Dictyostelium discoideum: Cell Culture and Molecular Tools

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).

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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.

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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]