Abstract
Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.
Highlights
The Dictyostelium genus are soil-living social amoeba that mainly feed on bacteria
The NF1 mutation is experimentally useful. This leads us to a second motivation for developing a transfection method for bacterially grown D. discoideum cells, since the increase in macropinocytosis rate makes axenic cells valuable for investigating fundamental aspects of this process[12]
The possibility of performing genetic manipulations with bacterial grown D. discoideum cells holds benefits for all Dictyostelium researchers, even if it is just the increased speed of the selection process due to faster growth of the amoeba (4 h doubling time) on bacteria compared to growth in axenic media (10 h doubling time)
Summary
The Dictyostelium genus are soil-living social amoeba that mainly feed on bacteria. Being placed in the phylum Amoebozoa, a large number of species have been isolated that can be grouped into four different clades[1]. This leads us to a second motivation for developing a transfection method for bacterially grown D. discoideum cells, since the increase in macropinocytosis rate makes axenic cells valuable for investigating fundamental aspects of this process[12]. Mutation in the genes required for macropinocytosis, such as Ras and PI3-kinases[10], have almost abolished axenic growth, making it necessary to manipulate these cells through growth on bacteria Another reason that renders bacteria-based transfections valuable is the increasing use of Dictyostelids to explore questions in the evolution of multi-cellularity[13,14], kin recognition[15,16], and altruistic cellular behaviour, which mainly depend on the use of freshly isolated wild-type isolates[17]. The possibility of performing genetic manipulations with bacterial grown D. discoideum cells holds benefits for all Dictyostelium researchers, even if it is just the increased speed of the selection process due to faster growth of the amoeba (4 h doubling time) on bacteria compared to growth in axenic media (10 h doubling time)
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