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Hideko Urushihara

Publications and source records attributed to Hideko Urushihara.

At least 19 recordsLinked to original sources
PubMed2006

Genes involved in Dictyostelium discoideum sexual reproduction.

Macrocyst formation in the cellular slime moulds is a sexual process induced under dark and humid conditions. Normal development life cycle in these organisms involves proliferation by cell division and, upon starvation, formation of multicellular aggregates and fruiting bodies, consisting of spores and stalk cells. Macrocyst formation, cell division by binary fission and spore formation are thus three alternative modes of reproduction, for which it is of interest to understand how a choice is made. The genetic basis of asexual development and fruiting body formation is well known, by contrast information on the genetic control of sexual reproduction during macrocyst formation is scarce. In Dictyostelium discoideum, the most widely used species, several cell-surface proteins relevant to sexual cell fusion have been identified using cell fusion-blocking antibodies, but isolation of the relevant genes has been unsuccessful. Analysis of sexually deficient mutants, some of which are normal for asexual development, has shown that sexual reproduction is regulated by both specific genes and genes that are also involved in asexual development. Reverse genetic analysis of 24 genes highly enriched in a gamete-specific subtraction library has revealed four genes involved in the regulation of sexual cell interactions. One of them was found to be a novel regulator of the cAMP signalling pathway specific to sexual development. Studies on the molecular genetic control of the sexual cycle will be reviewed and their contribution to our understanding of the organization and function of the D. discoideum genome as a whole discussed.

Animals↗
PubMed2006

Small GTPase RacF2 affects sexual cell fusion and asexual development in Dictyostelium discoideum through the regulation of cell adhesion.

Cells of Dictyostelium discoideum become sexually mature when submerged and in darkness, and fuse with opposite mating-type cells as gametes. The gene for a Rho GTPase, RacF2, is one of the extremely gamete-enriched genes (>100-fold) identified by us previously. Here, we isolated knockout, overexpression, constitutively active and dominant negative mutants of RacF2, and analyzed their phenotypes. These mutants showed anomalies in the extent of sexual cell fusion and asexual development as well as in EDTA-sensitive cell-cell adhesion. It is suggested that RacF2 controls the process of sexual and asexual development through the regulation of cellular adhesiveness. An analysis of the expression of all 18 rac family genes by real-time polymerase chain reaction revealed that four additional genes, rac1b, rac1c, racF1 and racG, were induced during maturation, suggesting their possible involvement in sexual cell interactions.

Animals↗
PubMed2006

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. pneumophilaDeltadotA. 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.

Animals↗
PubMed2006

Cultivation, spore production, and mating.

Dictyostelium discoideum proliferates as solitary amoebae, constitutes multicellular structures called fruiting bodies, and mates to form macrocysts depending on environmental conditions. All of these processes can be easily induced in the laboratory. The amoebae are normally cultured with food bacteria, but the strains with mutations in axe loci can proliferate in nutrient media without bacteria. The strains can be stored either as spores or amoebae. Synchronous development of fruiting bodies is initiated by depleting the culture media or food bacteria. Synchronous development of macrocysts is achieved by mixing the cells of heterothallic strains separately cultured in darkness to induce the sexual maturation.

Animals↗
PubMed2006

The cDNA sequencing project.

The Dictyostelium discoideum cDNA sequencing project started in 1995, preceding the genome sequencing project. Altogether, 14 cDNA libraries, including full-length ones, were constructed from five different stages of growth and asexual and sexual development, from which nearly 100,000 randomly chosen clones were sequenced to yield over 150,000 expressed sequence tags (ESTs). The data have been publicized online to facilitate clone distribution and collaboration using the whole clone set for microarray analyses. The EST reads were assembled to 6700 independent genes, which constitute about 55% of the total estimated Dictyostelium genes. Utilization of wet and dry resources have contributed to the understanding of the genetic system controlling the multicellular development in Dictyostelium.

Animals↗
PubMed2005

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

BACKGROUND: Formins 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. RESULTS: We 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. CONCLUSION: Our 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.

Actin Cytoskeleton↗
PubMed2005

Reverse genetic analyses of gamete-enriched genes revealed a novel regulator of the cAMP signaling pathway in Dictyostelium discoideum.

Sexual development in Dictyostelium discoideum is initiated by the fusion of opposite mating type cells to form zygote giant cells, which subsequently gather and phagocytose surrounding cells for nutrition to form macrocysts. Here we performed the targeting of 24 highly gamete-enriched genes we previously isolated, and successfully generated knockout mutants for 16 genes and RNAi mutants for 20 genes including 6 genes without disruptants. In the knockout mutants of two genes, cell aggregation toward the giant cells was much less extensive and many cells remained around poorly formed macrocysts. We named these genes tmcB and tmcC. Although macrocyst formation of wild type cells was suppressed by the addition of exogenous cAMP, that of knockout mutants of tmcB was much less sensitive. The mRNA level of phosphodiesterase (pde) was higher and that of its inhibitor (pdi) was lower in the latter cells compared to the parental strains during sexual development. Thus, tmcB appeared to be a novel regulator of the cAMP signaling pathway specific to sexual development. Knockout mutants of tmcC were indistinguishable from the wild type cells with respect to the cAMP response, suggesting that this gene is relevant to other processes.

Amino Acid Sequence↗
PubMed2005

Evidence that the Dictyostelium STAT protein Dd-STATa plays a role in the differentiation of inner basal disc cells and identification of a promoter element essential for expression in these cells.

Dd-STATa, a Dictyostelium homolog of the metazoan STAT (signal transducers and activators of transcription) proteins, is necessary in the slug for correct entry into culmination. Dd-STATa-null mutant fails to culminate and its phenotype correlates with the loss of a funnel-shaped core region, the pstAB core region, which expresses both the ecmA and ecmB genes. To understand how the differentiation of pstAB core cells is regulated, we identified an EST that is expressed in the core cells of normal slugs but down-regulated in the Dd-STATa-null mutant. This EST, SSK348, encodes a close homolog of the Dictyostelium acetyl-CoA synthetase (ACS). A promoter fragment of the cognate gene, aslA (acetyl-CoA synthetase-like A), was fused to a lacZ reporter and the expression pattern determined. As expected from the behavior of the endogenous aslA gene, the aslA::lacZ fusion gene is not expressed in Dd-STATa-null slugs. In parental cells, the aslA promoter is first activated in the funnel-shaped core cells located at the slug anterior, the "pstAB core." During culmination, the pstAB core cells move down, through the prespore cells, to form the inner part of the basal disc. As the spore mass climbs the stalk, the aslA gene comes to be expressed in cells of the upper and lower cups, structures that cradle the spore head. Deletion and point mutation analyses of the promoter identified an AT-rich sequence that is necessary for expression in the pstAB core. This acts in combination with repressor regions that prevent ectopic aslA expression in the pre-stalk regions of slugs and the stalks of culminants. Thus, this study confirms that Dd-STATa is necessary for the differentiation of pstAB core cells, by showing that it is needed for the activation of the aslA gene. It also identifies aslA promoter elements that are likely to be regulated, directly or indirectly, by Dd-STATa.

Amino Acid Sequence↗
PubMed2005

Gamete fusion and cytokinesis preceding zygote establishment in the sexual process of Dictyostelium discoideum.

Cells of Dictyostelium discoideum become sexually mature under submerged and dark conditions, and fuse with opposite mating-type cells to form zygote giant cells, which gather surrounding cells and finally develop into dormant structures called macrocysts. In the present study, we found that the multinuclear fused cells formed during this process frequently underwent cytokinesis driven by random local movements. The split cells were capable of re-fusion, and repeated cytokinesis. These radical behaviors continued until the extensive cell aggregation started around the giant cells. Thus, gamete fusion and initiation of zygote development do not coincide in the mating of D. discoideum. Analyses by confocal microscopy and flow cytometry indicated that the cessation of the random movement followed pronuclear fusion, and that microtubule organizing centers (MTOC), abundant in the fused cells at the beginning, gradually decreased and only one of them remained within the developed macrocyst. Some of the genes known to control cell movement, such as rasGEFB and rasS, increased shortly before the cessation of repeated fusion-cytokinesis and initiation of phagocytosis. These results suggest that the sequential molecular events are necessary in D. discoideum after gamete fusion to establish a new individuality of zygotes.

Animals↗
PubMed2004

Extracellular matrix family proteins that are potential targets of Dd-STATa in Dictyostelium discoideum.

Dd-STATa is a functional Dictyostelium homologue of metazoan STAT (signal transducers and activators of transcription) proteins, which is activated by cAMP and is thereby translocated into the nuclei of anterior tip cells of the prestalk region of the slug. By using in situ hybridization analyses, we found that the SLF308 cDNA clone, which contains the ecmF gene that encodes a putative extracellular matrix protein and is expressed in the anterior tip cells, was greatly down-regulated in the Dd-STATa-null mutant. Disruption of the ecmF gene, however, resulted in almost no phenotypic change. The absence of any obvious mutant phenotype in the ecmF-null mutant could be due to a redundancy of similar genes. In fact, a search of the Dictyostelium whole genome database demonstrates the existence of an additional 16 homologues, all of which contain a cellulose-binding module. Among these homologues, four genes show Dd-STATa-dependent expression, while the others are Dd-STATa-independent. We discuss the potential role of Dd-STATa in morphogenesis via its effect on the interaction between cellulose and these extracellular matrix family proteins.

Amino Acid Sequence↗
PubMed2004

An orderly retreat: Dedifferentiation is a regulated process.

Differentiation is a highly regulated process whereby cells become specialized to perform specific functions and lose the ability to perform others. In contrast, the question of whether dedifferentiation is a genetically determined process, or merely an unregulated loss of the differentiated state, has not been resolved. We show here that dedifferentiation in the social amoeba Dictyostelium discoideum relies on a sequence of events that is independent of the original developmental state and involves the coordinated expression of a specific set of genes. A defect in one of these genes, the histidine kinase dhkA, alters the kinetics of dedifferentiation and uncouples the progression of dedifferentiation events. These observations establish dedifferentiation as a genetically determined process and suggest the existence of a developmental checkpoint that ensures a return path to the undifferentiated state.

Animals↗
PubMed2004

Analyses of cDNAs from growth and slug stages of Dictyostelium discoideum.

Dictyostelium is a favored model for studying problems in cell and developmental biology. To comprehend the genetic potential and networks that direct growth and multicellular development, we are performing a large-scale analysis of Dictyostelium cDNAs. Here, we newly determine 7720 nucleotide sequences of cDNAs from the multicellular, slug stage (S) and 10 439 from the unicellular, vegetative stage (V). The combined 26 954 redundant ESTs were computer assembled using the PHRAP program to yield 5381 independent sequences. These 5381 predicted genes represent about half of the estimated coding potential of the organism. One-third of them were classified into 12 functional categories. Although the overall classification patterns of the V and S libraries were very similar, stage-specific genes exist in every category. The majority of V-specific genes function in some aspect of protein translation, while such genes are in a minority in the S-specific and common populations. Instead, genes for signal transduction and multicellular organization are enriched in the population of S-specific genes. Genes encoding the enzymes of basic metabolism are mainly found in the common gene population. These results therefore suggest major differences between growing and developing Dictyostelium cells in the nature of the genes transcribed.

Animals↗
PubMed2004

Control of cell type proportioning in Dictyostelium discoideum by differentiation-inducing factor as determined by in situ hybridization.

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.

Animals↗
PubMed2004

Identification of new modes of Dd-STATa regulation of gene expression in Dictyostelium by in situ hybridisation.

Signal Transducers and Activators of Transcription (STATs) are transcription factors which lie at the end of cytokine and growth signal transduction pathways. Dictyostelium Dd-STATa is a functional homologue of metazoan STATs. It is activated by cAMP and, at the slug stage, it translocates into the nuclei of the tip cells, which are a subset of the anterior, prestalk A (pstA) cells. Here we searched for novel Dd-STATa regulated genes by in situ hybridisation. A set of 54 cDNA clones whose gene expression patterns are known to be prestalk-specific (Maeda et al., 2003), were chosen as probes and we compared their expression patterns in parental and Dd-STATa-null strains. We identified 13 genes which are candidates for direct induction by Dd-STATa. In the parental strain, most of these genes are expressed in the cone shaped mass of pstAB cells which is located within the prestalk region. These cDNAs show little or no expression in the Dd-STATa-null strain. This contrasts markedly with the paradigmatic ecmB gene which is expressed in pstAB cells in parental cells, but which is expressed throughout the prestalk zone in the Dd-STATa-null strain. We also identified several genes which are normally expressed in pstA cells, or throughout the prestalk region, but whose expression is markedly down-regulated in the null mutant. Again, this contrasts with markers derived from the paradigmatic, ecmA gene which are expressed normally in the Dd-STATa-null strain. The identification of these novel genes provides valuable tools to investigate the role of Dd-STATa.

Animals↗
PubMed2003

Identification of a second member of the ponticulin gene family and its differential expression pattern.

We have identified a homologue (ponB) of the ponticulin gene (ponA), an F-actin binding protein, in the expressed sequence tag library generated to mRNA isolated from fusion-competent cells of Dictyostelium discoideum. PonB is predicted to have many of the same characteristics as ponticulin. Both proteins are predicted to possess a cleaved signal peptide, a glycosyl anchor, an amphipathic beta-strand structure and six conserved cysteines. Because of the sequence similarity and predicted conserved structures, this gene constitutes the second member of a ponticulin gene family. Unlike ponticulin, ponB is not expressed in axenically grown cells or during the asexual reproductive phase of D. discoideum. PonB is expressed by cells grown on bacterial lawns and by cells induced to be fusion-competent, i.e., gametes. The expression of ponB correlates with the appearance of a new F-actin binding activity in cell lysates of bacterially grown ponA(-) cells. By immunofluorescence microscopy, ponB appears to be localized to vesicles and to the plasma membrane of bacterially grown cells. Because ponticulin is the major high-affinity link between the plasma membrane and the cytoskeleton, the ponticulin gene family is likely to be part of the redundant system of proteins involved in connecting the cytoskeleton to the plasma membrane.

Amino Acid Sequence↗
PubMed2003

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.

Active Transport, Cell Nucleus↗