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The social amoebae (Dictyostelia) are a diverse group of Amoebozoa that achieve multicellularity by aggregation and undergo morphogenesis into fruiting bodies with terminally differentiated spores and stalk cells. There are four groups of dictyostelids, with the most derived being a group that contains the model species Dictyostelium discoideum.
Dictyostelium discoideum is an excellent system in which to study developmental decisions. Synchronous development is triggered by starvation and rapidly generates a limited number of cell types. Genetic and image analyses have revealed the elegant intricacies associated with this simple development system. Key signaling pathways identified as regulating cell fate decisions are likely to be conserved with metazoa and are providing insight into differentiation decisions under circumstances where considerable cell movement takes place during development.
Eukaryotic cells contain a large number of actin binding proteins of different functions, locations and concentrations. They bind either to monomeric actin (G-actin) or to actin filaments (F-actin) and thus regulate the dynamic rearrangement of the actin cytoskeleton. The Dictyostelium discoideum genome harbors representatives of all G-actin binding proteins including actobindin, twinfilin, and profilin. A phylogenetic analysis of all profilins suggests that two distinguishable groups emerged very early in evolution and comprise either vertebrate and viral profilins or profilins from all other organisms. The newly discovered profilin III isoform in D. discoideum shows all functions that are typical for a profilin. However, the concentration of the third isoform in wild type cells reaches only about 0.5% of total profilin. In a yeast-2-hybrid assay profilin III was found to bind specifically to the proline-rich region of the cytoskeleton-associated vasodilator-stimulated phosphoprotein (VASP). Immunolocalization studies showed similar to VASP the profilin III isoform in filopodia and an enrichment at their tips. Cells lacking the profilin III isoform show defects in cell motility during chemotaxis. The low abundance and the specific interaction with VASP argue against a significant actin sequestering function of the profilin III isoform.
Dictyostelium development begins with single-cell starvation and ends with multicellular fruiting bodies. Developmental morphogenesis is accompanied by sweeping transcriptional changes, encompassing nearly half of the 13,000 genes in the genome. We performed time-series RNA-sequencing analyses of the wild type and 20 mutants to explore the relationships between transcription and morphogenesis. These strains show developmental arrest at different stages, accelerated development, or atypical morphologies. Considering eight major morphological transitions, we identified 1371 milestone genes whose expression changes sharply between consecutive transitions. We also identified 1099 genes as members of 21 regulons, which are groups of genes that remain coordinately regulated despite the genetic, temporal, and developmental perturbations. The gene annotations in these groups validate known transitions and reveal new developmental events. For example, DNA replication genes are tightly coregulated with cell division genes, so they are expressed in mid-development although chromosomal DNA is not replicated. Our data set includes 486 transcriptional profiles that can help identify new relationships between transcription and development and improve gene annotations. We show its utility by showing that cycles of aggregation and disaggregation in allorecognition-defective mutants involve dedifferentiation. We also show sensitivity to genetic and developmental conditions in two commonly used actin genes, act6 and act15, and robustness of the coaA gene. Finally, we propose that gpdA is a better mRNA quantitation standard because it is less sensitive to external conditions than commonly used standards. The data set is available for democratized exploration through the web application dictyExpress and the data mining environment Orange.
Cells grow, move, expand, shrink and die in the process of generating the characteristic shapes of organisms. Although the structures generated during development of the social amoeba Dictyostelium discoideum look nothing like the structures seen in metazoan embryogenesis, some of the morphogenetic processes used in their making are surprisingly similar. Recent advances in understanding the molecular basis for directed cell migration, cell type specific sorting, differential adhesion, secretion of matrix components, pattern formation, regulation and terminal differentiation are reviewed. Genes involved in Dictyostelium aggregation, slug formation, and culmination of fruiting bodies are discussed.
Autophagic cell death (ACD) can be operationally described as cell death with an autophagic component. While most molecular bases of this autophagic component are known, in ACD the mechanism of cell death proper is not well defined, in particular because in animal cells there is poor experimental distinction between what triggers autophagy and what triggers ACD. Perhaps as a consequence, it is often thought that in animal cells a little autophagy is protective while a lot is destructive and leads to ACD, thus that the shift from autophagy to ACD is quantitative. The aim of this article is to review current knowledge on ACD in Dictyostelium, a very favorable model, with emphasis on (1) the qualitative, not quantitative nature of the shift from autophagy to ACD, in contrast to the above, and (2) random or targeted mutations of in particular the following genes: iplA (IP3R), TalB (talinB), DcsA (cellulose synthase), GbfA, ugpB, glcS (glycogen synthase) and atg1. These mutations allowed the genetic dissection of ACD features, dissociating in particular vacuolisation from cell death.
Blebs and F-actin-driven pseudopods are alternative ways of extending the leading edge of migrating cells. We show that Dictyostelium cells switch from using predominantly pseudopods to blebs when migrating under agarose overlays of increasing stiffness. Blebs expand faster than pseudopods leaving behind F-actin scars, but are less persistent. Blebbing cells are strongly chemotactic to cyclic-AMP, producing nearly all of their blebs up-gradient. When cells re-orientate to a needle releasing cyclic-AMP, they stereotypically produce first microspikes, then blebs and pseudopods only later. Genetically, blebbing requires myosin-II and increases when actin polymerization or cortical function is impaired. Cyclic-AMP induces transient blebbing independently of much of the known chemotactic signal transduction machinery, but involving PI3-kinase and downstream PH domain proteins, CRAC and PhdA. Impairment of this PI3-kinase pathway results in slow movement under agarose and cells that produce few blebs, though actin polymerization appears unaffected. We propose that mechanical resistance induces bleb-driven movement in Dictyostelium, which is chemotactic and controlled through PI3-kinase.
Rap1 is a key regulator of cell adhesion and cell motility in Dictyostelium. Here, we identify a Rap1-specific GAP protein (RapGAP3) and provide evidence that Rap1 signaling regulates cell-cell adhesion and cell migration within the multicellular organism. RapGAP3 mediates the deactivation of Rap1 at the late mound stage of development and plays an important role in regulating cell sorting during apical tip formation, when the anterior-posterior axis of the organism is formed, by controlling cell-cell adhesion and cell migration. The loss of RapGAP3 results in a severely altered morphogenesis of the multicellular organism at the late mound stage. Direct measurement of cell motility within the mound shows that rapGAP3(-) cells have a reduced speed of movement and, compared to wild-type cells, have a reduced motility towards the apex. rapGAP3(-) cells exhibit some increased EDTA/EGTA sensitive cell-cell adhesion at the late mound stage. RapGAP3 transiently and rapidly translocates to the cell cortex in response to chemoattractant stimulation, which is dependent on F-actin polymerization. We suggest that the altered morphogenesis and the cell-sorting defect of rapGAP3(-) cells may result in reduced directional movement of the mutant cells to the apex of the mound.
Previous studies using magnetic purification of Dictyostelium discoideum endocytic vesicles led us to the identification of some major vesicle proteins. Using the same purification procedure, we have now focused our interest on a 44 kDa soluble vesicle protein. Microsequencing of internal peptides and subsequent cloning of the corresponding cDNA identified this protein as the Dictyostelium homolog of mammalian cathepsins D. The only glycosylation detected on Dictyostelium cathepsin D (CatD) is common antigen 1, a cluster of mannose 6-sulfate residues on N-linked oligosaccharide chains. CatD intracellular trafficking has been studied, showing the presence of the protein throughout the entire endocytic pathway. During the differentiation process, the catD gene presents a developmental regulation, which is also observed at the protein level. catD gene disruption does not alter significantly the cell behaviour, either in the vegetative form or the differentiation stage. However, modifications in the SDS-PAGE profiles of proteins bearing common antigen 1 were detected, when comparing parental and catD(-) cells. These modifications point to a possible role of CatD in the maturation of a few Dictyostelium lysosomal proteins.
We share two simple modifications to enhance the fixation and imaging of relatively small, motile, and rounded model cells. These include cell centrifugation and the addition of trace amounts of glutaraldehyde to existing fixation methods. Though they need to be carefully considered in each context, they have been useful to our studies of the spatial relationships of the microtubule cytoskeletal system.
ATP-binding cassette (ABC) transporters can translocate a broad spectrum of molecules across the cell membrane including physiological cargo and toxins. ABC transporters are known for the role they play in resistance towards anticancer agents in chemotherapy of cancer patients. There are 68 ABC transporters annotated in the genome of the social amoeba Dictyostelium discoideum. We have characterized more than half of these ABC transporters through a systematic study of mutations in their genes. We have analyzed morphological and transcriptional phenotypes for these mutants during growth and development and found that most of the mutants exhibited rather subtle phenotypes. A few of the genes may share physiological functions, as reflected in their transcriptional phenotypes. Since most of the abc-transporter mutants showed subtle morphological phenotypes, we utilized these transcriptional phenotypes to identify genes that are important for development by looking for transcripts whose abundance was unperturbed in most of the mutants. We found a set of 668 genes that includes many validated D. discoideum developmental genes. We have also found that abcG6 and abcG18 may have potential roles in intercellular signaling during terminal differentiation of spores and stalks.
Dictyostelium, an amoeboid motile cell, harbors several paralogous Sec7 genes that encode members of three distinct subfamilies of the Sec7 superfamily of Guanine nucleotide exchange factors. Among them are proteins of the GBF/BIG family present in all eukaryotes. The third subfamily represented with three members in D. discoideum is the cytohesin family that has been thought to be metazoan specific. Cytohesins are characterized by a Sec7 PH tandem domain and have roles in cell adhesion and migration.
Chemotaxis, the directed motion of a cell toward a chemical source, plays a key role in many essential biological processes. Here, we derive a statistical model that quantitatively describes the chemotactic motion of eukaryotic cells in a chemical gradient. Our model is based on observations of the chemotactic motion of the social ameba Dictyostelium discoideum, a model organism for eukaryotic chemotaxis. A large number of cell trajectories in stationary, linear chemoattractant gradients is measured, using microfluidic tools in combination with automated cell tracking. We describe the directional motion as the interplay between deterministic and stochastic contributions based on a Langevin equation. The functional form of this equation is directly extracted from experimental data by angle-resolved conditional averages. It contains quadratic deterministic damping and multiplicative noise. In the presence of an external gradient, the deterministic part shows a clear angular dependence that takes the form of a force pointing in gradient direction. With increasing gradient steepness, this force passes through a maximum that coincides with maxima in both speed and directionality of the cells. The stochastic part, on the other hand, does not depend on the orientation of the directional cue and remains independent of the gradient magnitude. Numerical simulations of our probabilistic model yield quantitative agreement with the experimental distribution functions. Thus our model captures well the dynamics of chemotactic cells and can serve to quantify differences and similarities of different chemotactic eukaryotes. Finally, on the basis of our model, we can characterize the heterogeneity within a population of chemotactic cells.
Kinesin and dynein are the two families of microtubule-based motors that drive much of the intracellular movements in eukaryotic cells. Using a gene knockout strategy, we address here the individual function(s) of four of the 13 kinesin proteins in Dictyostelium. The goal of our ongoing project is to establish a minimal motility proteome for this basal eukaryote, enabling us to contrast motor functions here with the often far more elaborate motor families in the metazoans.
The amoeba Dictyostelium discoideum has been a valuable model organism to study numerous facets of eukaryotic cell biology, such as cell motility, cell adhesion, macropinocytosis and phagocytosis, host-pathogen interactions and multicellular development. However, the relative small size of the Dictyostelium community hampers the production and distribution of reagents and tools, such as antibodies, by commercial vendors.
Cells use phagocytosis and macropinocytosis to internalise bulk material, which in phagotrophic organisms supplies the nutrients necessary for growth. Wildtype Dictyostelium amoebae feed on bacteria, but for decades laboratory work has relied on axenic mutants that can also grow on liquid media. We used forward genetics to identify the causative gene underlying this phenotype. This gene encodes the RasGAP Neurofibromin (NF1). Loss of NF1 enables axenic growth by increasing fluid uptake. Mutants form outsized macropinosomes which are promoted by greater Ras and PI3K activity at sites of endocytosis. Relatedly, NF1 mutants can ingest larger-than-normal particles using phagocytosis. An NF1 reporter is recruited to nascent macropinosomes, suggesting that NF1 limits their size by locally inhibiting Ras signalling. Our results link NF1 with macropinocytosis and phagocytosis for the first time, and we propose that NF1 evolved in early phagotrophs to spatially modulate Ras activity, thereby constraining and shaping their feeding structures.
The presence of five P2X-like genes (p2xA-E) in Dictyostelium suggests that nucleotides other than cAMP may act as extracellular signalling molecules in this model eukaryote. However, p2xA was found to have an exclusively intracellular localisation making it unclear whether Dictyostelium utilise P2 receptors in a manner analogous to vertebrates. Using an apoaequorin expressing strain we show here that Dictyostelium do possess cell surface P2 receptors that facilitate Ca(2+) influx in response to extracellular ATP and ADP (EC(50)=7.5microM and 6.1microM, respectively). Indicative of P2X receptor activation, responses were rapid reaching peak within 2.91+/-0.04s, required extracellular Ca(2+), were inhibited by Gd(3+), modified by extracellular pH and were not affected by deletion of either the single Gbeta or iplA genes. Responses also remained unaffected by disruption of p2xA or p2xE showing that these genes are not involved. Cu(2+) and Zn(2+) inhibited purine-evoked Ca(2+) influx with IC(50) values of 0.9 and 6.3microM, respectively. 300microM Zn(2+) completely abolished the initial large rapid rise in intracellular Ca(2+) revealing the presence of an additional smaller, slower P2Y-like response. The existence of P2 receptors in Dictyostelium makes this organism a valuable model to explore fundamental aspects of purinergic signalling.
The development of new techniques to create gene knockouts and knock-ins is essential for successful investigation of gene functions and elucidation of the causes of diseases and their associated fundamental cellular processes. In the biomedical model organism Dictyostelium discoideum, the methodology for gene targeting with homologous recombination to generate mutants is well-established. Recently, we have applied CRISPR/Cas9-mediated approaches in Dictyostelium, allowing the rapid generation of mutants by transiently expressing sgRNA and Cas9 using an all-in-one vector. CRISPR/Cas9 techniques not only provide an alternative to homologous recombination-based gene knockouts but also enable the creation of mutants that were technically unfeasible previously. Herein, we provide a detailed protocol for the CRISPR/Cas9-based method in Dictyostelium. We also describe new tools, including double knockouts using a single CRISPR vector, drug-inducible knockouts, and gene knockdown using CRISPR interference (CRISPRi). We demonstrate the use of these tools for some candidate genes. Our data indicate that more suitable mutants can be rapidly generated using CRISPR/Cas9-based techniques to study gene function in Dictyostelium.
Dictyostelium intermedium is a member of dictyostelids, the unicellular eukaryotes with a unique life cycle, including a social cycle. Despite the high diversity of dictyostelids, only five species' complete mitochondrial genome sequences were reported. This study aimed to add the D. intermedium mitochondrial genome sequence to the list. The size of this genome is 58,627 bp, with 73.99% A/T, containing 62 genes located on one strand: 41 protein-coding genes, three ribosomal RNA genes, and 18 transfer RNA genes. The 41 protein-coding genes comprised 18 oxidative phosphorylation-related, 16 ribosomal, and seven hypothetical protein-coding genes. The cox1/2 and rnl gene contained introns, similar to other species of Dictyostelium. The phylogenetic tree built based on 34 protein sequences supported the monophyletic clade of Dictyostelium and the dictyostelids' ancestor's position between the two dictyostelids orders: Dictyosteliales and Acytosteliales.
The mature fruiting body of Dictyostelium consists of stalk and spore cells but its construction, and the migration of the preceding slug stage, requires a number of specialized sub-types of prestalk cell whose nature and function are not well understood. The prototypic prestalk-specific gene, ecmA, is inducible by the polyketide DIF-1 in a monolayer assay and requires the DimB and MybE transcription factors for full inducibility. We perform genome-wide microarray analyses, on parental, mybE- and dimB- cells, and identify many additional genes that depend on MybE and DimB for their DIF-1 inducibility. Surprisingly, an even larger number of genes are only DIF inducible in mybE- cells, some genes are only inducible in DimB- cells and some are inducible when either transcription factor is absent. Thus in assay conditions where MybE and DimB function as inducers of ecmA these genes fall under negative control by the same two transcription factors. We have studied in detail rtaA, one of the MybE and DimB repressed genes. One especially enigmatic group of prestalk cells is the anterior-like cells (ALCs), which exist intermingled with prespore cells in the slug. A promoter fusion reporter gene, rtaA:gal(u), is expressed in a subset of the ALCs that is distinct from the ALC population detected by a reporter construct containing ecmA and ecmB promoter fragments. At culmination, when the ALC sort out from the prespore cells and differentiate to form three ancillary stalk cell structures: the upper cup, the lower cup and the outer basal disk, the rtaA:gal(u) expressing cells preferentially populate the upper cup region. This fact, and their virtual absence from the anterior and posterior regions of the slug, identifies them as a new prestalk sub-type: the pstU cells. PstU cell differentiation is, as expected, increased in a dimB- mutant during normal development but, surprisingly, they differentiate normally in a mutant lacking DIF. Thus genetic removal of MybE or DimB reveals an alternate DIF-1 activation pathway, for pstU differentiation, that functions under monolayer assay conditions but that is not essential during multicellular development.
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