosis in the cultured rabbit lens initiated by the addition of insulin to medium KEI-4

    Energy Technology Data Exchange (ETDEWEB)

    Reddan, J R; Unakar, N J; Harding, C V; Bagchi, M; Saldana, G

    1975-01-01

    The epithelium of lenses cultured in KEI-4, a completely defined medium formulated with specific reference to the biochemistry and physiology of the rabbit lens, exhibits a pattern of cell division similar to that noted for the organ in situ. Initial fluctuations in mitotic activity occurred in the area of the germinative zone during the first 24 hr of culture. Mitosis decreased at 1 hr, was extremely low at 3 hr and returned to values comparable for lens in vivo by 22 hr. The precipitous drop in mitosis noted at 3 hr is in part attributable to the isolation of the lens from adjoining tissue. The addition of insulin to KEI-4 triggers a parasynchronous burst of DNA synthesis throughout the central lens epithelium. The activation requires the intact hormone; neither proinsulin nor the A and/or B chains of insulin, nor glucagon nor zinc chloride can initiate mitosis. The gamma-globulin-rich fraction of rabbit serum can also stimulate mitosis. The addition of dibutyryl adenosine 3':5' cyclic monophosphate (DBeAMP) plus theophylline to KEI-4-insulin inhibits mitosis and prevents the cells from entering the synthetic phase of the cell cycle. Theophylline alone or DBeAMP alone brings about a 90 percent reduction in the insulin-induced mitotic responses. Lenses exposed to insulin show a marked increase in RNA synthesis and also exhibit an increased binding of tritiated actinomycin D at 1 and 3 hr of culture relative to KEI-4 controls. The hormone apparently activates the genome including those genes governing cell division. The system is amenable for long-term culture of the mammalian lens and since the constituents of the medium are known it should be possible to determine the factor(s) in the medium which, in conjunction with insulin, are needed for the induction of cell division.

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

    Directory of Open Access Journals (Sweden)

    Shan Wang

    2010-03-01

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

  14. The multiple roles of Bub1 in chromosome segregation during mitosis and meiosis

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Venkatachalam, Sundaresan

    2009-06-19

    Aneuploidy, any deviation from an exact multiple of the haploid number of chromosomes, is a common occurrence in cancer and represents the most frequent chromosomal disorder in newborns. Eukaryotes have evolved mechanisms to assure the fidelity of chromosome segregation during cell division that include a multiplicity of checks and controls. One of the main cell division control mechanisms is the spindle assembly checkpoint (SAC) that monitors the proper attachment of chromosomes to spindle fibers and prevents anaphase until all kinetochores are properly attached. The mammalian SAC is composed by at least 14 evolutionary-conserved proteins that work in a coordinated fashion to monitor the establishment of amphitelic attachment of all chromosomes before allowing cell division to occur. Among the SAC proteins, the budding uninhibited by benzimidazole protein 1 (Bub1), is a highly conserved protein of prominent importance for the proper functioning of the SAC. Studies have revealed many roles for Bub1 in both mitosis and meiosis, including the localization of other SAC proteins to the kinetochore, SAC signaling, metaphase congression and the protection of the sister chromatid cohesion. Recent data show striking sex specific differences in the response to alterations in Bub1 activity. Proper Bub1 functioning is particularly important during oogenesis in preventing the generation of aneuploid gametes that can have detrimental effects on the health status of the fetus and the newborn. These data suggest that Bub1 is a master regulator of SAC and chromosomal segregation in both mitosis and meiosis. Elucidating its many essential functions in regulating proper chromosome segregation can have important consequences for preventing tumorigenesis and developmental abnormalities.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

  16. Water droplet excess free energy determined by cluster mitosis using guided molecular dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Lau, Gabriel V.; Müller, Erich A.; Jackson, George [Department of Chemical Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ (United Kingdom); Hunt, Patricia A. [Department of Chemistry, Imperial College London, South Kensington Campus, London SW7 2AZ (United Kingdom); Ford, Ian J. [Department of Physics and Astronomy and London Centre for Nanotechnology, University College London, Gower Street, London WC1E 6BT (United Kingdom)

    2015-12-28

    Atmospheric aerosols play a vital role in affecting climate by influencing the properties and lifetimes of clouds and precipitation. Understanding the underlying microscopic mechanisms involved in the nucleation of aerosol droplets from the vapour phase is therefore of great interest. One key thermodynamic quantity in nucleation is the excess free energy of cluster formation relative to that of the saturated vapour. In our current study, the excess free energy is extracted for clusters of pure water modelled with the TIP4P/2005 intermolecular potential using a method based on nonequilibrium molecular dynamics and the Jarzynski relation. The change in free energy associated with the “mitosis” or division of a cluster of N water molecules into two N/2 sub-clusters is evaluated. This methodology is an extension of the disassembly procedure used recently to calculate the excess free energy of argon clusters [H. Y. Tang and I. J. Ford, Phys. Rev. E 91, 023308 (2015)]. Our findings are compared to the corresponding excess free energies obtained from classical nucleation theory (CNT) as well as internally consistent classical theory (ICCT). The values of the excess free energy that we obtain with the mitosis method are consistent with CNT for large cluster sizes but for the smallest clusters, the results tend towards ICCT; for intermediate sized clusters, we obtain values between the ICCT and CNT predictions. Furthermore, the curvature-dependent surface tension which can be obtained by regarding the clusters as spherical droplets of bulk density is found to be a monotonically increasing function of cluster size for the studied range. The data are compared to other values reported in the literature, agreeing qualitatively with some but disagreeing with the values determined by Joswiak et al. [J. Phys. Chem. Lett. 4, 4267 (2013)] using a biased mitosis approach; an assessment of the differences is the main motivation for our current study.

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

    Energy Technology Data Exchange (ETDEWEB)

    Moriuchi, Takanobu; Kuroda, Masaki; Kusumoto, Fumiya; Osumi, Takashi; Hirose, Fumiko, E-mail: fhirose@sci.u-hyogo.ac.jp

    2016-03-01

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

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

    Science.gov (United States)

    Walsh, Charles J

    2012-01-01

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

  19. Seed priming with iron and zinc in bread wheat: effects in germination, mitosis and grain yield.

    Science.gov (United States)

    Reis, Sara; Pavia, Ivo; Carvalho, Ana; Moutinho-Pereira, José; Correia, Carlos; Lima-Brito, José

    2018-07-01

    Currently, the biofortification of crops like wheat with micronutrients such as iron (Fe) and zinc (Zn) is extremely important due to the deficiencies of these micronutrients in the human diet and in soils. Agronomic biofortification with Fe and Zn can be done through different exogenous strategies such as soil application, foliar spraying, and seed priming. However, the excess of these micronutrients can be detrimental to the plants. Therefore, in the last decade, a high number of studies focused on the evaluation of their phytotoxic effects to define the best strategies for biofortification of bread wheat. In this study, we investigated the effects of seed priming with different dosages (1 mg L -1 to 8 mg L -1 ) of Fe and/or Zn in germination, mitosis and yield of bread wheat cv. 'Jordão' when compared with control. Overall, our results showed that: micronutrient dosages higher than 4 mg L -1 negatively affect the germination; Fe and/or Zn concentrations higher than 2 mg L -1 significantly decrease the mitotic index and increase the percentage of dividing cells with anomalies; treatments performed with 8 mg L -1 of Fe and/or 8 mg L -1 Zn caused negative effects in germination, mitosis and grain yield. Moreover, seed priming with 2 mg L -1 Fe + 2 mg L -1 Zn has been shown to be non-cytotoxic, ensuring a high rate of germination (80%) and normal dividing cells (90%) as well as improving tillering and grain yield. This work revealed that seed priming with Fe and Zn micronutrients constitutes a useful and alternative approach for the agronomic biofortification of bread wheat.

  20. Effects of alpha particles on zebrafish embryos

    International Nuclear Information System (INIS)

    Yum, E.H.W.; Choi, V.W.Y.; Yu, K.N.; Li, V.W.T.; Cheng, S.H.

    2008-01-01

    Full text: Ionizing radiation such as X-ray and alpha particles can damage cellular macromolecules, which can lead to DNA single- and double-strand breaks. In the present work, we studied the effects of alpha particles on dechorionated zebrafish embryos. Thin polyallyldiglycol carbonate (PADC) films with a thickness of 16 μm were prepared from commercially available PADC films (with thickness of 100 μm) by chemical etching and used as support substrates for holding zebrafish embryos for alpha-particle irradiation. These films recorded alpha-particle hit positions, quantified the number and energy of alpha particles actually incident on the embryo cells, and thus enabled the calculation of the dose absorbed by the embryo cells. Irradiation was made at 1.25 hours post fertilization (hpf) with various absorbed dose. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was performed on the embryos at different time stages after irradiation. Marked apoptosis was detected only in embryos at earlier time stages. The results showed that DNA double-strand break during zebrafish embryogenesis can be induced by alpha-particle irradiation, which suggests that zebrafish is a potential model for assessing the effects of alpha-particle radiation

  1. Cryopreservation of Arachis pintoi (leguminosae) somatic embryos.

    Science.gov (United States)

    Rey, H Y; Faloci, M; Medina, R; Dolce, N; Engelmann, F; Mroginski, L

    2013-01-01

    In this study, we successfully cryopreserved cotyledonary somatic embryos of diploid and triploid Arachis pintoi cytotypes using the encapsulation-dehydration technique. The highest survival rates were obtained when somatic embryos were encapsulated in calcium alginate beads and precultured in agitated (80 rpm) liquid establishment medium (EM) with daily increasing sucrose concentration (0.50, 0.75, and 1.0 M). The encapsulated somatic embryos were then dehydrated with silica gel for 5 h to 20% moisture content (fresh weight basis) and cooled either rapidly (direct immersion in liquid nitrogen, LN) or slowly (1 degree C per min from 25 degree C to -30 degree C followed by immersion in LN). Beads were kept in LN for a minimum of 1 h and then were rapidly rewarmed in a 30 degree C water-bath for 2 min. Finally, encapsulated somatic embryos were post-cultured in agitated (80 rpm) liquid EM with daily decreasing sucrose concentration (0.75 and 0.5 M) and transferred to solidified EM. Using this protocol, we obtained 26% and 30% plant regeneration from cryopreserved somatic embryos of diploid and triploid cytotypes. No morphological abnormalities were observed in any of the plants regenerated from cryopreserved embryos and their genetic stability was confirmed with 10 isozyme systems and nine RAPD profiles.

  2. The maternal nucleolus plays a key role in centromere satellite maintenance during the oocyte to embryo transition.

    Science.gov (United States)

    Fulka, Helena; Langerova, Alena

    2014-04-01

    The oocyte (maternal) nucleolus is essential for early embryonic development and embryos originating from enucleolated oocytes arrest at the 2-cell stage. The reason for this is unclear. Surprisingly, RNA polymerase I activity in nucleolus-less mouse embryos, as manifested by pre-rRNA synthesis, and pre-rRNA processing are not affected, indicating an unusual role of the nucleolus. We report here that the maternal nucleolus is indispensable for the regulation of major and minor satellite repeats soon after fertilisation. During the first embryonic cell cycle, absence of the nucleolus causes a significant reduction in major and minor satellite DNA by 12% and 18%, respectively. The expression of satellite transcripts is also affected, being reduced by more than half. Moreover, extensive chromosome bridging of the major and minor satellite sequences was observed during the first mitosis. Finally, we show that the absence of the maternal nucleolus alters S-phase dynamics and causes abnormal deposition of the H3.3 histone chaperone DAXX in pronuclei of nucleolus-less zygotes.

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

    International Nuclear Information System (INIS)

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

    1982-01-01

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

  4. Wash functions downstream of Rho1 GTPase in a subset of Drosophila immune cell developmental migrations

    Science.gov (United States)

    Verboon, Jeffrey M.; Rahe, Travis K.; Rodriguez-Mesa, Evelyn; Parkhurst, Susan M.

    2015-01-01

    Drosophila immune cells, the hemocytes, undergo four stereotypical developmental migrations to populate the embryo, where they provide immune reconnoitering, as well as a number of non–immune-related functions necessary for proper embryogenesis. Here, we describe a role for Rho1 in one of these developmental migrations in which posteriorly located hemocytes migrate toward the head. This migration requires the interaction of Rho1 with its downstream effector Wash, a Wiskott–Aldrich syndrome family protein. Both Wash knockdown and a Rho1 transgene harboring a mutation that prevents Wash binding exhibit the same developmental migratory defect as Rho1 knockdown. Wash activates the Arp2/3 complex, whose activity is needed for this migration, whereas members of the WASH regulatory complex (SWIP, Strumpellin, and CCDC53) are not. Our results suggest a WASH complex–independent signaling pathway to regulate the cytoskeleton during a subset of hemocyte developmental migrations. PMID:25739458

  5. Functions and Mechanisms of Fibroblast Growth Factor (FGF Signalling in Drosophila melanogaster

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    Hans-Arno J. Müller

    2013-03-01

    Full Text Available Intercellular signalling via growth factors plays an important role in controlling cell differentiation and cell movements during the development of multicellular animals. Fibroblast Growth Factor (FGF signalling induces changes in cellular behaviour allowing cells in the embryo to move, to survive, to divide or to differentiate. Several examples argue that FGF signalling is used in multi-step morphogenetic processes to achieve and maintain a transitional state of the cells required for the control of cell fate. In the genetic model Drosophila melanogaster, FGF signalling via the receptor tyrosine kinases Heartless (Htl and Breathless (Btl is particularly well studied. These FGF receptors affect gene expression, cell shape and cell–cell interactions during mesoderm layer formation, caudal visceral muscle (CVM formation, tracheal morphogenesis and glia differentiation. Here, we will address the current knowledge of the biological functions of FGF signalling in the fly on the tissue, at a cellular and molecular level.

  6. Characterization of a depurinated-DNA purine-base-insertion activity from Drosophila.

    Science.gov (United States)

    Deutsch, W A; Spiering, A L

    1985-01-01

    An activity that binds preferentially to depurinated DNA and inserts purines into those sites was partially purified from Drosophila melanogaster embryos. The protein has a sedimentation coefficient of 4.9 S and is devoid of AP (apurinic/apyrimidinic) endonuclease activity. Upon incorporation of purines into apurinic DNA, the number of alkali-labile sites decreases, thus establishing the conversion of depurinated sites into normal nucleotides. The activity requires K+, and is totally inhibited by caffeine or EDTA. Guanine is specifically incorporated into partially depurinated poly(dG-dC) and adenine is specifically incorporated into poly(dA-dT), thus demonstrating the apparent template specificity of the enzyme. PMID:2417589

  7. Transcripts of mobile element MDG1 during ontogenesis of Drosophila melanogaster

    International Nuclear Information System (INIS)

    Kuvakina, A.I.; Nurminskii, D.I.; Kogan, G.L.; Gvozdev, V.A.

    1989-01-01

    It has been demonstrated by Northern hybridization using a single-stranded labeled probes that the number of MDG1 transcripts as well as their size change during ontogenesis of Drosophila. The transcripts of MDG1 were not found in unfertilized eggs. The full-length transcript of MDG1 (about 7 kb long) appears in the embryonic and larval cells, and its quantity sharply increases in pupae and adults. A transcript of about 5 kb length is also found in the pupae and adults. Another, about 2 kb long transcript forms in the embryos, pupae and adults, which is absent in larvae. The main transcript in the larval cells, complementary to the inner part of the body of MDG1, is about 1 kb long. The transcription level of MDG1 and the mobile element copia do not change under heat shock at adult stage

  8. Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation.

    Science.gov (United States)

    Stigliani, S; Anserini, P; Venturini, P L; Scaruffi, P

    2013-10-01

    Is the amount of cell-free DNA released by human embryos into culture medium correlated with embryo morphological features? The mitochondrial DNA (mtDNA) content of culture medium is significantly associated with the fragmentation rate on Days 2 and 3 of embryo development, whether the oocyte came from women ≤ 35 or >35 years old. Cellular fragmentation is often utilized as one of the morphological parameters for embryo quality assessment. The amount of cellular fragments is considered to be an important morphological parameter for embryo implantation potential. It has been hypothesized that fragments are apoptotic bodies or anuclear cytoplasmatic pieces of blastomeres, although no definitive conclusion has been drawn about their pathogenesis. Human fertilized oocytes were individually cultured from Day 1 to Days 2 and 3. A total of 800 samples (166 spent media from Day 2 and 634 from Day 3) were enrolled into the present study. Double-stranded DNA (dsDNA) was quantified in 800 spent embryo culture media by Pico Green dye fluorescence assay. After DNA purification, genomic DNA (gDNA) and mtDNA were profiled by specific quantitative PCR. Statistical analyses defined correlations among DNA contents, embryo morphology and maternal age. Different independent tests confirmed the presence of DNA into embryo culture medium and, for the first time, we demonstrate that both gDNA and mtDNA are detectable in the secretome. The amount of DNA is larger in embryos with bad quality cleavage compared with high-grade embryos, suggesting that the DNA profile of culture medium is an objective marker for embryo quality assessment. In particular, DNA profiles are significantly associated with fragmentation feature (total dsDNA: P = 0.0010; mtDNA; P = 0.0247) and advanced maternal age. It is necessary to establish whether DNA profiling of spent embryo culture medium is a robust onsite test that can improve the prediction of blastulation, implantation and/or pregnancy rate. The

  9. Miniaturized embryo array for automated trapping, immobilization and microperfusion of zebrafish embryos.

    Directory of Open Access Journals (Sweden)

    Jin Akagi

    Full Text Available Zebrafish (Danio rerio has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP. The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale.

  10. Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis.

    Science.gov (United States)

    Douglas, Pauline; Ye, Ruiqiong; Trinkle-Mulcahy, Laura; Neal, Jessica A; De Wever, Veerle; Morrice, Nick A; Meek, Katheryn; Lees-Miller, Susan P

    2014-06-25

    The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs' role in mitosis may be mechanistically distinct from its well-established role in NHEJ.

  11. Automated high-throughput quantification of mitotic spindle positioning from DIC movies of Caenorhabditis embryos.

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    David Cluet

    Full Text Available The mitotic spindle is a microtubule-based structure that elongates to accurately segregate chromosomes during anaphase. Its position within the cell also dictates the future cell cleavage plan, thereby determining daughter cell orientation within a tissue or cell fate adoption for polarized cells. Therefore, the mitotic spindle ensures at the same time proper cell division and developmental precision. Consequently, spindle dynamics is the matter of intensive research. Among the different cellular models that have been explored, the one-cell stage C. elegans embryo has been an essential and powerful system to dissect the molecular and biophysical basis of spindle elongation and positioning. Indeed, in this large and transparent cell, spindle poles (or centrosomes can be easily detected from simple DIC microscopy by human eyes. To perform quantitative and high-throughput analysis of spindle motion, we developed a computer program ACT for Automated-Centrosome-Tracking from DIC movies of C. elegans embryos. We therefore offer an alternative to the image acquisition and processing of transgenic lines expressing fluorescent spindle markers. Consequently, experiments on large sets of cells can be performed with a simple setup using inexpensive microscopes. Moreover, analysis of any mutant or wild-type backgrounds is accessible because laborious rounds of crosses with transgenic lines become unnecessary. Last, our program allows spindle detection in other nematode species, offering the same quality of DIC images but for which techniques of transgenesis are not accessible. Thus, our program also opens the way towards a quantitative evolutionary approach of spindle dynamics. Overall, our computer program is a unique macro for the image- and movie-processing platform ImageJ. It is user-friendly and freely available under an open-source licence. ACT allows batch-wise analysis of large sets of mitosis events. Within 2 minutes, a single movie is processed

  12. Gut-associated microbes of Drosophila melanogaster

    Science.gov (United States)

    Broderick, Nichole; Lemaitre, Bruno

    2012-01-01

    There is growing interest in using Drosophila melanogaster to elucidate mechanisms that underlie the complex relationships between a host and its microbiota. In addition to the many genetic resources and tools Drosophila provides, its associated microbiota is relatively simple (1–30 taxa), in contrast to the complex diversity associated with vertebrates (> 500 taxa). These attributes highlight the potential of this system to dissect the complex cellular and molecular interactions that occur between a host and its microbiota. In this review, we summarize what is known regarding the composition of gut-associated microbes of Drosophila and their impact on host physiology. We also discuss these interactions in the context of their natural history and ecology and describe some recent insights into mechanisms by which Drosophila and its gut microbiota interact. “Workers with Drosophila have been considered fortunate in that they deal with the first multicellular invertebrate to be cultured monoxenically (Delcourt and Guyenot, 1910); the first to be handled axenically on a semisynthetic diet (Guyenot, 1917); and the first to be grown on a defined diet (Schultz et al., 1946). This list of advantages is somewhat embarrassing, since it implies an interest in nutrition that, in reality, was only secondary. The very first studies were concerned with the reduction of variability in genetic experiments (Delcourt and Guyenot, 1910) and standardization of the nutritional environment.” -James Sang, 1959 Ann NY Acad 1 PMID:22572876

  13. Wolbachia-induced cytoplasmic incompatibility is associated with decreased Hira expression in male Drosophila.

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    Ya Zheng

    Full Text Available BACKGROUND: Wolbachia are obligate endosymbiotic bacteria that infect numerous species of arthropods and nematodes. Wolbachia can induce several reproductive phenotypes in their insect hosts including feminization, male-killing, parthenogenesis and cytoplasmic incompatibility (CI. CI is the most common phenotype and occurs when Wolbachia-infected males mate with uninfected females resulting in no or very low numbers of viable offspring. However, matings between males and females infected with the same strain of Wolbachia result in viable progeny. Despite substantial scientific effort, the molecular mechanisms underlying CI are currently unknown. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression studies were undertaken in Drosophila melanogaster and D. simulans which display differential levels of CI using quantitative RT-PCR. We show that Hira expression is correlated with the induction of CI and occurs in a sex-specific manner. Hira expression is significantly lower in males which induce strong CI when compared to males inducing no CI or Wolbachia-uninfected males. A reduction in Hira expression is also observed in 1-day-old males that induce stronger CI compared to 5-day-old males that induce weak or no CI. In addition, Hira mutated D. melanogaster males mated to uninfected females result in significantly decreased hatch rates comparing with uninfected crosses. Interestingly, wMel-infected females may rescue the hatch rates. An obvious CI phenotype with chromatin bridges are observed in the early embryo resulting from Hira mutant fertilization, which strongly mimics the defects associated with CI. CONCLUSIONS/SIGNIFICANCE: Our results suggest Wolbachia-induced CI in Drosophila occurs due to a reduction in Hira expression in Wolbachia-infected males leading to detrimental effects on sperm fertility resulting in embryo lethality. These results may help determine the underlying mechanism of CI and provide further insight in to the important role

  14. mei-9/sup a/ mutant of Drosophila melanogaster increases mutagen sensitivity and decreases excision repair

    International Nuclear Information System (INIS)

    Boyd, J.B.; Golino, M.D.; Setlow, R.B.

    1976-01-01

    The mei-9/sup a/ mutant of Drosophila melanogaster, which reduces meiotic recombination in females, is deficient in the excision of uv-induced pyrimidine dimers in both sexes. Assays were performed in primary cultures and established cell lines derived from embryos. An endonuclease preparation from M. luteus, which is specific for pyrimidine dimers, was employed to monitor uv-induced dimers in cellular DNA. The rate of disappearance of endonuclease-sensitive sites from DNA of control cells is 10-20 times faster than that from mei-9/sup a/ cells. The mutant mei-218, which is also deficient in meiotic recombination, removes nuclease-sensitive sites at control rates. The mei-9/sup a/ cells exhibit control levels of photorepair, postreplication repair and repair of single strand breaks. In mei-9 cells DNA synthesis and possibly postreplication repair are weakly sensitive to caffeine. Larvae which are hemizygous for either of the two mutants that define the mei-9 locus are hypersensitive to killing by the mutagens methyl methanesulfonate, nitrogen mustard and 2-acetylaminofluorene. Larvae hemizygous for the mei-218 mutant are insensitive to each of these reagents. These data demonstrate that the mei-9 locus is active in DNA repair of somatic cells. Thus functions involved in meiotic recombination are also active in DNA repair in this higher eukaryote. The results are consistent with the earlier suggestions that the mei-9 locus functions in the exchange events of meiosis. The mei-218 mutation behaves differently in genetic tests and our data suggest its function may be restricted to meiosis. These studies demonstrate that currently recognized modes of DNA repair can be efficiently detected in primary cell cultures derived from Drosophila embryos

  15. Time to take human embryo culture seriously.

    Science.gov (United States)

    Sunde, Arne; Brison, Daniel; Dumoulin, John; Harper, Joyce; Lundin, Kersti; Magli, M Cristina; Van den Abbeel, Etienne; Veiga, Anna

    2016-10-01

    Is it important that end-users know the composition of human embryo culture media? We argue that there is as strong case for full transparency concerning the composition of embryo culture media intended for human use. Published data suggest that the composition of embryo culture media may influence the phenotype of the offspring. A review of the literature was carried out. Data concerning the potential effects on embryo development of culture media were assessed and recommendations for users made. The safety of ART procedures, especially with respect to the health of the offspring, is of major importance. There are reports from the literature indicating a possible effect of culture conditions, including culture media, on embryo and fetal development. Since the introduction of commercially available culture media, there has been a rapid development of different formulations, often not fully documented, disclosed or justified. There is now evidence that the environment the early embryo is exposed to can cause reprogramming of embryonic growth leading to alterations in fetal growth trajectory, birthweight, childhood growth and long-term disease including Type II diabetes and cardiovascular problems. The mechanism for this is likely to be epigenetic changes during the preimplantation period of development. In the present paper the ESHRE working group on culture media summarizes the present knowledge of potential effects on embryo development related to culture media, and makes recommendations. There is still a need for large prospective randomized trials to further elucidate the link between the composition of embryo culture media used and the phenotype of the offspring. We do not presently know if the phenotypic changes induced by in vitro embryo culture represent a problem for long-term health of the offspring. Published data indicate that there is a strong case for demanding full transparency concerning the compositions of and the scientific rationale behind the

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

    International Nuclear Information System (INIS)

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

    1994-01-01

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

  17. Meanings of the embryo in Japan: narratives of IVF experience and embryo ownership

    NARCIS (Netherlands)

    Kato, M.; Sleeboom-Faulkner, M.

    2011-01-01

    This article explores the sociocultural meanings of the embryo implied in the narratives of 58 women who have undergone in vitro fertilisation in Japan over a period from 2006 to 2008. We argue that a lack of sufficient analysis of the sociocultural meanings of the embryo result in a situation where

  18. Differentiated muscles are mandatory for gas-filling of the Drosophila airway system

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    Yiwen Wang

    2015-12-01

    Full Text Available At the end of development, organs acquire functionality, thereby ensuring autonomy of an organism when it separates from its mother or a protective egg. In insects, respiratory competence starts when the tracheal system fills with gas just before hatching of the juvenile animal. Cellular and molecular mechanisms of this process are not fully understood. Analyses of the phenotype of Drosophila embryos with malformed muscles revealed that they fail to gas-fill their tracheal system. Indeed, we show that major regulators of muscle formation like Lame duck and Blown fuse are important, while factors involved in the development of subsets of muscles including cardiac and visceral muscles are dispensable for this process, suggesting that somatic muscles (or parts of them are essential to enable tracheal terminal differentiation. Based on our phenotypic data, we assume that somatic muscle defect severity correlates with the penetrance of the gas-filling phenotype. This argues that a limiting molecular or mechanical muscle-borne signal tunes tracheal differentiation. We think that in analogy to the function of smooth muscles in vertebrate lungs, a balance of physical forces between muscles and the elasticity of tracheal walls may be decisive for tracheal terminal differentiation in Drosophila.

  19. Characterization of postreplication repair in mutagen-sensitive strains of Drosophila melanogaster

    International Nuclear Information System (INIS)

    Boyd, J.B.; Setlow, R.B.

    1976-01-01

    Mutants of Drosophila melanogaster, with suspected repair deficiencies, were analyzed for their capacity to repair damage induced by x-rays, and uv radiation. Analysis was performed on cell cultures derived from embryos of homozygous mutant stocks. Postreplication repair following uv radiation has been analyzed in mutant stocks derived from a total of ten complementation groups. Cultures were irradiated, pulse-labeled, and incubated in the dark prior to analysis by alkaline sucrose gradient centrifugation. Kinetics of the molecular weight increase in newly synthesized DNA were assayed after cells had been incubated in the presence or absence of caffeine. Two separate pathways of postreplication repair have been tentatively identified by mutants derived from four complementation groups. The proposed caffeine sensitive pathway (CAS) is defined by mutants which also disrupt meiosis. The second pathway (CIS) is caffeine insensitive and is not yet associated with meiotic functions. All mutants deficient in postreplication repair are also sensitive to nitrogen mustard. The mutants investigated display a normal capacity to repair single-strand breaks induced in DNA by x-rays, although two may possess a reduced capacity to repair damage caused by localized incorporation of high specific activity thymidine- 3 H. The data have been employed to construct a model for repair of uv-induced damage in Drosophila DNA. Implications of the model for DNA repair in mammals are discussed

  20. High-dose irradiation induces cell cycle arrest, apoptosis, and developmental defects during Drosophila oogenesis.

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    Hee Jin Shim

    Full Text Available Ionizing radiation (IR treatment induces a DNA damage response, including cell cycle arrest, DNA repair, and apoptosis in metazoan somatic cells. Because little has been reported in germline cells, we performed a temporal analysis of the DNA damage response utilizing Drosophila oogenesis as a model system. Oogenesis in the adult Drosophila female begins with the generation of 16-cell cyst by four mitotic divisions of a cystoblast derived from the germline stem cells. We found that high-dose irradiation induced S and G2 arrests in these mitotically dividing germline cells in a grp/Chk1- and mnk/Chk2-dependent manner. However, the upstream kinase mei-41, Drosophila ATR ortholog, was required for the S-phase checkpoint but not for the G2 arrest. As in somatic cells, mnk/Chk2 and dp53 were required for the major cell death observed in early oogenesis when oocyte selection and meiotic recombination occurs. Similar to the unscheduled DNA double-strand breaks (DSBs generated from defective repair during meiotic recombination, IR-induced DSBs produced developmental defects affecting the spherical morphology of meiotic chromosomes and dorsal-ventral patterning. Moreover, various morphological abnormalities in the ovary were detected after irradiation. Most of the IR-induced defects observed in oogenesis were reversible and were restored between 24 and 96 h after irradiation. These defects in oogenesis severely reduced daily egg production and the hatch rate of the embryos of irradiated female. In summary, irradiated germline cells induced DSBs, cell cycle arrest, apoptosis, and developmental defects resulting in reduction of egg production and defective embryogenesis.

  1. Characterization of the Drosophila ortholog of the human Usher Syndrome type 1G protein sans.

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    Fabio Demontis

    Full Text Available BACKGROUND: The Usher syndrome (USH is the most frequent deaf-blindness hereditary disease in humans. Deafness is attributed to the disorganization of stereocilia in the inner ear. USH1, the most severe subtype, is associated with mutations in genes encoding myosin VIIa, harmonin, cadherin 23, protocadherin 15, and sans. Myosin VIIa, harmonin, cadherin 23, and protocadherin 15 physically interact in vitro and localize to stereocilia tips in vivo, indicating that they form functional complexes. Sans, in contrast, localizes to vesicle-like structures beneath the apical membrane of stereocilia-displaying hair cells. How mutations in sans result in deafness and blindness is not well understood. Orthologs of myosin VIIa and protocadherin 15 have been identified in Drosophila melanogaster and their genetic analysis has identified essential roles in auditory perception and microvilli morphogenesis, respectively. PRINCIPAL FINDINGS: Here, we have identified and characterized the Drosophila ortholog of human sans. Drosophila Sans is expressed in tubular organs of the embryo, in lens-secreting cone cells of the adult eye, and in microvilli-displaying follicle cells during oogenesis. Sans mutants are viable, fertile, and mutant follicle cells appear to form microvilli, indicating that Sans is dispensable for fly development and microvilli morphogenesis in the follicle epithelium. In follicle cells, Sans protein localizes, similar to its vertebrate ortholog, to intracellular punctate structures, which we have identified as early endosomes associated with the syntaxin Avalanche. CONCLUSIONS: Our work is consistent with an evolutionary conserved function of Sans in vesicle trafficking. Furthermore it provides a significant basis for further understanding of the role of this Usher syndrome ortholog in development and disease.

  2. Cryopreservation of peach palm zygotic embryos.

    Science.gov (United States)

    Steinmacher, Douglas A; Saldanha, Cleber W; Clement, Charles R; Guerra, Miguel P

    2007-01-01

    Cryopreservation is a safe and cost-effective option for long-term germplasm conservation of non-orthodox seed species, such as peach palm (Bactris gasipaes). The objective of the present study was to establish a cryopreservation protocol for peach palm zygotic embryos based on the encapsulation-dehydration technique. After excision, zygotic embryos were encapsulated with 3 percent sodium alginate plus 2 M glycerol and 0.4 M sucrose, and pre-treated or not with 1 M sucrose during 24 h, followed by air-drying. Fresh weight water contents of beads decreased from 83 percent and 87 percent to 18 percent and 20 percent for pre-treated or non-pretreated beads, respectively, after 4 h of dehydration. Sucrose pre-treatment at 1 M caused lower zygotic embryo germination and plantlet height in contrast to non-treated beads. All the variables were statistically influenced by dehydration time. Optimal conditions for recovery of cryopreserved zygotic embryos include encapsulation and dehydration for 4 h in a forced air cabinet to 20 percent water content, followed by rapid freezing in liquid nitrogen (-196 degree C) and rapid thawing at 45 degree C. In these conditions 29 percent of the zygotic embryos germinated in vitro. However, plantlets obtained from dehydrated zygotic embryos had stunted haustoria and lower heights. Histological analysis showed that haustorium cells were large, vacuolated, with few protein bodies. In contrast, small cells with high nucleus:cytoplasm ratio formed the shoot apical meristem of the embryos, which were the cell types with favorable characteristics for survival after exposure to liquid nitrogen. Plantlets were successfully acclimatized and showed 41+/-9 percent and 88+/-4 percent survival levels after 12 weeks of acclimatization from cryopreserved and non-cryopreserved treatments, respectively.

  3. Developmental competence of porcine chimeric embryos produced by aggregation

    DEFF Research Database (Denmark)

    Li, Juan; Jakobsen, Jannik E.; Xiong, Qiang

    2015-01-01

    The purpose of our study was to compare the developmental competence and blastomere allocation of porcine chimeric embryos formed by micro-well aggregation. Chimeras were created by aggregating either two blastomeres originating from 2-cell embryos or two whole embryos, where embryos were produced...... either by parthenogenetic activation (PA) or handmade cloning (HMC). Results showed that the developmental competence of chimeric embryos, evaluated based on their blastocyst rate and total cell number per blastocyst, was increased when two whole 2-cell stage embryos (PA or HMC) were aggregated....... In comparison, when two blastomeres were aggregated, the developmental competence of the chimeric embryos decreased if the blastomeres were either from PA or from HMC embryos, but not if they were from different sources, i.e. one PA and one HMC blastomere. To evaluate the cell contribution in embryo formation...

  4. Patients' Attitudes towards the Surplus Frozen Embryos in China

    Directory of Open Access Journals (Sweden)

    Xuan Jin

    2013-01-01

    Full Text Available Background. Assisted reproductive techniques have been used in China for more than 20 years. This study investigates the attitudes of surplus embryo holders towards embryos storage and donation for medical research. Methods. A total of 363 couples who had completed in vitro fertilization (IVF treatment and had already had biological children but who still had frozen embryos in storage were invited to participate. Interviews were conducted by clinics in a narrative style. Results. Family size was the major reason for participants’ (discontinuation of embryo storage; moreover, the moral status of embryos was an important factor for couples choosing embryo storage, while the storage fee was an important factor for couples choosing embryo disposal. Most couples discontinued the storage of their embryos once their children were older than 3 years. In our study, 58.8% of the couples preferred to dispose of surplus embryos rather than donate them to research, citing a lack of information and distrust in science as significant reasons for their decision. Conclusions. Interviews regarding frozen embryos, including patients’ expectations for embryo storage and information to assist them with decisions regarding embryo disposal, are beneficial for policies addressing embryo disposition and embryo donation in China.

  5. Embryos, individuals, and persons: an argument against embryo creation and research.

    Science.gov (United States)

    Tollefsen, C

    2001-01-01

    One strategy for arguing that it should be legally permissible to create human embryos, or to use spare human embryos, for scientific research purposes involves the claim that such embryos cannot be persons because they are not human individuals while twinning may yet take place. Being a human individual is considered to be by most people a necessary condition for being a human person. I argue first that such an argument against the personhood of embryos must be rationally conclusive if their destruction in public places such as laboratories is to be countenanced. I base this argument on a popular understanding of the role that the notion of privacy plays in abortion laws. I then argue that such arguments against personhood are not rationally conclusive. The claim that the early embryos is not a human individual is not nearly as obvious as some assert.

  6. Viruses and Antiviral Immunity in Drosophila

    Science.gov (United States)

    Xu, Jie; Cherry, Sara

    2013-01-01

    Viral pathogens present many challenges to organisms, driving the evolution of a myriad of antiviral strategies to combat infections. A wide variety of viruses infect invertebrates, including both natural pathogens that are insect-restricted, and viruses that are transmitted to vertebrates. Studies using the powerful tools available in the model organism Drosophila have expanded our understanding of antiviral defenses against diverse viruses. In this review, we will cover three major areas. First, we will describe the tools used to study viruses in Drosophila. Second, we will survey the major viruses that have been studied in Drosophila. And lastly, we will discuss the well-characterized mechanisms that are active against these diverse pathogens, focusing on non-RNAi mediated antiviral mechanisms. Antiviral RNAi is discussed in another paper in this issue. PMID:23680639

  7. Receptor Tyrosine Kinases in Drosophila Development

    Science.gov (United States)

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

    Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

  8. Apoptosis in Drosophila: which role for mitochondria?

    Science.gov (United States)

    Clavier, Amandine; Rincheval-Arnold, Aurore; Colin, Jessie; Mignotte, Bernard; Guénal, Isabelle

    2016-03-01

    It is now well established that the mitochondrion is a central regulator of mammalian cell apoptosis. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, mainly because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and cell death in Drosophila occurs at the mitochondrial level. Numerous proteins, including RHG proteins and proteins of the Bcl-2 family that are key regulators of Drosophila apoptosis, constitutively or transiently localize in mitochondria. These proteins participate in the cell death process at different levels such as degradation of Diap1, a Drosophila IAP, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. Here, we review these mitochondrial events that might have their counterpart in human.

  9. Generation of single-copy transgenic mouse embryos directly from ES cells by tetraploid embryo complementation

    Directory of Open Access Journals (Sweden)

    Zhao Roong

    2001-12-01

    Full Text Available Abstract Background Transgenic mice have been used extensively to analyze gene function. Unfortunately, traditional transgenic procedures have only limited use in analyzing alleles that cause lethality because lines of founder mice cannot be established. This is frustrating given that such alleles often reveal crucial aspects of gene function. For this reason techniques that facilitate the generation of embryos expressing such alleles would be of enormous benefit. Although the transient generation of transgenic embryos has allowed limited analysis of lethal alleles, it is expensive, time consuming and technically challenging. Moreover a fundamental limitation with this approach is that each embryo generated is unique and transgene expression is highly variable due to the integration of different transgene copy numbers at random genomic sites. Results Here we describe an alternative method that allows the generation of clonal mouse embryos harboring a single-copy transgene at a defined genomic location. This was facilitated through the production of Hprt negative embryonic stem cells that allow the derivation of embryos by tetraploid embryo complementation. We show that targeting transgenes to the hprt locus in these ES cells by homologous recombination can be efficiently selected by growth in HAT medium. Moreover, embryos derived solely from targeted ES cells containing a single copy LacZ transgene under the control of the α-myosin heavy chain promoter exhibited the expected cardiac specific expression pattern. Conclusion Our results demonstrate that tetraploid embryo complementation by F3 hprt negative ES cells facilitates the generation of transgenic mouse embryos containing a single copy gene at a defined genomic locus. This approach is simple, extremely efficient and bypasses any requirement to generate chimeric mice. Moreover embryos generated by this procedure are clonal in that they are all derived from a single ES cell lines. This

  10. Metabolite profiling of somatic embryos of Cyclamen persicum in comparison to zygotic embryos, endosperm and testa

    Directory of Open Access Journals (Sweden)

    Traud eWinkelmann

    2015-08-01

    Full Text Available Somatic embryogenesis has been shown to be an efficient in vitro plant regeneration system for many crops such as the important ornamental plant Cyclamen persicum, for which this regeneration pathway of somatic embryogenesis is of interest for the vegetative propagation of parental lines as well as elite plants. However, somatic embryogenesis is not commercially used in many crops due to several unsolved problems, such as malformations, asynchronous development, deficiencies in maturation and germination of somatic embryos. In contrast, zygotic embryos in seeds develop and germinate without abnormalities in most cases. Instead of time-consuming and labor-intensive experiments involving tests of different in vitro culture conditions and plant growth regulator supplements, we follow a more directed approach. Zygotic embryos served as a reference and were compared to somatic embryos in metabolomic analyses allowing the future optimization of the in vitro system. The aims of this study were to detect differences in the metabolite profiles of torpedo stage somatic and zygotic embryos of C. persicum. Moreover, major metabolites in endosperm and testa were identified and quantified.Two sets of extracts of two to four biological replicates each were analyzed. In total 52 metabolites were identified and quantified in the different tissues. One of the most significant differences between somatic and zygotic embryos was that the proline concentration in the zygotic embryos was about 40 times higher than that found in somatic embryos. Epicatechin, a scavenger for reactive oxygen species, was found in highest abundance in the testa. Sucrose, the most abundant metabolite was detected in significantly higher concentrations in zygotic embryos. Also, a yet unknown trisaccharide, was significantly enriched in zygotic embryos.

  11. Microwave effects in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Dardalhon, M.; Averbeck, D.; Berteaud, A.J.

    1979-01-01

    Experiments were set up to investigate the effects of open space microwave irradiation of the millimeter (73 GHz) and the centimeter (17 GHz) range in Drosophila melanogaster. We used the wild type strain Paris and the strain delta carrying melanitic tumors in the 3rd larval stage, in the pupae and the adults. The power densities were up to 100mW.cm -2 for 73 GHz and about 60 mW.cm -2 for microwaves at 17 GHz. After 2h exposure to microwaves of 17 GHz or 73 GHz the hatching of the irradiated eggs and their development were normal. In a few cases there was a tendency towards a diminution of the survival of eggs treated at different stages, of larvae treated in the stages 1, 2 and 3 and of treated pupae. However, this was not always statistically significant. The microwave treatment did not induce teratological changes in the adults. A statistical analysis brought about slight diminutions in the incidence and multiplicity of tumors in adult flies. When wild type females were exposed to microwaves of 17 GHz for 16 or 21 h and crossed with untreated males we observed a marked increase in fertility as compared to untreated samples. The viability and tumor incidence in the offspring was not affected. Similar results were obtained when microwaves treated males were crossed with untreated females

  12. Olfactory memory traces in Drosophila.

    Science.gov (United States)

    Berry, Jacob; Krause, William C; Davis, Ronald L

    2008-01-01

    In Drosophila, the fruit fly, coincident exposure to an odor and an aversive electric shock can produce robust behavioral memory. This behavioral memory is thought to be regulated by cellular memory traces within the central nervous system of the fly. These molecular, physiological, or structural changes in neurons, induced by pairing odor and shock, regulate behavior by altering the neurons' response to the learned environment. Recently, novel in vivo functional imaging techniques have allowed researchers to observe cellular memory traces in intact animals. These investigations have revealed interesting temporal and spatial dynamics of cellular memory traces. First, a short-term cellular memory trace was discovered that exists in the antennal lobe, an early site of olfactory processing. This trace represents the recruitment of new synaptic activity into the odor representation and forms for only a short period of time just after training. Second, an intermediate-term cellular memory trace was found in the dorsal paired medial neuron, a neuron thought to play a role in stabilizing olfactory memories. Finally, a long-term protein synthesis-dependent cellular memory trace was discovered in the mushroom bodies, a structure long implicated in olfactory learning and memory. Therefore, it appears that aversive olfactory associations are encoded by multiple cellular memory traces that occur in different regions of the brain with different temporal domains.

  13. Mutations in new cell cycle genes that fail to complement a multiply mutant third chromosome of Drosophila.

    Science.gov (United States)

    White-Cooper, H; Carmena, M; Gonzalez, C; Glover, D M

    1996-11-01

    We have simultaneously screened for new alleles and second site mutations that fail to complement five cell cycle mutations of Drosphila carried on a single third chromosome (gnu, polo, mgr, asp, stg). Females that are either transheterozygous for scott of the antartic (scant) and polo, or homozygous for scant produce embryos that show mitotic defects. A maternal effect upon embryonic mitoses is also seen in embryos derived from females transheterozygous with helter skelter (hsk) and either mgr or asp. cleopatra (cleo), fails to complement asp but is not uncovered by a deficiency for asp. The mitotic phenotype of larvae heterozygous for cleo and the multiple mutant chromosome is similar to weak alleles of asp, but there are no defects in male meiosis. Mutations that failed to complement stg fell into two complementation groups corresponding to stg and a new gene noose. Three of the new stg alleles are early zygotic lethals, whereas the fourth is a pharate adult lethal allele that affects both mitosis and meiosis. Mutations in noose fully complement a small deficiency that removes stg, but when placed in trans to certain stg alleles, result in late lethality and mitotic abnormalities in larval brains.

  14. Drosophila Studies on Autism Spectrum Disorders

    Institute of Scientific and Technical Information of China (English)

    Yao Tian; Zi Chao Zhang; Junhai Han

    2017-01-01

    In the past decade,numerous genes associated with autism spectrum disorders (ASDs) have been identified.These genes encode key regulators of synaptogenesis,synaptic function,and synaptic plasticity.Drosophila is a prominent model system for ASD studies to define novel genes linked to ASDs and decipher their molecular roles in synaptogenesis,synaptic function,synaptic plasticity,and neural circuit assembly and consolidation.Here,we review Drosophila studies on ASD genes that regulate synaptogenesis,synaptic function,and synaptic plasticity through modulating chromatin remodeling,transcription,protein synthesis and degradation,cytoskeleton dynamics,and synaptic scaffolding.

  15. REDfly: a Regulatory Element Database for Drosophila.

    Science.gov (United States)

    Gallo, Steven M; Li, Long; Hu, Zihua; Halfon, Marc S

    2006-02-01

    Bioinformatics studies of transcriptional regulation in the metazoa are significantly hindered by the absence of readily available data on large numbers of transcriptional cis-regulatory modules (CRMs). Even the richly annotated Drosophila melanogaster genome lacks extensive CRM information. We therefore present here a database of Drosophila CRMs curated from the literature complete with both DNA sequence and a searchable description of the gene expression pattern regulated by each CRM. This resource should greatly facilitate the development of computational approaches to CRM discovery as well as bioinformatics analyses of regulatory sequence properties and evolution.

  16. Longevity and the stress response in Drosophila

    DEFF Research Database (Denmark)

    Vermeulen, Corneel J.; Loeschcke, Volker

    2007-01-01

    briefly review the state of the art of research on ageing and longevity in the model organism Drosophila, with focus on the role of the general stress response. We will conclude by contemplating some of the implications of the findings in this research and will suggest several directions for future...... research. Keywords: Ageing; Stress response; Hsp; Drosophila; Stress......The concept that lifespan is a function of the capacity to withstand extrinsic stress is very old. In concordance with this, long-lived individuals often have increased resistance against a variety of stresses throughout life. Genes underlying the stress response may therefore have the ability...

  17. Casein kinase 1 alpha regulates chromosome congression and separation during mouse oocyte meiotic maturation and early embryo development.

    Directory of Open Access Journals (Sweden)

    Lu Wang

    Full Text Available Casein kinase I alpha (CK1α is a member of serine/threonine protein kinase, generally present in all eukaryotes. In mammals, CK1α regulates the transition from interphase to metaphase in mitosis. However, little is known about its role in meiosis. Here we examined Ck1α mRNA and protein expression, as well as its subcellular localization in mouse oocytes from germinal vesicle to the late 1-cell stage. Our results showed that the expression level of CK1α was increased in metaphase. Immunostaining results showed that CK1α colocalized with condensed chromosomes during oocyte meiotic maturation and early embryo development. We used the loss-of-function approach by employing CK1α specific morpholino injection to block the function of CK1α. This functional blocking leads to failure of polar body 1 (PB1 extrusion, chromosome misalignment and MII plate incrassation. We further found that D4476, a specific and efficient CK1 inhibitor, decreased the rate of PB1 extrusion. Moreover, D4476 resulted in giant polar body extrusion, oocyte pro-MI arrest, chromosome congression failure and impairment of embryo developmental potential. In addition, we employed pyrvinium pamoate (PP, an allosteric activator of CK1α, to enhance CK1α activity in oocytes. Supplementation of PP induced oocyte meiotic maturation failure, severe congression abnormalities and misalignment of chromosomes. Taken together, our study for the first time demonstrates that CK1α is required for chromosome alignment and segregation during oocyte meiotic maturation and early embryo development.

  18. Tripolar mitosis and partitioning of the genome arrests human preimplantation development in vitro.

    Science.gov (United States)

    Ottolini, Christian S; Kitchen, John; Xanthopoulou, Leoni; Gordon, Tony; Summers, Michael C; Handyside, Alan H

    2017-08-29

    Following in vitro fertilisation (IVF), only about half of normally fertilised human embryos develop beyond cleavage and morula stages to form a blastocyst in vitro. Although many human embryos are aneuploid and genomically imbalanced, often as a result of meiotic errors inherited in the oocyte, these aneuploidies persist at the blastocyst stage and the reasons for the high incidence of developmental arrest remain unknown. Here we use genome-wide SNP genotyping and meiomapping of both polar bodies to identify maternal meiotic errors and karyomapping to fingerprint the parental chromosomes in single cells from disaggregated arrested embryos and excluded cells from blastocysts. Combined with time lapse imaging of development in culture, we demonstrate that tripolar mitoses in early cleavage cause chromosome dispersal to clones of cells with identical or closely related sub-diploid chromosome profiles resulting in intercellular partitioning of the genome. We hypothesise that following zygotic genome activation (ZGA), the combination of genomic imbalance and partial genome loss disrupts the normal pattern of embryonic gene expression blocking development at the morula-blastocyst transition. Failure to coordinate the cell cycle in early cleavage and regulate centrosome duplication is therefore a major cause of human preimplantation developmental arrest in vitro.

  19. Assay using embryo aggregation chimeras for the detection of nonlethal changes in X-irradiated mouse preimplantation embryos

    International Nuclear Information System (INIS)

    Obasaju, M.F.; Wiley, L.M.; Oudiz, D.J.; Miller, L.; Samuels, S.J.; Chang, R.J.; Overstreet, J.W.

    1988-01-01

    We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos

  20. The Ubx Polycomb response element bypasses an unpaired Fab-8 insulator via cis transvection in Drosophila.

    Science.gov (United States)

    Lu, Danfeng; Li, Zhuoran; Li, Lingling; Yang, Liping; Chen, Guijun; Yang, Deying; Zhang, Yue; Singh, Vikrant; Smith, Sheryl; Xiao, Yu; Wang, Erlin; Ye, Yunshuang; Zhang, Wei; Zhou, Lei; Rong, Yikang; Zhou, Jumin

    2018-01-01

    Chromatin insulators or boundary elements protect genes from regulatory activities from neighboring genes or chromatin domains. In the Drosophila Abdominal-B (Abd-B) locus, the deletion of such elements, such as Frontabdominal-7 (Fab-7) or Fab-8 led to dominant gain of function phenotypes, presumably due to the loss of chromatin barriers. Homologous chromosomes are paired in Drosophila, creating a number of pairing dependent phenomena including transvection, and whether transvection may affect the function of Polycomb response elements (PREs) and thus contribute to the phenotypes are not known. Here, we studied the chromatin barrier activity of Fab-8 and how it is affected by the zygosity of the transgene, and found that Fab-8 is able to block the silencing effect of the Ubx PRE on the DsRed reporter gene in a CTCF binding sites dependent manner. However, the blocking also depends on the zygosity of the transgene in that the barrier activity is present when the transgene is homozygous, but absent when the transgene is heterozygous. To analyze this effect, we performed chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) experiments on homozygous transgenic embryos, and found that H3K27me3 and H3K9me3 marks are restricted by Fab-8, but they spread beyond Fab-8 into the DsRed gene when the two CTCF binding sites within Fab-8 were mutated. Consistent with this, the mutation reduced H3K4me3 and RNA Pol II binding to the DsRed gene, and consequently, DsRed expression. Importantly, in heterozygous embryos, Fab-8 is unable to prevent the spread of H3K27me3 and H3K9me3 marks from crossing Fab-8 into DsRed, suggesting an insulator bypass. These results suggest that in the Abd-B locus, deletion of the insulator in one copy of the chromosome could lead to the loss of insulator activity on the homologous chromosome, and in other loci where chromosomal deletion created hemizygous regions of the genome, the chromatin barrier could be compromised. This study highlights