Tetsuya Muramoto

Live imaging of nascent RNA dynamics reveals distinct types of transcriptional pulse regulation

Transcription of genes can be discontinuous, occurring in pulses or bursts. It is not clear how properties of transcriptional pulses vary between different genes. We compared the pulsing of five housekeeping and five developmentally induced genes by direct imaging of single gene transcriptional events in individual living Dictyostelium cells. Each gene displayed its own transcriptional signature, differing in probability of firing and pulse duration, frequency, and intensity. In contrast to the prevailing view from both prokaryotes and eukaryotes that transcription displays binary behavior, strongly expressed housekeeping genes altered the magnitude of their transcriptional pulses during development. These nonbinary “tunable” responses may be better suited than stochastic switch behavior for housekeeping functions. Analysis of RNA synthesis kinetics using fluorescence recovery after photobleaching implied modulation of housekeeping-gene pulse strength occurs at the level of transcription initiation rather than elongation. In addition, disparities between single cell and population measures of transcript production suggested differences in RNA stability between gene classes. Analysis of stability using RNAseq revealed no major global differences in stability between developmental and housekeeping transcripts, although strongly induced RNAs showed unusually rapid decay, indicating tight regulation of expression.

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.

Live imaging of the Dictyostelium cell cycle reveals widespread S phase during development, a G2 bias in spore differentiation and a premitotic checkpoint

The regulation of the Dictyostelium cell cycle has remained ambiguous owing to difficulties in long-term imaging of motile cells and a lack of markers for defining cell cycle phases. There is controversy over whether cells replicate their DNA during development, and whether spores are in G1 or G2 of the cell cycle. We have introduced a live-cell S-phase marker into Dictyostelium cells that allows us to precisely define cycle phase. We show that during multicellular development, a large proportion of cells undergo nuclear DNA synthesis. Germinating spores enter S phase only after their first mitosis, indicating that spores are in G2. In addition, we demonstrate that Dictyostelium heterochromatin is copied late in S phase and replicates via accumulation of replication factors, rather than recruitment of DNA to pre-existing factories. Analysis of variability in cycle times indicates that regulation of the cycle manifests at a single random transition in G2, and we present the first identified checkpoint in Dictyostelium, which operates at the G2-M transition in response to DNA damage.

Digital nature of the immediate-early transcriptional response

Stimulation of transcription by extracellular signals is a major component of a cells decision making. Yet the quantitative relationship between signal and acute transcriptional response is unclear. One view is that transcription is directly graded with inducer concentration. In an alternative model, the response occurs only above a threshold inducer concentration. Standard methods for monitoring transcription lack continuous information from individual cells or mask immediate-early transcription by measuring downstream protein expression. We have therefore used a technique for directly monitoring nascent RNA in living cells, to quantify the direct transcriptional response to an extracellular signal in real time, in single cells. At increasing doses of inducer, increasing numbers of cells displayed a transcriptional response. However, over the same range of doses, the change in cell response strength, measured as the length, frequency and intensity of transcriptional pulses, was small, with considerable variation between cells. These data support a model in which cells have different sensitivities to developmental inducer and respond in a digital manner above individual stimulus thresholds. Biased digital responses may be necessary for certain forms of developmental specification. Limiting bias in responsiveness is required to reduce noise in positional signalling.

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.

Nuclear organization and transcriptional dynamics in Dictyostelium

The Dictyostelium model has a set of features uniquely well‐suited to developing our understanding of transcriptional control. The complete Dictyostelium discoideum genome sequence has revealed that many of the molecular components regulating transcription in larger eukaryotes are conserved in Dictyostelium, from transcription factors and chromatin components to the enzymes and signals that regulate them. In addition, the system permits visualization of single gene firing events in living cells, which provides a more detailed view of transcription and its relationships to cell and developmental processes. This review will bring together the available knowledge of the structure and dynamics of the Dictyostelium nucleus and discuss recent transcription imaging studies and their implications for stability and accuracy of cell decisions.

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.

Imaging Nascent RNA Dynamics in Dictyostelium

Dictyostelium cells have great utility for live imaging of single gene transcriptional dynamics. The cells allow efficient molecular genetics, for targeting of RNA reporters and fluorescent proteins to individual, defined loci. Dictyostelium cells share many signalling, chromatin and nuclear characteristics of larger eukaryotes, yet the cells have a relatively simple scattered differentiation programme, allowing imaging of transcriptional events in the context of stochastic developmental choices. This review will detail the methods and considerations for imaging nascent RNA dynamics at single genes in living Dictyostelium cells.