Overexpressing centriole-replication proteins in vivo induces centriole overduplication and de novo formation

BACKGROUND: Centrosomes have important roles in many aspects of cell organization, and aberrations in their number and function are associated with various diseases, including cancer. Centrosomes consist of a pair of centrioles surrounded by a pericentriolar matrix (PCM), and their replication is tightly regulated. Here, we investigate the effects of overexpressing the three proteins known to be required for centriole replication in Drosophila-DSas-6, DSas-4, and Sak. RESULTS: By directly observing centriole replication in living Drosophila embryos, we show that the overexpression of GFP-DSas-6 can drive extra rounds of centriole replication within a single cell cycle. Extra centriole-like structures also accumulate in brain cells that overexpress either GFP-DSas-6 or GFP-Sak, but not DSas-4-GFP. No extra centrioles accumulate in spermatocytes that overexpress any of these three proteins. Most remarkably, the overexpression of any one of these three proteins results in the rapid de novo formation of many hundreds of centriole-like structures in unfertilized eggs, which normally do not contain centrioles. CONCLUSIONS: Our data suggest that the levels of centriolar DSas-6 determine the number of daughter centrioles formed during centriole replication. Overexpression of either DSas-6 or Sak can induce the formation of extra centrioles in some tissues but not others, suggesting that centriole replication is regulated differently in different tissues. The finding that the overexpression of DSas-4, DSas-6, or Sak can rapidly induce the de novo formation of centriole-like structures in Drosophila eggs suggests that this process results from the stabilization of centriole-precursors that are normally present in the egg.     

The genetic effective and adult census size of an Australian population of tiger prawns (Penaeus esculentus)

This study compares estimates of the census size of the spawning population with genetic estimates of effective current and long-term population size for an abundant and commercially important marine invertebrate, the brown tiger prawn (Penaeus esculentus). Our aim was to focus on the relationship between genetic effective and census size that may provide a source of information for viability analyses of naturally occurring populations. Samples were taken in 2001, 2002 and 2003 from a population on the east coast of Australia and temporal allelic variation was measured at eight polymorphic microsatellite loci. Moments-based and maximum-likelihood estimates of current genetic effective population size ranged from 797 to 1304. The mean long-term genetic effective population size was 9968. Although small for a large population, the effective population size estimates were above the threshold where genetic diversity is lost at neutral alleles through drift or inbreeding. Simulation studies correctly predicted that under these experimental conditions the genetic estimates would have non-infinite upper confidence limits and revealed they might be overestimates of the true size. We also show that estimates of mortality and variance in family size may be derived from data on average fecundity, current genetic effective and census spawning population size, assuming effective population size is equivalent to the number of breeders. This work confirms that it is feasible to obtain accurate estimates of current genetic effective population size for abundant Type III species using existing genetic marker technology.     

Two different pathways are involved in peroxynitrite-induced senescence and apoptosis of human erythrocytes

CO(2) changes the biochemistry of peroxynitrite basically in two ways: (i) nitrating species is the CO(3)(-) / ()NO(2) radical pair, and (ii) peroxynitrite diffusion distance is significantly reduced. For peroxynitrite generated extracellularly this last effect is particularly dramatic at low cell density because CO(3)(-) and ()NO(2) are short-lived and decay mostly in the extracellular space or at the cell surface/membrane. This study was aimed to distinguish between peroxynitrite-induced extra- and intracellular modifications of red blood cells (RBC). Our results show that at low cell density and in the presence of CO(2) peroxynitrite induced the oxidation of surface thiols, the formation of 3-nitrotyrosine and DMPO-RBC adducts, and the down-regulation of glycophorins A and C (biomarkers of senescence). Reactivation of glycolysis reversed only the oxidation of surface thiols. Without CO(2) peroxynitrite also induced the oxidation of hemoglobin and glutathione, the accumulation of lactate, a decrease in ATP, the clustering of band 3, the externalization of phosphatidylserine, and the activation of caspases 8 and 3 (biomarkers of apoptosis). The latter biomarkers were all reversed by reactivation of glycolysis. We hypothesize that cell senescence could (generally) be derived by irreversible radical-mediated oxidation of membrane targets, while the appearance of apoptotic biomarkers could be bolstered by oxidation of intracellular targets. These results suggest that, depending on extracellular homolysis or diffusion to the intracellular space, peroxynitrite prompts RBCs toward either senescence or apoptosis through different oxidation mechanisms.     

Subcellular localization and function of alternatively spliced Noxo1 isoforms

Nox organizer 1 (Noxo1), a p47(phox) homolog, is produced as four isoforms with unique N-terminal PX domains derived by alternative mRNA splicing. We compared the subcellular distribution of these isoforms or their isolated PX domains produced as GFP fusion proteins, as well as their ability to support Nox1 activity in several transfected models. Noxo1alpha, beta, gamma, and delta show different subcellular localization patterns, determined by their PX domains. In HEK293 cells, Noxo1beta exhibits prominent plasma membrane binding, Noxo1gamma shows plasma membrane and nuclear associations, and Noxo1alpha and delta localize primarily on intracellular vesicles or cytoplasmic aggregates, but not the plasma membrane. Nox1 activity correlates with Noxo1 plasma membrane binding in HEK293 cells, since Noxo1beta supports the highest activity and Noxo1gamma and Noxo1alpha support moderate or low activities, respectively. In COS-7 cells, where Noxo1alpha localizes on the plasma membrane, the activities supported by the three isoforms (alpha, beta, and gamma) do not differ significantly. The PX domains of beta and gamma bind the same phospholipids, including phosphatidic acid. These results indicate that the variant PX domains are unique determinants of Noxo1 localization and Nox1 function. Finally, the overexpressed Noxo1 isoforms do not affect p22(phox) localization, although Nox1 is needed to transport p22(phox) to the plasma membrane.     

The Extracellular A-loop of Dual Oxidases Affects the Specificity of Reactive Oxygen Species Release

NADPH oxidase (Nox) family proteins produce superoxide (O₂^⨪) directly by transferring an electron to molecular oxygen. Dual oxidases (Duoxes) also produce an O₂^⨪ intermediate, although the final species secreted by mature Duoxes is H₂O₂, suggesting that intramolecular O₂^⨪ dismutation or other mechanisms contribute to H₂O₂ release. We explored the structural determinants affecting reactive oxygen species formation by Duox enzymes. Duox2 showed O₂^⨪ leakage when mismatched with Duox activator 1 (DuoxA1). Duox2 released O₂^⨪ even in correctly matched combinations, including Duox2 + DuoxA2 and Duox2 + N-terminally tagged DuoxA2 regardless of the type or number of tags. Conversely, Duox1 did not release O₂^⨪ in any combination. Chimeric Duox2 possessing the A-loop of Duox1 showed no O₂^⨪ leakage; chimeric Duox1 possessing the A-loop of Duox2 released O₂^⨪. Moreover, Duox2 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA2 showed enhanced O₂^⨪ release, and Duox1 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA1 acquired O₂^⨪ leakage. Although we identified Duox1 A-loop residues (His¹⁰⁷¹, His¹⁰⁷², and Gly¹⁰⁷⁴) important for reducing O₂^⨪ release, mutations of these residues to those of Duox2 failed to convert Duox1 to an O₂^⨪-releasing enzyme. Using immunoprecipitation and endoglycosidase H sensitivity assays, we found that the A-loop of Duoxes binds to DuoxA N termini, creating more stable, mature Duox-DuoxA complexes. In conclusion, the A-loops of both Duoxes support H₂O₂ production through interaction with corresponding activators, but complex formation between the Duox1 A-loop and DuoxA1 results in tighter control of H₂O₂ release by the enzyme complex.     

Male mating competitiveness of a Wolbachia-introgressed Aedes polynesiensis strain under semi-field conditions

Background

Lymphatic filariasis (LF), a global public health problem affecting approximately 120 million people worldwide, is a leading cause of disability in the developing world including the South Pacific. Despite decades of ongoing mass drug administration (MDA) in the region, some island nations have not yet achieved the threshold levels of microfilaremia established by the World Health Organization for eliminating transmission. Previously, the generation of a novel Aedes polynesiensis strain (CP) infected with an exogenous type of Wolbachia has been described. The CP mosquito is cytoplasmically incompatible (i.e., effectively sterile) when mated with wildtype mosquitoes, and a strategy was proposed for the control of A. polynesiensis populations by repeated, inundative releases of CP males to disrupt fertility of wild females. Such a strategy could lead to suppression of the vector population and subsequently lead to a reduction in the transmission of filarial worms.

Methodology/Principal Findings

CP males and F1 male offspring from wild-caught A. polynesiensis females exhibit near equal mating competitiveness with F1 females under semi-field conditions.

Conclusions/Significance

While laboratory experiments are important, prior projects have demonstrated the need for additional testing under semi-field conditions in order to recognize problems before field implementation. The results reported here from semi-field experiments encourage forward progression toward small-scale field releases.     

Characterization of a dipeptide motif regulating IFN-gamma receptor 2 plasma membrane accumulation and IFN-gamma responsiveness

The IFN-gammaR complex is composed of two IFN-gammaR1 and two IFN-gammaR2 polypeptide chains. Although IFN-gammaR1 is constitutively expressed on all nucleated cells, IFN-gammaR2 membrane display is selective and tightly regulated. We created a series of fluorescent-tagged IFN-gammaR2 expression constructs to follow the molecule's cell surface expression and intracellular distribution. Truncation of the receptor immediately upstream of Leu-Ile 255-256 (254X) created a receptor devoid of signaling that overaccumulated on the cell surface. In addition, this truncated receptor inhibited wild-type IFN-gammaR2 activity and therefore exerted a dominant negative effect. In-frame deletion (255Delta2) or alanine substitution (LI255-256AA) of these amino acids created mutants that overaccumulated on the plasma membrane, but had enhanced function. Single amino acid substitutions (L255A or I256A) had a more modest effect. In-frame deletions upstream (253Delta2), but not downstream (257Delta2), of Leu-Ile 255-256 also led to overaccumulation. A truncation within the IFN-gammaR2 Jak2 binding site (270X) led to a mutant devoid of function that did not overaccumulate and did not affect wild-type IFN-gammaR2 signaling. We have created a series of novel mutants of IFN-gammaR2 that have facilitated the identification of intracellular domains that control IFN-gammaR2 accumulation and IFN-gamma responsiveness. In contrast to IFN-gammaR1, not only dominant negative, but also dominant gain-of-function, mutations were created through manipulation of IFN-gammaR2 Leu-Ile 255-256. These IFN-gammaR2 mutants will allow fine dissection of the role of IFN-gamma signaling in immunity.     

Peroxiredoxin 6 translocates to the plasma membrane during neutrophil activation and is required for optimal NADPH oxidase activity

Neutrophils provide the first line of defense against microbial invasion in part through production of reactive oxygen species (ROS) which is mediated through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generating superoxide anion (O2-). The phagocyte oxidase (phox) has multiple protein components that assemble on the plasma membrane in stimulated neutrophils. We recently described a protein in neutrophils, peroxiredoxin 6 (Prdx6), which has both peroxidase and phospholipase A2 (PLA2) activities and enhances oxidase activity in an SDS-activated, cell-free system. The function of Prdx6 in phox activity is further investigated. In reconstituted phox-competent K562 cells, siRNA-mediated suppression of Prdx6 resulted in decreased NADPH oxidase activity in response to formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). In neutrophils stimulated with PMA, Prdx6 translocated to plasma membrane as demonstrated by Western blot and confocal microscopy. Translocation of Prdx6 in phox competent K562 cells required both p67phox and p47phox. In addition, plasma membrane from PMA-stimulated, oxidase competent K562 cells with siRNA-mediated Prdx6 suppression contained less p47phox and p67phox compared to cells in which Prdx6 was not decreased. Cell-free oxidase assays showed that recombinant Prdx6 did not alter the Km for NADPH, but increased the Vmax for O2- production in a saturable, Prdx6 concentration-dependent manner. Recombinant proteins with mutations in Prdx (C47S) and phospholipase (S32A) activity both enhanced cell-free phox activity to the same extent as wild type protein. Prdx6 supports retention of the active oxidase complex in stimulated plasma membrane, and results with mutant proteins imply that Prdx6 serves an additional biochemical or structural role in supporting optimal NADPH oxidase activity.     

Neurogenin 2 regulates progenitor cell-cycle progression and Purkinje cell dendritogenesis in cerebellar development

By serving as the sole output of the cerebellar cortex, integrating a myriad of afferent stimuli, Purkinje cells (PCs) constitute the principal neuron in cerebellar circuits. Several neurodegenerative cerebellar ataxias feature a selective cell-autonomous loss of PCs, warranting the development of regenerative strategies. To date, very little is known as to the regulatory cascades controlling PC development. During central nervous system development, the proneural gene neurogenin 2 (Neurog2) contributes to many distinct neuronal types by specifying their fate and/or dictating development of their morphological features. By analyzing a mouse knock-in line expressing Cre recombinase under the control of Neurog2 cis-acting sequences we show that, in the cerebellar primordium, Neurog2 is expressed by cycling progenitors cell-autonomously fated to become PCs, even when transplanted heterochronically. During cerebellar development, Neurog2 is expressed in G1 phase by progenitors poised to exit the cell cycle. We demonstrate that, in the absence of Neurog2, both cell-cycle progression and neuronal output are significantly affected, leading to an overall reduction of the mature cerebellar volume. Although PC fate identity is correctly specified, the maturation of their dendritic arbor is severely affected in the absence of Neurog2, as null PCs develop stunted and poorly branched dendrites, a defect evident from the early stages of dendritogenesis. Thus, Neurog2 represents a key regulator of PC development and maturation.