Dictyostelium discoideum: Cell Culture and Molecular Tools

Markus Maniak, Universität Kassel, Kassel, Germany
Wolfgang Nellen, Universität Kassel, Kassel, Germany

Published online: August 2014

DOI: 10.1002/9780470015902.a0002579.pub2

Abstract

Dictyostelium discoideum provides an easy‐to‐grow model system to study virtually all functions typical for higher eukaryotic cells. A variety of specific conditions can induce individual cells to aggregate and initiate a differentiation process resulting in a multicellular structure of low complexity. A plethora of molecular tools is available. These tools include gene disruptions, gene replacements, expression of heterologous proteins, cell type tagging and others, which allow easy genetic manipulation. Dictyostelium discoideum has proven to be suitable for basic research in chemotaxis, cell differentiation, infection biology, signal transduction, cytoskeletal organisation, nuclear architecture, nutrient uptake and metabolism and many other topics.

The Dictyostelium Stock Centre provides a large collection of genetically modified strains, mutants and transformation vectors. Genomic sequences, complementary deoxyribonucleic acid sequences, expression profiles throughout development, genomes of related species and other data are accessible on DictyBase, the Dictyostelium website.

Key Concepts:

  • D. discoideum is a suitable model organism to study fundamental biological functions like cell motility, chemotaxis, signal transduction and metabolic pathways.

  • The developmental cycle in D. discoideum is easy to induce and results in only a few different cell types, thus providing a simple system to study cell differentiation.

  • Conditions for growth and maintenance of strains are simple.

  • Gene regulation can be studied on various levels as mechanisms like RNAi, miRNA and chromatin remodelling are similar to those in higher eukaryotes.

  • The intermediate position of D. discoideum in the phylogenetic tree provides insights into the plant as well as the animal kingdom.

  • Molecular tools like gene disruption, gene replacement, restriction enzyme‐mediated integration (REMI), expression of tagged genes, RNAi and antisense knockdown are available for D. discoideum.

  • Because D. discoideum is haploid, genetic alterations are easy to perform.

Keywords: cell culture; gene disruption; REMI; development; screening of mutants; cell differentiation; transgene; knockout strain; proliferation; differentiation

Figure 1.

Conditions for growth and development. A D. discoideum cell (green, in the inner circle) can feed on bacterial particles (red) or by the uptake of liquid medium (brown). In the outer circle, different setups for cultivation (light blue background) and development (yellow background) are shown and discussed clockwise, starting from top. Cells grow in liquid medium by either adhering to the surface of a Petri dish or shaking in an Erlenmeyer flask. Cells in a flask can multiply in a suspension of bacteria. Alternatively, the bacteria are spread to produce a lawn on an agar plate. As the cells grow, they consume bacteria and development commences in areas cleared from food. Finally, fruiting bodies are formed (bottom, cyan background). When a sufficient amount of cells is spread on a filter or agar surface devoid of nutrients, fruiting bodies come up synchronously. Starved cells in suspension aggregate but do not proceed further in development.
Figure 2.

Molecular tools for genetic manipulation of D. discoideum. The options to investigate the function of a given gene (centre) are illustrated clockwise from top. Using a promoter (P) and a terminator (T) of choice, fusions with GFP (or other tags) can be expressed. Alternatively, GFP may be inserted into the resident gene by homologous recombination (knockin, not shown). The heavy arrow indicates overexpression from a strong promoter in a multicopy vector. Site‐specific mutations (red box) can be introduced to express dominant negative or other altered versions of the protein. The function of different domains, for example, their ability to rescue a mutant phenotype, can be examined by expression of partial genes, indicated by the two constructs representing the 5′ and the 3′ part of the gene. Gene knockdown can be achieved by RNA interference with the expression of an inverted repeat separated by a nonpairing (red) loop or by construction of an antisense gene (inverted arrow). Finally, a gene may be disrupted (knockout) by homologous recombination and insertion of a resistance marker (yellow R).
close

References

Cosson P and Soldati T (2008) Eat, kill or die: when amoeba meets bacteria. Current Opinion in Microbiology 11(3): 271–276.

Crowley TE, Nellen W, Gomer RH and Firtel RA (1985) Phenocopy of discoidin I minus mutants by antisense transformation in Dictyostelium. Cell 43(3): 633–641.

De Lozanne A and Spudich JA (1987) Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science 236(4805): 1086–1091.

Du X, Barisch C, Paschke P et al. (2013) Dictyostelium lipid droplets host novel proteins. Eukaryotic Cell 12(11): 1517–1529.

Durston AJ (2013) Dictyostelium: the Mathematician's Organism. Current Genomics 14(6): 355–360.

Eichinger L, Pachebat JA, Glöckner G et al. (2005) The genome of the social amoeba Dictyostelium discoideum. Nature 435(7038): 43–57.

Escalante R (ed.) (2011) Dictyostelium as a model for human disease. Seminars in Cell and Developmental Biology 22(1): 69–130 (Special Issue).

Faix J, Kreppel L, Shaulsky G, Schleicher M and Kimmel AR (2004) A rapid and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the Cre‐loxP system. Nucleic Acids Research 32(19): e143.

Heuser J, Zhu Q and Clarke M (1993) Proton pumps populate the contractile vacuoles of Dictyostelium amoebae. Journal of Cell Biology 121(6): 1311–1327.

Kruger A, Batsios P, Baumann O et al. (2012) Characterization of NE81, the first lamin‐like nucleoskeleton protein in a unicellular organism. Molecular Biology of the Cell 23(2): 360–370.

Kuhlmann M, Borisova BE, Kaller M et al. (2005) Silencing of retrotransposons in Dictyostelium by DNA methylation and RNAi. Nucleic Acids Research 33(19): 6405–6417.

Kuspa A and Loomis WF (1992) Tagging developmental genes in Dictyostelium by restriction enzyme‐mediated integration of plasmid DNA. Proceedings of the National Academy of Sciences of the USA 89(18): 8803–8807.

Leiting B and Noegel A (1988) Construction of an extrachromosomally replicating transformation vector for Dictyostelium discoideum. Plasmid 20(3): 241–248.

Maniak M, Saur U and Nellen W (1989) A colony blot technique for the detection of specific transcripts in eukaryotes. Analytical Biochemistry 176(1): 78–81.

Martens H, Novotny J, Oberstrass J et al. (2002) RNAi in Dictyostelium: the role of RNA‐directed RNA polymerases and double‐stranded RNase. Molecular Biology of the Cell 13(2): 445–453.

Myre MA (2012) Clues to gamma‐secretase, huntingtin and Hirano body normal function using the model organism Dictyostelium discoideum. Journal of Biomedical Sciences 19: 41.

Nellen W, Silan C and Firtel RA (1984) DNA‐mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion. Molecular and Cellular Biology 4(12): 2890–2898.

Raper KB (1935) Dictyostelium discoideum, a new species of slime mold from decaying forest leaves. Journal of Agricultural Research 50: 135–147.

Sadiq M, Hildebrandt M, Maniak M and Nellen W (1994) Developmental regulation of antisense‐mediated gene silencing in Dictyostelium. Antisense Research and Development 4(4): 263–267.

Veltman DM, Keizer‐Gunnink I and Van Haastert PJ (2009) An extrachromosomal, inducible expression system for Dictyostelium discoideum. Plasmid 61(2): 119–125.

Vervoort EB, van Ravestein A, van Peij NN et al. (2000) Optimizing heterologous expression in Dictyostelium: importance of 5′ codon adaptation. Nucleic Acids Research 28(10): 2069–2074.

Wallraff E, Schleicher M, Modersitzki M et al. (1986) Selection of Dictyostelium mutants defective in cytoskeletal proteins: use of an antibody that binds to the ends of α‐actinin rods. EMBO Journal 5(1): 61–67.

Wiegand S, Meier D, Seehafer C et al. (2013) The Dictyostelium discoideum RNA‐dependent RNA polymerase RrpC silences the centromeric retrotransposon DIRS‐1 post‐transcriptionally and is required for the spreading of RNA silencing signals. Nucleic Acids Research 42: 3330–3345.

Witke W, Nellen W and Noegel A (1987) Homologous recombination in the Dictyostelium α‐actinin gene leads to an altered mRNA and lack of the protein. EMBO Journal 6(13): 4143–4148.

Further Reading

DictyBase. http://dictybase.org/ (An online informatics resource for Dictyostelium.).

Eichinger L and Rivero F (eds) (2006) Dictyostelium discoideum Protocols, Methods in Molecular Biology, vol. 346, pp. 1–564. Totowa, NJ: Humana Press.

Eichinger L and Rivero F (eds) (2013) Dictyostelium discoideum Protocols, Methods in Molecular Biology, 2nd edn, vol. 983, pp. 1–479. New York, NY: Humana Press/Springer.

Kessin RH (2001) Dictyostelium – Evolution, Cell Biology, and the Development of Multicellularity. Cambridge: Cambridge University Press.

Related Sub-Topics:

Techniques in Cell Biology | Multicellularity | Techniques and Tools in Molecular Biology | Protozoa
Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

Article Title: Dictyostelium discoideum: Cell Culture and Molecular Tools
 
How to Cite close
Maniak, Markus, and Nellen, Wolfgang(Aug 2014) Dictyostelium discoideum: Cell Culture and Molecular Tools. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002579.pub2]