RESUMO
Understanding the processes that underlie the development of population genetic structure is central to the study of evolution. Patterns of genetic structure, in turn, can reveal signatures of isolation by distance (IBD), barriers to gene flow, or even the genesis of speciation. However, it is unclear how severe range restriction might impact the processes that dominate the development of genetic structure. In narrow endemic species, is population structure likely to be adaptive in nature, or rather the result of genetic drift? In this study, we investigated patterns of genetic diversity and structure in the narrow endemic Hayden's ringlet butterfly. Specifically, we asked to what degree genetic structure in the Hayden's ringlet can be explained by IBD, isolation by resistance (IBR) (in the form of geographic or ecological barriers to migration between populations), and isolation by environment (in the form of differences in host plant availability and preference). We employed a genotyping-by-sequencing (GBS) approach coupled with host preference assays, Bayesian modelling, and population genomic analyses to answer these questions. Our results suggest that despite their restricted range, levels of genetic diversity in the Hayden's ringlet are comparable to those seen in more widespread butterfly species. Hayden's ringlets showed a strong preference for feeding on grasses relative to sedges, but neither larval preference nor potential host availability at sampling sites correlated with genetic structure. We conclude that geography, in the form of IBR and simple IBD, was the major driver of contemporary patterns of differentiation in this narrow endemic species.
Assuntos
Borboletas , Variação Genética , Animais , Borboletas/genética , Teorema de Bayes , Deriva Genética , Geografia , Genética PopulacionalRESUMO
Effective population size affects the efficacy of selection, rate of evolution by drift and neutral diversity levels. When species are subdivided into multiple populations connected by gene flow, evolutionary processes can depend on global or local effective population sizes. Theory predicts that high levels of diversity might be maintained by gene flow, even very low levels of gene flow, consistent with species long-term effective population size, but tests of this idea are mostly lacking. Here, we show that Lycaeides butterfly populations maintain low contemporary (variance) effective population sizes (e.g. ~200 individuals) and thus evolve rapidly by genetic drift. However, populations harboured high levels of genetic diversity consistent with an effective population size several orders of magnitude larger. We hypothesized that the differences in the magnitude and variability of contemporary versus long-term effective population sizes were caused by gene flow of sufficient magnitude to maintain diversity but only subtly affect evolution on generational timescales. Consistent with this hypothesis, we detected low but nontrivial gene flow among populations. Furthermore, using short-term population-genomic time-series data, we documented patterns consistent with predictions from this hypothesis, including a weak but detectable excess of evolutionary change in the direction of the mean (migrant gene pool) allele frequencies across populations and consistency in the direction of allele frequency change over time. The documented decoupling of diversity levels and short-term change by drift in Lycaeides has implications for our understanding of contemporary evolution and the maintenance of genetic variation in the wild.
Assuntos
Borboletas , Fluxo Gênico , Animais , Borboletas/genética , Deriva Genética , Variação Genética , Genética Populacional , Genômica , HumanosRESUMO
The role of malate dehydrogenase (MDH) in the metabolism of various medically significant protozoan parasites is reviewed. MDH is an NADH-dependent oxidoreductase that catalyzes interconversion between oxaloacetate and malate, provides metabolic intermediates for both catabolic and anabolic pathways, and can contribute to NAD+/NADH balance in multiple cellular compartments. MDH is present in nearly all organisms; isoforms of MDH from apicomplexans (Plasmodium falciparum, Toxoplasma gondii, Cryptosporidium spp.), trypanosomatids (Trypanosoma brucei, T. cruzi) and anaerobic protozoans (Trichomonas vaginalis, Giardia duodenalis) are presented here. Many parasitic species have complex life cycles and depend on the environment of their hosts for carbon sources and other nutrients. Metabolic plasticity is crucial to parasite transition between host environments; thus, the regulation of metabolic processes is an important area to explore for therapeutic intervention. Common themes in protozoan parasite metabolism include emphasis on glycolytic catabolism, substrate-level phosphorylation, non-traditional uses of common pathways like tricarboxylic acid cycle and adapted or reduced mitochondria-like organelles. We describe the roles of MDH isoforms in these pathways, discuss unusual structural or functional features of these isoforms relevant to activity or drug targeting, and review current studies exploring the therapeutic potential of MDH and related genes. These studies show that MDH activity has important roles in many metabolic pathways, and thus in the metabolic transitions of protozoan parasites needed for success as pathogens.
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Course-based Undergraduate Research Experiences (CUREs) integrate active, discovery-based learning into undergraduate curricula, adding tremendous value to Biochemistry and Molecular Biology (BMB) education. There are multiple challenges in transforming a research project into a CURE, such as the readiness of students, the time commitment of the instructor, and the productivity of the research. In this article, we report a CURE course developed and offered in the University of Massachusetts Amherst BMB Department since 2018 that addresses these challenges. Our CURE focuses on fungal effectors which are proteins secreted by a destructive pathogenic fungus Fusarium oxysporum, one of the top five most devastating plant pathogens. By studying this group of proteins, students are connected to real-world problems and participate in the search for potential solutions. A 3-week "standard Boot Camp" is implemented to help students familiarize themselves with all basic techniques and boost their confidence. Next, molecular cloning, a versatile technique with modularity and repeatability, is used as the bedrock of the course. Our past 5 years of experience have confirmed that we have developed a novel and feasible CURE protocol. Measurable progress documented by students who took this course includes stimulated active learning and increased career trajectory to pursue hypothesis-based research to address societal needs. In addition, data generated through the course advance ongoing lab research. Collectively, we encourage the implementation of CURE among research-intensive faculty to provide a more inclusive research experience to undergraduate students, an important element in predicting career success.
Assuntos
Bioquímica , Estudantes , Humanos , Bioquímica/educação , Currículo , Aprendizagem Baseada em Problemas , Proteínas/químicaRESUMO
Malate dehydrogenase (MDH) is a key enzyme in mammalian metabolic pathways in cytosolic and mitochondrial compartments. Regulation of MDH through phosphorylation remains an underexplored area. In this review we consolidate evidence supporting the potential role of phosphorylation in modulating the function of mammalian MDH. Parallels are drawn with the phosphorylation of lactate dehydrogenase, a homologous enzyme, to reveal its regulatory significance and to suggest a similar regulatory strategy for MDH. Comprehensive mining of phosphorylation databases, provides substantial experimental (primarily mass spectrometry) evidence of MDH phosphorylation in mammalian cells. Experimentally identified phosphorylation sites are overlaid with MDH's functional domains, offering perspective on how these modifications could influence enzyme activity. Preliminary results are presented from phosphomimetic mutations (serine/threonine residues changed to aspartate) generated in recombinant MDH proteins serving as a proof of concept for the regulatory impact of phosphorylation. We also examine and highlight several approaches to probe the structural and cellular impact of phosphorylation. This review highlights the need to explore the dynamic nature of MDH phosphorylation and calls for identifying the responsible kinases and the physiological conditions underpinning this modification. The synthesis of current evidence and experimental data aims to provide insights for future research on understanding MDH regulation, offering new avenues for therapeutic interventions in metabolic disorders and cancer.
RESUMO
Many pathogenic bacteria, fungi, and protozoa achieve chronic infection through an immune evasion strategy known as antigenic variation. In the human malaria parasite Plasmodium falciparum, this involves transcriptional switching among members of the var gene family, causing parasites with different antigenic and phenotypic characteristics to appear at different times within a population. Here we use a genome-wide approach to explore this process in vitro within a set of cloned parasite populations. Our analyses reveal a non-random, highly structured switch pathway where an initially dominant transcript switches via a set of switch-intermediates either to a new dominant transcript, or back to the original. We show that this specific pathway can arise through an evolutionary conflict in which the pathogen has to optimise between safeguarding its limited antigenic repertoire and remaining capable of establishing infections in non-naïve individuals. Our results thus demonstrate a crucial role for structured switching during the early phases of infections and provide a unifying theory of antigenic variation in P. falciparum malaria as a balanced process of parasite-intrinsic switching and immune-mediated selection.
Assuntos
Variação Antigênica , Antígenos de Protozoários/genética , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Algoritmos , Perfilação da Expressão Gênica , Fenótipo , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Transcrição GênicaRESUMO
Course-based Undergraduate Research Experiences (CUREs) integrate active, discovery-based learning into undergraduate curriculums, adding tremendous value to Biochemistry and Molecular Biology (BMB) education. There are multiple challenges in transforming a research project into a CURE, such as the readiness of students, the time commitment of the instructor, and the productivity of the research. In this article, we report a CURE course developed and offered in the University of Massachusetts Amherst BMB Department since 2018 that addresses these challenges. Our CURE focuses on fungal effectors which are proteins secreted by a destructive pathogenic fungus Fusarium oxysporum , one of the top five most devastating plant pathogens. By studying this group of proteins, students are connected to real-world problems and participate in the search for potential solutions. A three-week "standard Bootcamp" is implemented to help students familiarize themselves with all basic techniques and boost their confidence. Next, molecular cloning, a versatile technique with modularity and repeatability, is used as the bedrock of the course. Our past five years of experience have confirmed that we have developed a novel and feasible CURE protocol. Measurable progress documented by students who took this course includes stimulated active learning and increased career trajectory to pursue hypothesis-based research to address societal needs. In addition, data generated through the course advance ongoing lab research. Collectively, we encourage the implementation of CURE among research-intensive faculty to provide a more inclusive research experience to all students, an important element in predicting career success.
RESUMO
Trypanosoma brucei is a protozoan flagellate that causes African sleeping sickness. Flagellar function in this organism is critical for life cycle progression and pathogenesis, however the regulation of flagellar motility is not well understood. The flagellar axoneme produces a complex beat through the precisely coordinated firing of many proteins, including multiple dynein motors. These motors are found in the inner arm and outer arm complexes. We are studying one of the inner arm dynein motors in the T. brucei flagellum: dynein-f. RNAi knockdown of genes for two components of dynein-f: DNAH10, the alpha heavy chain, and IC138, an intermediate chain, cause severe motility defects including immotility. To determine if motility defects result from structural disruption of the axoneme, we used two different flagellar preparations to carefully examine axoneme structure in these strains using transmission electron microscopy (TEM). Our analysis showed that inner arm dynein size, axoneme structural integrity and fixed central pair orientation are not significantly different in either knockdown culture when compared to control cultures. These results support the idea that immotility in knockdowns affecting DNAH10 or IC138 results from loss of dynein-f function rather than from obvious structural defects in the axoneme.
Assuntos
Axonema/metabolismo , Dineínas/química , Trypanosoma brucei brucei/metabolismo , Animais , Ciclo Celular , Movimento Celular , Dineínas/metabolismo , Flagelos/metabolismo , Microscopia Eletrônica de Transmissão/métodos , Modelos Biológicos , Interferência de RNARESUMO
Environmental stress can have a profound effect on inbreeding depression. Quantifying this effect is of particular importance in threatened populations, which are often simultaneously subject to both inbreeding and environmental stress. But while the prevalence of inbreeding-stress interactions is well known, the importance and broader applicability of such interactions in conservation are not clearly understood. We used seed beetles, Callosobruchus maculatus, as a model system to quantify how environmental stressors (here host quality and temperature stress) interact with inbreeding as measured by changes in the magnitude of inbreeding depression, δ, as well as the relative importance of inbreeding-stress interactions to overall fitness. We found that while both environmental stressors caused substantial inbreeding-stress interactions as measured by change in δ, the relative importance of these interactions to overall survival was modest. This suggests that assessing inbreeding-stress interactions within the framework of δ alone may give an inaccurate representation of the relevance of interactions to population persistence. Furthermore, we found that the effect of environmental stress on fitness, but not inbreeding depression, varied strongly among populations. These results suggest that the outcomes of inbreeding-stress interactions are not easily generalized, an important consideration in conservation settings.
RESUMO
The ability to adapt to a novel host plant may vary among insect populations with different genetic histories, and colonization of a marginal host may be facilitated by genetic admixture of disparate populations. We assembled populations of the seed beetle, Callosobruchus maculatus (F.), from four continents, and compared their ability to infest two hosts, lentil and pea. We also formed two cross-continent hybrids (Africa × N.A. and Africa × S.A.). In pre-selection assays, survival was only ~3% in lentil and ~40% in pea. For three replicate populations per line, colonization success on lentil was measured as cumulative exit holes after 75-175 d. On pea, we estimated the change in larval survival after five generations of selection. Females in all lines laid few eggs on lentil, and survival of F1 larvae was uniformly <5%. Subsequently, however, the lines diverged considerably in population growth. Performance on lentil was highest in the Africa × N.A. hybrid, which produced far more adults (mean > 11,000) than either parental line. At the other extreme, Asian populations on lentil appeared to have gone extinct. The Africa × N.A. line also exhibited the highest survival on pea, and again performed better than either parent line. However, no line displayed a rapid increase in survival on pea, as is sometimes observed on lentil. Our results demonstrate that geographic populations can vary substantially in their responses to the same novel resource. In addition, genetic admixtures (potentially caused by long-distance transport of infested seeds) may facilitate colonization of an initially poor host.
Assuntos
Besouros , Animais , Besouros/genética , Feminino , Larva/genética , ÓvuloRESUMO
Genes that affect adaptive traits have been identified, but our knowledge of the genetic basis of adaptation in a more general sense (across multiple traits) remains limited. We combined population-genomic analyses of evolve-and-resequence experiments, genome-wide association mapping of performance traits, and analyses of gene expression to fill this knowledge gap and shed light on the genomics of adaptation to a marginal host (lentil) by the seed beetle Callosobruchus maculatus. Using population-genomic approaches, we detected modest parallelism in allele frequency change across replicate lines during adaptation to lentil. Mapping populations derived from each lentil-adapted line revealed a polygenic basis for two host-specific performance traits (weight and development time), which had low to modest heritabilities. We found less evidence of parallelism in genotype-phenotype associations across these lines than in allele frequency changes during the experiments. Differential gene expression caused by differences in recent evolutionary history exceeded that caused by immediate rearing host. Together, the three genomic datasets suggest that genes affecting traits other than weight and development time are likely to be the main causes of parallel evolution and that detoxification genes (especially cytochrome P450s and beta-glucosidase) could be especially important for colonization of lentil by C. maculatus.
Assuntos
Besouros/genética , Fabaceae/parasitologia , Interações Hospedeiro-Parasita/genética , Seleção Genética , Adaptação Fisiológica/genética , Animais , Besouros/patogenicidade , Frequência do Gene/genética , Genômica , Larva/parasitologia , Fenótipo , Sementes/parasitologiaRESUMO
BACKGROUND: Var genes encode a family of virulence factors known as PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) which are responsible for both antigenic variation and cytoadherence of infected erythrocytes. Although these molecules play a central role in malaria pathogenesis, the mechanisms generating variant antigen diversification are poorly understood. To investigate var gene evolution, we compared the variant antigen repertoires from three geographically diverse parasite isolates: the 3D7 genome reference isolate; the recently sequenced HB3 isolate; and the IT4/25/5 (IT4) parasite isolate which retains the capacity to cytoadhere in vitro and in vivo. RESULTS: These comparisons revealed that only two var genes (var1csa and var2csa) are conserved in all three isolates and one var gene (Type 3 var) has homologs in IT4 and 3D7. While the remaining 50 plus genes in each isolate are highly divergent most can be classified into the three previously defined major groups (A, B, and C) on the basis of 5' flanking sequence and chromosome location. Repertoire-wide sequence comparisons suggest that the conserved homologs are evolving separately from other var genes and that genes in group A have diverged from other groups. CONCLUSION: These findings support the existence of a var gene recombination hierarchy that restricts recombination possibilities and has a central role in the functional and immunological adaptation of var genes.
Assuntos
Antígenos de Protozoários/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Animais , Mapeamento Cromossômico , Evolução Molecular , Genes de Protozoários , Variação Genética , Genoma de Protozoário , Filogenia , Plasmodium falciparum/classificação , Recombinação Genética/genética , Análise de Sequência de DNARESUMO
Motility in the protozoan parasite Trypanosoma brucei is conferred by a single flagellum, attached alongside the cell, which moves the cell forward using a beat that is generated from tip-to-base. We are interested in characterizing components that regulate flagellar beating, in this study we extend the characterization of TbIC138, the ortholog of a dynein intermediate chain that regulates axonemal inner arm dynein f/I1. TbIC138 was tagged In situ-and shown to fractionate with the inner arm components of the flagellum. RNAi knockdown of TbIC138 resulted in significantly reduced protein levels, mild growth defect and significant motility defects. These cells tended to cluster, exhibited slow and abnormal motility and some cells had partially or fully detached flagella. Slight but significant increases were observed in the incidence of mis-localized or missing kinetoplasts. To document development of the TbIC138 knockdown phenotype over time, we performed a detailed analysis of flagellar detachment and motility changes over 108 hours following induction of RNAi. Abnormal motility, such as slow twitching or irregular beating, was observed early, and became progressively more severe such that by 72 hours-post-induction, approximately 80% of the cells were immotile. Progressively more cells exhibited flagellar detachment over time, but this phenotype was not as prevalent as immotility, affecting less than 60% of the population. Detached flagella had abnormal beating, but abnormal beating was also observed in cells with no flagellar detachment, suggesting that TbIC138 has a direct, or primary, effect on the flagellar beat, whereas detachment is a secondary phenotype of TbIC138 knockdown. Our results are consistent with the role of TbIC138 as a regulator of motility, and has a phenotype amenable to more extensive structure-function analyses to further elucidate its role in the control of flagellar beat in T. brucei.
Assuntos
Dineínas/fisiologia , Flagelos/fisiologia , Proteínas de Protozoários/fisiologia , Trypanosoma brucei brucei/fisiologia , Axonema/fisiologia , Ciclo Celular , Núcleo Celular/ultraestrutura , Dineínas/deficiência , Dineínas/genética , Flagelos/genética , Flagelos/ultraestrutura , Mitocôndrias/ultraestrutura , Movimento , Fenótipo , Proteínas de Protozoários/genética , Interferência de RNA , Trypanosoma brucei brucei/ultraestruturaRESUMO
Cytoadherence of Plasmodium falciparum-infected erythrocytes is associated with severe malaria and is primarily mediated through binding of the variant surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1) to specific host ligands. Infected erythrocyte binding to Intercellular Adhesion Molecule 1 (ICAM-1) has been implicated as having a role in cerebral malaria, a major cause of death from P. falciparum infection. We have examined ICAM-1-binding PfEMP1 proteins in the cytoadhesive P. falciparum strain IT4/25/5 in order to extend our understanding of binding. For A4tres, the ICAM-1 binding region was previously shown to reside within contiguous DBL2beta and c2 domains. We determined the gene sequence encoding IT-ICAM var, and showed that ICAM-1 binding in this protein also maps to DBL2betac2 domains that have 48% amino acid identity to A4tres. By truncation and chimera analysis, most of the DBL2beta and the first half of the c2 region were required for A4tres binding to ICAM-1, suggesting this tandem should be considered a structural-functional combination for ICAM-1 binding. Of interest, a chimera formed between two different ICAM-1 binding domains did not bind ICAM-1, suggesting a functional interdependence between DBL2beta and c2 from the same protein. As gene recombination and gene conversion are important mechanisms for generating diversity in the PfEMP1 protein family, this finding implies an extra level of constraint on the functional evolution of binding traits. Knowledge about the PfEMP1::ICAM-1 interaction may allow the development of interventions to prevent binding and disease.
Assuntos
Molécula 1 de Adesão Intercelular/metabolismo , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Sequência Conservada , DNA de Protozoário/química , Dados de Sequência Molecular , Plasmodium falciparum/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Recombinação Genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Immobilization of proteins and other biological macromolecules on solid supports is a method suitable for purification or screening applications in life science research. Prolinx, Inc. has developed a novel chemical affinity system that can be used for specific immobilization of proteins and other macromolecules via interaction of two small synthetic molecules, phenyldiboronic acid (PDBA) and salicylhydroxamic acid (SHA). This report describes immobilization applications of activated microporous membranes that have been functionalized with SHA derivatives. These SHA-membranes exhibit high capacity and specificity for binding of PDBA-labeled nucleic acids and proteins. Conjugation of active protein with PDBA is performed in solution independent of the immobilization step on SHA membranes. The resulting PDBA-protein conjugate is immobilized directly without purification and retains biological activity. PDBA conjugates may also be released from these SHA-affinity membranes in a controlled manner. Capture and release of PBA-modified oligonucleotides is also demonstrated. SHA-membranes can be used as surfaces for microarrays, and are therefore compatible with high-throughput analyses. These properties make them useful for development of numerous preparative or screening applications.
Assuntos
Membranas Artificiais , Salicilamidas , Análise Serial de ProteínasRESUMO
Fluorescent dyes provide specific, sensitive, and multiplexed detection of nucleic acids. To maximize sensitivity, fluorescently labeled reaction products (e.g., cycle sequencing or primer extension products) must be purified away from residual dye-labeled precursors. Successful high-throughput analyses require that this purification be reliable, rapid, and amenable to automation. Common methods for purifying reaction products involve several steps and require processes that are not easily automated. Prolinx, Inc. has devel oped RapXtract superparamagnetic separation technology affording rapid and easy-to-perform methods that yield high-quality product and are easily automated. The technology uses superparamagnetic particles that specifically remove unincorporated dye-labeled precursors. These particles are efficiently pelleted in the presence of a magnetic field, making them ideal for purification because of the rapid separations that they allow. RapXtract-purified sequencing reactions yield data with good signal and high Phred quality scores, and they work with various sequencing dye chemistries, including BigDye and near-infrared fluorescence IRDyes. RapXtract technology can also be used to purify dye primer sequencing reactions, primer extension reactions for genotyping analysis, and nucleic acid labeling reactions for microarray hybridization. The ease of use and versatility of RapXtract technology makes it a good choice for manual or automated purification of fluorescently labeled nucleic acids.
Assuntos
Corantes Fluorescentes/isolamento & purificação , Ácidos Nucleicos/isolamento & purificação , Sequência de Bases , Precipitação Química , DNA Complementar/isolamento & purificação , Corantes Fluorescentes/química , Perfilação da Expressão Gênica , Genótipo , Haemophilus influenzae/química , Magnetismo , Dados de Sequência Molecular , Ácidos Nucleicos/química , Análise de Sequência com Séries de Oligonucleotídeos , RNA Bacteriano/isolamento & purificação , RNA Fúngico/genética , Saccharomyces cerevisiae/genética , Análise de Sequência de DNARESUMO
Because understanding the structure of biological macromolecules is critical to understanding their function, students of biochemistry should become familiar not only with viewing, but also with generating and manipulating structural representations. We report a strategy from a one-semester undergraduate biochemistry course to integrate use of structural representation tools into both laboratory and homework activities. First, early in the course we introduce the use of readily available open-source software for visualizing protein structure, coincident with modules on amino acid and peptide bond properties. Second, we use these same software tools in lectures and incorporate images and other structure representations in homework tasks. Third, we require a capstone project in which teams of students examine a protein-nucleic acid complex and then use the software tools to illustrate for their classmates the salient features of the structure, relating how the structure helps explain biological function. To ensure engagement with a range of software and database features, we generated a detailed template file that can be used to explore any structure, and that guides students through specific applications of many of the software tools. In presentations, students demonstrate that they are successfully interpreting structural information, and using representations to illustrate particular points relevant to function. Thus, over the semester students integrate information about structural features of biological macromolecules into the larger discussion of the chemical basis of function. Together these assignments provide an accessible introduction to structural representation tools, allowing students to add these methods to their biochemical toolboxes early in their scientific development.
Assuntos
Bioquímica/educação , Substâncias Macromoleculares/química , Software , Ensino/métodos , Universidades , Estrutura Molecular , EstudantesRESUMO
The Trypanosoma brucei flagellum controls motility and is crucial for cell polarity and division. Unique features of trypanosome motility suggest that flagellar beat regulation in this organism is unusual and worthy of study. The flagellar axoneme, required for motility, has a structure that is highly conserved among eukaryotes. Of the several dyneins in the axonemal inner arm complex, dynein f is thought to control flagellar waveform shape. A T. brucei gene predicted to encode the dynein f alpha heavy chain, TbDNAH10, was silenced using RNA interference in procyclic T. brucei cells. This resulted in immotile flagella, showing no movement except for occasional slight twitches at the tips. Cell growth slowed dramatically and cells were found in large clusters. Microscopic analysis of silenced cultures showed many cells with detached flagella, sometimes entangled between multiple cells. DAPI staining showed an increased frequency of mis-positioned kinetoplasts and multinucleate cells, suggesting that these cells experience disruption at an early cell cycle stage, probably secondary to the motility defect. TEM images showed apparently normal axonemes and no discernable defects in inner arm structure. This study demonstrates the use of RNAi as an effective method to study very large genes such as dynein heavy chains (HCs), and the immotility phenotype of these dynein knockdowns suggests that an intact inner arm is necessary for flagellar beating in T. brucei. Since analogous mutants in Chlamydomonas reinhardtii retain motility, this phenotype likely reflects differences in requirements for motility and/or dynein assembly between the two organisms and these comparative studies will help elucidate the mechanisms of flagellar beat regulation.
Assuntos
Dineínas/antagonistas & inibidores , Flagelos/fisiologia , Locomoção , Interferência de RNA , Trypanosoma brucei brucei/fisiologia , Núcleo Celular/ultraestrutura , Dineínas/genética , Flagelos/genética , Flagelos/ultraestrutura , Microscopia Eletrônica de Transmissão , Organelas/ultraestrutura , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/ultraestruturaRESUMO
The Duffy binding-like (DBL) domain is a key adhesive module in Plasmodium falciparum, present in both erythrocyte invasion ligands (EBLs) and the large and diverse P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of cytoadherence receptors. DBL domains bind a variety of different host receptors, including intercellular adhesion molecule 1 (ICAM-1), a receptor interaction that may have a role in infected erythrocyte binding to cerebral blood vessels and cerebral malaria. In this study, we expressed the nearly full complement of DBLbeta-C2 domains from the IT4/25/5 (IT4) parasite isolate and showed that ICAM-1-binding domains (DBLbeta-C2(ICAM-1)) were confined to group B and group C PfEMP1 proteins and were not present in group A, suggesting that ICAM-1 selection pressure differs between PfEMP1 groups. To further dissect the molecular determinants of binding, we modelled a DBLbeta-C2(ICAM-1) domain on a solved DBL structure and created alanine substitution mutants in two DBLbeta-C2(ICAM-1) domains. This analysis indicates that the DBLbeta-C2::ICAM-1 interaction maps to the equivalent glycan binding region of EBLs, and suggests a general model for how DBL domains evolve under dual selection for host receptor binding and immune evasion.