Simple system – substantial share: The use of Dictyostelium in cell biology and molecular medicine

Dictyostelium discoideum offers unique advantages for studying fundamental cellular processes, host–pathogen interactions as well as the molecular causes of human diseases. The organism can be easily grown in large amounts and is amenable to diverse biochemical, cell biological and genetic approaches. Throughout their life cycle Dictyostelium cells are motile, and thus are perfectly suited to study random and directed cell motility with the underlying changes in signal transduction and the actin cytoskeleton. Dictyostelium is also increasingly used for the investigation of human disease genes and the crosstalk between host and pathogen. As a professional phagocyte it can be infected with several human bacterial pathogens and used to study the infection process. The availability of a large number of knock-out mutants renders Dictyostelium particularly useful for the elucidation and investigation of host cell factors. A powerful armory of molecular genetic techniques that have been continuously expanded over the years and a well curated genome sequence, which is accessible via the online database dictyBase, considerably strengthened Dictyostelium's experimental attractiveness and its value as model organism.     

Dictyostelium discoideum—a model for many reasons

The social amoeba or cellular slime mould Dictyostelium discoideum is a “professional” phagocyte that has long been recognized for its value as a biomedical model organism, particularly in studying the actomyosin cytoskeleton and chemotactic motility in non-muscle cells. The complete genome sequence of D. discoideum is known, it is genetically tractable, readily grown clonally as a eukaryotic microorganism and is highly accessible for biochemical, cell biological and physiological studies. These are the properties it shares with other microbial model organisms. However, Dictyostelium combines these with a unique life style, with motile unicellular and multicellular stages, and multiple cell types that offer for study an unparalleled variety of phenotypes and associated signalling pathways. These advantages have led to its recent emergence as a valuable model organism for studying the molecular pathogenesis and treatment of human disease, including a variety of infectious diseases caused by bacterial and fungal pathogens. Perhaps surprisingly, this organism, without neurons or brain, has begun to yield novel insights into the cytopathology of mitochondrial diseases as well as other genetic and idiopathic disorders affecting the central nervous system. Dictyostelium has also contributed significantly to our understanding of NDP kinase, as it was the Dictyostelium enzyme whose structure was first determined and related to enzymatic activity. The phenotypic richness and tractability of Dictyostelium should provide a fertile arena for future exploration of NDPK’s cellular roles.     

A role for copper in protozoan grazing – two billion years selecting for bacterial copper resistance

The Great Oxidation Event resulted in integration of soft metals in a wide range of biochemical processes including, in our opinion, killing of bacteria by protozoa. Compared to pressure from anthropologic copper contamination, little is known on impacts of protozoan predation on maintenance of copper resistance determinants in bacteria. To evaluate the role of copper and other soft metals in predatory mechanisms of protozoa, we examined survival of bacteria mutated in different transition metal efflux or uptake systems in the social amoeba Dictyostelium discoideum. Our data demonstrated a strong correlation between the presence of copper/zinc efflux as well as iron/manganese uptake, and bacterial survival in amoebae. The growth of protozoa, in turn, was dependent on bacterial copper sensitivity. The phagocytosis of bacteria induced upregulation of Dictyostelium genes encoding the copper uptake transporter p80 and a triad of Cu(I)‐translocating P_IB‐type ATPases. Accumulated Cu(I) in Dictyostelium was monitored using a copper biosensor bacterial strain. Altogether, our data demonstrate that Cu(I) is ultimately involved in protozoan predation of bacteria, supporting our hypothesis that protozoan grazing selected for the presence of copper resistance determinants for about two billion years.     

The biochemical differentiation of Enterobacter sakazakii genotypes

Background

Development 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.

Results

Here 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.

Conclusion

This 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.     

Evidence for sub-families of actin genes in Dictyostelium as determined by comparisons of 3' end sequences

We have sequenced the 3′ end of five actin genomic clones and three actin complementary DNA clones from Dictyostelium. Comparison of the sequences shows that the protein coding regions are highly conserved, while the region corresponding to the 3′ untranslated regions are divergent. Additional analysis indicates regions of homology in the 3′ untranslated region between sets of actin genes. Southern DNA blot hybridization studies using labeled 3′ ends suggest that there are sub-families of actin genes that are related within the 3′ untranslated regions. No homology is found in the sequences outside the messenger RNA encoding regions. Analysis of the sequence data has shown that the difference in length between the ~1.25 × 10³ and ~1.35 × 10³ base actin messenger RNAs is in the lengths of the 3′ untranslated region.     

Mfsd8 localizes to endocytic compartments and influences the secretion of Cln5 and cathepsin D in Dictyostelium

The neuronal ceroid lipofuscinoses (NCLs) are a family of neurodegenerative diseases that affect people of all ages and ethnicities, yet many of the associated genes/proteins are not well characterized. Mutations in MFSD8 (major facilitator superfamily domain-containing 8) cause an infantile form of NCL referred to as CLN7 disease. In this study, we revealed the localization and binding partners of an ortholog of human MFSD8 (Mfsd8) in the social amoeba Dictyostelium discoideum. Putative lysosomal targeting motifs are conserved in Dictyostelium Mfsd8, as are several residues mutated in CLN7 disease patients. Mfsd8 tagged with GFP localizes to endocytic compartments, which includes acidic intracellular vesicles and late endosomes. We pulled-down GFP-Mfsd8 and used mass spectrometry to reveal the Mfsd8 interactome during Dictyostelium growth and starvation. Among the identified hits were the Dictyostelium ortholog of human cathepsin D (CtsD), as well as proteins linked to the functions of the CLN3 (Cln3) and CLN5 (Cln5) orthologs in Dictyostelium. To study the function of Mfsd8, we validated a publically available mfsd8⁻ cell line (GWDI Project) and then used this knockout cell line to show that Mfsd8 influences the secretion of Cln5 and CtsD. This information is then integrated into an emerging model describing the molecular networking of NCL proteins in Dictyostelium. In total, this study identifies Dictyostelium as a new model system for studying CLN7 disease.     

Genomic database resources for Dictyostelium discoideum

Dictyostelium is an attractive model system for the study of mechanisms basic to cellular function or complex multicellular developmental processes. Recent advances in Dictyostelium genomics have generated a wide spectrum of resources. However, much of the current genomic sequence information is still not currently available through GenBank or related databases. Thus, many investigators are unaware that extensive sequence data from Dictyostelium has been compiled, or of its availability and access. Here, we discuss progress in Dictyostelium genomics and gene annotation, and highlight the primary portals for sequence access, manipulation and analysis (http://genome.imb-jena.de/dictyostelium/; http://dictygenome.bcm.tmc.edu/; http://www.sanger. ac.uk/Projects/D_discoideum/; http://www.csm.biol. tsukuba.ac.jp/cDNAproject.html).     

c-di-GMP induction of Dictyostelium cell death requires the polyketide DIF-1

Cell death in the model organism Dictyostelium, as studied in monolayers in vitro, can be induced by the polyketide DIF-1 or by the cyclical dinucleotide c-di-GMP. c-di-GMP, a universal bacterial second messenger, can trigger innate immunity in bacterially infected animal cells and is involved in developmental cell death in Dictyostelium. We show here that c-di-GMP was not sufficient to induce cell death in Dictyostelium cell monolayers. Unexpectedly, it also required the DIF-1 polyketide. The latter could be exogenous, as revealed by a telling synergy between c-di-GMP and DIF-1. The required DIF-1 polyketide could also be endogenous, as shown by the inability of c-di-GMP to induce cell death in Dictyostelium HMX44A cells and DH1 cells upon pharmacological or genetic inhibition of DIF-1 biosynthesis. In these cases, c-di-GMP–induced cell death was rescued by complementation with exogenous DIF-1. Taken together, these results demonstrated that c-di-GMP could trigger cell death in Dictyostelium only in the presence of the DIF-1 polyketide or its metabolites. This identified another element of control to this cell death and perhaps also to c-di-GMP effects in other situations and organisms.     

Dictyostelium protein kinase C-delta-like protein is localized in the cell nucleus

The molecular mechanism whereby protein kinase C (PKC) molecules transduce signals into the cell nucleus is unknown. In this study, we provide evidence that Dictyostelium discoideum contains PKCδ-like protein that is localized in the nucleus. The Dictyostelium PKCδ-like protein has an apparent molecular mass of 76 kDa. This protein is already highly expressed in vegetative Dictyostelium cells. The expression level remained constant up to 12 h of development, and sharply decreased after 16 h. The PKCδ-like protein is phosphorylated in vivo in response to cAMP and phorbol ester stimulation. Immunofluorescent studies, as well as subcellular fractionation experiments, have indicated that Dictyostelium PKCδ-like protein is permanently located in the nucleus. Our results may indicate that PKCδ-like protein in Dictyostelium functions as a link between cAMP and the tumor-promoting phorbol esters, and events that take place in the nucleus.     

Activation of Balbiani ring genes inChironomus tentans after a pilocarpine-induced depletion of the secretory products from the salivary gland lumen

We have constructed recombinant plasmid libraries containing complementary DNA (cDNA) inserts made to poly(A)⁺ RNA isolated from two stages of Dictyostelium development. The procedure utilized for the cloning allows the excision of the cDNA inserts free of vehicle sequences. The two libraries were screened for inserts complementary to moderately abundant and abundant poly(A)⁺ RNA whose genes are differentially modulated during Dictyostelium development. Several of these plasmids were then further examined by hybridization techniques to determine the reiteration frequencies of their genes, the relative rate of complementary RNA synthesis during development, and the relative accumulation and disappearance of complementary RNA during the Dictyostelium life cycle. RNA complementary to two sequences was found to accumulate from approximately one molecule per cell during vegetative growth to several hundred molecules during preaggregation.     

A rapid and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the Cre-loxP system

Dictyostelium discoideum has proven an exceptionally powerful system for studying numerous aspects of cellular and developmental functions. The relatively small (~34 Mb) chromosomal genome of Dictyostelium and high efficiency of targeted gene disruption have enabled researchers to characterize many specific gene functions. However, the number of selectable markers in Dictyostelium is restricted, as is the ability to perform effective genetic crosses between strains. Thus, it has been difficult to create multiple mutations within an individual cell to study epistatic relationships among genes or potential redundancies between various pathways. We now describe a robust system for the production of multiple gene mutations in Dictyostelium by recycling a single selectable marker, Blasticidin S resistance, using the Cre-loxP system. We confirm the effectiveness of the system by generating a single cell carrying four separate gene disruptions. Furthermore, the cells remain sensitive to transformation for additional targeted or random mutagenesis requiring Blasticidin selection and for functional expression studies of mutated or tagged proteins using other selectable markers.     

Rapid generation of gene disruption constructs by in vitro transposition and identification of a Dictyostelium protein kinase that regulates its rate of growth and development

We describe a rapid method for creating Dictyo stelium gene disruption constructs, whereby the target gene is interrupted by a drug resistance cassette using in vitro transposition. A fragment of genomic DNA containing the gene to be disrupted is amplified by PCR, cloned into a plasmid vector using topoisomerase and then employed as the substrate in an in vitro Tn5 transposition reaction. The transposing species is a fragment of DNA containing a Dictyostelium blasticidin S resistance (bs^r) cassette linked to a bacterial tetracycline resistance (tet^r) cassette. After transposition the plasmid DNA is transformed into Escherichia coli and clones in which the bs^r-tet^r cassette is inserted into the Dictyostelium target DNA are identified. To demonstrate its utility we have employed the method to disrupt the gene encoding QkgA, a novel protein kinase identified from the Dictyostelium genome sequencing project. QkgA is structurally homologous to two previously identified Dictyostelium kinases, GbpC and pats1. Like them it contains a leucine-rich repeat domain, a small GTP-binding (ras) domain and a MEKK domain. Disruption of the qkgA gene causes a marked increase in growth rate and, during development, aggregation occurs relatively slowly to form abnormally large multicellular structures.     

The Central Role of AMP-Kinase and Energy Homeostasis Impairment in Alzheimer’s Disease: A Multifactor Network Analysis

Alzheimer’s disease is the most common cause of dementia worldwide, affecting the elderly population. It is characterized by the hallmark pathology of amyloid-β deposition, neurofibrillary tangle formation, and extensive neuronal degeneration in the brain. Wealth of data related to Alzheimer’s disease has been generated to date, nevertheless, the molecular mechanism underlying the etiology and pathophysiology of the disease is still unknown. Here we described a method for the combined analysis of multiple types of genome-wide data aimed at revealing convergent evidence interest that would not be captured by a standard molecular approach. Lists of Alzheimer-related genes (seed genes) were obtained from different sets of data on gene expression, SNPs, and molecular targets of drugs. Network analysis was applied for identifying the regions of the human protein-protein interaction network showing a significant enrichment in seed genes, and ultimately, in genes associated to Alzheimer’s disease, due to the cumulative effect of different combinations of the starting data sets. The functional properties of these enriched modules were characterized, effectively considering the role of both Alzheimer-related seed genes and genes that closely interact with them. This approach allowed us to present evidence in favor of one of the competing theories about AD underlying processes, specifically evidence supporting a predominant role of metabolism-associated biological process terms, including autophagy, insulin and fatty acid metabolic processes in Alzheimer, with a focus on AMP-activated protein kinase. This central regulator of cellular energy homeostasis regulates a series of brain functions altered in Alzheimer’s disease and could link genetic perturbation with neuronal transmission and energy regulation, representing a potential candidate to be targeted by therapy.     

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.