Macropinocytosis is a fundamental mechanism that allows cells to take up extracellular liquid into large vesicles. It critically depends on the formation of a ring of protrusive actin beneath the plasma membrane, which develops into the macropinocytic cup. We show that macropinocytic cups in Dictyostelium are organised around coincident intense patches of PIP3, active Ras and active Rac. These signalling patches are invariably associated with a ring of active SCAR/WAVE at their periphery, as are all examined structures based on PIP3 patches, including phagocytic cups and basal waves. Patch formation does not depend on the enclosing F-actin ring, and patches become enlarged when the RasGAP NF1 is mutated, showing that Ras plays an instructive role. New macropinocytic cups predominantly form by splitting from existing ones. We propose that cup-shaped plasma membrane structures form from self-organizing patches of active Ras/PIP3, which recruit a ring of actin nucleators to their periphery.DOI: http://dx.doi.org/10.7554/eLife.20085.001

Cells use phagocytosis and macropinocytosis to internalise bulk material, which in phagotrophic organisms supplies the nutrients necessary for growth. Wildtype Dictyostelium amoebae feed on bacteria, but for decades laboratory work has relied on axenic mutants that can also grow on liquid media. We used forward genetics to identify the causative gene underlying this phenotype. This gene encodes the RasGAP Neurofibromin (NF1). Loss of NF1 enables axenic growth by increasing fluid uptake. Mutants form outsized macropinosomes which are promoted by greater Ras and PI3K activity at sites of endocytosis. Relatedly, NF1 mutants can ingest larger-than-normal particles using phagocytosis. An NF1 reporter is recruited to nascent macropinosomes, suggesting that NF1 limits their size by locally inhibiting Ras signalling. Our results link NF1 with macropinocytosis and phagocytosis for the first time, and we propose that NF1 evolved in early phagotrophs to spatially modulate Ras activity, thereby constraining and shaping their feeding structures.DOI: http://dx.doi.org/10.7554/eLife.04940.001

Macropinocytosis is a means by which eukaryotic cells ingest extracellular liquid and dissolved molecules. It is widely conserved amongst cells that can take on amoeboid form and, therefore, appears to be an ancient feature that can be traced back to an early stage of evolution. Recent advances have highlighted how this endocytic process can be subverted during pathology -certain cancer cells use macropinocytosis to feed on extracellular protein, and many viruses and bacteria use it to enter host cells. Prion and prion-like proteins can also spread and propagate from cell to cell through macropinocytosis. Progress is being made towards using macropinocytosis therapeutically, either to deliver drugs to or cause cell death by inducing catastrophically rapid fluid uptake. Mechanistically, the Ras signalling pathway plays a prominent and conserved activating role in amoebae and in mammals; mutant amoebae with abnormally high Ras activity resemble tumour cells in their increased capacity for growth using nutrients ingested through macropinocytosis. This Commentary takes a functional and evolutionary perspective to highlight progress in understanding and use of macropinocytosis, which is an ancient feeding process used by single-celled phagotrophs that has now been put to varied uses by metazoan cells and is abused in disease states, including infection and cancer.

The heterotetrameric AP and F-COPI complexes help to define the cellular map of modern eukaryotes. To search for related machinery, we developed a structure-based bioinformatics tool, and identified the core subunits of TSET, a 'missing link' between the APs and COPI. Studies in Dictyostelium indicate that TSET is a heterohexamer, with two associated scaffolding proteins. TSET is non-essential in Dictyostelium, but may act in plasma membrane turnover, and is essentially identical to the recently described TPLATE complex, TPC. However, whereas TPC was reported to be plant-specific, we can identify a full or partial complex in every eukaryotic supergroup. An evolutionary path can be deduced from the earliest origins of the heterotetramer/scaffold coat to its multiple manifestations in modern organisms, including the mammalian muniscins, descendants of the TSET medium subunits. Thus, we have uncovered the machinery for an ancient and widespread pathway, which provides new insights into early eukaryotic evolution.DOI: http://dx.doi.org/10.7554/eLife.02866.001

The genetics of sex determination remains mysterious in many organisms including some that are otherwise well-studied. Here we report the discovery and analysis of the mating-type locus of the model organism Dictyostelium discoideum. Three forms of a single genetic locus specifies this species' three mating-types: two versions of the locus are entirely different in sequence, and the third resembles a composite of the other two. Single, unrelated genes are sufficient to determine two of the mating-types, while homologues of both these genes are required in the composite type. The key genes encode polypeptides that possess no recognisable similarity to established protein families. Sex determination in the social amoebae thus appears to use regulators unrelated to any currently known.Most eukaryotes are sexual, but little is known in molecular detail about sex across most branches of the eukaryotic tree. One aspect, the genetic basis of sex determination, is well understood in several animal, fungal and plant lineages (1-5), but across the protozoan kingdoms we know little, and nothing in comparable detail. The social amoebae are members of the Amoebozoa, and have an unusual sexual cycle that leads to the formation of dormant, walled macrocysts (6) ( Fig. 1 A and B). To produce a macrocyst, a pair of haploid amoebae of different sexes fuse (7) to form a diploid zygote, which then attracts surrounding haploid cells (8). These help to lay down external layers of cellulose around the developing mass of cells before being cannibalized by the zygote (9). After a period of dormancy the cyst germinates, releasing haploid progeny that arise most likely after meiosis and multiple mitoses (10). Population genetics of wild isolates indicate that mating and recombination are probably frequent in the wild (11).The most-studied species of social amoeba, Dictyostelium discoideum, is notable for having three sexes (hereafter called mating-types I, II, and III; supporting online text, S1), as well as uncommon self-fertile homothallic strains (12)(13)(14). Each of the three sexes can pair with each of the other two, but not with itself, giving three possible classes of zygote: type-I/type-II, type-I/type-III, and type-II/type-III. Although several genes are known to be involved during the sexual cycle (15), the determinant of mating-type has proved elusive. Genetic analysis suggested that mating-type is stable, and determined by a single locus with two or * To whom correspondence should be addressed. garethb@mrc-lmb.cam.ac.uk Phone: +44 (0) (10,14,16). We argued that it might be possible to identify this postulated locus by searching for genes which are present in any member of one mating-type but absent (or highly diverged) in any member of another. For this purpose we performed comparative genomic hybridizations using DNA microarrays composed of probes for around 8500 of the 10500 predicted genes in the sequenced type-I D. discoideum genome (17).We analysed ten strains derived from independent wild isolates (table S1) usi...

Background: Phagocytosis plays a major role in the defense of higher organisms against microbial infection and provides also the basis for antigen processing in the immune response. Cells of the model organism Dictyostelium are professional phagocytes that exploit phagocytosis of bacteria as the preferred way to ingest food, besides killing pathogens. We have investigated Dictyostelium differential gene expression during phagocytosis of non-pathogenic bacteria, using DNA microarrays, in order to identify molecular functions and novel genes involved in phagocytosis.

Background: Duplications of stretches of the genome are an important source of individual genetic variation, but their unrecognized presence in laboratory organisms would be a confounding variable for genetic analysis.

Reactive oxygen species are known to have a signalling role in many organisms. In bacteria and yeast various response systems have evolved to combat oxidative stress which are triggered by reactive oxygen species. Mammals and plants are known to actively generate reactive oxygen species such as superoxide during signalling responses to a variety of extracellular factors. We report here the generation of superoxide as a signalling molecule in early development of Dictyostelium discoideum. Dictyostelium grows as single amoebae but, on starvation, the single cells aggregate to form a multicellular organism. Superoxide is generated in response to a secreted factor during the transition to the multicellular phase of development. Scavenging superoxide, either pharmacologically or by overexpressing the enzyme superoxide dismutase, inhibits the formation of the aggregate. This report of the use of superoxide as a signalling molecule in a lower eukaryote as it switches to a multicellular phase suggests that this signalling mechanism arose early in the evolution of multicellular organisms, perhaps as a necessary consequence of the need to diversify the number and type of signalling pathways available to facilitate intercellular communication.