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A mechanism for the elimination of the female gamete centrosome in Drosophila melanogaster
No full textThe 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.An important feature of fertilization is the asymmetric inheritance of centrioles. In most species it is the sperm that contributes the initial centriole, which builds the first centrosome that is essential for early development. However, given that centrioles are thought to be exceptionally stable structures, the mechanism behind centriole disappearance in the female germ line remains elusive and paradoxical. We elucidated a program for centriole maintenance in fruit flies, led by Polo kinase and the pericentriolar matrix (PCM): The PCM is down-regulated in the female germ line during oogenesis, which results in centriole loss. Perturbing this program prevents centriole loss, leading to abnormal meiotic and mitotic divisions, and thus to female sterility. This mechanism challenges the view that centrioles are intrinsically stable structures and reveals general functions for Polo kinase and the PCM in centriole maintenance. We propose that regulation of this maintenance program is essential for successful sexual reproduction and defines centriole life span in different tissues in homeostasis and disease, thereby shaping the cytoskeleton.Fundação Para a Ciência e Tecnologia grants: (SFRH/BD/33565/2008, SFRH/BPD/79680/2011, SFRH/BPD/87479/2012, PTDC/SAU-BD/105616/2008); Ciencia2007; EMBO Installation grant; ERC grant: (ERC-2010-StG-261344).info:eu-repo/semantics/publishedVersio
Trade-Offs of Escherichia coli Adaptation to an Intracellular Lifestyle in Macrophages
Get PDFThe bacterium Escherichia coli exhibits remarkable genomic and phenotypic variation, with some pathogenic strains having evolved to survive and even replicate in the harsh intra-macrophage environment. The rate and effects of mutations that can cause pathoadaptation are key determinants of the pace at which E. coli can colonize such niches and become pathogenic. We used experimental evolution to determine the speed and evolutionary paths undertaken by a commensal strain of E. coli when adapting to intracellular life. We estimated the acquisition of pathoadaptive mutations at a rate of 10-6 per genome per generation, resulting in the fixation of more virulent strains in less than a hundred generations. Whole genome sequencing of independently evolved clones showed that the main targets of intracellular adaptation involved loss of function mutations in genes implicated in the assembly of the lipopolysaccharide core, iron metabolism and di- and tri-peptide transport, namely rfaI, fhuA and tppB, respectively. We found a substantial amount of antagonistic pleiotropy in evolved populations, as well as metabolic trade-offs, commonly found in intracellular bacteria with reduced genome sizes. Overall, the low levels of clonal interference detected indicate that the first steps of the transition of a commensal E. coli into intracellular pathogens are dominated by a few pathoadaptive mutations with very strong effects.LAO/ITQB, FCT
Sepsis: the need for tolerance not complacency
Get PDFSepsis is a life-threatening condition that arises as a systemic inflammatory response syndrome to an infection. Its uncontrolled progression can in frequent cases lead to multiple organ failure, which is still associated with high mortality rates. Modern antibiotics made clear that the infection is only an initiating, and not always necessary, event of this syndrome as many patients with sepsis die despite effective eradication of the inciting pathogen. This observation critically contributed to a paradigm shift that focused the pathogenesis of sepsis on the host and not on the pathogen. However, therapeutic strategies based on the inhibition of proinflammatory critical mediators of sepsis or immunostimulation have so far failed to improve sepsis outcome and, therefore, this condition urgently needs transformative therapeutic ideas and strategies. Here we argue that the induction of tolerance, a defence strategy that minimises the impact of an infection on organ function without directly affecting the pathogen burden, is perhaps the missing but essential element to add to the current components of sepsis care and treatment.IGC Integrative Biology and Biomedicine (IBB) PhD Program, FCT, European Research Council (ERC-2014-CoG 647888-iPROTECTION)
Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation
Get PDFSince years, research on SnRK1, the major cellular energy sensor in plants, has tried to define its role in energy signalling. However, these attempts were notoriously hampered by the lethality of a complete knockout of SnRK1. Therefore, we generated an inducible amiRNA::SnRK1α2 in a snrk1α1 knock out background (snrk1α1/α2) to abolish SnRK1 activity to understand major systemic functions of SnRK1 signalling under energy deprivation triggered by extended night treatment. We analysed the in vivo phosphoproteome, proteome and metabolome and found that activation of SnRK1 is essential for repression of high energy demanding cell processes such as protein synthesis. The most abundant effect was the constitutively high phosphorylation of ribosomal protein S6 (RPS6) in the snrk1α1/α2 mutant. RPS6 is a major target of TOR signalling and its phosphorylation correlates with translation. Further evidence for an antagonistic SnRK1 and TOR crosstalk comparable to the animal system was demonstrated by the in vivo interaction of SnRK1α1 and RAPTOR1B in the cytosol and by phosphorylation of RAPTOR1B by SnRK1α1 in kinase assays. Moreover, changed levels of phosphorylation states of several chloroplastic proteins in the snrk1α1/α2 mutant indicated an unexpected link to regulation of photosynthesis, the main energy source in plants.Austrian Science Fund FWF Projects: (P 26342, P 25488, P 28491)
CYR61 and TAZ Upregulation and Focal Epithelial to Mesenchymal Transition May Be Early Predictors of Barrett’s Esophagus Malignant Progression
Get PDFBarrett's esophagus is the major risk factor for esophageal adenocarcinoma. It has a low but non-neglectable risk, high surveillance costs and no reliable risk stratification markers. We sought to identify early biomarkers, predictive of Barrett's malignant progression, using a meta-analysis approach on gene expression data. This in silico strategy was followed by experimental validation in a cohort of patients with extended follow up from the Instituto Português de Oncologia de Lisboa de Francisco Gentil EPE (Portugal). Bioinformatics and systems biology approaches singled out two candidate predictive markers for Barrett's progression, CYR61 and TAZ. Although previously implicated in other malignancies and in epithelial-to-mesenchymal transition phenotypes, our experimental validation shows for the first time that CYR61 and TAZ have the potential to be predictive biomarkers for cancer progression. Experimental validation by reverse transcriptase quantitative PCR and immunohistochemistry confirmed the up-regulation of both genes in Barrett's samples associated with high-grade dysplasia/adenocarcinoma. In our cohort CYR61 and TAZ up-regulation ranged from one to ten years prior to progression to adenocarcinoma in Barrett's esophagus index samples. Finally, we found that CYR61 and TAZ over-expression is correlated with early focal signs of epithelial to mesenchymal transition. Our results highlight both CYR61 and TAZ genes as potential predictive biomarkers for stratification of the risk for development of adenocarcinoma and suggest a potential mechanistic route for Barrett's esophagus neoplastic progression.Ophiomics Precision Medicine
REST is a hypoxia-responsive transcriptional repressor
Get PDFCellular exposure to hypoxia results in altered gene expression in a range of physiologic and pathophysiologic states. Discrete cohorts of genes can be either up- or down-regulated in response to hypoxia. While the Hypoxia-Inducible Factor (HIF) is the primary driver of hypoxia-induced adaptive gene expression, less is known about the signalling mechanisms regulating hypoxia-dependent gene repression. Using RNA-seq, we demonstrate that equivalent numbers of genes are induced and repressed in human embryonic kidney (HEK293) cells. We demonstrate that nuclear localization of the Repressor Element 1-Silencing Transcription factor (REST) is induced in hypoxia and that REST is responsible for regulating approximately 20% of the hypoxia-repressed genes. Using chromatin immunoprecipitation assays we demonstrate that REST-dependent gene repression is at least in part mediated by direct binding to the promoters of target genes. Based on these data, we propose that REST is a key mediator of gene repression in hypoxia.Science Foundation Ireland grant: (06/CE/B1129)
Dynamics of Wolbachia pipientis gene expression across the Drosophila melanogaster life cycle
Get PDFSymbiotic interactions between microbes and their multicellular hosts have
manifold impacts on molecular, cellular and organismal biology. To identify
candidate bacterial genes involved in maintaining endosymbiotic associations
with insect hosts, we analyzed genome-wide patterns of gene expression in the
alpha-proteobacteria Wolbachia pipientis across the life cycle of Drosophila
melanogaster using public data from the modENCODE project that was generated in
a Wolbachia-infected version of the ISO1 reference strain. We find that the
majority of Wolbachia genes are expressed at detectable levels in D.
melanogaster across the entire life cycle, but that only 7.8% of 1195 Wolbachia
genes exhibit robust stage- or sex-specific expression differences when studied
in the "holo-organism" context. Wolbachia genes that are differentially
expressed during development are typically up-regulated after D. melanogaster
embryogenesis, and include many bacterial membrane, secretion system and
ankyrin-repeat containing proteins. Sex-biased genes are often organised as
small operons of uncharacterised genes and are mainly up-regulated in adult
males D. melanogaster in an age-dependent manner suggesting a potential role in
cytoplasmic incompatibility. Our results indicate that large changes in
Wolbachia gene expression across the Drosophila life-cycle are relatively rare
when assayed across all host tissues, but that candidate genes to understand
host-microbe interaction in facultative endosymbionts can be successfully
identified using holo-organism expression profiling. Our work also shows that
mining public gene expression data in D. melanogaster provides a rich set of
resources to probe the functional basis of the Wolbachia-Drosophila symbiosis
and annotate the transcriptional outputs of the Wolbachia genome.National Environment Research Council Ph.D. studentship (F.G.), Fundação para a Ciência e a Tecnologia postdoctoral fellowship: (SFRH/BPD/73420/2010), Stowers Institute for Medical Research award (R.S.H.), National Science Foundation grant: (NSF-IOS-1456545), Human Frontier Science Program grant: (RGY0093/
2012), Biotechnology and Biological Sciences Research Council grant: (BB/L002817/1)
Multiple Resistance at No Cost: Rifampicin and Streptomycin a Dangerous Liaison in the Spread of Antibiotic Resistance
Get PDFEvidence is mounting that epistasis is widespread among mutations. The cost of carrying two deleterious mutations, or the advantage of acquiring two beneficial alleles, is typically lower that the sum of their individual effects. Much less is known on epistasis between beneficial and deleterious mutations, even though this is key to the amount of genetic hitchhiking that may occur during evolution. This is particularly important in the context of antibiotic resistance: Most resistances are deleterious, but some can be beneficial and remarkably rifampicin resistance can emerge de novo in populations evolving without antibiotics. Here we show pervasive positive pairwise epistasis on Escherichia coli fitness between beneficial mutations, which confer resistance to rifampicin, and deleterious mutations, which confer resistance to streptomycin. We find that 65% of double resistant strains outcompete sensitive bacteria in an environment devoid of antibiotics. Weak beneficial mutations may therefore overcome strong deleterious mutations and can even render double mutants strong competitors.LAO/ITQB, FCT
Drosophila melanogaster larvae make nutritional choices that minimize developmental time
No full textOrganisms from slime moulds to humans carefully regulate their macronutrient intake to optimize a wide range of life history characters including survival, stress resistance, and reproductive success. However, life history characters often differ in their response to nutrition, forcing organisms to make foraging decisions while balancing the trade-offs between these effects. To date, we have a limited understanding of how the nutritional environment shapes the relationship between life history characters and foraging decisions. To gain insight into the problem, we used a geometric framework for nutrition to assess how the protein and carbohydrate content of the larval diet affected key life history traits in the fruit fly, Drosophila melanogaster. In no-choice assays, survival from egg to pupae, female and male body size, and ovariole number - a proxy for female fecundity - were maximized at the highest protein to carbohydrate (P:C) ratio (1.5:1). In contrast, development time was minimized at intermediate P:C ratios, around 1:2. Next, we subjected larvae to two-choice tests to determine how they regulated their protein and carbohydrate intake in relation to these life history traits. Our results show that larvae targeted their consumption to P:C ratios that minimized development time. Finally, we examined whether adult females also chose to lay their eggs in the P:C ratios that minimized developmental time. Using a three-choice assay, we found that adult females preferentially laid their eggs in food P:C ratios that were suboptimal for all larval life history traits. Our results demonstrate that D. melanogaster larvae make foraging decisions that trade-off developmental time with body size, ovariole number, and survival. In addition, adult females make oviposition decisions that do not appear to benefit the larvae. We propose that these decisions may reflect the living nature of the larval nutritional environment in rotting fruit. These studies illustrate the interaction between the nutritional environment, life history traits, and foraging choices in D. melanogaster, and lend insight into the ecology of their foraging decisions.Fundação Calouste Gulbenkian; Instituto Gulbenkian de Ciência
SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants
Get PDFMetabolic adjustment to changing environmental conditions, particularly balancing of growth and defense responses, is crucial for all organisms to survive. The evolutionary conserved AMPK/Snf1/SnRK1 kinases are well-known metabolic master regulators in the low-energy response in animals, yeast and plants. They act at two different levels: by modulating the activity of key metabolic enzymes, and by massive transcriptional reprogramming. While the first part is well established, the latter function is only partially understood in animals and not at all in plants. Here we identified the Arabidopsis transcription factor bZIP63 as key regulator of the starvation response and direct target of the SnRK1 kinase. Phosphorylation of bZIP63 by SnRK1 changed its dimerization preference, thereby affecting target gene expression and ultimately primary metabolism. A bzip63 knock-out mutant exhibited starvation-related phenotypes, which could be functionally complemented by wild type bZIP63, but not by a version harboring point mutations in the identified SnRK1 target sites.Austrian Science Fund (FWF) grant: (P23435); Deutsche Forschungsgemeinschaft grant (HA2146/8-2); FCT grants: (PTDC/BIA-PLA/3937/2012, UID/Multi/0455½013)