Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.
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.
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 clades1. The species Dictyostelium discoideum (D. discoideum) has become a popular model organism to study complex cellular processes such as cell migration and phagocytosis. To control and standardize experimental conditions, axenic cell lines have been developed that are able to grow in complex or defined liquid medium in the absence of bacteria2. Of particular importance are the Ax2, Ax3, and Ax4 strains, which were all generated in the 1970s and ultimately derived from a single wild isolate NC43. Tools for genetic engineering were developed in these axenic strains, resulting in the first published knockout in 19874,5. Protocols were further developed and optimized for use under axenic conditions6,7.
Adaptation of these protocols to wild-type D. discoideum strains that are not able to grow in liquid broth have been attempted by several laboratories. However, this has not become fully successful since the transfection protocols are complex and lack efficiency, in part due to the capacity of the bacteria to act as a sink for the selective reagents8,9. As a result, essentially all molecular data on D. discoideum comes from descendants of a single wild-type isolate. We wanted to overcome this limitation and develop a method to genetically modify D. discoideum cells independent of their ability to grow in liquid medium. The need for such a method can be explained by the observation that it was assumed in the past that the mutations allowing axenic growth were mainly neutral and did not impair cell physiology. This supposition is only partially correct. In general, there are two notable differences; first, between the different isolated axenic strains, and second, when these axenic strains are compared with non-axenic wild isolates8,9.
Perhaps the most critical factor is the key axenic gene, axeB, that was identified recently as RasGAP NF1. The major function of NF1 as a RasGAP is to restrain Ras activity3. The deletion of the enzyme in all axenic strains leads to excessive Ras activity manifested as the formation of large active Ras patches. These enlarged Ras patches lead to the accumulation of PIP3 in the plasma membrane. Those coincidental appearing patches of PIP3 and active Ras are a template for the formation of a circular ruffle that eventually closes and leads to the formation of macropinosomes10. The consequence is an excessive increase in macropinocytic activity. Macropinocytosis is an actin-driven process. A competition for cytoskeletal components for the formation of either macropinosomes or pseudopods is the result. Its impact on cell behaviour is reflected in the almost complete prevention of chemotaxis of vegetative cells to folate11. The massively enlarged PIP3 patches are very persistent. Even in starved cells, the PIP3 patches remain and can be misinterpreted as pseudopods, which can cause problems interpreting studies on chemotaxis to cAMP.
In some cases, 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 process12. However, mutation in the genes required for macropinocytosis, such as Ras and PI3-kinases10, 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-cellularity13,14, kin recognition15,16, and altruistic cellular behaviour, which mainly depend on the use of freshly isolated wild-type isolates17. All mentioned research areas can be facilitated by efficient methods for genetic manipulation of wild isolates, which are non-axenic and do not grow in liquid broth.
Our protocols allow for overcoming of the described limitations. Taken together, 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).
The use of non-axenic, wild-type Dictyostelium cells has been very limited so far in molecular research. Available methods for genetic engineering of these strains have lacked reliability and efficiency23, preventing their general adoption. The generated materials and protocols presented here can be used for any D. discoideum strain independent of its ability to grow in liquid medium. It should be mentioned that this protocol is optimized for NC4-derived cell lines. Transfection efficiencies for freshly isolated strains from the wild differ from NC4, as we have observed before and are shown here for V12M2 and WS21628,9. The electroporation conditions seem in particular to have a considerable influence on transfection efficiencies and may require further optimization for some strains. In general, a sufficient amount of transfectants have been observed in all strains tested so far, showing that these methods are workable. The number of positive transfectants obtained using NC4-derived strains is higher compared to other non-axenic wild-type strains, but in all cases, sufficient numbers of transfectants are obtained to allow for further experiments. This, plus the simplicity of our protocol, is the major improvement compared to earlier attempts8,9.
These new non-axenic protocols cover all standard genetic procedures and add further advantages, since transfections can be performed rapidly and efficiently in parallel. Positive clones can be obtained in days rather than weeks, since growth on bacteria halves the division time. The newly established plasmids also work under axenic conditions and can be routinely used under both growth conditions, which is another advancement of this methology22. As introduced in the protocol, the plasmid used for selection on bacteria have special requirements that are crucial for the success of the transfection. In particular, the promoters driving the expression and resistance cassettes are important for successful transfection. The often used act6 (actin 6) promoter24 for driving the resistance or expression cassettes lacks efficiency when cells are grown on bacteria. In our plasmid system, the highly active act15 (actin 15) promoter drives all expression cassettes, while the resistance cassettes are under the control of a coA (coactosin A) promoter, both of which are active under axenic conditions and in cells grown on bacteria. Requirements for the expression of reporter constructs as well as knock-in and knock-out constructs make it necessary to use our plasmid repertoire, but unfortunately limits the use of vectors already created that use the inefficient act6 promoter.
Promoter efficiencies are especially critical for transfections that depend on a single integration event into the correct genomic locus. Due to our improvement of promoters driving the resistance gene expression, enough resistance protein is produced from single locus integrations. A major concern was the favoring of multiple integrations into the genome when using hygromycin or G418 as described. No favoring of multiple integrations has been observed so far, as reported previously for G418 selections using older promoter systems25. This means that both hygromycin and G418 are suitable selectable markers for the generation of clean knock-outs and knock-ins. Unfortunately, the selectable marker blasticidin does not work under non-axenic conditions. This is a major disadvantage of our method, since the blasticidin resistance cassette is routinely used to generate knock-out constructs in D. discoideum. Constructs that were already generated with blasticidin resistance cassettes will need to be rederived using one of the workable selectable markers. Another possibility to overcome this limitation is to combine the recently established axenic D. discoideum CRISPR technology with this transfection protocol26. The generation of suitable, single guide RNAs (sgRNAs) is simpler and faster than the reconstruction of complete knock-out constructs. For future directions, the possibility of generating multiple knock-outs using CRISPR/Cas9 combined with this transfection protocol is appealing and may pave the way for many researchers in the Dictyostelium community. However, the workability of the established transient expression system used for CRISPR/Cas9 in axenic grown cells should be carefully examined.
The act5 knock-in system presented offers a reliable and safe integration system for the generation of stable cell lines in D. discoideum, with the advantage of similar sites established in other organisms22. It also depends on a single integration event in the genome and offers many possibilities for different research directions. The act5 promoter is strongly active and guarantees almost homogenous expression27 independent of the cultivation conditions. Fluorescent reporter proteins can easily be integrated into this safe haven locus using the engineered targeting plasmids. This can be useful, for example, for cell-tracking purposes, as shown here in a mix experiment. The cells show minimal cell-to-cell expression variability, which can assist in automated cell tracking. Importantly, the insertion of a desired sequence into the act5 locus appears phenotypically neutral28,29. As the expression does not depend on a selectable marker to maintain protein expression, as seen in random integrants or extrachromosomal vector-bearing cell lines, the act5 system can be useful for rescue experiments, as well. Since the cell lines are homogenous, all cells can be analyzed. This is not possible using an extrachromosomal plasmid for expression, in which there is considerable heterogeneity in expression.
In addition to these general improvements for all strains, the ability to use non-axenic wild-type cells for molecular research allows an assessment of the effects of accumulated mutations in current laboratory strains. Multiple genetic rearrangements has been found since the adoption of the Ax2 strain in 1970, as shown by previously published microarray analyses26. Synthetic phenotypes are observed because of the presence of these mutations. For example, a reported phg2 mutant shows decreased adhesion in one laboratory strain background and increased adhesion in another3. Such conflicting data can now be resolved by repeating the experiment in the common ancestor strain (in this case, DdB). The strength of this approach has been recently shown for the small GTPase RasS30,31.
The ability to perform genetic experiments in any D. discoideum strain expands the potential of new research directions that depend on the use of wild isolates, notably those involving social evolution, kin recognition, and the sexual cycle22.
The authors have nothing to disclose.
The authors thank the Kay lab for input to this method and the LMB microscopy and flow cytometry facilities for excellent scientific and technical support. This work was funded by Medical Research Council grant MC_U105115237 to R. R. Kay, BBSRC (Biotechnology and Biological Sciences Research Council) grant BB/K009699/1 to R. R. Kay, Cancer Research UK grant A15672 to R. H. Insall, Wellcome Trust Senior Fellowship 202867/Z/16/Z to Jonathan R Chubb, and MRC funding (MC_U12266B) to the MRC LMCB University Unit at UCL.
Equipment | |||
Eppendorf Microcentrifuges 5424/5424R | ThermoScientific | 05-400-002 | |
Eppendorf 5702R Centrifuges with A-4-38 Model Rotor | ThermoScientific | 12823252 | |
Eppendorf Mastercycler Nexus Thermal Cyclers | Sigma Aldrich | EP6331000025 | |
Gene Pulser X Cell, including CE & PC modules | BioRad | 1652660 | |
Safety Cabinet Foruna – SCANLAF | Labogene | ||
BioPhotometer Plus | Eppendorf | ||
CLSM 710 | Zeiss | ||
Media & Agaroses | |||
LoFlo Medium | Formedium | LF0501 | |
Seakem GTG Agarose | Lonza | 50071 | |
Low EEO Agarose | BioGene | 300-200 | |
Purified Agar | Oxoid | LP0028 | |
SM broth | Formedium | SMB0102 | |
2x LB broth | Formedium | LBD0102 | |
Chemicals | |||
SYBR Safe DNA Gel Stain | ThermoScientific | S33102 | |
1 Kb Plus DNA Ladder | ThermoScientific | 10787018 | |
1 Kb DNA Ladder | NEB | N3232L | |
HEPES free acid | Sigma Aldrich/Merck | 391340 EMD | |
KH2PO4 | VWR | P/4800/53 | |
Na2HPO4 * 2 H2O | VWR | 10028-24-7 | |
MgCl2 * 6 H2O | Sigma Aldrich/Merck | 5982 EMD | |
CaCl2 * 2 H2O | Sigma Aldrich | 442909 | |
Folic Acid | Sigma Aldrich | F7876 | |
KOH | VWR | 26668.263 | |
Cultur Dishes | |||
96 Well Cell Culture Cluster, Flat Bottom with Low Evaporation Lid, Tissue Culture Treated | Corning Incorporated | 3595 | |
6 Well Cell Culture Cluster, Flat Bottom with Lid, Tissue Culture Treated | Corning Incorporated | 3516 | |
100 x 20 mm style Tissue Culture Dish, Tissue Culture Treated | Corning Incorporated | 353003 | |
50 mm Glass Bottom Microwell Dishes No1.5 | MatTek Corporation | P50G-1.5-30-F | |
Antibiotics | |||
Geneticin G418-sulphate | Gibco by Life Technologies | 11811-023 | |
Hygromycin B Gold | InvivoGen | ant-hg-1 | |
Additional Consumables | |||
2mm gap electroporation cuvettes, long electrode | Geneflow Limited | E6-0062 | |
10 µl Inoculating Loop, Blue 10 Micro L | ThermoScientific | 129399 | |
Spreader, L-shaped, sterile | greiner bio-one | 730190 | |
Combitips advanced 5 ml | Eppendorf BIOPUR | 30089669 | |
Kits | |||
ZR Plasmid Miniprep Classic | Zymoresearch | D4016 | |
Quick DNA Miniprep Kit | Zymoresearch | D3025 | |
Zymoclean DNA Recovery Kit | Zymoresearch | D40002 | |
Enzymes | |||
Restriction enzymes | NEB | ||
OneTaq 2X Master Mix with Standard Buffer | NEB | M0482L | |
T4 DNA Ligase | NEB | M0202S | |
PrimeSTAR Max DNA Polymerase | TakaraBio | R045A |