Comparative genome and transcriptome analyses of the social amoeba Acytostelium subglobosum that accomplishes multicellular development without germ-soma differentiation

Background Social amoebae are lower eukaryotes that inhabit the soil. They are characterized by the construction of a starvation-induced multicellular fruiting body with a spore ball and supportive stalk. In most species, the stalk is filled with motile stalk cells, as represented by the model organism Dictyostelium discoideum, whose developmental mechanisms have been well characterized. However, in the genus Acytostelium, the stalk is acellular and all aggregated cells become spores. Phylogenetic analyses have shown that it is not an ancestral genus but has lost the ability to undergo cell differentiation. Results We performed genome and transcriptome analyses of Acytostelium subglobosum and compared our findings to other available dictyostelid genome data. Although A. subglobosum adopts a qualitatively different developmental program from other dictyostelids, its gene repertoire was largely conserved. Yet, families of polyketide synthase and extracellular matrix proteins have not expanded and a serine protease and ABC transporter B family gene, tagA, and a few other developmental genes are missing in the A. subglobosum lineage. Temporal gene expression patterns are astonishingly dissimilar from those of D. discoideum, and only a limited fraction of the ortholog pairs shared the same expression patterns, so that some signaling cascades for development seem to be disabled in A. subglobosum. Conclusions The absence of the ability to undergo cell differentiation in Acytostelium is accompanied by a small change in coding potential and extensive alterations in gene expression patterns

Defects in the synthetic pathway prevent DIF-1 mediated stalk lineage specification cascade in the non-differentiating social amoeba, Acytostelium subglobosum

Separation of somatic cells from germ-line cells is a crucial event for multicellular organisms, but how this step was achieved during evolution remains elusive. In Dictyostelium discoideum and many other dictyostelid species, solitary amoebae gather and form a multicellular fruiting body in which germ-line spores and somatic stalk cells differentiate, whereas in Acytostelium subglobosum, acellular stalks form and all aggregated amoebae become spores. In this study, because most D. discoideum genes known to be required for stalk cell differentiation have homologs in A. subglobosum, we inferred functional variations in these genes and examined conservation of the stalk cell specification cascade of D. discoideum mediated by the polyketide differentiation-inducing factor-1 (DIF-1) in A. subglobosum. Through heterologous expression of A. subglobosum orthologs of DIF-1 biosynthesis genes in D. discoideum, we confirmed that two of the three genes were functional equivalents, while DIF-methyltransferase (As-dmtA) involved at the final step of DIF-1 synthesis was not. In fact, DIF-1 activity was undetectable in A. subglobosum lysates and amoebae of this species were not responsive to DIF-1, suggesting a lack of DIF-1 production in this species. On the other hand, the molecular function of an A. subglobosum ortholog of DIF-1 responsive transcription factor was equivalent with that of D. discoideum and inhibition of polyketide synthesis caused developmental arrest in A. subglobosum, which could not be rescued by DIF-1 addition. These results suggest that non-DIF-1 polyketide cascades involving downstream transcription factors are required for fruiting body development of A. subglobosum

A RabGAP Regulates Life-Cycle Duration via Trimeric G-protein Cascades in <i>Dictyostelium discoideum</i>

<div><p>Background</p><p>The life-cycle of cellular slime molds comprises chronobiologically regulated processes. During the growth phase, the amoeboid cells proliferate at a definite rate. Upon starvation, they synthesize cAMP as both first and second messengers in signalling pathways and form aggregates, migrating slugs, and fruiting bodies, consisting of spores and stalk cells, within 24 h. In <i>Dictyostelium discoideum</i>, because most growth-specific events cease during development, proliferative and heterochronic mutations are not considered to be interrelated and no genetic factor governing the entire life-cycle duration has ever been identified.</p><p>Methodology/Principal Findings</p><p>Using yeast 2-hybrid library screening, we isolated a <i>Dictyostelium discoideum</i> RabGAP, Dd Rbg-3, as a candidate molecule by which the <i>Dictyostelium</i> Gα2 subunit directs its effects. Rab GTPase-activating protein, RabGAP, acts as a negative regulator of Rab small GTPases, which orchestrate the intracellular membrane trafficking involved in cell proliferation. Deletion mutants of <i>Dd rbg-3</i> exhibited an increased growth rate and a shortened developmental period, while an overexpression mutant demonstrated the opposite effects. We also show that Dd Rbg-3 interacts with 2 Gα subunits in an activity-dependent manner <i>in vitro</i>. Furthermore, both human and <i>Caenorhabditis elegans rbg-3</i> homologs complemented the <i>Dd rbg-3</i>–deletion phenotype in <i>D. discoideum</i>, indicating that similar pathways may be generally conserved in multicellular organisms.</p><p>Conclusions/Significance</p><p>Our findings suggest that Dd Rbg-3 acts as a key element regulating the duration of <i>D. discoideum</i> life-span potentially via trimeric G-protein cascades.</p></div

Adenylyl cyclase activity and cellular localization of Dd Rbg-3.

<p>(A) Adenylyl cyclase activity was measured in intact AX2 (filled circles, •), <i>Dd rbg-3</i>-null (filled triangles, ▴), and <i>Dd rbg-3</i>-overexpression (filled squares, ▪) cells following cAMP stimulation. Starved cells were stimulated with 10 µM deoxy-cAMP. At specific time-points, aliquots of cells were lysed and the cAMP levels measured. (B) Adenylyl cyclase activity in cell lysates of the parental and mutant strains measured in the presence of 30 µM GTPγS. (C) Fluorescent microscopy image of AX2 cells expressing Dd Rbg-3-Venus. Scale bar, 10 µm. (D) Fluorescent microscopy images of migration of Dd Rbg-3-Venus in AX2 cells toward a micropipette releasing 1 µM cAMP. Stars represent the position of the tip of the micropipette. Scale bar, 10 µm. (E) Translocation of CRAC. Aggregating cells were pretreated with 5 µM latrunculin A. Fluorescence intensity of CRAC-GFP in the cytosol was measured at the indicated time after stimulation with 1 µM cAMP. Cells were excited at 440 nm and viewed through a cut-off filter of 500–550 nm to assess emission before and after cAMP stimulation. In each experiment, fluorescence intensity of an area in the cytosol was measured at the indicated time-point and was normalized to the average intensity measured 18 s before cAMP stimulation (n = 102 for AX2, n = 102 for the null mutant, n = 143 for the overexpression mutant).</p

Endocytosis and lysosomes in AX2, <i>Dd rbg-3</i>-null, and <i>Dd rbg-3-</i>overexpression strains.

<p>(A) Fluorescence microscopy images of endocytosis of rhodamine-dextran. Vegetative cells were incubated with rhodamine-dextran [0.4 mg/ml] for 10 min, and then washed 3 times with PB. Scale bar, 10 µm. (B) Fluorescence microscopy images of lysosomes visualized using LysoTracker® Green. Vegetative cells were incubated with 75 nM LysoTracker® Green (Invitrogen, USA) for 30 min, and then washed 3 times with PB. Arrowheads indicate swollen lysosomes in the overexpression cells. Scale bar, 10 µm. (C) Relative fluorescent intensity of cell suspension treated with rhodamine-dextran [0.4 mg/ml] in 5×10⁷ cells/ml. (D) Relative fluorescent intensity of cell suspension stained with LysoTracker® Green in 5×10⁷ cells/ml. **P<0.05 between AX2 and <i>Dd rbg-3</i> OE (<i>t</i>-test).</p

Caffeine does not inhibit the development, or adenylyl cyclase activity, of <i>Dd rbg-3</i>-null mutant cells and cells expressing the R364A-mutant <i>Dd rbg-3</i>.

<p>(A) Development of AX2 and <i>Dd rbg-3-null</i> cells starved at a density of 1×10⁶ cells/cm² on non-nutrient agar plates containing 4 mM caffeine. In the parental cells, slugs and fruiting bodies were not observed. Scale bar, 10 µm. (B) Adenylyl cyclase activity was measured, following stimulation with 10 µM deoxy-cAMP, in intact cells grown under starvation conditions for 8 h in the presence of 4 mM caffeine. (C) Adenylyl cyclase activity was measured in the presence of 30 µM GTPγS, in cell lysates grown in medium containing 4 mM caffeine.</p

<i>In vivo</i> interaction of Dd Rbg-3 with activated Gα2 and Gα9.

<p>(A) Co-immunoprecipitation of Gα2 with Dd Rbg-3. Lysates of AX2 cells and cells overexpressing dominant gα2 were used. (B) Co-immunoprecipitation of Gα8 and Gα9 with Dd Rbg-3. Lysates of AX2 cells and cells expressing eGFP-fused Gα8 and Gα9 were used. In each experiment, cell lysates were incubated with or without 30 µM GTPγS before the pull-down experiments.</p