Dictyostelium Discoideum Development Research Articles

Noncanonical usage of stop codons in ciliates expands proteins with structurally flexible Q-rich motifs.

Serine(S)/threonine(T)-glutamine(Q) cluster domains (SCDs), polyglutamine (polyQ) tracts and polyglutamine/asparagine (polyQ/N) tracts are Q-rich motifs found in many proteins. SCDs often are intrinsically disordered regions that mediate protein phosphorylation and protein-protein interactions. PolyQ and polyQ/N tracts are structurally flexible sequences that trigger protein aggregation. We report that due to their high percentages of STQ or STQN amino acid content, four SCDs and three prion-causing Q/N-rich motifs of yeast proteins possess autonomous protein expression-enhancing activities. Since these Q-rich motifs can endow proteins with structural and functional plasticity, we suggest that they represent useful toolkits for evolutionary novelty. Comparative Gene Ontology (GO) analyses of the near-complete proteomes of 26 representative model eukaryotes reveal that Q-rich motifs prevail in proteins involved in specialized biological processes, including Saccharomyces cerevisiae RNA-mediated transposition and pseudohyphal growth, Candida albicans filamentous growth, ciliate peptidyl-glutamic acid modification and microtubule-based movement, Tetrahymena thermophila xylan catabolism and meiosis, Dictyostelium discoideum development and sexual cycles, Plasmodium falciparum infection, and the nervous systems of Drosophila melanogaster, Mus musculus and Homo sapiens. We also show that Q-rich-motif proteins are expanded massively in 10 ciliates with reassigned TAAQ and TAGQ codons. Notably, the usage frequency of CAGQ is much lower in ciliates with reassigned TAAQ and TAGQ codons than in organisms with expanded and unstable Q runs (e.g. D. melanogaster and H. sapiens), indicating that the use of noncanonical stop codons in ciliates may have coevolved with codon usage biases to avoid triplet repeat disorders mediated by CAG/GTC replication slippage.

Noncanonical usage of stop codons in ciliates expands proteins with structurally flexible Q-rich motifs

Serine(S)/threonine(T)-glutamine(Q) cluster domains (SCDs), polyglutamine (polyQ) tracts and polyglutamine/asparagine (polyQ/N) tracts are Q-rich motifs found in many proteins. SCDs often are intrinsically disordered regions that mediate protein phosphorylation and protein-protein interactions. PolyQ and polyQ/N tracts are structurally flexible sequences that trigger protein aggregation. We report that due to their high percentages of STQ or STQN amino acid content, four SCDs and three prion-causing Q/N-rich motifs of yeast proteins possess autonomous protein expression-enhancing activities. Since these Q-rich motifs can endow proteins with structural and functional plasticity, we suggest that they represent useful toolkits for evolutionary novelty. Comparative Gene Ontology (GO) analyses of the near-complete proteomes of 26 representative model eukaryotes reveal that Q-rich motifs prevail in proteins involved in specialized biological processes, including Saccharomyces cerevisiae RNA-mediated transposition and pseudohyphal growth, Candida albicans filamentous growth, ciliate peptidyl-glutamic acid modification and microtubule-based movement, Tetrahymena thermophila xylan catabolism and meiosis, Dictyostelium discoideum development and sexual cycles, Plasmodium falciparum infection, and the nervous systems of Drosophila melanogaster, Mus musculus and Homo sapiens. We also show that Q-rich-motif proteins are expanded massively in 10 ciliates with reassigned TAAQ and TAGQ codons. Notably, the usage frequency of CAGQ is much lower in ciliates with reassigned TAAQ and TAGQ codons than in organisms with expanded and unstable Q runs (e.g. D. melanogaster and H. sapiens), indicating that the use of noncanonical stop codons in ciliates may have coevolved with codon usage biases to avoid triplet repeat disorders mediated by CAG/GTC replication slippage.

The Structure-Activity Relationship of MPBD and Dictyoquinone Analogs in <i>Dictyostelium discoideum</i> Cell Aggregation

4-Methyl-5-pentylbenzene-1,3-diol (MPBD) (1) and dictyoquinone (DQ or 2-hydroxy-5-methyl-6-pentyl-1,4-benzoquinone) (2) are two polyketides involved in the cell development of Dictyostelium discoideum. We synthesized several MPBD and DQ analogs and tested their ability to recover the cell aggregation delay in the stlA null strain. According to the findings, the alkyl side chain of both compounds facilitates their activity. The parent n-pentyl side chain is required for the cell aggregation.

Identification and characterization of Poly(ADP-ribose) polymerase-1 interacting proteins during development of Dictyostelium discoideum

Poly (ADP-ribose) polymerase-1 (PARP-1) is a multifunctional protein that is associated with various biological processes like chromatin remodeling, DNA damage, cell death etc. In Dictyostelium discoideum, PARP-1 has also been implicated in cellular differentiation and development. However, its interacting proteins during multicellular development are not yet explored. Hence, the present study aims to identify PARP-1 interacting proteins during multicellular development of D. discoideum.BRCA1 C-terminus (BRCT) domain of PARP-1, which is mainly involved in protein-protein interactions was cloned in pGEX4T1 vector and developmental interactome of PARP-1 were analyzed by affinity purification-mass spectrometry. These interactions were further confirmed by in-silico protein-protein docking analysis, which led to identification of the proteins that show high affinity for BRCT domain. Initially, the protein structures were modeled on SWISS MODEL and PHYRE2 servers, refined by 3Drefine and validated by PROCHECK. Further, interaction sites of BRCT and the conserved regions in all interacting proteins were predicted using cons-PPISP and ConSurf, respectively. Finally, protein-protein docking analysis was done by HADDOCK.Our results identified 19 possible BRCT interacting proteins during D. discoideum development. Furthermore, interacting residues involved in the interactions and functional regions were explored. This is the first report where PARP-1's developmental interactome in D. discoideum is well established. The current findings demonstrate PARP-1's developmental interactome in D. discoideum and provide the groundwork to understand its regulated functions in developmental biology which would undoubtedly extend our perception towards developmental diseases in higher complex organisms and their treatment.

Changes of the tRNA Modification Pattern during the Development of Dictyostelium discoideum.

Dictyostelium discoideum is a social amoeba, which on starvation develops from a single-cell state to a multicellular fruiting body. This developmental process is accompanied by massive changes in gene expression, which also affect non-coding RNAs. Here, we investigate how tRNAs as key regulators of the translation process are affected by this transition. To this end, we used LOTTE-seq to sequence the tRNA pool of D. discoideum at different developmental time points and analyzed both tRNA composition and tRNA modification patterns. We developed a workflow for the specific detection of modifications from reverse transcriptase signatures in chemically untreated RNA-seq data at single-nucleotide resolution. It avoids the comparison of treated and untreated RNA-seq data using reverse transcription arrest patterns at nucleotides in the neighborhood of a putative modification site as internal control. We find that nucleotide modification sites in D. discoideum tRNAs largely conform to the modification patterns observed throughout the eukaroytes. However, there are also previously undescribed modification sites. We observe substantial dynamic changes of both expression levels and modification patterns of certain tRNA types during fruiting body development. Beyond the specific application to D. discoideum our results demonstrate that the developmental variability of tRNA expression and modification can be traced efficiently with LOTTE-seq.

Mitochondrial Processes during Early Development of Dictyostelium discoideum: From Bioenergetic to Proteomic Studies.

The slime mold Dictyostelium discoideum’s life cycle includes different unicellular and multicellular stages that provide a convenient model for research concerning intracellular and intercellular mechanisms influencing mitochondria’s structure and function. We aim to determine the differences between the mitochondria isolated from the slime mold regarding its early developmental stages induced by starvation, namely the unicellular (U), aggregation (A) and streams (S) stages, at the bioenergetic and proteome levels. We measured the oxygen consumption of intact cells using the Clarke electrode and observed a distinct decrease in mitochondrial coupling capacity for stage S cells and a decrease in mitochondrial coupling efficiency for stage A and S cells. We also found changes in spare respiratory capacity. We performed a wide comparative proteomic study. During the transition from the unicellular stage to the multicellular stage, important proteomic differences occurred in stages A and S relating to the proteins of the main mitochondrial functional groups, showing characteristic tendencies that could be associated with their ongoing adaptation to starvation following cell reprogramming during the switch to gluconeogenesis. We suggest that the main mitochondrial processes are downregulated during the early developmental stages, although this needs to be verified by extending analogous studies to the next slime mold life cycle stages.

Assessing the necessity of a family of genes that encode small proteins in Dictyostelium discoideum development.

Dictyostelium discoideum’s genome encodes for a large class of small proteins that are developmentally regulated. We deleted six of the genes that encode these proteins to determine if they play an essential role in Dictyostelium discoideum development. Deletion of these genes had no significant effect on Dictyostelium discoideum development. These results suggest that the selected genes do not play an essential role in Dictyostelium discoideum development.

Investigating the Role of Translationally Control Tumor Protein in Growth, Development and Differentiation of Dictyostelium discoideum.

Translationally controlled tumor protein (TCTP) is a multifunctional protein implicated in various types of cellular processes involving growth and development of an organism. Here, we identified tctp gene in Dictyostelium discoideum and unraveled its function. The sequence analysis of D. discoideum TCTP (DdTCTP) showed its conservation among eukaryotes. Transcript of DdTCTP was highly expressed at the initial time points of development and protein is localized both in the cytoplasm and nucleus. Disruption of tctp was achieved by BSR cassette using double homologous recombination method. Abrogation of tctp resulted in reduced cell proliferation but increased cell size. Additionally, development was delayed by 4 h wherein small-sized aggregates and fruiting bodies were produced by tctp– cells while larger aggregates and fruiting bodies were produced by tctpOE cells concordant with the fact that TCTP regulates prestalk/prespore ratio and cell-type differentiation. tctp– cells produced round spores with reduced viability and stalk cells are arranged in septate pattern as compared to polyhedral manner of wild type. Abrogation of tctp resulted in aberrant localization of cell type specific markers and show low proclivity toward prespore/spore region, in presence of wild type cells.

Talin B regulates collective cell migration via PI3K signaling in Dictyostelium discoideum mounds

Collective cell migration is a key process during the development of multicellular organisms, in which the migrations of individual cells are coordinated through chemical guidance and physical contact between cells. Talin has been implicated in mechanical linkage between actin-based motile machinery and adhesion molecules, but how talin contributes to collective cell migration is unclear. Here we show that talin B is involved in chemical coordination between cells for collective cell migration at the multicellular mound stage in the development of Dictyostelium discoideum. From early aggregation to the mound formation, talB-null cells exhibited collective migration normally with cAMP relay. Subsequently, talB-null cells showed developmental arrest at the mound stage, and at the same time, they had impaired collective migration and cAMP relay, while wild-type cells exhibited rotational cell migration continuously in concert with cAMP relay during the mound stage. Genetic suppression of PI3K activity partially restored talB-null phenotypes in collective cell migration and cAMP relay. Overall, our observations suggest that talin B regulates chemical coordination via PI3K-mediated signaling in a stage-specific manner for the multicellular development of Dictyostelium cells.

A Heat Shock Protein 48 (HSP48) Biomolecular Condensate Is Induced during Dictyostelium discoideum Development.

The social amoeba Dictyostelium discoideum's proteome contains a vast array of simple sequence repeats, providing a unique model to investigate proteostasis. Upon conditions of cellular stress, D. discoideum undergoes a developmental process, transitioning from a unicellular amoeba to a multicellular fruiting body. Little is known about how proteostasis is maintained during D. discoideum's developmental process. Here, we have identified a novel α-crystallin domain-containing protein, heat shock protein 48 (HSP48), that is upregulated during D. discoideum development. HSP48 functions in part by forming a biomolecular condensate via its highly positively charged intrinsically disordered carboxy terminus. In addition to HSP48, the highly negatively charged primordial chaperone polyphosphate is also upregulated during D. discoideum development, and polyphosphate functions to stabilize HSP48. Upon germination, levels of both HSP48 and polyphosphate dramatically decrease, consistent with a role for HSP48 and polyphosphate during development. Together, our data demonstrate that HSP48 is strongly induced during Dictyostelium discoideum development. We also demonstrate that HSP48 forms a biomolecular condensate and that polyphosphate is necessary to stabilize the HSP48 biomolecular condensate.IMPORTANCE During cellular stress, many microbes undergo a transition to a dormant state. This includes the social amoeba Dictyostelium discoideum that transitions from a unicellular amoeba to a multicellular fruiting body upon starvation. In this work, we identify heat shock protein 48 (HSP48) as a chaperone that is induced during development. We also show that HSP48 forms a biomolecular condensate and is stabilized by polyphosphate. The findings here identify Dictyostelium discoideum as a novel microbe to investigate protein quality control pathways during the transition to dormancy.

A terpene synthase-cytochrome P450 cluster in Dictyostelium discoideum produces a novel trisnorsesquiterpene.

Terpenoids are enormously diverse, but our knowledge of their biosynthesis and functions is limited. Here we report on a terpene synthase (DdTPS8)-cytochrome P450 (CYP521A1) gene cluster that produces a novel C12 trisnorsesquiterpene and affects the development of Dictyostelium discoideum. DdTPS8 catalyzes the formation of a sesquiterpene discoidol, which is undetectable from the volatile bouquet of wild type D. discoideum. Interestingly, a DdTPS8 knockout mutant lacks not only discoidol, but also a putative trisnorsesquiterpene. This compound was hypothesized to be derived from discoidol via cytochrome P450 (CYP)-catalyzed oxidative cleavage. CYP521A1, which is clustered with DdTPS8, was identified as a top candidate. Biochemical assays demonstrated that CYP521A1 catalyzes the conversion of discoidol to a novel trisnorsesquiterpene named discodiene. The DdTPS8 knockout mutant exhibited slow progression in development. This study points to the untapped diversity of natural products made by D. discoideum, which may have diverse roles in its development and chemical ecology.

Poly(ADP-ribose) polymerase-1 (PARP-1) regulates developmental morphogenesis and chemotaxis in Dictyostelium discoideum.

Poly(ADP-ribose) polymerase-1 (PARP-1) has been attributed to varied roles in DNA repair, cell cycle, cell death, etc. Our previous reports demonstrate the role of PARP-1 during Dictyostelium discoideum development by its constitutive downregulation as well as by PARP-1 ortholog, ADP ribosyl transferase 1 A (ADPRT1A) overexpression. The current study analyses and strengthens the function of ADPRT1A in multicellular morphogenesis of D. discoideum. ADPRT1A was knocked out, and its effect was studied on cAMP signalling, chemotaxis and development of D. discoideum. We report that ADPRT1A is essential in multicellular development of D. discoideum, particularly at the aggregation stage. Genetic alterations of ADPRT1A and chemical inhibition of its activity affects the intracellular and extracellular cAMP levels during aggregation along with chemotaxis. Exogenous cAMP pulses could rescue this defect in the ADPRT1A knockout (ADPRT1A KO). Expression analysis of genes involved in cAMP signalling reveals altered transcript levels of four essential genes (PDSA, REGA, ACAA and CARA). Moreover, ADPRT1A KO affects prespore- and prestalk-specific gene expression and prestalk tendency is favoured in the ADPRT1A KO. ADPRT1A plays a definite role in regulating developmental morphogenesis via cAMP signalling. This study helps in understanding the role of PARP-1 in multicellular development and differentiation in higher complex organisms.

4-Methyl-5-Pentylbenzene-1,3-Diol Regulates Chemotactic Cell Aggregation and Spore Maturation Via Different Mechanisms in Dictyostelium discoideum.

4-Methyl-5-pentylbenzene-1,3-diol (MPBD), a product of the polyketide synthase SteelyA, is a signaling molecule that regulates Dictyostelium discoideum development. During early development, MPBD controls chemotactic cell aggregation by regulating the expression of genes in the cAMP signaling pathway; however, during culmination at late development, it induces spore maturation. In the present study, we analyzed the effects of MPBD, its derivatives, and a putative MPBD-derived metabolite on developmental defects in the MPBD-less stlA null mutant. Using structure-activity relationship studies, it was observed that in MPBD, the functional groups that were essential for induction of spore maturation were different from those essential for induction of cell aggregation. Dictyoquinone, a putative MPBD metabolite rescued the aggregation defect in stlA null mutant in early development, but not the spore maturation defect at the later stage. Our data suggest that MPBD regulates chemotactic cell aggregation and spore maturation via different mechanisms.

D-Serine Metabolism and Its Importance in Development of Dictyostelium discoideum.

In mammals, D-Ser is synthesized by serine racemase (SR) and degraded by D-amino acid oxidase (DAO). D-Ser acts as an endogenous ligand for N-methyl-D-aspartate (NMDA)- and δ2 glutamate receptors, and is involved in brain functions such as learning and memory. Although SR homologs are highly conserved in eukaryotes, little is known about the significance of D-Ser in non-mammals. In contrast to mammals, the slime mold Dictyostelium discoideum genome encodes SR, DAO, and additionally D-Ser specific degradation enzyme D-Ser dehydratase (DSD), but not NMDA- and δ2 glutamate receptors. Here, we studied the significances of D-Ser and DSD in D. discoideum. Enzymatic assays demonstrated that DSD is 460- and 1,700-fold more active than DAO and SR, respectively, in degrading D-Ser. Moreover, in dsd-null cells D-Ser degradation activity is completely abolished. In fact, while in wild-type D. discoideum intracellular D-Ser levels were considerably low, dsd-null cells accumulated D-Ser. These results indicated that DSD but not DAO is the primary enzyme responsible for D-Ser decomposition in D. discoideum. We found that dsd-null cells exhibit delay in development and arrest at the early culmination stage. The efficiency of spore formation was considerably reduced in the mutant cells. These phenotypes were further pronounced by exogenous D-Ser but rescued by plasmid-borne expression of dsd. qRT-PCR analysis demonstrated that mRNA expression of key genes in the cAMP signaling relay is perturbed in the dsd knockout. Our data indicate novel roles for D-Ser and/or DSD in the regulation of cAMP signaling in the development processes of D. discoideum.

Calcineurin Silencing in Dictyostelium discoideum Leads to Cellular Alterations Affecting Mitochondria, Gene Expression, and Oxidative Stress Response

Calcineurin is involved in development and cell differentiation of the social amoeba Dictyostelium discoideum. However, since knockouts of the calcineurin-encoding genes are not possible in D. discoideum it is assumed that the phosphatase also plays a crucial role during vegetative growth of the amoebae. Therefore, we investigated the role of calcineurin during vegetative growth in D. discoideum. RNAi-silenced calcineurin mutants showed cellular alterations with an abnormal morphology of mitochondria and had increased content of mitochondrial DNA (mtDNA). In contrast, mitochondria showed no substantial functional impairment. Calcineurin-silencing led to altered expression of calcium-regulated genes as well as mitochondrially-encoded genes. Furthermore, genes related to oxidative stress were higher expressed in the mutants, which correlated to an increased resistance towards reactive oxygen species (ROS). Most of the changes observed during vegetative growth were not seen after starvation of the calcineurin mutants. We show that impairment of calcineurin led to many subtle, but in the sum crucial cellular alterations in vegetative D. discoideum cells. As these alterations were not observed after starvation we propose a dual role for calcineurin during growth and development. Our results imply that calcineurin is one player in the mutual interplay between mitochondria and ROS during vegetative growth.

Overexpression of S-adenosylmethionine decarboxylase impacts polyamine homeostasis during development of Dictyostelium discoideum.

The polyamines putrescine, spermidine and spermine are essential polycations involved in the regulation of cellular proliferation. They exert dynamic effects on nucleic acids and macromolecular synthesis in vitro but their specific functions in vivo are poorly understood. Here, we have modulated the spermidine levels either by overexpressing the S-adenosylmethionine decarboxylase (samdc) gene or treating the cells with methylglyoxal-bis (guanylhydrazone) (MGBG), an inhibitor of SAMDC. In Dictyostelium, overexpression of SAMDC slowed cell proliferation, delayed development and arrested cells in the S-phase of the cell cycle. Treatment with MGBG reduced cell proliferation and stimulated development, but in samdcOE cells, it increased cell proliferation suggesting critical levels of spermidine to be important. In samdcOE cells, spermidine levels remained high throughout development but only small changes in the spermine levels were observed. Initial putrescine levels did increase but reverted to wild-type levels after the mound stage. As tight regulation of polyamine homeostasis is required, we identified genes that could be involved in its maintenance. In conclusion, we characterised samdcOE cells and observed the maintenance of polyamine homeostasis during the development of Dictyostelium cells.

Pheromones do regulate sexual development in Dictyostelium discoideum

Based on negative data, Douglas et al refuted earlier work by the O'Day laboratory on the role of pheromones in the sexual development of Dictyostelium discoideum. This letter addresses some of the issues with their study.

Evidence that differentiation-inducing factor-1 controls chemotaxis and cell differentiation, at least in part, via mitochondria in D. discoideum.

ABSTRACTDifferentiation-inducing factor-1 [1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one (DIF-1)] is an important regulator of cell differentiation and chemotaxis in the development of the cellular slime mold Dictyostelium discoideum. However, the entire signaling pathways downstream of DIF-1 remain to be elucidated. To characterize DIF-1 and its potential receptor(s), we synthesized two fluorescent derivatives of DIF-1, boron-dipyrromethene (BODIPY)-conjugated DIF-1 (DIF-1-BODIPY) and nitrobenzoxadiazole (NBD)-conjugated DIF-1 (DIF-1-NBD), and investigated their biological activities and cellular localization. DIF-1-BODIPY (5 µM) and DIF-1 (2 nM) induced stalk cell differentiation in the DIF-deficient strain HM44 in the presence of cyclic adenosine monosphosphate (cAMP), whereas DIF-1-NBD (5 µM) hardly induced stalk cell differentiation under the same conditions. Microscopic analyses revealed that the biologically active derivative, DIF-1-BODIPY, was incorporated by stalk cells at late stages of differentiation and was localized to mitochondria. The mitochondrial uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP), at 25–50 nM, and dinitrophenol (DNP), at 2.5–5 µM, induced partial stalk cell differentiation in HM44 in the presence of cAMP. DIF-1-BODIPY (1–2 µM) and DIF-1 (10 nM), as well as CCCP and DNP, suppressed chemotaxis in the wild-type strain Ax2 in shallow cAMP gradients. These results suggest that DIF-1-BODIPY and DIF-1 induce stalk cell differentiation and modulate chemotaxis, at least in part, by disturbing mitochondrial activity.

Glutathione S-transferase 4 is a putative DIF-binding protein that regulates the size of fruiting bodies in Dictyostelium discoideum

In the development of the cellular slime mold Dictyostelium discoideum, two chlorinated compounds, the differentiation-inducing factors DIF-1 and DIF-2, play important roles in the regulation of both cell differentiation and chemotactic cell movement. However, the receptors of DIFs and the components of DIF signaling systems have not previously been elucidated. To identify the receptors for DIF-1 and DIF-2, we here performed DIF-conjugated affinity gel chromatography and liquid chromatography–tandem mass spectrometry and identified the glutathione S-transferase GST4 as a major DIF-binding protein. Knockout and overexpression mutants of gst4 (gst4– and gst4OE, respectively) formed fruiting bodies, but the fruiting bodies of gst4– cells were smaller than those of wild-type Ax2 cells, and those of gst4OE cells were larger than those of Ax2 cells. Both chemotaxis regulation and in vitro stalk cell formation by DIFs in the gst4 mutants were similar to those of Ax2 cells. These results suggest that GST4 is a DIF-binding protein that regulates the sizes of cell aggregates and fruiting bodies in D. discoideum.

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells.

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.