Hideko Urushihara

The genome of the social amoeba Dictyostelium discoideum

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal–fungal lineage after the plant–animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

Dictyostelium transcriptional host cell response upon infection with Legionella.

Differential gene expression of Dictyostelium discoideum after infection with Legionella pneumophila was investigated using DNA microarrays. Investigation of a 48 h time course of infection revealed several clusters of co‐regulated genes, an enrichment of preferentially up‐ or downregulated genes in distinct functional categories and also showed that most of the transcriptional changes occurred 24 h after infection. A detailed analysis of the 24 h time point post infection was performed in comparison to three controls, uninfected cells and co‐incubation with Legionella hackeliae and L. pneumophilaΔdotA. One hundred and thirty‐one differentially expressed D. discoideum genes were identified as common to all three experiments and are thought to be involved in the pathogenic response. Functional annotation of the differentially regulated genes revealed that apart from triggering a stress response Legionella apparently not only interferes with intracellular vesicle fusion and destination but also profoundly influences and exploits the metabolism of its host. For some of the identified genes, e.g. rtoA involvement in the host response has been demonstrated in a recent study, for others such a role appears plausible. The results provide the basis for a better understanding of the complex host‐pathogen interactions and for further studies on the Dictyostelium response to Legionella infection.

Changing Patterns of Gene Expression in Dictyostelium Prestalk Cell Subtypes Recognized by In Situ Hybridization with Genes from Microarray Analyses

ABSTRACT We used microarrays carrying most of the genes that are developmentally regulated in Dictyostelium to discover those that are preferentially expressed in prestalk cells. Prestalk cells are localized at the front of slugs and play crucial roles in morphogenesis and slug migration. Using whole-mount in situ hybridization, we were able to verify 104 prestalk genes. Three of these were found to be expressed only in cells at the very front of slugs, the PstA cell type. Another 10 genes were found to be expressed in the small number of cells that form a central core at the anterior, the PstAB cell type. The rest of the prestalk-specific genes are expressed in PstO cells, which are found immediately posterior to PstA cells but anterior to 80% of the slug that consists of prespore cells. Half of these are also expressed in PstA cells. At later stages of development, the patterns of expression of a considerable number of these prestalk genes changes significantly, allowing us to further subdivide them. Some are expressed at much higher levels during culmination, while others are repressed. These results demonstrate the extremely dynamic nature of cell-type-specific expression in Dictyostelium and further define the changing physiology of the cell types. One of the signals that affect gene expression in PstO cells is the hexaphenone DIF-1. We found that expression of about half of the PstO-specific genes were affected in a mutant that is unable to synthesize DIF-1, while the rest appeared to be DIF independent. These results indicate that differentiation of some aspects of PstO cells can occur in the absence of DIF-1.

The mitochondrial DNA of Dictyostelium discoideum: Complete sequence, gene content and genome organization

Abstract We present an overview of the gene content and organization of the mitochondrial genome of Dictyostelium discoideum. The mitochondria genome consists of 55,564 bp with an A + T content of 72.6%. The identified genes include those for two ribosomal RNAs (rnl and rns), 18 tRNAs, ten subunits of the NADH dehydrogenase complex (nad1, 2, 3, 4, 4L, 5, 6, 7, 9 and 11), apocytochrome b (cytb), three subunits of the cytochrome oxidase (cox1/2 and 3), four subunits of the ATP synthase complex (atp1, 6, 8 and 9), 15 ribosomal proteins, and five other ORFs, excluding intronic ORFs. Notable features of D. discoideum mtDNA include the following. (1) All genes are encoded on the same strand of the DNA and a universal genetic code is used. (2) The cox1 gene has no termination codon and is fused to the downstream cox2 gene. The 13 genes for ribosomal proteins and four ORF genes form a cluster 15.4 kb long with several gene overlaps. (3) The number of tRNAs encoded in the genome is not sufficient to support the synthesis of mitochondrial protein. (4) In total, five group I introns reside in rnl and cox1/2, and three of those in cox1/2 contain four free-standing ORFs. We compare the genome to other sequenced mitochondrial genomes, particularly that of Acanthamoeba castellanii.

A comparative sequence analysis reveals a common GBD/FH3-FH1-FH2-DAD architecture in formins from Dictyostelium , fungi and metazoa

BackgroundFormins are multidomain proteins defined by a conserved FH2 (formin homology 2) domain with actin nucleation activity preceded by a proline-rich FH1 (formin homology 1) domain. Formins act as profilin-modulated processive actin nucleators conserved throughout a wide range of eukaryotes.ResultsWe present a detailed sequence analysis of the 10 formins (ForA to J) identified in the genome of the social amoeba Dictyostelium discoideum. With the exception of ForI and ForC all other formins conform to the domain structure GBD/FH3-FH1-FH2-DAD, where DAD is the Diaphanous autoinhibition domain and GBD/FH3 is the Rho GTPase-binding domain/formin homology 3 domain that we propose to represent a single domain. ForC lacks a FH1 domain, ForI lacks recognizable GBD/FH3 and DAD domains and ForA, E and J have additional unique domains. To establish the relationship between formins of Dictyostelium and other organisms we constructed a phylogenetic tree based on the alignment of FH2 domains. Real-time PCR was used to study the expression pattern of formin genes. Expression of forC, D, I and J increased during transition to multi-cellular stages, while the rest of genes displayed less marked developmental variations. During sexual development, expression of forH and forI displayed a significant increase in fusion competent cells.ConclusionOur analysis allows some preliminary insight into the functionality of Dictyostelium formins: all isoforms might display actin nucleation activity and, with the exception of ForI, might also be susceptible to autoinhibition and to regulation by Rho GTPases. The architecture GBD/FH3-FH1-FH2-DAD appears common to almost all Dictyostelium, fungal and metazoan formins, for which we propose the denomination of conventional formins, and implies a common regulatory mechanism.

Evolutionary linkage between eukaryotic cytokinesis and chloroplast division by dynamin proteins

Chloroplasts have evolved from a cyanobacterial endosymbiont and been retained for more than 1 billion years by coordinated chloroplast division in multiplying eukaryotic cells. Chloroplast division is performed by ring structures at the division site, encompassing both the inside and the outside of the two envelopes. A part of the division machinery is derived from the cyanobacterial cytokinetic activity based on the FtsZ protein. In contrast, other parts of the division machinery involve proteins specific to eukaryotes, including a member of the dynamin family. Each member of the dynamin family is involved in the division or fusion of a distinct eukaryotic membrane system. To gain insight into the kind of ancestral dynamin protein and eukaryotic membrane activity that evolved to regulate chloroplast division, we investigated the functions of the dynamin proteins that are most closely related to chloroplast division proteins. These proteins in the amoeba Dictyostelium discoideum and Arabidopsis thaliana localize at the sites of cell division, where they are involved in cytokinesis. Our results suggest that the dynamin for chloroplast division is derived from that involved in eukaryotic cytokinesis. Therefore, the chloroplast division machinery is a mixture of bacterial and eukaryotic cytokinesis components, with the latter a key factor in the synchronization of endosymbiotic cell division with host cell division, thus helping to establish the permanent endosymbiotic relationship.

A STAT-regulated, stress-induced signalling pathway in Dictyostelium

The Dictyostelium stalk cell inducer differentiation-inducing factor (DIF) directs tyrosine phosphorylation and nuclear accumulation of the STAT (signal transducer and activator of transcription) protein Dd-STATc. We show that hyperosmotic stress, heat shock and oxidative stress also activate Dd-STATc. Hyperosmotic stress is known to elevate intracellular cGMP and cAMP levels, and the membrane-permeant analogue 8-bromo-cGMP rapidly activates Dd-STATc, whereas 8-bromo-cAMP is a much less effective inducer. Surprisingly, however, Dd-STATc remains stress activatable in null mutants for components of the known cGMP-mediated and cAMP-mediated stress-response pathways and in a double mutant affecting both pathways. Also, Dd-STATc null cells are not abnormally sensitive to hyperosmotic stress. Microarray analysis identified two genes, gapA and rtoA, that are induced by hyperosmotic stress. Osmotic stress induction of gapA and rtoA is entirely dependent on Dd-STATc. Neither gene is inducible by DIF but both are rapidly inducible with 8-bromo-cGMP. Again, 8-bromo-cAMP is a much less potent inducer than 8-bromo-cGMP. These data show that Dd-STATc functions as a transcriptional activator in a stress-response pathway and the pharmacological evidence, at least, is consistent with cGMP acting as a second messenger.

Control of Cell Type Proportioning in Dictyostelium discoideum by Differentiation-Inducing Factor as Determined by In Situ Hybridization

ABSTRACT We have determined the proportions of the prespore and prestalk regions in Dictyostelium discoideum slugs by in situ hybridization with a large number of prespore- and prestalk-specific genes. Microarrays were used to discover genes expressed in a cell type-specific manner. Fifty-four prespore-specific genes were verified by in situ hybridization, including 18 that had been previously shown to be cell type specific. The 36 new genes more than doubles the number of available prespore markers. At the slug stage, the prespore genes hybridized to cells uniformly in the posterior 80% of wild-type slugs but hybridized to the posterior 90% of slugs lacking the secreted alkylphenone differentiation-inducing factor 1 (DIF-1). There was a compensatory twofold decrease in prestalk cells in DIF-less slugs. Removal of prespore cells resulted in cell type conversion in both wild-type and DIF-less anterior fragments. Thus, DIF-1 appears to act in concert with other processes to establish cell type proportions.

Analysis of phenotypic evolution in Dictyostelia highlights developmental plasticity as a likely consequence of colonial multicellularity

Colony formation was the first step towards evolution of multicellularity in many macroscopic organisms. Dictyostelid social amoebas have used this strategy for over 600 Myr to form fruiting structures of increasing complexity. To understand in which order multicellular complexity evolved, we measured 24 phenotypic characters over 99 dictyostelid species. Using phylogenetic comparative methods, we show that the last common ancestor (LCA) of Dictyostelia probably erected small fruiting structures directly from aggregates. It secreted cAMP to coordinate fruiting body morphogenesis, and another compound to mediate aggregation. This phenotype persisted up to the LCAs of three of the four major groups of Dictyostelia. The group 4 LCA co-opted cAMP for aggregation and evolved much larger fruiting structures. However, it lost encystation, the survival strategy of solitary amoebas that is retained by many species in groups 1–3. Large structures, phototropism and a migrating intermediate ‘slug’ stage coevolved as evolutionary novelties within most groups. Overall, dictyostelids show considerable plasticity in the size and shape of multicellular structures, both within and between species. This probably reflects constraints placed by colonial life on developmental control mechanisms, which, depending on local cell density, need to direct from 10 to a million cells into forming a functional fructification.

A membrane protein with possible relevance to sexual cell fusion in Dictyostelium discoideum

The molecular mechanism of sexual cell fusion in Dictyostelium discoideum was studied using the heterothallic strains HM1 and NC4. Monovalent antibodies (Fab) prepared from rabbit antiserum against a crude membrane preparation of fusion-competent HM1 cells inhibited fusion between HM1 and NC4 cells. Six out of 43 antigenic proteins were found in fusion-competent HM1 cells but not in fusion-incompetent cells. Among them, only one protein with a molecular mass of 70 kDa was able to neutralize the fusion-inhibiting activity of Fab, suggesting its possible participation in sexual cell fusion.