Assets of the non-pathogenic microorganism Dictyostelium discoideum as a model for the study of eukaryotic extracellular vesicles

Dictyostelium discoideum microvesicles have recently been presented as a valuable model for eukaryotic extracellular vesicles. Here, the advantages of D. discoideum for unraveling important biological functions of extracellular vesicles in general are detailed. D. discoideum, a non-pathogenic eukaryotic microorganism, belongs to a billion-year-old Amoeboza lineage, which diverged from the animal-fungal lineage after the plant animal-split. During growth and early starvation-induced development, it presents analogies with lymphocytes and macrophages with regard to motility and phagocytosis capability, respectively. Its 6-chromosome genome codes for about 12,500 genes, some showing analogies with human genes. The presence of extracellular vesicles during cell growth has been evidenced as a detoxification mechanism of various structurally unrelated drugs. Controls led to the discovery of constitutive extracellular vesicle secretion in this microorganism, which was an important point. It means that the secretion of extracellular vesicles occurs, in the absence of any drug, during both cell growth and early development. This constitutive secretion of D. discoideum cells is very likely to play a role in intercellular communication. The detoxifying secreted vesicles, which can transport drugs outside the cells, can also act as "Trojan horses", capable of transferring these drugs not only into naïve D. discoideum cells, but into human cells as well. Therefore, these extracellular vesicles were proposed as a new biological drug delivery tool. Moreover, Dictyostelium, chosen by the NIH (USA) as a new model organism for biomedical research, has already been used for studying some human diseases. These cells, which are much easier to manipulate than human cells, can be easily designed in simple conditioned medium experiments. Owing to the increasing consensus that extracellular vesicles are probably important mediators of intercellular communication, D. discoideum is here suggested to constitute a convenient model for tracking as yet unknown biological functions of eukaryotic extracellular vesicles.

Introduction A short four-decade story of extracellular vesicles Four decades ago, the extracellular medium was considered to be no more than a waste reservoir of cell life, exclusively occurring inside the eukaryotic cell delimited by the plasma membrane.
Here are a few landmarks of the story of extracellular vesicles (EVs): "Exosomes" were first mentioned by R. Johnstone in 1970 and further, thoroughly studied during maturation of reticulocytes 1,2 . "Shed" vesicles were observed in 1981 3 . Exosomes in blood cells and their function in the transfer of immunity were thoroughly studied in the early 2000s 4,5 . Clinical observations of exosomes, as normal and pathogenic biomarkers in urine, were first mentioned in 2004 6 . The presence of nucleic acids, mRNAs and microRNAs in exosomes, was pointed out in 2007 7 , as a first strong indication of vesicle-mediated intercellular communication.

Why do cells expel extracellular vesicles?
In the absence of a consensus on the nomenclature for various kinds of extracellular vesicles, the term "extracellular vesicles" is used throughout this paper to designate secreted vesicles in general, with no qualification based on defined criteria. For well known subtypes, like exosomes, the more precise qualification is mentioned, when appropriate.
After a widespread observation of many different kinds of extracellular vesicles, emerging from quite different fields of biology and medicine, the modern puzzling questions deal about the life functions of these vesicles.
Why do so many cells expand their action field beyond the cell membrane? Why do cells release vesicles? 11 . What are the physiological functions of all these EVs? The current state of the art about the role of extracellular vesicles has recently been presented 12 , whereas exosomes seem to be appealing candidates for mediating intercellular communication 13 .
Human complexity is indeed much too high to suggest one particular human cell type as a model for understanding the respective roles of the "inside/outside" parts of the cell. To address such a fundamental question requires a convenient well-known eukaryotic model, easy to handle and to follow in a variety of extracellular environments.
A model for the study of eukaryotic extracellular vesicles: Dictyostelium discoideum About Dictyostelium discoideum Dictyostelium discoideum came to scientific life in 1935, when it was discovered by Raper 14 in the USA Virginia forest, but belongs to a billion year-old Amoeboza lineage, which diverged from the animal-fungal lineage after the plant animal-split 15 . To survive for such a long time as a unicellular protist, it had to develop a wellconserved life strategy. One of its original biological tricks was to fight against starvation through the first known eukaryotic tentative step towards multicellularity, that is, the aggregation of undifferentiated cells. This aggregation is followed by differentiation into two main cell species: dying stalk cells and "everlasting" spores. Thus, D. discoideum experiences specific time-separated physiological states of growth and differentiation during its life cycle. During growth, as unicellular non-pathogenic amoebae, D. discoideum cells, about 10 µm in diameter, are very similar to some human blood cells; they are often compared to lymphocytes with regard to motility, and to macrophages concerning their capability for phagocytosis. After about 24h of starvation-induced development, through cell aggregation (of about 10 5 cells), followed by differentiation and morphogenesis, it ends up as a "mushroom-like" organism of 1-2 mm height, forming a fruiting body, composed of a stalk (about 20% of the cells) and a spore bag containing viable spores (about 80% of the cells) 16 .
Thus, whereas differentiation pulls the microorganism towards a plant-like structure, growing and aggregating cells remain as animal-like cells. Therefore, D. discoideum can offer a simple model of individual eukaryotic cells during growth and also mimic an undifferentiated "tissue" during starvation-induced aggregation. The above-defined "animal" state of D. discoideum during growth and aggregation, completely separated from differentiation, is the first asset of D. discoideum as a model for eukaryotic cells. Some molecular approaches to cell biology using D. discoideum are detailed in Methods in Cell Biology 17 .
Another advantage of D. discoideum is the good knowledge of its genetic material 18 . Its 6-chromosome genome has been completely sequenced 15 . The relatively small genomic DNA (3.4 × 10 7 bp) is efficiently transcribed (90%) into about 12,500 genes, some of which show analogies with human genes. Its 55.5 kb mitochondrial genome has also been sequenced 19 . A multicopy 90 kb extrachromosomal element that carries the ribosomal RNA genes, and some plasmids complete the DNA components. The RNA amount is about 10 times the DNA amount and due to this RNA excess, the cell cycle does not require a G1 RNA-synthesis step before the DNA-synthesis S phase. Moreover, Dictyostelium cells can be easily transformed by restriction enzyme-mediated integration (REMI) 20 .
A third asset of D. discoideum lies in its simple growth conditions. In nature, D. discoideum cells grow by bacterial phagocytosis, with a 3h generation time. A widely used laboratory double axenic mutant, Ax-2, can grow in semi-synthetic HL5-medium 21 , either as adhering cells or in agitated suspension, with a 8 -11h generation time. A completely defined medium has also been elaborated 22 and is now commercialised, as are many other Dictyostelium growth media (Foremedium, UK). Moreover, D. discoideum cells can reach a density of 2-3 × 10 7 cells ml -1 in the stationary phase and can also be grown in bioreactors 23 , when huge amounts of cell materials are needed.
It is worth noting that, owing to all these advantages, D. discoideum extracellular vesicles are not only a cell detoxifying tool. These vesicles are constitutively released during both growth 30 and starvation-induced aggregation 35 , and are thus candidates for the mediation of intercellular communication.
Quite recently, C. A. Parent found and characterised exosomes during Dictyostelium aggregate formation, suggesting that these organelles are conserved in both Dictyostelium and mammals 36,37 (personal communication).
A model to unravel biological functions of eukaryotic extracellular vesicles In order to use D. discoideum EVs as a model for the functional study of eukaryotic EVs, their characterisation is needed. Recently, these vesicles were used to propose a fast characterisation of EVs by means of three joined techniques: nanoparticle tracking analysis, cryo-electron microscopy and Raman tweezer microspectroscopy 38 .
With regard to the precise characterisation of Dictyostelium EVs, much remains to be done.
First chacterisations of EVs lipids and nucleic acids 30 and of EVs proteins 31 have been performed. A more precise protocol for the purification of these extracellular vesicles should be devised, followed by a thorough characterisation of the components of all the different subclasses of D. discoideum extracellular vesicles.
However, before performing a specialised study of each subclass of D. discoideum extracellular vesicles, it is worth questioning the whole distribution panel of EVs accompanying each important physiological state of D. discoideum cell life. R. Gomer was a pioneer in stressing the importance of the conditioned extracellular medium of D. discoideum cells as a source of secreted autocrine factors during both growth and early starvation 39,40 . He showed that the aggregate sizes were controlled by autocrine protein factors in the extracellular medium 41 and showed the presence of autocrine cell growth inhibition 42 .
Experiments are currently undertaken to test the plausible hypothesis that cells are not programmed to grow, differentiate or die by an apoptotic "clean" death, by their only genomic content and that cells would need intercellular communications to define their individual fate. The necessary signals would be conditioned by all the surrounding cells, according to their own history, and the most convenient way to communicate should indeed be searched for in the cell extracellular medium.
Previously, we found that an apoptotic-like death could be induced with full efficiency by starving a naïve D. discoideum cell population in a conditioned medium. This conditioned medium (t 22 ) was prepared by starving a first D. discoideum cell population in agitated suspension in a phosphate buffer (pH 6.8) for 22 h. When starved in this cell-depleted t 22 conditioned medium, the second D. discoideum cell population completely lost its aggregation competence and was directed to an apoptosis-like death 35,43 . Preliminary (unpublished) experiments indicated that the extracellular vesicles present in the t 22 conditioned medium might be responsible for changing the fate of D. discoideum cells from "social" aggregation to mitochondrialmediated apoptotic death.
In order to understand the important biological communication signals potentially carried by D. discoideum EVs, it is interesting to take advantage of the relative simplicity of this eukaryotic model. D. discoideum is indeed a wonderful tool to question the respective functions of and interplay between extracellular vesicles and soluble secreted autocrine factors in conditioned medium experiments. This should help in understanding the main, still mostly unknown, biological functions of the secreted vesicles.

Conclusion
In 2012, Dictyostelium discoideum was recognized as an interesting model for the study of eukaryotic extracellular vesicles 10,44 . Due to the important knowledge accumulated on this eukaryotic microorganism since 1935, and to the wide panel of well-defined mutants available in the Stock Center (see dictyBase), a lot of simple experiments can be designed to question the physiological functions of EVs during cell growth, aggregation or apoptotic-like cell death. Moreover, the findings obtained with D. discoideum are likely to be extrapolated to higher eukaryotic cells, in the same way as the extracellular vesiclemediated detoxification mechanism of D. discoideum cells was a clue towards the introduction of a new multidrug resistance mechanism against antitumoral chemotherapy 45 .

Competing interests
No relevant competing interests were disclosed.

Grant information
The author(s) declared that no grants were involved in supporting this work.