Dictyostelium Genome Research Articles

Iron metabolism in the social amoeba Dictyostelium discoideum: A role for ferric chelate reductases

Iron is the most abundant transition metal in all living organisms and is essential for several cellular activities, including respiration, oxygen transport, energy production and regulation of gene expression. Iron starvation is used by professional phagocytes, from Dictyostelium to macrophages, as a form of defense mechanism against intracellular pathogens. Previously, we showed that Dictyostelium cells express the proton-driven iron transporter Nramp1 (Natural Resistance-Associated Macrophage Protein 1) and the homolog NrampB (Nramp2) in membranes of macropinosomes and phagosomes or of the contractile vacuole network, respectively. The Nramp-driven transport of iron across membranes is selective for ferrous ions. Since iron is mostly present as ferric ions in growth media and in engulfed bacteria, we have looked for proteins with ferric reductase activity. The Dictyostelium genome does not encode for classical STEAP (Six-Transmembrane Epithelial Antigen of Prostate) ferric reductases, but harbors three genes encoding putative ferric chelate reductase belonging to the Cytochrome b561 family containing a N terminus DOMON domain (DOpamine β-MONooxygenase N-terminal domain). We have cloned the three genes, naming them fr1A, fr1B and fr1C. fr1A and fr1B are mainly expressed in the vegetative stage while fr1C is highly expressed in the post aggregative stage. All three reductases are localized in the endoplasmic reticulum, but Fr1A is also found in endolysosomal vesicles, in the Golgi and, to a much lower degree, in the plasma membrane, whereas Fr1C is homogeneously distributed in the plasma membrane and in macropinosomal and phagosomal membranes. To gain insight in the function of the three genes we generated KO mutants, but gene disruption was successful only for two of them (fr1A and fr1C), being very likely lethal for fr1B. fr1A- shows a slight delay in the aggregation stage of development, while fr1C- gives rise to large multi-tipped streams during aggregation and displays a strong delay in fruiting body formation. The two single mutants display altered cell growth under conditions of ferric ions overloading and, in the ability to reduce Fe3+, confirming a role of these putative ferric reductases in iron reduction and transport from endo-lysosomal vesicles to the cytosol.

WASP Restricts Active Rac to Maintain Cells' Front-Rear Polarization.

SummaryEfficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP’s Cdc42 and Rac interacting binding (“CRIB”) motif has been thought to be essential for its activation. However, we show that the CRIB motif’s biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought—Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif.

Mapping and characterization of G-quadruplexes in the genome of the social amoeba Dictyostelium discoideum.

G-quadruplexes (G4) are non-canonical DNA and/or RNA secondary structures formed in guanine-rich regions. Given their over-representation in specific regions in the genome such as promoters and telomeres, they are likely to play important roles in key processes such as transcription, replication or RNA maturation. Putative G4-forming sequences (G4FS) have been reported in humans, yeast, bacteria, viruses and many organisms. Here we present the first mapping of G-quadruplex sequences in Dictyostelium discoideum, the social amoeba. ‘Dicty’ is an ameboid protozoan with a small (34 Mb) and extremely AT rich genome (78%). As a consequence, very few G4-prone motifs are expected. An in silico analysis of the Dictyostelium genome with the G4Hunter software detected 249–1055 G4-prone motifs, depending on G4Hunter chosen threshold. Interestingly, despite an even lower GC content (as compared to the whole Dicty genome), the density of G4 motifs in Dictyostelium promoters and introns is significantly higher than in the rest of the genome. Fourteen selected sequences located in important genes were characterized by a combination of biophysical and biochemical techniques. Our data show that these sequences form highly stable G4 structures under physiological conditions. Five Dictyostelium genes containing G4-prone motifs in their promoters were studied for the effect of a new G4-binding porphyrin derivative on their expression. Our results demonstrated that the new ligand significantly decreased their expression. Overall, our results constitute the first step to adopt Dictyostelium discoideum as a ‘G4-poor’ model for studies on G-quadruplexes.

Recent Insights into NCL Protein Function Using the Model Organism Dictyostelium discoideum.

The neuronal ceroid lipofuscinoses (NCLs) are a group of devastating neurological disorders that have a global distribution and affect people of all ages. Commonly known as Batten disease, this form of neurodegeneration is linked to mutations in 13 genetically distinct genes. The precise mechanisms underlying the disease are unknown, in large part due to our poor understanding of the functions of NCL proteins. The social amoeba Dictyostelium discoideum has proven to be an exceptional model organism for studying a wide range of neurological disorders, including the NCLs. The Dictyostelium genome contains homologs of 11 of the 13 NCL genes. Its life cycle, comprised of both single-cell and multicellular phases, provides an excellent system for studying the effects of NCL gene deficiency on conserved cellular and developmental processes. In this review, we highlight recent advances in NCL research using Dictyostelium as a biomedical model.

The past, present and future of Dictyostelium as a model system.

The social amoeba Dictyostelium discoideum has been a preferred model organism during the last 50 years, particularly for the study of cell motility and chemotaxis, phagocytosis and macropinocytosis, intercellular adhesion, pattern formation, caspase-independent cell death and more recently autophagy and social evolution. Being a soil amoeba and professional phagocyte, thus exposed to a variety of potential pathogens, D. discoideum has also proven to be a powerful genetic and cellular model for investigating host-pathogen interactions and microbial infections. The finding that the Dictyostelium genome harbours several homologs of human genes responsible for a variety of diseases has stimulated their analysis, providing new insights into the mechanism of action of the encoded proteins and in some cases into the defect underlying the disease. Recent technological developments have covered the genetic gap between mammals and non-mammalian model organisms, challenging the modelling role of the latter. Is there a future for Dictyostelium discoideum as a model organism?

Cyclic AMP signaling in Dictyostelium promotes the translocation of the copine family of calcium-binding proteins to the plasma membrane

BackgroundCopines are calcium-dependent phospholipid-binding proteins found in many eukaryotic organisms and are thought to be involved in signaling pathways that regulate a wide variety of cellular processes. Copines are characterized by having two C2 domains at the N-terminus accompanied by an A domain at the C-terminus. Six copine genes have been identified in the Dictyostelium genome, cpnA – cpnF.ResultsIndependent cell lines expressing CpnA, CpnB, CpnC, CpnE, or CpnF tagged with green fluorescent protein (GFP) were created as tools to study copine protein membrane-binding and localization. In general, the GFP-tagged copine proteins appeared to localize to the cytoplasm in live cells. GFP-tagged CpnB, CpnC, and CpnF were also found in the nucleus. When cells were fixed or when live cells were treated with calcium ionophore, the GFP-tagged copine proteins were found associated with the plasma membrane and vesicular organelles. When starved Dictyostelium cells were stimulated with cAMP, which causes a transitory increase in calcium concentration, all of the copines translocated to the plasma membrane, but with varying magnitudes and on and off times, suggesting each of the copines has distinct calcium-sensitivities and/or membrane-binding properties. In vitro membrane binding assays showed that all of the GFP-tagged copines pelleted with cellular membranes in the presence of calcium; yet, each copine displayed distinct calcium-independent membrane-binding in the absence of calcium. A lipid overlay assay with purified GFP-tagged copine proteins was used to screen for specific phospholipid-binding targets. Similar to other proteins that contain C2 domains, GFP-tagged copines bound to a variety of acidic phospholipids. CpnA, CpnB, and CpnE bound strongly to PS, PI(4)P, and PI(4,5)P2, while CpnC and CpnF bound strongly to PI(4)P.ConclusionsOur studies show that the Dictyostelium copines are soluble cytoplasmic and nuclear proteins that have the ability to bind intracellular membranes. Moreover, copines display different membrane-binding properties suggesting they play distinct roles in the cell. The transient translocation of copines to the plasma membrane in response to cAMP suggests copines may play a specific role in chemotaxis signaling.

CRISPR/Cas9 mediated targeting of multiple genes in Dictyostelium

CRISPR/Cas9 has emerged in various organisms as a powerful technology for targeted gene knockout; however, no reports of editing the Dictyostelium genome efficiently using this system are available. We describe here the application of CRISPR/Cas9-mediated gene modification in Dictyostelium. The endogenous tRNA-processing system for expressing sgRNA was approximately 10 times more effective than the commonly used U6 promoter. The resulting sgRNA affected the sub-nuclear localisation of Cas9, indicating that the expression level of sgRNA was sufficiently high to form Cas9 and sgRNA complexes within the nucleus. The all-in-one vector containing Cas9 and sgRNA was transiently expressed to generate mutants in five PI3K genes. Mutation detective PCR revealed the mutagenesis frequency of the individual genes to be between 72.9% and 100%. We confirmed that all five targeting loci in the four independent clones had insertion/deletion mutations in their target sites. Thus, we show that the CRISPR/Cas9 system can be used in Dictyostelium cells to enable efficient genome editing of multiple genes. Since this system utilises transient expression of the all-in-one vector, it has the advantage that the drug resistance cassette is not integrated into the genome and simple vector construction, involving annealing two oligo-DNAs.

Dictyostelium Erk2 is an atypical MAPK required for chemotaxis

The Dictyostelium genome encodes only two MAPKs, Erk1 and Erk2, and both are expressed during growth and development. Reduced levels of Erk2 expression have been shown previously to restrict cAMP production during development but still allow for chemotactic movement. In this study the erk2 gene was disrupted to eliminate Erk2 function. The absence of Erk2 resulted in a complete loss of folate and cAMP chemotaxis suggesting that this MAPK plays an integral role in the signaling mechanisms involved with this cellular response. However, folate stimulation of early chemotactic responses, such as Ras and PI3K activation and rapid actin filament formation, were not affected by the loss of Erk2 function. The erk2− cells had a severe defect in growth on bacterial lawns but assays of bacterial cell engulfment displayed only subtle changes in the rate of bacterial engulfment. Only cells with no MAPK function, erk1−erk2− double mutants, displayed a severe proliferation defect in axenic medium. Loss of Erk2 impaired the phosphorylation of Erk1 in secondary responses to folate stimulation indicating that Erk2 has a role in the regulation of Erk1 activation during chemotaxis. Loss of the only known Dictyostelium MAPK kinase, MekA, prevented the phosphorylation of Erk1 but not Erk2 in response to folate and cAMP confirming that Erk2 is not regulated by a conventional MAP2K. This lack of MAP2K phosphorylation of Erk2 and the sequence similarity of Erk2 to mammalian MAPK15 (Erk8) suggest that the Dictyostelium Erk2 belongs to a group of atypical MAPKs. MAPK activation has been observed in chemotactic responses in a wide range of organisms but this study demonstrates an essential role for MAPK function in chemotactic movement. This study also confirms that MAPKs provide critical contributions to cell proliferation.

Retrotransposon Domestication and Control in Dictyostelium discoideum.

Transposable elements, identified in all eukaryotes, are mobile genetic units that can change their genomic position. Transposons usually employ an excision and reintegration mechanism, by which they change position, but not copy number. In contrast, retrotransposons amplify via RNA intermediates, increasing their genomic copy number. Hence, they represent a particular threat to the structural and informational integrity of the invaded genome. The social amoeba Dictyostelium discoideum, model organism of the evolutionary Amoebozoa supergroup, features a haploid, gene-dense genome that offers limited space for damage-free transposition. Several of its contemporary retrotransposons display intrinsic integration preferences, for example by inserting next to transfer RNA genes or other retroelements. Likely, any retrotransposons that invaded the genome of the amoeba in a non-directed manner were lost during evolution, as this would result in decreased fitness of the organism. Thus, the positional preference of the Dictyostelium retroelements might represent a domestication of the selfish elements. Likewise, the reduced danger of such domesticated transposable elements led to their accumulation, and they represent about 10% of the current genome of D. discoideum. To prevent the uncontrolled spreading of retrotransposons, the amoeba employs control mechanisms including RNA interference and heterochromatization. Here, we review TRE5-A, DIRS-1 and Skipper-1, as representatives of the three retrotransposon classes in D. discoideum, which make up 5.7% of the Dictyostelium genome. We compile open questions with respect to their mobility and cellular regulation, and suggest strategies, how these questions might be addressed experimentally.

The Parkinson's disease-associated protein DJ-1 plays a positive nonmitochondrial role in endocytosis in Dictyostelium cells.

ABSTRACTThe loss of function of DJ-1 caused by mutations in DJ1 causes a form of familial Parkinson's disease (PD). However, the role of DJ-1 in healthy and in PD cells is poorly understood. Even its subcellular localization in mammalian cells is uncertain, with both cytosolic and mitochondrial locations having been reported. We show here that DJ-1 is normally located in the cytoplasm in healthy Dictyostelium discoideum cells. With its unique life cycle, straightforward genotype-phenotype relationships, experimental accessibility and genetic tractability, D. discoideum offers an attractive model to investigate the roles of PD-associated genes. Furthermore, the study of mitochondrial biology, mitochondrial genome transcription and AMP-activated protein kinase-mediated cytopathologies in mitochondrial dysfunction have been well developed in this organism. Unlike mammalian systems, Dictyostelium mitochondrial dysfunction causes a reproducible and readily assayed array of aberrant phenotypes: defective phototaxis, impaired growth, normal rates of endocytosis and characteristic defects in multicellular morphogenesis. This makes it possible to study whether the underlying cytopathological mechanisms of familial PD involve mitochondrial dysfunction. DJ-1 has a single homologue in the Dictyostelium genome. By regulating the expression level of DJ-1 in D. discoideum, we show here that in unstressed cells, DJ-1 is required for normal rates of endocytic nutrient uptake (phagocytosis and, to a lesser extent, pinocytosis) and thus growth. Reduced expression of DJ-1 had no effect on phototaxis in the multicellular migratory ‘slug’ stage of the life cycle, but resulted in thickened stalks in the final fruiting bodies. This pattern of phenotypes is distinct from that observed in Dictyostelium to result from mitochondrial dyfunction. Direct measurement of mitochondrial respiratory function in intact cells revealed that DJ-1 knockdown stimulates whereas DJ-1 overexpression inhibits mitochondrial activity. Together, our results suggest positive roles for DJ-1 in endocytic pathways and loss-of-function cytopathologies that are not associated with impaired mitochondrial function.

The two Dictyostelium autophagy eight proteins, ATG8a and ATG8b, associate with the autophagosome in succession

Autophagy is an ancient cellular pathway that is conserved from yeast to man. It contributes to many physiological and pathological processes and plays a major role in the degradation of proteins and/or organelles in response to starvation and stress. In the autophagic process cytosolic material is captured into double membrane-bound vesicles, the autophagosomes. After fusion with lysosomes, the cargo is degraded in the generated autolysosomes and then recycled for further use.Autophagy 8 (ATG8, in mammals LC3), a well-established marker of autophagy, is covalently linked to phosphatidylethanolamine on the autophagic membrane during autophagosome formation. Bioinformatic analysis of the Dictyostelium genome revealed two atg8 genes which encode the ATG8a and ATG8b paralogs. They are with around 14kDa similar in size, 54 % identical to one another and more closely related to the corresponding proteins in fungi and plants than in animals. For ATG8a we found a strong up-regulation throughout the 24h developmental time course while ATG8b expression was highest in vegetative cells followed by a moderate reduction during early development. Confocal microscopy of fluorescently tagged ATG8a and ATG8b in vegetative AX2 wild-type and in ATG9− cells showed that both proteins mainly co-localized on vesicular structures with a diameter above 500nm while those smaller than 500nm were predominantly positive for ATG8b. In ATG9− cells we found a strong increase in the relative abundance of ATG8a-positive large vesicular structures and of total ATG8b-positive structures per cell indicating autophagic flux problems in this mutant. We also found that vesicular structures positive for ATG8a and/or ATG8b were also positive for ubiquitin. Live cell imaging of AX2 and ATG9− cells co-expressing combinations of red and green tagged ATG8a, ATG8b or ATG9 revealed transient co localizations of these proteins. Our results suggest that ATG8b associates with nascent autophagosomes before ATG8a. We further find that the process of autophagosome formation in Dictyostelium is highly dynamic. We infer from our data that Dictyostelium ATG8a and ATG8b have distinct functions in autophagosome formation and that ATG8b is the functional orthologue of the mammalian LC3 subfamily and ATG8a of the GABARAP subfamily.

Iron metabolism and resistance to infection by invasive bacteria in the social amoeba Dictyostelium discoideum

Dictyostelium cells are forest soil amoebae, which feed on bacteria and proliferate as solitary cells until bacteria are consumed. Starvation triggers a change in life style, forcing cells to gather into aggregates to form multicellular organisms capable of cell differentiation and morphogenesis. As a soil amoeba and a phagocyte that grazes on bacteria as the obligate source of food, Dictyostelium could be a natural host of pathogenic bacteria. Indeed, many pathogens that occasionally infect humans are hosted for most of their time in protozoa or free-living amoebae, where evolution of their virulence traits occurs. Due to these features and its amenability to genetic manipulation, Dictyostelium has become a valuable model organism for studying strategies of both the host to resist infection and the pathogen to escape the defense mechanisms. Similarly to higher eukaryotes, iron homeostasis is crucial for Dictyostelium resistance to invasive bacteria. Iron is essential for Dictyostelium, as both iron deficiency or overload inhibit cell growth. The Dictyostelium genome shares with mammals many genes regulating iron homeostasis. Iron transporters of the Nramp (Slc11A) family are represented with two genes, encoding Nramp1 and Nramp2. Like the mammalian ortholog, Nramp1 is recruited to phagosomes and macropinosomes, whereas Nramp2 is a membrane protein of the contractile vacuole network, which regulates osmolarity. Nramp1 and Nramp2 localization in distinct compartments suggests that both proteins synergistically regulate iron homeostasis. Rather than by absorption via membrane transporters, iron is likely gained by degradation of ingested bacteria and efflux via Nramp1 from phagosomes to the cytosol. Nramp gene disruption increases Dictyostelium sensitivity to infection, enhancing intracellular growth of Legionella or Mycobacteria. Generation of mutants in other “iron genes” will help identify genes essential for iron homeostasis and resistance to pathogens.

Identification and recombinant expression of anandamide hydrolyzing enzyme from Dictyostelium discoideum

BackgroundAnandamide (Arachidonoyl ethanolamide) is a potent bioactive lipid studied extensively in humans, which regulates several neurobehavioral processes including pain, feeding and memory. Bioactivity is terminated when hydrolyzed into free arachidonic acid and ethanolamine by the enzyme fatty acid amide hydrolase (FAAH). In this study we report the identification of a FAAH homolog from Dictyostelium discoideum and its function to hydrolyze anandamide.ResultsA putative FAAH DNA sequence coding for a conserved amidase signature motif was identified in the Dictyostelium genome database and the corresponding cDNA was isolated and expressed as an epitope tagged fusion protein in either E.coli or Dictyostelium. Wild type Dictyostelium cells express FAAH throughout their development life cycle and the protein was found to be predominantly membrane associated. Production of recombinant HIS tagged FAAH protein was not supported in E.coli host, but homologous Dictyostelium host was able to produce the same successfully. Recombinant FAAH protein isolated from Dictyostelium was shown to hydrolyze anandamide and related synthetic fatty acid amide substrates.ConclusionsThis study describes the first identification and characterisation of an anandamide hydrolyzing enzyme from Dictyostelium discoideum, suggesting the potential of Dictyostelium as a simple eukaryotic model system for studying mechanisms of action of any FAAH inhibitors as drug targets.

Salmonella typhimurium is pathogenic for Dictyostelium cells and subverts the starvation response

In unicellular amoebae, such as Dictyostelium discoideum, bacterial phagocytosis is a food hunting device, while in higher organisms it is the first defence barrier against microbial infection. In both cases, pathogenic bacteria exploit phagocytosis to enter the cell and multiply intracellularly. Salmonella typhimurium, the agent of food-borne gastroenteritis, is phagocytosed by both macrophages and Dictyostelium cells. By using cell biological assays and global transcriptional analysis with DNA microarrays covering the Dictyostelium genome, we show here that S. typhimurium is pathogenic for Dictyostelium cells. Depending on the degree of virulence, which in turn depended on bacterial growth conditions, Salmonella could kill Dictyostelium cells or inhibit their growth and development. In the early phase of infection in non-nutrient buffer, the ingested bacteria escaped degradation, induced a starvation-like transcriptional response but inhibited selectively genes required for chemotaxis and aggregation. This way differentiation of the host cells into spore and stalk cells was blocked or delayed, which in turn is likely to be favourable for the establishment of a replicative niche for Salmonella. Inhibition of the aggregation competence and chemotactic streaming of aggregation-competent cells in the presence of Salmonella suggests interference with cAMP signalling.

DNA double-strand break repair pathway choice inDictyostelium

DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). The mechanisms that govern whether a DSB is repaired by NHEJ or HR remain unclear. Here, we characterise DSB repair in the amoeba Dictyostelium. HR is the principal pathway responsible for resistance to DSBs during vegetative cell growth, a stage of the life cycle when cells are predominantly in G2. However, we illustrate that restriction-enzyme-mediated integration of DNA into the Dictyostelium genome is possible during this stage of the life cycle and that this is mediated by an active NHEJ pathway. We illustrate that Dclre1, a protein with similarity to the vertebrate NHEJ factor Artemis, is required for NHEJ independently of DNA termini complexity. Although vegetative dclre1(-) cells are not radiosensitive, they exhibit delayed DSB repair, further supporting a role for NHEJ during this stage of the life cycle. By contrast, cells lacking the Ku80 component of the Ku heterodimer that binds DNA ends to facilitate NHEJ exhibit no such defect and deletion of ku80 suppresses the DSB repair defect of dclre1(-) cells through increasing HR efficiency. These data illustrate a functional NHEJ pathway in vegetative Dictyostelium and the importance of Ku in regulating DSB repair choice during this phase of the life cycle.

The secreted proteome profile of developing Dictyostelium discoideum cells

Dictyostelium discoideum is a unicellular eukaryote that, when starved, aggregates to form multicellular structures. In this report, we identified the proteins secreted by developing Dictyostelium cells using MS-based proteomics. A total of 349 different secreted proteins were identified, indicating that at least 2.6% of the 13 600 predicted proteins in the Dictyostelium genome are secreted. Gene ontology analysis suggests that many of the secreted proteins are involved in protein and carbohydrate metabolism, and proteolysis.

Structural Analysis of a Metazoan Nuclear Pore Complex Reveals a Fused Concentric Ring Architecture

The sole gateway for molecular exchange between the cytoplasm and the nucleus is the nuclear pore complex (NPC). This large supramolecular assembly mediates transport of cargo into and out of the nucleus and fuse the inner and outer nuclear membranes to form an aqueous translocation channel. The NPC is composed of eight proteinaceous asymmetric units forming a pseudo-8-fold symmetric passage. Due to its shear size, complexity, and plastic nature, dissecting the high-resolution three-dimensional structure of the NPC in its hydrated state is a formidable challenge. Toward this goal, we applied cryo-electron tomography to spread nuclear envelopes from Xenopus oocytes. To compensate for perturbations of the 8-fold symmetry of individual NPCs, we performed symmetry-independent asymmetric unit averaging of three-dimensional tomographic NPC volumes to eventually yield a refined model at 6.4 nm resolution. This approach revealed novel structural features, particularly in the spoke–ring complex and luminal domains. Fused concentric ring architecture of the spoke–ring complex was found along the translocation channel. Additionally, a comparison of the refined Xenopus model to that of its Dictyostelium homologue yielded similar pore diameters at the level of the three canonical rings, although the Xenopus NPC was found to be 30% taller than the Dictyostelium pore. This discrepancy is attributed primarily to the relatively low homology and different organization of some nucleoporins in the Dictyostelium genome as compared to that of vertebrates. Nevertheless, the experimental conditions impose a preferred axial orientation of the NPCs within spread Xenopus oocyte nuclear envelopes. This may at least in part explain the increased height of the reconstructed vertebrate NPCs compared to those obtained from tomographic reconstruction of intact Dictyostelium nuclei.

Expression of zinc transporter family genes in Dictyostelium

Regulation of the zinc ion concentration is physiologically important to control the activities of a variety of cellular molecules. A BLAST search against a conserved domain of known zinc transporters identified twelve putative zinc transporter family genes in the Dictyostelium genome. Phylogenetic analysis revealed the presence of three zinc transporter subfamilies in Dictyostelium. One subfamily of proteins, consisting of the ZntA-D proteins, has weak homology to the STAT3-inducible LIV-1 protein. In addition, in situ hybridization revealed that the zntA-D genes are expressed in the pstAB cells, this expression being absent in the Dd-STATa null mutant. Thus, Dd-STATa may control stalk cell differentiation through some members of the zinc transporter family genes during Dictyostelium development.

An anatomy ontology to represent biological knowledge in Dictyostelium discoideum.

BackgroundDictyostelium discoideum is a model system for studying many important physiological processes including chemotaxis, phagocytosis, and signal transduction. The recent sequencing of the genome has revealed the presence of over 12,500 protein-coding genes. The model organism database dictyBase hosts the genome sequence as well as a large amount of manually curated information.ResultsWe present here an anatomy ontology for Dictyostelium based upon the life cycle of the organism.ConclusionAnatomy ontologies are necessary to annotate species-specific events such as phenotypes, and the Dictyostelium anatomy ontology provides an essential tool for curation of the Dictyostelium genome.

Optimization of a large-scale gene disruption protocol in Dictyostelium and analysis of conserved genes of unknown function

BackgroundDevelopment of the post-genomic age in Dictyostelium will require the existence of rapid and reliable methods to disrupt genes that would allow the analysis of entire gene families and perhaps the possibility to undertake the complete knock-out analysis of all the protein-coding genes present in Dictyostelium genome.ResultsHere we present an optimized protocol based on the previously described construction of gene disruption vectors by in vitro transposition. Our method allows a rapid selection of the construct by a simple PCR approach and subsequent sequencing. Disruption constructs were amplified by PCR and the products were directly transformed in Dictyostelium cells. The selection of homologous recombination events was also performed by PCR. We have constructed 41 disruption vectors to target genes of unknown function, highly conserved between Dictyostelium and human, but absent from the genomes of S. cerevisiae and S. pombe. 28 genes were successfully disrupted.ConclusionThis is the first step towards the understanding of the function of these conserved genes and exemplifies the easiness to undertake large-scale disruption analysis in Dictyostelium.