The cytosolic lncRNA dutA affects STATa signaling and developmental commitment in Dictyostelium.

STATa is a pivotal transcription factor for Dictyostelium development. dutA is the most abundant RNA transcribed by RNA polymerase II in Dictyostelium, and its functional interplay with STATa has been suggested. This study demonstrates that dutA RNA molecules are distributed as spot-like structures in the cytoplasm, and that its cell type-specific expression changes dramatically during development.

Loss of PIKfyve Causes Transdifferentiation of Spores Into Basal Disc Cells.

The 1-phosphatidylinositol-3-phosphate 5-kinase PIKfyve generates PtdIns3,5P2 on late phagolysosomes, which by recruiting the scission protein Atg18, results in their fragmentation in the normal course of endosome processing. Loss of PIKfyve function causes cellular hypervacuolization in eukaryotes and organ failure in humans. We identified as the defective gene in a mutant that failed to form spores.

Role of LrrkA in the Control of Phagocytosis and Cell Motility in .

LrrkA is a kinase with leucine-rich repeats. LrrkA stimulates Kil2 and intra-phagosomal killing of ingested bacteria in response to folate. In this study, we show that genetic inactivation of also causes a previously unnoticed phenotype: KO cells exhibit enhanced phagocytosis and cell motility compared to parental cells.

LrrkA, a kinase with leucine-rich repeats, links folate sensing with Kil2 activity and intracellular killing.

Phagocytic cells ingest bacteria by phagocytosis and kill them efficiently inside phagolysosomes. The molecular mechanisms involved in intracellular killing and their regulation are complex and still incompletely understood. Dictyostelium discoideum has been used as a model to discover and to study new gene products involved in intracellular killing of ingested bacteria.

Generation of deletions and precise point mutations in Dictyostelium discoideum using the CRISPR nickase.

The CRISPR/Cas9 system enables targeted genome modifications across a range of eukaryotes. Although we have reported that transient introduction of all-in-one vectors that express both Cas9 and sgRNAs can efficiently induce multiple gene knockouts in Dictyostelium discoideum, concerns remain about off-target effects and false-positive amplification during mutation detection via PCR. To minimise these effects, we modified the system to permit gene deletions of greater than 1 kb via use of paired sgRNAs and Cas9 nickase.

Analysis of DrkA kinase's role in STATa activation.

Dictyostelium STATa is a homologue of metazoan signal transducers and activators of transcription (STATs) and is important for morphogenesis. STATa is activated by phosphorylation on Tyr702 when cells are exposed to extracellular cAMP. Although two tyrosine kinase-like (TKL) proteins, Pyk2 and Pyk3, have been definitively identified as STATc kinases, no kinase is known for STATa activation.

Recent Advances in CRISPR/Cas9-Mediated Genome Editing in .

In the last 30 years, knockout of target genes via homologous recombination has been widely performed to clarify the physiological functions of proteins in . As of late, CRISPR/Cas9-mediated genome editing has become a versatile tool in various organisms, including , enabling rapid high-fidelity modification of endogenous genes. Here we reviewed recent progress in genome editing in and summarised useful CRISPR vectors that express sgRNA and Cas9, including several microorganisms.

CRISPR/Cas9 mediated targeting of multiple genes in Dictyostelium.

CRISPR/Cas9 has emerged in various organisms as a powerful technology for targeted gene knockout; however, no reports of editing the Dictyostelium genome efficiently using this system are available. We describe here the application of CRISPR/Cas9-mediated gene modification in Dictyostelium. The endogenous tRNA-processing system for expressing sgRNA was approximately 10 times more effective than the commonly used U6 promoter.

Regulation of ecmF gene expression and genetic hierarchy among STATa, CudA, and MybC on several prestalk A-specific gene expressions in Dictyostelium.

STATa, a Dictyostelium homologue of metazoan signal transducer and activator of transcription, is important for the organizer function in the tip region of the migrating Dictyostelium slug. We previously showed that ecmF gene expression depends on STATa in prestalk A (pstA) cells, where STATa is activated. Deletion and site-directed mutagenesis analysis of the ecmF/lacZ fusion gene in wild-type and STATa null strains identified an imperfect inverted repeat sequence, ACAAATANTATTTGT, as a STATa-responsive element.

Implications of expansin-like 3 gene in Dictyostelium morphogenesis.

Dictyostelium harbors multiple expansin-like genes with generally unknown functions. Thus, we analyzed the expansin-like 3 (expL3) gene and found that its expression was reduced in a null mutant for a STATa gene encoding a transcription factor. The expression of expL3 was developmentally regulated and its transcript was spliced only in the multicellular stages.

Two Dictyostelium tyrosine kinase-like kinases function in parallel, stress-induced STAT activation pathways.

When Dictyostelium cells are hyperosmotically stressed, STATc is activated by tyrosine phosphorylation. Unusually, activation is regulated by serine phosphorylation and consequent inhibition of a tyrosine phosphatase: PTP3. The identity of the cognate tyrosine kinase is unknown, and we show that two tyrosine kinase-like (TKL) enzymes, Pyk2 and Pyk3, share this function; thus, for stress-induced STATc activation, single null mutants are only marginally impaired, but the double mutant is nonactivatable.

Identification of the kinase that activates a nonmetazoan STAT gives insights into the evolution of phosphotyrosine-SH2 domain signaling.

SH2 domains are integral to many animal signaling pathways. By interacting with specific phosphotyrosine residues, they provide regulatable protein-protein interaction domains. Dictyostelium is the only nonmetazoan with functionally characterized SH2 domains, but the cognate tyrosine kinases are unknown.

Evidence for a functional link between Dd-STATa and Dd-PIAS, a Dictyostelium PIAS homologue.

Several mammalian protein families inhibit the activity of signal transducer and activator of transcription (STAT) proteins. The protein inhibitor of activated STAT (PIAS) was initially identified through its ability to interact with human STAT proteins. We isolated a gene (pisA) encoding a Dictyostelium orthologue of PIAS, Dd-PIAS, which possesses almost all the representative motifs and domains of mammalian PIAS proteins.

STAT signaling in Dictyostelium development.

Signal transducers and activators of transcription (STAT) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. These proteins are components of JAK/STAT signal transduction pathways, which regulate immune responses, cell fate, proliferation, cell migration, and programmed cell death in multicellular organisms. The cellular slime mould, Dictyostelium discoideum, is the simplest multicellular organism using molecules homologous to STATs, Dd-STATa-d.

SunB, a novel Sad1 and UNC-84 domain-containing protein required for development of Dictyostelium discoideum.

A gene, sunB, encoding a novel class of Sad1 and UNC-84 (SUN) domain, was isolated from a cDNA screen for suppressors of a mutation in Dd-STATa - a Dictyostelium homologue of metazoan STAT (signal transducers and activators of transcription). The SunB protein localized in the area around the nucleus in growing cells, but in the multicellular stages it was predominantly found in prespore vacuoles (PSVs). A disruptant of sunB was multinucleated in the vegetative phase; during development it formed mounds with multiple tips and failed to culminate.

Prespore cell inducing factor, psi factor, controls both prestalk and prespore gene expression in Dictyostelium development.

Prespore cell-inducing (psi, psi) factor (PsiA), encoded by the psiA gene of Dictyostelium, is a secreted signal glycoprotein that induces prespore cell differentiation when added to monolayer cultures. In situ hybridization during normal development showed that the psiA gene is highly expressed in scattered cells at the mound stage and in prespore cells at the onset of culmination. The conventional prespore-cell marker genes, cotC and pspA, were expressed normally in psiA(-) and psiA overexpressing strains.

Substituent effect on electrophoretic mobility for a series of poly(N-acryloyl-amino acid)s.

In this study, the electrophoretic mobilities (mu) for a series of poly(N-acryloyl-amino acid)s were determined by capillary zone electrophoresis to investigate the effect of substituent on the electrophoretic behavior of polyelectrolytes. The mu values determined showed a strong correlation with the molar volume of the corresponding amino acids.

Role of an expansin-like molecule in Dictyostelium morphogenesis and regulation of its gene expression by the signal transducer and activator of transcription protein Dd-STATa.

Expansins are proteins involved in plant morphogenesis, exerting their effects on cellulose to extend cell walls. Dictyostelium is an organism that possesses expansin-like molecules, but their functions are not known. In this study, we analyzed the expL7 (expansin-like 7) gene, which has been identified as a putative target of Dd-STATa, a Dictyostelium homolog of the metazoan signal transducer and activator of transcription (STAT) proteins.

Expression of zinc transporter family genes in Dictyostelium.

Regulation of the zinc ion concentration is physiologically important to control the activities of a variety of cellular molecules. A BLAST search against a conserved domain of known zinc transporters identified twelve putative zinc transporter family genes in the Dictyostelium genome. Phylogenetic analysis revealed the presence of three zinc transporter subfamilies in Dictyostelium.

GBF-dependent family genes morphologically suppress the partially active Dictyostelium STATa strain.

Transcription factor Dd-STATa, a functional Dictyostelium homologue of metazoan signal transducers and activators of transcription proteins, is necessary for culmination during development. We have isolated more than 18 putative multicopy suppressors of Dd-STATa using genetic screening. One was hssA gene, whose expression is known to be G-box-binding-factor-dependent and which was specific to prestalk A (pstA) cells, where Dd-STATa is activated.

Evidence that noncoding RNA dutA is a multicopy suppressor of Dictyostelium discoideum STAT protein Dd-STATa.

Dd-STATa, a Dictyostelium discoideum homologue of metazoan STAT transcription factors, is necessary for culmination. We created a mutant strain with partial Dd-STATa activity and used it to screen for unlinked suppressor genes. We screened approximately 450,000 clones from a slug-stage cDNA library for their ability to rescue the culmination defect when overexpressed.

Analysis of a homologue of the adducin head gene which is a potential target for the Dictyostelium STAT protein Dd-STATa.

A Dd-STATa-null mutant, which is defective in expression of a Dictyostelium homologue of the metazoan STAT (signal transducers and activators of transcription) proteins, fails to culminate and this phenotype correlates with the loss of expression of various prestalk (pst) genes. An EST clone, SSK395, encodes a close homologue of the adducin amino-terminal head domain and harbors a putative actin-binding domain. We fused promoter fragments of the cognate gene, ahhA (adducin head homologue A), to a lacZ reporter and determined their expression pattern.

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.

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.