DNA content of a functioning chicken kinetochore

© The Author(s) 2014. In order to understand the three-dimensional structure of the functional kinetochore in vertebrates, we require a complete list and stoichiometry for the protein components of the kinetochore, which can be provided by genetic and proteomic experiments. We also need to know how the chromatin-containing CENP-A, which makes up the structural foundation for the kinetochore, is folded, and how much of that DNA is involved in assembling the kinetochore. In this MS, we demonstrate that functioning metaphase kinetochores in chicken DT40 cells contain roughly 50 kb of DNA, an amount that corresponds extremely closely to the length of chromosomal DNA associated with CENP-A in ChIP-seq experiments. Thus, during kinetochore assembly, CENP-A chromatin is compacted into the inner kinetochore plate without including significant amounts of flanking pericentromeric heterochromatin. © 2014 The Author(s).Wellcome Trust [grant number 073915]; Wellcome Trust Centre for Cell Biology (core grant numbers 077707 and 092076); Darwin Trust of Edinburg

CSAP localizes to polyglutamylated microtubules and promotes proper cilia function and zebrafish development

The diverse populations of microtubule polymers in cells are functionally distinguished by different posttranslational modifications, including polyglutamylation. Polyglutamylation is enriched on subsets of microtubules including those found in the centrioles, mitotic spindle, and cilia. However, whether this modification alters intrinsic microtubule dynamics or affects extrinsic associations with specific interacting partners remains to be determined. Here we identify the microtubule-binding protein centriole and spindle–associated protein (CSAP), which colocalizes with polyglutamylated tubulin to centrioles, spindle microtubules, and cilia in human tissue culture cells. Reducing tubulin polyglutamylation prevents CSAP localization to both spindle and cilia microtubules. In zebrafish, CSAP is required for normal brain development and proper left–right asymmetry, defects that are qualitatively similar to those reported previously for depletion of polyglutamylation-conjugating enzymes. We also find that CSAP is required for proper cilia beating. Our work supports a model in which polyglutamylation can target selected microtubule-associated proteins, such as CSAP, to microtubule subpopulations, providing specific functional capabilities to these populations.National Institutes of Health (U.S.) (Grant no. GM074746)American Cancer Society. Research Scholar Grant (121776)National Institute of General Medical Sciences (U.S.) (GM088313

Unique genome-wide transcriptome profiles of chicken macrophages exposed to Salmonella-derived endotoxin

<p>Abstract</p> <p>Background</p> <p>Macrophages play essential roles in both innate and adaptive immune responses. Bacteria require endotoxin, a complex lipopolysaccharide, for outer membrane permeability and the host interprets endotoxin as a signal to initiate an innate immune response. The focus of this study is kinetic and global transcriptional analysis of the chicken macrophage response to <it>in vitro </it>stimulation with endotoxin from <it>Salmonella </it><it>typhimurium</it>-798.</p> <p>Results</p> <p>The 38535-probeset Affymetrix GeneChip Chicken Genome array was used to profile transcriptional response to endotoxin 1, 2, 4, and 8 hours post stimulation (hps). Using a maximum FDR (False Discovery Rate) of 0.05 to declare genes as differentially expressed (DE), we found 13, 33, 1761 and 61 DE genes between endotoxin-stimulated versus non-stimulated cells at 1, 2, 4 and 8 hps, respectively. QPCR demonstrated that endotoxin exposure significantly affected the mRNA expression of <it>IL1B</it>, <it>IL6</it>, <it>IL8</it>, and <it>TLR15</it>, but not <it>IL10 </it>and <it>IFNG </it>in HD 11 cells. Ingenuity Pathway Analysis showed that 10% of the total DE genes were involved in inflammatory response. Three, 9.7, 96.8, and 11.8% of the total DE inflammatory response genes were significantly differentially expressed with endotoxin stimulation at 1, 2, 4 and 8 hps, respectively. The <it>NFKBIA, IL1B, IL8 and CCL4 </it>genes were consistently induced at all times after endotoxin treatment. <it>NLRC5 </it>(CARD domain containing, NOD-like receptor family, RCJMB04_18i2), an intracellular receptor, was induced in HD11 cells treated with endotoxin.</p> <p>Conclusions</p> <p>As above using an <it>in vitro </it>model of chicken response to endotoxin, our data revealed the kinetics of gene networks involved in host response to endotoxin and extend the known complexity of networks in chicken immune response to Gram-negative bacteria such as <it>Salmonella</it>. The induction of <it>NFKBIA, IL1B, IL8, CCL4 </it>genes is a consistent signature of host response to endotoxin over time. We make the first report of induction of a NOD-like receptor family member in response to <it>Salmonella </it>endotoxin in chicken macrophages.</p

Genome-wide analysis of ivermectin response by Onchocerca volvulus reveals that genetic drift and soft selective sweeps contribute to loss of drug sensitivity

Treatment of onchocerciasis using mass ivermectin administration has reduced morbidity and transmission throughout Africa and Central/South America. Mass drug administration is likely to exert selection pressure on parasites, and phenotypic and genetic changes in several Onchocerca volvulus populations from Cameroon and Ghana-exposed to more than a decade of regular ivermectin treatment-have raised concern that sub-optimal responses to ivermectin's anti-fecundity effect are becoming more frequent and may spread.Pooled next generation sequencing (Pool-seq) was used to characterise genetic diversity within and between 108 adult female worms differing in ivermectin treatment history and response. Genome-wide analyses revealed genetic variation that significantly differentiated good responder (GR) and sub-optimal responder (SOR) parasites. These variants were not randomly distributed but clustered in ~31 quantitative trait loci (QTLs), with little overlap in putative QTL position and gene content between the two countries. Published candidate ivermectin SOR genes were largely absent in these regions; QTLs differentiating GR and SOR worms were enriched for genes in molecular pathways associated with neurotransmission, development, and stress responses. Finally, single worm genotyping demonstrated that geographic isolation and genetic change over time (in the presence of drug exposure) had a significantly greater role in shaping genetic diversity than the evolution of SOR.This study is one of the first genome-wide association analyses in a parasitic nematode, and provides insight into the genomics of ivermectin response and population structure of O. volvulus. We argue that ivermectin response is a polygenically-determined quantitative trait (QT) whereby identical or related molecular pathways but not necessarily individual genes are likely to determine the extent of ivermectin response in different parasite populations. Furthermore, we propose that genetic drift rather than genetic selection of SOR is the underlying driver of population differentiation, which has significant implications for the emergence and potential spread of SOR within and between these parasite populations

The Aurora Kinase in Trypanosoma brucei Plays Distinctive Roles in Metaphase-Anaphase Transition and Cytokinetic Initiation

Aurora B kinase is an essential regulator of chromosome segregation with the action well characterized in eukaryotes. It is also implicated in cytokinesis, but the detailed mechanism remains less clear, partly due to the difficulty in separating the latter from the former function in a growing cell. A chemical genetic approach with an inhibitor of the enzyme added to a synchronized cell population at different stages of the cell cycle would probably solve this problem. In the deeply branched parasitic protozoan Trypanosoma brucei, an Aurora B homolog, TbAUK1, was found to control both chromosome segregation and cytokinetic initiation by evidence from RNAi and dominant negative mutation. To clearly separate these two functions, VX-680, an inhibitor of TbAUK1, was added to a synchronized T. brucei procyclic cell population at different cell cycle stages. The unique trans-localization pattern of the chromosomal passenger complex (CPC), consisting of TbAUK1 and two novel proteins TbCPC1 and TbCPC2, was monitored during mitosis and cytokinesis by following the migration of the proteins tagged with enhanced yellow fluorescence protein in live cells with time-lapse video microscopy. Inhibition of TbAUK1 function in S-phase, prophase or metaphase invariably arrests the cells in the metaphase, suggesting an action of TbAUK1 in promoting metaphase-anaphase transition. TbAUK1 inhibition in anaphase does not affect mitotic exit, but prevents trans-localization of the CPC from the spindle midzone to the anterior tip of the new flagellum attachment zone for cytokinetic initiation. The CPC in the midzone is dispersed back to the two segregated nuclei, while cytokinesis is inhibited. In and beyond telophase, TbAUK1 inhibition has no effect on the progression of cytokinesis or the subsequent G1, S and G2 phases until a new metaphase is attained. There are thus two clearly distinct points of TbAUK1 action in T. brucei: the metaphase-anaphase transition and cytokinetic initiation. This is the first time to our knowledge that the dual functions of an Aurora B homolog is dissected and separated into two clearly distinct time frames in a cell cycle

In vitro anti-malarial interaction and gametocytocidal activity of cryptolepine

YesBackground: Discovery of novel gametocytocidal molecules is a major pharmacological strategy in the elimination and eradication of malaria. The high patronage of the aqueous root extract of the popular West African anti-malarial plant Cryptolepis sanguinolenta (Periplocaceae) in traditional and hospital settings in Ghana has directed this study investigating the gametocytocidal activity of the plant and its major alkaloid, cryptolepine. This study also investigates the anti-malarial interaction of cryptolepine with standard anti-malarials, as the search for new anti-malarial combinations continues. Methods: The resazurin-based assay was employed in evaluating the gametocytocidal properties of C. sanguinolenta and cryptolepine against the late stage (IV/V) gametocytes of Plasmodium falciparum (NF54). A fixed ratio method based on the SYBR Green I fluorescence-based assay was used to build isobolograms from a combination of cryptolepine with four standard anti-malarial drugs in vitro using the chloroquine sensitive strain 3D7. Results: Cryptolepis sanguinolenta ( IC50 = 49.65 nM) and its major alkaloid, cryptolepine ( IC50 = 1965 nM), showed high inhibitory activity against the late stage gametocytes of P. falciparum (NF54). In the interaction assays in asexual stage, cryptolepine showed an additive effect with both lumefantrine and chloroquine with mean ΣFIC50s of 1.017 ± 0.06 and 1.465 ± 0.17, respectively. Cryptolepine combination with amodiaquine at therapeutically relevant concentration ratios showed a synergistic effect (mean ΣFIC50 = 0.287 ± 0.10) whereas an antagonistic activity (mean ΣFIC50 = 4.182 ± 0.99) was seen with mefloquine. Conclusions: The findings of this study shed light on the high gametocytocidal properties of C. sanguinolenta and cryptolepine attributing their potent anti-malarial activity mainly to their effect on both the sexual and asexual stages of the parasite. Amodiaquine is a potential drug partner for cryptolepine in the development of novel fixed dose combinations

Caenorhabditis elegans Cyclin B3 Is Required for Multiple Mitotic Processes Including Alleviation of a Spindle Checkpoint–Dependent Block in Anaphase Chromosome Segregation

The master regulators of the cell cycle are cyclin-dependent kinases (Cdks), which influence the function of a myriad of proteins via phosphorylation. Mitotic Cdk1 is activated by A-type, as well as B1- and B2-type, cyclins. However, the role of a third, conserved cyclin B family member, cyclin B3, is less well defined. Here, we show that Caenorhabditis elegans CYB-3 has essential and distinct functions from cyclin B1 and B2 in the early embryo. CYB-3 is required for the timely execution of a number of cell cycle events including completion of the MII meiotic division of the oocyte nucleus, pronuclear migration, centrosome maturation, mitotic chromosome condensation and congression, and, most strikingly, progression through the metaphase-to-anaphase transition. Our experiments reveal that the extended metaphase delay in CYB-3–depleted embryos is dependent on an intact spindle assembly checkpoint (SAC) and results in salient defects in the architecture of holocentric metaphase chromosomes. Furthermore, genetically increasing or decreasing dynein activity results in the respective suppression or enhancement of CYB-3–dependent defects in cell cycle progression. Altogether, these data reveal that CYB-3 plays a unique, essential role in the cell cycle including promoting mitotic dynein functionality and alleviation of a SAC–dependent block in anaphase chromosome segregation

MVB-12, a Fourth Subunit of Metazoan ESCRT-I, Functions in Receptor Downregulation

After ligand binding and endocytosis, cell surface receptors can continue to signal from endosomal compartments until sequestered from the cytoplasm. An important mechanism for receptor downregulation in vivo is via the inward budding of receptors into intralumenal vesicles to form specialized endosomes called multivesicular bodies (MVBs) that subsequently fuse with lysosomes, degrading their cargo. This process requires four heterooligomeric protein complexes collectively termed the ESCRT machinery. In yeast, ESCRT-I is a heterotetrameric complex comprised of three conserved subunits and a fourth subunit for which identifiable metazoan homologs were lacking. Using C. elegans, we identify MVB-12, a fourth metazoan ESCRT-I subunit. Depletion of MVB-12 slows the kinetics of receptor downregulation in vivo, but to a lesser extent than inhibition of other ESCRT-I subunits. Consistent with these findings, targeting of MVB-12 to membranes requires the other ESCRT-I subunits, but MVB-12 is not required to target the remaining ESCRT-I components. Both endogenous and recombinant ESCRT-I are stable complexes with a 1:1:1:1 subunit stoichiometry. MVB-12 has two human homologs that co-localize and co-immunoprecipitate with the ESCRT-I component TSG101. Thus, MVB-12 is a conserved core component of metazoan ESCRT-I that regulates its activity during MVB biogenesis

Genetic control of chicken heterophil function in advanced intercross lines: associations with novel and with known Salmonella resistance loci and a likely mechanism for cell death in extracellular trap production

Heterophils, the avian polymorphonuclear leukocyte and the counterpart of mammalian neutrophils, generate the primary innate response to pathogens in chickens. Heterophil performance against pathogens is associated with host disease resistance, and heterophil gene expression and function are under genetic control. To characterize the genomic basis of heterophil function, heterophils from F13 advanced intercross chicken lines (broiler × Leghorn and broiler × Fayoumi) were assayed for phagocytosis and killing of Salmonella enteritidis, oxidative burst, and extracellular trap production. A whole-genome association analysis of single nucleotide polymorphisms at 57,636 loci identified genomic locations controlling these functional phenotypes. Genomic analysis revealed a significant association of extracellular trap production with the SAL1 locus and the SLC11A1 gene, which have both been previously associated with resistance to S. enteritidis. Fine mapping supports SIVA1 as a candidate gene controlling SAL1-mediated resistance and indicates that the proposed cell-death mechanism associated with extracellular trap production, ETosis, likely functions through the CD27/Siva-1-mediated apoptotic pathway. The SLC11A1 gene was also associated with phagocytosis of S. enteritidis, suggesting that the Slc11a1 protein may play an additional role in immune response beyond depleting metal ions to inhibit intracellular bacterial growth. A region of chromosome 6 with no characterized genes was also associated with extracellular trap production. Further characterization of these novel genes in chickens and other species is needed to understand their role in polymorphonuclear leukocyte function and host resistance to disease

Karyotypic Determinants of Chromosome Instability in Aneuploid Budding Yeast

Recent studies in cancer cells and budding yeast demonstrated that aneuploidy, the state of having abnormal chromosome numbers, correlates with elevated chromosome instability (CIN), i.e. the propensity of gaining and losing chromosomes at a high frequency. Here we have investigated ploidy- and chromosome-specific determinants underlying aneuploidy-induced CIN by observing karyotype dynamics in fully isogenic aneuploid yeast strains with ploidies between 1N and 2N obtained through a random meiotic process. The aneuploid strains exhibited various levels of whole-chromosome instability (i.e. chromosome gains and losses). CIN correlates with cellular ploidy in an unexpected way: cells with a chromosomal content close to the haploid state are significantly more stable than cells displaying an apparent ploidy between 1.5 and 2N. We propose that the capacity for accurate chromosome segregation by the mitotic system does not scale continuously with an increasing number of chromosomes, but may occur via discrete steps each time a full set of chromosomes is added to the genome. On top of such general ploidy-related effect, CIN is also associated with the presence of specific aneuploid chromosomes as well as dosage imbalance between specific chromosome pairs. Our findings potentially help reconcile the divide between gene-centric versus genome-centric theories in cancer evolution