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Regulation of Autophosphorylation Controls PLK4 Self-Destruction and Centriole Number
Get PDFThe deposited article is a post-print version and has been submitted to peer review.This publication hasn't any creative commons license associated.This deposit is composed by the main article plus the supplementary materials of the publication.Polo-like kinase 4 (PLK4) is a major player in centriole biogenesis: in its absence centrioles fail to form, while in excess leads to centriole amplification. The SCF-Slimb/βTrCP-E3 ubiquitin ligase controls PLK4 levels through recognition of a conserved phosphodegron. SCF-Slimb/βTrCP substrate binding and targeting for degradation is normally regulated by phosphorylation cascades, controlling complex processes, such as circadian clocks and morphogenesis. Here, we show that PLK4 is a suicide kinase, autophosphorylating in residues that are critical for SCF-Slimb/βTrCP binding. We demonstrate a multisite trans-autophosphorylation mechanism, likely to ensure that both a threshold of PLK4 concentration is attained and a sequence of events is observed before PLK4 can autodestruct. First, we show that PLK4 trans-autophosphorylates other PLK4 molecules on both Ser293 and Thr297 within the degron and that these residues contribute differently for PLK4 degradation, the first being critical and the second maximizing auto-destruction. Second, PLK4 trans-autophosphorylates a phospho-cluster outside the degron, which regulates Thr297 phosphorylation, PLK4 degradation, and centriole number. Finally, we show the importance of PLK4-Slimb/βTrCP regulation as it operates in both soma and germline. As βTrCP, PLK4, and centriole number are deregulated in several cancers, our work provides novel links between centriole number control and tumorigenesis.Fundação para a Ciência e Tecnologia grants: (SFRH/BD/33213/2007, PTDC/SAU-OBD/73194/2006, PTDC/SAU-OBD/105616/2008); ERC grant:(ERC-2010-StG grant number 261344); EMBO installation grant.info:eu-repo/semantics/publishedVersio
The Liver Plays a Major Role in Clearance and Destruction of Blood Trypomastigotes in Trypanosoma cruzi Chronically Infected Mice
Get PDFThis deposit is composed by a publication in which the IGC's authors have had the role of collaboration (it's a collaboration publication). This type of deposit in ARCA is in restrictedAccess (it can't be in open access to the public), and can only be accessed by two ways: either by requesting a legal copy from the author (the email contact present in this deposit) or by visiting the following link: http://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0000578Intravenous challenge with Trypanosoma cruzi can be used to investigate the process and consequences of blood parasite clearance in experimental Chagas disease. One hour after intravenous challenge of chronically infected mice with 5x10(6) trypomastigotes, the liver constituted a major site of parasite accumulation, as revealed by PCR. Intact parasites and/or parasite remnants were visualized at this time point scattered in the liver parenchyma. Moreover, at this time, many of liver-cleared parasites were viable, as estimated by the frequency of positive cultures, which considerably diminished after 48 h. Following clearance, the number of infiltrating cells in the hepatic tissue notably increased: initially (at 24 h) as diffuse infiltrates affecting the whole parenchyma, and at 48 h, in the form of large focal infiltrates in both the parenchyma and perivascular spaces. Phenotypic characterization of liver-infiltrating cells 24 h after challenge revealed an increase in Mac1(+), CD8(+) and CD4(+) cells, followed by natural killer (NK) cells. As evidence that liver-infiltrating CD4(+) and CD8(+) cells were activated, increased frequencies of CD69(+)CD8(+), CD69(+)CD4(+) and CD25(+)CD122(+)CD4(+) cells were observed at 24 and 48 h after challenge, and of CD25(-)CD122(+)CD4(+) cells at 48 h. The major role of CD4(+) cells in liver protection was suggested by data showing a very high frequency of interferon (IFN)-gamma-producing CD4(+) cells 24 h after challenge. In contrast, liver CD8(+) cells produced little IFN-gamma, even though they showed an enhanced potential for secreting this cytokine, as revealed by in vitro T cell receptor (TCR) stimulation. Confirming the effectiveness of the liver immune response in blood parasite control during the chronic phase of infection, no live parasites were detected in this organ 7 days after challenge
Early skin immunological disturbance after Plasmodium-infected mosquito bites
Get PDFThis deposit is in restrictedAccess (it can't be in open access to the public), and can only be accessed by two ways: either by requesting a legal copy from the author (the email contact present in this deposit) or by visiting the following link: https://www.sciencedirect.com/science/article/pii/S0008874912001153?via%3DihubAlthough the role of regulatory T cells (Tregs) during malaria infection has been studied extensively, such studies have focused exclusively on the role of Treg during the blood stage of infection; little is known about the detailed mechanisms of Tregs and sporozoite deposition in the dermis by mosquito bites. In this paper we show that sporozoites introduced into the skin by mosquito bites increase the mobility of skin Tregs and dendritic cells (DCs). We also show differences in MHC class II and/or CD86 expression on skin-resident dendritic cell subtypes and macrophages. From the observed decrease of the number of APCs into draining lymph nodes, suppression of CD28 expression in conventional CD4 T cells, and a low homeostatic proliferation of skin-migrated CD4 T found in nude mice indicate that Tregs may play a fundamental role during the initial phase of malaria parasite inoculation into the mammalian hos
Nucleozin Targets Cytoplasmic Trafficking of Viral Ribonucleoprotein-Rab11 Complexes in Influenza A Virus Infection
Get PDFNovel antivirals are needed to supplement existing control strategies for influenza A virus (IAV). A promising new class of drug, exemplified by the compound nucleozin, has recently been identified that targets the viral nucleoprotein (NP). These inhibitors are thought to act as "molecular staples" that stabilize interactions between NP monomers, promoting the formation of nonfunctional aggregates. Here we detail the inhibitory mechanism of nucleozin, finding that the drug has both early- and late-acting effects on the IAV life cycle. When present at the start of infection, it inhibited viral RNA and protein synthesis. However, when added at later time points, it still potently blocked the production of infectious progeny but without affecting viral macromolecular synthesis. Instead, nucleozin blocked the cytoplasmic trafficking of ribonucleoproteins (RNPs) that had undergone nuclear export, promoting the formation of large perinuclear aggregates of RNPs along with cellular Rab11. This effect led to the production of much reduced amounts of often markedly smaller virus particles. We conclude that the primary target of nucleozin is the viral RNP, not NP, and this work also provides proof of the principle that IAV replication can be effectively inhibited by blocking cytoplasmic trafficking of the viral genome.MRC grant: (G0700815), University of Cambridge/Trinity College grant: (Newton Trust), RGC Hong Kong grant: (GRF 768010 M)
Genome architecture is a selectable trait that can be maintained by antagonistic pleiotropy
Get PDFChromosomal rearrangements are mutations contributing to both within and between species variation; however their contribution to fitness is yet to be measured. Here we show that chromosomal rearrangements are pervasive in natural isolates of Schizosaccharomyces pombe and contribute to reproductive isolation. To determine the fitness effects of chromosome structure, we constructed two inversions and eight translocations without changing the coding sequence. We show that chromosomal rearrangements contribute to both reproductive success in meiosis and growth rate in mitosis with a strong genotype by environment interaction. These changes are accompanied by alterations in gene expression. Strikingly, we find several examples leading to antagonistic pleiotropy. Even though chromosomal rearrangements may have a deleterious effect during sexual reproduction, some compensate with a strong growth advantage in mitosis. Our results constitute the first quantification of fitness effects caused by de novo mutations that result in chromosomal rearrangement variation and suggest a mechanism for their maintenance in natural populations.FCT PhD and Postdoctoral fellowships
Bringing Dicynodonts Back to Life: Paleobiology and Anatomy of a New Emydopoid Genus from the Upper Permian of Mozambique
Get PDFDicynodontia represent the most diverse tetrapod group during the Late Permian. They survived the Permo-Triassic extinction and are central to understanding Permo-Triassic terrestrial ecosystems. Although extensively studied, several aspects of dicynodont paleobiology such as, neuroanatomy, inner ear morphology and internal cranial anatomy remain obscure. Here we describe a new dicynodont (Therapsida, Anomodontia) from northern Mozambique: Niassodon mfumukasi gen. et sp. nov. The holotype ML1620 was collected from the Late Permian K5 formation, Metangula Graben, Niassa Province northern Mozambique, an almost completely unexplored basin and country for vertebrate paleontology. Synchrotron radiation based micro-computed tomography (SRµCT), combined with a phylogenetic analysis, demonstrates a set of characters shared with Emydopoidea. All individual bones were digitally segmented allowing a 3D visualization of each element. In addition, we reconstructed the osseous labyrinth, endocast, cranial nerves and vasculature. The brain is narrow and the cerebellum is broader than the forebrain, resembling the conservative, "reptilian-grade" morphology of other non-mammalian therapsids, but the enlarged paraflocculi occupy the same relative volume as in birds. The orientation of the horizontal semicircular canals indicates a slightly more dorsally tilted head posture than previously assumed in other dicynodonts. In addition, synchrotron data shows a secondary center of ossification in the femur. Thus ML1620 represents, to our knowledge, the oldest fossil evidence of a secondary center of ossification, pushing back the evolutionary origins of this feature. The fact that the specimen represents a new species indicates that the Late Permian tetrapod fauna of east Africa is still incompletely known.Mozambique (Ministério dos Recursos Minerais), National Geographic Society, TAP airlines and other anonymous patrons, financial support from DESY through the I-20110184 EC project
miRNAs mediate SnRK1-dependent energy signaling in Arabidopsis
Get PDFThe SnRK1 protein kinase, the plant ortholog of mammalian AMPK and yeast Snf1, is activated by the energy depletion caused by adverse environmental conditions. Upon activation, SnRK1 triggers extensive transcriptional changes to restore homeostasis and promote stress tolerance and survival partly through the inhibition of anabolism and the activation of catabolism. Despite the identification of a few bZIP transcription factors as downstream effectors, the mechanisms underlying gene regulation, and in particular gene repression by SnRK1, remain mostly unknown. microRNAs (miRNAs) are 20-24 nt RNAs that regulate gene expression post-transcriptionally by driving the cleavage and/or translation attenuation of complementary mRNA targets. In addition to their role in plant development, mounting evidence implicates miRNAs in the response to environmental stress. Given the involvement of miRNAs in stress responses and the fact that some of the SnRK1-regulated genes are miRNA targets, we postulated that miRNAs drive part of the transcriptional reprogramming triggered by SnRK1. By comparing the transcriptional response to energy deprivation between WT and dcl1-9, a mutant deficient in miRNA biogenesis, we identified 831 starvation genes misregulated in the dcl1-9 mutant, out of which 155 are validated or predicted miRNA targets. Functional clustering analysis revealed that the main cellular processes potentially co-regulated by SnRK1 and miRNAs are translation and organelle function and uncover TCP transcription factors as one of the most highly enriched functional clusters. TCP repression during energy deprivation was impaired in miR319 knockdown (MIM319) plants, demonstrating the involvement of miR319 in the stress-dependent regulation of TCPs. Altogether, our data indicates that miRNAs are components of the SnRK1 signaling cascade contributing to the regulation of specific mRNA targets and possibly tuning down particular cellular processes during the stress response.FCT fellowships: (SFRH/BPD/47280/2008, SFRH/BD/33563/2008), EMBOLong-Term Fellowship, Gottfried Wilhelm Leibniz Award, Max Planck Society funds, Marie Curie IRG grant, EMBO Installation grant (FCT), Marie Curie Actions FP7-People-2010-ITN
Activation-Induced Cytidine Deaminase Does Not Impact Murine Meiotic Recombination
Get PDFActivation-Induced Cytidine Deaminase (AID) was first described as the triggering enzyme of the B-cell-specific reactions that edit the immunoglobulin genes, namely somatic hypermutation, gene conversion and class switch recombination. Over the years, AID was also detected in cells other than lymphocytes, and it has been assigned additional roles in the innate defense against transforming retroviruses, in retrotransposition restriction and in DNA demethylation. Notably, AID expression was found in germline tissues, and in heterologous systems it can induce the double-strand breaks required for the initiation of meiotic recombination and proper gamete formation. However, since AID deficient mice are fully fertile, the molecule is not essential for meiosis. Thus, the remaining question that we addressed here is whether AID influences the frequency of meiotic recombination in mice. We measured the recombination events in the meiosis of male and female mice F1 hybrids of C57BL/6J and BALB/c, in Aicda(+/+) and Aicda(-/-) background using a panel of SNPs that distinguishes C57BL/6J from BALB/c genome across the 19 autosomes. In agreement with the literature, we found that the frequency of recombination in the female germline was higher than in male germline, both in the Aicda(+/+) and the Aicda(-/-) backgrounds. No statistical difference was found in the average recombination events between Aicda(+/+) and Aicda(-/-) animals, either in females or males. In addition, the recombination frequency between SNPs flanking the IgH and Igκ loci was also not different. We conclude that AID has a minor impact, if any, on the overall frequency of meiotic recombination.Marie Curie Reintegration grant: (PIRG03-GA-2008230967), FCT fellowship: (SFRH/BPD/66824/2009)
Comparative Transcriptomic Analysis of the Burkholderia cepacia Tyrosine Kinase bceF Mutant Reveals a Role in Tolerance to Stress, Biofilm Formation, and Virulence
Get PDFThe bacterial tyrosine-kinase (BY-kinase) family comprises the major group of bacterial enzymes endowed with tyrosine kinase activity. We previously showed that the BceF protein from Burkholderia cepacia IST408 belongs to this BY-kinase family and is involved in the biosynthesis of the exopolysaccharide cepacian. However, little is known about the extent of regulation of this protein kinase activity. In order to examine this regulation, we performed a comparative transcriptome profile between the bceF mutant and wild-type B. cepacia IST408. The analyses led to identification of 630 genes whose expression was significantly changed. Genes with decreased expression in the bceF mutant were related to stress response, motility, cell adhesion, and carbon and energy metabolism. Genes with increased expression were related to intracellular signaling and lipid metabolism. Mutation of bceF led to reduced survival under heat shock and UV light exposure, reduced swimming motility, and alteration in biofilm architecture when grown in vitro. Consistent with some of these phenotypes, the bceF mutant demonstrated elevated levels of cyclic-di-GMP. Furthermore, BceF contributed to the virulence of B. cepacia for larvae of the Greater wax moth, Galleria mellonella. Taken together, BceF appears to play a considerable role in many cellular processes, including biofilm formation and virulence. As homologues of BceF occur in a number of pathogenic and plant-associated Burkholderia strains, the modulation of bacterial behavior through tyrosine kinase activity is most likely a widely occurring phenomenon.FCT contract: (PTDC/BIA-MIC/66977/2006), FCT postdoctoral grant and a doctoral grant, Fundação Calouste Gulbenkian scholarship
Reciprocal virulence and resistance polymorphism in the relationship betweenToxoplasma gondiiand the house mouse
Get PDFVirulence in the ubiquitous intracellular protozoon Toxoplasma gondii for its natural intermediate host, the mouse, appears paradoxical from an evolutionary standpoint because death of the mouse before encystment interrupts the parasite life cycle. Virulent T. gondii strains secrete kinases and pseudokinases that inactivate the immunity-related GTPases (IRG proteins) responsible for mouse resistance to avirulent strains. Such considerations stimulated a search for IRG alleles unknown in laboratory mice that might confer resistance to virulent strains of T. gondii. We report that the mouse IRG system shows extraordinary polymorphic complexity in the wild. We describe an IRG haplotype from a wild-derived mouse strain that confers resistance against virulent parasites by interference with the virulent kinase complex. In such hosts virulent strains can encyst, hinting at an explanation for the evolution of virulence polymorphism in T. gondii. DOI:http://dx.doi.org/10.7554/eLife.01298.001.Deutsche Forschungsgemeinschaft (SFB 680, SFB 635, SFB 670, SPP 1399), International Graduate
School in Development Health and Disease